dietary gangliosides positively modulate the percentages of th1 and th2 lymphocyte subsets in small...

BioFactors 15 (2001) 1–9 1IOS Press

Dietary gangliosides positively modulate thepercentages of Th1 and Th2 lymphocytesubsets in small intestine of mice at weaning

Enrique Vazqueza,b,∗, Angel Gilb and Ricardo Ruedaa

aResearch and Development Department, Abbott Laboratories, 18004 Granada, SpainbDepartment of Biochemistry and Molecular Biology, University of Granada, 18071 Granada, Spain

Received 25 October 2000

Revised 22 January 2001

Accepted 23 January 2001

Abstract. We studied the influence of dietary gangliosides on the number of spontaneous cytokine-secreting cells from twointestinal lymphocyte populations: lamina propria lymphocytes and Peyer’s patches lymphocytes in Balb/c mice for 28 daysafter weaning. Weanling mice were separated into two groups, designated as Control and BG. The Control group was fed witha semipurified diet without gangliosides and the BG group was fed with the semipurified diet supplemented with 47 mg/kg of amixture of bovine brain gangliosides. Intestinal lymphocytes were isolated from mice killed at 3, 7, 14 and 28 days after weaning,and the percentages of spontaneous Th1 as well as Th2 cytokine-secreting lymphocytes were determined using the ELISPOTassay. The BG group animals showed an earlier development in the number of cytokine-secreting cells, which appeared oneweek later in Control animals. In addition, mice fed with the ganglioside-supplemented diet showed a significantly highernumber of Th1 and Th2 cytokine-secreting lymphocytes than Control mice in lamina propria and Peyer’s patches lymphocytesat the end of the experimental period (28 days). Our results suggest that dietary gangliosides influence the maturation processof the intestinal immune system that take place during weaning.

Keywords: Mouse, weaning, dietary ganglioside, spontaneous cytokine-secreting lymphocytes, intestinal immunity

Abbreviations: BG, brain gangliosides supplemented diet; IL-2, interleukin 2; IL-5, interleukin 5; IL-6, interleukin 6; γIFN,gamma interferon; LPL, lamina propria lymphocytes; PPL, Peyer’s patches lymphocytes; Th, T helper

1. Introduction

Gangliosides, which are complex acid glycosphingolipids, form integral components of the cellularlipid bilayer. In addition, gangliosides have been identified in many animal fluids including milk of mostanimals and of humans [20]. The distribution pattern of milk gangliosides selectively changes duringlactation, differing different in colostrum, transitional milk and mature milk [23]; likewise, this patternchanges in milk from mothers delivering preterm and term infants [19] as well as in milk from mothersof different geographic and cultural origins [21]. Therefore milk gangliosides may participate in the

∗Corresponding author: Dr. Enrique Vazquez, R & D Department, Abbott Laboratories, Cno Purchil, No. 68, 18004 Granada,Spain. Tel.: +34 958 248666; Fax: +34 958 248660; E-mail: [email protected].

0951-6433/01/$8.00 2001 – IOS Press. All rights reserved

2 E. Vazquez et al. / Dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets

physiological processes that take place in the newborn development during lactation [20]. In addition, theprebiotic character of gangliosides in the gastrointestinal tract of the newborn has recently been reported;the supplementation of adapted milk formulas with brain gangliosides alters levels of fecal Escherichiacoli and Bifidobacterium in preterm newborn infants [24]. Likewise, the immunomodulatory propertiesof gangliosides on the systemic immune system are well known, acting as suppressive agents [5,13,15]as well as activator molecules [28,30].

The intestinal immune system exhibits several particularities that distinguish it from the systemicone. In fact, lymphocytes lining the gut are able to defend the organism against infection and promotethe development of the oral tolerance to food antigens [1]. Lamina propria lymphocytes (LPL) arephenotypically similar to peripheral blood lymphocytes (PBL) [25], and functionally the intestinallamina propria is a major mucosal effector site [3]. The B-lineage cells represent 20–40% of LPL butthere is also a substantial number of T cells (nearly 50% of LPL are CD3+, and the CD4+ to CD8+ratio is 3 : 1) [17]. The Peyer’s patches are germinative centers the predominant role of which is tocooperate in the differentiation of B cells towards IgA-producing cells [26]. This process involves Tcells, predominately CD4+, are involved [16], probably through the secretion of specific cytokines.

