Effect of thymosin on the mitogenic responses of guinea pig thymocytes with different maturation stages

DEVELOPKENTALAND COWARATIVfI IXIKJNOLOGY, Vol. 3, pp. 489-498, 0145-305X179/030491-10$02.00/O Printed in the USA. Copyright (c) 1979 Pergamon Press Ltd. All rights reserved.

1979.

EFFECT OF THYMOSIN ON THE MITOGENIC RESPONSESOF GUINEA PIG THYMOCYTES WITH DIFFERENT MATURATION STAGES

E. Soppi, J. Eskola, A. L. Goldstein* and 0. Ruuskanen Departments of Anatomy, Medical Microbiology and Pediatrics, University of Turku, SF-20520 Turku 52, Finland, and *Division of Biochemistry, The University of Texas Medical Branch, Galveston, Texas, USA

ABSTRACT Unfractionated guinea pig thymocytes were preincubated for two hours with 14 different concentrations of thymosin fraction 5 and thereafter the thymocytes were stimulated with mitogens. Three concentrations (0.05, 10 and 200 us/ml) of thymosin significantly increased the PHA response of thymocytes; one concentration (0.05 ug/ ml) significantly increased the Con A response. The results were not due to thymosin-induced changes in the PHA or Con A dose-response curves. Thymosin fraction 5 was also tested on two subpopulations of thymocytes at different stages of maturation. Thymosin did not increase PHA or Con-A responses in the immature subpopulation. In contrast, it induced a significant inhibition of proliferation in these cells. Thymosin concentrations of 0.05 and 10 pg/ml significantly increased the PHA and Con A responses of the more mature subpopulation of thymocytes. The results support the concept that thymosin fraction 5 at concentrations of 0.05 and 10 ug/ml convert thymocytes into more mature PHA and Con A reactive cells. Higher concentration of thymosin (200 pg/ml) probably induces maturation of thymocytes to PHA reactive cells. These findings suggest that various concentrations of thymosin act to mature thymocytes at different stages of maturation; higher concentrations of thymosin are required to affect immature thymocytes than are required to affect more mature cells.

INTRODUCTION Extensive studies on the maturation

have been carried out on the effects of T cells both in vivo and in vitro.

of thymosin fraction The maturation in-

5

This research was supported by Turku University Foundation, The'Lllke Dy Research Foundation, and The National Cancer Institute Grant Number CA16964.

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duced by thymosin is expressed in augmented mitogenic responses ( I ) , graft versus host and mixed lymphocyte reactions ( I , 2) and suppressor cell activity (3). Thymosin converts human (4, 5) and murine bone marrow (6, 7), and murine spleen Thy-1 negative lymphocytes into more mature T cells (7, 8). In addition, the incubation of peripheral blood lymphocytes with thymosin increases the number of E-rosette forming cells in certain human diseases ( I , 9). Treatment of murine thymocytes with thymosin fraction 5 converts immature thymocytes into functionally immunocompetent cells in the murine system as judged by markedly enhanced one-way allogeneic mixed lymphocyte culture reaction (10). On the basis of earlier reports i t appears possible that at different stages of maturation the T cell precursors within the th~/mus require various concentrations of thymosin for biological effect (10, I I ) . In order to test this possibility, we studied the effect of preincubation with different concentrations of thymosin on the maturation of unfractionated and two subpopulations (immature and more mature) of thymocytes as reflected by PHA and Con A responses. In addition, the effect of thymosin was determined on the number of thymocytes with different histochemical activities of alkaline phosphatase (AP) which enzyme reflects guinea pig thymocytes at different degree of maturation (12). MATERIALS AND METHODS Animals: Outbred strain Dunkin-Hartley guinea pigs of both sexes, 2.5 - 3.5 months of age (experimentally determined, optimum) were used as experimental animals. Cell preparation: Guinea pigs were etherized, and thymuses were prepared using sterile techniques. All cell suspensions were prepared by teasing the thymuses with forceps in Hanks' balanced salt solution (HBSS, Orion Laboratories, Helsinki, Finland). The cells were washed once and counted using B~rker-T~rk's chamber. Total cell counts per thymus were 500-1000 x 106; 8595 % were viable determined by the trypan blue (0.25 %) exclusion test. All cell counts given are based on the number of viable cells. Histochemical demonstration of alkaline phosphatase activity: The calcium cobalt method (13) with B-glycerophosphate (Merck, Darmstadt, West Germany) as a substrate was used for the demonstration of alkaline phosphatase (AP) activity in cell smears by light microscopy. Thymocytes were classified as t o t a l l y AP negative (AP - ) , weakly AP positive (AP +) and strongly AP positive (AP ++) cells (14). Gradient centrifugation: In separation of thymocytes with different maturation stages, a method described earlier (14) was used with slight modifications. Discontinuous albumin gradients were centrifugated to equilibrium for 120 min. Cell fractions were harvested by aspiration, numbered from FI to F6 from the top to the bottom, washed twice with HBSS and resuspended in RPMI 1640 (Grand Island Biological Company, Grand Island, N.Y.) containing100 U/ml penicillin and 100 ~g/ml streptomycin. In order to obtain a sufficient number of cells the fractions 3 and 4 were combined and this population w i l l be referred to as F4. The cells of F4 and F6 were stimulated by PHA and Con A. After stimulation of thymocytes by PHA the ratio between the responses of the F4 and F6 cells higher than 20 was chosen as the criterion of successful separation of thymocytes at different maturation stages (14). Thymosin preparation: Thymosin fraction 5 (lot 11B) was used throughout the study. Lot BPM390 was also tested and the results were identical with those

