Influence of vitamin E supplementation on nutritional statusand immune response in ethanol-fed mice and murine AIDS.

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Authors Wang, Yuejian.

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Influence of vitamin E supplementation on nutritional status and immune response in ethanol-fed mice and murine AIDS

Wang, Yuejian, Ph.D.

The University of Arizona, 1994

U·M·I 300 N. Zeeb Rd Ann Arbor, MI48106

INFLUENCE OF VITAMIN E SUPPLEMENTATION ON

NUTRITIONAL STATUS AND IMMUNE RESPONSE

IN ETHANOL-FED MICE AND MURINE AIDS

by

YUEJIAN WANG

A Dissertation Submitted to the Faculty of the

COMMIITEE ON NUTRITIONAL SCIENCES

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1994

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have

read the dissertation prepared by ___ Y~u~e~j~,~·a~n~W~a~n~g~ ____________________ __

entitled Influence of Dietary Vitamin E Supplementation on Nutritional

Status and Immune Response In Ethanol-Fed Mice and

Murine AIDS

and recommend that it be accepted as fulfilling the dissertation

Doctor of Philosophy

Rona 1 d R. Watson

lli v i d K. Y. Le i

Cl eamond D.

Harris Bernstein Date/ i

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I read this dissertation prepared under my

2

direction and requirement.

it be accepted as fulfilling the dissertation

Ro na 1 dR. Ha tso n Dissertation Dir

3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author.

SIGNE~~~

4

ACKNOWLEDGl\1ENTS

It would not have been possible to achieve my dream of earning a Ph.D and becoming a professional scientist without the assistance and support of a number of people. lowe a debt of gratitude and many thanks to them for helping to make my stay and learning experience enjoyable in Dr. Ronald Ross Watson's laboratory in the Department of the Family and Community Medicine.

First of all, I would like to express my deep gratitude to my advisor, Dr. Ronald R. Watson, for advising, understanding, supporting and guiding in the past years. He has provided continuing interest and support throughout my graduate research and his constant encouragement also helped this research run smoothly.

I wish to express my appreciation to Dr. David K. Y. Lei and Dr. Cleamond D. Eskelson for valuable advice and guidance in my graduate research. I also wish to express my appreciation to Dr. Harris Bernstein and Dr. Lynn Joens for serving as members of my committee and for their availability in time of need. In addition, my sincere thanks to Dr. Dennis S. Huang and Dr. Steve M. Wood for valuable discussion and their help in my entire research.

My research assistants should be acknowledged. My special thanks are extended to Mr. Bailin Liang, Mr. Paul T. Giger, Mr. Tony Stazzone, Ms. Andrea Herbert and Mr. Lee Murphy for their tremendous experimental support.

The guidance and support of the above individuals, and others too numerous to list here, made this work a pleasure.

5

DEDICATION

This dissertation is dedicated to all of my family: my wife, Li; my future

children, my parents, my parents-in-law, brothers, sisters-in-law and brothers-in-law

whose love and encouragement have been the major motivation for me in working toward

the Ph.D. degree. They have always been very supportive and understanding throughout

my life.

This dissertation is also specially dedicated to our American host family: Mr. and

Mrs. William and Hellen Arthur, whose love and caring enabled us to get through our

toughest time in the United States finishing this dissertation.

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

ABSTRACT

CHAPTER 1: REVIEW OF LITERATURE ................................ . Section I: The Animal Model of Human AIDS: Murine AIDS ..

Introduction ............................................. . Etiology of AIDS .................................... . Immune Functions in AIDS ........................... . Nutritional Status in AIDS ........................... . Conclusion .................................... .

Section II: Alcohol, Immune Response, Nutrition and AIDS .................................... .

Introduction ............................................. . Ethanol and Immune Response .......................... . Ethanol and Nutrition .......................... . Ethanol and AIDS ............................................. . Conclusion .................................... .

Section III: Vitamin E, Immune Response, Nutrition and AIDS .................................... .

Introduction .................................... . Vitamin E and Immune Response .......................... .. Vitamin E and Nutrition .......................... .. Vitamin E and AIDS Therapy .......................... .. Conclusion .................................... .

CHAPTER 2: ALTERATION OF CYTOKINE PRODUCTION, IMMUNE RESPONSE AND NUTRITIONAL STATUS I)~(J lVI~ l\IDS ............................................. . Abstract ................................................ . Introduction ...................................................... . Materials and Methods ............................................. .

LP-BM5 Murine Leukemia Virus Infection Preparation of Peritoneal Resident Macrophages Preparation of Splenocytes and Thymocytes

Page

14

18

22

24 24 24 25 28 32 35

36 36 36 39 42 44

45 45 45 47 47 49

50 50 52 56 56 . 56 56

6

TABLE OF CONTENTS (continued)

Preparation of T Cells from Splenocytes .................. . Cytokine Standards and Antisera .................. . Production of Cytokines ........................... . Preparation of IgG Production ........................... . ELISA for Cytokines ........................... . ELISA for IgG ........................... . Mitogenesis of Splenocytes and Thymocytes ............... . Measurement of Vitamin A and E ....................... . Serum Vitamin A and E .................................... . Measurement of Zinc and Copper ........................... . Statistics .................................... .

Results ............................................. . IL-6 and TNF-a Production by Peritoneal Macrophages ........................... . IL-2 and IFN--y Production by Thl cells ......... . IL-4,IL-5,IL-6 and IL-lO Production by Th2 Cell ......... . Mitogenesis of Splenocytes ........................... . Production of IgG ........................... . Mitogenesis of Thymocytes .................. . Cytokine Production by Thymocytes ......... . Hepatic Levels of Nutrients ........................... . Intestinal Levels of Nutrients ........................... . Splenic Levels of Nutrients ........................... . Thymic Levels of Nutrients ........................... . Serum Levels of Vitamins ........................... .

Discussion

CHAPTER 3: ALCOHOL CONSUMPTION SUPPRESSES CYTOKINE PRODUCTION AND IMMUNE RESPONSE

Page

57 57 58 58 58 59 60 60 61 61 61 62

62 62 62 62 62 62 63 63 64 64 64 64 65

IN C57BL/6 MICE ................................. 85 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Introduction . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 86 Materials and Methods . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 88

Animals and EtOH Diet .... ..... ......... . . . ..... ... .. ...... 88 Standard Cytokines and Their Antibodies ...... . ... ..... .... 88 Preparation of Splenocytes and Thymocytes . . . . . . . . . . 88 Preparation of Cytokine Production ...... .... . ........ 88

7

TABLE OF CONTENTS (continued) Page

Preparation of IgA and IgG Production . ..... .... ... .. . ... 89 Natural Killer (NK) Cell Activity . . ....... ..... . ..... . ....... 89 ELISA for Cytokines ............................ 89 ELISA for IgA and IgG .................................. '" 89 Mitogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Statistics . .... .... ...... .. .. .... ... . ... . . ... .. .. .. . .... 89

Results ......... ..... ..... .... ... ........... .... ....... ....... 90 Production of Splenic Cytokines ..... ..... ... . ..... 90 Production of IL-6 and TNF-a by Splenocytes ........ . . 90 Production of Thymic Cytokine . .... . . .. ..... ..... 90 Splenic NK Cell Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 IgA and IgG Production ............. ....... .... ... ... ....... 90 Mitogenesis of Splenocytes and Thymocytes . ...... . . . 90

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

CHAPTER 4: ALCOHOL CONSUMPTION MODULATION OF CYTOKINE PRODUCTION AND IMMUNE RESPONSE DURING MURINE AIDS ............................................. . Abstract Introduction ...................................................... . Materials and Methods .............................. .

Animals ...................................................... . EtOH Diets ...................................................... . LP-BM5 MuL V Infection .................................... . Cytokine Standards and Antisera .......................... .. Preparation of Splenocytes and Thymocytes ......... . Preparation of Cytokine Production .................. . ELISA for Cytokines .................................... . Mitogenesis of Splenocytes and Thymocytes ......... . Statistics ...................................................... .

Results ....................................... . Production of Cytokines by Splenocytes .................. . Production of Cytokines by Thymocytes .................. . Mitogenesis of Splenocytes and Thymocytes ......... . Production of Splenic Cytokine Prior to Retrovirus Infection

101 101 103 105 105 105 106 106 106 106 106 107 107 108 108 108 109

109

8

TABLE OF CONTENTS (continued)

Production of IgA and IgG Prior to Retrovirus Infection Mitogenesis of Splenocytes Prior to Retrovirus Infection Body and Thymus Weight Prior to Retrovirus Infection ........................... . Thymocyte Number and Proliferation Prior to Retrovirus Infection Production of Thymic Cytokines Prior to Retrovirus Infection

Discussion

CHAPTER 5: ALCOHOL CONSUMPTION ALTERS THE NUTRITIONAL STATUS DURING MURINE AID .................................... . Abstract ........................................................ . Introduction ...................................................... . Materials and Methods .................................... .

Animals and LP-BM5 Retrovirus Infection ......... . EtOH Diet ............................................. . Measurement of Tissue Vitamin A and E .......... .. Measurement of Serum Vitamin A and E ......... . Measurement of Tissue Zinc and Copper ......... . Statistics ............................................. .

Results ........................................................ . Nutrient Concentrations In the Liver .................. . Nutrient Concentrations in the Intestine .................. . Nutrient Concentrations in the Spleen .................. . Nutrient Concentrations in the Thymus .................. . Nutrient Concentrations in the Serum .................. .

Discussion

CHAPTER 6: VITAMIN E SUPPLEMENTATION NORMALIZES IMMUNE FUNCTIONS AND CYTOKINE PRODUCTION IN ALCOHOL-FED MICE .................................... . Abstract Introduction ...................................................... . Materials and Methods ................................... .

Page

110

110

111

111

111 113

130 130 131 133 133 133 133 134 134 134 135 135 135 135 135 135 137

143 143 144 146

9

TABLE OF CONTENTS (continued)

Animals ...................................... . Diet and Treatment ................... . Standard Cytokines and Their Antibodies ......... . Preparation of Splenocytes and Thymocytes ......... . Cytokine Production .................................... . IgA and IgG Production .................................... . ELISA for Cytokine Detection ............................ . ELISA for IgA and IgG .................................... . Mitogenesis of Splenocytes and Thymocytes ......... . Statistics ...................................................... .

Results ...................................................... . Production of IL-2 and IFN--y by Splenocytes ......... . Production of Cytokines by Splenocytes .................. . Production of IL-6 and TNF-a by Splenocytes ......... . IgA and IgG Production .................................... . Mitogenesis of Splenocytes ........................... . Production of Cytokines by Thymocytes ......... . Mitogenesis of Thymocytes ........................... .

Discussion ............................................. .

CHAPTER 7: VITAMIN E SUPPLEMENTATION ASSISTS IN THE RESTORATION OF UNDERNUTRITION AND IMMUNE DYSFUNCTIONS DURING MURINE AIDS ......... . Abstract Introduction Materials and Methods

Animals ...................................................... . Diet and Treatment .................................... . LP-BM5 Murine Leukemia Retrovirus Infection ......... . Tissue Vitamin A and E .................................... . Serum Vitamin A and E .................................... . Tissue Zinc and Copper .................................... . Preparation of Splenocytes ........................... . Standard Cytokines and Their Antibodies ......... . Cytokine production .................................... . ELISA Assays for Cytokines ............................... . Cytotoxicity for NK Cells .................................... .

Page

146 146 147 147 147 147 147 147 147 147 148 148 148 148 148 149 149 149 150

162 162 163 165 165 165 166 166 166 166 166 166 166 166 166

10

TABLE OF CONTENTS (continued)

Mitogenesis of Splenocytes Production of IgA and IgG Statistics

Results ...................................................... . Hepatic Levels of Nutrients ........................... . Intestinal Levels of Nutrients ........................... . Splenic Levels of Nutrients ........................... . Thymic Levels of Nutrients .......................... .. Serum Levels of Vitamin A and E ................. .. Production of IL-2 and IFN--y ........................... . Production of IL-4, IL-5 and IL-6 .................. . Production of IL-6 and TNF-a ................. .. ~ ~ell Activity .................................... . Mitogenesis of Splenocytes ........................... . Production of JgA and IgG .......................... ..

Discussion ............................................. .

~HAPTER 8: ALTERATION OF KINETI~S OF ~YTOKINE PRODU~TION BY VITAMIN E SUPPLEMENT A TION DURING MURINE AIDS .................................... . Abstract Introduction Materials and Methods

Animals Diet and Treatment .................................... . LP-BM5 Murine Leukemia Retrovirus Infection ......... . Tissue Vitamin E .................................... . Preparation of Splenocytes and Thymocytes ......... . Standard ~ytokines and Their Antibodies ......... . ~ytokine Production .................................... . ELISA for ~ytokine Detection ..................... .. Mitogenesis of Splenocytes and Thymocytes ......... . ELISA for IgG Detection ................................... . Statistics ............................................. .

Results ..................................................... . Body and Spleen Weights ........................... . Hepatic and Serum Levels of Vitamin E ......... .

Page

167 167 167 168 168 168 168 168 169 169 169 169 170 170 170 171

183 183 185 188 188 188 188 188 189 189 189 189 189 189 189 190 190 190

11

TABLE OF CONTENTS (continued)

Production of IL-2 and IFN-'Y ........................... . Production of IL-4, IL-5 and IL-6 .................. . Production of IL-6 and TNF-a ........................... . Mitogenesis of Splenocytes ........................... . Production of IgG ................................... . Thymus Weight ............................................. . Vitamin E in the Thymus and Serum .................. . Production of Thymic Cytokines .................. . Mitogenesis of Thymocytes ........................... .

Discussion ............................................. .

CHAPTER 9: MODULATION OF CYTOKINE SECRETION, IMMUNE RESPONSE AND NUTRITIONAL STATUS BY VARIOUS LEVELS OF VITAMIN E SUPPLEMENTATION DURING MURINE AIDS Abstract Introduction ...................................................... . Materials and Methods .................................... .

Animals ............................................. . Diet and Treatment .................................... . LP-BM5 Retrovirus Infection ........................... . Hepatic Vitamin A and E .......................... . Serum Vitamin E .................................... . Measurement of Zinc and Copper ........................... . Preparation of Splenocytes and Thymocytes ......... . Standard Cytokines and Their Antibodies .................. . Cytokine Production .................................... . ELISA for Cytokine Detection .......................... .. Mitogenesis of Splenocytes and Thymocytes ......... . NK Cell Cytotoxicity ........................... . IgA and IgG Production .................................... . ELISA for IgA and IgG .................................... . Statistics ............................................. .

Results ...................................................... . Body and Spleen Weights .................................... . Serum Vitamin A and E ........................... . Hepatic Nutrients .................................... .

Page

190 191 191 192 192 192 192 193 194 195

220 220 222 226 226 226 227 227 227 227 227 227 227 227 227 227 227 227 227 228 228 228 228

12

13

TABLE OF CONTENTS (continued) Page

Intestinal Nutrients .................................... . 229 Production of IL-2 and IFN-'Y ........................... . 230 Production of IL-6 and IL-1O ........................... . 230 Production of IL-6 and TNF-a ........................... . 231 Mitogenesis of Splenocytes ........................... . 231 NK Cell Cytotoxicity ................................... . 232 Production of IgA and IgG ........................... . 232 Thymus Weight .................................... . 232 Production of Cytokines by Thymocytes .................. . 232

Discussion ....................................................... . 235

CHAPTER 10: OVERALL CONCLUSIONS 261

REFERENCES 264

14

LIST OF FIGURES Page

FIG. 2.1 IL-6 and TNF-a Production by Macrophages ................... 75

FIG. 2.2 IL-2 and IFN--y Production by Splenic T cells ................... 76

FIG. 2.3 IL-4 and IL-5 Production by Splenic T Cells ................... 77

FIG. 2.4 IL-6 and IL-lO Production by Splenic T cells ................... 78

FIG. 2.5 IL-2 and IL-4 Production by Thymocytes ................... 79

FIG. 2.6 IL-6 and IFN--y Production by Thymocytes ................... 80

FIG. 3.1 Effect of Dietary EtOH on Splenocyte and Thymocyte Proliferation ..................................... 100

FIG. 4.1 Effects of Dietary EtOH on Splenocyte and Thymocyte Proliferation During Retrovirus Infection .... . . .. .. . . .. . . ... 127

FIG. 4.2 Effect of EtOH Consumption Prior to Retrovirus Infection on ConA-Induced Splenic Proliferation ............................ 128

FIG. 4.3 Effect of EtOH Consumption Prior to Retrovirus Infection on LPS-Induced Splenic Proliferation ............................ 129

FIG. 6.1 Effect of Vitamin Eon ConA-Induced Splenocyte Proliferation From EtOH-Fed Mice ............................ 159

FIG. 6.2 Effect of Vitamin Eon LPS-Induced Splenocyte Proliferation From EtOH-Fed Mice ............................ 160

FIG. 6.3 Effect of Vitamin E on Con A-Induced Thymocyte Proliferation.From EtOH-Fed Mice ............................ 161

FIG. 7.1 Effect of Vitamin E on NK Cell Activity During Retrovirus Infection . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 180

15

LIST OF FIGURES (continued) Page

FIG. 7.2 Effect of Vitamin E on Con A-Induced Splenocyte Proliferation During Retrovirus Infection .. .. .. .. .. .. .. .. .. .. .. .. .. .. 181

FIG. 9.3 Effect of Vitamin E Supplementation on LPS-Induced Splenocyte Proliferation During Retrovirus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182

FIG. 8.1 Effect of Vitamin E Supplementation on Kinetics of IL-2 Production By Splenocytes During Retrovirus Infection . .... .... ... . .. .... 206

FIG. 8.2 Effect of Vitamin E Supplementation on Kinetics of IFN--y Production By Splenocytes During Retrovirus Infection .... .. ... . .... .... . 207

FIG. 8.3 Effect of Vitamin E Supplementation on Kinetics of IL-4 Production By Splenocytes During Retrovirus Infection .... .. . . ... . .. . .... 208

FIG. 8.4 Effect of Vitamin E Supplementation on Kinetics of IL-5 Production By Splenocytes During Retrovirus Infection . . .. . . . . . . . . . . . . . . . 209

FIG. 8.5 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production By Splenocytes During Retrovirus Infection ................... 210

FIG. 8.6 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production By Splenocytes During Retrovirus Infection ................... 211

FIG. 8.7 Effect of Vitamin E Supplementation on Kinetics of TNF-a Production By Splenocytes During Retrovirus Infection ................... 212

FIG. 8.8 Effect of Vitamin E Supplementation on Kinetics of Con A-Induced Splenocyte Proliferation During Retrovirus Infection .......... 213

FIG. 8.9 Effect of Vitamin E Supplementation on Kinetics of LPS-Induced Splenocyte Proliferation During Retrovirus Infection .......... 214

FIG. 8.10 Effect of Vitamin E Supplementation on Kinetics of IL-2 Production by Thymocytes During Retrovirus Infection ................... 215

FIG. 8.11 Effect of Vitamin E Supplementation on Kinetics of IL-4 Production by Thymocytes During Retrovirus Infection ................... 216

LIST OF FIGURES (continued) Page

FIG. 8.12 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production by Thymocytes During Retrovirus Infection ................... 217

FIG. 8.13 Effect of Vitamin E Supplementation on Kinetics of IFN--y Production by Thymocytes During Retrovirus Infection ................... 218

FIG. 8.14 Effect of Vitamin E Supplementation on Kinetics of ConA-Induced Thymocyte Proliferation During Retrovirus Infection ................... 219

FIG. 9.1 Effects of Vitamin E Supplementation on Body and Spleen Weights During Retrovirus Infection .. ..... ..................... ...... ... 242

FIG. 9.2 Effects of Various Levels of Vitamin E Supplementation on Levels of Serum Vitamin A and E During Retrovirus Infection . .. . . . . . . . . . . . . . . . . 243

FIG. 9.3 Effects of Various Levels of Vitamin E Supplementation on Hepatic Vitamin A and E During Retrovirus Infection· . . .. . . .. . . . . . . . . . . . . . . . . . . . . 244

FIG. 9.4 Effects of Various Levels of Vitamin E Supplementation on Hepatic Copper and Zinc During Murine AIDS . . .. .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 245

FIG. 9.5 Effects of Various Levels of Vitamin E Supplementation on Intestinal Vitamin A and E During Retrovirus Infection . . . .. . . . . . . . . . . . . . . . . . . . . . . . 246

FIG. 9.6 Effects of Various Levels of Vitamin E Supplementation on Intestinal Zinc and Copper During Retrovirus Infection ............................ 247

FIG. 9.7 Effects of Various Levels of Vitamin E Supplementation on IL-2 and IFN--y Production by Splenocytes During Retrovirus Infection .......... 248

FIG. 9.8 Effects of Various Levels of Vitamin E Supplementation on IL-6 and IL-lO Production by Splenocytes During Retrovirus Infection .......... 249

FIG. 9.9 Effects of Various Levels of Vitamin E Supplementation on IL-6 and TNF-a Production by Splenocytes During Retrovirus Infection .......... 250

FIG. 9.10 Effects of Various Levels of Vitamin E Supplementation on Splenocyte Proliferation During Retrovirus Infection . . . ... . . . . . . . . . . . . . 251

16

17

LIST OF FIGURES (continued) Page

FIG. 9.11 Effects of Various Levels of Vitamin E Supplementation on NK Cell Activity During Retrovirus Infection ......... ..... . .. ... . .. . .... . 252

FIG. 9.12 Effects of Various Levels of Vitamin E Supplementation on IgA and IgG Production by Splenocytes During Retrovirus Infection . .... .. . .. 253

FIG. 9.13 Effects of Various Levels of Vitamin E Supplementation on Thymus Weight During Retrovirus Infection ............................ 254

FIG. 9.14 Effects of Various Levels of Vitamin E Supplementation on IL-2 Production by Thymocytes During Retrovirus Infection ... " .......... '" . . 255

FIG. 9.15 Effects of Various Levels of Vitamin E Supplementation on IL-4 Production by Thymocytes During Retrovirus Infection .......... 256

FIG. 9.16 Effects of Various Levels of Vitamin E Supplementation on IL-6 Production by Thymocytes During Retrovirus Infection .......... ....... .. 257

FIG. 9.17 Effects of Various Levels of Vitamin E Supplementation on IL-lO Production by Thymocytes During Retrovirus Infection ............ .. . .... 258

FIG. 9.18 Effects of Various Levels of Vitamin E Supplementation on IFN-'Y Production by Thymocytes During Retrovirus Infection . ...... .. . ... ... . .. 259

FIG. 9.19 Effects of Various Levels of Vitamin E Supplementation on Thymocyte Proliferation During Retrovirus Infection ... .. ... .. .. .. . .. . . 260

18

LIST OF TABLES Page

TABLE 1.1 Comparison of Immune Changes Between Human and Murine AIDS . . ... .. ...... ...... . .. ... ....... ... . .. . ..... . . . ... .. . .. 33

TABLE 2.1 Splenocyte Proliferation Induced By Con A During Murine AIDS ... . ..... . .... ......... .. . . ..... .... . .. ... .... . ... . . .. . 81

TABLE 2.2 Splenocyte Proliferation Induced By LPS During Murine AIDS . .... ..... . ............ ... . . .. .. . . ... . . . . .. ... . . . ... ... 81

TABLE 2.3 IgG Production During Murine AIDS 82

TABLE 2.4 Thymocyte Proliferation During Murine AIDS 82

TABLE 2.5 Hepatic Levels of Nutrients During Murine AIDS 83

TABLE 2.6 Intestinal Levels of Nutrients During Murine AIDS 83

TABLE 2.7 Splenic Levels of Nutrients During Murine AIDS 83

TABLE 2.8 Thymic Levels of Nutrients During Murine AIDS 84

TABLE 2.9 Serum Levels of Vitamin A and E During Murine AIDS ............... ..... ......... .... .. ... ... ... ... 84

TABLE 3.1 Effect of Dietary EtOH on Production of Cytokines By Splenocytes .. .. .. .. .... .... .. .. .... ........ .... . 98

TABLE 3.2 Effect of Dietary EtOH on IL-6 and TNF-O! by Splenocytes ...... .. .......... ...... .... ........ . 98

TABLE 3.3 Effect of Dietary EtOH on Production of Cytokines By Thymocytes ........ ...... .......... .... .. ...... . 99

TABLE 3.4 Effect of Dietary EtOH on Splenic NK Cell Activity .... ........ ..... . . . . . . . . ..... . .... ... ... .... .. ... . .. . . 99

TABLE 3.5 Effect of Dietary EtOH on Production of IgA and IgG By Splenocytes ........ ...... ........ .. ...... ....... 99

LIST OF TABLES (continued)

TABLE 4.1 Effect of EtOH on Production of Cytokines by Splenocytes During Retrovirus Infection

TABLE 4.2 Effect of EtOH on IL-6 and TNF-Q! by Splenocytes During Retrovirus Infection

TABLE 4.3 Effect of EtOH on Production of Cytokines by Thymocytes During Retrovirus Infection

TABLE 4.4 Effect of EtOH Consumption Prior to Retrovirus Infection

Page

121

121

122

on Production of IL-2 and IFN-'Y by Splenocytes ................... 122

TABLE 4.5 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-5, IL-6 and IL-lO by Splenocytes .......... 123

TABLE 4.6 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-6 and TNF-Q! by Splenocytes .. .. . .... ... . . . . . .. 123

TABLE 4.7 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IgA and IgG by Splenocytes ................... 124

TABLE 4.8 Effect of EtOH Consumption Prior to Retrovirus Infection on Body and Thymus Weight ..................................... 124

TABLE 4.8 Effect of EtOH Consumption Prior to Retrovirus Infection on Number of Thymocytes and Its Proliferation ................... 125

TABLE 4.9 Effect of EtOH Consumption Prior to Retrovirus Infection on IL-2 and IL-4 by Thymocytes ..................................... 125

TABLE 4.10 Effect of EtOH Consumption Prior to Retrovirus Infection on IL-6 and IFN-'Y by Thymocytes ............................ 126

TABLE 5.1 Effect of EtOH on Hepatic Nutrient Levels in Murine AIDS ....................................................... 140

19

20

LIST OF TABLES (continued) Page

TABLE 5.2 Effect of EtOH on Intestinal Nutrient Levels in Murine AIDS ....................................................... 140

TABLE 5.3 Effect of EtOH on Splenic Nutrient Levels in Murine I\.IDS ....................................................... 141

TABLE 5.4 Effect of EtOH on Thymic Nutrient Levels in Murine AIDS ....................................................... 141

TABLE 5.5 Effect of EtOH on Serum Vitamin A and E Levels in Murine AIDS .............................................. 142

TABLE 6.1 Effect of Vitamin E on IL-2 and IFN-'}' Production by Splenocytes From Ethanol-Fed Mice ............................ 156

TABLE 6.2 Effect of Vitamin Eon IL-4, IL-5, IL-6 and IL-lO Production by Splenocytes From Ethanol-Fed Mice ................... 156

TABLE 6.3 Effect of Vitamin Eon IL-6 and TNF-O! Production by Splenocytes From Ethanol-Fed Mice ................... 157

TABLE 6.4 Effect of Vitamin E on Immunoglobulin Production by Splenocytes From Ethanol-Fed Mice ............................ 157

TABLE 6.5 Effect of Vitamin E on IL-2, IL-4, IL-6 and IFN-,}, Production by Thymocytes From Ethanol-Fed Mice ................... 158

TABLE 7.1 Effect of Vitamin E on Hepatic Nutritional Levels During Retrovirus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

TABLE 7.2 Effect of Vitamin E on Intestinal Nutritional Levels During Retrovirus Infection 175

TABLE 7.3 Effect of Vitamin E on Splenic Nutritional Levels During Retrovirus Infection ... .... ..... .. ............ .... . ..... . 176

TABLE 7.4 Effect of Vitamin E on Thymic Nutritional Levels During Retrovirus Infection ... . .... .... . . ......... .... .... ... ... 176

21

LIST OF TABLES (continued) Page

TABLE 7.S Effect of Vitamin E on Serum Vitamin Levels During Retrovirus Infection . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

TABLE 7.6 Effect of Vitamin E on Production of IL-2 and IFN--y During Retrovirus Infection . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

TABLE 7.7 Effect of Vitamin E on Production of IL-4, IL-S and IL-6 During Retrovirus Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

TABLE 7.8 Effect of Vitamin E on Production of IL-6 and TNF-a During Retrovirus Infection . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

TABLE 7.9 Effect of Vitamin E on Immunoglobulin Production During Retrovirus Infection . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

TABLE 8.1 Effect of Vitamin E Supplementation on Spleen Weight During Murine AIDS ......... . . . .. . .. ..... .. .. .... . .. .. ... . .. .... . 203

TABLE 8.2 Effect of Vitamin E Supplementation on Hepatic Vitamin E Level During Murine AIDS .............. ........ ........ .. .. .. . 203

TABLE 8.3 Effect of Vitamin E Supplementation on Serum Vitamin E Level During Murine AIDS .... .... .. ...... ... .... .... .... ... ... 204

TABLE 8.4 Effect of Vitamin E Supplementation on IgO Production During Murine AIDS .......................................... " . . 204

TABLE 8.S Effect of Vitamin E on Thymus weight During Murine AIDS ... ......... ... . .. . ... ..... .... .. . .. ...... ... . 20S

TABLE 8.6 Effect of Vitamin E Supplementation on Thymic Vitamin E Level During Murine AIDS ............. ... . .... .. . .. .. 20S

22

ABSTRACT

LP-BM5 murine leukemia retrovirus infection in C57BL/6 mice rapidly produces

murine AIDS with many functional similarities to human AIDS, including progressive

lymphoprolifertaion and increasing severe immunodeficiency. The present studies

indicated that retrovirus infection induces immune dysfunctions via modulating the

cytokine production, and affects the thymus, producing altered T cell differentiation via

the dysregulation of thymocyte cytokine secretion. In addition, retrovirus infection can

directly cause malnutrition, possible via damaging gastrointestinal cells, thereby leading

to malabsorption. Such malnutrition has the theoretical potential to accelerate

development of AIDS via immunosuppression secondary to nutritional deficiency.

Chronic ethanol consumption in the mice altered the cytokine release, and impaired

immune response, and disrupted T cell maturation, which increase host susceptibility to

infection. Chronic ethanol consumption may be one of the co-factors accelerating

development of human AIDS after retrovirus infection. The results from this study

suggest that dietary ethanol, upon retrovirus infection or prior to retrovirus infection,

aggravates progression of immune dysfunction and affects T cell maturation in the

thymus, leading to AIDS as dietary ETOH modifies production of immunological

regulatory cytokines by splenocytes and thymocytes. Furthermore, ethanol can directly

aggravate undernutrition initiated by retrovirus infection. Such ethanol-induced

malnutrition in AIDS may also be a cofactor, accelerating development of AIDS via

immunosuppression secondary to nutritional deficiencies. Vitamin E supplementation

enhances immune responses. The immunostimulatory nature of vitamin E does provide

23

a basis for its use in the modulation of the various cell components and immune

functions, and its consequent therapeutic use during AIDS and alcoholics. The findings

in the study clearly demonstrated that dietary vitamin E supplementation can modulate

dysregulation of cytokines initiated by dietary EtOH and restore immune dysfunctions

induced by EtOH ingestion. The potential therapeutics of vitamin E supplementation

for AIDS treatment has also been determined in this study. Vitamin E supplementation,

even at extremely high levels, can help to restore levels of tissue nutrients, cytokine

dysregulation and some immune dysfunctions initiated by retrovirus infection during

murine AIDS. Thus, the vitamin E supplementation may provide additional therapeutic

approaches for treatment of HIV infected patients or alcoholics without additional

immunotoxicity.

Chapter 1: REVIEW OF LITERATURE

SECTION I: THE ANIMAL MODEL OF HUMAN AIDS: MURINE AIDS

Introduction

24

Acquired immune deficiency syndrome (AIDS) is a clinical disorder representing

the end point in a progression sequence of immune suppressive changes that render the

body highly susceptible to life-threatening tumors and opportunistic infections. AIDS has

been identified as a major public health priority in the United States with heavy social

and economic impact. By mid-1990, approximately 140,000 children and adults in the

United States had developed AIDS. The Centers for Disease Control reported that

85,430 persons died from this condition as of June 1990 (1). Projections of U.S. health

authorities estimated in 1990 that by the end of 1991, a total of 300,000 to 485,000 of

AIDS cases would be found in the United States with the number of deaths increasing

at a proportional rate (2). In addition, 1 to 1.5 million persons are reported to be

infected with the human immunodeficiency virus (HIV) as of mid-1990 (3). The unique

pathogenicity of HIV, its long latent period and the infection, the AIDS epidemic has

steadily increased in the United States. The cases of AIDS in the first three months in

1993 are three times more than that reported for the same period last year. Immune and

other physiological defects induced by HIV infection appear to be progressive and

irreversible with a high mortality rate that approaches 100% (4). Therefore, there are

pressing needs to understand immunopathogenesis of HIV, cofactor involvement in the

disease, anti-retroviral drug testing, and immunoenhancing nutritional therapy. Ethical,

economical and logistical considerations, however, have limited these AIDS studies in

25

humans.

Animal model systems for AIDS that simulate many aspects of the human

diseases, yet have the benefits of large numbers of subjects, low cost, and a well-defined

immunological system, are critically needed for mechanistic studies, cofactor in

development, and AIDS therapeutics. There are no doubts that knowing in one model

system the precise cellular and molecular mechanisms that lead to immunodeficiency

would represent a considerable step forward in the understanding of retrovirus-induced

immunodeficiency in general. Retroviruses appear to be widespread, and have been

isolated from fish, mammals, birds, and reptiles. However, the most promising

opportunities to study AIDS and its related therapeutics is murine AIDS, which is caused

by LP-BM5 murine leukemia retrovirus (MuLV) infection in a genetically susceptible

strain female C57BL/6 mice. Recent advances on murine AIDS further strengthen it as

an excellent animal model for study of the human AIDS. The similarities between

murine AIDS and the early stage of human AIDS are striking in many respects.

Etiology of AIDS

The family of Retroviridae is characterized by RNA containing viruses which

replicates via a DNA intermediate that is integrated with the host genome. Animal

retrovirus-induced diseases are usually not associated with cancer, but instead they are

manifested as nonneoplastic disorders which may include chronic degenerative conditions

or entirely pathogenic events. Immunosuppression, associated with certain retrovirus

mutants, confers a significant increase survival value of the virus in the host. There are

at least four mechanisms by which human and murine retrovirus modulate the host

26

immune apparatus: 1) direct lysis or functional impairment of virus-infected lymphoid

cells as consequence of viral replication; 2) activation and release of novel soluble

mediators of viral and/or host origin from virus-infected cells; 3) damage to host cellular

defense mechanisms involved in phagocytosis, antigen presentation, and nonspecific

effector aspects of cell-mediated immunity by virus infection; 4) disruption of the

immunoregulatory cytokine balance normally sustained by T suppressor or helper cell

cytokine production.

The virus mixture, LP-BM5 MuLV, was originally isolated by Latarjet and

Duplan and produces non-neoplastic lymphoproliferative disease in adult mice (5). LP­

BM5 MuL V was obtained from a non-thymic lymphoma which had been induced by

irradiation of mice. Then bone marrow stromal cells were isolated from the infected

mice and grown as cell lines yielded the agent. The cluster of MuL V viruses includes:

1) a 4.8-kbp replication-defective BM5d, 2) a B-tropic mink cell focus-inducing virus

(MCFV), and 3) a replication-competent B-tropic ectropic virus (BEV). None of these

MuLV isolates induced murine AIDS. The immunological disorders are apparently

induced with defective-genome BMSd, but not after infection by BEV and MCFV which

serve to facilitate the transmission of BM5d defective virus in mouse tissues. The murine

major histocompatibility complex has been shown to influence the development of murine

AIDS and the extent of virus spread in adult mouse (6). This murine retrovirus infection

provokes an enlargement of lymphoid organs, primarily spleen and lymph nodes.

Although lentiviruses are associated with human AIDS and C-type retroviruses appear

to be the causative agents in murine AIDS, both are very similar in that they cause

27

profound immunosuppression as well as reduced resistance to opportunistic pathogens and

neoplasia in their respective hosts. It is well established that the development of disease

reflects complicate interactions among cells of the immune system. The major immune

cell target for LP-BM5 MuLV is B cells and macrophage, which is also a major target

for HIV. CD4 + T cells and B cells are required for the development of murine AIDS

infection and the associated immunosuppression (7,8). Using molecular hybridization

techniques and molecular cloning, two studies recently have identified that a defective

viral DNA of 4.8-kbp was pathogenic (9,10). Sequencing of this 4.8-kbp defective viral

genome revealed a unique structure: the gag gene, normally found in helper MuLV, was

conserved but the pol and env genes were largely deleted (9). The single long open

reading frame found was in the gag gene and could encode a putative Pr6~18 protein,

which has been suggested to playa key role in murine AIDS pathogenesis (11). Pr6O:18,

which would be present on the surface of virus-infected B cells, would stimulate

polyclonal activation of helper T cells, leading to aberrant the production of cytokines

(11). It is hypothesized that Pr6~ag, acting as a superantigen, is different from other

antigens thus its presence would eventually lead to defect of the entire immune system

instead of induction of a more usual immune response. It may also stimulate some

primary proliferation of infected target cells in the very early stage of LP-BM5 MuLV

infection. Alternatively, immunodeficiency may arise as a paraneoplasmic syndrome, and

be consequence of the proliferation of the virus infected cells (12,13). Infection of these

cells could lead to an increased cytokine production that is detrimental to the immune

response, or less likely, to a decreased production of a factor essential to keep intact the

28

immune system. Alternatively the infected target cells, possible through the Pr6()1lag,

could themselves interact directly or indirectly with other immune cells of and lead to the

entirely immunodeficiency.

Immune Functions in AIDS

HIV infection in humans progresses to significant immunosuppression, as does

the LP-BM5 MuLV infection in mice (14). The role of T cells is critical to AIDS

development in humans. HIV causes abnormalities in the immune system by depleting

the CD4 + T helper cells, and by changing their functions (14). In addition, mv

activates B cells and infects macrophages which can be critical to antigen presentation

(15). The pathogenesis of LP-BM5 MuLV is characterized by T and B cell dysregulation

with many changes similar to those reported in human HIV infection (15). While T

cells are not the main source of viral replication and not viral components or the various

mixtures directly immunosuppressive, they are essentially required for early pathogenesis

and diseases (16). The temporal sequence of T-cell function changes is similar between

the two diseases, with helper T cell function lost well before dysfunctions of

cytotoxic/suppressor T cells (17). The quantitative loss of overall T-cell function occurs

far in advance of the reduction in CD4 + T helper cell numbers (17); however, the extent

of T cell depletion varies from organ to organ. Although T helper cell numbers stay

relatively constant during early stages of infection, it has been proposed that CD4 + T cell

populations shift from a heterogenous phenotype, characteristic of a mixed population of

naive and memory cells, to a more homogenous phenotype that broadly shares

characteristics of both memory and activated cells (18). The initial resting

29

immunocompetent CD4 + T cell population, in other words, is replaced by a polyclonal

population described as being in an anergic state (18). The state of anergy has been

demonstrated to be related to defects in the signal transduction pathway (PKC, calcium

mobilization, and phospholipase C ) induced by LP-BM5 MuLV during murine AIDS

(18-21). Furthermore, the state of anergy is apparently induced by a soluble, diffusible,

LP-BM5 MuL V -encoded superantigen-like component that is capable of stimulating most

CD4 + T cells (18,19). However, murine AIDS does not result in the generalized

depression of bone marrow function nor the death of infected T -helper cells, as occurs

in human AIDS.

Both viruses induce an early phase of B cell hyperactivity and polyclonal

activation (17). B cells and their production of immunoglobulin (lg) show a significant

number of alterations due to retroviral infection in man (14). Activation of both T and

B lymphocytes can be detected within one week of infection, as judged by flow

cytometric analysis of size of T and B cells and measurements of the percentage of B

cells secreting IgM. There is an absolute increase in the number of B cells in the spleen

and lymph nodes, and a three-fold increase in the fraction of B cells in cell cycle (16).

