Thymic hormone containing cells—IX. Steroids in vitro modulate thymulin secretion by human and murine thymic epithelial cells

J.s/mdEioche~. Vol. 30,No. l-6,pp.479-484,1988

0022-4731/88 $3.00+0.00

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THYMIC HORMONE CONTAINING CELLS-IX. STEROIDS IN VITRO MODULATE THYMULIN SECRETION BY HUMAN AND MURINE THYMIC EPITHELIAL CELLS W. SnvINo*t, E. BARTOCCIONI*,F. HOMO-DELARCHE*,M.CI. GAGNERAULT*,T. ITOHS and M. DARDENNE*$ *INSERM U25 and CNRS UA I22 , HBpital Necker, 75743 Paris Cedex 15,France and /Department of Anatomy, Tohoku University School of Medicine, Seiryo-Cho, Sendai 980, Japan Summary-We investigated the in vitro effects (kinetics and dose-response) of adrenal and sexual steroid hormones on the secretion of thymulin, a thymic hormone, by human thymic epithelial cells in primary cultures as well as in a rat epithelial cell line. We demonstrated that all steroids tested, in a range of physiological doses, stimulated thymulin production to various extents. Progesterone and estradiol, however, were revealed to be the most efficient. Specific steroid antagonists abrogated the steroid-induced stimulation of thymulin production. These findings confirm our previous in viva results and demonstrate that steroid hormones can act directly on thymic epithelial cells to modulate their endocrine production.

INTRODUCTION

now well established that thymic hormones play a role in intra- and extra-thymic steps of T cell differentiation [ 11. These hormones are polypeptides, and in some of them amino acid sequences have already been determined [2-41. Concerning thymulin, one of the well-defined thymic hormones, we have previously demonstrated that the peptide is biologically active when coupled with zinc [5] in an equimolecular ratio [6]. This zinc-containing tridimensional conformation of thymulin is quite specific, as revealed by NMR analysis [7], and yields a new monoclonal antibody-defined epitope [8]. Data obtained by several groups definitely demonstrated that thymic epithelial cells (TEC) are responsible for thymic hormone secretion [9-121, and actually the same TEC appear to simultaneously release distinct thymic hormones [ 131. Immunocytochemical data using both polyclonal and monoclonal antibodies revealed that thymulin (and/or its precursor) is stored within cytoplasmic vacuoles [ 141. Recent findings after two-dimensional immunoblot analysis suggest the existence of high molecular weight precursor molecules [16] that are synthesized at the rough endoplasmic reticulum, then move towards the Golgi complex, from which secretory vesicles (into which zinc is incorporated) are released, eventually fusing with the cell membrane, thus characterizing the exocytosis of the hormone [ 171. The mechanisms controlling thymulin secretion appear to be multi-factorial. In a first set of experiIt is

Proceedings of the 8th International Symposium of The Journal of Steroid Biochemistry “Recent Advances in Steroid Biochemistry” (Paris, 24-27 May 1987). tPresent address: Department of Immunology, Institute Oswald0 Cruz, Fiocruz, Mangbuinhos, Av. Brazil 4365, Cx Postal 926, 21040 Rio de Janeiro, Brazil. $To whom correspondence should be addressed. 479

ments we experimentally depleted circulating thymulin by either injecting exogenous anti-thymulin monoclonal antibodies or immunizing mice with synthetic thymulin (coupled to bovine serum albumin). These procedures resulted in an increase in the thymic production of the hormone, indicating that a feedback system controlling thymulin secretion was triggered [ 181. These data were further confirmed in vitro using primary cultures of human TEC [ 191. Nonetheless, thymulin secretion could also be influenced by the endocrine system. For example, we showed that in vivo treatment of normal rats or mice by thyroid hormones augmented the production of thymulin, whereas the injection of propyl-thiouracyl, an inhibitor of thyroid hormone biosynthesis, promoted the opposite effect [20]. The results by Fabris et a1.[21] are similar. Using T3 injections, they succeeded in restoring thymulin levels in old mice to the values found in young animals. In addition, the same group demonstrated that hypothyroidian subjects present low circulating levels of thymulin and conversely hyperthyroidian individuals have thymulin seric level higher than age-matched controls [22]. Concerning the influence of steroid hormones on thymulin secretion, we recently reported a series of in vivo experiments showing that the ablation of either gonads or adrenals resulted in a transient decrease in the circulating levels of thymulin which returned to normal values 1 month post-surgery [23]. Interestingly, the numbers of thymulin-containing cells initially increased within the thymus and then gradually returned to the normal range after 1 month.. This bulk of evidence again suggested that a feedback response was triggered. Such response was also observed when the two operations (castration and adrenalectomy) were performed simultaneously. However, it took 3-4 months to return to normal values of thymulin secretion [23]. All the effects described above were prevented when the operated

