GENERAL AND COMPARATIVE ENDOCRINOLOGY 67, 270-276 (1987)

Organ Weights, T-Cell Proliferation, and Graft vs Host Capabilities of Hypothyroidic Chickens

THOMAS SCOTT~ANDBRUCE GLICK'

Department of Poultry Science, Mississippi State University, Mississippi State, Mississippi 39762

Accepted April 19, 1987

Thiouracil-induced hypothyroidism reduced the thymus weights of young chickens. Thy- roid weights were greater in thiouracil-fed (TF) chicks, and the circulating levels of triiodo- thyronine and thyroxine were lower. Thiouracil treatment resulted in higher relative adrenal weights and serum corticosterone concentrations. Absolute weights of adrenals were equiv- alent between control and TF groups. The TF chicks showed depressions of total white blood cell counts and numbers of lymphocytes. Monocyte and granulocyte numbers were unchanged from those of controls. Thiouracil treatment lowered the phytohemagglutinin M responsiveness of peripheral blood leukocytes (PBL). On the other hand, the PBL from TF chicks produced the higher graft vs host (GvH) response. Therefore, while the hypothy- roidic state impaired cycling activity in T cells, this condition did not significantly influence a functional parameter of T cells, the GvH response. o 1987 Academic PKSS, I~C.

Diminished thyroid activity negatively affects the development of lymphoid tissues and immune responsiveness in mice (Baroni et al., 1969; Fabris et al., 1971; Pierpaoli et al., 1969) and rats (Fabris, 1973). In birds, bursa, spleen, and thymus, weights were reduced with thioureylene antithyroid treatment (Jackson and La- bisky, 1971; Mellen and Wentworth, 1962; Raheja and Snedecor, 1970; Snedecor, 1971; Yam et al., 1981). Lowered humoral immune responses of hypothyroid birds to sheep red blood cell challenges have been observed (Keast and Ayre, 1980; Yam et al., 1981), but the role of the thyroid in reg- ulating antibody production of chickens has been questioned (Mashaly et al., 1983). Humoral immune responsiveness of hypo- thyroid chickens were more than adequate (Scott et al., 1985). It was the kinetics of the responses which were affected by thy- roid deprivation.

Depression of T-cell mitogenic respon- siveness occurred with thyroidectomy of

t Present address: Poultry Science Department, 129 Poole Agricultural Center, Clemson University, Clemson, SC 29634-0379.

rats (Chatterjee and Chandel, 1983; Fabris, 1973) and propylthiouracil treatment of young chickens (Yam et al., 1981). In those studies, only cellular proliferation of T cells was examined as an indicator of cell-me- diated immunity. We have chosen to ex- amine both cellular proliferation (phytohe- magglutinin M stimulation) and a functional test (graft vs host potential) of T cells from hypothyroid chickens.

MATERIALS AND METHODS

General procedure. Chicks were hatched from eggs obtained from the Lester J. Dreesen strain of New Hampshire chickens maintained at Mississippi State University. At hatch, chicks were wingbanded and placed in battery brooder units. One-half of the chicks were fed a standard unmediated chick diet, and the other half were fed the same diet containing 0.1% thiouracil (ICN Nutritional Biochemicals, Cleveland, OH). Feed and water were provided ad libitum throughout the experiments.

In experiment 1, chicks were drawn from the treat- ment groups for measurement of body and gland (thy- roid and thymus) weights at 4 and 8 weeks of age. In a second experiment, body and gland (thyroid and adrenal) weights were recorded at 6 weeks of age.

Radioimmunoassay. Blood samples were taken from the cutanea ulnaris of chicks at 6 weeks of age in both experiments. Collected blood was allowed to stand until clotted and then centrifuged. Serum

270 0016-6480187 $1.50 Copyright 8 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

HYPOTHYROIDISM AND CELL-MEDIATED IMMUNITY 271

samples were assayed for 3,5,3’-triiodothyronine (T,) and thyroxine (I.,) in experiments 1 and 2 and for cor- ticosterone (B) in experiment 2. The procedure de- scribed by May (1978) was used to determine serum T, concentrations, and commercially available reagents (Antibodies, Inc., Davis, CA) were employed to assay the concentrations of serum T,. Etches’ (1976) radio- immunoassay procedure for corticosterone was fol- lowed to determine serum concentrations of B.

