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Alteration of lymphocyte reactivities by thyroid hormones
Int. J. lmmunopharmac., Vol. 8, No. 7, pp. 755-762, 1986. Printed in Great Britain.
0192-0561/86 $3.00+ .00 International Society for lmmunopharmacology.
A L T E R A T I O N OF L Y M P H O C Y T E REACTIVITIES BY T H Y R O I D H O R M O N E S M. L. ONG, D. G. MALKIN and A. MALKIN Department of Clinical Biochemistry, Sunnybrook Medical Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, Canada, M4N 3MS (Received 2 December 1985 and in final form 3 April 1986)
Abstract - - The effects of thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit
peripheral blood lymphocytes to T-cell mitogens, PHA and Con A, and B cell specific goat anti-rabbit light chain antibodies (Anti-L) were investigated. It was observed that (a) L-T4 potentiated the lymphocyte response to mitogens and Anti-L in a dose-dependent manner: 10-gM and 10-SM had no effect while 10-7- 10-SM significantly enhanced the lymphocyte response. (b) L-T3 (10-11- 10-SM) had no effect on the lymphocyte response to PHA and Con A. At 10-TM, L-T3 inhibited the response to PHA but not Con A. (c) L-T3 (10-'J-10-TM) suppressed the lymphocyte response to Anti-L. The suppression was directly proportional to the L-T3 concentration. (d) rT3 (10-'1 - 10-TM) inhibited the proliferative response to PHA and Anti-L in a dose-related manner. Its effect on the lymphocyte response to Con A was stimulatory at 10-11M but inhibitory at higher concentrations (10 -8 and 10-TM). (e) rT3 suppressed the enhancement by L-T4 of the lymphocyte response to the mitogens and Anti-L. The degree of suppression was proportional to its concentration. This data indicated that thyroid hormones can alter the reactivities of lymphocytes. The direction and magnitude of the alteration appear to depend on the concentration of a specific thyroid hormone ericountered by the responding cells.
Several studies have indicated that thyroid hormones may exert an influence on the immune system. Treatment with thyroxine can restore the depressed humoral and cellular immune response in thyroidectomized newborn or young adult rats (Fabris, 1973). Thyroid hormone-treated mice have increased peripheral blood lymphocyte and T cell counts as well as increased thymic incorporation of tritiated thymidine (Aoki, Wakisaka & Nagata, 1976). Antibody-forming capacity o f splenic cells from thyroxine treated mice were shown by Chen (1980) to be increased and by Gupta, Chiang & Deodhar (1983) to be suppressed. With regard to mitogen-induced lymphocyte transformation, thyroid hormones have been shown to have either an enhancing (Keast & Taylor, 1982; Bal~izs, Le6vey, Szab6 & Bak6, 1980) or suppressing (Gupta et al., 1983; Chatterjee & Chandel, 1983) effect on the response of T cell to P H A . However, the effect of thyroid hormones on the proliferative response to B cells has not been well established. In the rabbit, B cells can readily be transformed in the presence o f anti-immunoglobulin antibodies, e.g. anti-light chain antiserum (Anti-L) (Sell, 1970; Fanger, Pelley &
It has been recognized for some time that the immune system may be under h o r m o n a l regulation. For example, cortiscosteroids have been observed to have p r o f o u n d effects on lymphocytes (Claman, 1972) and h u m a n chorionic gonadotrophin can suppress mitogen-induced lymphocyte transformation (Contractor & Davies, 1973; Han, 1974; Huang, 1981). A direct action of growth hormone on the immune response has also been reported (Fabris, Pierpaoli & Sorkin, 1971; M a o r & Alexander, 1972; Comsa, Schwarz & Neu, 1974). In some pathological conditions, alteration in h o r m o n e levels is accompanied by abnormal immunological findings. Most patients with Graves disease have elevated thyroid hormones (Werner, 1978) associated with thyroid stimulating immunoglobulins and other antithyroid antibodies (Adams & Purves, 1957; A d a m s & Kennedy, 1971; Mori & Kriss, 1971; Debruin & Van der Heide, 1983). In the advanced stage of H a s h i m o t o ' s thyroiditis, alteration in serum thyroid hormone is usually accompanied by the presence of various anti-thyroid antibodies, such as the antithyroglobulin and the anti-microsomal antibodies (Beall & Solomon, 1978; Volp6, 1978). 755
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Reese, 1972) while T cells can be transformed by PHA or Con A (Ozer & Waksman, 1974; Teodorescu, Mayer, Reiter & Sheldon, 1976). In this study we compare, within one system, the effect of various thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit T and B lymphocytes to PHA, Con A and Anti-L.
