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Relationship between circulating thyroid hormones and humoral immunity in immature male chickens
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. I0, pp. 395-403, 0145-305X86 $3.00 + .00 Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd. All rights reserved.
1986.
RELATIONSHIP BETWEEN CIRCULATING THYROID HORMONES AND HUMORAL IMMUNITY IN IMMATURE MALE CHICKENS I
Suzanne E. Bachman and Magdi M. Mashaly 2 Department of Poultry Science The Pennsylvania State University University Park, PA 16802
ABSTRACT The objective of this experiment was to examine the relationship between levels of circulating T3, T4, and humoral immunity in immature male chickens. Three week old Single Comb White Leghorn male chicks were used as the experimental animals. In order to produce a wide range of circulating thyroid hormone concentrations, birds were divided into groups and received one of nine treatments including surgical thyroidectomy; 0.I~ propylthiouracil (PTU) in the feed; i ppm T 3 and I0 ppm T 4 in the feed. Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (thymus-independent antigen) was tested at 6 weeks of age. Concentrations of T 3 and T 4 were measured in birds from each treatment group at 7 and ii weeks of age. At ii weeks of age, birds were weighed, sacrificed and lymphoid organs removed and weighed. There were positive correlations between circulating thyroid hormones and weights of bursa of Fabricius and spleens. There were no significant correlations between circulating thyroid hormones and antibody production. It was concluded that physiological levels of thyroid hormones are needed to maintain normal weights of bursa and spleen. Furthermore, we conclude that lower than physiological levels might be sufficient for normal antibody production. Finally, stimulation of antibody production using thyroid hormones may require different doses than what were utilized in this study.
ipaper No. 7340 in the Journal Series of the Pennsylvania Agricultural Experiment
Station.
2To whom correspondence
should be addressed to:
Dr. Magdi M. Mashaly 202 W. L. Henning Building University Park, PA 16802 395
396
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INTRODUCTION The relationship between the endocrine and immune systems has been the subject of much research in current years and has been reviewed recently (5,6). Humoral immunity or antibody production is an important part of the immune system which is essential in maintaining normal function and adaptation in an organism. Bursa of Fabricius is a lymphoid organ which plays an important role in antibody production by c~ickens (4). The thyroid gland is known to have significant effects upon different physiological parameters in birds (14). There has been increased interest in studying the effects of the thyroid gland and its hormones on the bursa of Fabricius of birds and on antibody production in avian as well as in mammalian species. Hypothyroidism resulted in reduced bursal weights (16) and in smaller bursal follicles (18). Antibody production was either decreased (7) or inconsistently affected (Ii) in hypothyroid birds. Thyroxine enhanced antibody production in mice (2) and in rats (3), however, it had no effect in chickens (i0). In previous studies related to the effect of the thyroid gland and thyroid hormones on humoral immunity, the correlation between the levels of circulating thyroid hormones and antibody production was not evaluated. This information is important in order to better understand the physiological as well as pharmacological effects of thyroid hormones on humoral immunity. Therefore, this study was conducted to determine the relationship between circulating T 3 and T 4 concentrations and humoral immunity in immature male chickens.
MATERIAL AND METHODS Animals. Single Comb White Leghorn male chicks were used in this study. The chicks were housed in batteries and maintained on food and water a d libitum. They were exposed to 14 hours of light and i0 hours of darkness (lights on between 0600-2000 hours). Treatment. At three weeks of age, the birds were randomly divided into nine different groups with 20 birds in each group which received different treatments in order to produce a wide range of circulating T 3 and T 4 concentrations. These treatment groups included: i. Control group (C). 2. Sham-operated group (S). 3. A group that was surgically thyroidectomized (Tx) under a general inhalation anesthesia (Halothane) for 1-2 minutes and then under local anesthesia (2% Lidocaine). 4. Chemically thyroidectomized (PTU) group by supplementing the feed with 0.1% propylthiouracil (Sigma, St. Louis, MO). 5. T x + PTU group. 6. A group which was surgically thyroidectomized and the feed was supplemented by i ppm T 3 (Sigma, St. Louis, MO) (T x + T3). 7. A group which was surgically thyroidectomized group and the feed was supplemented by i0 ppm D,L-thyroxine (T x + T4). 8. A group that received i ppm T 3 in their feed (T3). 9. A group that received I0 ppm T 4 in their feed (T4). The birds were weighed and sacrificed at ii weeks of age when the experiment was terminated. The bursa of Fabricius, spleen, and thyroid glands were removed and weighed. Thyroidectomized birds were examined for the presence of residual thyroidal tissue. Hormone assays. Serum concentration of T 3 and T 4 were measured using double antibody radioimmunoassay kits purchased from Antibodies Incorporated (Davis, California). The antibodies were tested by the
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company and found not to crossreact with any of the other iodothyronines. The serum samples were obtained from all birds at 7 and II weeks of age. Humoral immunity. Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (dead microorganisms - Difco, Detroit, MI, U.S.A.) (thymus-independent antigen) was tested at 6 weeks of age. Ten chicks from each of the treatment groups were immunized intravenously with i ml of saline suspension of SRBC (5%) and another i0 chicks with 0.i ml of BA solution. These antigen doses are optimal for antibody production. Seven days following the antigen challenge, blood samples were collected and sera were frozen for later use in measuring the primary response. To measure the secondary response, the same antigens were injected 4 weeks following the first challenge, and birds were bled 7 days later. Complement in all serum samples was inactivated by incubation at 56oc for 30 minutes before measuring the antibody titer. Total as well as ME-resistant (IgG) and ME-senstiive (IgM) antibodies were measured for both primary and secondary responses by an agglutination tests using the microtiter technique described previously
(1). Statistical Analysis. Data were analyzed using one way analysis of variance to determine if there was a difference between treatment groups. Significant differences between treatment means were analyzed using Duncan's multiple range test.