White blood cell determination. Peripheral blood was obtained from the cutanea ulnaris of chicks from both treatments at 6 weeks of age. The blood samples were diluted with Natt-Herrick (1952) stain for total counts, and blood smears were stained with Mac- Neal’s (1922) tetrachrome stain for relative counts of mononuclear and granulocytic cells.

Culture medium. Medium RPM1 1640 with L-gluta- mine and without sodium bicarbonate (GIBCO Labo- ratories, Grand Island, NY) was supplemented with 100 units/ml penicillin, 100 &ml streptomycin, 0.25 &ml fungizone, 1 rr& L-glutamine, and 0.2% sodium bicarbonate and used as cell culture medium for lym- phocyte blast transformation microassays. The pH was adjusted to 6.7-6.9 with a 5% sodium bicarbonate solution.

Collection and preparation of peripheral blood leu- kocytes (PBL). Heparinized blood was collected from the cutanea ulnaris of 4-, 6-, and I-week-old chickens. Blood samples (3 ml) then were centrifuged at 5Og for 10 to 12 min at 4”. The buffy coat supernatant was removed and washed twice with either supplemented RPM1 1640 or sterile phosphate-buffered saline (PBS, pH 7.0). Mononuclear cells were enumerated by trypan blue exclusion, and viable cells were adjusted to a concentration of 1 x 10’ cells/ml with RPM1 1640 for blasting or PBS for graft vs host response.

Lymphocyte blast transformation microassay. One- hundred microliters of peripheral blood leukocytes (PBL) suspended in supplemented RPM1 1640 (1 x

10’ cells/ml) was added to the individual wells of flat- bottom microculture plates (96-well MicroTest III tissue culture plate with lid, Becton-Dickinson, Ox- nard, CA). To triplicate sets of wells was added 100 ~1 of 500 ug/ml phytohemagglutinin M (PHA-M, Difco Laboratories, Detroit, MI) which had been reconsti- tuted with supplemented RPM1 1640. Plates were in- cubated for 52 br in a humidified CO, (5%) incubator at 40”. After this period of incubation, 100 ~1 of RPM1 1640 containing 1 &i of [3H]thymidine (sp act 6.7 Ci/ mmol; New England Nuclear, Boston, MA) was added and the cultures were allowed to incubate an additional 16 hr. Cell cultures were harvested onto glass fiber filters with a multiple sample harvester (Mash II, Microbiological Associates, Walkersville, MD) and were washed repeatedly with distilled water. Filters were dried and placed in 10 ml scintillation fluid, and the radioactivity was measured in a liquid

scintillation counter (Beckman Instruments, Ful- lerton, CA).

Graft vs host response (GvH). The GvH was per- formed as described previously (Glick et al., 1983). Briefly, PBL suspended in sterile PBS (1 x 10’ cells/ ml) were injected (0.1 ml) into the vein of a 12-day-old White Leghorn embryo; 8 to 10 embryos were injected with each PBL suspension, and 8 to 10 embryos, each injected with 0.1 ml PBS, served as controls. Embryos were sacrificed 6 days after injection and body and spleen weights were recorded. A splenic index was calculated by dividing the spleen to body weight ratio of each PBL-injected embryo by the mean spleen to body weight ratio of the PBS-injected group.

Statistical analysis. Statistical analyses were per- formed using t tests. All statements of significance are based on a probability of 0.05.

RESULTS

At 4,6, and 8 weeks of age, body weights of thiouracil-fed (TF) chicks were lower than those of controls (Tables 1 and 2). Thymus weights were reduced by feeding a thiouracil diet (Table 1). Upon examining glands from both experimental groups, we observed obvious differences in the appear- ance of the thymi. Control thymi were pink in color and exhibited definite lobes which filled the neck spaces adjacent to the jug- ular veins. The thymi of TF chicks were dark red, thin strands of tissue buried in adipose tissue and possessed little sign of lobulation.

The goitrogenic effect of thiouracil on the thyroid was evident at all ages (Table 3). The thyroid glands of TF chicks were greater in weight. During experiment 1, it was observed that the relative size of the adrenal glands of TF chicks appeared to be larger than those of controls. This observa- tion was made again in experiment 2, and measurement of adrenal weights verified this finding (Table 2).