EXPERIMENTAL PROCEDURES
Experimental animals New Zealand white female rabbits, 2 - 3 lb, were obtained from Riemens Fur Ranches, Ste. Agathe, Ontario. Thyroid hormones L-thyroxine (L-T4), free acid, was purchased from Sigma Chemical Co., St. Louis, MO. Chemically purified L-3,3', 5-triiodothyronine (L-T3) and 3 , 3 ' 5 ' triiodothyronine (rT3) were obtained from Calbiochem-Behring, San Diego, CA. Isotope (6-3H)-thymidine (specific activity: 2 Ci/mmol) was obtained from Amersham Corporation, Arlington Heights, IL. Stimulants Goat Anti-L chain antiserum was obtained from Cappel Laboratories, Cochraneville, PA. Phytohaemagglutinin (PHA) 2 x crystallized, was from Pharmacia (Canada) Ltd., Dorval, Quebec. Concanavalin A (Con A), 3 × crystallized, was from Miles Laboratories, Elkhart, IN. Culture media Alpha medium, DNA- and RNA-depleted, was obtained from the Media Department of Princess Margaret Hospital, Toronto, Ontario, McCoy's 5A medium was purchased from Grand Island Biological Co., Grand Island, NY. Gelatine Knox unflavoured gelatine was purchased from Thomas J. Lipton Inc., Toronto, Ontario. Preparation o f peripheral blood mononuclear cells (PBMC) Peripheral blood mononuclear cells were prepared according to the method of Fanger, Hart, Wells &
Nisonoff (1970) with some modifications. Briefly, 25 - 30 ml of freshly drawn venous blood, obtained by ear vein puncture of rabbits, were defibrinated with glass beads. The defibrinated blood was mixed with an equal volume of 3070 gelatine in McCoy's 5A medium and incubated in a water bath at 37°C for 1 h. The lymphocyte-enriched supernatant was then separated, mixed with an equal volume of McCoy's 5A medium and centrifuged at 300 g for 10 min. The resulting pellet was washed with alpha medium and centrifuged again at 300 g for 10 min. Finally, the washed PBMC were resuspended in alpha medium, enumerated, and adjusted to 4 z 106 cells/ml. The viability of the recovered mononuclear cells, as determined by trypan blue exclusion, was invariably greater than 98070.
Assay o f the proliferative response Peripheral blood mononuclear cells were assayed for their responsiveness to mitogens and Anti-L antiserum using the microculture method. The cell concentration was adjusted to 4 x 106/ml in alpha medium. A volume of 100 ~1 of the cell suspension was delivered to the appropriate well of a microculture plate (Flow Laboratories, McClean, VA). Each well contained 100/al of medium with or without stimulant. A range of stimulant concentrations (PHA and Con A: 0.5 - 10 tag/ml; Anti-L: 1/256 to 1/8 dilution of the reconstituted volume) was used for the stimulation of each cell sample. The concentrations of the different hormones are indicated in the appropriate Figures. The ceils were cultured at 37°C in a humidified atmosphere with 5°70 CO2 and 95070 air for 48 h; 1.0 /aCi of 3H-thymidine (specific activity: 2.0 Ci/mmol) was added to each well 18 h before the culture was terminated. At the end of the culture period, cells were harvested on glass-fibre filters (Reeve Angel) using an automatic multiple cell harvester (MiniMash, M.A. Bioproducts, Wakersville, MD). After appropriate drying, radio-activity was determined by liquid scintillation counting in a Packard liquid scintillation spectrometer (Model 3375). Each cell culture was performed in triplicate and the proliferative response to each stimulant was expressed as counts/min per culture. RESULTS
Proliferative response o f rabbit P B M C stimulated by T cell mitogens, Anti-L and thyroid hormones Rabbit PBMC are highly reactive to the stimulation by Con A, PHA and Anti-L as indicated
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Fig. 1. Dose - response curves of rabbit peripheral blood lymphocytes cultured in the presence of different concentrations of PHA, Con A, Anti-L or thyroid hormones (L-T4, L-T3 and rT3). Each point represents the mean counts/min _+S.E. of triplicate cultures. Each inverted arrow indicates negligible (less than 200 counts/min/culture) 3H-thymidine incorporation by cells cultured in the presence of the indicated thyroid hormone, and this incorporation is not significantly different from that of control cultures without added thyroid hormone.
by the d o s e - r e s p o n s e curves shown in Fig. 1. In contrast, the three thyroid hormones (L-T4, L-T3 and rT3), over a wide range of doses tested, were found to be ineffective in inducing a significant stimulation of the same cell cultures. Although the thyroid hormones appeared to be non-stimulatory in terms o f the proliferative response, their possible effects on the lymphocyte reactivities to mitogenic stimulants were examined in the following experiments.
Enhancement o f the proliferative response by L-T4 The presence o f L-T4 in cell cultures stimulated by P H A , Con A or Anti-L was found to promote the resulting response. The magnitude o f enhancement is
dependent on the concentration of L-T4 added (Fig. 2). The maximal potentiation of the T cell response to Con A and P H A as well as the B cell response to Anti-L occurred at a concentration of 10-6M L-T4. Lower concentrations of the hormone (10 -8 and 10-gM) had an insignificant effect in potentiating the response of the stimulated lymphocytes.
