Journal of Autoimmunity (1991) 4,307-314
Inhibition of T Lymphocyte Proliferation by Retinal Glial Miiller Cells: Reversal of Inhibition by Glucocorticoids
Francois G. Roberge, Rachel R. Caspi, Chi-Chao Robert B. Nussenblatt Laboratory
of Immunology,
National
Chan and
Eye Institute, NIH, Bethesda, MD, USA
(Received 5 June 1990 and accepted 8 August 1990) Study of interactions between retinal glial Miiller cells and T lymphocytes have revealed a wide array of reciprocal influenceson the functionsof these cells. In the present study we show that these interactions can be further modified by corticosteroid hormones. The primary effect of MUler cells on T lymphocytes is an inhibition of the T-cell proliferative response, and it is exerted via a membrane-bound factor. In this report we show that glucocorticoids can reverse the inhibition by suppressingthe expressionof the Miiller cell inhibitory factor. This effect was independentof the action of glucocorticoids on arachldonic acid metabolism. --
Xntroduction
Cell differentiation and function are largely determined by direct contact with adjacent cells as well as with their secretory products. This action is bidirectional as the modified state of one may in a recurrent manner induce changes in the other until this dynamic process reaches equilibrium. The highly mobile and responsive nature of immune cells makes them prone to these kinds of interaction with most cells throughout the organism. In the context of uveoretinal inflammation we have studied such interactions between organ-specific retinal glial cells (Miiller cells) and T helper lymphocytes. It was found that secretory products of activated mononuclear cells could induce Miiller cells to proliferate and express MHC class II determinants [ I]. In turn, Miiller cells were shown to exert a profound inhibitory effect on the IL-Z dependent antigen as well as mitogen driven proliferation of T-helper cells [2, 31. This inhibition was mediated through a membrane-bound factor. After proteolytic Correspondence to: Francois G. Roberge, MD, HBpital Notre-Dame, Dept. d’Ophtalmologie, rue Sherbrookeest, MontrOal,Quebec, Canada H2L 4Ml.
1560
307 0896-841 l/91/020307+08
$03.00/O
0 1991 Academic Press Limited
308
F. G. Roberge et al.
removal of this inhibitory factor, Muller cells became capable of functioning as antigen-presenting cells (AK) for the proliferation of the T cells [4]. At a higher level of complexity, hormones from distant cells can influence the state of a cellular system. Glucocorticoids (GC) exert a broad range of effects on many cell types (reviewed in [5] and [6]). Cells of the immune system in particular are profoundly influenced by GC. Associated with an inhibition of interferon gamma secretion by T cells, the production of IL-l and the expression of class II determinants by macrophages is reduced [7-91. In addition GC directly inhibit IL-2 production and T-cell proliferation [lo, 111. Another type of cell that has shown sensitivity to GC is the glial cell. For example, in astrocytes insulin receptor expression and DNA synthesis are decreased by GC [12, 131. In the retina, Muller cell differentiation is dependent on GC action [ 14-161. The study reported here concerns the effect of GC on the interaction between Muller cells and a uveitogenic T-helper cell line (Th.S) specific for the retinal S-antigen (S-Ag). It is shown that various GC can reverse the inhibitory effect of Muller cells on the proliferation of T cells by suppressing the expression of the inhibitory factor in Miiller cell membrane. This effect of GC could not be reproduced with specific inhibitors of arachidonic acid (AA) metabolism. Materials and methods Cell lines and reagents Miiller cell cultures from adult rat retinae were prepared as described and characterized by immunostaining with an antiserum against glial fibrillary acidic protein (gift of Dr Lawrence Eng, Stanford, CA), and with a monoclonal antibody, specific for Miiller cells in the retina, prepared in our laboratory [l]. On average, one in three preparations yields pure Miiller cells by these criteria. These selected cells were grown in minimal essential medium (MEM) (M. A. Bioproducts, Walkersville, MD), supplemented with lOO& v/v of fetal bovine serum (FBS) (Hyclone, Logan, UT) containing low GC levels (corticosterone 0.16 ng/ml, cortisol 1 ng/ml), 50 pg/ ml gentamycin and 209; v/v of spleen conditioned medium (SCM). SCM was prepared by a 65-h stimulation of naive rat spleen cells with Concanavalin A (Con A). The Con A was removed by adsorption with Sephadex G-25, and alpha-methyl mannoside 0.04% was added as described [4]. The Muller cells used in experiments were between 3 and 10 doubling passages. Where indicated, Muller cells were washed with Earle’s Balanced Salt Solution (EBSS) and fixed with glutaraldehyde (Sigma, St Louis, MO) 0.03gb for 30 s, the reaction stopped with 2 M L-Lysine, and thecells washed with EBSS as reported [4]. In some experiments the Miiller cells were cultured in the presence of dexamethasone (DEX) (crystalline form from Sigma) at 1O-6 M for a period of 72 h before fixation. A T-helper lymphocyte line specific for the retinal S-Ag (Th. S) was prepared as described and has been characterized [ 171. It is cultured in RPM1 1640 (M. A. Bioproducts) supplemented with 10% v/v FBS (Hyclone), 50 pg/ml gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 5 x lop5 M 2-mercaptoethanol (cRPM1). The cells are maintained through alternate cycles of antigen stimulation followed by expansion in IL-2-containing medium. Accessory cells (AC) consisted of unselected syngeneic thymic cells prepared from naive rats and irradiated at 3000 rads.