While T cells from LP-BM5 MuLV-infected mice will not provide helper activity to

normal B cells for a specific antibody response in vitro, they do stimulate elevated

polyclonal IgM secretion by normal B cells (16). During the early part of the LP-BM5

MuL V infection the B cells produce large amounts of Igs with increased number of

plasma or Ig-producing cells. There is widespread, polyclonal activation of B cells (22).

The cells become unresponsive to new antigens, either due to B or T cell defects. The

30

later stages of the murine AIDS-like disease involve continued expansion of members of

the B lymphocyte lineage including all stages from pre-B cells to plasma cells, increased

numbers of the monocyte-macrophage lineage, and appearance of immature T cells in the

periphery (1). Natural killer (NK) cell function is also reduced early in both diseases,

but lymphokine-activated killer cell (LAK) function is maintained for 8-10 weeks after

initiation of LP-BM5 MuLV infection (18). Number and function of macrophages are

suppressed in both diseases. The similarities between human and murine AIDS are

summarized in Table 1.1.

Cytokines mediate a variety of biological and physiological processes providing

an interactive complexity of potentially immunomodulating agents which integrate cellular

and humoral function within higher mammals. This network of pleiotropic cell

regulators are secreted primarily from immunocompetent cells (23). They playa crucial

role in transmitting and regulating signals for proliferation, differentiation, and

expression of cellular function in a variety of targets especially in relation to immune

responses. Target cell functions are usually affected by the interactions of one or more

of these regulatory molecules. Cytokine dysfunction may be a key component to induce

AIDS in retrovirus-infected subjects. Interleukin 2 (IL-2) is a pivotal cytokine for the

growth and differentiation of T and B cells, and for the enhancement of B cell growth

and Ig production (24). It is secreted mainly by the Th1 subset of activated T helper

cells. In addition to T cell proliferation, subsequent studies have shown that B cells, NK

cells (25), and LAK cells also respond to IL-2 (25). IL-4, IL-5 and IL-6 are synthesized

predominantly by stimulated T cells (26, 27). IL-4, IL-5 and IL-6, respectively, promote

31

the activation, growth, and differentiation ofB lymphocytes into antibody-secreting cells,

but have also been found to have various effects on T cell activation and growth (25,28-

35). IL-10 is produced during a variety of immune activated conditions by ThO and Th2

cell subsets as well as by monocytes, macrophages, and B cells (36). It suppresses Th1

cytokine production (IL-2, IFN-'Y, and lymphotoxin) and antigen-specific proliferation

of Th1 cell subsets after activation in an accessory cell-dependent manner. It also

diminishes delayed-type hypersensitivity reactions and other Th 1 cell-mediated responses.

Acting indirectly, IL-10 suppresses the capacity of certain accessory cells to promote Th 1

cell development (32). Furthermore, it suppresses murine macrophage functions such

as the inhibition of macrophage-mediated immunity and the production of reactive

nitrogen oxides which are involved in the elimination of intra- and extra-cellular

parasites. Interferon-gamma (IFN-'Y) is a multiple immunoregulatory cytokine that is

secreted primarily by activated T lymphocytes (CD4 +, CD8+, and CD4-8-), cells of the

monocyte lineage, and NK cells (37-40). The production of IFN-'Y is modulated by the

simultaneous production of IL-1, IL-2, IL-4, and tumor necrosis factor-cy (TNF). It

primes macrophages for microbicidal and tumoricidal activity and enhances NK

cytotoxicity (41,42). Moreover, its functions include the inhibition of some IL-4-induced

B cell activations, the modulation of B cell differentiation, the augmentation of Ig

secretion in B lymphocytes (43-45), and the up-regulation of expression of the secretory

component required for binding and transport of secreted IgA and IgM (46).

Furthermore, it down-regulates cytokine production (IL-4, IL-6, and IL-lO) and cell

proliferation of Th2 cell clone. Finally, TNF is a cytokine that express activities

32

including inflammation and anti-tumor activity (42,47,48). Activated

monocytes/macrophages are the primary source of TNF-~ (49,50). The production of

TNF is regulated by IFN-')' released from stimulated T cells (51). Thus, the cytokine

production by immune cells will be a major focus in this research project.

Nutritional Status in AIDS

Since progression to AIDS is often complicated by various nutritional disorders,

the additional immune dysfunction due to undernutrition may have precluded recovery

from infectious events which earlier represented survivable episodes. This hypothesis

is based on findings that malnutrition has been associated with immunological dysfunction

(52, 53); development of infectious processes (97,99) and vital organ dysfunction (55).

Thus, the severe malnutrition seen in patients with AIDS may set up a vicious cycle in

which the underlying immunological defects related to HIV infection are accentuated by

the malnutrition-induced immune dysfunction.

Although the roles of vitamins and minerals in the clinical manifestation of HIV

infection has not been well defined (56), a growing number of studies have suggested

important links between vitamins or minerals and HIV infection (57). Vitamin B12 and

B6 deficiencies are common in patients with HIV infection (59-62). The patients at

various stages of HIV disease has serum vitamin A and E deficiencies (63-65).

Malabsorption secondary to HIV-induced intestinal dysfunction appears to be the likely

case of vitamin A deficiency, but no further study about pathogenesis has been reported.

The case of vitamin E deficiency is unclear, although it may relate to the malabsorption

associated with worsening HIV infection. A recent study which found that a major

33

TABLE 1.1 Comparison of Immune Changes Between Human and Murine AIDS

Immunological Parameters Retrovirus Infection with HIV LP-BM5 MuLV

Abnormalities of T-Lymphocytes

T -helper (Th) cell functions decreased decreased

Numbers of CD4 + (Th) cells decreased variable

Spontaneous proliferation increased increased

Abnormalities of B-Lymphocytes

Responsiveness to antigens from immunization and infection decreased decreased

Immunoglobulin production increased increased

Spontaneous proliferation increased increased

Abnormalities of Macrophages

Numbers of activated macrophages variable decreased

Numbers of macrophages decreased decreased

Abnormalities of Natural Killer Cells

Tumor cell cytotoxicity decreased decreased

Abnormalities of Cytokine Release

IL-2 and IFN-'Y decreased decreased

IL-4, IL-5 and IL-lO increased increased

IL-6 and TNF-cx increased increased

Modified from Review by Dr. R.R. Watson (58).

34

percentage of patients with AIDS (50%), ARC (58%) and HIV (38%) had a vitamin E

intake of less than 50% the Recommended Daily Allowance (56). Zinc deficiency in

HIV infection has been identified consistently (65-68). One study reported marginal

copper deficiency in 22 % of their AIDS patients (69). Selenium deficiency also occurs

in AIDS-related malnutrition, though its mechanism has not been determined (69, 70).

Thus, patients with HIV infection can be increasingly compromised nutritionally as the

disease progresses. Indeed, it has been suggested that HIV infection might play

pathogenic role in gastrointestinal cells. In situ hybridization studies have localized HIV

infection in various types of epithelial cells of the bowel mucosa (71, 72). HIV infection

of gastrointestinal cell lines has been documented in vitro (73-75). Since the lymphoid

tissue, the main target of HIV, is present throughout of the gastrointestinal tract, and

CD4-related receptors on the epithelial cells have been demonstrated (76), it is

conceivable that AIDS patients have dysfunction of some portion of gastrointestinal tract

due to retrovirus infection, thereby leading to malabsorption and malnutrition.

Since serum vitamin and mineral levels may not always be sensitive or specific

indicators of states deficiency or excess, levels do not always reliably reflect nutrient

status. Thus, the studies in the animal model are necessary to define the nutritional

status in the different organs during the progression to AIDS. Since some of the

deficiency states seen in HIV -infected patients could result from the alcoholism, drug

use, infections, and other extraneous variables, which are usually associated with AIDS

patients and contribute malnutrition and immune dysfunctions (77-78), it is har.d to

correlate the relationship between retrovirus infection and malnutrition, and between

35

malnutrition and immune dysfunction in human subjects as AIDS advances. Thus, the

studies in murine AIDS may help to clarify the relationship between malnutrition state

and retrovirus infection, and between undernutrition and immune dysfunction. In this

project, we will investigate the nutritional status during murine AIDS.

Conclusion

Studies of murine AIDS have already been instrumental in formulating novel

hypotheses on human AIDS, some of them testable (80). This animal model appears,

therefore, to fulfill its role efficiently. Practically, LP-BM5 MuLV is not considered as

a human pathogen and carries no risk of transmission to other mice (58). The model is

economical, simple to implement and manipulate and offers a practical and ethical

alternative to AIDS studies using primates or humans. The use of this murine AIDS

model, therefore, should provide a fruitful and exciting experimental opportunity to

elucidate the pathogenesis of retrovirus infection, cofactor involvement such as alcohol

user, drug abuse and nutrition, and anti-retroviral drug testing.

SECTION II: ALCOHOL, IMMUNE RESPONSE, NUTRITION AND AIDS

Introduction

36

Excessive alcohol (EtOH) consumption is a major health problem in the United

States. There are 18 million EtOH-dependent people and another 40 million heavy

drinkers in the United States. The cost to individuals and society, in terms of medical

care and other financial and social costs, are tremendous. Between 20-40% of hospital

beds in the United States are filled by patients whose illnesses are EtOH-related.

According to an estimate of the National Institute of Alcohol Abuse and Alcoholism

yearly economic costs for EtOH-related problem amount to $117 billion (81).

Historically, EtOH use has been associated with infectious disease and lowered

resistance of host. As early as 1884, Robert Koch suggested that most victims of

endemic cholera were heavy alcohol drinkers (82). A 1923's report from Cook County

Hospital associated with mortality from pneumonia with heavy alcohol use (83).

Alcoholism has also been closely associated with lung abscesses, bacteremia and

peritonitis, and tuberculosis (84). It thus appears that prolonged consumption of EtOH

results in alterations of immune responses, ultimately manifested by increasing

susceptibility to infectious agents (85). Such changes can be due to the direct effects of

EtOH or its metabolites on immune cells, as well as to nutritional deficiencies found

frequently with alcohol abuse.

Ethanol and Immune Response

Alcohol use is immunotoxic, and affects most components of the immune defense

mechanism. Chronic alcohol users have a number of similar alterations in

37

immunocompetence including a depression of serum bacteriocidal activity, alterations of

immunoglobulin production, leukopenia, defects in chemotaxis, decreased antigen

trapping and processing, and decreased T-cell mitogenesis (81, 82). A loss in the

number of circulating leukocytes has been associated with EtOH use in humans (86, 87),

with detrimental effects on humoral immune response, including antibody production

(88). In addition, EtOH suppresses several aspects of cell-mediated immunity such as

delayed-type hypersensitivity reactions and lymphocyte proliferative response to mitogens

in humans (89). EtOH administration to animals has also a profound effect on decreasing

the weight of peripheral lymphoid organs as measured by a decrease number of

thymocytes and splenocytes (90-92). In mice, EtOH use produces thymic and splenic

atrophy, alterations in circulating lymphocytes and lymphocyte subpopulations, and in

cellular and humoral immunity and impaired cytokine production (93-96). Mice fed

EtOH for a short period of time have shown both normal and decreased natural killer cell

activity (97, 98). Antibody dependent cellular cytotoxicity (99), lymphocytes

proliferation (100), B lymphocyte functions (100, 101) and cytokine production by

lymphoid cells (94, 102) are also impaired by dietary EtOH. Thus, EtOH-induced

immunosuppression may render alcoholics more susceptible to tumorigenesis and

infections seen frequently in alcoholics.

Since any perturbation in tightly controlled cytokine regulatory system can result

in immune alterations modifying host resistance to infectious disease and cancer, the

influence of EtOH consumption on cytokine secretion has been considerably investigated.

Several studies have indicated a correlation between circulating levels of macrophage-

38

derived cytokines and disease progression during chronic BtOH consumption. Increased

plasma concentrations ofTNF-a have been observed in cases of ALD and, interestingly,

relate significantly to decreased long-term survival; plasma IL-1a is also significantly

increased in these patients (relative to healthy controls) but does not correlate with

increased mortality (103). McClain and Cohen (104) reported that both spontaneous and

LPS-stimulated release of TNF-a was increased significantly by monocytes of ALD

patients compared to normals controls. An earlier study indicated that an increase in IL-

6 production by LPS-stimulated monocytes could also be related to ALD, and that

monocytes from alcoholics without ALD did not release IL-6 above control levels (105).

Another report links ALD with increased serum levels of IL-6 and TNF-a by LPS­

stimulated monocytes over controls. IL-la, IL-6 and TNF-a govern the production of

certain acute-phase reactants by hepatocytes (106-109), a characteristic response of the

liver to systemic tissue damage and inflammation. They also induce temperature

elevation and fever via cyclooxygenase-dependent pathway (110). Thus, BtOH-mediated

tissue damage may have a significant basis in the effects of BtOH on cytokine secretion

in vivo, especially of the primarily macrophage-derived cytokines. Patients with ALD

also have depressed IL-2 and IFN-'Y production (101, 111), suggesting a possible defect

in cell-mediated immunity.

The results from animal studies differ from data obtained from in human

alcoholics with ALD. Intravenous application of LPS caused comparable serum TNF-a

peaks after 90 min in both normal and chronic alcoholic rats (112). In contrast, acute

BtOH injections confirm the in vitro data (104), that acute BtOH exposure suppresses

39

TNF-a release, independent of chronic EtOH exposure. In a further study, marked

suppression of TNF-a in serum and bronchoalveolar fluid occurred after short-term

EtOH exposure of rats (113). The underlying mechanisms by which EtOH consumption

affects cytokine secretion are listed as follow. It could be a direct effect of EtOH on

cytokine-producing cells by affecting signal transduction and changing fatty acid

composition in cell membrane; It could be affected by some hormonal regulators such

as ACTH, fi-endorphin and prostaglandin(PG) altered by EtOH consumption; It could be

directly affected by tissue damage such as ALD with consecutive cytokine release.

However, a paucity of information, regarding the role of EtOH consumption on other

important immunoregulatory cytokines such as 11-2, IL-4, IL-5, IL-lO and IFN--y is

available.

Ethanol and Nutrition

EtOH depresses the appetite, replaces other food in the diet, and decrease the

value of food by interfering with digestion and absorption (114). EtOH-induced

undernutrition could be due to an inadequate and imbalanced intake of nutrients,

maldigestion, and malabsorption, as well as impaired metabolism, decreased tissue uptake

and storage, increased loss of nutrients through the urine, increased nutritional

requirements, or combination of these factors (115). EtOH is transported throughout the

body system giving it the potential to affect most organs and tissues. EtOH affects the

gastrointestinal tract by alterations in stomach acid secretion and delayed gastric

emptying. Alterations in the mucosa of the small intestine leads to alterations in the

production of enzymes, decreased absorptive capacity, malabsorption, and altered

40

transport of nutrients. EtOH also negatively affects pancreatic function with the potential

for maldigestion of foods (116). Therefore, EtOH intake can affect every stage in

nutrient metabolism including nutrient intake, digestion, absorption, activation, storage,

utilization, degradation and excretion.

Since some nutrient deficiencies are well known to be associated with immune

dysfunction (79, 117), reduction in micro- and macro-nutrient intakes can be related to

significant immunosuppression (118, 119). Chronic EtOH consumption increases the

catabolism of retinol leading to a depletion of existing hepatic vitamin A stores. In

addition, there is a decrease of hepatic storage of vitamin A (120). Deficiency of vitamin

A has been associated with EtOH ingestion in humans (121). Vitamin A deficiency

depresses lymphocyte activation by mitogen in rats, leads to a depletion of mature T -cells

from the spleen, decreases thymus weight, and depresses levels of lymphocytes and

plasma cells (122). Repletion with retinoic acid results in a rapid increase in the

peripheral lymphocyte count (123). Vitamin A deficiency has also been associated with

an increased incidence in a variety of cancers and to an increased susceptibility to

infection (124). Therefore, the EtOH-induced vitamin A deficiency can contribute to

immunosuppression found in EtOH users.

Animal studies have correlated a decreased serum vitamin E level with EtOH use

(125). The decreased serum levels of vitamin E may affect the integrity of cell structure

and function as indicated by an increase in lipid peroxidation and enhance free radical

activity. In rats, depressed antibody-dependent cell mediated cytotoxicity, decreased

lymphocytes blastogenesis in response to mitogens, and depressed NK cell mediated

41

cytotoxicity (126) have been reported in vitamin E deficient states. In humans, a strong

correlation has been shown between decreased serum levels of vitamin E and decreased

number and metabolic functions of T cells (127). Therefore, deficiencies,of vitamin E

in immune organs would be expected to partly contribute to the immune dysfunctions

initiated seen in alcohol-fed mice: depressed proliferation of T and B cells, suppressed

IgA and IgO production, impaired cytotoxicity of splenic NK cell, and dysregulated

cytokine production (94, 102).

Zinc deficiency has great influence on barrier immunity by causing skin lesions,

gastrointestinal alteration, damage to the microvilli, and impaired pulmonary functions.

Zinc has also an important role in immunocompetence and exerts a crucial regulatory

effect on specific immune functions. Zinc deficiency results in the adverse effects on the

immune system of a depression of cell-mediated immunity with decreased T -cells and B­

cells in lymphoid tissue, decreased T -helper cell number and activity, an inverted ratio

of T -helper to T -suppressor cells, a depressed response of T-cell to tumor cells, delayed

cutaneous hypersensitivity, decreased lymphokine production, decreased production of

cytotoxic T cells, reduced antibody response requiring T -helper cells, decreased natural

killer cells activity, depressed IgM production, and depression of thymic hormones (128).

In the rabbit, both epithelial and stromal herps simplex keratitsare more severe when zinc

is deficient (129). A dose of trypanosoma cruzi, which would normally be sublethal,

causes the death of 80% of zinc-deficient mice who also exhibit a 50-fold higher

parasitemia than pair-fed controls (130). In such zinc-deficient rats, the expUlsion of

trichinella spiralis (130) and Strongyloides ratti is impaired (131).

42

Zinc deficiency is common with chronic EtOH use. Zinc deficiency may occur

with chronic EtOH use due to inadequate dietary intake, altered absorption and increased

urinary excretion. One study found that 90% of alcoholics with or without liver disease

had inadequate dietary zinc intGke. In animals EtOH intake decreased zinc absorption.

Plasma zinc levels can be decreased 60% below normal in chronic EtOH users (132).

Zinc deficiency can affect vitamin A status due to alterations in vitamin A metabolism,

manifesting with impaired visual function. Zinc plays a role in the storage and

mobilization of vitamin A (133). Zinc is also a part of a metalloenzyme that is

responsible for EtOH metabolism. In animals zinc deficiency impairs EtOH metabolism,

resulting in increased levels of EtOH in the blood and tissues for longer times. This

findings also support the notion that the malnourished state of alcoholics may aggravate

the immune dysfunctions initiated by EtOH ingestion, e.g. depressed proliferation of T

and B cells, impaired cytotoxicity of splenic NK cell, and dysregulated cytokine

production observed in EtOH-fed mice by us (94, 102). These evidence concomitantly

explain the loss of host resistance to the tumors and opportunistic infections observed in

EtOH-fed mice by us (134, -136). Taken together, deficiencies of nutrients induced by

EtOH intakes may render more susceptible to peroxidative injuries, increased appearance

of cancers, immuno- suppression and subsequent .increased infections.

Ethanol and AIDS

Since the association of EtOH use with an increased incidence of a variety of

infectious diseases and cancers (137), EtOH-induced changes in immune response have

been presumed to increase susceptibility to tumorigenesis and opportunistic infections

43

found frequent in AIDS subjects. EtOH use has been associated with some high risk

social practices for HIV infection and because EtOH is often used concomitantly with

other drugs by the high risk group of intravenous drug users (138). A number of

researches in vitro indicated that EtOH may increase both the susceptibility of cells to

mv infection and the extend of viral replication in infected monocytes/macrophages and

T cells (139-141). However, a recent study of EtOH ingestion in mY-positive persons

did not demonstrate an increase in the development of AIDS (142). Since the dosage,

frequency and duration of EtOH exposure in the various stage of mv -positive patients

may be a key role in the progression to AIDS, the results of this study is questionable

when all these factors in EtOH exposure were not well defined. In order to comprehend

the roles of EtOH consumption in development of AIDS, we must address these two

questions. First, are alcoholics more predisposed to HIV infection? Second, can EtOH

ingestion at the special stage of mv infection ( e.g. following seroconversion) aggravate

the development of AIDS? Thus, human studies on the role of EtOH use in AIDS

development should focus attention on those patients who have had prolonged, very high

intakes of EtOH and would therefore be most predisposed to immunosuppression due to

HIV infection. In addition, since significant progression to AIDS with loss of most

peripheral CD4 + T helper cells is necessary to cause immunosuppression, CD4 + T

helper cells (less than 250/mm3) would be most likely to be at risk of further

immunosuppression by EtOH factor. Thus, it is necessary to perform these studies in

animal AIDS model, defining the role of EtOH consumption in development of AIDS.

44

Conclusion

EtOH ingestion broadly suppresses the various arms of immune response as

described above, seriously impairing the body's normal host defense to invading

microbes and tumorigenesis. There is little doubt that alcohol abuse exerts a disruptive

effect on the immune system. These disruptions are the combined result of direct

immunotoxic effects and indirect effects such as malnutrition. The alcoholic's

predisposition to extracellular and intracellular infection indicates the effects of EtOH

consumption at the local, humoral, and cellular levels, inhibiting immune response and

host defense.

SECTION III: VITAMIN E, IMMUNE RESPONSE, NUTRITION AND AIDS

Introduction

45

Since AIDS and alcoholics are diseases associated with immune dysfunctions, one

of the most attractive nutrients is vitamin E, which functions as an antioxidant and an

immunoenhancer. High doses of vitamin E decrease CD8+ T cells and increase

CD4 + /CD8+ ratio, increase total lymphocyte count, stimulate activity of cytotoxic cells,

natural killer (NK) cell activity, phagocytosis of macrophages and mitogen responsiveness

(143). The immunostimulatory nature of vitamin E does provide a basis for its use in

the modulation of the various cell components and immune functions in humans, and the

consequent therapeutic use during AIDS and alcoholics. Furthermore, high doses of

vitamin E supplementation provide essentially no toxicity (144-146). Thus, the

combination of existing medical therapy with vitamin E supplementation may provide a

more successful and novel therapeutic approach for treatment of mv infected individuals

and alcoholics.

Vitamin E and Immune Response

Vitamin E supplementation exerts a positive effect upon the immune system while

its deficiency compromises the immune system (143, 147, 148). Vitamin E has been

found to stimulate Th cells, antibody response, delayed cutaneous hypersensitivity

reaction, phagocytosis, mitogen responsiveness, the reticuloendothelial system, and host

resistance in animal models (149-151). In hemodialysis patients with low concentrations

of vitamin E in the mononuclear cells, parenteral administration of physiological doses

vitamin E, repleting body stores, decreased the number of CD8+ T cells and increased

46

the CD4+/CD8+ ratio (152). Prasad demonstrated a significant increase in bactericidal

activity in human leukocytes after ingestion of 300 mg of vitamin E daily for 3 weeks

(153). Vitamin E deficiency in dogs leads to depression of lymphocyte activation by

mitogens (154). Vitamin E supplementation has also been shown to be beneficial in

reducing the incidence or severity of infectious diseases in elderly persons and in rats

(155). In elder human subjects, vitamin E has been shown to increase IL-2 production

while decreasing that of prostaglandin E2 (PG), to increase the response to delayed

hypersensitivity skin testing, and to enhance lymphocyte proliferating in response to

mitogens (156). In patients with SLE, supplementation with vitamin E decreased in the

severity of this autoimmune disease with the plasma globulin level decreased and the

plasma albumin level increased (157). Therefore, vitamin E supplementation may

decrease the heightened levels of gammaglobulin from polyc1onal B cell activation that

occur in AIDS patients and alcoholics.

Vitamin E may also protect the immune system from and retrovirus infection and

EtOH-induced damage through intervention by hormonal changes. EtOH can increase

the levels of prostaglandin and corticosteroids (158, 159), which are elevated in

alcoholics and AIDS, and are potent inhibitors of the immune cells' activity, including

cytokine release (114). Vitamin E could decrease its levels (160, 156), thereby

enhancing immune response and increasing host resistance. Taken together, vitamin E

supplementation, which normalizes immune responses, could improve host resistance to

tumorigenesis and opportunistic infections seen frequently in alcoholics and AIDS.

47

Vitamin E and Nutrition

Since AIDS and alcoholics are diseases associated with malnutrition, the vitamin

E supplementation may improve the nutritional status. Vitamin E supplementation has

been shown to increase hepatic level of vitamin A in the rat. However, the role of

vitamin E in the absorption or transportation of minerals is still unclear clear. The

hypothesis is that vitamin E may protect gastrointestinal mucosa cells from damages of

retrovirus infection and EtOH use, thereby restoring the malnutrition occurred often in

AIDS and alcoholics. The hypothesis will be tested in this research project.

Vitamin E and AIDS Therapy

AIDS therapies involve pharmacological interventions (e.g. Ganciclovir and

Zidovudine, AZT) to inhibit HIV replication (161). However, these drugs are

accompanied by deleterious toxic side effects. For example, Ganciclovir toxicities

include bone marrow suppression, atrophy of the gastrointestinal tract mucosa and

inhibition of spermatogenesis, nausea, vomiting, reduced white blood cell count and

headaches. These anti-HIV drugs do not also repair damaged immune functions and

improve nutritional deficiencies or other pathological symptoms caused by retrovirus

infection in AIDS individuals. Furthermore, a strain of AZT -resistant HIV was reported

in human individual possibly due to high dose and prolonged use of AZT (162). Thus,

new strategies including specific dietary nutrient supplementation could provide additional

approaches to ameliorate malnutrition status and thereby immune dysfunctions in infected

individuals, eventually slowing the progression of disease to AIDS.

Since AIDS is a disease associated with immune dysfunctions, malnutrition,

48

oxidative stress. one of the most attractive nutrients is vitamin E, which functions as an

antioxidant and an immunoenhancer. Vitamin E may also directly inhibit viral

replication through inhibition of protein kinase C (PKC). The HIV coat protein gp120

induces PKC activity in lymphocytes (163). In tum, PKC activation results in the

transition of HIV from a state of latency to active replication in lymphocytes, and leads

to upregulation of HIV replication in cells with non-dormant HIV (53-55). This notion

is strongly supported by the demonstration that HIV transcription is redox regulated, and

inhibited by antioxidants such as N-acetylcysteine, a well-known antioxidant used to

replenish intracellular glutathione (56, 57, 59, 60). Collectively, vitamin E

supplementation not only enhances the immune response and restore tissue vitamin E

deficiencies but also directly inhibit retrovirus replication, thereby retarding the

progression to AIDS.

Vit2.min E should not be considered as a direct antiretroviral drug but rather as

a potentially immunomodulating and antioxidant agent that achieves its antiretroviral

effect through indirect mechanisms, possible through immunoenhancing, modulation of

signal transduction, restoration of undernutrition and anti-oxidative stress. Its use as

combination therapy with other pharmacologic agents such as AZT could have several

advantages. First, it could lead to more complete viral suppression by deceasing

oxidative stress and enhancing immunity, associated with this retrovirus infection, than

any single drug used alone. Second, it may allow each drug to be used in lower doses,

thus limiting drug toxicities, and decreased frequency of production of drug-resistant

HIV strains. Third, such immunorestorative treatment may be given during maintenance

49

therapy of the AIDS patient following the control of tumors and immune dysfunction by

chemotherapy. Fourth, the use of vitamin E may be important in forestalling initial

episodes of general immune disorders in some patients by extending the period between

mv infection and the appearance of clinical symptoms of AIDS.

Conclusion

Experimental results from animal or human studies have implied that vitamin E

supplementation may act on various immune components to repair immune defects and

lessen malnutrition status induced by EtOH intakes and retrovirus infection. Therefore,

vitamin E supplementation has theoretically therapeutic effects in human AIDS and

alcoholics. I will define in this research project the potential therapeutics of vitamin E

supplementation in alcohol-fed mice and murine AIDS by monitoring immune functions,

cytokine secretion and nutritional status. Although the animal stat may not be totally

extrapolated to alcoholics and mY-infected individuals, information obtained from

murine model may be potentially serve as a basis for the study of therapeutic roles of

vitamin E in humans.

50

CHAPTER 2: ALTERATION OF CYTOKINE PRODUCTION,

IMMUNE RESPONSE AND NUTRITIONAL STATUS DURING MURINE AIDS

Abstract

In present study, we used this model to investigate release of cytokines by splenic

T cells, macrophages and thymocytes, regulatory factors of the immune response and T

cell maturation, and some immune functions, and nutritional status. Our results indicate

secretion of TNF-a and IL-6 by peritoneal resident macrophages was significantly

enhanced beginning at 2 and 4 weeks, respectively, and continuing to 22 weeks post­

infection. Release of IL-2 and IFN-'Y by splenic T cells was significantly inhibited from

4 week to 22 week post-infection, whereas secretion of IL-6 and IL-lO splenic T cells

was enhanced from 4 week to 22 week post-infection. Release of IL-4 and IL-5 by

splenic T cells was significantly enhanced at the 8-10 and 4 week post-infection,

respectively and declines progressively thereafter. The splenocyte proliferation

stimulated by mitogens was significantly inhibited after 4 weeks post infection, whereas

IgG production by splenocytes was significantly enhanced after 4 weeks post infection.

The proliferation of thymocyte induced by ConA was significantly enhanced by retrovirus

infection at 4 weeks post-infection compared to control, but significantly inhibited during

8-12 weeks post-infection. Release of IL-2 by thymocytes was significantly increased

by retrovirus infection during 2-5 weeks post-infection and 11-18 weeks post infection,

but significantly decreased during 7-9 weeks post-infection. Secretion of IL-4 by

thymocyte was significantly enhanced by retrovirus infection from 5 to 18 weeks post­

infection. The level of IL-6 produced by was significantly inhibited by retrovirus

51

infection at the beginning of retrovirus infection (2-9 weeks), but significantly elevated

after 11 weeks post-infection. Release of IFN--y by thymocytes, however, was

significantly enhanced during the whole period of retrovirus infection compared to

control, while it surged at the 13 weeks post-infection. The levels of vitamin A, E and

copper in the liver in murine AIDS were significantly reduced compared to controls,

whereas the level of zinc in the liver was not affected. Intestinal level of vitamin A was

significantly reduced by retrovirus infection, whereas copper level in the small intestine

was significantly increased compared to controls. Intestinal levels of zinc and vitamin

E were not affected. The levels of vitamin A, E and zinc in the spleen in murine AIDS

were significantly rebated compared to controls, whereas the splenic level of copper was

not influenced. The levels of vitamin A, E and copper in the thymus in murine AIDS

were significantly lessened by retrovirus infection, whereas thymic level of zinc was

significantly elevated. The levels of vitamin A and E in the serum in murine AIDS were

significantly decreased by retrovirus infection compared to controls. We conclude

retrovirus infection induces immune dysfunctions via modulating the cytokine production,

and affects the thymus, producing altered T cell differentiation via the dysregulation of

thymocyte cytokine secretion. Furthermore, the data indicate that retrovirus infection can

directly cause malnutrition, possible via damaging gastrointestinal cells, thereby leading

to malabsorption. Such malnutrition has the theoretical potential to accelerate

development of AIDS via immunosuppression secondary to nutritional deficiency.

52

Introduction

LP-BM5 murine leukemia retrovirus (MuLV) infection induces murine acquired

immune deficiency syndrome (AIDS) characterized by lymphadenopathy, splenomegaly,

hypergammaglobulinemia, deficient B cell responses to T -independent antigens or

depressed allogenic cytotoxic T cell response (58). It produces immune dysfunctions

via dysregulation of cytokine production (164) and possesses many functional similarities

to human AIDS, including changes in T cell number and functions (58). LP-BM5 MuLV

infection in mice is a potentially useful model to study the consequence of human

immunodeficiency virus (HIV) infection in humans (58).

The balance of cellular and humoral immune response to antigens is regulated

by soluble mediators, termed cytokines, mostly produced by T cells and macrophages.

Therefore it is likely that dysregulated release of cytokines is responsible for some of

these changes. Recently the secretion of cytokines has been reported to be altered in

murine AIDS (164). Because of the change in the T cell population of spleen cells

during progression of murine AIDS and interaction between CD4 + and CD8+ T cells that

could affect level and kinetics of cytokine production, we evaluated cytokine production.

We measured during development of murine AIDS Thl-related cytokines [interleukin-2

(IL) and interferon-gamma (IFN--y)] and Th2-related cytokines (IL-4, IL-5, IL-6 and IL-

10) by ConA-stimulated T cells, and monokines (IL-6 and Tumor necrosis factor-a

,TNF) by LPS-stimulated peritoneal macrophages.

The generation of functional competent T cells from precursor populations within

the thymus involves several stages of cellular proliferation and differentiation (165, 166).

53

It has been proposed that the control of these processes is mediated by cytokines (167).

Consequently, the alteration in T cell subsets or T cell functions observed after

retrovirus infection in human peripheral blood cells or mouse spleen cells could be the

consequence of changes at the thymus level, including thymocyte cytokine production.

Histopathological studies of thymic glands from HIV -I-infected children revealed

involuted architecture, reduced size and weight, and fewer Hassal's corpuscles (168,

169). mV-l causes changes in thymic hormone and cytokine levels such as thymosin

lex and IL-6, which act on T cell differentiation (170, 171). Human fetal thymocyte cell

lines were infected with mv -1 in vitro and altered expression of T cell differentiation

markers (172). However, no any detailed information about cytokine production by

thymocytes in the progression of AIDS was reported. Therefore we examined the

changes of cytokine production (IL-2, IL-4, IL-6 and IFN-'Y) by in vitro ConA-stimulated

thymocytes to understand how retrovirus infection alters thymic cytokine secretion, and

thereby adversely affects thymocyte development and function. These studies in murine

AIDS would facilitate the understanding of immunopathagenesis of mv infection.

The human immunodeficiency virus (HIV) is a key causative agent of AIDS,

although malnutrition could contribute to the progression to AIDS. Profound weigh

loss, cachexia, multiple nutrient deficiencies and protein calorie malnutrition are

frequently nutritional disorders found in AIDS patients (198). They have the theoretical

potential to accelerate development of AIDS via immunosuppression secondary to

nutritional deficiency. Therefore, the malnourished state of the AIDS patients may have

precluded recovery from infectious events which earlier represented survivable episodes.

54

Gastrointestinal dysfunctions including diarrhea and malabsorption are also common

manifestations of the AIDS (199, 200). It has been suggested that HIV infection might

play pathogenic role in gastrointestinal cells. This current view of an HIV -induced

enteropathy is supported by two main kinds of data. On the one hand, in situ

hybridization studies have localized HIV infection in various types of epithelial cells of

the bowel mucosa (71, 72). On the other hand, HIV infection of gastrointestinal cell

lines has been documented in vitro (73-75). Such complications in AIDS contribute to

malnutrition with increased susceptibility to infection and malabsorption to further

compromise nutritional status.

Although the roles of vitamins and minerals in the clinical manifestation of HIV

infection has not been well defined (57), a growing number of studies have suggested

important links between vitamins or minerals and HIV infection. Bogend et al. found

that 12 % of their 30 patients at various stages of HIV disease has serum vitamin A

deficiency (63). In their study of AIDS patients, Malcolm et al. reported low vitamin

A levels in seven of 24 patients (29 %) (65). Bogden et al. found that 12 % of 30

patients, at varying stages of mv infection, had low plasma levels of vitamin E (63).

In another study, Passi et al. also noted deficient levels of plasma vitamin E in patients

with HIV (65). Zinc deficiency in HIV infection has been identified consistently. Falutz

et al. reported that serum zinc decreased progressively with advancing mv disease

among 150 patients, compared with 50 HIV-seronegative controls (201). Fordyce-Baum

et al., Malcolm et al., and Bogden et al., all found that marginal or clear zinc deficiency

in any where from 23% to virtually all of the patients tested (63-67). The prevalence of

55

copper deficiency is less clearly set up since there are only two available studies.

Fordyce-Baum et al. found marginal copper deficiency in 22 % of their patients (61). In

contrast, Bogden et al. noticed low levels in only 3% of patients (63). Thus preliminary

data support the clinical importance of vitamin A, E and zinc and copper deficiencies in

HIV infection, although their different extends of deficiency in mv infection have been

reported. There is little doubt that patients with HIV infection will be increasingly

compromised nutritionally as the disease progresses. Thus, two questions were addressed

in the present research. Since serum vitamin and mineral levels may not always be

sensitive or specific indicators of deficient or excess states, levels do not always reliably

reflect nutrient status of the body. Thus, the studies in the animal model are necessary

to define the nutritional status in the different organs during the progression of AIDS.

In addition, since some of the deficient states seen in HIV -infected patients may result

from the alcoholism, infections and drug use or other extraneous variables, which are

usually associated with AIDS patients and contribute malnutrition (77, 78), it is hard to

correlate the relationship between retrovirus infection and malnutrition in human subjects

as AIDS advances. Thus, the studies in the animal model may help to clarify the

relationshIp between undernutrition and retrovirus infection.

56

Materials and Methods

LP-BM5 murine leukemia virus infection Female mice, 4-6 months old, were assigned

to one of two groups. The first group of mice was injected once intraperitoneal with

0.10 ml of LP-BM5 inoculum which had an ecotropic titer of 4.5 10glO PFU/ml which

induced the disease with a time course of immune dysfunction as previously published

(11).

Preparation of peritoneal resident macrophages Peritoneal resident cells (PRC) were

obtained by washing the peritoneal cavity with 7.5 ml of PBS (0.01 M, pH 7.2) three

times after sacrifice under ether anesthesia. After washing PRC with cold RPMI-I640

containing 10% FBS (complete Medium, CM), the cell suspension in 5 ml (5 x 1()6

cells/ml) was added to FBS-coated petri dish (60x15 mm) and incubated for 2 hours at

37°C, 5% CO2 incubator. Non-adherent cells were removed by washing with cold CM

three times. Adherent cells were removed from petri dish with cell scraper. Then the

cell concentration was adjusted to Ixl()6/ml for cell culture. More than 95 % of adherent

cells had morphological characteristics macrophages, judged by a microscope using 2.0%

acetic acid as diluent (1:1). Cell viability was always more than 95% as determined by

trypan blue exclusion.

Preparation of Splenocytes and Thymocytes Spleens and thymi from mice receiving

ETOH or isocaloric control diets were gently teased with forceps in RPMI-I640 medium

(Sigma) containing 10 % FBS (Complete Medium, CM), producing a single cell

suspension of spleen cells. Red blood cells were lysed by the addition of a lysis buffer

(0.16 M ammonia chloride Tris buffer, pH 7.2) at 37° C for 2 minutes. Then the cells

57

were washed twice with CM. Cell concentration was counted and adjusted to 1x107

cellslml. Splenocytes viability was always more than 95 % as determined by trypan blue

exclusion.

Preparation of T cells from Splenocytes. Spleens from mice were gently teased with

forceps in CM. Red blood cells in a single-cell suspension were lysed by the addition of

a lysis buffer (0.16 M ammonia chloride Tris Buffer, pH 7.2). Then the cells were

washed twice with cold CM. The cell concentration was adjusted to Ix 107 Iml in low­

cytotoxicity medium. Purified rat anti-mouse Ia monoclonal was added into the cell

suspension (lx1061J£g antibody) at 4 °c for 1 hour. After removing supernatant, cell

pellet was re-suspended into low-cytotoxicity medium and added with rabbit complement

(1:12.5) at 37°C for 1 hour. Then the cell suspension was loaded on the Lymphocyte-M

gradient and centrifuged at 1,650 rpm and 20°C for 20 minutes. Viable cells were

collected with a pasteur pipet and washed twice with cold CM. Viability of purified cells

was more than 95 % as determined by trypan blue exclusion. More than 85 % of the

purified cells were Thyl,2+ T cells and less than 1 % of the purified cells were total of

IgM and Mac-l positive cells as determined by indirect immunofluorescence staining

using monoclonal antibodies (173).