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Chemical Co., St Louis, MO. RU 38486 with both antiglucocorticoid and antiprogestative properties was a gift from Roussel UCLAF (Paris). All these agents were dissolved in ethanol to make 10e3 M stock solutions. The final concentration of ethanol in the samples was inferior or equal to 0.1 O/aand did not itself modify cell secretion.

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Fig. 1. Dose dependency of steroid hormone effects on thymulin secretion by human (upper panel) and rat (lower panel) thymic epithelial cells. Each value represents the average ( f SE) of four different experiments at day 8 and day 6 for human and rat epithelial cells respectively. Thymulin levels are expressed as log-2 reciprocal titer of the highest active dilution.

animals received the appropriate hormonal replacement. In spite of the data showing that the reticuloepithelial matrix of the thymus and/or TEC contain specific receptors for glucocorticoid [24] and sexual hormones [25-271, it could be questioned whether the above-described effects resulted from the direct steroid action on TEC, or from an effect on another cell type which in turn might respond eventually modulating TEC endocrine function. In order to further approach this question, we analysed the in vitro effects of adrenal or sexual steroid hormones on the secretion of thymulin, using primary cultures of human TEC or a rat TEC line as experimental models. In the present investigation, we show evidence that both human and murine cultured TEC can be influenced by the addition of various steroid types, in their ability to enhance the production of thymulin. These data clearly demonstrate that steroid hormones act directly on thymic epithelial cells and modulate their endocrine function. EXPERIMENTAL

Reagents

Culture medium (RPM1 1640) and supplements (Lglutamine and penicillin-streptomycin solutions) were obtained from Gibco and fetal calf serum (FCS) as well as trypsin-EDTA from Flow Lab. The steroids, dexamethasone, 17P-estradiol, progesterone and testosterone, as well as the estradiol antagonists, tamoxifen and nafoxidine, were obtained from Sigma

The anti-thymulin monoclonal antibody (MAb) was raised after immunization with synthetic thymulin coupled to BSA [28]. This antibody was proved to specifically bind to murine and human TEC [29,30]. The fluorescent second antibody used was a GAMI FITC (goat serum anti-mouse Ig, coupled to fluorescein isothiocyanate), provided by Biosys (Compiegne, France). For the experiments with human TEC cultures, an antikeratin rabbit immune serum (Clinisciences, Paris) and its respective fluorescent second antibody (GAR/TRITC; tetramethylrhodamineisothiocyanate-labeled goat-anti rabbit immunoglobulins) were used. Cultures of thymic epithelial

cells

Primary cultures of human TEC were established according to the protocol initially devised by Papiernik et a1.[31]. Briefly, thymus fragments (kindly provided by Dr J. Y. Neveux) from children undergoing cardiac surgery were minced with fine scissors and left to adhere in slide flasks (Nunc Co., Denmark). The culture medium was RPM1 1640, supplemented with 1% L-glutamine 1% sodium pyruvate, penicillinstreptomycin 2% and 10% FCS. The primary cultures of human TEC were allowed to grow for 4 days before steroid treatment. Steroid hormones and/or their antagonists were added daily and the culture medium was also replaced daily. The rat TEC line IT 45Rl was established by Itoh et a1.[32]. It is able to promote proliferation of prethymocytes [33] and to produce thymic hormones [34]. The epithelial nature of this cell line was determined by the presence of desmosomes and tonofilaments [32] and by its keratin content 1341. The cells were grown in 75-ml culture flasks, as previously described for human epithelial cells. On day 4 after plating, they were incubated for 10 min in PBS and then for 1Omin in trypsin-EDTA (0.05% trypsin l/250 and 0.02% EDTA). The cells were washed in RPM1 medium supplemented with 10% FCS, counted and put into slide flasks (25,000 cells per flask, 2 ml complete medium) where they were left to adhere for 24 h. Steroids were then added as described for human epithelial cells. Kinetics of the steroid effect was followed by daily removal of the medium, with or without the different drugs tested. Immunojluorescence