Thiouracil treatment was much more ef- fective in lowering circulating levels of T, than those of T3 (Table 3). The serum con- centrations of T, in controls were approxi- mately 2.6 times greater than those in TF chicks. However, the T3 concentrations of controls were about 1.3 times greater. Fol-

272 SCOTT AND GLICK

TABLE 1 BODY(~)ANDRELATIVETHYMUS WEICHTS(~~/~)OF~-ANDY-WEEK-OLDCHICKSFEDCONTROLOR

THIOURACIL DIETS

Control

4 Weeks 8 Weeks

Thiouracil Control Thiouracil

Body 508.9 + 23.2" 318.8 k 20.0b 1406.3 t 66.4" 701.9 k 60.0b Thymus’ 1.44 + 0.13” 0.41 2 0.06b 1.10 ” 0.07” 0.90 2 0.13”

a~b Diet means within ages with different superscripts are significantly different at P c 0.05 c Left thymus.

lowing the initial observations of larger adrenal size in TF chicks, it was decided that serum levels of B should be deter- mined for chicks in experiment 2. There- fore, at the time of blood collection, care was taken not to introduce any stressor ef- fects (i.e., excessive handling). In doing so, we were able to measure the resting levels of serum B in both treatment groups. The values in Table 2 indicate that TF chicks had significantly higher serum levels of B.

Total white blood cell counts were higher in controls compared with those in TF chicks (Table 4). Of the major leukocytes in circulation, the number of lymphocytes was reduced with thiouracil treatment, but heterophil counts were equivalent between control and TF groups. Treatment differ- ences were not observed for the number of monocytes, eosinophils, and basophils.

The mitogenic responses of PBL to PHA-M are found in Table 5. Control PBL responded better to mitogenic challenge.

TABLE 2 BODY(~) ANDRELATIVE ADRENAL WEIGHTS(~~/~)

ANDSERUMCORTICOSTERONE(B)CONCENTRATIONS (ng/ml)O~6-WEEK-OLDCHICKSFEDCONTROLOR

THIOURACILDIETS

Control Thiouracil

Body 1282.6 + 44.9” 464.1 ‘- 63.66 Adrenalc 0.03 _' O.oob 0.09 2 0.01" B 2.95 f 0.41b 5.89 " 0.78"

a*b Diet means with different superscripts are signiti- cantly different at P s 0.05.

e Left adrenal gland.

Although the mitogen response was statis- tically different only for 4-week-old chicks in experiment 1, control PBL had consis- tently greater responsiveness to PHA-M in both experiments. Unlike the mitogenic re- sponses, PBL from TF chicks gave a slightly better GvH (Table 5). This higher response from TF chicks’ PBL was also consistent for the two experiments.

DISCUSSION

Feeding a 0.1% thiouracil diet reduced the size of the thymus. This agrees with previous reports on thioureylene anti- thyroid treatment of birds (Jackson and La- bisky, 1971; Yam et al., 1981). Thyroidec- tomized rats exhibited reduced thickness of the thymic cortex (Fabris, 1973). Yam et al.

TABLE 3 RELATIVETHYROIDWEIGHTS(~~/~)ANDSERUM

TRIIODOTHYRONINE (T,) ANDTHYROXINE CONCENTRATIONS (&ml) OFCHICKSFEDCONTROL

ORTHIOURACIL DIETS

Control Thiouracil

Experiment 1 Thyroid (4 weeks) 0.05 k O.Olb 1.34 + 0.22” Thyroid (8 weeks) 0.07 t O.oob 1.10 k 0.19" T, (6 weeks) 2.48 f 0.07" 1.96 -c 0.409 T4 (6 weeks) 23.65 + 2.13" 8.91 t 0.57b

Experiment 2 Thyroid (6 weeks) 0.08 2 O.Olb 0.96 f 0.19 T, (6 weeks) 3.01 2 0.10” 2.10 * 0.306 T4(6 weeks) 23.93 f 2.02" 9.49 f 0.74b

o.b Diet means within experiments with different su- perscripts are significantly different at P s 0.05.