RESULTS Since the results for organ weights as well as serum hormone concentrations were similar for the two groups of birds that were injected with either SRBC or BA, the data presented here are only for the SRBC group. Thyroid and body weights. Data for thyroid and body weights of all treatment groups at ii weeks of age are shown in Table I. Tx, TABLE I Body and relative thyroid weight of ii week old male chickens in all treatment groups injected with sheep red blood cells.
Treatment Group* Control Sham Thyroidectomy (Tx) Thiouracil (PTU) (0.1%) T x + PTU T X + T 3 (i ppm) T X + T 4 (I0 ppm) T 3 (i ppm) T 4 (i0 ppm)
Body Weight (gm) 1164.10 1154.50 898.44 781.70 680.90 1092.20 1153.82 1152.67 1134.73
+ ± ± ± ± ± i ± ±
43.67 a 49.01 a 54.80 b 34.19 c 25.75 c 55.70 a 33.88a 33.37 a 32.39 a
Relative Thyroid Weight (mg/100 gm BW) 8.24 + 0.58 b 8.83 ± 1.38 b --46.56 ± 3.62 a ------1.29 ± 0.25 c 1.22 ± 0.22 c
* Values are mean + S.E. a,b,c Means in the same column having different superscripts are statistically different (P<0.05) PTU, and T X + PTU birds had significantly (P
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Thyroid hormone replacement in groups T x + T 3 and T x + T 4 increased body weights to values comparable to the controls. To verify the absence of thyroid tissue in the T x groups, the birds were sacrificed and autopsied. No thyroid tissue was present in any of the T x birds. Thyroid gland enlargement in PTU birds were apparent (Table I). Birds in the T 3 and T 4 groups had significantly (P<0.05) smaller thyroids than control groups (Table i). Furthermore, there were negative correlations between circulating levels of T3, T4, and relative thyroid weights (Table 3), however, the correlation was significant (P<0.05) only for T 4 . Hormone concentrations. Concentrations of serum T 3 and T 4 for all treatment groups at 7 and Ii weeks of age are shown in Figure I. It is apparent that treatments have resulted in producing a wide range of circulating T 3 and T4, which was the intention of using these treatments. At 7 weeks of age, serum concentrations ranged from 3.62-11.58 ng/ml and from 10.50-373.51 ng/ml for T 3 and T4, respectively. Similar ranges were also found at Ii weeks of age (Figure i). Lymphoid organ weights. Bursa of Fabric±us and spleen weights of all treatment groups at II weeks of age are shown in Table 2. Bursa and spleen relative weights were significantly (P<0.05) reduced in Tx, PTU and T X + PTU birds. Hormone replacement increased these weights to values comparable to controls (Table 2). Birds in T 3 or T 4 groups TABLE 2 Relative bursa and spleen weight of ii week old male chickens all treatment injected groups with sheep red blood cells.