Effect o f L-T3 and rT3 on the proliferative response The response o f rabbit peripheral blood T cells to P H A and Con A was not significantly affected by the presence o f different concentrations of L-T3 except at 10-'M where the response to P H A was depressed by about 17% (Fig. 3, upper panel). However, the addition of L-T3 to the cultures
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Fig. 2. Thyroxine-induced enhancement of the proliferative response stimulated by mitogens (PHA and Con A) and an anti-immunoglobulin reagent (Anti-L). Different c o n c e n t r a t i o n s (10 - 9 - 10-SM) of L-T4 were added to cell cultures each containing a pre-determined optimal dose of Con A (2 /ag/ml), PHA (2 /ag/ml), or Anti-L (1/32 dilution). The results are expressed as "°7o change in response", i.e. mean . . . . t s / m i n i n the presence o f thyroid Shormo mean c o u n t s / m i n in the absence o f thyroid hormone
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Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.05 to P < 0.001, Student's t-test) between the response of cultures with and without L-T4. mediated a d o s e - d e p e n d e n t depression o f the proliferative response o f i m m u n o g l o b u l i n - b e a r i n g B cells to Anti-L (Fig. 3, lower panel). Reverse T3 (rT3), at a dose o f 10-" to 10-TM, progressively depressed the l y m p h o c y t e response to P H A and Anti-L (Fig. 4). The effect o f rT3 on the T cell response to C o n A was f o u n d to be variable and was related to the h o r m o n e concentration. The response to C o n A was e n h a n c e d at a low
Fig. 3. Effect of L-T3 on rabbit lymphocyte response to PHA, Con A and Anti-L. Different concentrations (10-'-10-TM) of L-T3 were added to cell cultures each containing a pre-determined optimal dose of Con A (2/ag/ ml) PHA (2/ag/ml), or Anti-L (1/32 dilution). The results are expressed as "07o change in response" as defined in Fig. 2. Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.02 to P < 0.005, Student's t-test) between the response of cultures with and without L-T3. concentration o f 10-'IM and depressed at higher concentrations o f 10-" and 10-TM.
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Fig. 4. D o s e - response curves for the effect of rT3 on the lymphocyte transformation induced by mitogens and AntiL. Indicated quantities of freshly diluted rT3 were added to triplicate cultures of 4 x l0 s cells just prior to the addition of mitogens (PHA or Con A, 2 tag/ml) and Anti-L (1/32 dilution). The results are expressed as "07o change in response" as defined in Fig. 2. Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.05 to P < 0.0005, Student's t-test) between the response of cultures with and without rT3. DISCUSSION In the present study, n o n e o f the thyroid h o r m o n e s (L-T4, L-T3 a n d rT3) investigated exerted any mitogenic effect o n the unstimulated lymphocytes (Fig. 1, right panel). High c o n c e n t r a t i o n s o f L-T3 (10-TM) a n d L-T4 (10-'M) had been reported by Lundell & Blomgren (1975) to be mitogenic for
Fig. 5. Combined effects of L-T4 and rT3 on rabbit lymphocyte response to PHA, Con A and Anti-L. Different concentrations (10- ' t - 10-TM) of rT3 were added to cell cultures each containing an enhancing dose (10-6M) of L-T4 and an optimal dose of Con A (2/ag/ml), PHA (2/~g/ml), or Anti-L (1/32 dilution). The results are expressed as "o70 change in response", i.e. mean counts/rain in the presence of rT3
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Each point represents the mean change in response ± S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.05 to P < 0.001, Student's t-test) between the responses of cultures with and without rT3.
h u m a n lymphocytes. The highest c o n c e n t r a t i o n o f L-T4 used in this study was 10-SM. A higher c o n c e n t r a t i o n o f L-T4, e.g. 10-'M, was f o u n d not to be soluble at the p H o f our culture system. In o r d e r to render L-T4 soluble at 10-4M, an alkaline p H , which is not a p p r o p r i a t e for tissue culture, needs to
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be used. Furthermore, 10-4M L-T4 is about three orders of magnitude higher than its physiological concentration. Thus, the mitogenicity of high L-T4 concentrations on lymphocyte transformation remains to be established. At 10-TM, L-T3 did not stimulate rabbit lymphocyte proliferation (Fig. t, right panel). This is in contrast to the results observed by Lundell & Blomgren (1975) using human lymphocytes. This apparent discrepancy may be explained by a possible species difference in terms of the sensitivity of their respective lymphocytes to L-T3 stimulation. L-thyroxine was found to exert a dose-related potentiating effect on the rabbit lymphocyte response to PHA, Con A and anti-light chain antibodies (Fig. 2). Low L-T4 concentrations (10-9 and 10-SM) had insignificant effect, while higher concentrations (10 - 7 - 10-SM) of the hormone significantly potentiated the response. The