Cytokine Standards and Antisera Rat anti-murine IL-2, IFN-'Y, IL-6 monoclonal

antibodies and standard recombinant murine IL-2, IFN-r and IL-6 were obtained from

Genzyme (Boston, MA). Rabbit anti-murine IL-2 polyclonal antibody was obtained from

Collaborative (Bedford, MA). Rat anti-murine IL-4,IL-5 and IL-10, biotin-rat anti-murine

IL-4, IL-5 and IL-lO monoclonal antibodies, and recombinant murine IL-4, IL-5 and IL-

58

10 standard were obtained from PharMingen (San Diego, CA). Goat anti-murine IL-6

polyc1onal antibody was obtained from R&D System (Minneapolis, MN). Rabbit anti­

murine IFN--y serum was prepared in our lab.

Production of Cytokines 100 J.(.l of lxia' macrophages/ml, 5x106 T cells/ml or Ix107

thymocytes were added into 96-well cell culture plate. Then 100 J.(.l of LPS (10 J.(.g/ml)

for macrophage culture and 100 J.(.l of ConA (10 J.(.g/ml) for T cell culture were added and

incubated in 37°C, 5% CO2 incubator. After 24 hours of incubation for IL-2, IL-4, IL-

6, IL-lO and TNF-a production, and 72 hours for IL-5 and IFN--y, cell culture

supernatants were collected for cytokine detection.

Preparation ofIgG Production Splenocytes (lx107/ml) from mice of the same experiment

group were individually cultured in triplicate on 96-well tissue culture plates (0.1

mllwell, Falcon) with CM. Then splenocytes were stimulated with LPS (Gibco, 5

J.(.g/ml) diluted in CM. After 120 hour incubation at 37°C, 5% CO2 incubator, the plates

were centrifuged for 10 minutes at 800 g, and supernatant fluids were collected and

stored at -70° C until detection. Triplicate cultures were set up for each individual

mouse.

ELISA for Cytokines The wells of 96-well microtiter plates were coated overnight at

4°C with 50 J.(.l of anti-cytokine rat or hamster monoclonal antibody specific for the

measured cytokines, diluted to 1-4 J.(.g/ml in 0.05 M bicarbonate buffer (PH 9.6). Plates

were washed one time with PBS (0.01 M, pH 7.2-7.4) containing 0.05% (v/v) Tween-20

(PBST). Then 100 J.(.1 of standard or sample cytokines diluted in culture medium were

added. Plates were incubated for two hours, and washed three times. 50 J.(.1 of diluted

59

anti-IL-2, TNF-a or IFN--y polyclonal serum (1: 100), or goat anti-IL-6 polyclonal, or

biotinylated rat anti-IL-4,5, 10 monoclonal detecting antibodies in PBS (1-4 ",g/ml) were

added into each well. Plates were incubated as above for 1.5 hours and washed four

times with PBST. Then SO ",I of diluted strepavidin-HRP (1:5000) for IL-4,S, 10, goat

anti-rabbit IgG-HRP (1:5000) for IL-2 and IFN--y or donkey anti-goat IgG-HRP (1:5000)

for IL-6 conjugates were added to each well. Plates were incubated as above for one

hour, and washed five times with PBST and once with PBS. Finally, 100 ",I of substrate

buffer (ABTS in 0.1 M citrate buffer, pH 4.2 containing 0.03% H20:z) were added to

each well, and the color was allowed to develop for 20-30 minutes at room temperature.

Optical density was determined at 405 nm. Sensitivity of the ELISAs for IL-2, IL-4, IL-

5, IL-6, IL-lO, IFN--yand TNF-a were determined to be 156pg, 156pg, 32Opg, 200pg,

2U, 78pg and lS6pg per ml, respectively. No cross-reactions between these measured

cytokines in the ELISA kits were observed.

ELISA for IgG. The wells of 96-well microtiter plates (Immulon,II) were coated

overnight at 4 C with 50 ",I of Goat anti-murine IgG gamma-chain (Sigma), diluted to

2 ",g/ml in 0.05 M bicarbonate buffer (PH 9.6). Plates were washed one time with PBS

(0.01 M, Ph 7.2-7.4) containing 0.05%( v/v) Tween-20 (PBST). Plates were washed

twice as above, and 50 ",I of standards and sample murine standard IgA and IgG (Sigma)

diluted in CM were added to plates. Plates were incubated as above for 1 hour, and

washed three times as above. 50 ",I of diluted biotinylated goat anti-murine IgG (2

",g/ml) in PBS were added and incubated at 37° C incubator for another hour. Then 50

",I of diluted strepavidin-HRP (1:5,000, Jackson, West Grove, PA) was added for another

60

hour incubation at 37° C. Finally, 100 #-,1 of substrate buffer of ABTS (Sigma, in citrate

buffer, O.lM, pH 4.2 containing 0.03% H20 2) were added to each well, and the plates

were allowed to develop for 20-30 minutes at room temperature. Optical density was

determined at 405 nm by Titertek Multiscan (Flow Lab, Mclean, VA). Sensitivity of

ELISAs for IgG were determined to be 40 pg/ml, respectively. No cross-reaction of

ELISA kit with IgA and IgM were observed (data not shown).

Mitogenesis of splenocytes and thymocytes Thymocytes(1x107/ml) and splenocytes

(lx107) in 0.1 ml were cultured in 96-well flat-bottom cultured plates (Falcon) with

ConA ( #-,g/ml) in CM at 37° C , 5% CO2 for 20 hours for ConA-induced T Cell

proliferation. Then they were pulsed with 3H-thymidine ( 0.5 #-,Ci/well,New England

Nuclear, Boston, MA). After 4 hours, they were harvested by an a cell sample

harvester(Cambridge Technology, Cambridge, MA). Radioactivity was determined by

a liquid scintillation counter (Tri-Carb, 2200CA, Packand, Lagunahills, CA). Data are

presented as count per minute (cpm).

Measurement of Vitamin A and E. Tissue concentrations of total tocopherol and retinol

were determined by the flu oro metric method described by Dugan et al (202). Briefly,

about 0.1 g of tissues was homogenized in 5 ml water and 5 ml ethanol, and vitamin A

and E extracted with 5.0 ml n-hexane by shaking for 1 minutes and centrifugation at

3000 rpm for 5 minutes. The n-hexane layer was removed and measured at an emission

of 430 nm and an excitation of maximum of 365 nm for retinol measurement. Then,

0.5ml of 60% sulfuric acid added, the solution mixed thoroughly and the fluorescence

intensity for vitamin E determined in the hexane at an emission of 340 nm and an

61

excitation maxima of 295 nm using a fluorescence spectrophotometer (Hitachi F-2000,

Hitachi, Tokyo, Japan) and using alpha-tocopherol or all-trans retinol (Eastman

Chemical, Kingsport, TN) as a standard. All levels of vitamins were represented by

J.l.g/wet g tissue.

Serum Vitamin A and E. Serum vitamin A and E were measured by mixing 0.2 ml

of serum with 2 ml of ethanol, extracting with 4.0 ml n-hexane by shaking for 5 minutes

and centrifugation at 300 rpm for 5 minutes. The vitamin A and E levels in n-hexane

layers were be measured as indicated above. Serum vitamin levels were represented by

J.l.g/ml serum.

Measurement of Zinc and Copper. About 0.1 gram of tissues is dried at 56°C in a

oven for overnight. After cooling, 5 ml of 70% nitric acid is then added. After mixing

and predigestion in a 60°C water bath for an hour, the water bath was brought to

boiling. Five drops of 30% hydrogen peroxide were then added the sample. Before the

tubes were removed from the water bath, another drop of hydrogen peroxide was added.

The solution is either measured directly or diluted to the appropriate concentrations for

elemental analysis by flame atomic absorption (Hitachi, 180-70 Spectrophotometer) at

213.8 nm for zinc and at 324.8 nm for copper. The same nitric acid containing 30%

hydrogen peroxide was used for the standard and blank controls in the mineral analysis.

All levels of minerals were represented by J.l.g/ g dry tissue.

Statistics Two-tailed student t test was applied. p < 0.05 was considered

significant difference between two groups.

62

Results

IL-6 and TNF-O! Production by Peritoneal Macrophages From Fig. 2.1, secretion of

IL-6 and TNF-O! by LPS-stimulated peritoneal resident macrophages was significantly

enhanced from 2 and 4 weeks after infection, respectively, to 22 week post-infection

compared with control.

IL-2 and IFN-y Production by Th 1 Cells From Fig.2.2, release of IL-2 and IFN--y

by ConA-stimulated Thl cells was significantly inhibited from 4 week to 22 week post­

infection.

IL-4. IL-5. IL-6 and IL-IO Production by Th2 Cells From Fig. 2.3, secretion of IL-6

and IL-I0 by ConA-stimulated Th2 cells was significantly enhanced from 4 week to 22

week post-infection compared with control, while release of IL-4 and IL-5 by ConA­

stimulated Th2 cells was significantly enhanced at the 8-10 week and 4 week post­

infection, respectively, but declines progressively thereafter.

Mitogenesis of Splenocytes. As shown in Table 2.1 and 2.2, the ConA- and LPS­

induced splenocyte proliferation was significantly ( p < 0.05) reduced by retrovirus

infection during whole period of study (4-16 weeks post infection).

Production of IgG. As shown in Table 2.3, The IgA and IgG production by LPS­

stimulated splenocytes was significantly (p < 0.05) enhanced by retrovirus infection

compared to controls from 4-weeks post infection to 16-weeks post infection.

Mitogenesis of Thymocytes As shown in Table 2.4, in vitro thymocyte proliferation

was significantly increased at 4 week post retrovirus infection compared with uninfected

control (p < 0.05). Thereafter the proliferation was significantly inhibited by retrovirus

63

infection at 8 and 12 week post-infection (p < 0.05). However, the proliferation was

no significant change by retrovirus infection at 16 week post-infection (p > 0.05).

Cytokine Production by Thymocytes As shown in Fig. 2.4, the release of IL-2

produced by in vitro ConA-stimulated thymocytes was significantly higher during the

initial weeks of retrovirus infection compared with uninfected control ( p < 0.05).

Thereafter in vitro production of IL-2 was significantly reduced by retrovirus infection

(p < 0.05). Then, level of IL-2 was significantly elevated again by retrovirus infection

after 11 week until 18 week post-infection (p < 0.05), while that of thymocytes from

uninfected mice was gradually decreased as age increased. As shown in Fig. 2.5,

secretion of JL-4 by thymocytes from retrovirus infected mice was significantly higher

than that by thymocytes from uninfected mice from 5 week to 18 week post-infection (p

< 0.05). There was a significantly decline in IL-4 production by thymocytes from

uninfected mice as age increased. As shown in Fig. 2.6, IL-6 secreted by thymocytes

was significantly inhibited by retrovirus infection compared with uninfected controls up

to 11 week post-infection ( p < 0.05), while IL-6 production by thymocytes from

uninfected mice had significantly declined as age increased (P < 0.05). However the

release of IL-6 was significantly enhanced by retrovirus infection after 11 week post­

infection compared to continuing reduced production of IL-6 by controls ( p < 0.05).

As shown in Fig. 2.7, IFN--y production by thymocytes was significantly elevated by

retrovirus infection compared with uninfected controls through beginning 2 week post

infection (p < 0.05), and then secretion of IFN--y surged at 13 week post-infection.

Hepatic Levels of Nutrients As shown in Table 2.5, the levels of vitamin A, E and

64

copper in the liver in murine AIDS were significantly (p < 0.05) reduced compared to

uninfected mice. The level of zinc in the liver was not affected by retrovirus infection.

Intestinal Levels of Nutrients The Copper level in the small intestine was significantly

(p < 0.05) increased by retrovirus infection compared to controls, whereas the vitamin

A level was significantly (p < 0.05) reduced by the retrovirus infection. The zinc and

vitamin E level was not affected by retrovirus infection (Table 2.6).

Splenic Levels of Nutrients As shown in Table 2.7, the levels of vitamin A, E and zinc

in the spleen after development murine AIDS were significantly (p < 0.05) reduced

compared to controls, whereas the splenic level of copper was slightly increased by

retrovirus infection.

Thymic Levels of Nutrients The levels of vitamin A, E and copper in the thymus after

development of murine AIDS were significantly (p < 0.05) decreased by retrovirus

infection compared to controls, whereas thymic level of zinc was significantly elevated

by retrovirus infection (Table 2.8).

Serum Levels of Vitamins The levels of vitamin A and E in the serum in murine AIDS

were significantly reduced by retrovirus infection compared to controls (Table 2.9).

65

Discussion

Our results confirm and expand those of Gazzinelli et al (164) showing altered

cytokine expression caused by LP-BM5 murine retrovirus infections. We conclude that

lymphoid cell dysfunction modified cytokine production by mitogen-stimulated

immunocompetent cells which may be induced by a superantigen encoded by defective

genome in the LP-BM5 retrovirus mixture. We demonstrated, for the first time, that

retrovirus infection can cause undernutrition, reducing tissue levels of nutrients during

murine AIDS.

Imbalanced release of cytokines appears responsible for much of the

immunopathogenesis of MAIDS. Release of IL-6 and TNF-o: by LPS-stimulated

macrophages was significantly enhanced from 4 weeks after virus infection. Elevated

levels of plasma IL-6 and TNF-o: are also seen in HIV-infected human subjects (174, ,

175). Elevated levels of IL-6 may play an important role in the polyclonal B cell

activation and Immunoglobulin secretion, which are characteristically seen in both HIV

infection and LP-BM5 infection. This notion is supported by the elevated level of IgG

produced by splenocytes we obs,~rved. This increase of non-specific antibodies may

contribute to development of AIDS. Elevated level of TNF-o: may be involved lipid

metabolism: inducing hypertriglyceridemia and wasting in both human AIDS (176) and

murine AIDS (177).

CD4 + T cells can be divided into two major subsets on the basis of cytokine

expression. Thl cells produce IL-2, IFN--yand lymphotoxin, whereas Th2 cells produce

IL-4, IL-5, IL-6 and IL-I0 (178). The relevance of these patterns of cytokine secretion

66

and reciprocal regulation by Th clones also have been recently demonstrated (179). IL-

10 can inhibit Th1 differentiation and its cytokine production, and conversely IFN--y can

inhibit Th2 differentiation and its cytokine production. IL-4 induces ThO cells to

become Th2 cells producing Th2-related cytokines. The capability of T cells in spleen

to produce the Th1 cytokines, IL-2 and IFN--y, in response of ConA stimulation is

diminished. IFN--y activates macrophages, neutrophils and natural killer cells, and

stimulates IL-1 secretion by monocytes. IL-2 is a major factor in the T cell growth and

proliferation. Thus the decreased release of IL-2 and IFN--y appears responsible for the

immune defects observed in the progress of MAIDS, which may contribute to enhanced

susceptibility to pathogen infections. Increased production ofIL-10 during murine AIDS

can be a contributing factor to inhibition of Th1-type cells in MAIDS. Th2-type

responses would be dominated in the progression of murine AIDS due to decreased IFN­

-y. Elevated production of Th2 cytokines, IL-4, IL-5 and IL-6 can efficiently stimulate

B cell growth and differentiation to immunoglobulin secretion , a central feature of

murine AIDS. Cellular immune dysfunction in monokine and cytokine production during

progression of murine AIDS has been shown to parallel the observation in HN infection.

The continuing elucidation of the mechanism of immunomodulation during murine AIDS

may help us to understand how infection with HIV induce these immune defects in

human subjects, perhaps via preferential activation of Th2 cells.

We demonstrated for the first time that murine retrovirus infection modified

production of cytokines by in vitro ConA-stimulated thymocytes. The mammalian

thymocytes can be subdivided into 4 major sUbpopulations based on expression of the

67

differentiation-related antigens CD4 and CDS. "Double-negative" (DN) CD4-CDS-

thymocytes (2-4% of total in the normal adult mouse) represent immature cells with the

capacity to give rise to the other subsets in irradiated host (ISO). CD4+S+ cells in the

thymic cortex account for the majority (SO-S5 %) of thymocytes and most of such

"double-positive" (DP) cells are destined to die in situ (1S1). The other two" single­

positive" (SP) CD4-S+ and CD4+S- subsets (5-10%) in the thymic medulla represent

mature T cells seen in the peripheral tissue (1S1). SP subsets could be further divided

into CD3 + and CD3- subset. CD3 + cells resemble mature peripheral T cells in that they

are functionally immunocompetent, including responding to a variety of mitogen stimuli

and secreting cytokines, and originate from CD3-4-8+ and CD3-4 +S- immature T cells in

the thymus (ISO, lSI). Our previous report found murine retrovirus infection alters T

cell differentiation by reducing CD4 + CDS + cells. This population (SO-S5 % in normal

mice) dramatically dropped to only 30% and other populations, CD4 +S- and CD4-S+ (SP

subsets) were elevated respectively from controls 1.99% and 7.67% to 33.9S% and

15.5% of thymocytes in retrovirus infected mice at 16 week post-infection (lS2). The

continuing analysis of CD3 marker of two SP subsets by fluorescence cytometry in our

lab indicated that the percentage of CD3+CD4 +CDS- subsets in the thymus from

retrovirus infected mice for 16 weeks dramatically dropped from control 74% to 7.9%

of CD4+CDS- subset, while percentage of CD3+CD4-CDS+ thymocytes of CD4-CDS+

subsets did not change during the murine AIDS (2.3% in murine AIDS vs 2.2% in

(.::ontrol mice, Lopez et al., unpublished data). Therefore the increased SP thymocytes

by the retrovirus infection were immature CD3- SP T cells, not mature CD3 + SP T cells.

68

Since we did not study the kinetics of thymocyte subsets during the progression of

murine AIDS, we speculated that the increased or decreased thymocytes proliferation and

thymic cytokine release induced by retrovirus infection might partly reflect the fluctuation

of populations of CD3 + mature T cells in the medulla and CD3- immature T cells in the

cortex of thymus. The changes in thymocytes differentiation may explain the dysfunction

of T cells seen in AIDS.

How could thymocyte differentiation be altered during the progression of murine

AIDS? One possibility appears that activated peripheral T cells by retrovirus infection

re-enter to the adult thymus, thereby they secret some cytokines and adversely affect

regulation or differentiation of T maturation in the thymus. Agus et al found that

homing of mature T cells to the thymus of adult mice was substantial and exclusively

restricted to Con A-activated T cells. However, mature resting T cells have virtually no

capacity to migrate to the thymus (183). The physiological significance of "back

migration II of activated T cells into the thymus may be that presentation of retrovirus

antigens in the thymus may trigger self-tolerance induction, causing the loss of immune

response to retrovirus antigen. Alternatively, release of cytokines by retrovirus-activated

mature T cells may impair T cell differentiation, causing secondary acquired T Cell

deficiency. This notion that circulating T cells activated by retrovirus migrate into the

adult thymus is in accord with the some reported (184-187). This speculation is also

supported by the recent observation in our lab that mature mesenteric lymph node

lymphocytes from mice infected with retrovirus LP-BM5 migrated back to thymus of

normal mice by adoptive transfer assay ( Lopez et al, unpublished data).

69

Production and action of cytokines in the human and mouse thymus have been

associated with an essential role in T cell development (167). In mice, expressing a

human IL-2Ra chain transgene, thymocytes express a nonfunctional murine IL-2RB­

human IL-2Ra heterodimer resulting in the accumulation of T cell precursors in the

thymus and periphery (188). Also, anti-IL-2R a chain antibodies abrogate T cell

development which can be reversed by addition of IL-2 (189). Addition of IL-2 to intact

lobes immersed in culture medium promotes the selective outgrowth of T cells expression

'YO TCR (190). Consequently, the dysregulation of IL-2 release by retrovirus infection

we observed may be responsible for the changes in T cell subpopulations and

differentiation in the thymus during the progression to murine AIDS. The addition of

IL-4 blocks T cell development by reducing the number of DP thymocytes (191). The

constitutive production of IL-4 in IL-4 transgenic mice also result in the inhibition of DP

thymocytes and mature peripheral T cell development (192, 193). Consequently we

conclude that consistently increased level of IL-4 induced by retrovirus infection in the

thymus may contribute the impairment of T cell development in the murine or human

AIDS, thereby causing secondary acquired T cell deficiency. IL-6 has been shown to

promote the differentiation of Thy-l +IL-2R + donor thymocytes after intrathymic transfer

into irradiated hosts (194). Thus, the dysregulation of IL-6 secretion induced by

retrovirus infection at the beginning of infection may also contribute the abrogation of

T cell development in the thymus. The physiological significance of increased level of

IFN-'Y by thymocytes induced by retrovirus infection may be involved in the up­

regulation of MHC class I and II expression in the surface of thymic stromal cells, which

70

playa crucial roles in the positive and negative selection during T cell education (Le.

recognization 'self and non-self) in the thymus. Thus, the dysregulation of IFN--y

release by thymocytes may reflect the failure of T cell education, leading to loss of

tolerance to self antigens or reaction to non-self antigens. The source of IFN--y in vivo

has been suggested to be mature CD4 + T cells and NK cells in the peripheral tissues

(195). The decreased number of CD3+CD4+CDS- T subset or no changed CD3+CD4-

CDS+ T subset in the thymus during the progression of murine AIDS can not account

for consistently increased secretion of IFN--y by thymocytes. Thus, we measured thymic

NK cell activity by fluorescence release assay (192). Surprisingly, NK cells activity was

increased after 12 week retrovirus infection (data not shown), because NK cells are

present at a very low frequency ( < 0.1 % of thymocytes) in both the human and the

mouse thymus (196, 197). This result suggested that the impairment of T cell

differentiation and changes of T subpopulations in the thymus during the progression of

murine AIDS could be caused by back migration of other peripheral lymphoid cells (e.g.

NK cells) as well as mature peripheral T cells. The continuing elucidation of the

physiological roles of cytokines in the T cell maturation and the mechanism of these

changes induced by retrovirus infection may facilitate the understanding the

immunopathogenic mechanism of HIV infection on the thymus in human AIDS.

Vitamin A deficiency depresses lymphocyte activation by mitogen in rats (122),

and leads to a depletion of mature T -cells from the spleen. Repletion with retinoic acid

results in a rapid increase in the peripheral lymphocyte count (123). Vitamin A

deficiency may decrease antigen binding due to alterations in surface membrane

71

glycoproteins (203). In large populations, vitamin A deficiency has been found to be

associated with an increased incidence of a variety of cancers, and severity of infection

(203). Similar depression of the general immune functions associated with vitamin E

deficiencies have also been observed in both animals and humans. In rats, depressed

antibody-dependent cell mediated cytotoxicity (204), decreased lymphocytes blastogenesis

in response to mitogens (205), and depressed NK cell mediated cytotoxicity (126) have

been reported in vitamin deficient states. In guinea pigs, dietary vitamin E deficiency

depressed spleen T-and B-lymphocyte immune responses to mitogens to about 50% of

vitamin E adequate animals (133). The macrophages of these vitamin E deficient guinea

pigs also lacked accessory cell functions and acted only as suppressor cells (206). In

humans, a strong correlation of decreased serum levels of vitamin E and decreased

number and metabolic functions of T-lymphocytes have been shown in athletes (207).

Therefore, the deficiencies of vitamin A and vitamin E in the tissues including immune

organs should contribute immune dysfunctions initiated by retrovirus infection in murine

AIDS, e.g. depressed proliferation of T and B cells in the spleen and mesenteric lymph

nodes, impaired cytotoxicity of splenic NK cell, and dysregulated cytokine production

by lymphocytes from spleen and mesenteric lymph node observed in murine AIDS by us

(102, 208, 209).

Zinc has an important role in immunocompetence and exerts a crucial regulatory

effect on specific immune functions. Zinc deficiency results in the adverse effects on the

immune system of a depression of cell-mediated immunity with decreased T -cells and B­

cells in lymphoid tissue, decreased T-helper cell number and activity, an inverted ratio

72

of T-helper to T-suppressor cells, a depressed response of T-cell to tumor cells, delayed

cutaneous hypersensitivity, decreased lymphokine production, decreased production of

cytotoxic T cells, reduced antibody response requiring T-helper cells, decreased natural

killer cells activity, depressed IgM, and depression of thymic hormones (123, 210). Zinc

deficiency may be particularly important because of indications that zinc may play an

important role in immune function, particularly in T-cell response, in mv infection.

Fallutz et al. reported that serum zinc concentration correlated with the

lymphoproliferative responses of mononuclear cells to PHA stimulation and that zinc

supplementation augmented this response to PHA in HIV-infected patients (201). In the

rabbit, both epithelial and stromal herps simplex keratitsare more severe when zinc is

deficient (129). A dose of trypanosoma cruzi, which would normally be sublethal,

causes the death of 80% of zinc-deficient mice who also exhibit a 50-fold higher

parasitemia than pair-fed. controls (131). In such zinc-deficient rats, the expulsion of

trichinella spiralis (130) and Strongyloides ratti is impaired (130, 211). Findings

indicating an impaired host defense system in copper deficiency have been reported in

humans and animals. Children with the Menkes syndrome, a rare congenital disease

resulting in copper deficiency, frequently die from pneumonia (212). Copper deficiency

in humans is accompanied by bacterial infections, diarrhea, and bronchopneumonia (213-

216). Bactericidal activity by neutrophils is decreased on copper-deficient cattle,ewes,

and lambs (217, 218). In rat, increased suscep.tibility to Salmonella typhimurium in

copper deficiency was caused by decreased macrophage activity. Reduced numbers of

splenic antibody-producing cells and T cells, reduced proliferation of T and B cells,

73

reduced responsiveness and stimulator activity of splenic T cells in one-way mixed

lymphocyte reaction (219-224), and impaired cell-mediated immunity have been observed

in copper deficient mice (221). Therefore, nutrient deficiencies in AIDS could further

predispose HIV -infected patients to more frequent or severe infections. Thus, the severe

malnutrition seen in patients with AIDS may set up a vicious cycle in which the

underlying immunological deficit related to HIV infection is exacerbated by the immune

dysfunction associated with malnutrition. This finding also supports the notion that the

malnourished state of AIDS may aggravate the immune dysfunctions initiated by

retrovirus infection, e.g. depressed proliferation of T and B cells in the spleen and

mesenteric lymph node, impaired cytotoxicity of splenic NK cell, and dysregulated

cytokine production by lymphocytes from spleen and mesenteric lymph node observed

in murine AIDS by us (102, 208, 209). These evidence concomitantly explain the loss

of host resistance to the tumor and opportunistic infections observed in murine AIDS by

us (225-227). It is not clear why zinc level in the thymus and level of copper in the small

intestine were elevated by retrovirus infection during murine AIDS. They may reflect

another kind of pathological symptom of AIDS as these nutrient deficiencies in other

tissues, possible originating from an abnormal pathway of their absorption into the body.

In summary, results in a murine model of AIDS induced by LP-BM5 murine

leukemia retrovirus show a undernutrition after development of AIDS, possible due to

the retrovirus infection causing significant gastrointestinal damage, thereby inducing

malabsorption. Nutritional deficiencies could facilitate the progression of the disease

initiated by retrovirus infection, and suppress immune responses, and speed loss of

74

resistance to the tumors and opportunistic infections. Obviously the mechanism by which

retrovirus infection ca.uses malnutrition should be further elucidated, which would

facilitate the understanding of HIV -induced enteropathy in AIDS individuals. The

alteration of immune response, cytokine productions, and the enhanced susceptibility to

opportunistic infections during the progression of LP-BM5 MuLV infection has been

shown in parallel with that of HIV-infected patients (195). Therefore, to continue

elucidating of the mechanism of immunomodulation during LP-BM5 MuLV infection

should help us to understand HIV -induced immune dysfunction in humans.

75

-e. Control

10 -E 8 -Cl 6 c -c 4 I

U. Z 2 I-

0 0 2 4 ' 10 16 22

35

::::- 28

/~ E -- 21 ~ Cl C -to 14 ~

I

~

7

0 0 2 4 10 16 22

Post-Infection (weeks)

Fig. 2.1 IL-6 and TNF-cx Production by Peritoneal Macrophages. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mice. Cytokines were secreted by I x HP macrophages in 0.2 ml volume.

4

E 3 -en S 2 C\I

I

....I

_ 2

E -C)

c: -<IS E 1 E ('(J OJ •

Z u.

-e. Centrel -0- LP.BMS

o 2 4 6 B 10 16 22

0---------__ _ o 2 4 6 B 1 0 16 22

Post-Infection (Weeks)

76

Fig. 2.2 IL-2 and IFN--y Production by Splenic T cells. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mice. Cytokines were secreted by 1 x 106 T cells in 0.2 ml volume.

77

-e. Control -0. lP.BM5

5

E 4 ~O - 3 Cl

c: -0I;f' 2 , ~ 1

0 0 2 4 6 8 10 16 22

8

:::: 6 E -C) c: 4 -U')

I

...J 2

a a 2 4 6 8 10 16 22

Post-Infection (Weeks)

Fig. 2.3 IL-4 and IL-5 Production by Splenic T cells. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mice. Cytokines were secreted by 1 x 106 T cells in 0.2 ml volume.

78

.. -- Control _0_ LP.8M5

21

- c=/ E 14 -C)

c:: -<0 7 I

-!

0 0 2 4 6 8 10 16 22

320

Q - 240 E -:::J - 160 0 ,..

I

~ 80 ~- ----- -'-...... a

0 2 4 6 8 10 16

Post-Infection (\Veeks)

Fig. 2.4 IL-6 and IL-lO Production by Splenic T cells. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mIce. Cytokines were secreted by 1 x 106 T cells in 0.2 ml volume.

79

-e. Control -0_ LP.8MS

1.00

-E

0.75 -en ..s 0.50

C\I I

~ 0.25

0.00 0

0.50 2 5 7 9 11 13 15 18 20

::::- 0.40

E - 0.30 C)

C -~ 0.20

I

-1 0.10

0.00 a 2 5 7 9 11 13 15 1 B 20

Post-I nfection (Weeks)

Fig. 2.5 IL-2 and IL-4 Production by thymocytes. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mice. Cytokines were secreted by I x las thymocytes in 0.2 ml volume.

80

-.- Control _0_ LP.8M5

2

0 • 0 2 5 7 9

9 11 13 15 18 20

::::-e 6 -CD C -l-

I

Z :3 U.

0- 0--0

0 0 2 5 7 9 11 13 15 18 20

Post·lnfection (Weeks)

Fig. 2.6 IL-6 and IFN--y Production by thymocytes. Every sample was determined in triplicate. The values are Mean + SD of 6-8 mice. Cytokines were secreted by 1 x 1<f thymocytes in 0.2 ml volume.

Table 2.1 Splenocyte Proliferation Induced by Con A During Murine AIDS#

Retrovirus CPM ( x 1()3)

Infection 4 8 12

16.5 ± 0.54 19.0 + 0.5 20.8 ± 0.7 28.7 + 1.1

+ 11.6 ± 2.3* 16.7 ± 0.24* 5.2 ± 0.49* 4.2 ± 0.75*

# The values are mean + SD for 6 individual culture wells from a pool of 4 mouse spleens. The proliferation was evaluated by 1 x 106 splenocytes/well with Con A (5 J.tg/ml) stimulation.

* P < 0.05 compared to controls.

Table 2.2 Splenocyte Proliferation Induced by LPS During Murine AIDS#

Retrovirus CPM ( x 1()3)

Infection 4 8 12 16 (weeks)

28.6 + 2.0 8.5 + 0.56 8.4 ± 0.2 6.3 + 0.9

+ 21.6 + 1.6* 2.0 + 0.67* 1.7 ± 0.31 * 1.6 + 0.35*

# The values are mean + SD for 6 individual culture wells from a pool of 4 mouse spleens. The proliferation was evaluated by 1 x 106 splenocytes/well with LPS (5 J.tg/ml) stimulation.

* P < 0.05 compared to controls.

81

Table 2.3 IgG Production During Murine AIDS#

Retrovirus IgG (ng/ml)

Infection 4 8 12 16 (weeks)

3.1 + 0.15 5.5 + 0.4 7.6 + 0.45 8.0 + 0.58

+ 10.0 + 1.2* 23.5 + 1.5* 30.0 + 2.9* 75.9 + 9.6*

# The values are mean + SD for 6 individual culture wells from a pool of 4 mouse spleens. The content of IgG was secreted by 1 x 1()6 splenocytes/well with LPS stimulations (5 I-'g/ml) stimulation.

* P < 0.05 compared to controls.

Table 2.4 Thymocyte Proliferation During Murine AIDS#

Retrovirus CPM (x 103)

Infection 4 8 12 16 (weeks)

7.66 + 1.06 10.63 + 2.05 11.63 + 2.59 9.44 + 2.83

+ 9.62 + 0.96* 7.95 + 0.32* 5.04 + 1.48* 11.23 + 1.48

# The values are mean ± SD for 6 individual culture wells from a pool of 4 mouse thymuses. The proliferation was evaluated by 1 x 1()6 thymocytes/well with Con A (5 I-'g/ml) stimulation.

* P < 0.05 compared to controls.

82

Table 2.5 Hepatic Levels of Nutrients During Murine AIDS*

Groups Nutrients (p,gig liver)

a-retinol a-tocopherol zinc copper

Control 450.0+60.1 67.3±8.5 86.4+ 10.1 15.9+ 1.4

Murine AIDS 150.0±39.0@ 43.3±4.0@ 89.7±11.8 12.8±1.9@ * Values are mean + SD for 8-10 mice. @ p < 0.05.

Table 2.6 Intestinal Levels of Nutrients During Murine AIDS*

Groups Nutrients (p,gig intestine)

a-retinol a-tocopherol zinc

Uninfected 57.1 +2.4 145.2+30.0 53.3+ 15.0

Murine AIDS 46.8+6.7@ 154.8+46.7 50.3+15.8 * Values are mean + SD for 8-10 mlce. @ p < 0.05.

Table 2.7 Splenic Levels of Nutrients During Murine AIDS*

Groups Nutrients (p,gig spleen)

a-retinol a-tocopherol zinc

Uninfected 23.7+3.2 396.6+37.6 13.9+1.4

copper

6.3+0.4

8.5+0.2@

copper

7.38+1.4

Murine AIDS 7.4±1.9@ 85.8±33.3@ 7.17±1.2@ 9.13±0.6@ * Values are mean + SD for 8-10 mlce. @ p < 0.05.

83

Table 2.8 Thymic Levels of Nutrients During Murine AIDS*

Groups Nutrients (p.g/g thymus)

a-retinol a-tocopherol zinc copper

Uninfected 48.6+ 10.8 53.5±9.0 1.46+0.2 15.48+4.8

Murine AIDS 30.6±4.0@ 38.1±7.9@ 6.38±2.4@ 8.54± 1.8@ * Values are mean + SD for 8-10 mIce. @ p < 0.05.

Table 2.9 Serum Levels of Vitamins During Murine AIDS*

Groups Vitamins

a-retinol

Uninfected 1.57+0.33

Murine AIDS 0.71 +0.2@ * Values are mean + SD for 8-10 mIce.

(JLg/ml)

a-tocopherol

3.3+0.36

0.73+0.19@ @ p < 0.05.

84

CHAPTER 3: ALCOHOL CONSUMPTION SUPPRESSES

CYTOKINE PRODUCTION AND IMMUNE RESPONSE IN C57BL/6 MICE

Abstract

85

In the present study, effects of chronic dietary ethanol (EtOH) on cytokine

production by splenocytes and thymocytes, splenocyte and thymocyte proliferation

induced by mitogens, splenic NK cell activity and antibody production (IgA and IgG)

were examined. C57BL\6 mice were fed 5% EtOH v/v in the Lieber-DeCarli liquid diet

for 11 weeks. Release of interleukin-2 (IL), IL-5, IL-6, IL-lO and Interferon-gamma

(IFN) produced by ConA-stimulated splenocytes was significantly decreased, while

secretion of IL-4 was slightly decreased by chronic dietary EtOH compared to controls.

Production of TNF-a and IL-6 by LPS-stimulated splenocytes was significantly and

slightly increased by EtOH compared with controls, respectively. Splenocyte and

thymocyte proliferation induced by ConA was significantly inhibited by EtOH, while

splenocyte proliferation induced by LPS was not affected. NK cell activity was

significantly inhibited by EtOH compared with controls. The production of IgA and IgG

by splenocytes were also significantly decreased by EtOH compared with controls. The

levels of IL-2, IL-4 and IL-6 produced by ConA-stimulated thymocytes were significantly

reduced by dietary EtOH compared with control, while production of IFN-,), by

thymocytes was not affected. Our results suggest that chronic dietary EtOH alters the

cytokine release, thereby impairing immune response and T cell maturation, which

increase host susceptibility to infection.

86

Introduction

Prolonged consumption of ethanol (EtOH) results in alterations of host defense

mechanisms manifested ultimately by increasing susceptibility to infectious agents (84).

The deleterious effects of EtOH on the ability to generate immune responses may play

a crucial role in this increased susceptibility to infection. In experimental animals and

humans EtOH has an inhibitory effect on many aspects of the immune response (85).

A loss of number of circulating leukocytes has been associated with EtOH use (86, 87)

with detrimental effects on humoral immune response, including antibody production

(88). It also suppressed aspects of cell-mediated immunity such as delayed-type

hypersensitivity reactions (86, 88) and lymphocyte proliferative response to mitogens

(89). EtOH administration to experimental animals has a profound effect on peripheral

lymphoid organs as measured by a decrease number of thymocytes and splenocytes as

well as in weighs of the spleen and thymus (90-92, 228, 299). Mice fed EtOH for short

periods of time have shown both normal and decreased NK cell activity (97, 98, 230).

Antibody dependent cellular cytotoxicity (99), lymphocytes proliferation (100), and B

lymphocyte functions (100, 101) are also impaired by dietary EtOH. However little is

known about the immunomodulatory mechanisms that cause these various immune

changes, especially EtOH-dysregulated cytokine production (231).

Pro-thymocytes leave the bone marrow and arrive at the thymus where they find

the appropriate environment that let them complete their differentiation to become

functional T cells. Thus, the thymus provides a unique environment for the development

of T cells, supporting both precursor cell proliferation and differentiation. Production

87

and action of cytokines in the human and mouse thymus have been associated with an

essential role in T cell development. To our best knowledge, there is no any papr~rs to

report the effects of chronic dietary EtOH on cytokine release by thymocytes.

Interference of EtOH with alterations of immune mechanisms may be one of the

most important factors that predispose alcohol abusers to an increased risk of infections.

Any perturbation in tightly controlled cytokine regulated system can result in immune

alterations modifying host resistance to infectious disease and cancer. We have therefore

designated this study to investigate the chronic effects of dietary EtOH on the production

of major regulatory cytokines (interleukin-2 (IL), IL-4, IL-5, IL-6, IL-lO, tumor necrosis

factor-ex (TNF) and interferon-gamma [IFN-'Y)] by splenocytes and cytokines (IL-2, IL-4,

IL-6 and IFN-'Y by thymocytes, and some immune functions (splenocyte and thymocyte

proliferation, splenic NK cell activity, IgG and IgA production).

88

Materials and Methods

Animals and EtOH Diet Female C57BL\6 mice, 3 to 5 weeks old, were obtained

from the Charles River Laboratories Inc. (Wilmington, DE). The mice were housed in

transparent plastic cages with stainless steel wire lids. Mice were housed to 4 mice a

cage. The housing facility was maintained at 20 to 22° C and 60 to 80% relative

humidity and the animals were exposed to a 12: 12 hr light-dark cycle. Water and

mouse chow (Texland, Madison, WI) were provided ad libitum. After 3 week housing

in the animal facility in the Arizona Health Sciences Center, mice were randomly

assigned to one of the following two treatments: control and EtOH (5% v/v, Lieber­

DeCarli liquid diet, #710260, Dyetes, Bethlehem, PA). All diets were made isocaloric

by substituting 26% of the total calories with ethanol for the ethanol diet and dextrose

for the control diet. In previous study in our lab, we have found that higher dietary

EtOH (e.g. more than 6% v/v) significantly increased mortality of mice, and lower

dietary EtOH (e.g. less than 4% v/v) did not cause significant change on immune

functions. Mice were fed the respective diets for 11 weeks. Pair-fed study was

performed in our lab, and no difference between pair-fed group and EtOH-fed ad-lib

group was observed in the weight of thymus and spleen, and some immunoparameters

and cytokine secretion (IL-2, IFN-'Y and TNF-a) (232). Thus, we did not set up another

group for pair-fed group in this study.

Standard Cytokines and their Antibodies See Materials and Methods in Ch. 2.

Preparation of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Preparation of Cytokine Production See Materials and Methods in Ch. 2.