HYPOTHYROIDISM AND CELL-MEDIATED IMMUNITY 273

TABLE 4 TOTAL WHITE BLOOD CELLS (TWBC), ABSOLUTE

LYMPHOCYTES (AL), AND ABSOLUTE HETEROPHILS (AH) OF ~-WEEK-OLD CHICKS FED CONTROL OR

THIOURACIL DIETS

TWBC x 103/mm3 AL x 103/mm3 AH x 103/mm3

Control Thiouracil

37.8 f 2.9“ 26.7 f 2.1b 25.4 f 2.2” 13.9 f 1.76 8.0 f 1.4” 8.8 * 0.9”

a,b Diet means with different superscripts are signifi- cantly different at P s 0.05.

(1981), however, observed enlargement of the cortex and decreased medulla size in thymi from propylthiouracil (PTU)-treated chickens. Thymi of our TF chicks were found to have reduced cortical areas and poor cellularity of both cortex and medulla. Furthermore, the number of observable mi- totic figures in cortices was greater for con- trol chicks, while TF chicks had more Has- sail’s corpuscles in their medulla (un- published observation). According to Ken- dall’s review (1980), a drop in the number of cortical mitotic figures and the appear- ance of Hassall’s corpuscles in the medulla have been used as indicators of a regressing thymus. Therefore, our TF chicks experi-

TABLE 5 PHYTOHEMAGGLUTININ M (PHA-M) PROLIFERATION

RESWNSES (INCORPORATION OF [3H]T~~~~~~; cpm x 10-3) AND GRAFT vs HOST (GvH)

POTENTIALS (EMBRYONIC SPLENOMEGALY; SPLENIC INDEX) OF PERIPHERAL BLOOD LEUK~CVTES ( lo6

CELLS) FROM CHICKS FED CONTROL OR THIOUEUCIL DIETS

Control Thiouracil

PHA-M 4 weeks 8 weeks

Experiment 1 81.9(0.8) t 12.2” 57.4 (1.0) f 20.46 77.8 (3.0) f 20.7” 64.4 (4.2) k 15.6”

GvH 6 weeks 5.95 f 0.71n 7.01 k 1.09”

PHA-M Experiment 2 4 weeks 188.0 (1.0) 2 25.8” 159.9 (0.8) f 40.0”

GvH 4 weeks 5.53 k 0.90” 6.04 + 0.84”

Note. PHA-M, 50 pg/well. Means of cells treated with 0 &well PHA-M are in parentheses.

o-* Diet means within experiments with different super- scripts are significantly different at P c 0.05.

enced thymic regression at an earlier age than did controls.

The goitrogenic effect on the thyroid glands was observed and the serum con- centrations of thyroid hormones were lower in TF chicks. The latter was in agreement with May (1978) and Yam et al. (1981). Rudas and Pethes (1984) showed that short-term administration of PTU blocked the peripheral conversion of T, to T, in chickens. Furthermore, PTU and 2- thiouracil both were effective in inhibiting the activity of type I 5’-monodeiodinase in quail liver homogenates (McNabb et al., 1986). Therefore, the lower serum thyroid hormone levels of our TF chicks would be explained by the action of thiouracil at the level of the thyroid gland and the peripheral tissues (e.g., liver).

Relative weights of adrenal glands and serum concentrations of B were greater in TF chicks. Based on these results, it would appear that thiouracil treatment produced a stressful state. Jackson and Labisky (1971) found the absolute weights of adrenals from thiouracil-treated pheasants to be equivalent to those from controls. The adrenal glands from our treatment groups were also very similar in weight (36.4 + 3.3 mg, control; 36.6 ? 2.9 mg, TF). The ability to produce and release B could have been the same for control and TF chicks, but the smaller cardiovascular space of TF chicks, which had smaller body size than did controls, may have resulted in greater serum concentrations of B.