technique

The classical indirect immunofluorescence procedure was used. Cultured cells were fixed in absolute methanol for 5 min, washed in phosphate-buffered saline (PBS) for 15 min and subjected to the anti-

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oaysof CUltWe Fig. 2. Kinetics of steroid stimulatory effects on thymulin production by human (left panel) and rat (right panel) TEC. Cells were treated with vehicle alone (control) (o--o) lo-’ M dexamethasone (A-A), 10-s M progesterone (A-A), testosterone (o-o), or 17/I-estradiol (o-o). The results represent the mean (k SE) of six different experiments, three individual determinations are performed by experiments.

thymulin Mab. After a 30-min incubation, the cultures were again washed in PBS and exposed to the fluorescent second antibody for 30 min. A further PBS wash was carried out and the specimens were mounted under coverslides to be analysed. For each experiment, at least 200 cells (grown in at least two distinct coverslides) were analysed. A given experiment was carried out 2-3 times.

Thymulin levels The levels of thymulin were measured in culture supernatants according to the rosette inhibition bioassay as detailed elsewhere [35]. This test is based on the ability of natural or synthetic thymulin to render splenocytes from thymectomized mice sensitive to azathioprine or anti-Thy-l serum. Results were expressed as the log of the highest reciprocal dilution able to produce rosette inhibition.

in human primary cultures [ 191 and from day 3 to 8 after plating in rat TEC cultures [34]. The addition of steroids significantly stimulated thymulin production in human as well as rat epitheha1 cell supematants. This stimulation was already

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In vitro release of thymulin after steroid hormone action The dose-dependency of the steroid hormone enhancement effect on thymulin secretion by primary cultures of human epithelial cells is shown in Fig. 1 (upper panel) for each steroid tested. The results show that the in vitro stimulation of thymulin secretion was observed in the range of physiological concentrations for all steroids tested, with no significant difference between males and females (results not shown). In addition, progesterone and estradiol appeared to be the most efficient in stimulating epithelial cell production. Similar results were obtained with pure rat epithelial cell cultures (Fig. 1, lower panel). Kinetics of the steroid effect on thymulin secretion by human and rat TEC are shown in Fig. 2. As already demonstrated the secretion of thymulin into the culture medium by the thymic epithelial cells increases gradually as a function of time, from day 5 to day 10

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Fig. 3. Effect of steroid antagonists on the steroid-induced stimulation of thymulin production by human TEC. The cells were incubated in the absence (control) (0) or presence of 1O-’ M dexamethasone (Dex) (a), 10e8 M progesterone (Pro) (H), lo-* M 17B-estradiol (E), (a) either alone or in association with respective antagonists: RU 38486 used at 10V6 and lo-’ M in the presence of dexamethasone (1) and progesterone (&Z?j)as well as 1O-’ M tamoxifen and 10e6 M nafoxidine in the presence of 17/J-estradiol (B, El respectively). Antagonists were incubated alone at the concentrations: RU 38486 at lo-‘M (1) and 10e6M (m), tamoxifen lo-‘M (B) and nafoxidine 10m6 M (a).