89

Preparation of IgA and IgG Production See Materials and Methods in Ch. 2.

Natural Killer (NK) Cell Activity See Materials and Methods in Ch. 2.

ELISA For Cytokines See Materials and Methods in Ch. 2.

ELISA for IgA and IgG See Materials and Methods in Ch. 2.

Mitogenesis of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Statistics Data are presented as the mean + SD for each group. Two tail t-student

test was applied. p < 0.05 was considered significant difference between two groups.

90

Results

Production of Splenic Cytokines As shown in Table 3.1, the secretion of IL-2, IL-5,

IL-6, IL-10 and IFN--y by conA-stimulated splenocytes was significantly suppressed by

chronic dietary EtOH compared to the controls (p < 0.05), while the release of IL-4

by splenocytes was slightly enhanced ( p > 0.05).

Production of IL-6 and TNF-O! The production of TNF-O! by LPS-stimulated

splenocytes was significantly increased by dietary EtOH compared to the controls (p <

0.02), while secretion of IL-6 was slightly increased (Table 3.2, P > 0.05 ).

Production of Thymic Cytokines The levels ofIL-2, IL-4 and IL-6 by thymocytes were

significantly reduced by dietary EtOH compared to the control (p < 0.05), while IFN--y

production was not affected (Table 3.3, p > 0.05).

Splenic NK Cell Activity As shown Table 3.4, The NK cell activity was significantly

inhibited by dietary EtOH at ratio of effector and target cells: 100: 1, 50: 1, 25: 1, 12.5: 1

compared to the control (p < 0.05).

IgA and IgG Production As shown in Table 3.5, production of IgA and IgG by LPS­

stimulated splenocytes was significantly inhibited by dietary EtOH compared to controls

(p < 0.05).

Mitogenesis of Splenocytes and Thymocytes Con A-induced splenocyte (T cells) and

thymocytes proliferation were significantly inhibited by dietary EtOH compared to

controls (p < 0.05), while LPS-induced splenocyte (B cells) proliferation was not

affected (Fig. 3.1).

91

Discussion

Medical complications related to the ingestion of large amounts of alcoholic

beverages are a major health problem. The emphasis of this study was to evaluate if

EtOH consumption could impair cytokine secretion, which is a critical factor in immune

modulation (233). Our data demonstrated that chronic dietary EtOH had a general

suppressive effect on in vitro cytokine production by splenocytes and thymocytes. This

could lead to immune dysfunction, favoring pathogenic or opportunistic infection as seen

in the chronic alcohol user (82, 84, 195).

Several mechanisms have been suggested for immunomodulatory effect of EtOH

(233a, 234). Some immunological parameters are directly influenced by EtOH

interacting with cell membranes while others are changed through intervention by

hormonal changes caused by central nervous system effects, nutritional modulation or

physiological modification by EtOH, and alcoholic liver diseases (235, 236). Model of

murine EtOH-consumption used Lieber-DeCarli liquid diet. It is supplemented with

nutrients largely, minimizing the interference of alcoholic liver diseases and EtOH­

induced malnutrition. Thus, the suppression mechanism of dietary EtOH on cytokine

production is partly caused by a direct effects of EtOH or its metabolites' effects.

Although it is not an accurate picture of changes associated with the totality of dietary

EtOH effects in human alcoholism, it hopefully will aid in the definition of

immunomodulatory role of chronic dietary EtOH on immune response. Our in vitro data

indicated that EtOH at physiological concentrations (1 %, 0.2%, 0.04%, v/v in

medium)directly suppress cytokine production in a concentration-dependent manner

92

(Wang et al. in manuscript). We also performed experiments to test for the effects of

BtOH before or after mitogen (ConA) stimulation in order to understand the possible

mechanism of BtOH direct effects on cytokine production. First, we exposed BtOH to

splenocytes for 4 hours, then washed this BtOH off the splenocytes with CM. ConA was

added and plates were allowed to incubate for 24 hours in order to induce cytokine

production. No suppressive effect of BtOH on these cytokines was observed (Wang et

al. in manuscript). Alternatively, first Con A is added for 4 hour stimulation, and then

ConA was washed off from splenocytes. BtOH was then added into the culture plates

for another 24 hour incubation for the production of cytokine production. The results

showed a kind of the suppression of IL-2, IL-4 and IL-6 production in a concentration­

dependent manner (Wang et al. in manuscript). Taken together, we speculated that

suppression of cytokine secretion directly induced by BtOH was perhaps caused by

impairment of signal transduction and inhibition of expression of mRNA and translation

of cytokines. Resting cells were not affected by BtOH exposure before mitogen activates

them. Since BtOH could directly interfere with cytokine producing cells by altering

signal transduction and membrane fluidity (237, 238), we speculated that chronic dietary

BtOH may dysregulate cytokine production via perturbing signal transduction when

immunocompetent cells are activated. In addition, Chronic BtOH consumption can

decrease the percentage of T cells and macrophages in the spleen (95), explaining part

of the reduction in splenocyte cytokine production.

In the immune system, IL-2 is a pivotal cytokine in the growth and differentiation

of T and B cells and activation of natural killer (NK) cells and lymphokine-activated

93

killer cells (231). IL-2 also can act as a growth and differentiation factor for B cells and

can activate macrophages (23). IL-4 was initially described as a costimulator of

proliferation of B cells treated with anti-IgM antibody. IL-4 has since been shown to

playa critical role in immunoglobulin class switching. IL-4 also acts as a growth factor

for T cells and may also influence T cells by inducing expression of high affinity IL-2

receptor and the production of IL-2. In addition, IL-4 has been shown to induce

macrophage to express MHC class and FcR, and activate them to kill tumor cells (239-

241). IL-5 is a B cell growth factor because of its ability to stimulate proliferation and

antibody production by B cells. IL-5 increase the amount of IgA secreted by murine

splenic B cells (242, 243). IL-6 is a major inducer of terminal differentiation of B cells

into plasma cells. In relation to T cells, IL-6 has been shown to play an important role

in the T response to alloantigen and development of effector T cytotoxicity cells (244).

IFN-'Y has mUltiple distinct biological activity including anti-viral activity, activating

phagocytosis of macrophages and neutrophil cells, cytotoxicity of NK cells, augmenting

the development of Thl (T helper) cells and inhibits differentiation of Th2 cells and

production of Th2 cytokines (245). IL-IO inhibits Thl differentiation of Thl cells and

production of Th 1 cytokines via indirect action of IL-I 0 on a subpopulation of accessary

cells (195). TNF-a can not only stimulate IL-I and IL-6 secretion by macrophages, but

also induces IL-2 receptor expression and T cell proliferation (246, 237). This study

confirm and expand the understanding of the substantial immune dysfunction caused by

dietary EtOH. The suppression of T cell proliferation by chronic dietary EtOH we

observed confirmed the reports (89, 91, 248). It could be due to the decreased level of

94

IL-2 induced by dietary EtOH we observed. The decreased production of IgA and IgG

by LPS-stimulated splenocytes by chronic dietary EtOH we observed is consistent with

inhibition of B cell antibody response to T-cell dependent antigenic stimulation by

dietary EtOH (248). That could be caused by decreased levels of IL-5 and IL-6 induced

by EtOH observed here. However no effect of dietary EtOH on LPS-stimulated B cell

proliferation was observed, which is consistent with previous finding that dietary EtOH

has no effect on B cell response to T -independent antigens (248). This may be due to

the absence in change of IL-4 secretion and decreased production of IFN--y induced by

dietary EtOH we observed. Our results confirmed that chronic dietary EtOH exerts a

suppressive effect on NK cell activity, which is consistent with the previous reports (92,

230). The suppression of NK cell activity could have been caused by the reduction of

IL-2 and IFN--y we observed, which are potent inducers and simulators of NK cells.

Impaired immune functions of polymorphonuclear neutrophils (PMN) and

macrophages/monocytes were also observed in alcoholism and EtOH-fed animal models

(249, 250). With regards to functions as hematopoiesis, chemotaxis, adherence and

activation of PMN of macrophages/monocytes are critically dependent on the presence

ofIL-l and IFN--y (251). IFN--y release by ConA-stimulated splenocytes from the EtOH­

treated mice were significantly inhibited. IL-la release by murine peritoneal

macrophages was also significantly suppressed by dietary EtOH (Wang et al. unpublished

data). Therefore, suppression of macrophages functions could be caused decreased

levels of IL-la and IFN-,}" which is responsible for initiating macrophage activation and

augment macrophage-related immune responses, including antigen presentation,

95

phagocytosis and release of soluble factors.

The mammalian thymocytes can be subdivided into 4 major subpopulations based

on expression of the differentiation-related antigens CD4 and CD8. "Double-negative

" (DN) CD4-CD8- thymocytes (2-4% of total in the normal adult mouse) represent

immature cells with the capacity to give rise to the other subsets in irradiated host (180).

CD4 +8+ cells in the thymic cortex account for the majority (80-85 %) of thymocytes and

most of such "double-positive" (DP) cells are destined to die in situ (181). The other

two" single-positive" (SP) CD4-8+ and CD4+8- subsets (5-10%) in the thymic medulla

represent mature T cells seen in the peripheral tissue, which originate from DP

thymocytes (181). The control of generation of functional competent T cells from

precursor population within the thymus was mediated by cytokines (167). In mice,

expressing a human IL-2Ra chain transgene, thymocytes express a nonfunctional murine

IL-2Rl3-human IL-2Ra heterodimer resulting in the accumulation of T cell precursors in

the thymus and periphery (167). Also, anti-IL-2R ex chain antibodies abrogate T cell

development which can be reversed by addition ofIL-2 (189). Addition of IL-2 to intact

lobes immersed in culture medium promotes the selective outgrowth of T cells expression

'YO TCR (190). The addition of IL-4 blocks T cell development by reducing the number

of DP thymocytes (191). The constitutive production of IL-4 in IL-4 transgenic mice

also result in the inhibition of DP thymocytes and mature peripheral T cell development

(192, 193). IL-6 has been shown to promote the differentiation of Thy-1 +IL-2R + donor

thymocytes after intrathymic transfer into irradiated hosts (194). The physiological

significance of IFN-'Y is involved in the up-regulation of MHC class I and II expression

96

in the surface of thymic stromal cells, which play a crucial role in the positive and

negative selection during T cell education (Le. recognization 'self' and non-self) in the

thymus. The BtOH-induced suppression of IL-2, IL-4 and IL-6 by thymocytes may be

responsible for the changes in T cell subpopulations and differentiation in the thymus

resulting from BtOH ingestion (253). There were no difference in the percentage of a6-

TCR+, CD4+ and CD8+ thymocytes between Lieber-DeCarli fed mice with or without

ethanol (5 %, v/v) (62). Therefore, the functional change of mature T cells in the thymus

may be responsible for the change of cytokine secretion by thymocytes we observed here.

BtOH-induced corticosteroid may play some role in this process (254). The another

possible mechanism has been proposed whereby ethanol interacts with the endogenous

opioid system by : (a) production of certain ethanol metabolites, for example the

isoquinolines which bind to opiate receptors, (b) altering the binding properties of opiate

receptors and (c) altering the release, synthesis and post-translational processing of

endogenous opioid peptide (255, 256). Thus, BtOH-induced changes in corticosteroid

and opiate could be responsible for the changes in cytokine release by thymocytes,

thereby altering T cell maturation. Abnormalities ofT cell maturation in the thymus may

contribute the impairment of T cell response we and others observed in the peripheral

tissues in animals or humans.

In summary, chronic BtOH ingestion via liquid diet generally caused marked

suppression in the cytokine production induced by mitogens in vitro by splenocytes and

thymocytes, concomitant with a general suppression of immune response. Our results

suggest that the suppressive effects of BtOH on immune responses could be directly

97

caused by dysregulation of cytokine r€?lease by splenocytes and thymocytes, which

controls the T cell maturation and general immune response.

Table 3.1 Effect of Dietary EtOH on Production of Cytokines by Splenocytes*

Cytokines

(ng/ml) Control

IL-2 1.90 + 0.65

IL-4 0.088 + 0.031

IL-5 1.71 + 0.85

IL-6 3.84 + 0.031

IFN--y 21.0 + 5.60

IL-IO(U/ml) 9.76 + 4.89

Treatment

EtOH

0.82 + 0.19

0.056 + 0.033

0.41 + 0.07

1.44 + 1.25

14.7 + 0.97

1.65 + 0.10

P Values

< 0.02

N.S.

< 0.002

< 0.0002

< 0.02

< 0.001

* Every sample was determined in triplicate. Values are mean + SD for 8 mice per group. Cytokine production was secreted by 1 x 1()6 splenocytes in 0.2ml volumes.

N.S. Not Significant

Table 3.2 Effect of Dietary EtOH on IL-6 and TNF-O! By Splenocytes*

Cytokines

(ng/ml)

IL-6

TNF-O!

Control

3.60 ± 0.64

1.32 + 0.04

Treatment

EtOH

2.95 + 0.46

0.9 + 0.18

P Values

N.S.

< 0.05

* Values are mean + SD for 8 mice per group. Cytokine production was secreted by 1 x 1<r splenocytes in a 0.2 ml volume.

N.S. Not Significant

98

Table 3.3 Effect of Dietary EtOH on Production of Cytokines by Thymocytes*

Cytokines

(ng/ml)

IL-2

IL-4

Control

0.157 + 0.020

0.130 + 0.112

Treatment

EtOH

0.104 + 0.009

0.049 + 0.013

P Values

< 0.05

< 0.05

IL-6 1.07 + 0.56 0.478 + 0.30 < 0.01

IFN-" 0.185 + 0.06 0.182 + 0.03 N.S. * Every sample was determined in triplicate. Values are mean + SD for 8 mice per group.Cytokine production was secreted by 1 x IO' thymocytes in 0.2 ml volumes.

N.S. Not Significant.

Table 3.4 Effect of Dietary EtOH on Splenic NK Cell activity *

Treatment Cytotoxicity (%)

100:1 50:1 25:1 12.5: 1 (E:T)

Control 42.3 + 2.3 34.6 + 3.9 29.7 + 2.9 25.7 + 3.7

EtOH 26.7 ± 3.6# 24.6 ± 2.9# 18.3 ± 4.6# 16.8 ± 2.4# * Every sample was determinea in triplicate. Values are mean + SD for 8 mice per group. Activity of NK cell was evaluated in a 0.2 ml volume with 1 x lOS of target cells (YAC-l).

# p < 0.05

Table 3.5 Effect of Dietary EtOH on Production of IgA and IgG By Splenocytes*

Immunoglobulins

(ng/ml) Control

Treatment P Values

EtOH

IgA 55.7 + 21.1 26.6 + 14.8 < 0.05

IgG 479.0 + 117.0 279.0 + 130.8 < 0.05 * Every sample was determmed m tnphcate. Values are mean + SD for 8 mice per group. Immunoglobulin production was secreted by 1 x 10' splenocytes in a 0.2 ml volume.

99'

0 0 0 ..->C

::E D-O

100

* P< 0.05 140 50

124

108 1 44

3B

* 92 32

76 26

60 -"'"

control ETOH control ETOH B cell Proliferation T cell Proliferation .

60

52

control eTOH Thymocyte Proliferation

Fig. 3.1 Effects of dietary EtOH on splenocyte and thymocyte proliferation. Every sample was determined in triplicate. Values are mean ± SD for 6-8 mice each group. The proliferation was evaluated in 0.2 ml volume with 1()6 cells.

101

CHAPTER 4: ALCOHOL CONSUMPTION MODULATION OF

CYTOKINE PRODUCTION AND IMMUNE RESPONSE DURING MURINE AIDS

Abstract

Chronic dietary EtOH 5% (v/v) in the Lieber-DeCarli liquid diet was fed

C57BL/6 mice inoculated with LP-BM5 retrovirus causing murine AIDS for 11 weeks.

Cytokine production by splenocytes and thymocytes, and some immune functions were

measured. Decreased levels of IL-2 caused by retrovirus infection remained unchanged.

Elevated levels of IL-5 and IL-6 produced in vitro by splenocytes during retrovirus

infection were further increased by EOH. Elevated IL-4 due to retroviral infection was

not affected by EtOH. Increased production of IL-10 induced by retrovirus infection,

however, was significantly inhibited by EtOH while decreased release of IFN--y induced

by retrovirus infection was significantly enhanced. Elevated levels of TNF-c:x produced

by splenocytes from retrovirus infected mice were significantly further increased by

EtOH, while levels of IL-6 were not affected. Suppressed T cell proliferation caused by

retrovirus infection was significantly reduced further by EtOH. However, no effect of

EtOH was observed on B cell decreased proliferation by retrovirus infection.

Retrovirus-suppressed IL-2 production and retrovirus-elevated IFN--y secretion by

thymocytes were not further affected by EtOH. However, decreased IL-5 levels and

increased IL-5 during murine AIDS were significantly increased and reduced by EtOH,

respectively. Unchaged IL-4 during murine AIDS was significantly reduced by EtOH.

IL-2 production produced by ConA-stimulated splenocytes was suppressed during murine

AIDS. It was further inhibited in EtOH-fed mice compared to controls at 6 weeks post-

102

infection, whereas decreased level of IFN-'Y during murine AIDS was not further affected

in EtOH-fed mice. The levels of IL-S, IL-6 and IL-1O by secreted by ConA-induced

splenocytes, elevated during murine AIDS, were significantly further enhanced in EtOH­

fed mice compared to controls at 6 weeks post-infection, while retrovirus-induced

elevated release of IL-6 and TNF-a, by produced by LPS-stimulated splenocytes, were

further increased in EtOH-fed mice compared to controls at 6 and 9 weeks post-infection,

respectively. ConA- and LPS-induced splenocytes proliferation, inhibited by retrovirus

infection, was significantly further suppressed in EtOH-fed mice compared to controls.

The body and thymus weight were not affected by EtOH consumption, whereas

thymocyte number and proliferation, which had been reduced during murine AIDS, were

significantly further reduced by EtOH use. The production of IL-2 and IL-6 by

thymocytes, which has been lessened during murine AIDS, were significantly further

suppressed by dietary EtOH at 6 weeks post- infection, whereas levels of IL-4 and IL-6

by thymocytes, which had been elevated during murine AIDS, were significantly further

increased by EtOH consumption. These data also suggest that dietary EtOH consumption

can modulate cytokine production by thymocytes, adversely affecting T cell

differentiation, especially during retrovirus infection. These results suggest that dietary

EtOH aggravates progression of immune dysfunction and affects T cell maturation in the

thymus, leading to AIDS as dietary EtOH modifies production of immunological

regulatory cytokines by splenocytes and thymocytes.

103

Introduction

Because of overwhelming evidence that the deleterious effects of chronic EtOH

consumption on immune response (84, 85, 287), it could further enhance susceptibility

to infection and development of AIDS (288, 17). EtOH increased in vitro both the

susceptibility of cells to HIV infection and their replication of HIV in infected

monocytes/macrophages and T cells (139-141). However, a recent study of EtOH

ingestion in HIV -positive persons did not demonstrate an increase in the development of

AIDS (142). Since the dosage, frequency and duration of EtOH exposure in the various

stage of HIV -positive patients may be a key role as a cofactor in the progression to

AIDS, the results of this study are difficult to be interpreted when EtOH exposure was

not well defined. Thus it remains unclear if alcoholics more predisposed to HIV

infection and whether EtOH ingestion at the special stage of HIV infection (e.g.

following seroconversion) aggravates the development of AIDS. Ethical, economical

and logistical considerations, however, have precluded well-defined studies in mv+

humans using large amounts of EtOH. As the rate of development of human AIDS

after retrovirus infection is variable (17), some cofactors may exacerbate the effects of

retrovirus infection on development of severe immunosuppression initiated by retrovirus.

Alcohol (BtOH) use, which has suppressive effects on immune responses (85, 248, 257),

could synergize with retrovirus infection to accelerate the onset of immune dysfunctions

(17, 269). We found that dietary EtOH reduced in vitro cytokine production by cells

from normal, uninfected animals (269, 270). Dietary EtOH also synergized with murine

AIDS to promote growth of tumors (271), death rate after Streptococcus pneumoniae

104

infection (272), and intestinal parasite colonization (273, 226). Therefore we further

defined the effects of chronic dietary EtOH consumption on in vitro production of

regulatory cytokines (IL-2, IL-4, IL-5, IL-6, IL-lO, IFN-'Y and TNF-a) by mitogen­

stimulated splenocytes and thymocytes, and some immune functions during murine

retrovirus infection. Then in order to assert whether EtOH users prior to retrovirus

infection are more predisposed to immunological susceptibility to retrovirus infection,

chronic EtOH-fed mice were infected with LP-BM5 retrovirus infections, and immune

dysfunctions and cytokine dsyregulation were monitored in T and B cell proliferation,

antibody [immunoglobulin A and G (Ig)] production and cytokine [Interleukin-2, 5, 6,

10 (IL), interferon-'Y (IFN) and tumor necrosis factor-a (TNF)] production. In addition,

we proposed EtOH use prior to retrovirus infection may act as a cofactor in the

development of AIDS via affecting T cell maturation in the thymus. In order to test this

hypothesis, chronic EtOH-fed mice were infected with LP-BM5 retrovirus infections

causing murine AIDS, which functionally simulates human AIDS (58). Thymocyte

proliferation and its cytokine [interleukin-2, 4, 6 (IL), and IFN-'Y ] production were

monitored.

105

Materials and Methods

Animals Female C57BL\6 mice, 3 to 4 weeks old were obtained from the Charles

River Laboratories Inc. (Wilmington, DE). The mice were housed in transparent plastic

cages with stainless steel wire lids. Mice were housed 4 mice to a cage. The housing

facility was maintained at 20 to 22° C and 60 to 80% relative humidity and animals were

exposed to a 12: 12 hr light-dark cycle. Water and mouse chow (Texland, Madison, WI)

were provided ad libitum. After 1 week housing in the animal facility in the Arizona

Health Sciences Center, mice were infected with retrovirus LP-BM5, and then randomly

assigned to the following two treatments in the first study: control and EtOH. These

treatments were initiated simultaneously. There was no observed difference at the time

of mouse sacrifice between the two treatments on mouse body weight (Control: 26.61 ±

2.35 g vs EtOH: 27.31 ± 1.75 g, values are mean + S.D. of 8 mice per group). In the

second study, one group received control liquid diet as a control. Another group

received EtOH liquid diet. After 10 weeks feeding, all mice received retrovirus

infection. Upon infection, all mice received the control liquid diet until sacrifice. The

schedule of experiment is following:

Weeks: 0 Dietary Treatment (4.5 % EtOH and and Control)

10 LP-BM5 Infection and

all mice received control liquid diet.

16 19 Sacrifice for Analysis

EtOH Diets In the first study, infected mice were fed with 5 % EtOH (v/v) in Lieber-

106

DeCarli liquid diet (#710260,Dyets, Bethlehem, PA). All diets were made isocaloric by

substituting 26% of the total calories with ethanol for the ethanol diet and dextrose for

the control diet. Mice were fed the respective diets for 11 weeks. In the second study,

all mice were fed the National Research Council (NRC) liquid diet (20). Dietary

ingredients were obtained from Dytes (#710279, Bethlehem, PA). All diets were made

isocaloric by substituting 26% of the total calories with ethanol in the ethanol diet and

with dextrose in the control diet. In previous studies in our lab, we have found that

higher dietary EtOH (e.g. more than 5% v/v) significantly increased mortality of mice,

and lower dietary EtOH (e.g. less than 3% v/v) did not cause significant change in

immune functions. Therefore, the concentration of EtOH in a NRC liquid diet was 4.5 %

(v/v) in this study. A pair-fed study was previously performed in our lab with no

significant differences between the pair-fed group and ad-lib group observed in diet

consumption, body weight, the weight of thymus and spleen, immune functions and

cytokine production (294). Therefore, the diets were provided ad libitum in this study.

The average consumed volume of liquid diet was 12-15 ml per mouse per day for every

diet. No significant difference of consumption of the diets and body weight before

sacrifice between the two groups were observed (data not shown).

LP-BM5 MULV Infection See Materials and Methods in Ch. 2.

Cytokine Standards and Antisera See Materials and Methods in Ch. 2.

Preparation of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Preparation of Cytokine Production See Materials and Methods in Ch. 2.

ELISA for Cytokines See Materials and Methods in Ch. 2.

107

Mitogenesis of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Statistics Two-tailed student T-test was applied. p < 0.05 was considered a

significant difference between two groups.

108

Results

Production of Cytokines by Splenocytes. As shown in Table 4.1, the elevated levels

of IL-S and IL-6 by splenocytes induced by retrovirus infection ( in normal mice IL-S:

1.71 + 0.85 ng/ml; IL-6: 3.84 + 0.031) were significantly and further increased by

dietary BtOH ( p < O.OS), while the release of decreased IL-2 and IL-4 levels by

splenocytes in the murine AIDS ( in normal mice IL-2: 1.90 + 0.65 ng/ml; IL-4: 0.088

+ 0.031 ng/ml) was not further affected by BtOH. The decreased secretion of IFN-'Y

by splenocytes during retrovirus infection ( in normal mice: 21.0 + S.6 ng/ml) was

significantly increased by dietary BtOH (p < 0.05), while increased release of IL-lO

in murine AIDS ( in normal mice 9.76 + 4.89 U/ml) was significantly reduced (p <

0.05). As shown in Table 4.2, the elevated production of TNF-a by LPS-stimulated

splenocytes during murine AIDS ( in normal mice 1.32 + 0.34 ng/ml) was significantly

and further increased by dietary BtOH (p < O.OS), while increased level of IL-6

secretion during retrovirus infection ( in normal mice 3.6 + 0.64 ng/ml) was slightly

increased ( p> 0.05 ).

Production of Cytokines by Thymocytes. As shown in Table 4.3, The decreased IL-2

and IL-S production by thymocytes during retrovirus infection was significantly ( p <

O.OS) further reduced by dietary BtOH (in normal mice: IL-2: 0.157 + 0.053; IL-S: 1.70

+ 0.014 ng/ml). Increased IL-4 and IL-6 production by thymocytes during retrovirus

infection was significantly (P < O.OS) reduced by BtOH ( in normal mice: IL-4: 0.064

+ 0.035; IL-6: 1.07 + 0.S6 ng/ml). Increased IFN-'Y level during retrovirus infection

was not affected by BtOH (in normal mice: 0.18S + 0.056 ng/ml).

109

Mitogenesis of Splenocytes and Thymocytes. The suppressed splenocytes proliferative

response to the T-cell mitogen ConA during murine AIDS ( in normal mice 9300 + 2400

cpm) was significantly and further inhibited by dietary EtOH, while suppressed

splenocyte proliferation induced by the B-cell mitogen LPS during murine AIDS ( in

normal mice 44000 + 2500 cpm) was not affected (Fig. 5.1). As shown in Fig. 5.1,

increased thymocyte proliferation during murine AIDS was not significnatly affected by

EtOH.

Production of Splenice Cytokine Prior to Retrovirus Infection. T helper type I cells

(Thl) produce IL-2 and IFN-'Y which are responsible for cell-mediated immunity.

Secretion of IL-2 and IFN-'Y produced by ConA-stimulated splenocytes during murine

AIDS declined due to retrovirus infection compared to uninfected normal mice (IL-2:

3.54 + 0.43; IFN-'Y: 30.0 + 5.0 ng/ml). IL-2 production during murine AIDS was

significantly (p < 0.05) further inhibited in EtOH-fed mice at 6 and 9 weeks post­

infection compared to their controls (Table 4.4). However, the decreased levels of IFN­

'Y produced by ConA-stimulated splenocytes were not affected by EtOH use (Table 4.4).

Th2 cells produce IL-5, IL-6 and IL-lO that are responsible for humoral immunity.

Secretion of IL-5, IL-6 and IL-lO by ConA-stimulated splenocytes during murine AIDS

is enhanced due to retrovirus infection compared to uninfected, normal mice (IL-5: 0.32

+ 0.015 ng/ml; IL-6: 2.0 + 0.05 ng/ml; IL-lO: 0.45 + 0.05 ng/ml). As shown in

Table 4.5, elevated levels ofIL-5, IL-6 and IL-lO during murine AIDS were significantly

(p < 0.05) further increased in EtOH-fed group at 6 weeks post-infection, but not at

9 weeks post-infection. Macrophages and B cells produce IL-6 and TNF-a, which are

110

involved in immune reactions and immunopathogenesis. The levels of IL-6 and TNF-a

produced by LPS-stimulated splenocytes during murine AIDS were elevated due to

retrovirus infection compared to uninfected, normal mice (IL-6: 1.75 + 0.04 ng/ml;

TNF-a: 1.46 + 0.034 ng/ml). The levels ofIL-6 and TNF-a, increased during murine

AIDS, were significantly (p < 0.05) further enhanced in EtOH-fed mice compared to

control group at 6 and 9 weeks post-infection (fable 4.6).

Production of IgA and IgG Prior to Retrovirus Infection. Hypergammaglobulinemia is

a feature of AIDS. The in vitro production of IgA and IgG by splenocytes was enhanced

by retrovirus infection during murine AIDS compared to uninfected, nonnal mice (IgA:

50.1 + 4.3 ng/ml; IgG: 479.0 ± 77.0 ng/ml). As shown in Table 4.7, elevated IgA and

IgG production by LPS-stimulated splenocytes during murine AIDS was significantly (p

< 0.05) increased in EtOH-fed mice compared to controls at 6 weeks post-infection, but

not at 9 weeks post-infection. These results are in accord with the findings of increased

IL-5 and IL-6 production (Table 4.5), which are major simulators of IgA and IgG

secretion.

Mitogenesis of Splenocytes Prior to Retrovirus Infection. T and B cell proliferation

response to mitogens during murine AIDS are suppressed compared to uninfected,

nonnal mice (f cells: 26,855 + 1,300 cpm; B cells: 48,566 + 2,459 cpm). Decreased

ConA-induced splenocyte proliferation was significantly (p < 0.05) further suppressed

in EtOH-fed mice at 6 weeks post-infection, but not at 9 weeks (Fig. 4.2). This results

are in accord with the findings of decreased IL-2 release (fable 4.1), which is a major

T cell growth factor. Inhibited LPS-induced splenocytes proliferation was significantly

111

further (p < 0.05) reduced in EtOH-fed mice compared to control group at 6 and 9

weeks post-infection (Fig. 4.3).

Body and thymus weight Prior to Retrovirus Infection. As shown in Table 4.7, The

body weight and thymus weight were not affected by dietary EtOH consumption.

Thymocyte number and proliferation Prior to Retrovirus Infection. Thymocyte number

was slightly decreased by retrovirus infection compared to that from uninfected, normal

mice (4.8). EtOH consumption significantly (p < 0.05) further reduced thymocyte

number compared to controls at 6 or 9 weeks post-infection, while decreased thymocyte

proliferation during murine AIDS was significantly ( P < 0.05) reduced by EtOH use

compared to controls at 6 weeks post-infection, but not at 9 weeks post-infection (fable

4.9).

Production of thymic cytokines Prior to Retrovirus Infection. IL-2 production by

thymocytes was decreased during murine AIDS at 6 post-infection compared to that from

uninfected, normal mice and was elevated at 9 weeks post infection, whereas IL-6

production was suppressed at 6 weeks post-infection and was enhanced at 9 weeks post­

infection. IL-4 and IFN--y levels produced by thymocytes were elevated during murine

AIDS. EtOH consumption significantly (p < 0.05) further reduced IL-2 production

compared to controls at 6 weeks post-infection, but it had no effect on IL-2 secretion at

9 weeks post-infection (fable 4.10). Increased IL-4 release by thymocytes during murine

AIDS was significantly (p < 0.05) further elevated by EtOH compared to controls at

6 and 9 weeks post-infection (fable 4.10). EtOH consumption significantly (p < 0.05)

further reduced IL-6 production compared to controls at 6 weeks post-infection, but it

112

had no effect on retrovirus-induced increased IL-6 secretion at 9 weeks post-infection

(Table 4.11). Increased IFN--y release by thymocytes during murine AIDS was sightly

further elevated by EtOH compared to controls at 6 and 9 weeks post-infection (fable

4.11).

113

Discussion

Murine AIDS has been shown to increase certain cytokines (lL-4, IL-5, IL-6, IL-

10 and TNF-a), while suppressing others (lL-2 and IFN-'Y) (208). Dietary EtOH fed

normal, uninfected mice (94) and rats (270) usually suppresses cytokine production.

Thus, there is potential to normalize or exacerbate retrovirus-induced immune

dysfunction. However, for the first time, EtOH was shown to exacerbate, as well as

normalize, immune dysfunctions in cytokine production induced by murine retrovirus

infection. IL-5 and IL-6 production increased by murine AIDS (208) was further

enhanced by EtOH while IL-lO secretion, increased by infection (208), was reduced by

EtOH. IFN-'Y production, suppressed by retrovirus infection (208), was partially

normalized by dietary EtOH. Thus, retrovirus-induced modulation of cytokine

production was exacerbated in some cases and counteracted in others. Chronic EtOH

consumption prior to retrovirus infection more yielded immune dysfunctions and cytokine

dysregulation initiated by retrovirus infection, even though EtOH use was discontinued

for the 6 weeks after infection. Thus, chronic consumption of EtOH can act as a

cofactor to aggravate immune dysfunction and cytokine dysregulation, supporting the

concept that alcoholics may be predisposed to accelerated immune dysfunctions during

HIV infection. The chronic EtOH diet induced immunosuppression in normal mice (94)

appears responsible for the speeding development of murine AIDS, indicating a greater

loss of host resistance after EtOH consumption. Clearly dietary EtOH functioned as a

co-factor by further modulating retrovirus-induced immune dysfunction in cytokine

production.

114

Murine retrovirus infection or dietary EtOH consumption alone has been shown

to have a suppressive effect on T cell proliferation (22, 94). Chronic dietary EtOH

consumption could exacerbate decreased T cell proliferation induced by retrovirus

infection. Decreased IL-2 production in murine AIDS (274) was not further affected by

EtOH. This could be due to the limitation of ELISA kit for IL-2, because the level of

IL-2 in murine AIDS is around the detection limit of the kit. Decreased production of

IFN-,), by Th2 cells in the spleen (208) was significantly increased by EtOH. This may

be caused by increased NK cell activity induced by EtOH via B-endorphin and

norepinephrine, potent simulators of NK cell activity (274-276), because NK cells was

suggested to be a major source ofIFN-')' in vivo (164). Increased release ofIFN-')' could

shut off production of IL-IO by Thl cells, because it is a reciprocal regulator (277),

leading to the imbalance of Th 1 and Th2 cell development and cytokine production, a

critical factor in the resistance to infection. While chronic EtOH can modify spleen T

subsets, the changes (278, 95) are not dramatic enough to significantly explain the

changes we observed in cytokine production. Consequently, we conclude that the effects

of chronic EtOH consumption on cytokine production was also functional, perhaps via

disturbing the signal transduction of immunocompetent cells (237, 238). An imbalance

in the Thl and Th2 response may contribute to the immune dysfunction associated with

HIV infection, and resistance to HIV infection and progression to AIDS may be

dependent on a Thl > Th2 dominance (89, 295, 296). This notion is supported by the

evidence that knockout of IL-4 gene in murine AIDS prevented development of immune

dysfunction and loss of Thl cytokine production (80). The levels of IL-5, IL-6 and IL-

115

10 (Th2 cytokines) by ConA-stimulated splenocytes, elevated during murine AIDS, were

further increased in EtOH-fed mice. This suggests that chronic EtOH consumption could

worsen the imbalanced ofThI and Th2 cytokine production occurred often in AIDS (80).

Inhibited production of IL-2 (Thl cytokine) during murine AIDS was further suppressed

in EtOH-fed mice, supporting the notion that preferential activation of Th2 cells due to

EtOH consumption in murine AIDS. Production IFN--y (Thl cytokine) by cells from

EtOH-fed mice was not affected. This may be due to increased NK cell activity induced

by EtOH via B-endorphin and norepinephrine, potent simulators of NK cell activity (297,

298), as NK cells are major sources of IFN--y in vivo (178, 195). Thus, many of the

features of chronic EtOH use before retrovirus infection are consistent with a decrease

in Thl function and activation of Th2 function seen often in AIDS, thus exacerbating to

the development of AIDS as a cofactor.

In vivo activated B cells from HIV patents spontaneously produce IL-6 and TNF­

Ol (279), which was what we found with LPS-stimulated spleen cells for LP-BM5

retrovirus infected mice (208). IL-6 production would explain the

hypergammaglobulinemia and global B cell dysfunction seen with both pathogens.

Chronic dietary EtOH consumption slightly enhanced this increased level ofIL-6 by LPS­

stimulated splenocytes, which may aggravate hypergammaglobulinemia and global B cell

dysfunction. Enhancement of increased IL-5 levels in murine AIDS by dietary EtOH

may also contribute to this exacerbation. Furthermore, IL-6 also is involved in

governing the production of certain acute-phase reactants by hepotocytes, which results

in a series of tissue damage (107). Elevated levels of TNF-Ol may be involved with lipid

116

metabolism: inducing hypertriglyceridemia and wasting in both human and murine AIDS

(176, 177). Thus enhancement of elevated levels of TNF-a and IL-6 in murine AIDS

by dietary BtOH consumption will aggravate pathological symptoms of the host initiated

by retrovirus infection. In the other hand, increased enhancement by chronic BtOH

consumption before retrovirus infection of elevated levels of TNF-a and IL-6 in murine

AIDS may aggravate pathological symptoms of the host initiated by retrovirus infection,

thus suggesting that BtOH use could be an important cofactor to facilitate the

development of AIDS.

Several mechanisms have been suggested for EtOH-containing diets that suppress

immunological parameters (280, 234). Some immunological parameters are directly

influenced by BtOH (281) or its metabolites interacting with the cell membranes (231),

or perhaps via BtOH-induced hormonal changes caused by the central nervous system.

Second, chronic BtOH use causes nutritional deficiencies which are very

immunosuppressive and the cause of some cytokine suppression (282) by BtOH. Finally,

immune dysfunction is a part of major physiological modification during alcoholic liver

disease (117, 283). In this study, the model of murine BtOH-consumption, by using

Lieber-DeCarli liquid diet which is supplemented with nutrients well above the

requirements for growth in non-EtOH fed mice, minimized the interference of alcoholic

liver diseases and alcohol-induced malnutrition. Thus, the mechanism of dietary BtOH

action on cytokine production is likely a direct effect of BtOH or its metabolites' effect,

or via hormonal and central nervous system changes. Chronic consumption of dietary

BtOH was shown to have a consistently suppressive effect on the cytokine productioa in

117

non-retrovirus infected mice (279) and rats (270). However, we found that chronic

dietary EtOH consumption results in stimulation of several cytokines including IFN-'Y,

IL-5, IL-6 and TNF-a during retrovirus-induced immune dysfunction. This distinction

may reflect that significant changes in drug metabolism (284), response of cytokine

secretion to EtOH exposure and hormonal status in murine AIDS. The elucidation of

these mechanisms will help to explain dietary EtOH modulation on cytokine production

in murine AIDS mice, which will facilitate understanding the immunopathegenic

mechanism of dietary EtOH in human AIDS.

Altered responsiveness may be due to the essential elimination of mature

thymocytes with CD surface markers in the thymus, while the number of immature

lymphoid cells increased dramatically. Because of the well-known role of

adrenocorticosteroids in thymic atrophy, we hypothesize that alteration of cytokine

production from the thymus was due to cortisteroids induced by EtOH and retrovirus.

The EtOH consumption reduced the thymus weight (289, 248). However, the results

from this study indicated EtOH use prior to retrovirus infection did not reduce thymus

weight during murine AIDS. The m3:~or reason is thymus weight increases with lower

number ofthymocytes in retrovirus-infected mice (182). The histological studies showed

the disappearance of defined areas (like the cortex and medulla) in these thymuses from

murine AIDS. Moreover, there are empty spaces between groups of cells that do not

correspond to collagen fibers. Thus, the increase of thymus induced by retrovirus

infection may offset the decrease induced by EtOH consumption. These evidences

consistently explain EtOH consumption reduced thymocyte number with no change of

118

thymus weight.