Treatment Groups* Control Sham Thyroidectomy (Tx) Thiouracil (PTU) (0.1%) T x + PTU (0.1%) T x + T 3 (i ppm) T x + T 4 (i0 ppm) T 3 (i ppm) T 4 (i0 ppm)
Relative Bursa Weight (mg/100 g BW) 370.22 378.50 275.11 137.91 143.75 395.79 375.90 350.69 352.75
± + ± ± ± ± ± ± ±
32.54 ab 30.23 ab 42.58 b 16.64 c 15.10 c 64.26 a 31.88 ab 31.91 ab 36.92 ab
*Values are mean ± S.E. a,b Means in the same column having different superscripts statistically different (P<0.05).
in
Relative Spleen Weight (mg/100 g BW) 264 227 206 127 145 316 280 246 261
16 33 71 93 77 42 69 ii 16
+ + + ± + + + + +
19.59 abc 16 24 bc 27 56 cd 8 93 e 9 76 de 55 91 a 30 00 ab 12 01 bc 16 94 abc
are
did not have larger bursa or spleens than did birds in the control groups. However, there were significant (P<0.05) correlations between circulating T3, T4, and both lymphoid organ weights (Table 3). Humoral immunity. Correlation coefficients between circulating T3, T 4 at 7 weeks of age and antibodies of the primary response against BA and SRBC are shown in Table 4. There were no significant correlations between concentrations of T 3 or T 4 and antibodies against BA. Similar results were found between T 4 ~nd antibodies against SRBC. On the other hand, there were significant negative correlations between T 3 and total as well as IgM
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T 3 CONCENTRATIONS AT
T 4 CONCENTRATIONS AT
7 WEEKS OF AGE
7 WEEKS OF AGE
ng/ml n,l,~
1.5 400 B
E
E
30(
10
,i~, D
BC
D
200
N'I i', :<
V.
lOC A PTU Tx
Tx ,4-
C
S
T4
PTU
Tx
T3
T4
Tx •
A
A
A
PTU T3
Tx
T3
A
A
A
Tx
Tx
C
S
T3
PTU
,I
Tx
T4
T4
TREATMENT TREATMENT
T 3 CONCENTRATIONS AT
T 4 CONCENTRATIONS AT
ii WEEKS OF AGE
Ii WEEKS OF AGE ng/ml
nglml
300j lO
E
8
C
200
B
6
• AB
i
AB ~ ~
4
100.
0 T4
~x T4
PTU
TX
S
Tx
PTU TR.E~TMENT
C
T3
Tx
T~
Tx + T3
T3
Tx
Tx
PTU
$
PTU
C
T4
Tx T4
TREATMENT
FIGURE i. Concentrations (ng/ml) of serum T 3 and T 4 at 7 and ii weeks of age of male chickens in all treatment groups injected with sheep red blood cells. Within each age and hormone, treatments having different letters are statistically different (P
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TABLE 3 Correlation coefficients between levels of circulating T3, T 4 at Ii weeks of age and organ weights of male chickens of all treatment groups injected with sheep red blood cells. Organ
T3
Body Weight Thyroid Weight Relative Bursa Weight Relative Spleen Weight
+0.185 -0.075 +0.252 +0.262
T4 (NS) (NS) (P<0.05) (P<0.05)
0.307 -0.249 +0.273 +0.271
(P
TABLE 4 Correlation coefficients between circulating T3, T 4 at 7 weeks of age and the antibodies of the primary response against Brucella abortus (BA) and sheep red blood cells (SRBC). BA
SRBC
Antibody
Total IgG IgM
T3
T4
T3
T4
+0.080 (NS) -0.039 (NS) +0.105 (NS)
-0.172 (NS) -0.016 (NS) -0.155 (NS)
-0.216 (P<0.05) -0.041 (NS) -0.264 (P<0.05)
-0.023 (NS) +0.058 (NS) -0.078 (NS)
antibodies against SRBC. As for the secondary response (Table 5), there were no significant correlations between T 3 and T 4 and antibodies against SRBC. However, T 4 and not T 3 was positively (P
DISCUSSION Birds in Tx, PTU and T x + PTU groups displayed body weights and thyroid status which are characteristic of hypothyroidism. It has been TABLE 5 Correlation coefficients between circulating T3, T 4 at ii weeks of age and antibodies of the secondary response against Brucella abortus (BA) and sheep red blood cells (SRBC). BA
SRBC
Antibody
Total IgG IgM
T3
T4
T3
T4
+0.146 (NS) +0.175 (NS) -0.057 (NS)
+0.219 (P<0.05) +0.358 (P<0.05) -0.176 (NS)
-0.054 (NS) +0.036 (NS) -0.077 (NS)
+0.168 (NS) +0.089 (NS) +0.059 (NS)
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previously reported that surgical thyroidectomy (11,17) or goiterogen treatment (11,17,18) results in reduced body weight. The data of this experiment demonstrates that PTU is more effective in reducing body weight than surgical thyroidectomy and when both methods are applied, body weight is reduced further. Birds in PTU group possessed enlarged thyroids which is a characteristic of goiterogen treated birds (7,11). Even though these treatments produced a wide range of circulating T 3 and T 4 concentrations, the lowest values, which were found in Tx, PTU and T x + PTU birds, still represented a considerable amount of hormone. T 3 and T 