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Fig. 4. Percentage of thymulin-containing cells in human TEC, at day 6 of the culture, in absence (control) (C) or presence of iOm8M progesterone, (Pro), 17rSestradiol (E2f. testosterone (Testo), or IO-’ M dexamethasone (Dex). Each value represents the average of three different experiments. In each experiment, at least 200 cells are analysed.

significant at days 6 and 4 in human and rat supematants respectively, and persisted until the end

of the culture. The specificity of the stimulation observed was assessed by studying the effects of various steroid antagonists. We used RU 38486, an antagonist of glucocorticoid [36] as well. as progesterone action [ 371, and tamoxifen [ 381 and nafoxidine [ 391 which exhibit anti-oestrogen properties. As shown in Fig. 3, these compounds did not exert, when used alone at 1Oa6and 1O-’ M for RU38486, and 1O-’ and 10m6M for tamoxifen and nafoxidine respectively, any inhibition of thymulin secretion but, when added to steroids in the culture medium abrogated the stimulation induced by 1O-’ M dexamethasone, 1O-* M progesterone and 1Ovs M 17~-estradiol, respectively (Fig. 3).

of thymulin

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As previously mentioned, the percentage of thymulin-positive cells, counted by indirect immunofluorescence, among the keratin-positive cell population increases gradually in control human TEC cultures from day 5 to day 12. The percentage of positive cells reaches approx. 20°h on day 550% on day 8 and 80~ on day 12, in parallel with thymulin activity in the supernatants [ 191. The immunohistochemical analysis on cultures of thymic epithelial cells that had grown under steroid influence revealed a significant increase in the percentage of thymulin-containing cells, already detectable at day 6 of the culture of human TEC. This increase was in strict parallelism with the levels of thymulin released in the culture supernatants (Fig. 4). Again, progesterone appeared to be the most efficient in increasing the number of thymulin-producing cells. In addition, the fluorescent labeling observed with the anti-thymulin MAb (Fig. 5a) was also more pronounced in steroid-treated cells than in control cultures (Fig. Sb). As described above, the steroid effects on intracellular thymulin contents were abrogated by the addition of appropriate antagonists to the culture medium. DISCUSSION The findings reported here represent strong evidence that steroid hormones can exert a stimulatory effect in vitro on the secretion of the thymic hormone, thymulin, by cultured thymic epithelial cells. Moreover, our data extend previous in vivo results in which we showed that surgical abrogation of steroid-producing glands, namely adrenals and/or gonads, promotes a transient depletion of seric thymulin levels [24]. These results also demonstrate that the different types of steroid hormones can act directly on thymic

Fig. 5. Immunotluorescence labeling of the rat thymic epithelial cell line (IT-45Rl) with the anti-thymulin monoctonal antibody. On day 4 of culture, (a) shows a progesterone-treated culture, revealing a much higher number of labeled cells as compared with control culture (b). x 600.

In vitro modulation of thymulin secretion by steroids

epithelial cells, causing them to increase thymic hormone production. In our hands, physiological concentrations of glucocorticoid and sexual hormones were quite efficient in stimulating thymic epithelial cells. These findings are in keeping with previous demonstration of the presence of estrogen and androgen receptors in the cytosol of the reticuloepithelial matrix of the rat thymus[25,26,40], whereas progesterone and glucocorticoid receptors have been more precisely localized in thymic epithelial cells [24,27]. Recently, we were able to characterize androgen receptors in primary culture of human with high affinity epithelial cells thymic (&= 1.5 x 10e9M) and low capacity (not shown), Moreover, the use of steroid receptor antagonists such as RU 38486, with both antiglucocorticoid and antiprogestative properties and of tamoxifen and nafoxidine with antiestrogenic properties were shown to be able to counteract the effect of dexamethasone and progesterone, as well as of estradiol respectively. In this regard, the effect of in viva estradiol treatment on the lymphoid cell population of the fetal guineapig thymus has also been shown to be antagonized by tamoxifen [41]. A last point to be underlined is that the different types of steroids tested act in the same way both in vivo[23] and in vitro and that, among them, progesterone appears very effective. If the role of androgens and estrogens in the immune system is well documented 142-441, the action of progesterone is less well defined, despite its possible role in thymusrelated immunosuppression in pregnancy ]45]. It is interesting to note that a striking parallelism between thymus and uterus has been described, for various manipulations, such as estradiol ti progesterone treatment (oestrus, pregnancy and pseudopregnancy) upon tissue levels of progesterone and their distribution between cytosol and nuclear compartments [45]. Progesterone receptors have also been characterized in the bursa of Fabricius of normal and obese strain chickens spontaneously developing autoimmune thyroiditis [46]. Taken together, these data may emphasize the role of progesterone in the immune system. Finally, as recently reported, thymocyte function is regulated by thymic serum factors, which are modulated by gonadal steroids [47-491. As the source of various thymic factors appears to be TEC, a tentative link between sex steroids, release of various thymic factors and thymocyte function may be established. In conclusion, these findings indicate that in viva steroids are likely to play a role in the complex multifactorial system for controlling thymulin production and release by TEC. Moreover, our results lead to the general concept that the thymic epithelium should be considered a physiological target tissue for both adrenal and sexual steroid hormones. Acknowledgements-The authors wish to thank V. Alves and J. Coulaud for their skilful technical assistance and Mrs D. Broneer for reviewing the manuscript.