Reversal of Miiller cell inhibition of T lymphocyte proliferation 309 Table 1. Inhibition of T-helper proliferation
[‘H]TdR Added culture’
cpm
by Mdler
cells
x lo-’
to MuTh.SACAg*
Medium SCM IL-1 IL-2 IL- 1+ IL-2
Th.S
0.3 0.9 0.3 0.5 0.2
AC Ag
77.6 82.2 62.5 150.9 155.0
Th.S
AC
2.6 22.4 3.0 32.7 39.3
‘SCM 20”” v/v, or human rIL-1 (100 U/ml), or human rIL-2 (200U/ml) were added at the beginning of the incubation period. ‘Muher cells (Mu) 1 x 10’ cells/well irradiated at 3,000 rads; Th.S 2 x 10’ cells/well; accessory cells (AC) 5 x lo5 cells/well irradiated at 3,000 rads; and S-Ag (Ag) 5 ug/ml. The results of the average of triplicate cultures are in counts per minute (cpm) of [‘Hlthymidine incorporation during the last 16 h of a 65 h incubation period. The standard deviation from the mean of the data was less than 200,, .
T lymphocyte proliferation
assay
T-helper proliferation assays were conducted in triplicate in flat bottom microtiter plates in cRPM1 200 ul/well, with 2 x lo4 Th.S cells/well and 5 x lo5 AC/well. Where indicated, Miiller cells irradiated at 3,000 rads were added at 1 x lo4 cells/ well. For Ag stimulation, S-Ag prepared following Dorey et al. [18] was added at 5 pg/ml. For lymphokine stimulation, human rIL-2 (gift from Cetus, Emeryville, CA) was added at 200 U/ml, and human rIL-1 (gift from J. J. Oppenheim, NIH, Frederick, MD) at 100 U/ml. The incubation was for 65 h, the last 16 h in the presence of [3H]thymidine (New England Nuclear, Boston, MA) 1 pCi/well. The cells were harvested on glass fibre filters and the incorporated radioactivity counted by scintillation spectroscopy. When GC (Sigma) were added, it was at the beginning of the assay at the following concentrations: DEX lop5 to lo-l5 M, corticosterone 10e5 M, deoxycorticosterone 10e5 M, hydrocortisone 1O-5 M, prednisone low5 M. Inhibitors of AA metabolism were purchased from Sigma and used at the following concentrations: p-bromo phenacetyl bromide lop5 to lOPi M, indomethacin 10e6 to 10w8M, nor-dihydro guaiaretic acid (NDGA) 1O-5 to lo-*M. All dilutions were made from stock solutions freshly prepared in ethanol at 2 x lo-’ M. Controls for a possible effect of ethanol were conducted at the corresponding dilutions and showed no influence on the assay. Results Inhibition of Th.S proliferation
by Miiller cells
In confirmation of previous results, it can be seen in Table 1 that in the presence of Miiller cells there is a complete inhibition of antigen as well as IL-2 driven proliferation of Th.S cells. Evaluation of the mechanism of the inhibition had shown that it is contact dependent [2]. In addition, examination of supernatants from Miiller cell
310
F. G. Roberge et al.
Th.S+AC+Ag : 0
70
;
60
e
50
Mu+Th.S+AC+Ag
Dex concentration
(t41
Figure 1. Proliferative response of Th.S (2 x 10’ cells/well) to S-Ag (5 pg/ml) presented by thymic AC (5 x 10’ cells/well) irradiated at 3,000 rads, in counts per minute (cpm) of [‘Hlthymidine incorporation of triplicate cultures incubated 65 h, the last 16 h with [)H]thymidine 1 pCi/well, in the presence or absence of Miiller cells (1 x 10’ cells/well) irradiated at 3,000 rads; 200 U/ml of human rIL-2 were added to the culture medium. In (a), graded concentration of DEX produced a reversal of the inhibition of Th.S proliferation by Miiller cells. The results are expressed in (b) as percent of inhibition reversal relative to DEX concentration.