4 have been detected previously in thyroidectomized birds despite the absence of thyroidal tissue (8,11,13). In the present study, bursa and spleen weights were reduced in Tx, PTU and T x + PTU birds. Furthermore, there were positive correlations between circulating T3, T4, and bursa and spleen weights. These results indicate that thyroid gland and thyroid hormones are essential for maintaining normal weights of these lymphoid organs. It was found previously that hypothyroidism resulted in hypoplasia of these lymphoid organs (3,15). Using 250 ppm T4, Yam e t al. (18) found enhancement in chicken spleen weight. In the present study there was no increase in lymphoid organ weights when I0 ppm T 4 was used. It is possible, therefore, that higher doses of T 3 and T 4 than what were used in this study are needed in order to stimulate lymphoid organ weights. The results of the present study did not demonstrate any consistent relationships between circulating T 3 and T 4 concentrations and antibody production against both thymus-dependent and thymus-independent antigens. This is in agreement with our previous finding (ii). One possible explanation is that although hormone concentrations were lower in hypothyroid groups, the amount of hormone present was adequate for maintaining normal antibody production. It has been demonstrated that the number of lymphocyte receptors for T 3 and T 4 are increased in hypothyroidism but remains unchanged in hyperthyroidism (9). Thus, while the hormone levels were lower in hypothyroid groups, lymphocytes may be able to better utilize T 3 and T 4. Yam et al. (18) found that thyroid hormones stimulated antibody production in chickens. Our present results do not confirm the findings by Yam et al. (18). This could be due to the use of different doses of thyroid hormones. It has been shown that the effect of T 3 and T 4 on antibody production may be dose dependent (2,12). Therefore, it is conceivable that the stimulatory effect of thyroid hormones on antibody production occurs at either lower or higher levels than what were used in the present study. In conclusion, we suggest that physiological levels of thyroid hormones are needed to maintain normal weights of bursa and spleen. Furthermore, we conclude that lower than physiological levels might be sufficient for normal antibody production. Finally, stimulation of antibody production using thyroid hormones may require different doses than what were utilized in this study.
ACKNOWLEDGEMENTS The authors are indebted to Dr. Robert F. Wideman, Jr. for his technical assistance in the surgical procedures, Ms. Jeannette M. Bachman for her technical assistance in hormone assay and statistical procedures, and Ms. Dee Ann Longenecker for her expert secretarial assistance.
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REFERENCES
i.
Benedict, A. A., R. J. Brown, and R. Hersh. Inactivation of high and low molecular weight chicken antibodies by mercaptoethanol. Proc. Soc. Exp. Biol. and Med., 113, 136-138 (1963).
2.
Chen, Y. Effect of thyroxine on the immune response of mice in vivo and in vitro. Immunol. Comm., 9, 269-276 (1980).
3.
Fabris, N. Immunodepression in thyroid deprived animals. Immunol., 15, 601-611 (1973).
4.
Glick, B. The bursa of Fabricius: 602-604 (1970).
5.
Glick, B. Interrelation of avian immune and neuroendocrine systems. J. Exp. Zool., 232, 671-682 (1984).
6.
Grossman, C. J. Regulation of the immune system by sex steroids. Endo. Rev., 5, 435-455 (1984).
7.
Keast, D. and D. J. Ayre. Antibody regulation in birds by thyroid hormones. Dev. Comp. Immunol., 4, 323-330 (1980).
8.
Klandorf, H., P. J. Sharp, and M. G. Macleod. The relationship between heat production and concentrations of plasma thyroid hormones in the domestic hen. Gen. Comp. Endo., 45, 513-520 (1981).
9.
Lemarchand-Beraud, T., A. C• Holm, and B. R. Scazziga. Triiodothyronine and thyroxine nuclear receptors in lymphocytes from normal, hyper- and hypothyroid subjects. Acta. Endocrinol., 85, 44-54 (1977).
i0.
Marsh, J. Assessment of antibody production in sex-linked and autosomal dwarf chickens. Dev. Comp. Immunol., 7, 535-544 (1983).
Ii.
Mashaly, M. M., S. L. Youtz, and R. F. Wideman, Jr. Hypothyroidism and antibody production in immature male chickens. Immunol. Comm., 12, 551-563 (1983).
12.