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REFERENCES 1. Bach J. F. (Ed.): Thymic hormones. Clin. Immunol. Allergy. Vol. 3 (1983) pp. 133-157. 2. Goldstein A. L., Low T. L. K., MacDoo M., McClure J., Thurman G., Rossio J., Lai C., Chang D., Wang S., Harvey C., Ramel A. H. and Melenhofer J.: Thymosin crl: isolation and sequence analysis of an immunologically active thymic polypeptide. Proc. natn. Acud. Sci. U.S.A. 74 (1977) 725-729. 3. Schlesinger D. H. and Goldstein G.: The amino acid sequence of thymopoietin II. Cell 5 (1975) 36 l-365. 4. Bach J. F., Dardenne M., P&au J. M. and Rosa J.: Biochemical characterization of a serum thymic factor. Nature 266 (1977) 55-57.

5. Dardenne M., Pleau J. M., Nabarra B., Lefrancier P., Derrien M., Choay J. and Bach J. F.: Contribution of zinc and other metals to the biological activity of the serum thymic factor. Proc. natn. Acad. Sci. U.S.A. 79 (1982) 5370-5373. 6. Gastinel L., Dardenne M., Pleau J. M. and Bach J. F.: Studies on the zinc binding to the serum thymic factor. Biochim. biophys. Acta 797 ( 1984) 147-l 55. 7. Laussac J. P., Haran R., Dardenne M., Lefrancier P. and Bach J. F.: Nuclear magnetic resonance study on the interaction of zinc with thymulin. 0. hebd. SPanc. Acad. Sci. Paris, series III, 30 (1985) 471-475. 8. Dardenne M., Savino W., Berrih S. and Bach J. F.: A zinc-dependent epitope on the molecule of thymulin. Proc. natn. Acad. Sci. U.S.A. 82 (1985) 7035-7038. 9. Monier J. C., Dardenne M., Pldau J. M., Schmitt P., Deschaux P. and Bach J. F.: Characterization of the “facteur thymique serique” (MS) in the thymus. I. Fixation of anti-FTS antibodies on thymic reticulum epithelial cells. C&n. exp. Zmmun. 42 (I 980) 470-476. 10. Jambon B., Montagne P., Bene M. C., Brayer M. P., Faure G. and Duheille J. F.: ImmunohistoIogic localization of “facteur thymique strique” (FIS) in human thymic epithelium. 1. Immun. 127 (198 1) 2055-2059. II. Dalakas M., Engel W. K., McLure J. E., Goldstein A. L. and Askanas V.: Immunocytochemical localization of thymosin (~1 in thymic epithelial cells of normal and myasthenia gravis patients and in thymic cultures. J. neur. Sci. 50 (1981) 239-247. 12. Haynes B. F., Shimisu K. and Eisenbarth G. E.: Identification of human and rodent thymic epithelium using tetanus toxin and monoclonal antibody A2B5. J. cl&r. Invest. 71 (1983) 9-14. 13. Savino W. and Dardenne M.: Thymic hormone containing cells. VI. Immunohistolog~c evidence for the simultaneous presence of thymulin, thymopoietin and thymosin oil in normal and pathological human thymuses. Eur. J. Immun. 14 (1984) 987-991.