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of lot 1lB. Thymosin was dissolved in distilled water, sterilized by Millipore (0.22 pm) filtration and aliquoted. Thereafter, thymosin was frozen at -7O'C until use, when it was diluted with RPM1 1640 to appropriate concentrations. Preincubation of thymocytes with thymosin: The unfractionated (UF), F4 and k6 thymocytes were suspended in RPM1 164D at a concentration of 10 x 106ce11s per ml and preincubated with 14 concentrations (0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5, 10, 50, 100, 200, 300 and 400 ug/ml) of thymosin in plastic tubes (no. 2001, Falcon Plastics, Los Angeles, Ca.) at 37OC in a humidified 5 % co2, 95 % air atmosphere. In preliminary experiments preincubation times of 30 min, 2 hr, 4 hr and 6 hr were used. The 30 min incubation of thymocytes was ineffective. The incubation time of 2 hr was equal to 4 hr and 6 hr and:.was used in further experiments. After incubation the cells were diluted and washed twice with large amounts of RPM1 1640. Before stimulation of the thymocytes with mitogens, cell smears were also prepared. Stimulation of thymocytes with PHA and Con A: tion of thymocytes with mitqgens was used (14). phytohemagglutinin (PHA M, Difco Laboratories, and 250 ug/ml and those of concanavalin A (Con Uppsala, Sweden) were 0.2, 1.0 and 5.0 pg/ml. cpm (corrected to the reference day of 5-iodo-2'Radiochemical Centre, Amersham) of incorporated

The micromethod for stimulaThe final concentrations of Detroit, Mich.) were 10, 50 A, Pharmacia Fine Chemicals, The results are ex ressed as 129 IUdR, The oxyuridine, l!@ IUdR/culture.

RESULTS Effect of different mitogen doses and thymosin concentrations on the PHA and Con A responses of unfractionated thymocytes: Unfractionated thymocytes were cultured wi'th three different doses of PHA and Con A after a two hour preincubation with 14 thymosin concentrations. The results are presented in Fig. 1. The thymosin pretreatment increased the PHA and Con A responses of thymocytes to optimal mitogen doses (50 pg/ml of PHA and 1 pg/ml of Con A). At sub- or supraoptimal mitogen doses thymosin was ineffective or the response was only slightly increased (Fig. 1). The results indicate that the incubation of thymocytes with various thymosin concentrations did not change their dose response curve to mitogens. Fig. 2 summarizes the results using optimal mitogen doses after preincubation with 14 different thymosin concentrations. The results confirm the findings in Fig. 1: different thymosin concentrations had various capabilities to increase the PHA and Con A response of the thymocytes. The arrows from B to F in Fig. 2 indicate the five critical thymosin concentrations; A Summarized results using these thymosin concenindicates the RPM1 control. trations are presented in Table I. Three thymosin concentrations, 0.05, 10.0 and 200 pg/ml, significantly augmented the response of unfractionated thymoOne of these thymosin concentrations, cytes to PHA stimulation (P < 0.05). 0.05 pg/ml, significant1 augmented the response to Con A (P < 0.05). In some animals (4 out of 9 3 a thymosin concentration of 10 pg/ml also significantly increased the Con A responses, but the concentration of 200 ug/ml was ineffective. None of the tested thymosin concentrations induced a significant inhibition in cell proliferation. The five th mosin concentrations (B = 0.05, C = 1.0, D = 2.5, E = 10.0 and F = 200 pg/ml f were chosen for further studies. Effect of thymosin on the PHA and Con A res onses of the two sub o ulations 0; thymocyteg: i t ymocyte su populations (14) were incubated with the five thymosin concen-