cultures by radioreceptor competition binding assay on A549 cell line did not detect any production of TGF-beta (data not shown). Reversal of Miiller cell inhibition of Th.S proliferation by DEX Addition of graded concentrations of DEX to culture similar to the one described in Table 1 produced a dose-dependent reversal of inhibition of Th.S proliferation, that was maximal at 10V6 to 10P5~ DEX concentration (Figure 1). The assay was conducted in the presence of 200 U/ml of human rIL-2 because of the DEX-induced suppression of IL-2 production by T cells. A comparison of various GC was also
Reversal of Miiller cell inhibition of T lymphocyte proliferation
311
Prednisone Hydrocortisone Ii-deoxycorticosterone Corticosterone Dexomethosone 0
IO
20 % inhibition
30
3
reversal
Figure 2. Comparison of the relative efficiency of various GC in reversing the inhibition of Th.S proliferation by Miiller cell. Culture done as in Figure 1 in the presence of Miiller cells, but without adding rIL-2 to the medium. GC were added at the initiation of culture at the following concentrations: prednisone 10 5M, hydrocortisone 10 ‘M, 11-deoxycorticosterone 10 5M, corticosterone 10 5M, and DEX 10 ‘M.
made and the results are shown in Figure 2. It can be seen that hydrocortisone and prednisolone at optimal concentration had an effect comparable to DEX. Corticosterone, a natural GC in rats, was also very effective in reversing the inhibition. Products of AA metabolism, in particular prostaglandin E,, are known to inhibit T-lymphocyte proliferation. Because GC are potent inhibitors of AA metabolism, we investigated whether that action could contribute to the reversal of inhibition. However the addition to the cultures of specific inhibitors of AA catabolic pathways over a wide range of concentrations (p-bromo phenacetyl bromide at 1O-5 to lo- lo M, a phospholipase A2 inhibitor, indomethacin at 10e6 to 10m8M, a cycle-oxygenase inhibitor, or NDGA at 10M5to lOPa M, a lipoxygenase inhibitor) had no effect on the inhibition of Th.S proliferation by Miiller cells (results not shown). GC suppress the expression of the T-cell proliferation inhibitory factor in Mdler membrane
cell
In the next set of experiments we investigated whether the GC would affect the membrane expression of the Muller cell inhibitory factor. Muller cells were cultured for 72 h in the presence of DEX, fixed with glutaraldehyde to stop metabolism, and added to an Ag-driven Th.S proliferation assay. As shown in Table 2, while fixed Miiller cells cultured in normal medium profoundly inhibited the T-helper cell response, preincubating the Miiller cells with 10e6~ DEX completely abolished their inhibitory action. Discussion
The observation that many somatic cells can be induced to express class II major histocompatibility complex (MHC) determinants has generated a sustained interest in the study of interactions taking place between organ-specific cells and cells of the immune system. In particular, many studies have concentrated on the possibility that organ resident cells could present autoantigens. Different findings have come out of these investigations. In some studies organ-specific cells, such as astrocytes or vascular endothelial cells, were able to function as APC to stimulate the proliferation
312 F. G. Roberge et al.
Table 2. Suppression of inhibitory factor expression on fixed Miiller cells by pretreatment with dexamethasone Fixed Miiller cells’ None DEX pretreatment No pretreatment
Indicator system’
cpm k SEM3
Th.SACAg Th.S AC Ag Th.S AC Ag
68,574+ 1,913 68,953 k 3,639 13,837&2,841
‘Miiller cells were fixed with glutaraldehyde as described in materials and methods after 72 h of culture in the presence or absence of dexamethasone 10 ‘M and added where indicated at 1 x 104/well. 2Th.S cell line (2 x 10’ cells/well) responding to S-Ag (5 ug/ml) presented by AC (thymic accessory cells, irradiated at 3,000 rads) 5 x lo5 cells/well. Counts per minute of [rH]thymidine incorporation during the last 16 h of a 65 h culture; average of triplicate cultures.