One particularity of the intestinal immune system is the high cytokine production released by intestinallymphocytes both in humans and mice [4,12,27] a characteristic that could reflect the chronic activatedstatus of the intestinal lymphocytes [14,29]. In the gut, cytokines are involved in a wide spectrum ofprocesses for which cytokines play a relevant role; among the total pool of intestinal cytokines, IL-2,IL5, and IL-6 have been shown to promote the maturation of murine IgA-expressing cells to IgA-secreting cells. Th1 cells secrete IL-2 and γIFN whereas Th2 produce, among other cytokines, IL-5and IL-6. Thus, cytokines from both Th1 and Th2 populations influence the differentiation of murineIgA-expressing cells [14]. Recently, our group has reported a high number of murine spontaneous Th1and Th2 cytokine-secreting cells from LPL as well as Peyer’s patches lymphocytes (PPL) during the firstmonth after weaning [29]. In addition, these cytokine-producing lymphocytes exhibit an ontogeneticdevelopment consisting of a peak in the percentage of cytokine-secreting cells between 7 and 14 days afterweaning that could be related to the intestine maturation/differentiation processes in early weaning [29].

The aim of the present study is to assess the influence of dietary gangliosides on the evolution of thespontaneous cytokine-secreting lymphocytes belonging to both Th1 and Th2 profiles from murine LPLas well as PPL for 28 days after weaning.

2. Material and methods

2.1. Experimental design

The Animal Welfare Committee of the University of Granada approved the protocol of this study.BALB/c male mice (three weeks old) were purchased from Iffa Credo, (Lyon, France). The mice wereweighed and housed in an air-conditioned room at 22◦C on a 12 hour light-dark cycle in groups of sixanimals per cage. The animals were randomly separated in two groups. The Control group was fed asemipurified diet without gangliosides following the AIN-76 guidelines [2] and the other group called BGwas fed with the semipurified diet supplemented with a complex mixture of bovine-brain gangliosides(SIGMA, St Louis, USA) at 47 mg/kg (BG diet). Diets were prepared and packaged by the R&DDepartment of Abbott Laboratories Granada, Spain. The overall composition of the semipurified diet isshown in Table 1. All animals had free access to fresh diet and water and received humane treatmentaccording to EU regulations for laboratory animals. At 3, 7, 14 and 28 days after the beginning of the

E. Vazquez et al. / Dietary gangliosides positively modulate the percentages of Th1 and Th2 lymphocyte subsets 3

Table 1Composition of the experimental control diet

Ingredient Amount Composition Amountg/kg g/kg

Calcium caseinate1 191.14 Protein 170.00Saccharose 150.00 Carbohydrate 579.60Oil2 97.84 Fat 100.0Wheat starch 431.80 Moisture 66.5Cellulose 50.00 Ash 24.2DL methionine 3.00Choline 1.10Minerals3 16.01Vitamins4 0.12

Footnotes: 1Calcium caseinate consisted of casein(88.94%), minerals (2.36%), water (5.77%).2The oil mixture consisted of olive oil (66.5%), soybean oil(23%) and refined coconut oil (10.5%).3American Institute of Nutrition [2].4Composition of the vitamin supplement was as follows(mg/kg): retinol 1.20, thiamine hydrochloride 6.00, ri-boflavine 6.00, nicotinamide 30.00, calcium pantothen-ate 16.00, pyridoxine HCL 7.00, biotin 0.20, folic acid2.00, cyanocobalamine 0.01, cholecalciferol 0.03, alpha-tocopherol 50.00, phyloquinone 0.05.

experiment, mice (n = 8) were killed by neck fracture and the intestinal lymphocytes were inmediatelyisolated. The isolated cells were used for the ELISPOT assay to determine the number of Th1 and Th2cytokine-secreting intestinal lymphocytes.