Paavonen, T. Enhancement of human E-lymphocyte differentiation i_nn vitro by thyroid hormone. Scand. J. Immunol., 15, 211-215 (1982).
13.
Peczely, P., G. Pethes, and P. Rudas. Interrelationship between thyroid and gonadal function in female Japanese quail kept under short and long photoperiods. J. Endocrinol., 87, 55-63 (1980).
14
Ringer, R. K. and D. C. Meyer. Thyroids, in Avian Physiology, P. D. Sturkie, ed., Springer-Verlag, New York, (1976) pp. 348-358.
15
Snedecor, J. G. Liver hypertrophy, liver glycogen accumulation and organ-weight changes in radiothyroidectomized and goiterogen-treated chicks. Gen. Comp. Endocrinol., i0, 277-291 (1968).
16.
Snedecor, J. G. Responses of normal and goiterogen-fed cockerels to different environmental temperatures. Poultry Sci., 50, 237-243 (1971).
A central issue.
Clin. Exp.
BioScience 20,
Vol. I0, No. 3
17.
18.
THYROID HORMONES AND IMMUNITY IN CHICKENS
403
Snedecor, J. G. and M. F. Comyre. Interaction of thyroid hormone and androgen on body weight, comb, and liver in cockerels. Gen. Comp. Endocrinol., 6, 276-287 (1966).
Yam, D., D. Heller, and N. Snapir. The effect of the thyroidal state on the immunological state of the chicken. Dev. Comp° Immunol., 5, 483-490 (1981). Received: March, 1986 Accepted: July, 1986
1986.
RELATIONSHIP BETWEEN CIRCULATING THYROID HORMONES AND HUMORAL IMMUNITY IN IMMATURE MALE CHICKENS I
Suzanne E. Bachman and Magdi M. Mashaly 2 Department of Poultry Science The Pennsylvania State University University Park, PA 16802
ABSTRACT The objective of this experiment was to examine the relationship between levels of circulating T3, T4, and humoral immunity in immature male chickens. Three week old Single Comb White Leghorn male chicks were used as the experimental animals. In order to produce a wide range of circulating thyroid hormone concentrations, birds were divided into groups and received one of nine treatments including surgical thyroidectomy; 0.I~ propylthiouracil (PTU) in the feed; i ppm T 3 and I0 ppm T 4 in the feed. Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (thymus-independent antigen) was tested at 6 weeks of age. Concentrations of T 3 and T 4 were measured in birds from each treatment group at 7 and ii weeks of age. At ii weeks of age, birds were weighed, sacrificed and lymphoid organs removed and weighed. There were positive correlations between circulating thyroid hormones and weights of bursa of Fabricius and spleens. There were no significant correlations between circulating thyroid hormones and antibody production. It was concluded that physiological levels of thyroid hormones are needed to maintain normal weights of bursa and spleen. Furthermore, we conclude that lower than physiological levels might be sufficient for normal antibody production. Finally, stimulation of antibody production using thyroid hormones may require different doses than what were utilized in this study.
ipaper No. 7340 in the Journal Series of the Pennsylvania Agricultural Experiment
Station.
2To whom correspondence
should be addressed to:
Dr. Magdi M. Mashaly 202 W. L. Henning Building University Park, PA 16802 395
396
THYROID HORMONES AND IMMUNITY IN CHICKENS
Vol.
i0, No. 3
INTRODUCTION The relationship between the endocrine and immune systems has been the subject of much research in current years and has been reviewed recently (5,6). Humoral immunity or antibody production is an important part of the immune system which is essential in maintaining normal function and adaptation in an organism. Bursa of Fabricius is a lymphoid organ which plays an important role in antibody production by c~ickens (4). The thyroid gland is known to have significant effects upon different physiological parameters in birds (14). There has been increased interest in studying the effects of the thyroid gland and its hormones on the bursa of Fabricius of birds and on antibody production in avian as well as in mammalian species. Hypothyroidism resulted in reduced bursal weights (16) and in smaller bursal follicles (18). Antibody production was either decreased (7) or inconsistently affected (Ii) in hypothyroid birds. Thyroxine enhanced antibody production in mice (2) and in rats (3), however, it had no effect in chickens (i0). In previous studies related to the effect of the thyroid gland and thyroid hormones on humoral immunity, the correlation between the levels of circulating thyroid hormones and antibody production was not evaluated. This information is important in order to better understand the physiological as well as pharmacological effects of thyroid hormones on humoral immunity. Therefore, this study was conducted to determine the relationship between circulating T 3 and T 4 concentrations and humoral immunity in immature male chickens.