optimal enhancement was mediated by L-T4 at 10-6M. Our observation that L-T4 can potentiate the reactivities of T and B cells is in agreement with the observation by Chen (1980) who showed that L-T4 could promote the in vivo antibody response to a T cell dependent antigen, sheep erythrocytes. In contrast to the stimulatory effect of L-T4, L-T3 was found to exert no effect on the response of T cells except at 10-TM, which significantly depressed the response to PHA (Fig. 3, upper panel). The action of L-T3 on the response of B cells was predominantly suppressive (Fig. 3, lower panel). Parallel to our current observations in lymphocytes, a differential effect of L-T4 and L-T3 has also been observed in studies examining the ability of each hormone to stimulate the incorporation of amino acids into proteins in liver cells (Sokoloff, Roberts, Januska & Kline, 1968). Although deiodination of L-T4 has been shown to occur in lymphoid cells (Holm, Lemarchand-Ber~ud, Scazziga & Cuttelod, 1975), it is not known whether the conversion product is L-T3. Since L-T4 by itself is capable of potentiating the proliferative response of lymphocytes (Fig. 2), it is possible that the conversion of L-T4 to L-T3 may not be obligatory in mediating certain biological reactions. Reverse T3 was found to have a dose-dependent inhibitory effect on the rabbit lymphocyte response to PHA and Anti-L (Fig. 4). Its effect on the lymphocyte response to Con A was stimulatory at low concentrations, e.g. 10-11M, and inhibitory at higher concentrations, e.g. 10-s and 10-TM. In experiments where an enhancing dose of L-T4 and varying doses of rT3 were added to cell culture stimulated with PHA, Con A or Anti-L, the
inhibitory effect of rT3 was dominant (Fig. 5). Thus rT3 appears to be antagonistic to the action of L-T4 on lymphocyte reactivities. The opposing effect of rT3 on L-T4 action has also been reported in other studies. For example, rT3 was found to be capable of decreasing the calorigenic action of L-T4 (Pittman and Barker, 1959; Pittman, Tingley, Nickerson & Hill, 1960). Based on these findings and that of others, it is reasonable to suggest that rT3 may not be just an inactivation product of T4. Instead, rT3 may exert a regulatory role in modulating the biological action of L-T4. In the present study, rT3 was observed to be more potent than T3 in suppressing the lymphocyte response to mitogens and Anti-L (Figs 3 and 4). Other studies have also demonstrated rT3 to be either more potent or equally as effective as T3 in various biological systems. For example, rT3 is more potent than L-T3 in inhibiting cyclic nucleotide phosphodiesterase activity (Marcus, Lundquist & Chopra, 1975) as well as in stimulating erythropoiesis (Golde, Bersch, Chopra & Cline, 1976). The maximum effect of rT3 on growth hormone production and glucose consumption in cultured rat pituitary tumor cells is equal to that produced by L-T3 (Papavasiliou, Martial, Latham & Baxter, 1977). Thus, the effect of rT3 can impose a significant influence on specific biological activities. The physiological concentrations of different thyroid hormones in the circulation are equivalent to 10-TM for L-T4, and 10-gM each for L-T3 and rT3 (Larsen, 1978; Chopra, 1974). Results of the present study indicate that this physiological range of hormone levels can alter lymphocyte reactivities. Thus, L-T4 (10-TM) was found to potentiate the proliferative response of T and B lymphocytes (Fig. 2). Both L-T3 and rT3, at 10-gM, were able to suppress the B cell response to Anti-L (Fig. 3 and 4, lower panels). Furthermore, the same rT3 concentration (10-gM) was also found to suppress the T cell response to PHA (Fig. 4, upper panel) as well as to antagonize the potentiating effect of L-T4 on the PHA, Con A or Anti-L stimulated cell cultures (Fig. 5). These data suggest that circulating thyroid hormones, at physiological concentration may play a role in modulating lymphocyte functions. The exact mechanism by which thyroid hormones may influence the response of lymphocytes is not clear at present. However, lymphocytes have been demonstrated to possess nuclear receptors for T3 and T4 (Tsai & Samuels, 1974; Holm, Scazziga & Lemarchand-Ber~iud, 1976; Liewendahl, Rosenghrd & Lamberg, 1978). Thus the change in lymphocyte response induced by L-T4 (Fig. 2) and L-T3 (Fig. 3)
Alteration of Lymphocyte Reactivities may well be triggered by an interaction o f the thyroid h o r m o n e s with its specific receptors. In conclusion, based on the data presented in this study, it is clear that thyroid h o r m o n e s can alter the reactivities o f lymphocytes. The direction and magnitude o f alterations appear to d e p e n d on the
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concentration o f a specific thyroid h o r m o n e e n c o u n t e r e d by the responding cells. The implication o f these results with respect to altered lymphocyte function a m o n g patients suffering f r o m Graves' disease and other thyroid disorders remains to be established.