2.2. Isolation of intestinal lamina propria lymphocytes and Peyer’s patches lymphocytes

LPL and PPLwere isolated following the procedure of Gautreaux et al. [8] with some modifications.Briefly, the small intestine was removed and the lumin content was flushed with Hanks Balanced SaltSolution, (HBSS, Sigma, St. Louis, USA), the visible Peyer’s patches were excised, and the intestine wasopened longitudinally and cut into small pieces, which were placed in 25 ml of HBSS , with 5 mmol/Ldithiotreitol (DTT, Roche Molecular Biochemicals, Indianapolis, USA), 2 mmol/L EDTA (Sigma, St.Louis, USA) and 25 mmol/L Tris buffer (Sigma, St. Louis, USA) (HBSS-DTT-EDTA-Tris). For removalof the epithelium, the mixture was incubated for 15 min at 37◦C in a shaking water bath (100 strokesper min). The supernatant was decanted, fresh HBSS-DTT-EDTA-Tris was added, and the incubationprocedure was repeated. LPL were liberated from the remaining intestinal debris by placing them inRPMI 1640 medium (Sigma, St.Louis, USA) containing 10% heat-inactivated fetal calf serum (Sigma,St.Louis, USA), 20 mM HEPES (Sigma, St.Louis, USA), 2 mmol/L L-glutamine, 500 U penicillin and100 µg/ml streptomycin (Sigma, St.Louis, USA) (complete medium), 0.02 U/ml collagenase, 0.12 U/mldispase, and, 200 U/ml Dnase I (Roche Molecular Biochemicals, Indianapolis, USA) for 90 min in a37◦C shaking water bath at 100 strokes per min. The LPL suspension was washed by centrifugation andsubjected to discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradients to enrich the cellsuspension for lymphocytes.

The excised Peyer’s patches were placed in complete medium and dissected with scalpels. Theywere subjected to enzymatic digestion for 30 min, and centrifuged in discontinuous Percoll densitygradients to obtain a purified suspension of PPL. Finally, LPL and PPL were washed by centrifugationand resuspended in complete medium at 1 × 106 cells/ml.


2.3. ELISPOT assay for detection of cytokine-secreting cell

The ELISPOT assay was performed basically as described by Fujihashi [7]. This method is designed todetect specific cytokine-secreting cells at a single level. In the present work, we have tested the frequencyof Th1 (secreting interleukin 2, IL-2, or gamma interferon, γIFN) and Th2 (secreting interleukin 5, IL-5, or interleukin 6, IL-6) cytokine-secreting lymphocytes in LPL and PPL. Nitrocellulose-bottomedmicrotiter wells (Millipore Co., Bedford, MA) were coated with cytokine-specific monoclonal captureantibodies (PharMingen, San Diego, CA, 100 µl of 5 µg/ml in PBS per well) overnight at roomtemperature. After removal of the unadsorbed antibody the wells were blocked with complete medium.The cells were resuspended in complete medium and were added to individual wells (n = 8) in serialdilutions (5×102-5×103-5×104 cells/100 µl) and incubated for 20 h at 37◦C in a humidified atmosphereof 5% CO2 in air. Following incubation, plates were extensively washed with PBS and then with PBScontaining 0.05% Tween 20 (PBST) to remove all cells. Individual wells were reacted with 100 µl ofbiotin-labeled cytokine specific mAb (PharMingen, San Diego, CA), (2 µg/ml in PBST 1% BSA), andincubated in a humidified chamber overnight at 4◦C. PBST 1% BSA was added to the wells for negativecontrols. After five washes with PBST, 100 µl of avidin-peroxidase (Sigma, St. Louis, USA) diluted1 : 400 in PBST 1% FCS was added and the plates were incubated for two hours at room temperature.After extensive washing, spots were developed by adding the substrate 3-amino-9-ethylcarbazole (Sigma,St. Louis, USA) in 0.1 mol/L sodium acetate, pH 5.0, 200 µl/well and incubating for one hour. Finally,plates were washed with tap water and dried with a hair-drier. Spots were counted with the aid of abinocular lens, (SZ-PT, Olympus, Lake Suecess, USA).

2.4. Statistical analysis

The number of lymphocytes belonging to each Th profile was determined by the average of IL-2and γIFN-secreting cells for Th1 and the average of IL-5 and IL-6-secreting lymphocytes for Th2.Results were expressed as mean percentages ± standard deviation. The homogeneity of variances wastested by Levene’s test and a two-way Anova was conducted to assess the effects of weaning time anddietary gangliosides on Th1 and Th2 percentages. The Bonferroni test was used a posteriori to evaluatesignificant differences (p < 0.05) between 3, 7, 14 and 28 days.