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The effect of preincubation with 14 different thymosin concentrations on the PHA dose response curve of unfractionated thymocytes. Each curve expresses the mean ~ s.e.m, of three animals.

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trations mentioned above. The summarized results are presented in Table I I . Thymosin concentrations of 0.05 and 10.0 ~g/ml increased significantly the responses of F4 th~mocytes to PHA (P < 0.01 and P < 0.05) and to Con A (P < 0.001 and P < 0.05), respectively. These concentrations are the same which significantly enhanced PHA and Con A responses of unfractionated thymocytes, although the Con A response was not significantly increased on every occasion after incubation with 10.0 ~g/ml thymosin. Thymosin at 200 ug/ml, which significantly augmented the PHA response of unfractionated thymocytes, did not affect markedly the mitogen responses of F4 thymocytes. None of the tested thymosin concentrations increased the PHA and Con A responses of F6 thymocytes (Table I I ) . On the contrary, thymosin concentrations of 2.5, 10.0 and 200 ug/ml, which significantly increased the PHA response of unfractionated thymocytes, caused a significant inhibition in the proliferation of F6 thymic cells. The inhibition was not due to the toxicity of thymosin because the thymosin concentrations used did not reduce the number of viable thymocytes during the 2 hr's preincubation period. Effect of different thymosin concentrations on the histochemical alkaline phosphatase activity of thymocytes: Preincubation of unfractionated thymocytes with thymosin did not change the number of AP- thymocytes. The number of AP+ thymocytes was slightly increased (from 23 ± 2 % to 27 ± 2) after pretreatment with thymosin (0.05 ~g/ml, Table I ) . Incubation of F6 thymocytes with thymosin induced no changes in the number of thymocytes with different AP activities. After incubation of F4 thymocytes with thymosin (0.05, 10.0 and 200 ug/ml) the number of AP++ cells, which are immature thymocytes, showed a slight tendency to decrease and correspondingly, the number of more mature thymocytes (AP+ cells) were increasing. However, these changes were not s t a t i s t i c a l l y significant (Table I I ) . DISCUSSION In this work the thymosin induced increases in the mitogenic responses of guinea pig thymocytes are of the same magnitude as those induced by thymopoietin in the murine thymocytes (15). Our findings support the concept that low concentration(s) of thymosin convert UF thymocytes into more immunocompetent PHA and Con A responsive cells. The results are in agreement with several previous findings. Thymic humoral factor (THF) (16) and human serum thymic factor (17) have been found to increase the resistance of murine thymic cells to the cytolytic effect of hydrocortisone indicating the conversion of thymic cortical cells into medullary thymocytes. In addition, thymosin has been shown to convert immature thymocytes into functionally immunocompetent cells as judged by enhanced allogeneic mixed lymphocyte reaction (10). In mice, lymphocytes at various stages of maturation react differently to PHA and Con A suggesting that these mitogens effect on different populations of lymphocytes (18, 19). Interestingly, THF increases more the murine lymphocyte response to PHA than that to Con A, and the dose required for augmentation of the PHA responsive population is higher (20, 21). In accordance with these studies our results demonstrate that the high concentration (200 ~g/ml) of thymosin converts thymocytes to PHA reactive cells and smaller concentrations increase also the Con A response of thymic cells. To study in more detail the effects of thymosin on the thymocytes undergoing differentiation, we studied the effects of thymosin preincubation not only on unfractionated cells, but also on the two subpopulations (F4 and F6) of thymocytes with different stages of maturation (14). Noneof the tested thymosin concentrations increased the mitogen responses of the immature F6 thymocytes. On the contrary, thymosin concentrations of 2.5, 10 and 200 ug/ ml induced an inhibition in the proliferation of these cells. The failure of