Pro-thymocytes leave the bone marrow and arrive at the thymus where they find

the appropriate environment that let them complete their differentiation to become

functional T cell. The generation of functional competent T cells from precursor

populations within the thymus involves several stages of cellular proliferation and

differentiation (181). It has been proposed that the control of these processes is mediated

by cytokines (167). For example, in mice expressing a human IL-2RO! chain transgene,

thymocytes express a nonfunctional murine IL-2RJ3-human IL-2RO! heterodimer resulting

in the accumulation of T cell precursors in the thymus and periphery (167). Also, anti­

IL-2R O! chain antibodies abrogate T cell development which can be reversed by addition

of IL-2 (189). Addition of IL-2 to intact lobes immersed in culture medium promotes

the selective outgrowth ofT cells expression ')'0 TCR (190). Consequently, the inhibition

of IL-2 production by BtOH use we observed may partly contribute to the disruption of

T cell differentiation in the thymus, thereby adversely affecting pheripheral immune

responses. Consequently, the inhibition of IL-2 production by EtOH use we observed

may partly contribute to the changes in T cell subpopulations and differentiation in the

thymus. The addition of IL-4 blocks T cell development by reducing the number of

double-positive thymocytes (191). The constitutive production ofIL-4 in IL-4 transgenic

mice also result in the inhibition of double-positive thymocytes and mature peripheral T

cell development (193, 194). Consistently increased level of IL-4 induced by retrovirus

infection in the thymus was further enhanced by EtOH consumption. Thus, we

concluded that this increase may aggravate the impairment of T cell development in the

119

murine or human AIDS, thereby causing secondary acquired T cell deficiency. IL-6 has

been shown to promote the differentiation of Thy-l +IL-2R+ donor thymocytes after

intrathymic transfer into irradiated hosts (254). Thus, the suppression of IL-6 secretion

induced by retrovirus infection at the beginning of infection may also contribute the

abrogation of T cell development in the thymus. EtOH ingestion further increased IL-6

production, thus EtOH definitely contribute to the disruption of T cell maturation. The

physiological significance of increased level of IFN--y by thymocytes induced by

retrovirus infection may be involved in the up-regulation of MHC class I and II

expression in the surface of thymic stromal cells, which play a crucial roles in the

positive and negative selection during T cell education (i.e. recognition 'self and non­

self) in the thymus. Thus, the dysregulation of IFN--y release by thymocytes may reflect

the failure of T cell education, leading to loss of tolerance to self antigens or reaction to

non-self antigens. Dietary EtOH prior to retrovirus infection further increased IFN--y

levels, which would be expected to contribute the failure of T cell education. Thus,

abnormalities of T cell maturation in the thymus may contribute the impairment of T cell

response we and others observed in the peripheral tissues in animals or humans.

The underlying mechanisms by which EtOH uses are more predisposed to

disruption of T cell functions due to retrovirus infection is that EtOH-induced

corticosteroid may play some role in this process (254). The another possible mechanism

has been proposed whereby ethanol interacts with the endogenous opioid system by : (a)

production of certain ethanol metabolites, for example the isoquinolines which bind to

opiate receptors, (b) altering the binding properties of opiate receptors and (c) altering

120

the release, synthesis and post-translational processing of endogenous opioid peptide

(303, 304). Thus, EtOH-induced changes in corticosteroid and opiate could be

responsible for the changes in cytokine release by thymocytes, thereby altering T cell

maturation. In addition, EtOH consumption may facilitate the spread and replication of

retrovirus infection via inhibiting immune response and increasing oxidative stress (287).

In summary, chronic dietary EtOH consumption further aggravated the already

imbalanced immune response induced by the retrovirus infection. This would facilitate

the progression of AIDS and pathological damage caused by retrovirus infection,

explaining EtOH facilitation of retrovirus-induced loss of disease and cancer resistance.

Although the potential of EtOH consumption as a cofactor is human AIDS is not well

defined, our results indicated that the chronic EtOH consumption exacerbated the

imbalanced immune response induced by the retrovirus infection. It is conceivable that

this could facilitate the progression to AIDS and pathological damage caused by

retrovirus infection, explaining EtOH facilitation of retrovirus-induced loss of disease and

cancer resistance. Likewise, one of the mechanisms of EtOH use as a cofactor in

development of AIDS is preferential activation of Th2 cells. The adversely effects of

EtOH consumption on T cell differentiation in the thymus via modulating cytokine

secretion during murine AIDS may provide defect helper T cells, explaining aggravation

of immune dysfunction by EtOH during murine AIDS by EtOH consumption. The

continuing elucidation of the mechanism of EtOH effects on immune system during AIDS

may help us to understand how combination of EtOH use and HIV infection aggravate

the progression of AIDS in human subjects, perhaps via synergic effects.

121

TABLE 4.1 Effect of EtOH on Production of Cytokines by Splenocytes During Retrovirus Infection *

Cytokines Dietray

(ng/ml) Control

IL-2 0.124 + 0.12'

IL-4 0.232 + 0.13

IL-S 1.36 + 0.71

IL-6 18.6 + 9.38

IFN-'Y 8.8 + 1.4

IL-lO(U/ml) 17.3 + S.2

Supplement

EtOH

0.146 + 0.12

0.329 + 0.27

2.87 + 0.78

30.9 + 6.S

21.6 + 4.6

1.66 + 0.28

P Values

N.S.

N.S.

< 0.0001

< 0.02

< 0.0001

< 0.0001

* Values are mean + SD for 8 mice per group. Cytokine production was secreted by 1 x 1()6 splenocytes in a 0.2ml volume.

N.S. Not Significant

Table 4.2 Effect of EtOH on IL-6 and TNF-O! By Splenocytes During Retrovirus Infection *

Cytokines Dietary

(ng/ml) Control

IL-6 24.S + 6.8

TNF-O! 3.54 + 0.71

Supplement

EtOH

28.7 + 8.S

4.47 + 0.68

P Values

N.S.

< 0.02

* Every sample was determined in triplicate. Values are mean + SD for 8 mice per group. Cytokine production was secreted by 1 x 1()6 splenocytes in 0.2 ml volumes.

N.S. Not Significant

Table 4.3 Effect of EtOH on Production of Cytokines by Thymocytes During Retrovirus Infection *

Cytokines Dietray

(ng/ml) Control

IL-2 0.134 + 0.014

IL-4 0.130 + 0.112

IL-5 0.55 ± 0.05

IL-6 3.80 + 2.3

IFN--y 12.4 + 9.1

Supplement

EtOH

0.103 + 0.007

0.049 + 0.013

0.36 + 0.02

0.67 + 0.32

12.8 + 15.2

P Values

< 0.05

< 0.05

< 0.0001

< 0.02

N.S.

* Values are mean ± SD for 8 mice per group. Cytokine production was secreted by 1 x 1()6 thymocytes in a 0.2m1 volume.

N.S. Not Significant

Table 4.4 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-2 and IFN--y by Splenocytes*

Post-infection Groups Cytokines (ng/ml)

(weeks) IL-2 IFN--y

6 Control 1.80 + 0.34 15.6 + 7.06

EtOH 0.87 + 0.22# 18.8 + 3.6

9 Control 0.30 + 0.032 10.6 + 3.54

EtOH 0.061 + 0.03# 13.36 + 10.76

122

* Every sample was determmed m tnphcate. Values are mean + SD for 6-8 mice for each group. Cytokine was secreted by 1x1()6 splenocytes in 0.2 ml. The concentrations of IL-2 and IFN--y produced by ConA-stimulated splenocytes from uninfected, normal mice ( diet- and age-matched to experimental mice) are 3.54 + 0.43 and 30.0 + 5.0 ng/ml, respectively.

# p < 0.05 compared to age-matched control.

Table 4.5 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-5, IL-6 and IL-lO by Splenocytes*

Post-infection Groups Cytokines (ng/ml)

(weeks) IL-5 IL-6 IL-lO

6 Control 0.66 + 0.16 4.74 + 1.27 1.67 + 0.43

EtOH 1.14 + 0.58# 9.93 ± 1.5# 2.52 + 0.25#

9 Control 0.47 + 0.22 5.7 + 0.54 1.89 + 0.76

EtOH 0.56 + 0.29 5.6 + 1.57 1.81 + 0.40

* Every sample was determined in triplicate. Values are mean + SD for 6-8 mice for each group. Cytokine was secreted by lxlcYi splenocytes in 0.2 ml. The concentrations of IL-5, 11-6 and IL-lO produced by ConA-stimulated splenocytes from uninfected, normal mice (diet- and age-matched to experimental mice) are 0.32 + 0.015; 2.0 + 0.05 and 0.45 + 0.047 ng/ml, respectively.

# P < 0.05.

Table 4.6 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-6 and TNF-a by Splenocytes*

Post-infection Groups Cytokines (ng/ml)

(weeks) IL-6 TNF-a

6 Control 2.2 + 0.2 2.5 + 0.11

EtOH 10.0 + 1.69# 20.0 + 1.82#

9 Control 4.2 + 0.11 10.4 + 1.01

EtOH 7.5 ± 0.35# 18.8 + 1.11#

* Every sample was determined in triplicate. Values are mean + SD for 6-8 mice for each group. Cytokine was secreted by lxlcYi splenocytes in 0.2 ml. The concentrations of IL-2 and IFN-'Y produced by ConA­stimulated splenocytes from uninfected, normal mice ( diet- and age-matched to experimental mice) are 3.54 + 0.43 and 30.0 + 5.0 ng/ml, respectively.

# p < 0.05 compared to age-matched control.

123

Table 4.7 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IgA and IgG by Splenocytes*

Post-infection

(weeks)

6

9

Groups

Control

EtOH

Control

EtOH

Immunoglobulin

IgA (ng/ml)

60.8 + 4.1

71.7 + 6.7#

125.1 + 10.2

105.2 + 28.9

IgG (~g/ml)

1.01 + 0.52

2.12 ± 0.41#

8.8 + 2.78

8.86 + 2.59

* Every sample was determined in triplicate. Values are mean + SD for 6-8 mice for each group. IgA and IgG were secreted by 1x1<r splenocytes in 0.2 ml. The concentrations of IgA and IgG produced by LPS-stimulated splenocytes from uninfected, normal mice (diet- and

124

age- matched to experimental mice) are 50.1 + 4.3 and 479.0 + 77.0 ng/ml, respectively.

# p < 0.05 compared to age-matched control.

Table 4.8 Effect of EtOH Consumption Prior to Retrovirus Infection on Body and Thymus Weight*

Post-infection Groups Weight

(weeks) Body (gm) Thymus (mg)

6 Control 28.7 + 2.3 59.8 + 5.9

EtOH 29.1 ± 4.2 65.6 + 4.3

9 Control 30.6 ± 5.0 66.7 + 10.8

EtOH 31.6 + 2.5 68.5 + 13.2

* Values are mean + SD for 6-8 mice for each group. Body and thymus weight of uninfected, normal mouse (diet- and age-matched mice) are 30.2 + 3.2 gm and 52.9 + 2.3 mg, respectively.

Table 4.9 Effect of EtOH Consumption Prior to Retrovirus Infection on Number of Thymocytes and Thymocyte Proliferation*

Post-infection

(weeks)

6

9

Groups

Control

EtOH

Control

EtOH

Number Proliferation

(x 107/thymus) (CPM x 1,000)

7.23 + 0.56 13.3 + 3.8

4.03 + 1.23# 9.02 + 2.1#

6.21 + 1.63 15.8 + 2.8

4.36 + 1.17# 17.6 + 6.24

* Values are mean ± SD for 6-8 mice for each group. The thymocyte number and proliferation of uninfected, normal mice (diet- and age-matched mice) are 8.05 + 1.2 x 107 and 23,525 + 1,453 cpm, respectively.

# p < 0.05 compared to age-matched control.

Table 4.10 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-2 and IL-4 by Thymocytes*

Post-infection Groups Cytokines (ng/ml)

(weeks) IL-2 IL-4

6 Control 0.203 + 0.067 0.038 + 0.015

EtOH 0.120 + 0.038# 0.060 + 0.013#

9 Control 0.439 + 0.030 0.114 + 0.04

EtOH 0.438 + 0.025 0.153 + 0.066#

* Every sample was determined in triplicate. Values are mean + SD for 6-8 mice for each group. Cytokine was secreted by lxl(Yi thymocytes in 0.2 ml. The concentrations of IL-2 and IL-4 produced by ConA-stimulated thymocytes from uninfected, normal mice ( diet- and age-matched to experimental mice) are 0.354 + 0.069 and 0.015 + 0.009 ng/ml, respectively.

# p < 0.05 compared to age-matched control.

125

Table 4.11 Effect of EtOH Consumption Prior to Retrovirus Infection on Production of IL-6 and IFN--y by Thymocytes*

Post-infection Groups Cytokines (ng/m1)

(weeks) IL-6 IFN--y

6 Control 1.40 + 1.02 1.87 + 0.098

EtOH 0.32 + 0.039# 2.83 + 2.38

9 Control 4.58 + 1.54 6.45 + 2.0

EtOH 4.78 + 1.04 13.1 +9.6

* Every sample was determined in triplicate. Values are mean + SD for 6-8 mice for each group. Cytokine was secreted by 1x1<f thymocytes

126

in 0.2 ml. The concentrations of IL-2 and IL-4 produced by ConA-stimulated thymocytes from uninfected, normal mice ( diet- and age-matched to experimental mice) are 2.50 + 0.42 and 0.560 + 0.023 ng/ml, respectively.

# p < 0.05 compared to age-matched control.

127

20

-0 0 0 ,.-x 10 -:: a. (.)

0 control ETOH Thymocyte Proliferation

20 # P < 0.05

-0 0 o . ..-)( 10 -:: D-O

0

control ETOH

T cell Proliferation 20

-0 0 0 ..-

10 )(

1 1

-:: D-O

0

control ETOH B cell Proliferation

Fig. 4.1 Effects of Dietary EtOH on Splenocyte and Thymocyte Proliferation During Retrovirus Infection. Every sample was determined in triplicate. Values are mean + Sd of 8-10 mice. Proliferation was evaluated with 106 cells in 0.25 ml volume.

-:::a a.. 25 u -c: o ._-- U) tU"'C ~ c: o ca e-U) o ::::J () a c:..c: -I-m­e

"C

E >.

..c: I-

20

15

10

5

128

D Control EtOH

6 9

Post-I nfection (weeks)

Fig.4.2 Effect of EtOH Consumption Prior to Retrovirus Infection on Con A-Induced Splenic Proliferation. Every sample was determined in triplicate. Values are mean + SD of 10 mice. Thymidine incorporation was evaluated with 1<1 splenocytes in 0.25 ml volume.

-~ a. 25 o -c: .2_ - (/) CO-c '- c: o co ~(/) o ~ (J a c:..c:: -l-

CD -c: "'C

E >.

..c:: l-

20

15

10

5

o

129

D Control

#

#

6 9

Post-I nfection (weeks)

Fig. 4.3 Effect of EtOH Consumption Prior To Retrovirus Infection on LPS-Induced Splenic Proliferation. Every sample was determined in triplicate. Values are mean ± SD of 10 mice. Thymidine incorporation was evaluated with 1(JS splenocytes in 0.25 ml volume.

CHAPTER 5: ALCOHOL CONSUMPTION ALTERS THE NUTRITIONAL

STATUS DURING MURINE AIDS

Abstract

130

As alcohol (BtOH) abusers and AIDS patients have nutritional disorders, the

influence of chronic EtOH consumption (5%, v/v for 10 weeks) on levels of

immunomodulatory nutrients (vitamin A, E, zinc and copper) in the serum, liver, small

intestine, spleen and thymus was determined in murine AIDS. The hepatic levels of

vitamin A, E and zinc in both normal and LP-BM5 retrovirus infected female C56BL/6

mice fed EtOH were significantly reduced compared to controls, whereas the level of

copper in the liver was not affected. Intestinal levels of vitamin A and copper were not

affected by EtOH, whereas vitamin E and zinc were significantly reduced in both normal

mice and those with AIDS fed EtOH. The splenic levels of vitamin A and zinc in the

normal mice were significantly reduced by EtOH compared to controls, but not vitamin

E and copper. All splenic levels of nutrients we measured were reduced in EtOH-fed

mice with AIDS. The levels of vitamin A, E, zinc and copper in the thymus in murine

AIDS were also significantly affected by EtOH consumption. The serum levels of

vitamin A and E in both normal mice and murine AIDS were significantly decreased by

dietary EtOH. These data produced evidence that chronic EtOH can directly aggravate

undernutrition initiated by retrovirus infection. Such EtOH-induced malnutrition in AIDS

may be a cofactor, accelerating development of AIDS via immunosuppression secondary

to nutritional deficiencies.

131

Introduction

A growing number of studies have suggested important links between vitamins

or minerals deficiencies and HIV infection (57). Patients at various stages of mv

disease has serum vitamin A and E deficiencies (63, 95). Serum zinc decreased

progressively with advancing mv disease among 150 patients, compared with 50 HIV­

seronegative controls (201). Marginal or substantial zinc deficiency occurs one-quarter

to virtually all of the patients tested (63, 64, 67, 95). The prevalence of copper

deficiency is less clearly set up since there are only two available studies (63, 95). In

animal model for human AIDS, LP-BM5 retrovirus infection has been shown to cause

undernutrition by reducing tissue levels of immunomodulatory nutrients (e.g vitamin A,

E, zinc and copper) in the organs including immune organs during murine AIDS (305).

These findings provide evidence that undernutrition secondary to retrovirus infection seen

in AIDS individuals could play a forceful role in aggravating immune dysfunctions,

rendering the body more susceptible to tumors and opportunistic infections.

EtOH-induced undernutrition could be due to an inadequate and imbalanced intake

of nutrients, maldigestion, and malabsorption, as well as impaired metabolism, decreased

tissue uptake and storage, increased loss of nutrients through the urine, increased

nutritional requirements, or combination of these factors (98, 114). EtOH is transported

throughout the body system giving it the potential to affect most organs and tissues.

EtOH affects the gastrointestinal tract by alterations in stomach acid secretion and

delayed gastric emptying. Alterations in the mucosa of the small intestine reduce

production of enzymes, decrease absorptive capacity, induce malabsorption, and alter

132

transport of nutrients (116). Thus, EtOH intake can affect every stage of nutrient

metabolism including nutrient intake, digestion, absorption, activation , storage,

utilization, degradation and excretion. Therefore, it is conceivable that EtOH

consumption in AIDS can worsen the nutritional status, leading to deeper nutritional

deficiencies in AIDS. However, no direct studies in humans or animals have been

investigated EtOH's detrimental role on nutritional status during AIDS.

The onset of clinical symptoms of AIDS (low CD4 + T cells count and

opportunistic infections) is quite variable among HIV positive patients with a mean

incubation time 3-10 years following seroconversion (306). In this study, we investigated

if chronic EtOH consumption in murine AIDS as a cofactor aggravates undernutrition

initiated by retrovirus infection. The important immunomodulatory nutrients (vitamin A

and E, zinc and copper) in the liver, intestine and serum as well as immune organs

(spleen and thymus) were analyzed in this study.

133

Materials and Methods

Animals and LP-BM5 Retrovirus Infection See Materials and Methods in Ch. 2.

EtOH Diet All mice were fed the National Research Council (NRC) liquid diet (307).

Dietary ingredients were obtained from Dytes (#710279, Bethlehem, PA). All diets were

made isocaIoric by substituting 26% of the total calories with ethanol in the ethanol diet

and with dextrose in the control diet. In previous studies in our lab, we have found that

higher dietary EtOH (e.g. more than 5% v/v) significantly increased mortality of mice,

and lower dietary EtOH (e.g. less than 3% v/v) did not cause significant change in

physiological functions. Therefore, the concentration of EtOH in a NRC liquid diet was

4.5% (v/v) in this study. After 3 weeks housing in the animal facility in the Arizona

Health Sciences Center, mice were randomly assigned to one of the following four

treatments: normal mice with fed control diet, murine AIDS with control diet, normal

mice with EtOH diet, and murine AIDS with EtOH diet. A pair-fed study was

previously performed in our lab, and no difference between the pair-fed group and ad-lib

group was observed in the weight of thymus and spleen, nutritional status (307). Thus,

all diets were provided ad libitum in this study. The average consumed volume of liquid

diet was 12-15 ml for one mouse per day for every treatment. No significant difference

of consumption of the diets and body weight between the four groups was observed (data

not shown). Retrovirus infection and diet treatment began simultaneously. The total

treatment was for 10 weeks. Animals were cared for as required by the University of

Arizona Committee on Animal Research.

Measurement of Tissue Vitamin A and E See Materials and Methods in Ch. 2.

Measurement of Serum Vitamin A and E

134

See Materials and Methods in Ch. 2.

Measurement of Tissue Zinc (Zn) and Copper (Cu)

Ch.2.

See Materials and Methods in

Statistics All parameters were compared using one way ANOV A test, followed two-

tailed t student test for comparison between any two groups. p < 0.05 was considered

significant difference between two groups.

135

Results

Nutrient Concentrations in the Liver EtOH consumption significantly (p < 0.05)

reduced vitamin A and E concentrations in the liver in the uninfected, normal mice

(Table 7.1). In murine AIDS, EtOH further significantly (p < 0.05) decreased them,

which had been significantly (p < 0.05) lessened by retrovirus infection (Table 7.1).

Hepatic Zn concentration was also significantly (p < 0.05) decreased by EtOH ingestion

in both normal mice and those with AIDS, whereas hepatic Cu concentration was not

affected (Table 7.1).

Nutrient Concentrations in the Intestine As shown in Table 7.2, intestinal vitamin E

and Zn levels were significantly (p < 0.05) reduced by EtOH consumption in both

normal mice and those with AIDS, whereas levels of vitamin A and Cu were unaffected.

Nutrient Concentrations in the Spleen Splenic levels of vitamin A and Zn were

significantly (p < 0.05) lessened by EtOH in uninfected, normal mice and during murine

AIDS (Table 7.3), while vitamin E and Cu levels were significantly (p < 0.05) reduced

by EtOH only in murine AIDS. The reduction of splenic nutrients during murine AIDS

was not significantly exacerbated by EtOH consumption (Table 7.3).

Nutrient Concentrations in the Thymus As shown in Table 7.4, thymic levels of vitamin

E, Zn and Cu were significantly (p < 0.05) decreased by EtOH during murine AIDS,

while thymic concentrations of vitamin A and Zn were significantly (p < 0.05) lessened

by EtOH in normal mice. However thymic levels of vitamin E and Cu were not affected

by EtOH in normal mice.

Nutrient Concentrations in the Serum Serum vitamin A and E were significantly (p <

136

0.05) decreased by EtOH in uninfected mice and during murine AIDS (Table 7.5). But

EtOH ingestion did not further reduce serum vitamin A and E levels in murine AIDS,

which had been lessened by retrovirus infection.

137

Discussion

We demonstrated, for the first time, that EtOH consumption in murine AIDS can

aggravate undernutrition initiated by retrovirus infection in some tissues, but not others.

The levels of tissue nutrients were altered by EtOH consumption in normal mice and

during murine AIDS. These findings suggest that EtOH could be an important cofactor

in speeding the development of AIDS disease, via worsening retrovirus-induced

undernutrition in AIDS.

Since some nutrient deficiencies are associated with immune dysfunction,

reduction in tissue concentrations of nutrients can be related to significant

immunosuppression (118, 119). Vitamin A deficiency depresses lymphocyte activation

by mitogens (122). Vitamin A deficiency may decrease antigen binding due to alterations

in surface membrane glycoproteins (123). Vitamin A deficiency in humans has been

found to be associated with an increased incidence of a variety of cancers, and severity

of infection (123). Similar depression of the general immune functions associated with

vitamin E deficiencies have also been observed in both animals and humans. In rats,

depressed antibody-dependent cell mediated cytotoxicity (201), decreased lymphocytes

blastogenesis in response to mitogens (126), and depressed NK cell mediated cytotoxicity

(126) have been reported in vitamin deficient states. In guinea pigs, dietary vitamin E

deficiency depressed spleen T and B lymphocyte responses to mitogens to about 50% of

vitamin E adequate animals (133). The macrophages of these vitamin E deficient guinea

pigs also lacked accessory cell functions and acted only as suppressor cells (206). In

humans, a strong correlation of decreased serum levels of vitamin E and decreased

138

number and metabolic functions of T-Iymphocytes have been shown in athletes (207).

Therefore, the reduced levels of vitamin A and vitamin E in the tissues including immune

organs should exacerbate immune dysfunction initiated by retrovirus infection during

EtOH consumption. These results are in accord with our previous findings that

depressed proliferation of splenic and thymic T cells, and their dysregulated cytokine

production in murine AIDS were aggravated by EtOH consumption (102, 208, 209).

Zinc deficiency has great influence on barrier immunity by causing skin lesions,

gastrointestinal alteration, damage to the microvilli, and impaired pulmonary functions.

Zinc has also an important role in immunocompetence and exerts a crucial regulatory

effect on specific immune functions. Zinc deficiency results in the adverse effects on the

immune system with depression of cell-mediated immunity, decreased T -cells and B-cells

in lymphoid tissue, a depressed response of T-cell to tumor cells, decreased lymphokine

production, reduced antibody response, decreased natural killer cells activity and

depression of thymic hormones (123, 210). Zinc deficiency may be an important

cofactor suppressing T cell responses, especially in HIV infection with its reduction in

T cell number and functions. Fallutz et al. reported that serum zinc concentration

correlated with the lymphoproliferative responses of mononuclear cells to PHA

stimulation and that zinc supplementation augmented this response to PHA in HN­

infected patients (219). Reduced numbers of splenic antibody-producing cells and T

cells, reduced proliferation of T and B cells, reduced responsiveness and stimulator

activity of splenic T cells in one-way mixed lymphocyte reaction (219-224), and impaired

cell-mediated immunity have been observed in copper deficient mice (307). Therefore,

139

EtOH-induced nutrient deficiencies of zinc and copper in AIDS could further predispose

HIV -infected patients to more frequent or severe infections. This finding also supports

the notion that EtOH consumption as a cofactor may aggravate the immune dysfunctions

initiated by retrovirus infection via reducing nutrient levels in the tissues. These

evidence concomitantly explain the loss of host resistance to the tumor and opportunistic

infections in murine were exacerbated by EtOH consumption observed by us (225-227).

In conclusion, EtOH ingestion broadly suppresses the various nutrient levels in

the tissues including lymphoid organs as described above, seriously impairing the body's

nutritional status. These disruptions in nutritional status can significantly suppress

immune responses, suggesting the EtOH ingestion could be a potential cofactor in the

progression to AIDS.

140

Table 5.1 Effect of EtOH on Hepatic Nutrient Levels in Murine AIDS*

Groups Nutrients (p.g/g tissue)

EtOH Retrovirus Vitamin A Vitamin E Zinc Copper

962.2± 640.0+ 26.6± 3.1+0. 151.1 123.2 2.6 49

+ 149.0+ 237.3+ 19.5+ 2.65+ 51.1# 84.4# 0.96# 0.31

+ 718.1 + 326.8+ 24.6+ 2.58+ 54.1# 59.8# 1.3 0.3

+ + 107.2+ 173.5+ 19.9+ 2.55+ 38.2#@ 48.6#@ 1.8#@ 0.24

* Values are mean + SD of 6-8 mice. # p < 0.05 compared to the uninfected mice. @ p < 0.05 compared to the infected mice.

Table 5.2 Effect of EtOH on Intestinal Nutrient Levels in Murine AIDS*

Groups Nutrients (p.g/g tissue)

EtOH Retrovirus Vitamin A Vitamin E Zinc Copper

57.1 +2.4 145.2+30.0 53.3+ 6.3+0.4 5.0

+ 66.3+8.3 76.8+17.6# 35.6+ 7.5+ 1.4 6.3#

+ 46.8+6.7# 154.8+46.7 50.3+ 8.5+0.2 5.8

+ + 58.0+5.6 87.0+ 13.0#@ 25.6+ 8.2+ 1.9 9.5#@

* Values are mean + SD of 6-8 mice. # p < 0.05 compared to the uninfected mice. @ p < 0.05 compared to the infected mice.

141

Table 5.3 Effect of EtOH on Splenic Nutrient Levels in Murine AIOS*

Groups Nutrients (p.g/g tissue)

EtOH Retrovirus Vitamin A Vitamin E Zinc Copper

110.7+18.5 67.5+9.9 32.8+ 3.2+ 2.4 0.32

+ 53.9+9.0# 58.6+6.4 24.5+ 2.8+ 1.7# 0.53

+ 50.2+18.8# 47.4+10.9# 21.1+ 1.5+ 1.5# 0.63#

+ + 75.0+16.0# 44.3+9.8# 16.1+ 1.48+ 2.6#@ 0.58#

* Values are mean + SO of 6-8 mice. # p < 0.05 compared to the uninfected mice. @ p < 0.05 compared to the infected mice.

Table 5.4 Effect of EtOH on Thymic Nutrient Levels in Murine AIOS*

Groups Nutrients (p.g/g tissue)

EtOH Retrovirus Vitamin A Vitamin E Zinc Copper

48.6+1.8 84.1 + 11.7 24.1+ 2.03+ 2.3 0.13

+ 37.1+1.2# 90.2+14.5 17.3+ 1.75+ 1.5# 0.26

+ 30.6+4.0# 56.8+13.5# 28.2+ 1.91+ 2.6 0.14

+ + 37.1+2.8# 65.1±6.8# 16.1+ 1.57+ 2.5# 0.3#@

* Values are mean + SO of 6-8 mice. # p < 0.05 compared to the uninfected mice. @ p < 0.05 compared to the infected mice.

Table 5.5 Effect of EtOH on Serum Vitamin Levels in Murine AIDS*

Groups

ETOH

+

Retrovirus

+

Vitamins

Vitamin A

5.4 + 2.7

1.58 + 0.82#

2.08 + 0.99#

+ + 2.02 + 1.0# * Values are Mean + SD of 6-8 mice. # p < 0.05 compared with uninfected mice. @ p < 0.05 compared with infected mice.

(",g/ml serum)

Vitamin E

2.32 + 0.23

1.07 + 0.16#

1.19 + 0.09#

1.18 + 0.26#

142

143

CHAPTER 6: VITAMIN E SUPPLEMENTATION NORMALIZES IMMUNE

FUNCTIONS AND CYTOKINE PRODUCTION IN ALCOHOL-FED MICE.

Abstract

A fifteen-fold increase of vitamin E (160 lUlL) in a liquid diet (National

Council Research), with or without EtOH (4.5%, v/v), was fed to mice for 10 weeks.

Vitamin E supplementation restored production of interleukin-2, -5, -6, -10 and

interferon-'Y by ConA-stimulated splenocytes and interleukin-6 and tumor necrosis factor­

ex by LPS-stimulated splenocytes, which were suppressed by dietary EtOH. However,

it had no effect on interleukin-4 secretion, which was also reduced by splenocytes from

EtOH-fed mice. Vitamin E supplementation also restored EtOH-suppressed mitogen­

induced splenocyte proliferation, but not thymocyte proliferation, while it slightly

increased production of immunoglobulin A and G by LPS-stimulated splenocytes, which

were suppressed by dietary EtOH. Dietary vitamin E, furthermore, significantly

increased interleukin-2 and -6 secretion by ConA-stimulated thymocytes, which were

suppressed by dietary EtOH, while it had no effect on interleukin-4 and interferon-T

production by ConA-stimulated thymocytes from EtOH-fed mice. These data suggest

that dietary vitamin E supplementation can modulate dysregulation of cytokines initiated

by dietary EtOH and restore immune dysfunctions induced by EtOH ingestion.

144

Introduction

Prolonged consumption ofEtOH results in alterations of host defense mechanisms,

ultimately manifested by increasing susceptibility to infectious agents (84). In

experimental animals and humans, EtOH has an inhibitory effect on many aspects of the

immune response (85). A loss in the number of circulating leukocytes has been

associated with EtOH use in humans (86, 87), with detrimental effects on humoral

immune response, including antibody production (88). In addition, EtOH suppresses

several aspects of cell-mediated immunity such as delayed-type hypersensitivity reactions

(86, 88) and lymphocyte proliferative response to mitogens in humans (89). EtOH

administration to animals has a profound effect on decreasing the weight of peripheral

lymphoid organs as measured by a decrease number of thymocytes and splenocytes (91,

228, 308). Mice fed EtOH for a short period of time have shown both normal and

decreased natural killer cell activity (97, 98, 230). Antibody dependent cellular

cytotoxicity (97), lymphocytes proliferation (100), B lymphocyte functions (100, 101) and

cytokine production by lymphoid cells (94, 102) are also impaired by dietary EtOH.

In experimental animals and humans, dietary supplementation of vitamin E has

been associated with increased lymphoproliferative response to mitogens, T-helper

activity, antibody synthesis, neutrophil antimicrobial activity, lymphoblastic cell viability

and macrophage phagocytic activity (160, 308a, 308b). Dietary vitamin E

supplementation also stimulates splenocytes to release valuable cytokines, thereby

enhancing the overall immune response (309). Furthermore, high intakes of vitamin E

improves host resistance to pathogens (155, 310). Our previous finding indicated that

145

vitamin E supplementation in EtOH-fed mice also reduces the size and numbers of

carcinogen-induced tumors, which had been increased by dietary alcohol (135). If the

EtOH-induced immune dysfunction is normalized by vitamin E supplementation, then our

hypothesized mechanism of vitamin E-improved tumor resistance would be further

defined.

The generation of functional T cells from precursors within the thymus involves

several stages of cellular proliferation and differentiation (165, 166). Control of these

processes is mediated by cytokines, hormones, and by interactions with specific elements

of the thymic stroma (167). Thus, EtOH-induced alterations in the numbers of T cell

subsets and their functions as observed in human peripheral blood cells or mouse spleen

cells, could be the consequence of changes at the thymic level, particularly thymocyte

cytokine production. Our recent studies showed that dietary EtOH adversely affects

thymic cytokine secretion, while dietary vitamin E supplementation favorably affects

thymic cytokine release (311). However, nothing is known about the influence of

vitamin E on secretion of cytokines by thymocytes in the EtOH-fed animals or humans.

We have designated this study to investigate if dietary vitamin E supplementation

can normalize EtOH-induced reduction of major regulatory cytokines (lL-2, IL-4, IL-5,

IL-6, IL-lO, IFN--y and TNF-O! by splenocytes and IL-2, IL-4, IL-6 and IFN--y by

thymocytes), and some EtOH-induced immunosuppression including splenocyte and

thymocytes proliferation, immunoglobulin A and G (lg) production.

146

Materials and Methods

Animals Female C57BL\6 mice, 4 weeks old, were obtained from the Charles River

Laboratories Inc. (Wilmington, DE). The mice were housed 4 to a cage, in transparent

plastic cages with stainless steel wire lids. The housing facility was maintained at 20 to

22° C and 60 to 80% relative humidity with the animals exposed to a 12: 12 hr light-dark

cycle. Water and mouse chow (Texlnd, Madison, WI) were provided ad libitum.

Diet and Treatment All mice were fed the National Research Council (NRC) liquid

diet (294). Dietary ingredients were obtained from Dytes (#710279, Bethlehem, PA).

The vitamin E supplemented diet had 150 lUlL d-alpha-tocopherol acetate (Sigma, st.

Louis, MO), which was added to a liquid diet, in addition to the 10.5 lUlL d-alpha­

tocopherol acetate contained in the basal liquid diet. All diets were made isocaloric by

substituting 26% of the total calories with ethanol in the ethanol diet and with dextrose

in the control diet. In previous studies in our lab, we have found that higher dietary

EtOH (e.g. more than 5% v/v) significantly increased mortality of mice, and lower

dietary EtOH (e.g. less than 3% v/v) did not cause significant change in immune

functions (312). Therefore, the concentration of EtOH in a NRC liquid diet was 4.5%

(v/v) in this study. After 3 weeks housing in the animal facility in the Arizona Health

Sciences Center, mice were randomly assigned to one of the following four treatments:

untreated but fed liquid diet, liquid diets supplemented with (a) EtOH (4.5% v/v), (b)

Vitamin E (160 U/L) and (c) Vitamin E (160 U/L) + EtOH (4.5%, v/v). Mice were

fed the respective diets for 10 weeks. A pair-fed study was performed in our lab, and

no difference between the pair-fed group and ad-lib group was observed in the weight

147

of thymus and spleen, and some immunoparameters and cytokine secretion (29). Thus,

diets were provided ad libitum in this study. The average consumed volume of liquid

diet was 12-15 ml for one mouse per day for every treatment. No significance difference

of consumption of the diets and body weight between the four groups was observed (data

not shown). Animals were cared for as required by the University of Arizona Committee

on Animal Research.

Standard Cytokines and Their Antibodies See Materials and Methods in Ch. 2.

Preparation of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Cytokine Production See Materials and Methods in Ch. 2.

IgA and IgG Production See Materials and Methods in Ch. 2.

ELISA for Cytokine Detection See Materials and Methods in Ch. 2.

ELISA Assays for IgA and IgG See Materials and Methods in Ch. 2.

Mitogenesis of Splenocytes and Thymocytes See Materials and Methods in Ch. 2.

Statistics All parameters were compared at individual time points using one way

ANOV A test, followed by a two-tailed t student test between any two groups. p < 0.05

was considered a significant difference between two groups.

148

Results

Production of IL-2 and IFN--y by Splenocytes As shown in Table 8.1, dietary vitamin

E significantly (p<0.05) enhanced release of IL-2 and IFN--y by ConA-stimulated

splenocytes compared with the controls, and significantly (p < 0.05) restored the secretion

of IL-2 and IFN--y by ConA-stimulated splenocytes, which had been significantly

suppressed by chronic dietary EtOH (p<0.05).

Production of Cytokines by Splenocytes Vitamin E supplementation significantly

(p < 0.05) increased IL-4 production by ConA-stimulated splenocytes compared with the

controls, but not production ofIL-5, IL-6 and IL-lO. The decreased production ofIL-5,

IL-6 and IL-10 by splenocytes from EtOH-fed mice was significantly (p < 0.05) enhanced

by dietary vitamin E supplementation compared to EtOH-fed mice (Table 8.2).

However, dietary vitamin E had no effect on IL-4 secretion, which was significantly

(p<0.05) reduced by dietary EtOH (Table 8.2).

Production of IL-6 and TNF-a by Splenocytes As shown in Table 8.3, vitamin E

supplementation had no effect on the IL-6 and TNF-a production by LPS-stimulated

splenocytes compared to the contrcls. However, the decreased production of IL-6 and

TNF-a by LPS-stimulated splenocytes from EtOH-fed was significantly (p<0.05)

improved by dietary vitamin E supplementation compared to EtOH-fed mice.

IgA and IgG Production As shown in Table 8.4, production of IgA and IgG by LPS­

stimulated splenocytes was significantly inhibited by dietary ETOH compared to

controls(p < 0.05). Dietary vitamin E supplementation slightly improved these reduced

the levels of IgA and IgG, but not significantly (p > 0.05). However, vitamin E

149

supplementation did not show any enhancement of IgA and IgG production by

splenocytes compared to the controls.

Mitogenesis of Splenocytes Vitamin E supplementation significantly (p < 0.05) enhanced

the proliferation of ConA and LPS -induced splenocyte (Fig. 8.1 & Fig. 8.2). ConA and

LPS-induced splenocyte proliferations, significantly (p < 0.05) inhibited by dietary EtOH,

were significantly (p < 0.05) increased by dietary vitamin E supplementation (Fig. 1

& Fig. 2).

Production of Cytokines by Thymocytes As shown in Table 8.5, vitamin E

supplementation only significantly affected IL-4 production, but not production of IL-2,

IL-6 and IFN-'Y by ConA-induced thymocytes. However, suppressed secretion of IL-2

and IL-6 by thymocytes from EtOH-fed mice was significantly (p < 0.05) restored by

dietary vitamin E supplementation. No significantly effects of dietary vitamin E on IL-4

and IFN release by thymocytes in EtOH-fed mice were observed.

Mitogenesis of thymocytes Dietary vitamin E supplementation had no significant effect

on thymocyte proliferation from both normal mice and EtOH-fed mice, which was

significantly (p < 0.05) suppressed by dietary EtOH (Fig. 8.3).