MATERIAL AND METHODS Animals. Single Comb White Leghorn male chicks were used in this study. The chicks were housed in batteries and maintained on food and water a d libitum. They were exposed to 14 hours of light and i0 hours of darkness (lights on between 0600-2000 hours). Treatment. At three weeks of age, the birds were randomly divided into nine different groups with 20 birds in each group which received different treatments in order to produce a wide range of circulating T 3 and T 4 concentrations. These treatment groups included: i. Control group (C). 2. Sham-operated group (S). 3. A group that was surgically thyroidectomized (Tx) under a general inhalation anesthesia (Halothane) for 1-2 minutes and then under local anesthesia (2% Lidocaine). 4. Chemically thyroidectomized (PTU) group by supplementing the feed with 0.1% propylthiouracil (Sigma, St. Louis, MO). 5. T x + PTU group. 6. A group which was surgically thyroidectomized and the feed was supplemented by i ppm T 3 (Sigma, St. Louis, MO) (T x + T3). 7. A group which was surgically thyroidectomized group and the feed was supplemented by i0 ppm D,L-thyroxine (T x + T4). 8. A group that received i ppm T 3 in their feed (T3). 9. A group that received I0 ppm T 4 in their feed (T4). The birds were weighed and sacrificed at ii weeks of age when the experiment was terminated. The bursa of Fabricius, spleen, and thyroid glands were removed and weighed. Thyroidectomized birds were examined for the presence of residual thyroidal tissue. Hormone assays. Serum concentration of T 3 and T 4 were measured using double antibody radioimmunoassay kits purchased from Antibodies Incorporated (Davis, California). The antibodies were tested by the
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company and found not to crossreact with any of the other iodothyronines. The serum samples were obtained from all birds at 7 and II weeks of age. Humoral immunity. Antibody production against sheep red blood cells (SRBC) (thymus-dependent antigen) and Brucella abortus (BA) (dead microorganisms - Difco, Detroit, MI, U.S.A.) (thymus-independent antigen) was tested at 6 weeks of age. Ten chicks from each of the treatment groups were immunized intravenously with i ml of saline suspension of SRBC (5%) and another i0 chicks with 0.i ml of BA solution. These antigen doses are optimal for antibody production. Seven days following the antigen challenge, blood samples were collected and sera were frozen for later use in measuring the primary response. To measure the secondary response, the same antigens were injected 4 weeks following the first challenge, and birds were bled 7 days later. Complement in all serum samples was inactivated by incubation at 56oc for 30 minutes before measuring the antibody titer. Total as well as ME-resistant (IgG) and ME-senstiive (IgM) antibodies were measured for both primary and secondary responses by an agglutination tests using the microtiter technique described previously
(1). Statistical Analysis. Data were analyzed using one way analysis of variance to determine if there was a difference between treatment groups. Significant differences between treatment means were analyzed using Duncan's multiple range test.
RESULTS Since the results for organ weights as well as serum hormone concentrations were similar for the two groups of birds that were injected with either SRBC or BA, the data presented here are only for the SRBC group. Thyroid and body weights. Data for thyroid and body weights of all treatment groups at ii weeks of age are shown in Table I. Tx, TABLE I Body and relative thyroid weight of ii week old male chickens in all treatment groups injected with sheep red blood cells.
Treatment Group* Control Sham Thyroidectomy (Tx) Thiouracil (PTU) (0.1%) T x + PTU T X + T 3 (i ppm) T X + T 4 (I0 ppm) T 3 (i ppm) T 4 (i0 ppm)
Body Weight (gm) 1164.10 1154.50 898.44 781.70 680.90 1092.20 1153.82 1152.67 1134.73
+ ± ± ± ± ± i ± ±
43.67 a 49.01 a 54.80 b 34.19 c 25.75 c 55.70 a 33.88a 33.37 a 32.39 a
Relative Thyroid Weight (mg/100 gm BW) 8.24 + 0.58 b 8.83 ± 1.38 b --46.56 ± 3.62 a ------1.29 ± 0.25 c 1.22 ± 0.22 c
* Values are mean + S.E. a,b,c Means in the same column having different superscripts are statistically different (P<0.05) PTU, and T X + PTU birds had significantly (P
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Thyroid hormone replacement in groups T x + T 3 and T x + T 4 increased body weights to values comparable to the controls. To verify the absence of thyroid tissue in the T x groups, the birds were sacrificed and autopsied. No thyroid tissue was present in any of the T x birds. Thyroid gland enlargement in PTU birds were apparent (Table I). Birds in the T 3 and T 4 groups had significantly (P<0.05) smaller thyroids than control groups (Table i). Furthermore, there were negative correlations between circulating levels of T3, T4, and relative thyroid weights (Table 3), however, the correlation was significant (P<0.05) only for T 4 . Hormone concentrations. Concentrations of serum T 3 and T 4 for all treatment groups at 7 and Ii weeks of age are shown in Figure I. It is apparent that treatments have resulted in producing a wide range of circulating T 3 and T4, which was the intention of using these treatments. At 7 weeks of age, serum concentrations ranged from 3.62-11.58 ng/ml and from 10.50-373.51 ng/ml for T 3 and T4, respectively. Similar ranges were also found at Ii weeks of age (Figure i). Lymphoid organ weights. Bursa of Fabric±us and spleen weights of all treatment groups at II weeks of age are shown in Table 2. Bursa and spleen relative weights were significantly (P<0.05) reduced in Tx, PTU and T X + PTU birds. Hormone replacement increased these weights to values comparable to controls (Table 2). Birds in T 3 or T 4 groups TABLE 2 Relative bursa and spleen weight of ii week old male chickens all treatment injected groups with sheep red blood cells.