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thymosin to increase mitogenic response of the F6 thymocytes may reflect the lack of responsive thymocytes. On the other hand, the i n a b i l i t y of thymosin to increase the mitogenic response of F6 thymocytes may be due to the inhibitory effect of BSA on the proliferation of thymocytes (22). This does not necessarily mean that thymosin does not effect on the maturation of F6 cells; i t has been shown that the maturation of thymocytes does not require DNA synthesis or proliferation of cells (23). Two low concentrations of thymosin (0.05 and 10 ~g/ml) had an increasing effect on the mitogenic responses of both the unfractionated and the more mature, PHA and Con A responsive, F4 population. The high concentration (200 ~g/ml) of thymosin increased only the PHA response of UF thymocytes, but i t had no significant effect on the mitogenic responses of the F4 thymocytes. Apparently therefore the F4 population does not contain cells susceptible to the effect of this high concentration of thymosin, but these cells belong to the more immature population of thymic cells. The results support the sequence of maturation suggested by Cohen (10), Cantor (11) and their colleagues; the amount of thymosin required to affect immature thymocytes is higher than that required to affect more mature thymocytes. I t appears further that such a high concentration of thymosin used in the in v i t r o experiments of this work is found in vivo only at the site of production, the thymus, and the effects of the high concentration of thymosin robably reflect the f i r s t step(s) of intrathymic lymphoid cell maturation 11). On the other hand, one component (thymosin ~i) in the thymosin fraction 5 is not able to produce all the effects of fraction 5 (24), which contains more than 30 components. Therefore, the effects of different thymosin concentrations may also reflect the optimal doses of active components in the thymosin fration 5 as proposed by Stutman (25). However, in spite of the complex nature of the fraction 5 i t has high specificity. Control fractions prepared identical to the thymosin fraction 5 from spleens (2, 5, 6, 26) or other organs (27) have been shown to be ineffective in various test systems including mitogen responses (5, 27). In guinea pigs, cortical thymocytes exhibit on their cell surface a high activity of alkaline phosphatase which is diminished or lost during maturation into medullary thymocytes or peripheral T cells (12, 14, 28). Incubation of thymocytes with thymosin ind6ced no s t a t i s t i c a l l y significant changes in the numbers of thymocytes with different histochemical activities of AP (Tables I and I I ) . The results are in agreement with the previous reports where simultaneous aquisition of T cell surface markers and PHA and Con A responses was not either observed (5, 29). Altogether, our findings indicate that thymosin plays an important role in the intrathymic differentiation and maturation of thymocytes. Thymosin seems to convert thymocytes into functionally more immunocompetent cells as judged by their increased PHA and Con A responses. In addition, various concentrations of thymosin most probably increase the mitogenic responses of thymocytes which belong to different subpopulations of thymocytes; high concentration of thymosin affects immature thymocytes and lower concentrations affect more mature thymic cells.

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ACKNOWLEDGEMENTS We are grateful to Mrs. Marjo Ingman for excellent technical assistance. We are very much indebted to Dr. Auli Toivanen, who gave invaluable constructive criticism in revising this manuscript.

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REFERENCES GOLDSTEIN,A. L., THURMAN,G. B., COHEN, G. H., and HOOPER, J. A. The role of thymosin and the endocrine thymus on the ontogenesis and function.of T-cells. In: Molecular Approaches to In~nunology. New York: Academic Press, 1975, p. 243. GOLDSTEIN,A. L., GUHA, A., HOWE, M. L., and WHITE, A. Ontogenesis of cell-mediated immunity in murine thymocytes and spleen cells and its acceleration by thymosin, a thymic hormone. J. In~nunol. 106, 773, 1971. HOROWITZ,S., BORCHERDiNG,W., MOORTHY,A. V., CHESNEY, R., SCHULTEWISSERMANN, H., HONG, R., and GOLDSTEIN, A. Induction of suppressor T cells in systemic lupus erythematosus by thymosin and cultures thymic epithelium. Science. 197, 999, 1977. TOURAINE,J. L., INCEFY, G. S., TOURAINE, F., RHO, Y. M., and GOOD, R.A. Differentiation of human bone marrow cells into T lymphocytes by in vitro incubation with thymic extracts. Olin. Exp. In~nunol. 17, 151, 1974. TOURAINE,J. L., HADDEN,J. W., and GOOD, R. A. Sequential stages of human T lymphocyte differentiation. Proc. Natl. Acad. Sci. USA. 74, 3414, 1977.