of T cells [ 19,201. In other situations, presentation of Ag by tissue-specific cells was shown to induce a state of unresponsiveness of T cells to subsequent presentation of Ag by professional APC, because of the absence of costimulatory signals [21,22]. In the case of the Miiller cell, a retinal glial cell, there was an inhibition of T-cell proliferation even in the presence of these costimulatory factors [2]. This inhibition was exerted by cell contact through a factor sensitive to protease [4]. In the present study we have found that the expression of this membrane factor was negatively regulated by GC. The mode of action of GC on cellular metabolism is exerted at the gene level. Transformational changes of the steroid receptor upon binding of its ligand allow for interaction with transcriptional elements leading to a modification in the expression of many genes [23, 241. Our data are consistent with a probable suppression of synthesis of the Mi.iller cell inhibitory factor. In accordance with that conclusion, we have found in unpublished studies that in the profile of membrane protein preparations from Miiller cells, some bands were eliminated when the cells were cultured in the presence of DEX. However, an improper insertion of the factor in the cell membrane due to GC cannot be ruled out by these experiments. During the last decade many investigators have uncovered a large number of reciprocal influences between the neuroendocrine system and the immune system (reviewed in [25]). Recently GC have been implicated in the susceptibility of certain animal strains to autoimmunity. An inverse correlation was found between GC stress response and the susceptibility to experimental arthritis [26,27]. Our results bring a new aspect to the issue: that GC can alter the outcome of interaction between target organ cells and autoimmune T lymphocytes. These findings further underline the complex integration existing between the endocrine and the immune systems. References Roberge, F. G., R. R. Caspi, C.-C. Chan, T. Kuwabara, and R. B. Nussenblatt. 1985. Long-term culture of Miiller cells from adult rats in the presence of activated lymphocytes/monocytes products. Curr. Eye Res. 4: 975-981 2. Caspi R. R., F. G. Roberge, and R. B. Nussenblatt. 1987. Organ-resident non-lymphoid cells suppress proliferation of autoimmune T-lymphocytes. Science 237: 1029-1032 1.
Reversal of Miiller cell inhibition of T lymphocyte proliferation
313
of 3. Caspi, R. R. and F. G. Roberge. 1989. Glial cells as suppressor cells: Characterization the inhibitory function. J. Autoimmunity 2: 709-722 1988. Glial retinal Muller cells 4. Roberge, F. G., R. R. Caspi, and R. B. Nussenblatt. produce IL-1 activity and have a dual role in regulation of autoimmune T-lymphocytes: antigen presentation manifested after removal of suppressive activity. J. Zmmunol. 140: 2193-2196 hormone action: An overview. In 5. Baxter, J. D. and G. G. Rousseau. 1979. Glucocorticoid Monographs of Endocrinology, Vol. 12. J. D. Baxter and G. G. Rousseau, eds. SpringerVerlag, Berlin. pp. l-24 6. Munck, A., D. B. Mendel, L. I. Smith, and E. Orti. 1990. Glucocorticoid receptors and actions. Am. Rev. Respir. Dis. 141(2 Pt2): S2-SlO 7. Kelso A., and A. Munck. 1984. Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones. 3. Zmmunol. 133: 784-791 8. Jiayi, D., Y. Shikun, and X. Renbao. 1989. The inhibitory effect of hydrocortisone on the interferon production by rat spleen cel1s.J. Steroid Biochem. 33: 1139-l 141 9. Gillis, S., G. R. Crabtree, and K. A. Smith. 1979. Glucocorticoid-induced inhibition of T cell growth factor production. I‘. The effect on mitogen-induced lymphocyte proliferation.3. Zmmunol. 123: 1624-1631 10. Daynes, R. A. and B. A. Araneo. 1989. Contrasting effects of glucocorticoids on the capacity of T cells to produce the growth factors interleukin 2 and interleukin 4. Eur. J. Zmmunol. 19: 2319-2325 11. Snyder, D. S. and E. R. Unanue. 1982. Corticosteroids inhibit murine macrophage Ia expression and interleukin 1 production. 3. Zmmunol. 129: 1803-1805 12. Kniss, D. A. and R. W. Burry. 1985. Glucocorticoid hormones inhibit DNA synthesis in glial cells cultured in chemically defined medium. Exp. Cell Res. 161: 29-40 13. Piddington, R. and A. A. Moscona. 1967. Precocious induction of retinal glutamine synthetase by hydrocortisone in the embryo and in culture: age-dependent differences in tissue response. Biochim. Biophys. Acta 141: 429-432 14. Montiel, F., J. Ortiz-Caro, A. Villa, A. Pascual, and A. Aranda. 1987. Glucocorticoids regulate insulin binding in a rat glial cell line. Endocrinology 121: 258-265 15. Patejunas, G. and A. P. Young. 