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LUNDELL, G. & BLOMGREN, n . (1975). Thyroid hormones and lymphoid cells. Acta Radiol., 14, 552-560 MAOR, D. ,~¢ ALEXANDER, P. (1972). Possible role of growth hormone in the stimulation of thymus of rats following irradiation of the head. Experientia, 28, 957- 959. MARCUS, R., LUNQUIST, C. & CHOPRA, I. (1975). In vitro inhibition of cyclic nucleotide phosphodiesterase (PDE) by thyroid hormones. Clin. Res., 23, 94A. MORI, T. & KRISS, J. P. (1971). Measurements by competitive binding radio-assay of serum anti-microsomal and antithyroglobulin antibodies in Graves' disease and other thyroid disorders. J. clin. endocr. Metab., 33, 688- 698. OZER, H., JR. & WAKSMAN,B. H. (1974). The response of rabbit lymphocytes to mitogens and alloantigens. Evidence for T cell heterogeneity. J. I m m u n . , 113, 1780-1792. PAPAVASILIOU,S. S., MARTIAL, J. A., LATHAM,K. R. 8~ BAXTER,J. D. (1977). Thyroid hormonelike actions of 3, 3 ', 5'triiodothyronine and 3, 3'-diiodothyronine. J. clin. Invest., 60, 1230-1239. PITTMAN, C. S. & BARKER, S. B. (1959). Anti-thyroxine effects of some thyroxine analogues. A m . J. Physiol., 197, 1271 - 1274. PITTMAN, J. A., TINGLEY, J. O., NICKERSON,J. F. & HILL, S. R. JR., (1960). Anti-metabolic activity of 3, 3', 5'-triiodoDL-thyronine in man. Metabolism, 9, 293- 295. SELL, S. (1970). Stimulation of rabbit peripheral lymphocytes with F(ab')2 and Fab fragments of anti-lymphocytic and anti-lgG globulins. Fedn Proc. Fedn A m . Socs exp. Biol., 29, 301A. SOKOLOFF, L., ROBERTS, P. A., JANUSKA,M. M. & KLINE, J. E. (1968). Mechanism of stimulation of protein synthesis by thyroid hormones in vivo. Proc. natn. Acad. Sci. U.S.A., 60, 652-659. TEODORESCU, M., MAYER, E. P., REITER, n . & SHELDON,D. (1976). Rabbit lymphocyte subpopulations. I. Separation of lg ÷ and Ig- cells and their interaction in cultures stimulated by mitogens. Cell. I m m u n . , 22, 66-75. TSAI, J. S. & SAMUELS,H. H. (1974). Thyroid hormone action: Demonstration of putative nuclear receptors in human lymphocytes. J. clin. Endocr. Metab., 38, 919-922. VOLPE, R. (1978). Lymphocytic (Hashimoto's) thyroiditis. In The Thyroid (eds Werner, S.C. and Ingbar, S.H.) 4th edn, pp. 996- 1008. Harper & Row, New York. WERNER, S. C. (1978). Toxic goitre. In The Thyroid (eds Werner, S. C. & lngbar, S. H.) 4th edn, pp. 591-603. Harper & Row, New York.
0192-0561/86 $3.00+ .00 International Society for lmmunopharmacology.
A L T E R A T I O N OF L Y M P H O C Y T E REACTIVITIES BY T H Y R O I D H O R M O N E S M. L. ONG, D. G. MALKIN and A. MALKIN Department of Clinical Biochemistry, Sunnybrook Medical Centre, University of Toronto, 2075 Bayview Avenue, Toronto, Ontario, Canada, M4N 3MS (Received 2 December 1985 and in final form 3 April 1986)
Abstract - - The effects of thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit
peripheral blood lymphocytes to T-cell mitogens, PHA and Con A, and B cell specific goat anti-rabbit light chain antibodies (Anti-L) were investigated. It was observed that (a) L-T4 potentiated the lymphocyte response to mitogens and Anti-L in a dose-dependent manner: 10-gM and 10-SM had no effect while 10-7- 10-SM significantly enhanced the lymphocyte response. (b) L-T3 (10-11- 10-SM) had no effect on the lymphocyte response to PHA and Con A. At 10-TM, L-T3 inhibited the response to PHA but not Con A. (c) L-T3 (10-'J-10-TM) suppressed the lymphocyte response to Anti-L. The suppression was directly proportional to the L-T3 concentration. (d) rT3 (10-'1 - 10-TM) inhibited the proliferative response to PHA and Anti-L in a dose-related manner. Its effect on the lymphocyte response to Con A was stimulatory at 10-11M but inhibitory at higher concentrations (10 -8 and 10-TM). (e) rT3 suppressed the enhancement by L-T4 of the lymphocyte response to the mitogens and Anti-L. The degree of suppression was proportional to its concentration. This data indicated that thyroid hormones can alter the reactivities of lymphocytes. The direction and magnitude of the alteration appear to depend on the concentration of a specific thyroid hormone ericountered by the responding cells.