14. Schmitt D., Monier J. C., Dardenne M., Pleau J. M., Deschaux P. and Bach J. F.: Cytoplasmic localization of FTS (facteur thymique serique) in thymic epithelial cells. An immunoelectron microscopic study. Thymus 2 (1980) 177-186.

15. Auger C., Monier J. C., Savino W. and Dardenne M.: Localization of thymulin (FTS-Zn) in mouse thymus. Comparative data using monoclonal antibodies following different plastic embedding procedures. Biol. CeZZ. 52 (1984) 139-146.

16. Savino W., Gastinel L. N., Wolff A. and Dardenne M.: Immunochemical characterization of anti-thymulin monoclonal antibody defined molecules in human thymic epithelium. C.r. hebd. Seanc. Acad. Sci. Paris, series III, 301 (1985) 577-579. 17. Savino W. and Dardenne M.: Thymic hormone containing cells. VIII. Effects of colchicine, cytochalasin B and monensin on the secretion of thymulin by cultured human thymic epithelial cells. J. Nistochem. Cytochem. 34 (1986) 1719-1725.

18 Savino W., Dardenne

M. and Bach J. F.: Thymic

484

19.

20.

21.

22. 23.

24.

25.

26.

27.

28.

29.

30.

31. 32.

33.

34.

W.

S~vIt-40 et al.

hormone containing cells. III. Evidence for a feedback regulation of the secretion of the serum thymic factor (FIS) by thymic epithelial cells. Chn. exp. Immun. 52 (1983) 7-12. Cohen S., Berrih S., Dardenne M. and Bach J. F.: Feedback regulation of the secretion of a thymic hormone (thymulin) by human thymic epithelial cells in culture. Thymus 8 (1986) 109-l 19. Savino W., Wolff B., Arab-Spire S. and Dardenne M.: Thymic hormone containing cells. IV. ~uctuations in the thyroid hormone levels in vivo can modulate the secretion of thymulin by the epithelial cells of young mouse thymus. Clin. exp. Immun. 55 (1984) 629-635. Fabris N. and Mocchegiani E.: Endocrine control of thymic serum factor production in young adult and old mice. Cell Immun. 91 (1985) 325-335. Fabris N., Mocchegiani S., Pacini F. and Pinchera A.: Thyroid function modulates thymic endocrine function, Clin. Endocr. Metab. 62 (1986) 474-478. Dardenne M., Savino W., Duval D., Kaisedian D., Hassid J. and Bach J. F.: Thymic hormone containing cells. VII. Adrenals and gonads control the in viva secretion of thymulin and its plasmatic inhibitor. J. Immun. 136 (1986) 1303-I 308. Dardenne M., Itoh T. and Homo-Delarche F.: Presence of glucocorticoid receptors in cultured thymic epithelial cells. Cell Immun. 100 (I 986) 112-l 18. Grossman C. .I., Sholiton L. J. and Nathan P.: Rat thymic estrogen receptor. I. Preparation, localization and physiological properties. J. steroid Biochem. 11 (1979) 1233-1240. Grossman C. J., Nathan P., Taylor B. B. and Sholiton L. J.: Rat thymic dihydrotestosterone receptor. Preparation, localization and physiolo~c~ properties. Steroid 34 (1979) 539-553. Sakabe K., Seiki K. and Fuju-Hanamoto H.: Histochemica1 localization of progestin receptor cells in the rat thymus. Thymes 8 (1986) 97-107. Dardenne M., PlCau J. M., Savino W. and Bach J. F.: Monoclonal antibody against the serum thymic factor (ITS). Immun. Lett. (1982) 79-83. Savino W., Dardenne M., Papiernik M. and Bach J. F.: Thymic hormone containing cells. Characterization and localization of serum thymic factor in young mouse thymus studied by monoclonal antibodies. J. exp. Med. 156 (1982) 628-633. Berrih S., Savino W., Azoulay M., Dardenne M. and Bach J. F.: Production of anti-thymulin (ITS) monoclonal antibodies by immunization against human thymic epithelial cells. L Histochem. Cytochem. 32 (1984) 432-438. Papiernik M., Nabarra B. and Bach J. F.: In vitro cultures of functional thymic epithelium. Clin. exp. Immun. 19 (1985) 281-287. Itoh T., Aizu S., Kasahara S. and Mori T.: Establishment of a functional epithelial cell line from the rat thymus. A cell line that induces the differentiation of rat bone marrow cells into T cell lineage. Biomed. Res. 2 (1981) 1l-19. Itch T., Kasahara S. and Moni T.: A thymic epithelial cell line, IT-45R1, induces the differentiation of prethymic progenitor cells into post-thymic cells through direct contact. Thymus 4 (1982) 69-75. Savino W., Itoh T., Imhof B. A. and Dardenne M.: Immunohistochemicai studies on the phenotype of