All test were performed using the PC90 version of 7D BMDP program (BMDP PC 90 version,Statistical Software, Inc, Los Angeles, CA).

3. Results

No significant differences in body weight were detected between the group fed the semipurified diet(Control diet) and that fed the diet supplemented with bovine brain ganglioside supplemented diet atthe considered weaning periods (Table 2). The individual food intake was not quantified, but eachexperimental group consumed similar amounts of diet.

Table 3 and Table 4 show the mean percentages for IL-2, γIFN, IL-5 and IL-6 cytokine-secretinglymphocytes in LPL and PPL, respectively. Both populations, LPL and PPL, exhibited a very similarprofile for secreted cytokines, showing high percentages of Th1 (IL-2, γIFN) and Th2 (IL-5, IL-6)cytokine-secreting cells. In LPL and PPL from Control mice, the percentages of IL-2, IL-6 and γIFN-secreting cells after 3, 7 and 28 days of feeding did not significantly differ. However, these values weresignificantly lower (p < 0.05) than on day 14. Likewise, a peak in the percentage of IL-5-secreting cells


Table 2Weight (g) of mice fed with two diets differing intheir ganglioside contents

Time Diets

Days of feeding Control BG1

0 11.21 ± 0.48 11.31 ± 0.533 14.51 ± 0.62 14.53 ± 0.917 16.80 ± 0.51 16.72 ± 0.46

14 20.33 ± 0.61 20.29 ± 0.9328 24.40 ± 1.32 23.98 ± 0.75

Footnotes: Data are expressed as means ± standarddeviation in g. Experimental diets (Control and BG)(n = 8 for each period).1BG: diet supplemented with bovine brain ganglio-sides.

Table 3Percentage of IL-2, γIFN, IL-5 and IL-6 cytokine-secreting cells in lamina propria lymphocytes from micefed with two diets differing in their ganglioside content1

Days of feeding Diets CytokinesIL-2 γIFN IL-5 IL-6

3 Control 27.40 ± 2.90b 18.93 ± 7.16a 15.95 ± 1.97a 29.55 ± 1.72a

3 BG2 26.89 ± 15.95a 23.09 ± 13.42a 20.18 ± 10.88a 28.94 ± 4.36a

7 Control 15.12 ± 1.94a 24.33 ± 5.19a 36.23 ± 4.40b 20.93 ± 10.02a

7 BG 50.45 ± 8.41b∗ 55.4 ± 25.83b∗ 58.44 ± 21.78c∗ 44.69 ± 18.49b∗

14 Control 37.89 ± 5.86c 49.06 ± 6.57b 40.69 ± 3.28b 39.74 ± 2.18b

14 BG 26.13 ± 0.97a* 40.88 ± 6.96b 32.70 ± 4.81a 42.15 ± 3.10b

28 Control 24.14 ± 15.11b 14.33 ± 10.69a 27.11 ± 10.34a 18.43 ± 11.60a

28 BG 32.68 ± 5.48a 40.83 ± 20.83b∗ 40.31 ± 3.75b∗ 37.23 ± 19.29ab

Footnotes: 1Data are expressed as means ± standard deviation (n = 8 for each period). All assays wereperformed out in duplicate (total n = 16). a < b < c. Significant differences between sampling times forthe same experimental group (Control or BG) (p < 0.05).∗: significant differences between Control and BG groups (p < 0.05).2BG: diet supplemented with bovine-brain gangliosides.

appeared on days 7 and 14, and the IL-5-secreting-cell percentages proved significantly higher than ondays 3 and 28.

A notable difference was evident in the behavior of the cytokine-secreting cells of LPL and PPL fromControl mice as compared to those fed the BG. In fact, even though the global profile of the BG groupregarding the intestinal spontaneous cytokine-secreting lymphocytes was essentially the same as that ofthe Control group, in the former the ontogenetic peak in the number of cytokine-producing cells showedup significantly earlier (p < 0.05) than in the latter (day 7 versus day 14, respectively), both in LPL andPPL and in both T helper subsets.