Treatment Groups* Control Sham Thyroidectomy (Tx) Thiouracil (PTU) (0.1%) T x + PTU (0.1%) T x + T 3 (i ppm) T x + T 4 (i0 ppm) T 3 (i ppm) T 4 (i0 ppm)
Relative Bursa Weight (mg/100 g BW) 370.22 378.50 275.11 137.91 143.75 395.79 375.90 350.69 352.75
± + ± ± ± ± ± ± ±
32.54 ab 30.23 ab 42.58 b 16.64 c 15.10 c 64.26 a 31.88 ab 31.91 ab 36.92 ab
*Values are mean ± S.E. a,b Means in the same column having different superscripts statistically different (P<0.05).
in
Relative Spleen Weight (mg/100 g BW) 264 227 206 127 145 316 280 246 261
16 33 71 93 77 42 69 ii 16
+ + + ± + + + + +
19.59 abc 16 24 bc 27 56 cd 8 93 e 9 76 de 55 91 a 30 00 ab 12 01 bc 16 94 abc
are
did not have larger bursa or spleens than did birds in the control groups. However, there were significant (P<0.05) correlations between circulating T3, T4, and both lymphoid organ weights (Table 3). Humoral immunity. Correlation coefficients between circulating T3, T 4 at 7 weeks of age and antibodies of the primary response against BA and SRBC are shown in Table 4. There were no significant correlations between concentrations of T 3 or T 4 and antibodies against BA. Similar results were found between T 4 ~nd antibodies against SRBC. On the other hand, there were significant negative correlations between T 3 and total as well as IgM
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THYROID HORMONES AND IMMUNITY IN CHICKENS
399
T 3 CONCENTRATIONS AT
T 4 CONCENTRATIONS AT
7 WEEKS OF AGE
7 WEEKS OF AGE
ng/ml n,l,~
1.5 400 B
E
E
30(
10
,i~, D
BC
D
200
N'I i', :<
V.
lOC A PTU Tx
Tx ,4-
C
S
T4
PTU
Tx
T3
T4
Tx •
A
A
A
PTU T3
Tx
T3
A
A
A
Tx
Tx
C
S
T3
PTU
,I
Tx
T4
T4
TREATMENT TREATMENT
T 3 CONCENTRATIONS AT
T 4 CONCENTRATIONS AT
ii WEEKS OF AGE
Ii WEEKS OF AGE ng/ml
nglml
300j lO
E
8
C
200
B
6
• AB
i
AB ~ ~
4
100.