35.

36.

37.

38.

39.

40.

41.

42.

43. 44.

45.

46.

47.

48.

49.

murine and human thymic stromal cell lines. Thymus 8 (1986) 245-257. Dardenne M. and Bach J. F.: The sheep red blood cell rosette assay for the evaluation of thymic hormones. In Biological Activity of Thymic Hormones (Edited by Van Bekkum). Kooyer Scientific Publ., Leiden (1975) pp. 235-243. Duval D., Durant S. and Homo-Delarche F.: Effect of anti~ucoco~icoids on dexame~asone-induced inhibition of uridine inco~oration and cell lysis in isolated mouse thymocytes. J. steroid Biochem. 20 (I 984) 283-287. Horwitz K. B., Wei L. W., Sedlack S. M. and d’Arville C. N.: Progestin action and progesterone receptor structure in human breast cancer: a review. In Recent Progesss in Hormone Research. Academic Press, London, Vol. 41 (1985) pp. 249-316. Horwitz K. B., Koseki Y. and McGuire W. L.: Estrogen control of progesterone receptor in human breast cancer: role of estradiol and antiestrogen. Endocrinoiogy 103 (1978) 1742-1751. Butler W. B., Kelsey W. H. and Goran N.: Effects of serum and insulin on the sensitivity of the human breast cancer cell line MCF-7 to estrogen and antiestrogens. Cancer Res. 41 (198 I) 82-88. Stimson W. H.: Sex steroids, steroid receptors and immunity. In Hormones and Immunity (Edited by I. Berczi and K. Kovacs). MTP Press, Lancaster (1987) pp. 43-53. Gulino A., Screpanti I. and Pasqualini J. R.: Estrogen and antiestrogen effects on different lymphoid cell populations in the developing fetal thymus of guineapig. Endocrinology 113 (1983) 1754-l 762. Nelson J. L. and Steinberg A. D.: Sex steroids, autoimmunity and autoimmune diseases. In hormones and Immunity (Edited by I. Berczi and K. Kovacs). MTP Press, Lancaster (1987) pp. 93-119. Grossman C. J.: Regulation of the immune system by sex steroids. Endocr. Rev. (1984) 435-455. Ahmed S. A., Penhale W. J. and Talal N.: Sex hormones, immune responses and autoimmune diseases. Mechanisms of sex hormone action. Am. J. Path. 121 (1985) 531-551. Pearce P. and Funder J. W.: Cytosol and nuclear levels of thymic progesterone receptors in pregnant, pseudopregnant and steroid treated rats. J. steroid Biochem. 25 (1986) 65-69. Fiissler R., Schwarz S., Dietrich H. and Wick G.: Sex steroid and ghtcocorticoid receptors in the bursa of Fabricius of obese strain chickens spontaneously developing autoimmune thyroiditis. .I. steroid B&hem. 24 (1986) 405-411. Stimson W. H., Crilly P. J. and McGudden A. B.: Effects of the sex steroid on the synthesis of immunoregulatory factors by thymic epithelial cell cultures. Immunology 44 (1981) 401-407. Grossman C. J., Sholiton L. J. and Roselle G. A.: Estradiol regulation of thymic lymphocyte function in the rat: mediation by serum thymic factors. J. steroid Biochem. 16 (1982) 683-690. Grossman C. J., Sholiton L. J. and Rosette G. A.: Dihydrotestosterone regulation of thymocyte function in the rat: mediation by serum thymic factors. J. steroid Biochem. 5 (I 982) 683-690.