1987. Tissue-specific regulation of avian glutamine synthetase expression during development in response to glucocorticoid hormones. Mol. Cell. Biol. 7: 1070-1077 16. Vardimon, L., L. L. Fox, L. Degenstein, and A. A. Moscona. 1988. Cell contacts are required for induction by cortisol of glutamine synthetase gene transcription in the retina. Proc. Natl. Acad. Sci. USA. 85: 5981-5985 17. Caspi, R. R., F. G. Roberge, C. G. McAllister, M. El-Saied, T. Kuwabara, I. Gery, E. Hanna, and R. B. Nussenblatt. 1986. T cell lines mediating experimental autoimmune uveoretinitis (EAU) in the rat.3. Zmmunol. 136: 928933 18. Dorey, C., J. Cozette, and J. I’. Faure. 1982. A simple and rapid method for isolation of retinal S antigen. Ophthalmic Res. 14: 249-255 19. McCarron, R. M., 0. Kempski, M. Spatz, and D. E. McFarlin. 1985. Presentation of myelin basic protein by murine cerebral vascular endothelial cells. 3. Zmmunol. 134: 3100-3103 20. Fontana, A., W. Fierz, and H. Wekerle. 1984. Astrocytes present myelin basic protein to encephalitogenic T cell lines. Nature 307: 273-276 21. Gaspari, A. A., M. K. Jenkins, and S. I. Katz. 1988. Class II MHC-Bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific TH 1 clones. 3. Zmmunol. 141: 2216-2220 22. Mueller, D. L., M. K. Jenkins, and R. H. Schwartz. 1989. An accessory cell-derived costimulatory signal acts independently of protein kinase C activation to allow T cell proliferation and prevent the induction of unresponsiveness. 3. Zmmunol. 142: 2617-2628 23. Godowski, P. J., S. Rusconi, R. Miesfeld, and K. R. Yamamoto. 1987. Glucocorticoid receptor mutants that are constitutive activators of transcriptional enhancement. Nature 325: 365-368
314 F. G. Roberge et at. 24. Beato, M., G. Chalepakis, M. Schauer, and E. P. Slater. 1989. DNA regulatory elements for steroid hormones. J. Steroid Biochem. 32: 737-748 25. Weigert, D. A. and J. E. Blalock. 1987. Interactions between the neuroendocrine and the immune systems: Common hormone and receptors. Immunol. Rev. 100:79-108 26. Sternberg, E. M., J. M. Hill, G. P. Chrousos, T. Kamilaris, S. J. Listwak,P. W. Gold, and R. L. Wilder. 1989. Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptococcal cell wall arthritis-susceptible Lewis rats. Proc. Natl. Acad. Sci. USA. 86: 2374-2378 27. Sternberg, E. M., W. S. Young, R. Bernardini, A. E. Calogero, G. I?. Chrousos, P. W. Gold, and R. L. Wilder. 1989. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc. Natl. Acad. Sci. USA. 86: 4771-4775
Inhibition of T Lymphocyte Proliferation by Retinal Glial Miiller Cells: Reversal of Inhibition by Glucocorticoids
Francois G. Roberge, Rachel R. Caspi, Chi-Chao Robert B. Nussenblatt Laboratory
of Immunology,
National
Chan and
Eye Institute, NIH, Bethesda, MD, USA
(Received 5 June 1990 and accepted 8 August 1990) Study of interactions between retinal glial Miiller cells and T lymphocytes have revealed a wide array of reciprocal influenceson the functionsof these cells. In the present study we show that these interactions can be further modified by corticosteroid hormones. The primary effect of MUler cells on T lymphocytes is an inhibition of the T-cell proliferative response, and it is exerted via a membrane-bound factor. In this report we show that glucocorticoids can reverse the inhibition by suppressingthe expressionof the Miiller cell inhibitory factor. This effect was independentof the action of glucocorticoids on arachldonic acid metabolism. --
Xntroduction
Cell differentiation and function are largely determined by direct contact with adjacent cells as well as with their secretory products. This action is bidirectional as the modified state of one may in a recurrent manner induce changes in the other until this dynamic process reaches equilibrium. The highly mobile and responsive nature of immune cells makes them prone to these kinds of interaction with most cells throughout the organism. In the context of uveoretinal inflammation we have studied such interactions between organ-specific retinal glial cells (Miiller cells) and T helper lymphocytes. It was found that secretory products of activated mononuclear cells could induce Miiller cells to proliferate and express MHC class II determinants [ I]. In turn, Miiller cells were shown to exert a profound inhibitory effect on the IL-Z dependent antigen as well as mitogen driven proliferation of T-helper cells [2, 31. This inhibition was mediated through a membrane-bound factor. After proteolytic Correspondence to: Francois G. Roberge, MD, HBpital Notre-Dame, Dept. d’Ophtalmologie, rue Sherbrookeest, MontrOal,Quebec, Canada H2L 4Ml.