0 T4
~x T4
PTU
TX
S
Tx
PTU TR.E~TMENT
C
T3
Tx
T~
Tx + T3
T3
Tx
Tx
PTU
$
PTU
C
T4
Tx T4
TREATMENT
FIGURE i. Concentrations (ng/ml) of serum T 3 and T 4 at 7 and ii weeks of age of male chickens in all treatment groups injected with sheep red blood cells. Within each age and hormone, treatments having different letters are statistically different (P
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TABLE 3 Correlation coefficients between levels of circulating T3, T 4 at Ii weeks of age and organ weights of male chickens of all treatment groups injected with sheep red blood cells. Organ
T3
Body Weight Thyroid Weight Relative Bursa Weight Relative Spleen Weight
+0.185 -0.075 +0.252 +0.262
T4 (NS) (NS) (P<0.05) (P<0.05)
0.307 -0.249 +0.273 +0.271
(P
TABLE 4 Correlation coefficients between circulating T3, T 4 at 7 weeks of age and the antibodies of the primary response against Brucella abortus (BA) and sheep red blood cells (SRBC). BA
SRBC
Antibody
Total IgG IgM
T3
T4
T3
T4
+0.080 (NS) -0.039 (NS) +0.105 (NS)
-0.172 (NS) -0.016 (NS) -0.155 (NS)
-0.216 (P<0.05) -0.041 (NS) -0.264 (P<0.05)
-0.023 (NS) +0.058 (NS) -0.078 (NS)
antibodies against SRBC. As for the secondary response (Table 5), there were no significant correlations between T 3 and T 4 and antibodies against SRBC. However, T 4 and not T 3 was positively (P
DISCUSSION Birds in Tx, PTU and T x + PTU groups displayed body weights and thyroid status which are characteristic of hypothyroidism. It has been TABLE 5 Correlation coefficients between circulating T3, T 4 at ii weeks of age and antibodies of the secondary response against Brucella abortus (BA) and sheep red blood cells (SRBC). BA
SRBC
Antibody
Total IgG IgM
T3
T4
T3
T4
+0.146 (NS) +0.175 (NS) -0.057 (NS)
+0.219 (P<0.05) +0.358 (P<0.05) -0.176 (NS)
-0.054 (NS) +0.036 (NS) -0.077 (NS)
+0.168 (NS) +0.089 (NS) +0.059 (NS)
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previously reported that surgical thyroidectomy (11,17) or goiterogen treatment (11,17,18) results in reduced body weight. The data of this experiment demonstrates that PTU is more effective in reducing body weight than surgical thyroidectomy and when both methods are applied, body weight is reduced further. Birds in PTU group possessed enlarged thyroids which is a characteristic of goiterogen treated birds (7,11). Even though these treatments produced a wide range of circulating T 3 and T 4 concentrations, the lowest values, which were found in Tx, PTU and T x + PTU birds, still represented a considerable amount of hormone. T 3 and T 4 have been detected previously in thyroidectomized birds despite the absence of thyroidal tissue (8,11,13). In the present study, bursa and spleen weights were reduced in Tx, PTU and T x + PTU birds. Furthermore, there were positive correlations between circulating T3, T4, and bursa and spleen weights. These results indicate that thyroid gland and thyroid hormones are essential for maintaining normal weights of these lymphoid organs. It was found previously that hypothyroidism resulted in hypoplasia of these lymphoid organs (3,15). Using 250 ppm T4, Yam e t al. (18) found enhancement in chicken spleen weight. In the present study there was no increase in lymphoid organ weights when I0 ppm T 4 was used. It is possible, therefore, that higher doses of T 3 and T 4 than what were used in this study are needed in order to stimulate lymphoid organ weights. The results of the present study did not demonstrate any consistent relationships between circulating T 3 and T 4 concentrations and antibody production against both thymus-dependent and thymus-independent antigens. This is in agreement with our previous finding (ii). One possible explanation is that although hormone concentrations were lower in hypothyroid groups, the amount of hormone present was adequate for maintaining normal antibody production. It has been demonstrated that the number of lymphocyte receptors for T 3 and T 4 are increased in hypothyroidism but remains unchanged in hyperthyroidism (9). Thus, while the hormone levels were lower in hypothyroid groups, lymphocytes may be able to better utilize T 3 and T 4. Yam et al. (18) found that thyroid hormones stimulated antibody production in chickens. Our present results do not confirm the findings by Yam et al. (18). This could be due to the use of different doses of thyroid hormones. It has been shown that the effect of T 3 and T 4 on antibody production may be dose dependent (2,12). Therefore, it is conceivable that the stimulatory effect of thyroid hormones on antibody production occurs at either lower or higher levels than what were used in the present study. In conclusion, we suggest that physiological levels of thyroid hormones are needed to maintain normal weights of bursa and spleen. Furthermore, we conclude that lower than physiological levels might be sufficient for normal antibody production. Finally, stimulation of antibody production using thyroid hormones may require different doses than what were utilized in this study.
ACKNOWLEDGEMENTS The authors are indebted to Dr. Robert F. Wideman, Jr. for his technical assistance in the surgical procedures, Ms. Jeannette M. Bachman for her technical assistance in hormone assay and statistical procedures, and Ms. Dee Ann Longenecker for her expert secretarial assistance.
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Yam, D., D. Heller, and N. Snapir. The effect of the thyroidal state on the immunological state of the chicken. Dev. Comp° Immunol., 5, 483-490 (1981). Received: March, 1986 Accepted: July, 1986