150

Discussion

The emphasis of this study has been focused on whether vitamin E

supplementation could offset EtOH-induced suppression of cytokine secretion, which is

a critical factor in immune modulation (23). Our data demonstrates that dietary vitamin

E has a general enhancing effect on in vitro cytokine production by splenocytes and

thymocytes. This discovery has led to a normalization of some immune dysfunctions

induced by EtOH ingestion. It could, in addition, restore host resistance to pathogenic

or opportunistic infection and tumor in the chronic EtOH users (84).

Vitamin E supplementation has been associated with enhancement of the immune

response and increasing resistance to pathogenic infections and tumors in animal models

and humans. In this study, the results indicated that vitamin E supplementation enhanced

production of IL-2, IL-4 and IFN--y, which are the most important cytokines in immune

response, concomitant with the enhancement of mitogenesis of splenocytes. It is not

surprising, therefore, that vitamin E supplementation plays a role in preventing EtOH­

induced suppression of immune response. There are a number of mechanisms by which

vitamin E may protect the immune system from EtOH-induced damage. Some

immunological parameters are directly influenced by EtOH ingestion through intervention

by hormonal changes caused by central nervous system effects. For example, EtOH can

increase the levels of prostaglandin (pG) and corticosteroids (158, 159), which are potent

inhibitors of the immune cells' activity, including cytokine release (254). Vitamin E

could decrease its levels (160, 156), thereby enhancing immune response and increasing

host resistance. Some immunological responses could be directly affected by EtOH

151

ingestion via nutrient deficiencies (79, 117), which are well known to be associated with

immune dysfunction (118, 119). For example, deficiencies of hepatic vitamin A, E, and

zinc have been associated with EtOH ingestion (313-315). Vitamin E supplementation

can beneficially improve the hepatic levels of vitamin A , E, and zinc (208), which are

strongly correlated with their levels in the spleen and thymus. Thus dietary vitamin E

can improve secondary malnutrition to EtOH ingestion, thereby favorably affecting

immune response. Some immune cells are directly damaged by dietary EtOH through

free radical and lipid peroxide production. Vitamin E , functioning as antioxidant and

free radical scavenger, can inhibit lipid peroxidation and decrease the burden of free

radicals on immune cells (316). These should prevent immune cells from losing

functions due to reaction with free radical or lipid peroxidation in cell membranes.

Finally, dietary EtOH could directly interfere with cytokine producing cells via altering

transduction and membrane fluidity (237, 238) by increasing an alcohol metabolite:

acetaldehyde (316). Vitamin E, as an essential component of cell membrane, could

directly protect cytokine producing cells from EtOH disturbance via inhibiting the actions

of agents capable of alkylating or forming adduct with lipids in the surface of cell

membranes. Taken together, dietary vitamin E supplementation can help to overcome

tissue deficiencies of nutrients, and restore abnormalities of hormones release, and

modulate cytokine release in EtOH-fed mice, thereby favorably affecting immune

response and host defense.

Cytokines play crucial roles in the immune response. IL-2 is a pivotal cytokine

in the growth and differentiation of T and B cells, and activation of NK cells and

152

lymphokine-activated killer cells (231). IL-2 also can act as a growth and differentiation

factor for B cells and activate macrophages. Stimulation of IL-2 and IFN by vitamin E

may be part of the explanation for vitamin E's increased tumor resistance in EtOH-fed

mice (135). IFN--y plays a key role in T-cell mediated tumor regression (316) by its

anti-proliferative activity. IFN--y has multiple distinct biological activities including anti­

viral activity, activating phagocytosis of macrophages and neutrophil cells, cytotoxicity

of NK cells (23). Enhancement of IFN--y by dietary vitamin E in normal mice may be

due to the decrease of immunosuppressive product PG~ by immunocompetent cells

induced by dietary vitamin E (156). PG~ is a potent inhibitor of NK cells (48), a major

source of IFN--y production in vivo (195). In addition. EI-Hag et a1 showed that NK cell

activity was susceptible to oxidative injury by H20 2 (317). Seaman et a1 found that

phorbol ester-mediated suppression of NK cell activity was depressed on release of

reactive oxygen metabolites, especially of H20 2, by monocytes and polymorphonuclear

leukocytes (318). Since dietary vitamin E supplementation decreased H20 2 level (319),

increased NK cell activity in mice fed with vitamin E may be responsible for the

increased level of IFN--y. This idea is strengthened by the demonstration of vitamin E

stimulated NK cell activity in vivo and in vitro (320, 321). Vitamin E functions as an

antioxidant and free radicals scavenger, therefore it may act directly on macrophages in

several ways, including enhancement of receptor expression and reduction of PG~

production. Since PG~ can down regulate MHC molecule expression on macrophage

(322), reduced production of PG~ may be responsible for up-regulation of IL-l , an

inducer of IL-2. Subsequently IL-2 could stimulate the production of other cytokines,

153

including IL-5 and IL-6. IL-5 is a B cell growth factor because of its ability to stimulate

proliferation and antibody production by B cells. IL-5 increases the amount of IgA

secreted by murine splenic B cells (242, 243). IL-6 is a major inducer of terminal

differentiation of B cells into plasma cells. In relation to T cells, IL-6 has been shown

to play an important role in the T cell response to alloantigen and development of

effector T cytotoxicity cells (244). We observed that suppression of T cell proliferation

by chronic dietary EtOH was restored by vitamin E. This could be due to the increased

level of IL-2 and IL-6 induced by vitamin E. Decreased production of IgA and IgG by

LPS-stimulated splenocytes from chronic EtOH-fed mice we observed was slightly

restored by vitamin E, consistent with the vitamin E-induced increased of IL-5 and IL-6

by splenocytes in EtOH-fed mice. Suppressed IL-I0 release by EtOH also was enhanced

by vitamin E, explaining the enhancement of Th2 cytokines (IL-5 and IL-6) and IgA and

IgG production. However, it is still not clear why vitamin E supplementation stimulated

production of IL-2 and IFN-I', not production of IL-5, IL-6 and IL-lO by splenocytes

from normal mice. The possible reason is that vitamin E selectively enhance Thl cell

differentiation, which is responsible for IL-2 and IFN--y production.

The control of generation in functional competent T cells from precursor

population within the thymus was mediated by cytokines (167). In mice, expressing a

human IL-2Ra chain transgene, thymocytes express a nonfunctional murine IL-2R13-

human IL-2Ra heterodimer resulting in the accumulation of T cell precursors in the

thymus and periphery (167). Also, anti-IL-2R a chain antibodies abrogate T cell

development which can be reversed by addition of IL-2 (189). Addition of IL-2 to intact

154

lobes immersed in culture medium promotes the selective outgrowth of T cells expression

'Yo TCR (190). The addition of IL-4 blocks T cell development by reducing the number

of double positive thymocytes (191). The constitutive production of IL-4 in IL-4

transgenic mice also result in the inhibition of double positive thymocytes and mature

peripheral T cell development (192, 193). IL-6 has been shown to promote the

differentiation of Thy-l +IL-2R+ donor thymocytes after intrathymic transfer into

irradiated hosts (194). The vitamin E-induced enhancement of IL-2 and IL-6 by

thymocytes, suppressed by EtOH, may be responsible for the normalization in T cell

subpopulations and differentiation in the thymus after chronic EtOH ingestion (323).

Therefore, the change of cytokine secretion by thymocytes during vitamin E

supplementation in EtOH-fed mice we observed may be responsible for the functional

change of mature T cells in the thymus, contributing the general enhancement of immune

response. It is not clear why vitamin E supplementation selectively reduced IL-4

production by thymocytes. Vitamin E may alter the subpopulations of thymocytes in the

thymus or their functions (e.g. alteration of receptors and signal transduction) via

modulating some thymic hormone production. Possible underlying mechanisms we

propose include: (A) vitamin E supplementation reversing the deficiencies of vitamin A

and E in the thymus initiated by dietary EtOH, which may be essential for T cell

maturation; This notion is supported by our previous finding that dietary vitamin E

significantly normalized EtOH-induced deficiencies of hepatic vitamin A and E levels

(324), which have been strongly corrected with its level in the thymus (325); (B)

Vitamin E supplementation normalized the deficiency of hepatic zinc initiated by dietary

155

EtOH (326), which has been shown to be essential for T cell maturation (327); and (C)

Vitamin E supplementation reduced the level of corticosteroids, which are potent inducers

of thymocyte apoptosis (247). Taken together, abnormalities of T cell maturation by

dietary EtOH in the thymus, could be restored by dietary vitamin E, contributing to a

general enhancement of the immune response.

In summary, dietary vitamin E supplementation in a liquid diet generally caused

marked enhancement in the cytokine production induced by mitogens in vitro by

splenocytes and thymocytes in EtOH-fed mice, concomitant with a general enhancement

of immune response. Our results suggest that the suppressive effects of EtOH on the

immune response could be directly restored by dietary vitamin E supplementation,

thereby increasing host disease resistance to infections and tumors. Although the animal

data should not necessarily be extrapolated to EtOH users, information obtained from

such studies may be potentially transferable or serve as basis for the study of the

immunoenhancing properties of vitamin E in EtOH users.

156

Table 6.1 Effect of Vitamin E on IL-2 and IFN--y Production by Splenocytes From Ethanol-Fed Mice*

Supplementation Cytokines (ng/ml)

ETOH

+

+

Vitamin E

+

+

IL-2

1.0 + 0.046

0.5 + 0.029#

1.85 ± 0.03#

0.95 + 0.06@

IFN--y

30.0 + 5.0

18.0 + 2.5#

39.0 + 2.44#

27.0 + 0.43@

* # @

Every sample was determined in triplicate. Values are Mean + SD of 6-8 mice. p < 0.05 compared with untreated mice. p < 0.05 compared with ETOH-fed mice.

Table 6.2 Effect of Vitamin E on IL-4, IL-S, IL-6 and IL-lO Production by Splenocytes from Eathnol-Fed Mice*

Supplemen- Cytokines (ng/ml) tation

ETOH Vitamin E IL-4(pg/ml) IL-S IL-6 IL-lO(U/ml)

20.0+0.98 0.58+ 2.0+ 150.0+5.2 0.03 0.05

+ 15.0+1.3# 0.3± 1.0± 100+6.1# 0.04# 0.34#

+ 30.0±2.7# 0.59+ 1.8+ 144.0+4.3 0.03 0.04

+ + 17.0±2.0 1.2S± 1.7± 13S.0+7.3@ 0.16@ 0.24@

* Every sample was determined in triplicate. Values are mean + SD of 6-8 mice per group. # p < 0.05 compared with untreated mice. @ p < 0.05 compared with ETOH-fed mice.

157

Table 6.3 Effect of Vitamin E on IL-6 and TNF-a Production by Splenocytes From Ethanol-Fed Mice*

* # @

Supplementation Cytokines (ng/ml)

ETOH Vitamin E IL-6 TNF-a

1.75 + 0.4 2.46 + 0.81

+ 0.67 + 0.32# 0.71 + 0.037#

+ 1.12 + 0.46 2.28 + 0.66

+ + 1.33 + 0.036@ 2.58 + 0.52@

Every sample was determined in triplicate. Values are Mean + SD of 6-8 mice. p < 0.05 compared with untreated mice. p < 0.05 compared with ETOH-fed mice.

Table 6.4 Effect of Vitamin E on Immunoglobulins Production by Splenocytes from Ethanol-Fed Mice*

*

ETOH

+

+

Supplementation

Vitamin E

+ +

Immunoglobulins (ng/ml)

IgA IgG

55.7 ± 21.1 479.0 + 77.0

26.6 + 14.8 279.0 + 130.8

50.3 ± 15.6 584.0 + 95.5

42.5 + 9.5 390.0 + 50.6

Every sample was determined in triplicate. Values are Mean + SD of 6-8 mice.

158

Table 6.5 Effect of Vitamin E on IL-2, IL-4, IL-6 and IFN--y Production by

Thymocytes from Ethanol-Fed Mice*

Supple- Cytokines (ng/ml) mentation

ETOH Vitamin E IL-2 IL-4 IL-6 IFN--y

0.17+ 0.11+0.09 1.25+ 0.21+0.09 0.04 0.23

+ 0.09+ 0.09+0.06 0.63+ 0.20+0.02 0.01# 0.01#

+ 0.19+ 0.06+0.01# 1.35+ 0.23+0.09 0.01 0.045

+ + 0.12+ 0.10+0.02 1.0+ 0.25+0.09 0.1@ 0.03@

* Every sample was determined in triplicate. Values are mean + SD of 6-8 mice per group. # p < 0.05 compared with untreated mice.

@ p < 0.05 compared with ETOH-fed mice.

159

-0 0 0 30 .,.-x :E 25 a.. U # - 20 c 0 .--as 15 ~

0 a. ~

0 10 (J C

<D 5 c "C .-E 0 >- Control ETOH Vitamin E ETOH + Vit E ..c .-

Group

Fig. 6.1 Effect of Vitamin E on Con A-induced Splenocyte Proliferation from EtOH-Fed Mice. Every sample was determined in triplicate. The values are mean + SD of 6-8 mice. Thymidine incorporation was evaluated with las splenocytes in 0.25 ml volume.

-o o o· 66 ,.­>< E 55 a. o -c o -'" ... o a. ... o o c CD c .-

"C .-E ~ .c I-

44

33

22

11

Control

#

ETOH Vitamin E ETOH+Vit E

Group

160

Fig. 6.2 Effect of Vitamin Eon LPS-Induced Splenocyte Proliferation from EtOH-Fed Mice. Every sample was determined in triplicate. The values are mean + SD of 6-8 mice. Thymidine incorporation was evaluated with 1rf splenocytes in 0.25 ml volume.

-o o o 20 .,.­x ::E a.. U -c o

.';:: ca '-o a. '-o Cl C

CD C .-

'"C .-E :>. .c I-

16

12

8

4

o

161

Control ETOH Vitamin E ETOH + Vit E

Group

Fig. 6.3 Effect of Vitamin E on ConA-Induced Thymocyte ProHferation from EtOH-fed mice. Every sample was determined in triplicate. The values are mean + SD of 6-8 mice. Thymidine incorporation was evaluated with 10> thymocytes in 0.25 ml volume.

Abstract

CHAPTER 7: VITAMIN E SUPPLEMENTATION ASSISTS

IN THE RESTORATION OF UNDERNUTRITION AND

IMMUNE DYSFUNCTIONS DURING MURINE AIDS

162

Dietary supplementation, with a fifteen-fold increase of vitamin E (160

International Units/Liter) in a liquid diet, restored concentrations of vitamin A and E in

the liver, intestine, serum, spleen and thymus. It significantly normalized hepatic,

splenic concentrations of zinc and copper. Vitamin E supplementation also partially

restored production of interleukin-2 and interferon--y by splenocytes in vitro, which were

suppressed by murine AIDS. Retrovirus-induced elevated production of interleukin-4,

interleukin-5 and interleukin-6 by splenocytes in vitro was significantly reduced by

dietary vitamin E. Increased levels of interleukin-6, tumor necrosis factor-a and

immunoglobulin A and G produced by splenocytes in vitro during murine AIDS were

also significantly normalized by vitamin E. It also partially restored splenocyte

proliferation and splenic natural killer cell activity. These data indicate that vitamin E

supplementation can help to restore levels of tissue nutrients, cytokine dysregulation and

some immune dysfunctions initiated by retrovirus infection during murine AIDS.

163

Introduction

Acquired immune deficiency syndrome (AIDS) is a clinical disorder in humans

caused by human immunodeficiency virus (HIV) infection. It represents the end point

in a progressive sequence of immunosuppressive changes which render the body highly

susceptible to life-threatening tumors and opportunistic infections. AIDS has been

identified as a major public health priority in the United States with heavy social and

economic impact. Immune and other physiological defects induced by HIV infection

appear to be progressive and irreversible with a high mortality rate that approaches 100

% (328). Therefore, there is a pressing need for valid and effective therapeutic or

preventive interventions in AIDS individuals.

Gastrointestinal dysfunction including diarrhea and malabsorption, is a common

manifestation of the AIDS (200). mv infection may play a pathogenic role in

gastrointestinal cells, deleteriously affecting their nutritional absorption capacities. In

situ hybridization studies have localized HIV infection in various types of epithelial cells

of the bowel mucosa (72, 75). Although the roles of vitamins and minerals in the

clinical manifestation of HIV infection has not been well defined, a growing number of

studies have suggested important links between vitamins or minerals and HIV infection

(57). Patients at various stages of mv disease had serum vitamin A and E deficiencies

(63). A recent study which found that a major percentage of patients with AIDS (50%),

AIDS-related complex (58%) and HIV (38%) had a vitamin E intake of less than 50%

the Recommended Daily Allowance (56). Serum zinc decreased progressively with

advancing mv disease among 150 patients (201). Zinc and copper deficiencies in HIV

164

patients tested were also reported (67). Undernutrition has been associated with

immunological dysfunction, development of infectious processes and vital organ

dysfunctions. Thus, a vicious cycle may be set up in which the underlying

immunological defects related to HIV infection are exacerbated by the immune

dysfunction associated with undernutrition, initiated by retrovirus infection.

The immunostimulatory nature of vitamin E does provide a basis for its use in the

modulation of the various cell components and immune functions, and its consequent

therapeutic use during AIDS. Thus, we investigated if dietary vitamin E supplementation

would normalize undernutrition, cytokine dysregulation and immune dysfunctions

associated with retrovirus infection in murine AIDS. Such a system, with the potential

for the manipulation of immune cells and functions, may also be useful as a model for

clinical trials in humans and eventually for use as a therapy in human AIDS individuals.

165

Materials and Methods

Animals. Female C57BL\6 mice, 5 weeks old, were obtained from the Charles River

Laboratories Inc. (Wilmington, DE). The mice were housed in transparent plastic cages

with stainless steel wire lids with 4 mice per cage. The housing facility was maintained

at 20 to 22° C and 60 to 80% relative humidity, and animals were exposed to a 12:12

hr light-dark cycle. Water and mouse chow (Texland, Madison, WI) were provided ad

libitum. After 2 week housing in the animal facility in the Arizona Health Sciences

Center, mice were then randomly assigned to one of the following four treatments:

control (untreated and uninfected), vitamin E supplemented diet (treated and uninfected),

LP-BM5 infection (untreated and infected), and LP-BM5 infection plus vitamin E

supplemented diet (treated and infected). All diets were provided ad libitum. The

consumed volume of liquid diet was 12-15 x 1O-3L per mice. No significant difference

of consumption of the diets and body weight between four groups was observed (data not

shown). Animals were cared for as required by the University of Arizona Committee

on Animal Research.

Diet and Treatment. All mice were given the National Research Council liquid diet

(294), which meets the nutritional requirement of mice. Dietary ingredients were

obtained from Dytes (#710279, Bethlehem, PA). The vitamin E supplemented diet had

150 International Units (lU)/L of vitamin E as d-alpha-tocopherol acetate (Sigma, St.

Louis, MO) added to liquid diet in addition to the 10.5 lUlL d-alpha-tocopherol acetate

contained in the basal liquid diet. Vitamin E dietary supplementation was initiated the

day of retrovirus infection.

166

LP-BM5 Murine Leukemia Retrovirus Infection. See Materials and Methods in Ch. 2.

Tissue Vitamin A and E. See Materials and Methods in Ch. 2.

Serum Vitamin A and E. See Materials and Methods in Ch. 2.

Tissue Zinc and Copper. See Materials and Methods in Ch. 2.

Preparation of Splenocytes. See Materials and Methods in Ch. 2.

Standard Cytokines and Their Antibodies. See Materials and Methods in Ch. 2.

Cytokine Production. See Materials and Methods in Ch. 2.

ELISA Assays For Cytokines. See Materials and Methods in Ch. 2.

Cytotoxicity for NK Cells. Activity of NK cells will be measured using 51Cr release

described previously (294). NK cell sensitive Y AC-1 cells were used as target cells.

Briefly, YAC-1 target cells are labeled with 100 ILCi 51Cr at 37°C for 2 hours, washed,

and incubated in PBS for 30 minutes. After incubation, target cells were washed and

adjusted to 1x109 cells/L. Splenocytes for NK cell activity and target cells in triplicate

will be suspended in round-bottom microtiter plate wells producing effector/target ratio

of 100: 1 and 50: 1. The plates were then centrifuged (200 g, 2 min), incubated for 4

hours at 37°C and again c~ntrifuged (200 g, 10 min). 1x104 L of supernatant liquid was

harvested and radioactivity (release in counts per minutes, ER) is measured by a gamma

counter. Spontaneous release (SR) in cpm was obtained by incubating labeled target cells

in medium without splenocytes. Maximal release (MR) in cpm was induced by the

presence of 104 'L of 1 % NP-40. The amount of lysis was then calculated in terms of

the percentage of chromium release by the equation:

% specific chromium release=(ER-SR)/(MR-SR) X 100%

167

Mitogenesis of Splenocytes. See Materials and Methods in Ch. 2.

Preparation of IgA and IgG . See Materials and Methods in Ch. 2.

Statistics. All parameters were compared between four groups using one way

ANOV A, followed by two-tailed student t test between any two groups. p < 0.05 was

considered significant difference between two groups.

168

Results

Hepatic levels of Nutrients. Zn level in the liver was not affected by retrovirus

infection, whereas levels of vitamin A, E and eu were significantly (p < 0.05) reduced

(Table 7.1). Vitamin E supplementation significantly (p<0.05) increased the hepatic

levels of vitamin A and E and hepatic concentrations of Zn and eu in retrovirus-infected

mice (Table 7.1). It had no effect on the hepatic levels of Zn and eu in uninfected mice,

while significantly (p<0.05) increasing hepatic concentrations of vitamin A and E (Table

7.1).

Intestinal Levels of Nutrients. Intestinal levels of vitamin A and E were significantly

(p < 0.05) reduced by the retrovirus infection, whereas the Zn and eu concentrations

were not affected (Table 7.2). Vitamin E supplementation significantly (p<0.05)

increased the intestinal concentration of vitamin E in the retrovirus-infected mice, but it

had no effect on intestinal concentrations of vitamin A and Zn in the uninfected mice and

infected mice (Table 7.2).

Splenic Levels of Nutrients. The levels of vitamin A, E and Zn in the spleen were

significantly (p < 0.05) decreased by retrovirus infection, whereas eu level was

significantly (p<0.05) elevated in the infected mice (Table 7.3). Vitamin E

supplementation significantly (p < 0.05) normalized all splenic levels of nutrients (Table

7.3).

Thymic Levels of Nutrients. The levels of vitamin A, E in the thymus were

significantly (p<0.05) lessened by retrovirus infection (Table 7.4). Vitamin E

supplementation significantly (p < 0.05) normalized thymic vitamin A and E

169

concentrations (Table 7.4).

Serum Levels of Vitamin A and E. Serum vitamin A and E levels were significantly

(p<0.05) reduced by retrovirus infection (Table 7.5). Vitamin E significantly (p<0.05)

increased the serum levels of vitamin E in the retrovirus-infected mice (Table 7.5),

whereas serum vitamin A was not affected by vitamin E.

Production of IL-2 and IFN-=y. IL-2 and IFN--y are secreted by Thl cells, and modulate

cell-mediated immunity against virus infection. Dietary vitamin E supplementation

significantly (p < 0.05) increased release of IL-2 and IFN--y by splenocytes from

uninfected mice (Table 7.6). Production of IL-2 and IFN--y by splenocytes, suppressed

by retrovirus infection, was significantly (p < 0.05) restored by vitamin E

supplementation (Table 7.6). These results are in according with the enhancement of

Con A-induced T cell proliferation and NK cell activity in the spleen (Fig. 7.1 & 7.2).

Production of IL-4. IL-5 and IL-6. IL-4, IL-5 and IL-6 are produced by Th2 cells, and

modulate humoral responses. Release of IL-4 by splenocytes from uninfected mice was

significantly (p< 0.05) enhanced by dietary vitamin E (Table 7.7), whereas IL-5 and IL-

6 secretion was not affected by vitamin E (Table 7.7). While murine AIDS elevated IL-

4, IL-5 and IL-6 secretion, vitamin E significantly (p<0.05) decreased them (Table 7.7).

Production of IL-6 and TNF-a. Production of IL-6 and TNF-a by LPS-stimulated

splenocytes could be released by macrophages and B cells. Vitamin E supplementation

did not affect on IL-6 and TNF-a production by LPS-stimulated splenocytes in the

uninfected mice, while it significantly (p < 0.05) reduced their levels, which were

elevated by retrovirus infection (Table 7.8).

170

NK Cell Activity. Vitamin E supplementation had no effect on the splenic NK cell

activity in uninfected mice, while it significantly (p<0.05) restored the decreased NK

cell activity induced by retrovirus infection at a ratio of effectors/target cells: 100: 1 and

50:1 (Fig 7.1).

Mitogenesis of Splenocytes. Proliferation of ConA- and LPS-induced splenocytes was

significantly increased (p < 0.05) by dietary vitamin E, whereas suppressed T and B cell

proliferation in the spleen induced by retrovirus infection was significantly (p < 0.05)

restored by vitamin E supplementation (Fig 7.2 & 7.3).

Production of IgA and IgG. Vitamin E supplemental diet had no effect on IgA and IgG

production by LPS-induced splenocytes in the uninfected mice (Table 7.9). However,

increased IgA and IgG levels induced by retrovirus infection were significantly (p < 0.05)

reduced by vitamin E (Table 7.9).

171

Discussion

The present study investigated the effects of supplemental vitamin E during

murine AIDS on nutrients levels, cytokine secretion by splenocytes, and immune

functions. Increased dietary vitamin E normalized concentrations of vitamin A, E , zinc

and copper in the tissues, which were either reduced or elevated by murine AIDS. It

partially restored secretion of IL-2 and IFN-'Y, mitogenesis of splenocytes, and NK cell

activity, which were suppressed by retrovirus infection. Vitamin E treatment reduced

levels of IL-4, IL-5, IL-6 and TNF-a, which had been elevated by retrovirus infection.

These results suggest a possible role for vitamin E as a potential therapeutic nutrient to

help normalize undernutrition and immune dysfunction caused by retrovirus infection in

AIDS patients.

Vitamin A and E deficiencies have also been associated with suppression of

immune responses and loss of disease resistance. Vitamin A deficiency depresses

lymphocyte activation by mitogen in rats, and leads to a depletion of mature T-cells from

the spleen. Repletion with retinoic acid results in a rapid increase in the peripheral

lymphocyte count (119). In rats, depressed antibody-dependent cell mediated

cytotoxicity, decreased lymphocytes blastogenesis in response to mitogens, and depressed

NK cell mediated cytotoxicity have been reported in vitamin E deficient states. In

humans, a strong correlation of decreased serum levels of vitamin E and decreased

number and metabolic functions ofT cells have been shown (Odeleye and Watson 1991).

Therefore, deficiencies of vitamin A and E in immune organs would be expected to

partly contribute to the immune dysfunctions seen in murine AIDS: depressed

172

proliferation of T and B cells form spleen and mesenteric lymph nodes, impaired

cytotoxicity of splenic NK cell, and dysregulated cytokine production by lymphocytes

from spleen and mesenteric lymph node observed (208). Thus, vitamin E

supplementation in AIDS may enhance immune response in part via normalizing

nutritional status in the immune organs or tissues.

Zinc has an important role in immunocompetence and exerts a crucial regulatory

effect on specific immune functions. Zinc deficiency results in the adverse effects on the

immune system of a depression of cell-mediated immunity with decreased T -cells and B­

cells in lymphoid tissue, decreased T -helper cell number and activity, an inverted ratio

of T -helper to T -suppressor cells, a depressed response of T-cell to tumor cells,

decreased lymphokine production, decreased production of cytotoxic T cells, reduced

antibody response, and decreased NK cells activity (128). Serum zinc concentration

correlated with the lymphoproliferative responses of mononuclear cells to mitogen

stimulation and that zinc supplementation augmented this response to mitogen in mv­

infected patients (201). Copper deficiency has also been shown to be involved in

immune dysfunctions and defects of host defense. Reduced numbers of splenic

antibody-producing cells and T cells, reduced proliferation of T and B cells, and reduced

responsiveness and stimulator activity of splenic T cells in one-way mixed lymphocyte

reaction have been observed in copper deficient mice (330). Therefore, zinc and copper

deficiencies in AIDS could further predispose HIV -infected patients to more frequent or

severe infections. Such nutritional changes may help explain the loss of host resistance

to the tumor and opportunistic infections observed in murine AIDS (225-227). Thus,

173

vitamin E supplementation may help normalize host defenses via reversing zinc and

copper deficiencies in AIDS. It is not clear, however, why zinc level in the thymus and

level of copper in the spleen were elevated by retrovirus infection during murine AIDS.

They may reflect another kind of pathological symptom of AIDS as these nutrient

deficiencies in other tissues, possible originating from an abnormal pathway of their

absorption into the body. Clearly, murine retrovirus infection caused deficiencies of

nutrients, which play important roles in normal immune functions and antioxidant

activities.

Oxidants such as superoxide radicals, hydrogen peroxides, hydroxyl radicals, and

lipid peroxides play an important role in AIDS (337). TNF-a generates oxidants from

neutrophils at concentrations as low as 0.5 ng/ml (338). TNF-a levels are elevated in

the serum of HIV patients and murine AIDS, and thus TNF-a may use oxidative stress

as a second messenger. Oxidants can induce the expression ofHIV in human T cell lines

by activating transcription of NF-kB (339,340). Thus vitamin E, as an antioxidant, may

block activation of NF-kB via offsetting the oxidative stress, and reducing TNF-a level,

thereby inhibiting HIV replication and retarding the progression to AIDS. A unique

feature of the retrovirus is its persistence in a quiescent state, prior to activation, without

production of either viral mRNA or proteins. Vitamin E, which lowers oxidative stress,

may potentially inhibit retrovirus replication, keep retrovirus in a quiescent state, and

retard the progression to AIDS. Indeed, our previous results indicated that vitamin E

supplementation significantly reduced free radicals and hepatic lipid peroxidation, which

were enhanced by retrovirus infection in murine AIDS (225).

174

IFN-,}, has multiple distinct biological activity including anti-viral activity,

activating phagocytosis of macrophages and neutrophils and increasing cytotoxicity of NK

cells (231). Thus, stimulation of IFN-,}, production by vitamin E may explain vitamin

E's increased tumor resistance (225). IFN-'}' plays a key role in T-cell mediated tumor

regression (341) by its anti-proliferative activity of tumor cells. Enhancement of IFN-'}'

production by dietary vitamin E in normal mice may be due to the d~reased production

of immunosuppressive prostaglandin Hz (PG) by immunocompetent cells (156). PGHz

is a potent inhibitor of NK cells (148, 341), a major source of IFN-,}, production. Since

NK cell activity is susceptible to oxidative injury by H20 2 (342), and vitamin E

supplementation decreased H20 2 level (343), increased NK cell activity and IFN--y

production in mice fed vitamin E was expected.

Our murine AIDS studies imply that vitamin E supplementation acts on various

immune components to modify immune defects induced by retrovirus infection, and

effectively normalize the other important nutrient deficiencies initiated by retrovirus

infection in the immune organs. Thus, the combination of existing medical therapy with

nutritional supplementation may provide successful and novel therapeutic approaches for

treatment of HIV infected individuals. Although our animal data may not totally be

extrapolated to HIV -positive humans, information obtained from such studies using

murine AIDS may potentially serve as a basis for the study of immunoenhancing

properties of vitamin E in HIV -infected humans.

175

Table 7.1 Effect of Vitamin E on Hepatic Nutritional Levels During Retrovirus Infection*

Treatment Vitamins (p.g/g tissue) Minerals

Vitamin E Retroviurs Retinol Tocopherol Zinc Copper

886.1+ 66.8+7.9 84.6+ 18.8+ 108 5.5 2.1

+ 1180+ 87.4+7.7# 88.2± 16.9+ 303# 6.0 2.7

+ 678+ 23.0+7.2# 98.9+ 16.1+ 97.5# 17.2 2.3

+ + 1446+ 73.5+6.8$ 129+ 22+ 135$ 13.1$ 2.7$

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

Table 7.2 Effect of Vitamin E on Intestinal Nutritional Levels During Retrovirus Infection *

Treatment Vitamins (p.g/g tissue) Minerals

Vitamin E Retroviurs Retinol Tocopherol Zinc Copper

66.6± 55.2+5.4 54.7+ 5.9+ 5.1 9.1 1.2

+ 60.4+ 51.3+6.0 63.5+ 9.1+ 16.9 11.4 1.7

+ 50.6± 44.0±4.8# 56.1± 7.8+ 4.4# 9.2 2.4

+ + 49.6+ 56.7+3.9$ 74.8+ 9.2+ 15.5 8.9 1.4

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

176

Table 7.3 Effect of Vitamin E on Splenic Nutritional Levels During Retrovirus Infection *

Treatment Vitamins (p.g/g tissue) Minerals

Vitamin E Retroviurs Retinol Tocopherol Zinc Copper

23.7+ 396.6+37.6 13.9+ 7.4+ 3.2 1.4 1.3

+ 24.5+ 570+84.5# 14.7+ 14.7+ 4.5 1.2 3.9#

+ 7.4± 85.8+33.3# 7.2+ 11.7+ 4.5# 1.2# 2.9#

+ + 1O.4± 161±47.6$ 10.7+ 12.2± 1.1$ 1.4$ 1.6

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

Table 7.4 Effect of Vitamin E on Thymic Nutritional Levels During Retrovirus Infection*

Treatment Vitamins (p.g/g tissue) Minerals

Vitamin E Retroviurs Retinol Tocopherol Zinc Copper

48.6± 53.5+9.0 1.5±0.2 15.5+ 10.8 4.8

+ 42.6+ 65.0+4.5# 1.7+ 1.2 19.5+ 7.8 1.1

+ 30.6+ 38.1+7.9# 6.3+ 8.5+ 4.0# 2.4# 1.8#

+ + 45.9± 53.2+5.4$ 1.8± 13.4+ 6.5$ 0.3$ 1.1$

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

Table 7.5 Effect of Vitamin E on Serum Vitamin Levels During Retrovirus Infection *

Treatment Vitamins (j.tg/ml)

Vitamin E Retroviurs Retinol Tocopherol

2.0±0.4 1.2±0.5

+ 1.9+0.3 3.3+0.9#

+ 1.3+0.5# 0.72+0.1#

+ + 1.4+0.6 2.1+0.6$

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

Table 7.6 Effect of Vitamin E on Production of IL-2 and IFN--y During Retrovirus Infection*

Treatment Cytokines (ng/ml)

Vitamin E Retroviurs IL-2 IFN--y

0.50+0.03 15.0+1.9

+ 1.0±0.06# 29.0±2.4#

+ 0.02+0.001# 1.05+0.1#

+ + 0.35+0.03$ 18 +0.94$

* Values are SD + of 6-8 mice. # P < 0.05 compared to uninfected mice. $ p < 0.05 compared to retrovirus-infected mice.

177

Table 7.7 Effect of Vitamin E on Production of IL-4, IL-S and IL-6 During Retrovirus Infection*

Treatment Cytokines (ng/ml)

Vitamin E Retroviurs IL-4 IL-S IL-6

0.02+0.001 0.S8+0.03 2.1+0.0S

+ 0.03+0.001# 0.S9+0.033 1.8+0.08

+ 0.031 +0.001# 2.4+0.1S# 3.4+0.13#

+ + 0.017+0.002$ 0.4+0.012$ 2.3+0.13$

* Values are SD + of 6-8 mice. # P < O.OS compared to uninfected mice. $ p < O.OS compared to retrovirus-infected mice.

Table 7.S Effect of Vitamin E on Production of IL-6 and TNF-a During Retrovirus Infection *

Treatment Cytokines (ng/ml)

Vitamin E Retroviurs IL-6 TNF-a

20.S+3.2S 2.S2+0.89

+ 21.S+2.4 2.73+0.32

+ 32.4±4.32# 4.S1+0.S6#

+ + 24.S±I.S2$ 3.21 +0.089$

* Values are SD + of 6-8 mice. # P < O.OS compared to uninfected mice. $ p < O.OS compared to retrovirus-infected mice.

178

Table 7.9 Effect of Vitamin E on Immunoglobulin Production During Retrovirus Infection *

Treatment Immunoglobulin

Vitamin E Retrovirus IgA(ng/ml) IgG(~g/ml)

44.S±20.3 3.6+0.3

+ 40.0+1S.1 3.6+0.3

+ 16S+43.7# 24.5+9.9#

+ + 110.0+S.0$ 13.S+1I3$

* Values are SD + of 6-8 mice. # P < O.OS compared to uninfected mice. $ p < O.OS compared to retrovirus-infected mice.

179

o Control

60

-~ -~ 40 ~ .....

CJ ..... ~ 0 ~

0 ~

~ 20 U ~ Z

o L-..l.-_

-Vitamin E

100:1

Retrovirus

~ ~

Control Vitamin E

b

50:1

Ratiu of Effectors/Target Cells

180

Fig. 7.1 Effect of Vitamin E on NK Cell Activity During Retrovirus Infection. Bars are represented as mean + SD of 6-8 mice.

a p < 0.05 compared to uninfected mice b P < 0.05 compared to retrovirus-infected mice.

181

- 30 ~ ~ a U -.-== Q .... - 20 .... CIl ="0 &.. == Q = c..

CIl

&.. = Q Q CJ,.e: ~~ 10 ~-.-

== .... "0 .... S ~

,.e: ~ 0

Control Vitamin E Control Vitamin E

Retrovirus Treatments

Fig. 7.2 Effect of Vitamin E on eonA-Induced Splenocyte Proliferation During Retrovirus Infection. Every sample was determined in triplicate. Bars are represented as mean + SD of 6-8 mice. The proliferation was evaluated in 0.2 ml with l(r splenocytes.

a p < 0.05 compared to uninfected mice b P < 0.05 compared to retrovirus-infected mice.

15

10

5

o

a

Control Vitamin E Control Vitamin E

Retrovirus Treatments

182

Fig. 7.3 Effect of Vitamin E on LPS-induced Splenocyte Proliferation During Retrovirus Infection. Every sample was determined in triplicate. Bars are represented as mean + SD of 6-8 mice. The proliferation was evaluated in 0.2 ml with las splenocytes.

a p < 0.05 compared to uninfected mice b P < 0.05 compared to retrovirus-infected mice.

183

CHAPTER 8: ALTERATION OF KINETICS OF CYTOKINE

PRODUCTION BY VITAMIN E SUPPLEMENTATION DURING MURINE AIDS

Abstract

Dietary supplementation, with a IS-fold increase of vitamin E (160 IU/L) in a

liquid diet, normalized hepatic and serum levels of vitamin E, reduced by retrovirus

infection at 4 and 12 weeks post-infection. Vitamin E significantly dwindled

splenomegaly induced by retrovirus infection, while significantly restoring levels ofIL-2

and IFN-'Y produced by ConA-stimulated splenocytes at 4, 8 and 12 weeks post-infection,

respectively, suppressed by murine AIDS. Retrovirus infection elevated levels of IL-4,

IL-S and IL-6 produced by ConA-stimulated splenocytes, which were significantly

normalized by dietary vitamin E supplementation at 4, 8 and 12 weeks post-infection,

respectively. Levels of IL-6, TNF-~ and IgG produced by LPS-stimulated splenocytes

during progression to murine AIDS were also significantly normalized by vitamin Eat

4, 8 and 12 weeks post-infection, respectively. Vitamin E supplementation also restored

retrovirus-suppressed splenocyte proliferation at 8 and 12 weeks post-infection,

respectively. Vitamin E modulated IL-2 production by thymocytes in both uninfected

mice and retrovirus-infected mice. It significantly reduced level of IL-4 secretion by

thymocytes in the uninfected mice at 4 and 8 weeks, but not at 12 and 16 weeks. It also

significantly reduced IL-4 production by thymocytes, elevated by the retrovirus infection.

Vitamin E significantly reduced IL-6 and IFN-'Y production by thymocytes increased in

the murine AIDS. The effects of dietary vitamin E on conA-induced proliferation of

thymocytes were consistent with the finding on changes of IL-2 secretion. No effects of

184

dietary vitamin E on thymus weight were observed in both uninfected and retrovirus-

infected mice, whereas vitamin E significantly increased serum and thymic vitamin E

concentration, which had been reduced by retrovirus infection. These data indicate that

dietary vitamin E supplementation can overcome lowered levels of vitamin E, and

modulate cytokine release, normalize immune dysfunctions during progression to murine

AIDS, thereby favorably affecting host resistance. These changes help to explain

restoration of cancer resistance in murine AIDS by vitamin E.