1560
307 0896-841 l/91/020307+08
$03.00/O
0 1991 Academic Press Limited
308
F. G. Roberge et al.
removal of this inhibitory factor, Muller cells became capable of functioning as antigen-presenting cells (AK) for the proliferation of the T cells [4]. At a higher level of complexity, hormones from distant cells can influence the state of a cellular system. Glucocorticoids (GC) exert a broad range of effects on many cell types (reviewed in [5] and [6]). Cells of the immune system in particular are profoundly influenced by GC. Associated with an inhibition of interferon gamma secretion by T cells, the production of IL-l and the expression of class II determinants by macrophages is reduced [7-91. In addition GC directly inhibit IL-2 production and T-cell proliferation [lo, 111. Another type of cell that has shown sensitivity to GC is the glial cell. For example, in astrocytes insulin receptor expression and DNA synthesis are decreased by GC [12, 131. In the retina, Muller cell differentiation is dependent on GC action [ 14-161. The study reported here concerns the effect of GC on the interaction between Muller cells and a uveitogenic T-helper cell line (Th.S) specific for the retinal S-antigen (S-Ag). It is shown that various GC can reverse the inhibitory effect of Muller cells on the proliferation of T cells by suppressing the expression of the inhibitory factor in Miiller cell membrane. This effect of GC could not be reproduced with specific inhibitors of arachidonic acid (AA) metabolism. Materials and methods Cell lines and reagents Miiller cell cultures from adult rat retinae were prepared as described and characterized by immunostaining with an antiserum against glial fibrillary acidic protein (gift of Dr Lawrence Eng, Stanford, CA), and with a monoclonal antibody, specific for Miiller cells in the retina, prepared in our laboratory [l]. On average, one in three preparations yields pure Miiller cells by these criteria. These selected cells were grown in minimal essential medium (MEM) (M. A. Bioproducts, Walkersville, MD), supplemented with lOO& v/v of fetal bovine serum (FBS) (Hyclone, Logan, UT) containing low GC levels (corticosterone 0.16 ng/ml, cortisol 1 ng/ml), 50 pg/ ml gentamycin and 209; v/v of spleen conditioned medium (SCM). SCM was prepared by a 65-h stimulation of naive rat spleen cells with Concanavalin A (Con A). The Con A was removed by adsorption with Sephadex G-25, and alpha-methyl mannoside 0.04% was added as described [4]. The Muller cells used in experiments were between 3 and 10 doubling passages. Where indicated, Muller cells were washed with Earle’s Balanced Salt Solution (EBSS) and fixed with glutaraldehyde (Sigma, St Louis, MO) 0.03gb for 30 s, the reaction stopped with 2 M L-Lysine, and thecells washed with EBSS as reported [4]. In some experiments the Miiller cells were cultured in the presence of dexamethasone (DEX) (crystalline form from Sigma) at 1O-6 M for a period of 72 h before fixation. A T-helper lymphocyte line specific for the retinal S-Ag (Th. S) was prepared as described and has been characterized [ 171. It is cultured in RPM1 1640 (M. A. Bioproducts) supplemented with 10% v/v FBS (Hyclone), 50 pg/ml gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 5 x lop5 M 2-mercaptoethanol (cRPM1). The cells are maintained through alternate cycles of antigen stimulation followed by expansion in IL-2-containing medium. Accessory cells (AC) consisted of unselected syngeneic thymic cells prepared from naive rats and irradiated at 3000 rads.
Reversal of Miiller cell inhibition of T lymphocyte proliferation 309 Table 1. Inhibition of T-helper proliferation
[‘H]TdR Added culture’
cpm
by Mdler
cells
x lo-’
to MuTh.SACAg*
Medium SCM IL-1 IL-2 IL- 1+ IL-2
Th.S
0.3 0.9 0.3 0.5 0.2
AC Ag
77.6 82.2 62.5 150.9 155.0
Th.S
AC
2.6 22.4 3.0 32.7 39.3
‘SCM 20”” v/v, or human rIL-1 (100 U/ml), or human rIL-2 (200U/ml) were added at the beginning of the incubation period. ‘Muher cells (Mu) 1 x 10’ cells/well irradiated at 3,000 rads; Th.S 2 x 10’ cells/well; accessory cells (AC) 5 x lo5 cells/well irradiated at 3,000 rads; and S-Ag (Ag) 5 ug/ml. The results of the average of triplicate cultures are in counts per minute (cpm) of [‘Hlthymidine incorporation during the last 16 h of a 65 h incubation period. The standard deviation from the mean of the data was less than 200,, .