Several studies have indicated that thyroid hormones may exert an influence on the immune system. Treatment with thyroxine can restore the depressed humoral and cellular immune response in thyroidectomized newborn or young adult rats (Fabris, 1973). Thyroid hormone-treated mice have increased peripheral blood lymphocyte and T cell counts as well as increased thymic incorporation of tritiated thymidine (Aoki, Wakisaka & Nagata, 1976). Antibody-forming capacity o f splenic cells from thyroxine treated mice were shown by Chen (1980) to be increased and by Gupta, Chiang & Deodhar (1983) to be suppressed. With regard to mitogen-induced lymphocyte transformation, thyroid hormones have been shown to have either an enhancing (Keast & Taylor, 1982; Bal~izs, Le6vey, Szab6 & Bak6, 1980) or suppressing (Gupta et al., 1983; Chatterjee & Chandel, 1983) effect on the response of T cell to P H A . However, the effect of thyroid hormones on the proliferative response to B cells has not been well established. In the rabbit, B cells can readily be transformed in the presence o f anti-immunoglobulin antibodies, e.g. anti-light chain antiserum (Anti-L) (Sell, 1970; Fanger, Pelley &
It has been recognized for some time that the immune system may be under h o r m o n a l regulation. For example, cortiscosteroids have been observed to have p r o f o u n d effects on lymphocytes (Claman, 1972) and h u m a n chorionic gonadotrophin can suppress mitogen-induced lymphocyte transformation (Contractor & Davies, 1973; Han, 1974; Huang, 1981). A direct action of growth hormone on the immune response has also been reported (Fabris, Pierpaoli & Sorkin, 1971; M a o r & Alexander, 1972; Comsa, Schwarz & Neu, 1974). In some pathological conditions, alteration in h o r m o n e levels is accompanied by abnormal immunological findings. Most patients with Graves disease have elevated thyroid hormones (Werner, 1978) associated with thyroid stimulating immunoglobulins and other antithyroid antibodies (Adams & Purves, 1957; A d a m s & Kennedy, 1971; Mori & Kriss, 1971; Debruin & Van der Heide, 1983). In the advanced stage of H a s h i m o t o ' s thyroiditis, alteration in serum thyroid hormone is usually accompanied by the presence of various anti-thyroid antibodies, such as the antithyroglobulin and the anti-microsomal antibodies (Beall & Solomon, 1978; Volp6, 1978). 755
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Reese, 1972) while T cells can be transformed by PHA or Con A (Ozer & Waksman, 1974; Teodorescu, Mayer, Reiter & Sheldon, 1976). In this study we compare, within one system, the effect of various thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit T and B lymphocytes to PHA, Con A and Anti-L.
EXPERIMENTAL PROCEDURES
Experimental animals New Zealand white female rabbits, 2 - 3 lb, were obtained from Riemens Fur Ranches, Ste. Agathe, Ontario. Thyroid hormones L-thyroxine (L-T4), free acid, was purchased from Sigma Chemical Co., St. Louis, MO. Chemically purified L-3,3', 5-triiodothyronine (L-T3) and 3 , 3 ' 5 ' triiodothyronine (rT3) were obtained from Calbiochem-Behring, San Diego, CA. Isotope (6-3H)-thymidine (specific activity: 2 Ci/mmol) was obtained from Amersham Corporation, Arlington Heights, IL. Stimulants Goat Anti-L chain antiserum was obtained from Cappel Laboratories, Cochraneville, PA. Phytohaemagglutinin (PHA) 2 x crystallized, was from Pharmacia (Canada) Ltd., Dorval, Quebec. Concanavalin A (Con A), 3 × crystallized, was from Miles Laboratories, Elkhart, IN. Culture media Alpha medium, DNA- and RNA-depleted, was obtained from the Media Department of Princess Margaret Hospital, Toronto, Ontario, McCoy's 5A medium was purchased from Grand Island Biological Co., Grand Island, NY. Gelatine Knox unflavoured gelatine was purchased from Thomas J. Lipton Inc., Toronto, Ontario. Preparation o f peripheral blood mononuclear cells (PBMC) Peripheral blood mononuclear cells were prepared according to the method of Fanger, Hart, Wells &
Nisonoff (1970) with some modifications. Briefly, 25 - 30 ml of freshly drawn venous blood, obtained by ear vein puncture of rabbits, were defibrinated with glass beads. The defibrinated blood was mixed with an equal volume of 3070 gelatine in McCoy's 5A medium and incubated in a water bath at 37°C for 1 h. The lymphocyte-enriched supernatant was then separated, mixed with an equal volume of McCoy's 5A medium and centrifuged at 300 g for 10 min. The resulting pellet was washed with alpha medium and centrifuged again at 300 g for 10 min. Finally, the washed PBMC were resuspended in alpha medium, enumerated, and adjusted to 4 z 106 cells/ml. The viability of the recovered mononuclear cells, as determined by trypan blue exclusion, was invariably greater than 98070.
Assay o f the proliferative response Peripheral blood mononuclear cells were assayed for their responsiveness to mitogens and Anti-L antiserum using the microculture method. The cell concentration was adjusted to 4 x 106/ml in alpha medium. A volume of 100 ~1 of the cell suspension was delivered to the appropriate well of a microculture plate (Flow Laboratories, McClean, VA). Each well contained 100/al of medium with or without stimulant. A range of stimulant concentrations (PHA and Con A: 0.5 - 10 tag/ml; Anti-L: 1/256 to 1/8 dilution of the reconstituted volume) was used for the stimulation of each cell sample. The concentrations of the different hormones are indicated in the appropriate Figures. The ceils were cultured at 37°C in a humidified atmosphere with 5°70 CO2 and 95070 air for 48 h; 1.0 /aCi of 3H-thymidine (specific activity: 2.0 Ci/mmol) was added to each well 18 h before the culture was terminated. At the end of the culture period, cells were harvested on glass-fibre filters (Reeve Angel) using an automatic multiple cell harvester (MiniMash, M.A. Bioproducts, Wakersville, MD). After appropriate drying, radio-activity was determined by liquid scintillation counting in a Packard liquid scintillation spectrometer (Model 3375). Each cell culture was performed in triplicate and the proliferative response to each stimulant was expressed as counts/min per culture. RESULTS
Proliferative response o f rabbit P B M C stimulated by T cell mitogens, Anti-L and thyroid hormones Rabbit PBMC are highly reactive to the stimulation by Con A, PHA and Anti-L as indicated
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by the d o s e - r e s p o n s e curves shown in Fig. 1. In contrast, the three thyroid hormones (L-T4, L-T3 and rT3), over a wide range of doses tested, were found to be ineffective in inducing a significant stimulation of the same cell cultures. Although the thyroid hormones appeared to be non-stimulatory in terms o f the proliferative response, their possible effects on the lymphocyte reactivities to mitogenic stimulants were examined in the following experiments.