185

Introduction

AIDS therapies involve pharmacological interventions (e.g. Ganciclovir and

Zidovudine, AZT) to inhibit HIV replication (161). However, these drugs are

accompanied by deleterious toxic side effects. For example, Ganciclovir toxicities

include bone marrow suppression, atrophy of the gastrointestinal tract mucosa and

inhibition of spermatogenesis, nausea, vomiting, reduced white blood cell count and

headaches. These anti-HIV drugs do not repair damaged immune functions caused by

retrovirus infection. Furthermore, a strain of AZT -resistant HIV was reported in human

individuals possible due to the use of high dose and prolonged use of AZT (162). Thus,

new strategies including specific dietary nutrient supplementation with immunoenhancing

activity could provide supplemental approaches to ameliorate damaged immune system

in infected individuals, eventually slowing the progression to AIDS. The combination

of existing medical therapy with nutritional supplementation may provide much successful

and novel therapeutic approaches for treatment of HIV infected individuals.

The role of vitamin E in the clinical manifestation of HIV has not been well

defined (56). Some patients with varying stages of HIV infection had low plasma levels

of vitamin E (63). Thus, vitamin E supplementation may act as an immunoenhancing

agent to favorably modulate the immune response in HIV infection. Our previous study

(349) indicated that 8-weeks dietary vitamin E supplementation during murine retrovirus

infection prior to development of AIDS significantly restored NK cell activity and

mitogenesis of splenocytes, and production of IL-2 and IFN-'Y, suppressed by retrovirus

infection. Thus, we investigated if prolonged supplementation of dietary vitamin E more

186

extensively modulates cytokine production and restoration of some immune functions

altered by progression to AIDS. Such a system, with this potential for the manipulation

of immune cells and functions, may also be useful as a model for clinical trials in

humans and eventually for use as an adjunct therapy in human AIDS individuals.

Most studies have been focused on enhancement of vitamin E on immune changes

in peripheral immune organs such as spleen and lymph nodes. However nothing is

known about the influence of vitamin E on the thymus, a unique site of T cell

maturation. The generation of functional T cells from precursors within the thymus

involves several stages of cellular proliferation and differentiation (165, 166). Control

of these processes is mediated by cytokines (167). Thus, retrovirus-induced alterations

in the numbers of T cell subsets and their functions observed after in human peripheral

blood cells or mouse spleen cells could be the consequence of changes at the thymus

level, particularly thymocyte cytokine production. Histopathological studies of thymic

glands from HIV -I-infected children revealed involuted architecture, reduced size and

weight, and fewer Hassal's corpuscles (168, 169). HIV-1 causes changes in thymic

hormone and cytokine levels such as thymosin Icy and IL-6, which act on T cell

differentiation (170, 350). Human fetal thymocyte cell lines were infected with HIV-1

in vitro and altered expression ofT cell differentiation markers (351). Murine retrovirus

LP-BM5 infection also targeted the thymus during the progression to AIDS, by

dsyregulating cytokine production by thymocytes (354), and reducing double-positive

CD4+8+ and CD3+ CD4+CD8- thymocytes, and increasing CD3-CD4+CD8- thymocytes

(182). As vitamin E has low toxicity, it could be an immunoenahancing agent to

187

favorably modulate the cytokine secretion by thymocytes, thereby favorably affecting T

cell maturation in the thymus and improving immune responses. Therefore we examined

if dietary vitamin E supplementation would normalize dysregulation of cytokine

production (IL-2, IL-4, IL-6 and IFN-'Y) by in vitro ConA-stimulated thymocytes and

their proliferation during progression to murine AIDS.

188

Materials and Methods

Animals. Female CS7BL\6 mice, S weeks old, were obtained from the Charles River

Laboratories Inc. (Wilmington, DE). The mice were housed in transparent plastic cages

with stainless steel wire lids with 4 mice per cage. The housing facility was maintained

at 20 to 22° C and 60 to 80% relative humidity, and animals were exposed to a 12:12

hr light-dark cycle. Water and mouse chow (Texland, Madison, WI) were provided ad

libitum. After 2 week housing in the animal facility in the Arizona Health Sciences

Center, mice were then randomly assigned to one of the following four treatments:

control (untreated and uninfected), vitamin E supplemented diet (treated and uninfected),

LP-BMS infection (untreated and infected), and LP-BMS infection plus vitamin E

supplemented diet (treated and infected). All diets were provided ad libitum. The

consumed volume of liquid diet was 12-1S ml per mouse. No significance difference of

consumption of the diets between four groups was observed (data not shown). Animals

were cared for as required by the University of Arizona Committee on Animal Research.

Diet and Treatment. All mice were given the National Research Council liquid diet

(294). Dietary ingredients were obtained fr:om Dyets (#710279, Bethlehem, PA). The

vitamin E supplemented diet had ISO lUlL of vitamin E as d-alpha-tocopherol acetate

(Sigma, St. Louis, MO) added to liquid diet in addition to the IO.S lUlL d-alpha­

tocopherol acetate contained in the basal liquid diet. Vitamin E dietary supplementation

was initiated the day of retrovirus infection.

LP-BMS Murine Leukemia Retrovirus Infection. See Materials and Methods in Ch. 2.

Tissue Vitamin E. See Materials and Methods in Ch. 2.

189

Preparation of Splenocytes and Thymocytes. See Materials and Methods in Ch. 2.

Standard Cytokines and Their Antibodies. See Materials and Methods in Ch. 2.

Cytokine Production. See Materials and Methods in Ch. 2.

ELISA For Cytokine Detection. See Materials and Methods in Ch. 2.

Mitogenesis of Splenocytes and Thymocytes. See Materials and Methods in Ch. 2.

ELISA for IgG Detection. See Materials and Methods in Ch. 2.

Statistics. All parameters were compared at individual time points using one way

ANDV A test, following using two-tailed t student test between any two groups. p <

0.05 was considered significant difference between two groups.

190

Results

Body and Spleen Weights. The body weight of mice given vitamin E supplementation

during progression to murine AIDS were not significantly changed (data not shown).

Vitamin E supplementation significantly reduced increased spleen weight (splenomegaly)

induced by retrovirus infection during the whole period of study (p < 0.05, Table 8.1),

while vitamin E has no effect on spleen weight of non-infected mice (Table 8.1).

However, vitamin E can not completely normalized spleen weight after retrovirus

infection.

Hepatic and Serum Levels of Vitamin E. Hepatic level of vitamin E was significantly

reduced by the retrovirus infection at 4 and 12 weeks post-infection, respectively (Table

8.2, p < 0.05). Vitamin E supplementation significantly increased hepatic level of

vitamin E in the uninfected mice. Serum vitamin E level was significantly reduced by

retrovirus infection at 4 and 12 weeks post-infection, respectively (Table 8.3, p < 0.05).

Vitamin E significantly increased the serum levels of vitamin E in both non-infected mice

and retrovirus-infected mice (Table 8.3, p < 0.05).

Production of IL-2 and IFN-y. IL-2 and IFN--y are secreted by Th1 cells, which are

responsible for regulation of cell-mediated immunity against virus infections. Dietary

vitamin E supplementation significantly increased IL-2 release (Fig. 8.1) at 4, 8 and 12

weeks and IFN--y release (Fig. 8.2) during the whole period by splenocytes from

uninfected mice compared with that of controls (p < 0.05). However, at 16 weeks

production of IL-2 was significantly inhibited (p < 0.05, Fig. 8.1) compared with the

untreated controls. As retrovirus infection progressed to murine AIDS, production of

191

IL-2 and IFN--y was suppressed by retrovirus infection (p < 0.05, Fig. 8.1 & 8.2). IL-2

production was significantly partly restored by dietary vitamin Eat 4, 8 and 12 week (

P < 0.05), but no effect of dietary vitamin E was observed at 16 week. These results

were in agreement with the proliferation of ConA-induced splenocytes (Fig. 8. 8).

Decreased release of IFN--y induced by retrovirus infection was significantly (p < 0.05)

restored by dietary vitamin E (Fig. 8.2).

Production of IL-4. IL-5 and lL-6. IL-4, IL-5 and IL-6 are produced by Th2 cells,

which are responsible for regulation of humoral immune responses. Release of IL-4 by

splenocytes was significantly (p < 0.05, Fig. 8.3) enhanced by dietary vitamin Eat 8

and 12 week compared with uninfected controls, but no significantly effect by dietary

vitamin E was observed at 4 and 16 weeks (Fig. 8.3). IL-5 secretion was significantly

(p<0.05, Fig. 8.4) reduced and IL-6 production was not affected by vitamin E (Fig.

8.5). Retrovirus infection significantly (p < 0.05) increased production ofIL-4 and IL-6

occurred during the whole period, and IL-5 at 4 weeks post-infection. However,

elevated IL-4, IL-5 and IL-6 secretion by murine AIDS was significantly decreased ( p

< 0.05) by dietary vitamin E supplementation during the whole period relative to their

controls: untreated, retrovirus infected mice (Fig. 8.3, 8.4 & 8.5).

Production of lL-6 and TNF-~. Production of IL-6 and TNF-~ by LPS-induced

splenocytes could be released by macrophages or B cells. Dietary vitamin E

supplementation did not modify IL-6 and TNF-~ production by LPS-stimulated

splenocytes in the uninfected mice, while it significantly normalizes their levels elevated

by retrovirus infection after 4 weeks post-infection compared with retrovirus-infected

192

controls (Fig. 8.6 & 8.7).

Mitogenesis of Splenocytes. Proliferation of Con A-induced splenocytes was significantly

increased (p < 0.05) by feeding increase dietary vitamin Eat 8 and 12 weeks, while

proliferation was significantly inhibited by dietary vitamin E at 16 weeks(Fig. 8.8).

Higher dietary vitamin E significantly increased T-cell mitogenesis (p < 0.05)

proliferation, which was suppressed by retrovirus infection at 4, 8 and 12 weeks, but not

at 16 weeks when the retrovirus infection had progressed to murine AIDS. Dietary

vitamin E significantly enhanced (p < 0.05) proliferation LPS-induced splenocytes

compared with untreated controls at 8, 12 and 16 weeks, while no effect of dietary

vitamin E was observed at 4 weeks (Fig. 8.9). Vitamin E enhanced B-cell proliferation,

suppressed by retrovirus infection at 4, 8 and 12 weeks post-infection. However, by 16

weeks of dietary vitamin E supplementation had no effect on the suppressed proliferation

of B cells during the final stage of murine AIDS.

Production of IgG. Feeding the vitamin E supplemented diet had no effect on IgG

production in the uninfected mice during the whole period of this study (Table 8.4).

However, increased IgG levels induced by retrovirus infection in the murine AIDS were

significantly reduced by vitamin E at 4, 8 and 12 weeks post-infection, but not at 16

weeks post-infection (Table 8.4).

Thymus Weight. Vitamin E supplementation has no effect on the thymus weight in

uninfected and infected mice (Table 8.5). Retrovirus infection caused thymic atrophy at

4 weeks post-infection and increased the thymus weight at 12 weeks post-infection.

Vitamin E in the Thymus and Serum. As shown in Table 8.6, retrovirus infection

193

significantly (p < 0.05) reduced both serum and thymic vitamin E levels during murine

AIDS. Vitamin E supplementation significantly (p < 0.05) increased the serum and

thymic concentrations of vitamin E in both uninfected, normal mice and those with

murine AIDS.

Production of Thymic Cytokines. Vitamin E supplementation significantly (p < 0.05)

increased IL-2 production at 8 weeks (Fig. 8.10), while it significantly (p < 0.05)

reduced the levels of IL-2 at 12 and 16 weeks (Fig. 8.10). Dietary vitamin E

supplementation significantly (p < 0.05) enhanced IL-2 secretion, suppressed by

retrovirus infection, at 8 weeks post-infection (Fig. 8.10), while it significantly (p <

0.05) reduced the levels of IL-2 production at 12 and 16 weeks post-infection (Fig.

8.10). No significant effect of vitamin Eon IL-2 production was observed at 4 weeks

treatment. Dietary vitamin E supplementation significantly (p < 0.05) reduced levels

of IL-4 secretion in the uninfected mice at the 4, 8 and 16 weeks treatment, but not at

12 weeks (Fig. 8.11), while it significantly (p < 0.05) decreased IL-4 production,

elevated by retrovirus infection, during the whole period of murine AIDS development

(Fig. 8.11). There was no effect of vitamin E supplementation on production of IL-6 in

the uninfected mice, while it significantly (p < 0.05) reduced IL-6 levels, increased by

retrovirus infection, at'12 and 16 weeks post-infection in murine AIDS, but not before

(Fig. 8.12). There was no significant effect of dietary vitamin E supplementation on

IFN--y production was observed in the uninfected mice except at 16 weeks (p < 0.05),

while it significantly (p < 0.05) reduced production of IFN--y which was dramatically

increased by retrovirus infection, at 8 to 16 weeks post-infection in murine AIDS (Fig.

194

8.13).

Mitogenesis of thymocytes Vitamin E supplementation significantly (p < 0.05)

enhanced ConA-induced proliferation of thymocytes in the uninfected, normal mice at

4 and 8 weeks treatment, however, it significantly (p < 0.05) reduced the proliferation

at 16 weeks treatment (Fig. 8.14). Dietary vitamin E supplementation significantly (p

< 0.05) increased proliferation of thymocytes, suppressed by retrovirus infection, at 8

weeks post-infection and significantly reduced proliferation, enhanced by retrovirus

infection, at 16 weeks post-infection in murine AIDS, while no effect was observed on

proliferation at 4 and 12 weeks post-infection (Fig. 8.14).

195

Discussion

IL-2 is a pivotal cytokine in the growth and differentiation of T and B cells, the

activation of natural killer (NK) cells and lymphokine-activated killer cells (233). It also

acts as a growth and differentiation factor for B cells and activates macrophages (23).

Thus, stimulation of IL-2 production we observed by vitamin E supplementation in both

uninfected mice and retrovirus infected mice may be responsible for vitamin E's

immunoenhancing activity (147, 156) and reduction in tumor growth (225). Since

vitamin E functions as an antioxidant and free radicals scavenger, it may act directly on

macrophages in several ways, including enhancement of receptor expression and

reduction of prostaglandin (PO) production. As POE,z down-regulates Ia expression by

macrophages (355), reduced production of PG~ could be responsible for up-regulation

of IL-l , an inducer of IL-2. Subsequently IL-2 may stimulate the production of other

cytokines including IL-4. Our finding of enhancement by dietary vitamin E of mitogen­

induced splenic B proliferation is in accord with our finding of increased production of

IL-4 by Th cells. In addition, reduced levels of PO~ would lead to the elimination of

PGE,z dependent-T suppressor cells and thus permit mitogen-induced T cell proliferation

and IL-2 production by Th cells (155).

IFN--y has multiple biological functions including anti-viral activity, activating

phagocytosis of macrophages and neutrophils, increasing cytotoxicity of NK cells and

CTL, and augmenting the development of Thl cells. IFN--y inhibits differentiation of

Th2 cells and suppresses the production of Th2 cytokines (245). Stimulation of IFN--y

production by vitamin E may explain its increased tumor resistance during murine AIDS

196

(225). IFN-'Y plays a key role in T-cell mediated tumor regression (356) by its anti-

proliferative activity of tumor cells. The restoration of IFN-'Y production by vitamin E

during its suppression by retrovirus infection may explain the concomitant reduction Th2

cytokine production (IL-4, IL-5 and IL-6), elevated by murine AIDS. Enhancement of

IFN-'Y production by dietary vitamin E in normal mice may be due to the decrease

production of immunosuppressive PG~ by immunocompetent cells induced by dietary

vitamin E (156). PG~ is a potent inhibitor of NK cells (148, 357), a major source of

IFN-'Y production in vivo (195). In addition, El-Hag et al (358) showed that NK cell

activity was susceptible to oxidative injury by H20 2• Vitamin E should help to prevent

H20 2 toxicity. Seaman et al (359) found that phorbol ester-mediated suppression of NK

cell activity was depressed by reactive oxygen metabolites, especially the release of

H20 2, by monocytes and polymorphonuclear leukocytes. Since vitamin E dietary

supplementation decreased H20 2 level (360), the increased NK cell activity in mice fed

vitamin E may be responsible for the increased level ofIFN-'Y. This idea is strengthened

by the demonstration of vitamin E stimulated NK cell activity in vivo and in vitro in

uninfected, normal mice (361, 362). Due to the central regulatory roles of IL-2, IL-4

and IFN-'Y in immune regulation, the effects of supplemental vitamin E on macrophages

and T cells can cause general immunoenhancement through increasing levels of IL-2, IL-

4 and IFN-'Y. However it is not clear why longer duration of dietary vitamin E (16

weeks) supplementation results in mitogen-induced splenic T and B cell proliferation, in

accord with decreased IL-2 and IL-4. In vitro studies in our laboratory revealed that

higher vitamin E concentration ( e.g more than 10 ",g/ml) can suppress T and B cell

197

proliferation, while physiological levels of vitamin E (1-5 J.!g/ml) significantly enhanced

T and B cell proliferation from both uninfected mice and retrovirus-infected mice (Wang

et al. unpublished observation). Thus, the reduced cytokine production with long

exposure of vitamin E could be caused by increasing vitamin E in cell membranes

causing increased fluidity, which may adversely affect the immune response. The exact

mechanism by which very high vitamin E can suppress immune responses is still not

clear. The unexpected reduced release of IL-5 by Th2 cells in uninfected mice by

vitamin E supplementation could also be due to the decreased PGEz, a potent stimulator

of IL-5 production (363). In addition, previous studies have shown that high vitamin

E diet reduced serum corticosterone levels (160). Corti steroids suppress the numbers and

function of T -cells, which would explain the enhanced immune responses we observed

with supplemental vitamin E. Thus, high intake of vitamin E modulates cytokine

production, leading to modulation of immune response in vivo in uninfected mice, as

well as retrovirus infected mice with immune dysfunctions.

In vivo activated B cells from HIV patents produce high levels of IL-6 and TNF-a

(365), as we found with LPS-stimulated splenocytes for LP-BM5 retrovirus infected mice

(208). Increased IL-6 production would explain the hypergammaglobulinemia and global

B cell dysfunction seen with both pathogens (244). Dietary vitamin E supplementation

significantly reduced the increased production of IL-6 by LPS-stimulated splenocytes l,

suggesting how it alleviated hypergammaglobulinemia and global B cell dysfunction in

the murine AIDS. Suppression of increased IL-5 levels during murine AIDS by dietary

vitamin E may also contributed to this mitigation. Furthermore, IL-6 also is involved

198

in governing the production of acute-phase reactants by hepatocytes, which results in a

series of tissue damage (366). Elevated levels of TNF-~ may be involved with lipid

metabolism: inducing hypertriglyceridemia and wasting in both human and murine AIDS

(176, 177). Elevated levels of IL-6 and TNF-~ have also been associated with the

stimulation of HIV replication in macrophages/monocytes and T cells (364, 365). Thus

reduction by dietary vitamin E supplementation of elevated levels of TNF-~ and IL-6 in

murine AIDS should ameliorate pathological symptoms of the host initiated by retrovirus

infection including helping to explain the lower incidence and small size of esophageal

cancer (225).

Formation of highly reactive oxygen-containing molecules is a normal

consequence of a variety of essential immunological reactions. Oxidants such as

superoxide radicals, hydrogen peroxides, hydroxyl radicals, and lipid peroxides play

important roles in human diseases (360). Lower levels of antioxidants such as

glutathione and other acid soluble thiols correlated with the progression to AIDS (45).

As thiols including vitamin E were associated with the inhibition of HIV replication in

vitro (368, 369), oxidative stress may be a potent inducer of both viral activation and

DNA damage in infected cells, including immunosuppression (370, 371). As TNF-~

generates reactive oxygen species through neutrophils at concentration as low as 0.5

ng/ml (372). TNF-~ levels are elevated in the serum of HIV patients and murine AIDS

(233, 365). TNF-~, produced by retrovirus-infected macrophages/monocytes, could use

oxidative stress as a second messenger. Reactive oxygen species induce expression of

HIV in human T cell lines by activating transcription of NF-kB (373. 374). Thus higher

199

intake of vitamin E supplementation may block activation of NF-kB via offsetting the

oxidative stress (375), thereby retarding the progression of AIDS. The reduction by

vitamin E supplementation we observed of increased levels of TNF-a during murine

AIDS may also contribute to lower the oxidative burden initiated by the retrovirus

infection. A unique feature of the retrovirus life cycle is that persistence in a quiescent

state, prior to activation, without production of either viral mRNA or proteins. Agents

such as vitamin E, that function as antioxidants and lower oxidative stress, may slow

the retrovirus replication, thereby offsetting these AIDS-symptoms and retarding the

progression to AIDS in HIV -positive individuals. This idea is also supported by our

finding that dietary vitamin E supplementation reduced the splenomegaly and restored T

and B cell proliferation in murine AIDS.

While dietary vitamin E did not completely restore immune dysfunctions including

cytokine production induced by retrovirus, its use as combination therapy with other

pharmacologic agents such as AZT could have several advantages. First, it could lead

to more complete viral suppression by deceasing oxidative stress, associated with this

retrovirus infection, than any single drug used alone. Second, it may allow each drug

to be used in lower doses, thus limiting drug toxicities, and decreased frequency of

drug-resistant HIV production. Third, such immunorestorative treatment may be given

during maintenance therapy of the AIDS patient following the control of tumors and

immune dysfunction by chemotherapy, especially when tissue levels of vitamin E are

lowered. In addition, although the animal data may not totally be extrapolated to mv­

positive humans, information obtained from such studies using murine AIDS may be

200

potentially transferable or serve as a basis for the study of immunoenhancing properties

of vitamin E in HIV -infected humans.

Production and action of cytokines in the thymus have been associated with an

essential role in T cell development (167). In mice, expressing a human IL-2Ra chain

transgene, thymocytes expressed a nonfunctional murine IL-2RB-human IL-2Ra

heterodimer resulting in the accumulation of T cell precursors in the thymus and

periphery (167). Also, anti-IL-2R a chain antibodies abrogate T cell development which

can be reversed by addition of IL-2 (189). Addition of IL-2 to intact lobes immersed in

culture medium promotes the selective outgrowth of T cells expression 'YO TCR (190).

Consequently, the modulation of IL-2 release by dietary vitamin E we observed may be

responsible for favorable changes in T cell subpopulations and differentiation. The

addition of IL-4 blocks T cell development by reducing the number of "double-positive:

CD4 +CD8+ thymocytes (191). The constitutive production of IL-4 in IL-4 transgenic

mice also resulted in the inhibition of "double-positive" CD4 +CD8+ thymocytes and

mature peripheral T cell development (192, 193). As dietary vitamin E reduced

production of IL-4, increased by retrovirus infection in the thymus, these changes may

contribute to the restoration of abrogation of T cell development during progression to

murine or human AIDS, normalizing immune dysfunctions initiated by retrovirus

infection. IL-6 has been shown to promote the differentiation of Thy-l +IL-2R+ donor

thymocytes after intrathymic transfer into irradiated hosts (194). Thus, the reduction of

IL-6 secretion by dietary vitamin E in murine AIDS may also contribute to the

restoration of T cell development in the thymus abrogated by retrovirus infection. The

201

physiological significance of increased production of IFN-'Y by thymocytes, induced by

retrovirus infection, may be involved in the up-regulation of MHC class I and II

expression on the surface of thymic stromal cells. It plays a crucial role in the positive

and negative selection during T cell education (Le. recognition 'self and 'non-self) in

the thymus. Thus, the elevated levels of IFN-'Y release by thymocytes in murine AIDS

may reflect the failure of T cell education, leading to loss of tolerance to self antigens

or reaction to non-self antigens. Supplemental vitamin E reduced the IFN-'Y production

in murine AIDS, suggesting modulatory effects of dietary vitamin E on thymic cytokine

production with reversing of impairment of T cell maturation in the thymus initiated by

retrovirus infection. The exact physiological significance of vitamin E modulatory effects

on thymic cytokine production depends on the further elucidation of mechanism on how

cytokines regulate T cell development.

Changes in function and subpopulations of thymocytes appear to be responsible

for the modulation of cytokine production by vitamin E supplementation. Possible

underlying mechanisms we propose include: (A) vitamin E supplementation reversing

the deficiencies of vitamin E in the thymus initiated by retrovirus infection, which may

be essential for T cell maturation; (B) Vitamin E supplementation normalized the

deficiency of hepatic zinc initiated by retrovirus infection (10), which has been shown

to be essential for T cell maturation (379-381); (C) Vitamin E supplementation reduced

the level of corticosteroids (160), which are potent inducers of thymocyte apoptosis (38).

In summary, the favorably effects of dietary vitamin E supplementation on T cell

differentiation in the thymus via modulating cytokine secretion we observed during

202

murine AIDS may provide new mature helper T cells and replace retrovirus-infected and

damaged peripheral T helper cells, explaining restoration of immune response in murine

AIDS by dietary vitamin E. The effects of dietary vitamin E supplementation on T cell

maturation in the thymus in the uninfected, normal mice may also enhance the immune

response via regulating T cell differentiation, explaining enhancement of cell-mediated

immunity by vitamin E supplementation in animals and individuals (148).

Table 8.1 Effect of Vitamin E Supplementation on Spleen Weight During Murine AIDS*

Treatment Spleen Weight (mg)

Vitamin E Retrovirus 4 Weeks 8 Weeks 12 Weeks 16 Weeks

84.0± 84.4+2.9 79.0+ 85.0+ 2.0 12.0 17.0

+ 210+ 690.6+ 1240+ 2410+ 62.0# 36# 420# 250#

+ 77.0+ 88.4+9.0 75.0+ 90.0+8.0 7.0 16.0

+ + 140+ 341± 620+ 775+ 22.0@ 121@ 240@ 241@

* Values are mean + SD of 4 mice per group. # p < 0.05 compared with untreated and uninfected mice. @ p < 0.05 compared with infected mice.

Table 8.2 Effect of Vitamin E Supplementation on Hepatic Vitamin E level During Murine AIDS *

Treatment Ql-Tocopherol (p.g/ g liver)

Vitamin E Retrovirus 4 Weeks 12 Weeks

23.8 ± 2.5 67.3 ± 8.5

+ 13.9 ± 5.8# 43.3 + 4.0#

+ 49.4 ± 2.6# 180.0 + 13.2#

+ + 37.9 ± 5.3@ 107.0 + 11.4@

* Values are mean + SD of 4 mice. # @

p < 0.05 compared with untreated and uninfected mice. p < 0.05 compared with infected mice.

203

Table 8.3. Effect of Vitamin E Supplementation on Serum Vitamin E level During Murine AIDS*

Treatment Ol-Tocopherol (~g/ml)

Vitamin E Retrovirus 4 Weeks 12 Weeks

3.66 + 0.49 3.3 + 0.36

+ 2.2 + 0.56# 0.73 + 0.19#

+ 5.86 + 0.94# 13.9 + 5.1#

+ + 6.32 ± 204@ 7.17 + 304@

* Values are mean + SD of 4 mice. # p < 0.05 compared with untreated and uninfected mice. @ p < 0.05 compared with infected mice.

Table 8.4 Effect of Vitamin E Supplementatin on IgG Production During Murine AIDS*

Treatment IgG ~g/ml)

Vitamin E Retrovirus 4 Weeks 8 Weeks 12 Weeks 16 Weeks

3.1+ 5.5±Oo4 7.6+0.45 8.0+0.58 0.15

+ 10.0+ 23.5+ 30.0+2.9# 75.0+9.6# 1.2# 1.5#

+ 3.0+ 5.0+ 704+1.2 8.2+0.69 0.66 0.74

+ + 5.6± 8.4+ 18.0+1.8@ 74.0+ 1.8 0.17@ 0.47@

204

* Content of IgG was secreted by 1()6 splenocytes stimulated by LPS in a 0.2 ml volume. # p < 0.05 compared with untreated and uninfected mice. @ p < 0.05 compared with infected mice.

Table 8.S Effect of Vitamin E on Thymus Weight During Murine AIDS*

Treatment Thymus Weight (mg)

Vitamin E Retrovirus 4 Weeks 8 Weeks 12 Weeks 16 Weeks

56.0+ 55.0+3.0 53.0+5.0 55.0+7.0 10.0

+ 42.0+ 48.0+ 1.5# 90.0+ 99.0+ 16.0# 13.0# 23.0#

+ 53.0+ 51.0+4.0 56.0+ 58.2+7.0 8.0 10.0

+ + 52.1+ 54.5+10.0 91.0+ 93.4+2.0 20.0 11.0

* Values are mean + SD of 6-8 mice per group. # p < 0.05 compared with untreated and uninfected controls.

Table 8.6 Effect of Vitamin E Supplementation on Thymic Vitamin E level During Murine AIDS*

Treatment

Vitamin E

+

Retrovirus

+

Thymus (p.g/g wet tissue)

53.5 + 9.0

65.0 + 4.5#

38.1 ± 7.9#

+ + 53.2 + 5.4@ * The length of treatment of vitamin E and retrovirus infection was 8 weeks. Values are mean + SD of 4 mice. # p < 0.05 compared with untreated and uninfected mice. @ p < 0.05 compared with infected mice.

205

206

1.50 Tie -0- Control

-.- Vitamin E --E 1.00 -C)

c - -0.-C\1

I

-' 0.50 ~ • -.- Vit E+Virus

"- ~a a~l. - .1.

0.00 a 4 8 12 16 Post-Infection (Weeks)

Fig. 8.1 Effect of Vitamin E Supplementation on Kinetics of IL-2 Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1()6 splenocytes in 0.2 ml volume.

207

40

T T -0- Control

30 ~r-l--1 -E i'-.+ 1 -~- Virus -C) c:

20 -~ -... -e- Vitamin E I

Z U. 0

10 A~ _._ Vit E+Virus

~A 0

0 4 8 12 16 Post-Infection (Weeks)

Fig. 8.2 Effect of Vitamin E Supplementation on Kinetics of IFN-')' Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1<r splenocytes in 0.2 ml volume.

208

0.40

-0- Control

- Vitamin E -E -C) c 0.20 - Virus ~

-f).-

I

.....J

.L ..r.. -y- Vit E+Virus

0.00 o 4 8 12 16 Post-Infection (Weeks)

Fig. 8.3 Effect of Vitamin E Supplementation on Kinetics of IL-4 Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by l<r splenocytes in 0.2 ml volume.

209

1.50

-0- Control

T - 1.00 Vitamin E E -.--C) c: - Virus U')

-0.-I

~ 0.50 -~- Vit E+Virus

0.00 o 4 8 12 16 Post-Infection (Weeks)

Fig. 8.4 Effect of Vitamin E Supplementation on Kinetics of IL-5 Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1()6 splenocytes in 0.2 ml volume.

210

28 T

-0- Control

21 - -.- Vitamin E E -C) c: 14 - -~- Virus (Q

I

-1

7 Vit E+Virus

o o 4 8 12 16 Post-Infection (Weeks)

Fig. 8.5 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production by Splenocytes during Retrovirus infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1()6 splenocytes in 0.2 ml volume.

45

--E 35 -C)

c -CD

I

...J 25

15 a

T/,-1-0- Control

A~ yI -.- Vitamin E

T .-V r ~ -,,- Virus

V 1 - .. - Vit E+Virus

4 8 12 16 Post-I nfection (Weeks)

211

Fig. 8.6 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytoldne was secreted by 1()6 splenocytes in 0.2 ml volume.

212

10

-0- Control T T - / ~t-·-E Vitamin E -Cl

c y/L - 5 C -0.- Virus ,

LL .--- H z is ==~ 1 r--v- Vit E+Virus

a a 4 8 12 16 Post-Infection (Weeks)

Fig. 8.7 Effect of Vitamin E Supplementation on Kinetics of TNF-a Production by Splenocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1()6 splenocytes in 0.2 ml volume.

.,,-... o o o

'" ,...-4 30

~ 24 ~ ~

::s 1 8 d

• ...-4

:E 12

Control

c=J

4

Vitamin E

~

8

Virus

~

#

12

Vit E+Virus 1:::::::::::::1

16

Post-I nfection (Weeks)

213

Fig. 8.8 Effect of Vitamin E Supplementation on Kinetics of ConA-induced Splenocyte Proliferation During Retrovirus Infection. The values are mean + SD of 6 mice. The proliferation was evaluated by 1Q6 splenocytes in 0.25 ml volume.

214

Control Vitamin E Virus Vit E+Virus

D - - II§] ...... -0 0 0 32 .. ..-X -(J) 24 .... ::l c:

16 ~ ~

(J) 8 0.. tJJ .... c: 0 ::l 0 4 8 12 16 (,)

Post-Infection (Weeks)

Fig. 8.9 Effect of Vitamin E Supplementation on Kinetics of LPS-induced Splenocyte Proliferation During Retrovirus Infection. The values are mean + SD of 6 mice. The proliferation was evaluated by 1<fi splenocytes in 0.25 ml volume.

215

1.50

-0- Control

E 1.00 T • --C) c - -~- Virus C\I

I ....l 0.50 - -~- VitE+Virus

0.00 a 4 8 12 16

Treatment (Weeks)

Fig. 8.10 Effect of Vitamin E Supplementation on Kinetics of IL-2 Production by Thymocytes during Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1<r thymocytes in 0.2 ml volume.

216

0.80

-0- Control

-E -C)

c: 0.40 -~

I

~

0.00 a 4 8 12 16

Treatment (Weeks)

Fig. 8.11 Effect of Vitamin E Supplementation on Kinetics of IL-4 Production by Thymocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1()6 thymocytes in 0.2 ml volume.

217

2.40

-0- Control

1.80 v - / .... -.- Vitamin E

E - ~i-.-Virus Cl 1.20 c: -co

I ~ !><~ ~ -.- VitE+Virus 0.60

0.00 o 4 8 12 16

Treatment (Weeks)

Fig. 8.12 Effect of Vitamin E Supplementation on Kinetics of IL-6 Production by Thymocytes During Retrovirus Infection. The values are mean + SO of 6 mice. The cytokine was secreted by 1()6 thymocytes in 0.2 ml volume.

218

20

-E -0- Controt -... 15 C)

c: Virus -<U

E 10 E -\1- VitE+Virus <U (!l

I 5 -,,- Vitamin E Z LL

-y ~

0 0 4 8 12 16

Treatment (Weeks)

Fig. 8.13 Effect of Vitamin E Supplementation on Kinetics of IFN-'Y Production by Thymocytes During Retrovirus Infection. The values are mean + SD of 6 mice. The cytokine was secreted by 1cr thymocytes in 0.2 ml volume.

-o o o ,.. >< -en CD -::l C .-:! "-CD a.. -C ::l o (J

16

#

12 o Control

- Vitamin E 8

- Retrovirus

4 k::::;::::1 Vit E+Virus

o 4 8 12 16

Treatment (Weeks)

Fig. 8.14 Effect of Vitamin E supplementation on Kinetics of ConA-induced Thymocyte Proliferation During Retrovirus Infection. The values are mean + SD of 6 mice. The proliferation was evaluated by 1()6 thymocytes in 0.25 ml volume.

219

220

CHAPTER 9: MODULATION OF CYTOKINE SECRETION,

IMMUNE RESPONSE AND NUTRITIONAL STATUS BY VARIOUS LEVELS OF

VITAMIN E SUPPLEMENTATION DURING MURINE AIDS

Abstract

Dietary supplementation, with a 15-, 150- and 450-fold increase of vitamin E

in a liquid diet (National Council Research), significantly restored levels of interleukin-2

(lL) and interferon-r produced by splenocytes, which were suppressed by retrovirus

infection. Retrovirus infection elevated levels of IL-6 and IL-IO produced by

splenocytes, which were significantly normalized by all levels of vitamin E

supplementation, respectively. Increased levels of IL-6, tumor necrosis factor-a,

immunoglobulin A and G produced by splenocytes during progression to murine AIDS

were also significantly normalized by all levels of vitamin E supplementation. Vitamin

E supplementation also restored retrovirus-suppressed splenocyte proliferation and natural

killer cell cytotoxicity. 15-, 150- and 450-fold increase of vitamin E supplementation

modulated IL-2 production by thymocytes in both uninfected mice and retrovirus-infected

mice, causing murine AIDS. 150- and 450-fold vitamin E significantly reduced level of

IL-4 secretion by thymocytes in the uninfected, normal mice. Its supplementation at all

levels also significantly reduced IL-4 production by thymocytes, elevated by the

retrovirus infection. Vitamin E significantly reduced IL-6 and interferon-r production

by thymocytes increased in the murine AIDS. The effects of dietary vitamin E on conA­

induced proliferation of thymocytes were consistent with the finding on changes of IL-2

secretion. No effects of dietary vitamin E on thymus weight were observed in both

221

uninfected and retrovirus-infected mice. They also significantly restored serum and

haptic vitamin A and E which had been reduced by retrovirus infection. They also

significantly restored hepatic copper, which had been reduced by retrovirus infection,

whereas only 150- and 450-fold vitamin E improved hepatic zinc level. Vitamin E

supplementation at all levels had no effects on hepatic zinc and copper levels in normal

mice, whereas they significantly increased serum and hepatic vitamin A and E

concentrations. Vitamin E supplementation at all levels significantly increased intestinal

vitamin A and E levels during murine AIDS, whereas only intestinal vitamin E levels

was altered by various levels of vitamin E supplementation. Interestingly, vitamin E

supplementation had no effect on intestinal copper level, whereas they significantly

increased intestinal zinc level in the normal mice and only 450-fold vitamin E

supplementation significantly elevated intestinal level of zinc. These data indicate that

dietary vitamin E supplementation at extremely high levels was not immunotoxic, and

can modulate cytokine release and normalize immune dysfunctions during progression

to murine AIDS, which should favorably affect host resistance. These data also indicate

that dietary vitamin E supplementation at extremely high levels can modulate cytokine

production by thymocytes, affecting T cell differentiation, especially during murine

AIDS. Furthermore, thsy improved undernutrition initiated by retrovirus infection during

progression to murine AIDS, which should favorably affect immune response.

222

Introduction

Acquired immune deficiency syndrome (AIDS) is a clinical disorder causf!d by

human immunodeficiency virus (HIV), representing the end point in a progressive

sequence of immunosuppressive changes. HIV, the key causative agent of AIDS, induces

immunosuppression which results in host defense defects that render the body highly

susceptible to opportunistic infections and neoplasm (383). The unique pathogenicity of

HIV, the long latent period of HIV infection and the rapidity with which the AIDS

epidemic has steadily increased in the United States in recent years. Therefore there is

a pressing need for valid and effective therapeutic and preventive interventions in HIV­

infected individuals.

Since progression to AIDS is often complicated by various nutritional disorders,

the additional immune dysfunction due to undernutrition may exacerbate immune damage

due to the retrovirus infection, and have precluded recovery from infectious events which

earlier represented survivable episodes. This hypothesis is based on findings from

clinical and experimental situations where malnutrition has been associated with

immunological dysfunction (53, 163); development of infectious processes (53, 54) and

vital organ dysfunction (55). Thus, the severe malnutrition seen in patients with AIDS

may set up a vicious cycle in which the underlying immunological defects related to HIV

infection are aggravated by the malnutrition-induced immune dysfunction. Although the

roles of vitamins and minerals in the clinical manifestation of HIV infection has not been

well defined (56), a growing number of studies have suggested important links between

vitamins or minerals and HIV infection (57). Two studies found that 12%-29% of their

223

patients at various stages of HIV disease has serum vitamin A and E deficiencies (63,

65). A recent study which found that a major percentage of patients with AIDS (50%),

ARC (58%) and HIV (38%) had a vitamin E intake ofless than 50% the Recommended

Daily Allowance (56). Zinc deficiency in HN infection has been identified consistently

(67, 68, 421). One study reported marginal copper deficiency in 22% of their AIDS

patients (68). Thus, patients with HIV infection will be increasingly compromised

nutritionally as the disease progresses. Indeed, it has been suggested that HIV infection

might play pathogenic role in gastrointestinal cells. In situ hybridization studies have

localized HN infection in various types of epithelial cells of the bowel mucosa (72, 73).