BACH, J.-F., DARDENNE,M., GOLDSTEIN, A. L., GUHA, A., and WHITE, A. Appearance of T-cell markers in bone marrow rosette-forming cells after incubation with thymosin, a thymic hormone. Proc. Natl. Acad. Sci. USA. 68, 2734, 1971. 7. SCHEID,M. P., HOFFMAN,M. K., KOMURO,K., H~MMERLING,U., ABBOTT, J., BOYSE, E. A., COHEN, G. H., HOOPER,J. A., SCHULOF, R. S., and GOLDSTEIN, A . L . Differentiation of T cells induced by preparations from thymus and by nonthymic agents. The determined state of the precursor cell. J. Exp. Med. 138, 1027, 1973. 8. KOMURO,Ko, and BOYSE, E. A. In-vitro demonstration of thymic hormone in the mouse by conversion of p~ecursor cells into lymphocytes. Lancet. I, 740, 1973. 9. MOUTSOPOULOS,H., FYE, K. H., SAWADA, S., BECKER, M. J., GOLDSTEIN, A., and TALAL, N. In vitro effect of thymosin on T-lymphocyte rosette formation in rheumatic diseases. Olin. Exp. InTnunol. 26, 563, 1976. I0. COHEN, G. H., HOOPER, J. A., and GOLDSTEIN, A. L. Thymosin-induced differentiation of murine thymocytes in allogeneic mixed lymphocyte cultures. Ann. N.Y. Acad. Sci. 249, 145, 1975. II. CANTOR, H., and WEISSMAN, I. L. Development and function of subpopulations of thymocytes and T lymphocytes. Progr. Allergy. 20, I, 1976. 12. RUUSKANEN, 0., and KOUVALAINEN, K. Differentiation of thymus and thymocytes. A study in foetal guinea-pigs using alkaline phosphatase as a label of thymocytes. Immunology. 26, 187, 1974. 13. PEARSE, A. G. E. In: Histochemistry: Theoretical and Applied. 3rd ed. London: J. & A. Churchill Ltd., 1968, p. 710. 14. SOPPI, E., RUUSKANEN,0., ESKOLA, J., FRkKI. J., and KOUVALAINEN, K. Two subpopulations of guinea pig thymocytes with different maturation stages. Immunology. 33, 343, 1977. 15. BASCH, R. S., and GOLDSTEIN, G. Thymopoietin-induced aquisition of responsiveness to T cell mitogens. Cell. I~nunol. 20, 218, 1975. 6.

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16.

TRAININ, N., LEVO, Y., and ROTTER, V. Resistance to hydrocortisone conferred upon thymocytes by a thymic humoral factor. Eur. J. Immunol. 4, 634, 1974.

17.

ASTALDI, G. C. B., ASTALDI, A., GROENEWOUD,M., WIJERMANS, P., SCHELLEKENS, P. Th. A., and EIJSVOOGEL, V. P. Effect of a human thymic factor on hydrocortisone-treated thymocytes. Eur. J. Immunol. 7, 836, 1977. STOBO,J. D., and PAUL, W. E. Functional heterogenity of murine lymphoid cells. I I I . Differential responsiveness of T-cells to phytohemagglutinin and concanavalin A as a probe to T cell subsets. Immunology. 110, 362, 1973.

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JACOBSSON,H., and BLOMGREN,H. Responses of mouse thymic cells to mitogens. A comparison between phytohaemagglutinin and concanavalin A. Cell. Immunol. 11, 427, 1974.

20.

ROTTER,V., and TRAININ, N. Increased mitogenic reactivity of normal spleen cells to T lectins induced by thymus humoral factor (THF). Cell. I ~ u n o l . 16, 413, 1975.

21.

ROTTER,V., and TRAININ, N. Effect of thymic hormone on the response of different lymphoid cell populations to T mitogens. I s r a e l J. Med. Sci. 13, 363, 1977.

22.

PIGUET, P.-F., DEWEY, H. K., and VASSALI, P. Synergistic and suppressive interactions among mouse T lymphocytes in the response to phytohemagglutinin. J. Exp. Med. 142, 1591, 1975.

23.

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