Figures 1 and 2 show the percentages of Th1 (IL-2 or γIFN secreting cells) and Th2 (IL-5 and IL-6secreting cells) in lamina propria and Peyer’s patches of small intestine in mice, 3, 7, 14 and 28 daysafter weaning fed the control and BG. Changes in Th1 and Th2 percentages exhibited a quite similarpattern in both intestinal lymphocyte populations. Figures 1 and 2 indicate that irrespective of thelymphocyte population (LPL or PPL) and T helper subset (Th1 or Th2) in the BG group, the percentageof cytokine-secreting cells on day 7 was significantly higher than in control. In addition, whereas in theControl group the maximum percentage of cytokine-secreting cells appeared on day 14, the maximum


Table 4Percentage of IL-2, γIFN, IL-5 and IL-6 cytokine-secreting cells in Peyer’s patches lymphocytes from micefed with two diets differing in their ganglioside content1

Days of feeding Diets CytokinesIL-2 γIFN IL-5 IL-6

3 Control 26.55 ± 0.32b 19.69 ± 8.37a 21.88 ± 3.36a 26.58 ± 1.47a

3 BG2 26.64 ± 4.72a 24.58 ± 14.68a 23.35 ± 2.72a 26.21 ± 4.96a

7 Control 19.59 ± 7.92b 22.68 ± 11.10a 40.24 ± 6.85b 22.76 ± 9.44a

7 BG 55.15 ± 5.21b∗ 49.78 ± 15.03b∗ 62.83 ± 15.34b∗ 34.48 ± 5.39a

14 Control 37.48 ± 3.62c 42.08 ± 3.31b 42.05 ± 5.26b 40.91 ± 2.13b

14 BG 29.30 ± 1.22a∗ 40.33 ± 3.30b 28.20 ± 5.19a∗ 43.85 ± 1.96b

28 Control 13.79 ± 9.42a 13.15 ± 9.56a 22.24 ± 11.15a 18.64 ± 15.49a

28 BG 29.08 ± 8.98a∗ 36.04 ± 13.46a∗ 39.95 ± 3.28b∗ 36.68 ± 20.97ab∗

Footnotes: 1Data are expressed as means ± standard deviation (n = 8 for each period). All assays wereperformed in duplicate (total n = 16). a < b < c Significant differences between different sampling timesfor the same experimental group (Control or BG) (p < 0.05).∗: significant differences between control and BG groups (p < 0.05).2BG: diet supplemented with bovine brain gangliosides.

was reached earlier (7 days) in the BG group. Also, the percentages of LPL and PPL subsets remainedsignificantly higher later on (14 and 28 days) in the BG group compared with the Control group.

4. Discussion

The present study demonstrates high percentages of intestinal activated lymphocytes at weaning,which release cytokines belonging to Th1 and Th2 profiles. These results agree with those of otherauthors showing high percentages of intestinal cytokine-secreting cells in mice [27,29] as well as inhumans [4,11,12]. In fact, not only a high percentage of cytokine-producing cells but also high levelsof released cytokines and a high status of cell activation have been reported in murine [9] and humanintestinal lymphocytes [10]. These features suggest that the lamina propria and Peyer’s patches are areasof chronic antigenic stimulation [14,27], thereby supporting the concept of a high intestinal lymphocyteactivation during weaning. Our team has recently reported that spontaneous cytokine-producing cellsfrom both intestinal lymphocyte populations (LPL and PPL) in mice fed a chow diet reach a maximumafter weaning [29]. The results found here with Control mice, fed with a semipurified diet, essentiallyagree with this latter report, showing the mentioned ontogenetic peak in the percentage of spontaneousintestinal cytokine-secreting cells on day 14. It appears that, regardless of the type of diet, antigenicstimulation by food and microbial antigens could be responsible for the high percentage of intestinalcytokine-secreting lymphocytes and their behavior during the first month after weaning. This fact appearsstrongly linked with the maturation/differentiation processes of the intestinal immune system.