T lymphocyte proliferation
assay
T-helper proliferation assays were conducted in triplicate in flat bottom microtiter plates in cRPM1 200 ul/well, with 2 x lo4 Th.S cells/well and 5 x lo5 AC/well. Where indicated, Miiller cells irradiated at 3,000 rads were added at 1 x lo4 cells/ well. For Ag stimulation, S-Ag prepared following Dorey et al. [18] was added at 5 pg/ml. For lymphokine stimulation, human rIL-2 (gift from Cetus, Emeryville, CA) was added at 200 U/ml, and human rIL-1 (gift from J. J. Oppenheim, NIH, Frederick, MD) at 100 U/ml. The incubation was for 65 h, the last 16 h in the presence of [3H]thymidine (New England Nuclear, Boston, MA) 1 pCi/well. The cells were harvested on glass fibre filters and the incorporated radioactivity counted by scintillation spectroscopy. When GC (Sigma) were added, it was at the beginning of the assay at the following concentrations: DEX lop5 to lo-l5 M, corticosterone 10e5 M, deoxycorticosterone 10e5 M, hydrocortisone 1O-5 M, prednisone low5 M. Inhibitors of AA metabolism were purchased from Sigma and used at the following concentrations: p-bromo phenacetyl bromide lop5 to lOPi M, indomethacin 10e6 to 10w8M, nor-dihydro guaiaretic acid (NDGA) 1O-5 to lo-*M. All dilutions were made from stock solutions freshly prepared in ethanol at 2 x lo-’ M. Controls for a possible effect of ethanol were conducted at the corresponding dilutions and showed no influence on the assay. Results Inhibition of Th.S proliferation
by Miiller cells
In confirmation of previous results, it can be seen in Table 1 that in the presence of Miiller cells there is a complete inhibition of antigen as well as IL-2 driven proliferation of Th.S cells. Evaluation of the mechanism of the inhibition had shown that it is contact dependent [2]. In addition, examination of supernatants from Miiller cell
310
F. G. Roberge et al.
Th.S+AC+Ag : 0
70
;
60
e
50
Mu+Th.S+AC+Ag
Dex concentration
(t41
Figure 1. Proliferative response of Th.S (2 x 10’ cells/well) to S-Ag (5 pg/ml) presented by thymic AC (5 x 10’ cells/well) irradiated at 3,000 rads, in counts per minute (cpm) of [‘Hlthymidine incorporation of triplicate cultures incubated 65 h, the last 16 h with [)H]thymidine 1 pCi/well, in the presence or absence of Miiller cells (1 x 10’ cells/well) irradiated at 3,000 rads; 200 U/ml of human rIL-2 were added to the culture medium. In (a), graded concentration of DEX produced a reversal of the inhibition of Th.S proliferation by Miiller cells. The results are expressed in (b) as percent of inhibition reversal relative to DEX concentration.
cultures by radioreceptor competition binding assay on A549 cell line did not detect any production of TGF-beta (data not shown). Reversal of Miiller cell inhibition of Th.S proliferation by DEX Addition of graded concentrations of DEX to culture similar to the one described in Table 1 produced a dose-dependent reversal of inhibition of Th.S proliferation, that was maximal at 10V6 to 10P5~ DEX concentration (Figure 1). The assay was conducted in the presence of 200 U/ml of human rIL-2 because of the DEX-induced suppression of IL-2 production by T cells. A comparison of various GC was also
Reversal of Miiller cell inhibition of T lymphocyte proliferation
311
Prednisone Hydrocortisone Ii-deoxycorticosterone Corticosterone Dexomethosone 0
IO
20 % inhibition
30
3
reversal
Figure 2. Comparison of the relative efficiency of various GC in reversing the inhibition of Th.S proliferation by Miiller cell. Culture done as in Figure 1 in the presence of Miiller cells, but without adding rIL-2 to the medium. GC were added at the initiation of culture at the following concentrations: prednisone 10 5M, hydrocortisone 10 ‘M, 11-deoxycorticosterone 10 5M, corticosterone 10 5M, and DEX 10 ‘M.