Enhancement o f the proliferative response by L-T4 The presence o f L-T4 in cell cultures stimulated by P H A , Con A or Anti-L was found to promote the resulting response. The magnitude o f enhancement is
dependent on the concentration of L-T4 added (Fig. 2). The maximal potentiation of the T cell response to Con A and P H A as well as the B cell response to Anti-L occurred at a concentration of 10-6M L-T4. Lower concentrations of the hormone (10 -8 and 10-gM) had an insignificant effect in potentiating the response of the stimulated lymphocytes.
Effect o f L-T3 and rT3 on the proliferative response The response o f rabbit peripheral blood T cells to P H A and Con A was not significantly affected by the presence o f different concentrations of L-T3 except at 10-'M where the response to P H A was depressed by about 17% (Fig. 3, upper panel). However, the addition of L-T3 to the cultures
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Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.05 to P < 0.001, Student's t-test) between the response of cultures with and without L-T4. mediated a d o s e - d e p e n d e n t depression o f the proliferative response o f i m m u n o g l o b u l i n - b e a r i n g B cells to Anti-L (Fig. 3, lower panel). Reverse T3 (rT3), at a dose o f 10-" to 10-TM, progressively depressed the l y m p h o c y t e response to P H A and Anti-L (Fig. 4). The effect o f rT3 on the T cell response to C o n A was f o u n d to be variable and was related to the h o r m o n e concentration. The response to C o n A was e n h a n c e d at a low
Fig. 3. Effect of L-T3 on rabbit lymphocyte response to PHA, Con A and Anti-L. Different concentrations (10-'-10-TM) of L-T3 were added to cell cultures each containing a pre-determined optimal dose of Con A (2/ag/ ml) PHA (2/ag/ml), or Anti-L (1/32 dilution). The results are expressed as "07o change in response" as defined in Fig. 2. Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.02 to P < 0.005, Student's t-test) between the response of cultures with and without L-T3. concentration o f 10-'IM and depressed at higher concentrations o f 10-" and 10-TM.
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Fig. 4. D o s e - response curves for the effect of rT3 on the lymphocyte transformation induced by mitogens and AntiL. Indicated quantities of freshly diluted rT3 were added to triplicate cultures of 4 x l0 s cells just prior to the addition of mitogens (PHA or Con A, 2 tag/ml) and Anti-L (1/32 dilution). The results are expressed as "07o change in response" as defined in Fig. 2. Each point represents the mean change in response _+ S.E. of peripheral blood cells obtained from four animals. Each asterisk indicates statistically significant differences (P < 0.05 to P < 0.0005, Student's t-test) between the response of cultures with and without rT3. DISCUSSION In the present study, n o n e o f the thyroid h o r m o n e s (L-T4, L-T3 a n d rT3) investigated exerted any mitogenic effect o n the unstimulated lymphocytes (Fig. 1, right panel). High c o n c e n t r a t i o n s o f L-T3 (10-TM) a n d L-T4 (10-'M) had been reported by Lundell & Blomgren (1975) to be mitogenic for
Fig. 5. Combined effects of L-T4 and rT3 on rabbit lymphocyte response to PHA, Con A and Anti-L. Different concentrations (10- ' t - 10-TM) of rT3 were added to cell cultures each containing an enhancing dose (10-6M) of L-T4 and an optimal dose of Con A (2/ag/ml), PHA (2/~g/ml), or Anti-L (1/32 dilution). The results are expressed as "o70 change in response", i.e. mean counts/rain in the presence of rT3
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h u m a n lymphocytes. The highest c o n c e n t r a t i o n o f L-T4 used in this study was 10-SM. A higher c o n c e n t r a t i o n o f L-T4, e.g. 10-'M, was f o u n d not to be soluble at the p H o f our culture system. In o r d e r to render L-T4 soluble at 10-4M, an alkaline p H , which is not a p p r o p r i a t e for tissue culture, needs to
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be used. Furthermore, 10-4M L-T4 is about three orders of magnitude higher than its physiological concentration. Thus, the mitogenicity of high L-T4 concentrations on lymphocyte transformation remains to be established. At 10-TM, L-T3 did not stimulate rabbit lymphocyte proliferation (Fig. t, right panel). This is in contrast to the results observed by Lundell & Blomgren (1975) using human lymphocytes. This apparent discrepancy may be explained by a possible species difference in terms of the sensitivity of their respective lymphocytes to L-T3 stimulation. L-thyroxine was found to exert a dose-related potentiating effect on the rabbit lymphocyte response to PHA, Con A and anti-light chain antibodies (Fig. 2). Low L-T4 concentrations (10-9 and 10-SM) had insignificant effect, while higher concentrations (10 - 7 - 10-SM) of the hormone significantly potentiated the response. The optimal enhancement was mediated by L-T4 at 10-6M. Our observation that