One of the main results of the present work is the impact of the ganglioside-supplemented diet onthe behavior of the intestinal spontaneous cytokine-secreting lymphocytes. We suggest that dietarygangliosides are responsible for the stimulatory effects on the number of spontaneous Th1 and Th2cytokine-producing lymphocytes.

Another aspect that may be emphasized is the percentages of Th1 cells do not prevail over those ofTh2. This feature has been described in a previously report from Taguchi et al. [27]. Provided that oneof the main goals in the maturation of the intestinal immune system is the generation of IgA-producingB cells, both cytokine profiles (Th1 and Th2 subsets) may be involved in the induction of the maturationprocess, as stated by other authors [18].


L P L s ubs e t Th1

0

2 0

4 0

6 0

3 7 1 4 2 8

da ys of fe e ding a f te r w e a ning

% cy

tokin

e sec

reting

cells

*a

*

+ *a

L P L s ubs e t Th2

0

2 0

4 0

6 0

3 7 1 4 2 8

da ys of fe e ding a f te r w e a ning%

cyto

kine s

ecret

ingce

lls

*a

*+

*a

Control BG

Fig. 1. Percentages of Th1 and Th2 lymphocytes in small-intestine lamina propria in mice at 3, 7, 14 and 28 days after weaning,fed with a Control diet (dotted bars) and a diet supplemented with bovine brain ganglioside (BG) (solid bars). Bars show themean percentages ± standard deviation of the Th1 (secreting IL-2 or γIFN) and Th2 (secreting IL-5 or IL-6) cytokine-secretingcells from Peyer’s patches lymphocytes. (*) significant differences with respect to day 3 for BG group (p < 0.05). (+)significant differences with respect to day 3 for Control group (p < 0.05). (a) significant differences between Control and BGgroups for the same period of time (p < 0.05).

There are only a few studies related to the ganglioside influence on the cytokine production, andparticulary about the intestinal immune response to gangliosides. However, GD1α and GD1a have beenidentified as specific differentiation markers for Th1 and Th2 subsets, respectively [6]. On the other hand,it has been described that gangliosides in vitro work as regulator molecules not only for the cytokineproduction but also for the systemic activated T cell proliferative response, inhibiting the Th1 cytokineproduction and having no effect on the Th2 subset [13]. The apparent contradiction between these resultsand those found in our study might be justified taking into consideration that Irani et al. [13] studied thein vitro systemic immunity our research refers to in vivo mucosal immunity.

In conclusion, according to our results, there is an ontogenetic peak in the number of Th1 and Th2intestinal lymphocytes at weaning, which is developed earlier in mice fed on gangliosides. In addition,the higher number of Th1 and Th2 cytokine-secreting cells in mice fed on gangliosides remains at theend of the weaning period.

Acknowledgements

We are gratefully indebted to Mrs. Maria Luisa Jimenez Lopez for help and technical assistance.The first author, E Vazquez, was the recipient of a fellowship (Ayudas para el Intercambio de PersonalInvestigador entre Industrias y Centros Publicos de Investigacion) provided by the Spanish Ministry ofEducation.


*a

*+ *a

P P L s ubs e t Th2

0

2 0

4 0

6 0

3 7 1 4 2 8

da ys of fe e ding a fte r w e a ning%

cytok

ine se

cretin

g cell

s

P P L s ubs e t Th1

0

2 0

4 0

6 0

3 7 1 4 2 8

da ys of fe e ding a fte r w e a ning

% cy

tokine

secre

tingc

ells

*a

+*

+

a

Control BG

Fig. 2. Percentages of Th1 and Th2 lymphocyte in small-intestine Peyer’s patches in mice at 3, 7, 14 and 28 days after weaning,fed a Control diet (dotted) and a diet supplemented with bovine brain ganglioside (BG) (solid bars) . Bars show the meanpercentages ± standard deviation of the Th1 (secreting IL-2 or γIFN) and Th2 (secreting IL-5 or IL-6) cytokine-secreting cellsfrom Peyer’s patches lymphocytes. (*) significant differences with respect to day 3 for BG group (p < 0.05). (+) significantdifferences with respect to day 3 for Control group (p < 0.05). (a) significant differences between Control and BG groups forthe same period of time (p < 0.05).

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dietary gangliosides positively modulate the percentages of th1 and th2 lymphocyte subsets in small...

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