With the higher concentrations of B cir- culating in TF chicks, one might expect to see a slightly elevated total white blood cell count, relatively fewer numbers of lympho- cytes, and higher numbers of heterophils (Glick, 1958, 1961). Thiouracil treatment did not produce this type of white blood cell profile. In TF chicks, total counts and absolute numbers of lymphocytes were lower. Heterophil counts were similar for control and TF chicks. Chattejee and Chandel (1973) both reported that thy-

274 SCOTT AND GLICK

roidectomy of rats resulted in decreased numbers of total white cells and lympho- cytes. The lowering of lymphocyte numbers would seem to reflect the reduc- tion in size (cellularity) of the lymphoid tissues. Absolute eosinophil and basophil counts, like heterophils, were unchanged by thiouracil treatment in our study. The reduced total white blood count of thy- roidectomized rats was due to lower lym- phocyte numbers, since thyroidectomy did not affect the numbers of granulocytes (Fabris, 1973).

We have demonstrated previously that the kinetics of the primary and secondary humoral immune responses to sheep red blood cells were changed by thiouracil-in- duced hypothyroidism (Scott et al., 1985), while the overall antibody production of TF chicks was similar to that of controls. In the present study, indicators of cell-me- diated immune responsiveness were tested. The mitogen, PHA-M, was used to activate T cells to synthesize DNA and proliferate. Since the rate of antibody production was reduced in our previous study by thiouracil treatment, it was believed that T-cell acti- vation by a mitogen would be diminished in TF chicks. The GvH test was chosen be- cause it would determine whether the func- tional ability of T cells to recognize foreign cells would be altered by thiouracil treat- ment.

The T-cell response to a mitogen such as PHA-M leads to the generation of T-cell help, T-suppressor cells, and cytotoxic ef- fector cells (Clark, 1983). The response has been characterized as entry into the cell cycle and proliferation. The PHA-M re- sponse of PBL was lower for TF chicks at two ages and, therefore, the expansion of T-cell subpopulations occurred slowly for TF chicks. Mitogenic responsiveness of spleen and thymus cells to concanavalin A was reduced in young chickens fed a diet containing PTU (Yam et al., 1981). In studies with thyroidectomized rats, cells from the spleen, thymus, lymph nodes, and

peripheral blood also responded less to PHA in vitro (Chatterjee and Chandel, 1983; Fabris, 1973). The diminished incor- poration of [3H]thymidine by PHA-M- treated PBL from our TF chicks suggested that a normal thyroid state may be neces- sary for the synthetic phase of the cell cycle. The ability of cultured human lym- phocytes to take up and concentrate thy- roid hormones (Holm et al., 1980) was in- dicative of a significant biological role for these hormones in lymphocyte physiology.

In chick embryos, the GvH or spleno- megaly response has been shown to be brought about by donor immunocompetent cells acting primarily as stimulators to cause massive host cell proliferation (Biggs and Payne, 1959; Mun et al., 1962; Mun and Burns, 1964; Seto and Albright, 1965; Weber, 1970). It generally is accepted that donor T cells release various lymphokines upon antigenic stimulation and some of the factors stimulate host cells to proliferate (Clark, 1983). The GvH response of PBL from our chicks was not changed adversely by thiouracil treatment. In fact, we ob- served a slightly greater GvH response from our TF chicks. T-cell subpopulations may have been altered in the blood of our TF chicks such that a greater percentage of T cells involved in GvH were present in the PBL cell suspensions. Pacini et al. (1983) reported that thyroidectomy and PTU treatment of rats did not change the total number of T cells in the blood. However, the blood ratio of T-helper to T-suppressor (nonhelper cells as identified by a mono- clonal antibody) cells was decreased in the hypothyroid rats. A change in blood T-cell subpopulation numbers of TF chicks may explain our results, or the GvH ability of these cells was unchanged by thiouracil treatment.

In this study, consideration should be made concerning the possible infhrence of elevated corticosterone levels of TF chicks on the T-cell-involved responses. Diver- gent lines of chickens selected for plasma

HYPOTHYROIDISM AND CELL-MEDIATED IMMUNITY 275

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ACKNOWLEDGMENTS

We thank Janice Orr and Lillian Lappie for typing the manuscript and G. Todd Pharr, Joyce Hood, and Doris Thompson for their laboratory assistance. J. David May kindly provided the reagents for the thy- roid hormone radioimmunoassays. The corticosterone antiserum was a gift of R. J. Etches. This is Journal Article No. 6570 from the Mississippi Agricultural and Forestry Experiment Station.

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