L-T4 can potentiate the reactivities of T and B cells is in agreement with the observation by Chen (1980) who showed that L-T4 could promote the in vivo antibody response to a T cell dependent antigen, sheep erythrocytes. In contrast to the stimulatory effect of L-T4, L-T3 was found to exert no effect on the response of T cells except at 10-TM, which significantly depressed the response to PHA (Fig. 3, upper panel). The action of L-T3 on the response of B cells was predominantly suppressive (Fig. 3, lower panel). Parallel to our current observations in lymphocytes, a differential effect of L-T4 and L-T3 has also been observed in studies examining the ability of each hormone to stimulate the incorporation of amino acids into proteins in liver cells (Sokoloff, Roberts, Januska & Kline, 1968). Although deiodination of L-T4 has been shown to occur in lymphoid cells (Holm, Lemarchand-Ber~ud, Scazziga & Cuttelod, 1975), it is not known whether the conversion product is L-T3. Since L-T4 by itself is capable of potentiating the proliferative response of lymphocytes (Fig. 2), it is possible that the conversion of L-T4 to L-T3 may not be obligatory in mediating certain biological reactions. Reverse T3 was found to have a dose-dependent inhibitory effect on the rabbit lymphocyte response to PHA and Anti-L (Fig. 4). Its effect on the lymphocyte response to Con A was stimulatory at low concentrations, e.g. 10-11M, and inhibitory at higher concentrations, e.g. 10-s and 10-TM. In experiments where an enhancing dose of L-T4 and varying doses of rT3 were added to cell culture stimulated with PHA, Con A or Anti-L, the
inhibitory effect of rT3 was dominant (Fig. 5). Thus rT3 appears to be antagonistic to the action of L-T4 on lymphocyte reactivities. The opposing effect of rT3 on L-T4 action has also been reported in other studies. For example, rT3 was found to be capable of decreasing the calorigenic action of L-T4 (Pittman and Barker, 1959; Pittman, Tingley, Nickerson & Hill, 1960). Based on these findings and that of others, it is reasonable to suggest that rT3 may not be just an inactivation product of T4. Instead, rT3 may exert a regulatory role in modulating the biological action of L-T4. In the present study, rT3 was observed to be more potent than T3 in suppressing the lymphocyte response to mitogens and Anti-L (Figs 3 and 4). Other studies have also demonstrated rT3 to be either more potent or equally as effective as T3 in various biological systems. For example, rT3 is more potent than L-T3 in inhibiting cyclic nucleotide phosphodiesterase activity (Marcus, Lundquist & Chopra, 1975) as well as in stimulating erythropoiesis (Golde, Bersch, Chopra & Cline, 1976). The maximum effect of rT3 on growth hormone production and glucose consumption in cultured rat pituitary tumor cells is equal to that produced by L-T3 (Papavasiliou, Martial, Latham & Baxter, 1977). Thus, the effect of rT3 can impose a significant influence on specific biological activities. The physiological concentrations of different thyroid hormones in the circulation are equivalent to 10-TM for L-T4, and 10-gM each for L-T3 and rT3 (Larsen, 1978; Chopra, 1974). Results of the present study indicate that this physiological range of hormone levels can alter lymphocyte reactivities. Thus, L-T4 (10-TM) was found to potentiate the proliferative response of T and B lymphocytes (Fig. 2). Both L-T3 and rT3, at 10-gM, were able to suppress the B cell response to Anti-L (Fig. 3 and 4, lower panels). Furthermore, the same rT3 concentration (10-gM) was also found to suppress the T cell response to PHA (Fig. 4, upper panel) as well as to antagonize the potentiating effect of L-T4 on the PHA, Con A or Anti-L stimulated cell cultures (Fig. 5). These data suggest that circulating thyroid hormones, at physiological concentration may play a role in modulating lymphocyte functions. The exact mechanism by which thyroid hormones may influence the response of lymphocytes is not clear at present. However, lymphocytes have been demonstrated to possess nuclear receptors for T3 and T4 (Tsai & Samuels, 1974; Holm, Scazziga & Lemarchand-Ber~iud, 1976; Liewendahl, Rosenghrd & Lamberg, 1978). Thus the change in lymphocyte response induced by L-T4 (Fig. 2) and L-T3 (Fig. 3)
Alteration of Lymphocyte Reactivities may well be triggered by an interaction o f the thyroid h o r m o n e s with its specific receptors. In conclusion, based on the data presented in this study, it is clear that thyroid h o r m o n e s can alter the reactivities o f lymphocytes. The direction and magnitude o f alterations appear to d e p e n d on the
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concentration o f a specific thyroid h o r m o n e e n c o u n t e r e d by the responding cells. The implication o f these results with respect to altered lymphocyte function a m o n g patients suffering f r o m Graves' disease and other thyroid disorders remains to be established.
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