made and the results are shown in Figure 2. It can be seen that hydrocortisone and prednisolone at optimal concentration had an effect comparable to DEX. Corticosterone, a natural GC in rats, was also very effective in reversing the inhibition. Products of AA metabolism, in particular prostaglandin E,, are known to inhibit T-lymphocyte proliferation. Because GC are potent inhibitors of AA metabolism, we investigated whether that action could contribute to the reversal of inhibition. However the addition to the cultures of specific inhibitors of AA catabolic pathways over a wide range of concentrations (p-bromo phenacetyl bromide at 1O-5 to lo- lo M, a phospholipase A2 inhibitor, indomethacin at 10e6 to 10m8M, a cycle-oxygenase inhibitor, or NDGA at 10M5to lOPa M, a lipoxygenase inhibitor) had no effect on the inhibition of Th.S proliferation by Miiller cells (results not shown). GC suppress the expression of the T-cell proliferation inhibitory factor in Mdler membrane
cell
In the next set of experiments we investigated whether the GC would affect the membrane expression of the Muller cell inhibitory factor. Muller cells were cultured for 72 h in the presence of DEX, fixed with glutaraldehyde to stop metabolism, and added to an Ag-driven Th.S proliferation assay. As shown in Table 2, while fixed Miiller cells cultured in normal medium profoundly inhibited the T-helper cell response, preincubating the Miiller cells with 10e6~ DEX completely abolished their inhibitory action. Discussion
The observation that many somatic cells can be induced to express class II major histocompatibility complex (MHC) determinants has generated a sustained interest in the study of interactions taking place between organ-specific cells and cells of the immune system. In particular, many studies have concentrated on the possibility that organ resident cells could present autoantigens. Different findings have come out of these investigations. In some studies organ-specific cells, such as astrocytes or vascular endothelial cells, were able to function as APC to stimulate the proliferation
312 F. G. Roberge et al.
Table 2. Suppression of inhibitory factor expression on fixed Miiller cells by pretreatment with dexamethasone Fixed Miiller cells’ None DEX pretreatment No pretreatment
Indicator system’
cpm k SEM3
Th.SACAg Th.S AC Ag Th.S AC Ag
68,574+ 1,913 68,953 k 3,639 13,837&2,841
‘Miiller cells were fixed with glutaraldehyde as described in materials and methods after 72 h of culture in the presence or absence of dexamethasone 10 ‘M and added where indicated at 1 x 104/well. 2Th.S cell line (2 x 10’ cells/well) responding to S-Ag (5 ug/ml) presented by AC (thymic accessory cells, irradiated at 3,000 rads) 5 x lo5 cells/well. Counts per minute of [rH]thymidine incorporation during the last 16 h of a 65 h culture; average of triplicate cultures.
of T cells [ 19,201. In other situations, presentation of Ag by tissue-specific cells was shown to induce a state of unresponsiveness of T cells to subsequent presentation of Ag by professional APC, because of the absence of costimulatory signals [21,22]. In the case of the Miiller cell, a retinal glial cell, there was an inhibition of T-cell proliferation even in the presence of these costimulatory factors [2]. This inhibition was exerted by cell contact through a factor sensitive to protease [4]. In the present study we have found that the expression of this membrane factor was negatively regulated by GC. The mode of action of GC on cellular metabolism is exerted at the gene level. Transformational changes of the steroid receptor upon binding of its ligand allow for interaction with transcriptional elements leading to a modification in the expression of many genes [23, 241. Our data are consistent with a probable suppression of synthesis of the Mi.iller cell inhibitory factor. In accordance with that conclusion, we have found in unpublished studies that in the profile of membrane protein preparations from Miiller cells, some bands were eliminated when the cells were cultured in the presence of DEX. However, an improper insertion of the factor in the cell membrane due to GC cannot be ruled out by these experiments. During the last decade many investigators have uncovered a large number of reciprocal influences between the neuroendocrine system and the immune system (reviewed in [25]). Recently GC have been implicated in the susceptibility of certain animal strains to autoimmunity. An inverse correlation was found between GC stress response and the susceptibility to experimental arthritis [26,27]. Our results bring a new aspect to the issue: that GC can alter the outcome of interaction between target organ cells and autoimmune T lymphocytes. These findings further underline the complex integration existing between the endocrine and the immune systems. References Roberge, F. G., R. R. Caspi, C.-C. Chan, T. Kuwabara, and R. B. Nussenblatt. 1985. Long-term culture of Miiller cells from adult rats in the presence of activated lymphocytes/monocytes products. Curr. Eye Res. 4: 975-981 2. Caspi R. R., F. G. Roberge, and R. B. Nussenblatt. 1987. Organ-resident non-lymphoid cells suppress proliferation of autoimmune T-lymphocytes. Science 237: 1029-1032 1.
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