HIV infection of gastrointestinal cell lines has been documented in vitro (74, 75). Since

the lymphoid tissues, the main target of HIV, are present throughout of the

gastrointestinal tract, and CD4-related receptors have been demonstrated (76), it is

conceivable that AIDS patients have dysfunction of some portion of gastrointestinal tract

due to retrovirus infection, thereby leading to malabsorption and malnutrition. The

results from murine AIDS studies further confirmed that retrovirus infection induced

hepatic and serum nutrient deficiencies as well as splenic nutrients in the presence of

normal food intake, reducing important immunoregulatory nutrients such as vitamin A,

E, zinc and copper (422). Such retrovirus-induced malnutrition has the theoretical

potential to accelerate development of AIDS via immunosuppression secondary to

nutritional deficiency.

The role of vitamin E in the clinical manifestation of HIV has not been well

defined (57). Some patients with varying stages of HIV infection had low plasma levels

224

of vitamin E (63). The data from murine AIDS study further confirmed that the

deficiencies of vitamin E occurred in the liver as well as spleen and thymus (384). In

rats, depressed antibody-dependent cell mediated cytotoxicity (204), decreased

lymphocytes blastogenesis in response to mitogens (205), and depressed NK cell

mediated cytotoxicity (126) have been reported in vitamin deficient states. Therefore,

the deficiencies of vitamin E in the tissues including immune organs may aggravate

immune dysfunctions initiated by retrovirus infection, speeding the development of

AIDS. Thus, vitamin E supplementation could act as an immunoenhancing agent to

favorably modulate the immune response in HIV infection.

As vitamin E has low toxicity, it could be an immunoenhancing agent to favorably

modulate the cytokine secretion by thymocytes, thereby favorably affecting T cell

maturation in the thymus and improving immune responses. Consequently, vitamin E

supplementation has been proposed to be a useful additional therapy for AIDS treatment

(390). IS-fold increase of vitamin E during murine AIDS significantly restored NK cell

activity, T and B cell proliferation, and IL-2 and IFN--y, and nutritional status which had

been suppressed by retrovirus infection (391). The results from another study (392)

suggested that 8-12 weeks vitamin E consumption (1S-fold increase) can significantly

normalized immune dysfunctions and cytokine dysregulation, whereas 4 weeks vitamin

E supplementation was not effective for immunorestoration. Thus, the combination of

existing medical therapy with vitamin E supplementation may provide a more successful

and novel therapeutic approach for treatment of HIV infected individuals (393). The

strategy in this study was developed to determine whether supplementation of dietary

225

vitamin E at extremely high levels (150- and 450-fold) would more extensively modulate

cytokine production by splenocytes and thymocytes and restore some immune functions

, and improve nutritional status, altered by progression to AIDS, or if vitamin E at

extremely high oral supplementation is immunotoxic. Such a system, with this potential

for the manipulation of immune cells and functions, may also be useful as a model for

clinical trials in humans and eventually for use as an adjunct therapy in HIV-infected

individuals.

226

Materials and Methods

Animals. Female CS7BL\6 mice, 5 weeks old, were obtained from the Charles River

Laboratories Inc. (Wilmington, DE). The mice were housed in transparent plastic cages

with stainless steel wire lids with 4 mice per cage. The housing facility was maintained

at 20 to 22° C and 60 to 80% relative humidity. Animals were exposed to a 12:12 hr

light-dark cycle. Water and mouse chow (Texland, Madison, WI) were provided ad

libitum. Animals were cared for as required by the University of Arizona Committee

on Animal Research. After 1 week of adaptation in the animal facility in the Arizona

Health Sciences Center, mice were then randomly assigned to one of the following eight

treatments: control diets including uninfected and infected mice, vitamin E

supplemented diets (1S-fold increase) including uninfected and infected mice, vitamin E

supplemented diets (1S0-fold increase) including uninfected and infected mice, vitamin

E supplemented diets (4S0-fold increase) including uninfected and infected mice. All

diets were provided ad libitum. The mice consumed 14-16 mlliquid diet per day. No

significant differences of diet consumption between groups were observed (data not

shown).

Diet and Treatment. All mice were given the National Research Council liquid diet

(294). Dietary ingredients were obtained from Dyets (#710279, Bethlehem, PA). The

vitamin E supplemented diet had ISO (1S-fold increase), 1,SOO (ISO-fold increase) and

4,SOO (4S0-fold increase) lUlL of vitamin E as d-alpha-tocopherol acetate (Sigma, St.

Louis, MO) added to liquid diet in addition to the 1O.S lUlL d-alpha-tocopherol acetate

contained in the basal liquid diet. Vitamin E dietary supplementation was initiated the

227

day of retrovirus infection. The length of diet treatment and retrovirus infection was 10

weeks.

LP-BMS Retrovirus Infection. See Materials and Methods in Ch. 2.

Hepatic Vitamin A and E. See Materials and Methods in Ch. 2.

Serum Vitamin A and E. See Materials and Methods in Ch. 2.

Measurement of Zinc and Copper. See Materials and Methods in Ch.3

Preparation of Splenocytes and Thymocytes. See Materials and Methods in Ch. 2.

Standard Cytokines and Their Antibodies. See Materials and Methods in Ch. 2.

Cytokine Production. See Materials and Methods in Ch. 2.

ELISA For Cytokine Detection. See Materials and Methods in Ch. 2.

Mitogenesis of Splenocytes and Thymocytes. See Materials and Methods in Ch. 2.

NK CeIl Cytotoxicity. See Materials and Methods in Ch. 8.

IgA and IgG Production. See Materials and Methods in Ch. 2.

ELISA for IgA and IgG. See Materials and Methods in Ch. 2.

Statistics. The purpose of this study is to investigate if IS0- and 4S0-fold increase

of vitamin E supplementation is better for murine AIDS therapy. Thus comparison will

be focused on between two groups, e.g. uninfected vs infected; IS-fold vs control; IS0-

fold vs 4S0-fold and so on. All parameters were compared using two-tailed student's t

test between two groups. p < O.OS was considered significant difference between two

groups.

228

Results

Body and Spleen Weights. The body weight of mice given vitamin E supplementation

during progression to murine AIDS was not significantly different (Fig. 9.1A). Vitamin

E supplementation slightly reduced spleen weight, which had been induced to

splenomegaly by retrovirus infection (Fig. 9. !tB), but no statistic significance.

Serum Vitamin A and E. The concentrations of serum vitamin A and E were

significantly (p < 0.05) reduced by retrovirus infection (Fig. 9.2A&B). Vitamin E

supplementation at all levels significantly (p < 0.05) increased serum vitamin A and E

in both normal mice and murine AIDS, however, 150- and 4S0-fold vitamin E did not

further increase serum vitamin A and E in normal mice (Fig. 9.2A&B). ISO-fold

vitamin E supplementation significantly (p < 0.05) further increased serum vitamin A

compared to that increased by IS-fold vitamin E, but 4S0-fold vitamin E did not

compared to that increased by ISO-fold vitamin E.

Hepatic Nutrients. The concentrations of serum vitamin A, E and copper were

significantly (p < 0.05) lessened by retrovirus infection, whereas zinc was not affected

(Fig. 9.3A&B and Fig. 9.4A&B). Vitamin E supplementation at all levels significantly

(p < 0.05) elevated hepatic vitamin E levels in both normal mice and murine AIDS,

while ISO-fold vitamin E significantly (p < 0.05) further increased hepatic vitamin E

(Fig. 9.4A). However, 4S0-fold vitamin E did not further increase hepatic vitamin E

compared to that increased by ISO-fold vitamin E (Fig. 9.4A). 150- and 4S0-fold

vitamin E significantly (p < 0.05) increased hepatic vitamin A concentration in normal

mice, but IS-fold vitamin E failed to do (Fig. 9.4B). Vitamin E supplementation at all

229

levels significantly (p < 0.05) restored hepatic vitamin A, whereas ISO-fold vitamin E

significantly (p < 0.05) further increased vitamin A level compared to that increased

by IS-fold vitamin E. 4S0-fold vitamin E failed to further increase hepatic vitamin E

compared to that increased by ISO-fold vitamin E (Fig. 9.3B). Hepatic zinc and copper

were not affected by vitamin E supplementation at all levels, whereas vitamin E

supplementation at all levels significantly (p < 0.05) restored zinc levels, and 150- and

4S0-fold significantly (p < 0.05) increased hepatic copper (Fig. 9.4A&B). ISO-fold

vitamin E significantly (p < 0.05) further increased hepatic zinc compared to that

increased by IS-fold vitamin E, whereas 4S0-fold vitamin E failed to further increase

hepatic Zn and copper levels compared to that increased by ISO-fold vitamin E (Fig.

9.4A&B).

Intestinal Nutrients. Intestinal vitamin A and E levels was significantly ( p < 0.05)

reduced by retrovirus infection, whereas intestinal zinc and copper were not affected

(Fig. 9.SA&B and Fig. 9.6A&B). Vitamin E supplementation at all levels significantly

(p < 0.05) increased intestinal vitamin E and zinc concentrations, but not vitamin A and

copper (p < 0.05) in normal mice (Fig. 9.SA&B and Fig. 9.6A&B). 4S0-fold vitamin

E did not further increase vitamin E and zinc compared to that increased by ISO-fold

vitamin E in normal mice, even though ISO-fold vitamin E significantly (p < 0.05)

increased intestinal vitamin E and zinc compared to that increased by IS-fold vitamin E

(Fig. 9.SA&B and Fig. 9.6A&B). Vitamin E supplementation at all levels significantly

(p < 0.05) restored intestinal vitamin A and E levels in murine AIDS, whereas 150-

fold vitamin E significantly (p < 0.05) increase intestinal vitamin E compared to that

230

increased by IS-fold vitamin E, but not vitamin A (Fig. 9.SA&B). 4S0-fold vitamin E

had no effect on further increase of vitamin A and E in murine AIDS (Fig. 9.SA&B).

Vitamin E supplementation had no effects on intestinal copper in murine AIDS, whereas

4S0-fold vitamin E significantly ( p < O.OS) elevated intestinal zinc (Fig. 9.6A&B).

Production of IL-2 and IFN-'}'. IL-2 and IFN--y are secreted by Thl cells, and are

responsible for regulation of cell-mediated immunity against virus infections and tumors.

Dietary vitamin E supplementation significantly (p < O.OS) increased IL-2 release (Fig.

9.7A) and IFN--yrelease (Fig. 9.7B) by ConA-induced splenocytes from uninfected mice,

whereas ISO-fold and 4S0-fold vitamin E supplementation failed to further enhance

production of IL-2 and IFN--y compared to that increased by IS-fold vitamin E

supplementation. As the retrovirus infection progressed to murine AIDS, production of

IL-2 and IFN--y was suppressed (p < O.OS, Fig. 9.7A&B). IL-2 and IFN--y production

was significantly restored by dietary vitamin E at IS-, ISO- and 4S0-fold vitamin E

supplementation (p < O.OS), whereas ISO- and 4S0-fold vitamin E supplementation did

not further restore the levels of IL-2 and IFN--y produced by splenocytes compared to

that increased by IS-fold vitamin E supplementation (Fig. 9.7A&B).

Production of IL-6 and IL-lO. IL-6 and IL-lO are produced by Th2 cells, and are

responsible for regulation of humoral immune responses. Release of IL-6 by splenocytes

was not significantly affected by dietary vitamin E at any levels in uninfected mice (Fig.

9.8A), whereas IL-IO production could be significantly (p < O.OS) reduced by only

4S0-fold vitamin E supplementation (Fig. 9.8B). The retrovirus infection significantly

(p < O.OS) increased IL-6 and IL-IO production. However, elevated IL-6 and IL-lO

231

secretion during murine AIDS was significantly decreased (p < 0.05) by dietary vitamin

E supplementation at all levels of vitamin E supplementation (Fig. 9.8A&B), whereas

150- and 450-fold vitamin E supplementation did not further reduce the levels of IL-6

and IL-lO produced by splenocytes compared to that reduced by IS-fold vitamin E

supplementation.

Production of IL-6 and TNF-~. Production of IL-6 and TNF-~ by LPS-induced

splenocytes could be released by macrophages or B cells. Dietary IS-fold vitamin E

supplementation did not modify IL-6 by LPS-stimulated splenocytes in the uninfected

mice. However, 150- and 450-fold vitamin E supplementation significantly (p < 0.05)

reduced IL-6 production (Fig. 9.9A). All vitamin E supplementation had little effect on

the production of TNF-~ by LPS-stimulated splenocytes from uninfected mice (Fig.

9.9B). Increased secretion of IL-6 and TNF-~ during murine AIDS could be

significantly (p < 0.05) reduced by all levels of vitamin E supplementation, whereas

150- and 450-fold ~itamin E supplementation did not further reduce the levels of IL-6

and TNF-~ produced by splenocytes compared to that lessened by IS-fold vitamin E

supplementation (Fig. 9.9A&B).

Mitogenesis of Splenocytes. Proliferation of ConA- and LPS-induced splenocytes was

significantly (p < 0.05) increased by increased dietary vitamin E in a concentration

dependent manner in the uninfected mice (Fig. 9. lOA & B). Higher levels of dietary

vitamin E significantly (p < 0.05) increased T-cell and B-cell mitogenesis, which was

suppressed by retrovirus infection, whereas 450-fold increase of vitamin E

supplementation did not further enhance splenic T and B cell proliferation compared to

232

that increased by ISO-fold vitamin E supplementation (Fig. 9.lOA&B).

NK Cell Cytotoxicity. NK cell cytotoxicity was not affected by all levels of vitamin E

supplementation in uninfected mice (Fig. 9.11). The suppressed NK cell activity by

retrovirus infections were significantly (p < 0.05) restored by all levels of vitamin E

supplementation during murine AIDS, whereas 150- and 4S0-fold vitamin E

supplementation did not further increase splenic NK cell activity compared to that

increased by that IS-fold vitamin E supplementation, yet there was a trend to increase

NK cell activity as increase of vitamin E supplementation (Fig. 9.11).

Production of IgA and IgG. Supplemented vitamin E diets had no effect on IgA and

IgG production by LPS-stimulated splenocytes in the uninfected mice (Fig. 9.12A&B).

However, increased IgA and IgG levels induced by retrovirus infection were significantly

(p < 0.05) reduced by all levels of vitamin E supplementation in the murine AIDS,

whereas 150- and 4S0-fold vitamin E supplementation did not further decrease production

of IgA and IgG by splenocytes compared to that reduced by IS-fold vitamin E

supplementation (Fig. 9.12A&B).

Thymus Weight. As shown in Fig. 9.13, vitamin E supplementation has no effect on

the thymus weight in uninfected and infected mice.

Production of Cytokines by Thymocytes. IS-fold vitamin E slightly decreased IL-2

production, whereas 150- and 4S-fold vitamin E significantly (p < 0.05) reduced. IL-2

production by thymocytes (Fig. 9.14). The retrovirus-induced suppression ofIL-2 could

be significantly (p < 0.05) normalized by IS-fold vitamin E, however, 150- and 4S0-fold

vitamin E had no effects on that (Fig. 9.14). Vitamin E supplementation at all levels had

233

no effects on IL-4 production by thymocytes in the uninfected, normal mice, whereas

their supplementation in murine AIDS significantly (p < O.OS) reduced IL-4 production

during which had been elevated by retrovirus infection (Fig. 9.1S). However, IS0- and

4S0-fold vitamin E supplementation did not further reduce IL-4 production compared to

that reduced by IS-fold vitamin E during murine AIDS (Fig. 9.1S). IS-fold vitamin E

had no effect on thymic IL-6 production, whereas IS0- and 4S0-fold vitamin E

suppressed IL-6 production in the uninfected, normal mice (Fig. 9.16). Elevated IL-6

production by thymocytes during murine AIDS was significantly ( p < O.OS) reduced by

vitamin E supplementation at all levels, but IS0- and 4S0-fold vitamin E did not further

decrease IL-6 production compared to that lessened by IS-fold vitamin E supplementation

(Fig. 9.16). Retrovirus infection did not alter thymic IL-IO production (Fig. 9.17). IS­

fold vitamin E had no effect on thymic IL-IO production, whereas IS0- and 4S0-fold

vitamin E suppressed IL-IO production in both the uninfected, normal mice and those

with murine AIDS (Fig. 9.17). 4S0-fold vitamin E supplementation did not further

reduce IL-IO production compared to that decreased by IS-fold vitamin E

supplementation (Fig. 9.17). These was no changes in IFN-')' production by thymocytes

from the uninfected, normal mice fed vitamin E at all levels (Fig. 9.18), whereas

increased IFN-')' production during murine AIDS was significantly ( p < O.OS)

normalized by various levels of vitamin E supplementation (Fig. 9.18). lSO- and 4S0-

fold vitamin E supplementation slight decreased thymocyte proliferation, whereas IS-fold

vitamin E supplementation slight increased thymocyte proliferation in the uninfected,

normal mice (Fig. 9.19). The retrovirus-induced suppression of thymocyte proliferation

234

was significantly (p < 0.05) enhanced by IS-fold vitamin E supplementation, whereas

150- and 4S0-fold had no effects on thymocyte proliferation (Fig. 9.19).

235

Discussion

The present study investigated the effects of different supplemental vitamin E

levels during murine AIDS on cytokine secretion, immune functions and nutritional

status. The findings presented in this study confirm and expand our previous findings

that vitamin E supplementation could be effective treatment, favorably affecting immune

response and nutritional ststus during murine AIDS. Vitamin E supplementation at 150-

and 450-fold were not immunotoxic, even though this extremely high vitamin E

supplementation appears to only marginally further normalize immune dysfunction and

cytokine dysregulation. These results suggest a possible role for high vitamin E dosage

as a potential therapeutic nutrient to help normalize immune dysfunction and malnutrition

caused by HIV infection, thereby retarding the development of AIDS.

Cytokines play crucial roles in the immune response. IL-2 is a pivotal cytokine

in the growth and differentiation of T cells, and activation of NK cells and lymphokine­

activated killer cells (231). IL-2 also can act as a growth and differentiation factor for

B cells and activate macrophages. Stimulation of IL-2 by vitamin E is correlate with its

enhancement of T and B cell proliferation, and NK cell activity by vitamin E during

murine AIDS. Increased secretion of IFN-'Y by vitamin E may be part of the explanation

for its enhancement of tumor resistance during murine AIDS (225). IFN-'Y plays a key

role in T-cell mediated tumor regression by its anti-proliferative activity (341). IFN-'Y

has multiple distinct biological activities including anti-viral activity, activating

phagocytosis of macrophages and neutrophil cells, cytotoxicity of NK cells (397).

Enhancement of IFN-'Y by vitamin E in murine AIDS may be partly due to the decrease

236

of immunosuppressive product PGE,z produced by immunocompetent cells induced by

vitamin E (156). PGE,z is increased during murine AIDS (390) and is a potent inhibitor

of NK cells (397), which is a major source of IFN--y production in vivo (195). NK cells

were also injured by reactive oxygen species (ROS, 358, 359), which were significantly

enhanced during murine AIDS (225). Vitamin E supplementation in murine AIDS was

shown to be protective against AIDS-associated oxidative stress such as increased free

radicals and increased haptic lipid peroxidation (225). Thus vitamin E, acting as an

antioxidant, protects NK cells from attacks of ROS. This idea is strengthened by the fact

that vitamin E stimulated NK cell activity during murine AIDS in this study. Thus,

increased activity of NK cells by vitamin E supplementation is expected to be responsible

for the increased IFN--y. In turn, increased IFN--y depressed Th2 cytokine production,

explaining the decrease of IL-6 and IL-10 by vitamin E supplementation we observed in

this study. Elevated levels of IL-6 and IL-lO have been believed to be associated with

AIDS development. For instance, IL-lO can inhibit Th1 cytokine production (e.g. IL-2

and IFN--y), leading to imbalance of Th1 and Th2 cytokine profile. This imbalance is

believed to be a key point during development of AIDS (398). Increased IL-6 can also

stimulate mv replication in HIV-infected monocytes/macrophages and CD4+ T cells

(399,400), and contribute the hypergammaglobulinemia seen often in both AIDS diseases

(244). Therefore, immunomodulatory nature of vitamin E dose provide a basis for

vitamin E nutritional therapy in AIDS.

TNF-a levels are elevated in mv patients and murine AIDS (208, 365). The

physiological effects of TNF-a in the body include increasing phospholipase A2 activity

237

and generation of ROS (401), a potent inducer of HIY replication in monocytes (402-

405), and stimulating synthesis of PGEz, an immunosuppressive substance produced by

macrophages/monocytes and lymphocytes in human and murine AIDS (406). These

effects of TNF-a could also be directly lessened by vitamin E, because vitamin E

reduced TNF-a level in murine AIDS and inhibits phospholipase A2 and PGEz production

(148, 407). Thus, vitamin E supplementation during murine AIDS would contribute to

inhibition of retrovirus infection, via reducing generation of ROS, PGEz and TNF-a, and

inhibiting activity of phospholipase A2. Vitamin E may also directly inhibit viral

replication through inhibition of protein kinase C (PKC). The HIV coat protein gp120

induces PKC activity in lymphocytes (408). In turn, PKC activation results in the

transition of my from a state of latency to active replication in lymphocytes, and leads

to upregulation ofHIY replication in cells with non-dormant mv (409-411). This notion

is strongly supported by the fact that HIY transcription is redox regulated, and inhibited

by antioxidants such as N-acetylcysteine, a well-known antioxidant used to replenish

intracellular glutathione (412-415). Collectively, vitamin E supplementation not only

enhances the immune response and restor/! tissue vitamin E deficiencies but also directly

inhibit retrovirus replication, thereby retarding the progression to AIDS.

This result is consistent with our previous finding that longer duration ( more than

12 weeks) of vitamin E supplementation inhibited IL-2 production by thymocytes (416).

The exact physiological significance of high dosage of vitamin E supplementation on T

cell differentiation should be further defined. As dietary vitamin E reduced production

of IL-4, increased by retrovirus infection in the thymus, these changes may contribute

238

to the restoration of abrogation of T cell development during progression to murine or

human AIDS, normalizing immune dysfunctions initiated by retrovirus infection. Thus,

the reduction of IL-6 secretion by dietary vitamin E in murine AIDS may also contribute

to the restoration of T cell development in the thymus abrogated by retrovirus infection.

The physiological significance of increased production of IFN-'Y by thymocytes, induced

by retrovirus infection, may be involved in the up-regulation of MHC class I and II

expression on the surface of thymic stromal cells. It plays a crucial role in the positive

and negative selection during T cell education (Le. recognition 'self and 'non-self) in

the thymus. Thus, the elevated levels of IFN-'Y release by thymocytes in murine AIDS

may reflect the failure of T cell education, leading to loss of tolerance to self antigens

or reaction to non-self antigens. Supplemental vitamin E reduced the IFN-'Y production

in murine AIDS, suggesting modulatory effects of dietary vitamin E on thymic cytokine

production with reversing of impairment of T cell maturation in the thymus initiated by

retrovirus infection. Since the role of IL-lO in the T cell maturation is not elucidated so

far, the physiological significance of vitamin E-induced reduction of IL-lO production

is not clear.

Changes in function and subpopulations of thymocytes appear to be responsible

for the modulation of cytokine production by vitamin E supplementation. Possible

underlying mechanisms we propose include: (A) vitamin E supplementation reversing

the deficiencies of vitamin E in the thymus initiated by retrovirus infection, which may

be essential for T cell maturation; (B) Vitamin E supplementation normalized the

deficiency of hepatic zinc initiated by retrovirus infection (417), which has been shown

239

to be essential for T cell maturation (418-420); (C) Vitamin E supplementation reduced

the level of corticosteroids, which are potent inducers of thymocyte apoptosis (144).

Vitamin E supplementation is known to be not toxic in humans (144, 146). The

450-fold increase of vitamin E supplementation did not further increase the levels of

haptic and serum vitamin E. This may explain the nontoxicity of high dosage of vitamin

E supplementation observed in both human studies. The possible mechanism is increased

excretion of vitamin E in the body after increased oral vitamin E supplementation,

keeping vitamin E a reasonable levels. Therefore, extremely high vitamin E

supplementation (e.g. 150- and 450-fold) is not immunotoxic, even though it was not

more effective than that of 15-fold vitamin E supplementation.

Vitamin E supplementation has a positive effects on immune response in murine

AIDS (423). One of mechanisms of vitamin E immunoenhancing functions is that

vitamin E may improve tissue nutrient levels, which had been reduced by retrovirus

infection. Since some nutrient intakes are well known to be associated with immune

response, increase in tissue concentrations of nutrients can be related to significant

immunoenhancemen~ (119). Vitamin A supplementation was found to stimulate

lymphocyte proliferation response to Trichophyton and Candida and to stimulate cell

mediated cytotoxicity to allogenic tumor cells (424). Vitamin A supplementation in mice

stimulates immune functions and reduced death due to retrovirus infection (425).

Vitamin E supplementation has been found to stimulate T-helper lymphocytes, antibody

response, delayed cutaneous hypersensitivity reaction, the reticuloendothelial system, and

host resistance in animal models (426). Vitamin E has also been found to stimulate

240

phagocytosis and mitogen responsiveness, in addition to enhancement of humoral immune

response (424). Therefore, the increase of vitamin A and E in the tissues including

immune organs should contribute to ameliorate immune dysfunctions initiated by

retrovirus infection: increased proliferation of T and B in the spleen, and normalized

cytokine production by lymphocytes from spleen and thymus observed in murine AIDS

(423). Indeed, the improvement of nutritional status by various levels of vitamin E

supplementation is correlated with the enhancement of immune response by various levels

of vitamin E supplementation during murine AIDS we observed (427).

The underlying mechanism by which vitamin E supplementation restored hepatic

nutrients levels during murine AIDS is proposed as follow. Vitamin E may protect

gastrointestinal mucosal cells from retrovirus infection, possible via vitamin E's

inhibition of retrovirus replication (436). Thus, retrovirus-induced malabsorption would

be retarded and the improvement of nutritional status would be expected. High levels

of vitamin E supplementation is known to be nontoxic in humans (140, 144). The 450-

fold increase of vitamin E supplementation did not further increase the levels of haptic

and serum vitamin E. This may explain the non toxicity of high dosage of vitamin E

supplementation observed in both human and animal studies. The possible mechanism

is increased excretion of vitamin E in the body after increased oral vitamin E

supplementation, keeping vitamin E a reasonable levels. Therefore, extremely high

vitamin E supplementation (150- and 450-fold) is not toxic, even though it was not more

effective than that of 15-fold vitamin E supplementation at normalization of tissue

nutrient levels during murine retrovirus infection.

241

In conclusion, vitamin E supplementation in murine AIDS acts on various immune

components to modify immune defects and nutritional status induced by retrovirus

infection without any immunotoxicity at extreme high oral supplementation. It tends to

partially normalize immune dysfunction, cytokine dsyregulation and malnutrition during

murine AIDS. Thus, the vitamin E nutritional supplementation may provide additional

therapeutic approaches for treatment of HIV infected patients without additional

immunotoxicity.

..-. b!I -....

40

.c 30 b!I .... CI.I

~ :>-. 20

"0 o =

-bJl

E! --.... .c: bJl ....

10

800

600

IU 400

~ I:: IU 200 IU -~

C"I'.J o

242

II 1x .15x 1m 150x~ 450x

A

Unifected Murine AIDS b

Unifected Murine AIDS

Fig. 9.1 Effects of Vitamin E supplementation on Body and Spleen Weights During retrovirus infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

-r.. 81x • 15x m 150X~ 450x ~ ~ .­-btl -btl

800

.: 600

~

= .- 400 S CIS ... :> 200

CJ

a

a

.... ... CIS Co ~

o~--

= 5 --S -~ 4 -= 3 .-s !! 2 > 8 1 :: r.. ~

ell 0

Uninfected

a

Unifected

Murine AIDS

Murine AIDS

243

Fig. 9.2 Effects of Various Levels of Vitamin E Supplementation on Serum Vitamin A and E During Retrovirus Infection. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group.

a: p < 0.05 compared to control diet-fed uninfected, normal mice. b: p < 0.05 compared to control diet-fed infected mice.

Vitamin E

III 1x • 15x m 150x~ 450x

~ ~ 800 ac :::L. -~ == .... S CI:I ..... .... >

CJ .... .... CI:I Co. ~

==

600

400

200

2000

1700

~ 1400 bJ)

=.. -< c .• S c:I -.. >

1100

800

500

200

a

a

UninCected Infected

244

Fig. 9.3 Effects of Various Levels of Vitamin E supplementation on hepatic vitamin A and E during Retrovirus Infection. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group.

a: p < 0.05 compared to control diet-fed uninfected, normal mice. b: p < 0.05 compared to control diet-fed infected mice.

245

- 1x _ 15x m 150x ~ 450x

- 50 ~ QJ a ;;.. •• 40 -0.0 -0.0 :t. 30 -~ QJ

~ ~

20 0 u

10 120

a

'i:' 4.1 90 ;> .,.. -~ -~ 60 =. -Col

= 30 .,.. N

0 Uninfected Infected

Fig. 9.4 Effects of Various Levels of Vitamin E supplementation on hepatic zinc and copper during murine AIDS. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group.

a: p < 0.05 compared to control diet-fed uninfected, normal mice. b: p < 0.05 compared to control diet-fed infected mice.

-4J .S 420 ..... rn 4J

== 320 .... ~ -~ 220 :::1. -~ = .... S = .... .... i>_

4> CI ... ~ III 4> ~

CI ...

120

60

50

40

30

20

10

246

_ 1x _ 15x rmJ 150x ~ 450x

a

a

a

Unirected Infected

Fig. 9.5 Effects of Various Levels of Vitamin E Supplementation on Intestinal Vitamin A and E During Retrovirus Infection. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group.

a: p < 0.05 compared to control diet-fed uninfected, normal mice. b: p < 0.05 compared to control diet-fed infected mice.

-Q,I = .... ..... til Q,I

-= ....

~

== .... .... til Q,I .... == .... ~ -

40

30

20

10

o 140

120

100

~ 80 =. -~ 60 .... N

40

247

_ 1x _ 15x ~ 150x ~ 450x

a

Uninfected Infected

Fig. 9.6 Effects of Various Levels of Vitamin E Supplementation on Intestinal Zinc and Copper During Retrovims Infection. The values are mean + SD for 4 or 8 mice in each group. Mouse number of groups for 1 x and 15 x was 4 mice per group. Mouse number of groups for 150 x and 450 x was 8 mice per group.

a: p < 0.05 compared to control diet-fed uninfected, normal mice. b: p < 0.05 compared to control diet-fed infected mice.

_ 1x _ 15x m 150x ~ 450x

2 A

--8 -OJ)

,s 1 ~

I ....:I ....

--e -~ = -= 8 e = ~ I

Z ~ ....

o

15 a

B a a

10

5

0 UninCected Murine AIDS

248

Fig. 9.7 Effects of Various Levels of Vitamin E Supplementation on IL-2 and IFN--y Production by Splenocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

249

Vitamin E _ 1x _ 15x ml150x ~ 450x

4 b A

--e -bf)

= '-"

2

b B

o Uninfected Murine AIDS

Fig. 9.8 Effects of Various Levels of Vitamin E Supplementation on IL-6 and IL-lO Production by Splenocytes During Retrovirus Infection. The values are mean + SO for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

Vitamin E

24

- 24 i ~ -S 18 tS I

~ Z 12 E-4 ~ CI ~ 6 o CI o

- 1x _ 15x 1m 150x ~ 450x

A

b B

~ 0 L.cm~~~L-

Uninfected Murine AIDS

250

Fig. 9.9 Effects of Various Levels of Vitamin E Supplementation on IL-6 and TNF-a Production by Splenocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

251

-= _ 1x _ 15x m 150x ~ 450x

= ~ 1"'"4 40 ~ e A c.. (oJ 30 - ae

= 0 .... 20 ..... ~ J.4

~ 10 .... -0

J.4 =.. - 0 -QJ

_(oJ

gE-4 = 80 .. 1"'"4 ~

E! acd B ~ 60 CJ -= 0 .... 40 .... cu J.4

~ .... - 20 0

'" =.. -- 0 QJ CJ

== Unifected Murine AIDS

Fig. 9.10 Effects of Various Levels of vitamin E Supplementation on Splenocyte Proliferation During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

252

_1x _ 15x _ 150x _ 450x

~

~ 100

ac

a

50

o Uninfected Infected

Fig. 9.11 Effects of Various Levels of Vitamin E Supplementation on NK cell Avtivitu during retrovirus infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

Vitamin E

-~ 100 -:;... .... .... CJ .... ~ o .... .B 50 :;... U --11.1 U ~ Z

--e -boO =.. -~ boO

I-c

o

40

20

o

- 1x _ 15x 1m 150x ~ 450x

A Be

B b

Unifected Murine AIDS

253

Fig. 9.12 Effects of Various Levels of Vitamin E Supplementation on IgA and IgG Production by Splenocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

254

100 Vitamin E ~

b.Il - Ix a '-' .... --= I5x b.Il •• Q,)

50 ~ - I50x rI.l = -a 450x ~

-= ~ 0

Uninfected Infected

Fig. 9.13 Effects of Various Levels of Vitamin E Supplementation on Thymus weight During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

255

0.30 Vitamin E

- 1x

~ ,..... 0.20 -a 15x

-.... bf)

= - 150x ---M I 0.10 ~ -t-04 450x

0.00 Uninfected Infected

Fig. 9.14 Effects of Various Levels of Vitamin E Supplementation on IL-2 Production by Thymocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

256

0.30 Vitamin E:

b - 1x ~ ....... a 0.20 - 15x --btl

= -'-' 150x ~

I 0.10 ~

_ 450x 1-4

0.00 Uninfected Infected

Fig. 9.15 Effects of Various Levels of Vitamin E Supplementation on IL-4 Production by Thymocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

257

0.30 b Vitamin E - 1x

,-.. ,.... 0.20 -a 15x

~

bIl

== - 150x '-' \C

I 0.10 ~ - 450x '"""'"

0.00 Uninfected Murine AIDS

Fig. 9.16 Effects of Various Levels of Vitamin E Supplementation on IL-6 Production by Thymocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for 1x and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

258

4 Vitamin E:

3 ~ 1x --e ~ 15x ........ ~ = 2 - ~ = 150x

1""04 I

~ ~ 450x 1-1

1

o U ninfected Infected

Groups

Fig. 9.17 Effects of Various Levels of Vitamin E Supplementation on IL-lO Production by Thymocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

259

Vitamin E b 3 - 1x

2 - 15x

1m 150x

1 - 450x

o Uninfected Murine AIDS

Groups Fig. 9.18 Effects of Various Levels of Vitamin E Supplementation on IFN- y Production by Thymocytes During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and 15x was 4 mice per group. Mouse number of groups for 150x and 450x was 8 mice per group.

a p < 0.05 compared to control diet-fed uninfected, normal mice. b p < 0.05 compared to control diet-fed infected mice.

260

..-.. = = = .. ~

~ 20 Vitamin E a -~ 1x CJ '-"

= - 15x 0 .,. ~

~ 10 .. - 150x ~ •• ...... "'" -0 450x ~

Q,) ~

~ CJ 0 0 a Uninfected Murine AIDS ~

Groups ~ ~

Fig. 9.19 Effects of Various Levels of Vitamin E Supplementation on Thymocyte Proliferation During Retrovirus Infection. The values are mean + SD for 4 or 8 mice per group. Mouse number of groups for Ix and I5x was 4 mice per group. Mouse number of groups for I50x and 450x was 8 mice per group.

261

CHAPTER 10: OVERALL CONCLUSIONS

In present studies, we found that retrovirus infection altered the cytokine

production by T cells, macrophages and thymocytes. This cytokine dysregulation has

been shown in parallel with that of HIV -infected individuals. Thus, the elucidating of

the mechanisms of aberrant cytokine release by retrovirus infection should facilitate our

understanding HIV-induced immune dysfunction in humans. In addition, our present

results indicated that retrovirus infection also directly induced the nutritional deficiencies

in the various organs or tissues, including immune organs. Since the importance of these

nutrients in induction of immune responses, undernutrition has the theoretical potential

to accelerate the development of AIDS via immuno- suppression secondary to nutritional

deficiencies.

EtOH ingestion broadly suppresses the various arms of immune response. The

one of mechanisms of EtOH-induced immunosuppression is modulation of cytokine

release by EtOH. Our results indicated that EtOH consumption impaired the cytokine

release by splenocytes and thymocytes, which should contribute to suppression of

immune responses and impairment of T cell differentiation. Due to the overwhelming

evidence that the deleterious effects of chronic EtOH use on immune responses, it could

further enhances susceptibility to infection and development of AIDS. Our present data

suggested that dietary EtOH aggravated progression of immune dysfunction as it modified

production of immunological regulatory cytokines and immune functions. These data

also suggested that dietary EtOH consumption can modulate cytokine production by

thymocytes, adversely affecting T cell differentiation, especially during retrovirus

262

infection. These results produce evidences that EtOH consumption could be a cofactor

during development of AIDS, via producing altered immune responses and then

disrupting T cell differentiation. In addition, chronic EtOH can directly aggravate

undernutrition initiated by retrovirus infection. Such EtOH-induced malnutrition in AIDS

could be a cofactor, accelerating development of AIDS via immunosuppression secondary

to nutritional deficiencies.

Vitamin E supplementation restored the important immunoregulatory cytokine

production by splenocytes and thymocytes and immune response, which was suppressed

by dietary ETOH. Our results suggest that the suppressive effects of EtOH on the

immune response could be directly restored by dietary vitamin E supplementation,

thereby increasing host disease resistance to infections and tumors. Vitamin E

supplementation has been shown to normalize cytokine dysregulation by splenocytes and

thymocytes, and ameliorate immune dysfunctions, and improve undernutrition during

retrovirus infection. In addition, our data also indicated that dietary vitamin E

supplementation at extremely high levels was not immunotoxic, and can modulate

cytokine release and normalize immune dysfunctions during progression to murine AIDS,

which should favorably affect host resistance. These data also indicate that dietary

vitamin E supplementation at extremely high levels can modulate cytokine production by

thymocytes, affecting T cell differentiation, especially during murine AIDS. Thus, the

vitamin E nutritional supplementation may provide additional therapeutic approaches for

treatment of HIV infected patients without additional immunotoxicity. The clinical trials

for potential therapeutic role of vitamin E in AIDS individuals have not been performed.

263

Experimental results from murine AIDS studies have implied that vitamin E

supplementation may act on various immune components to repair immune defects

induced by retrovirus infection, and effectively normalize the other important nutrient

deficiencies initiated by retrovirus infection in the immune organs. Although the animal

data may not be totally extrapolated to HIV-positive humans, information obtained from

such studies using murine AIDS may be potentially transferable or serve as a basis for

the study of therapeutic roles of vitamin E in HIV -infected humans.

Vitamin E should not be considered as a direct anti-retroviral drug but rather as

a potentially immunomodulating antioxidant that achieves its anti-retroviral effect through

indirect mechanisms, possible through inactivation of triggers of HIV gene expression,

immunoenhancing, membrane fluidity, restoration of undernutrition and anti-oxidative

stress. Its use as combination therapy with other pharmacologic agents such as AZT

could have several advantages. First, it could lead to more complete viral suppression

by deceasing oxidative stress and enhancing immunity, associated with this retrovirus

infection, than any single drug used alone. Second, it may allow each drug to be used

in lower doses, thus limiting drug toxicities, and decreased frequency of drug-resistant

HIV production. Third, such immunorestorative treatment may be given during

maintenance therapy of the AIDS patient following the control of tumors and immune

dysfunction by chemotherapy. Fourth, the use of vitamin E may be important in

forestalling initial episodes of general immune disorders in some patients by extending

the period between HIV infection and the appearance of clinical symptoms of AIDS.

264

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2. Centers for Disease Control For AIDS, MMWR, 39:1, 1990.

3. Holmes, KK, Karon, JM, Kreiss, J. Am. Public Health 85:858, 1990.

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263. Chen, GJ, Watson, RR. Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine retrovirus. J. Leuk. BioI. 50:349, 1991.

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275. Liu, KD, Darrow, WW, Tutherfor, GW. III. A model-based estimate of the mean incubation period for AIDS in homosexual men. Sciences, 240: 1333, 1988.

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280. Wang, Y, Watson, RR. A key understanding retrovirus-induced immune dysfunction: murine AIDS. submitted, 1994.

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