Immunology 1992 76 344-347

BRIEF COMMUNICATION

Interferon-gamma-dependent immunosuppressive effects of human gingival fibroblasts Y. SHIMABUKURO, S. MURAKAMI & H. OKADA Dept. of Periodontology and Endodontology, Osaka University Faculty of Dentistry, Suita, Osaka, Japan

Acceptedfor publication 27 February 1992

SUMMARY fibroblasts (HGF) were examined. As in fibroblasts gingival human of Immunoregulatory functions isolated from other tissues, HGF were activated with interferon-gamma (IFN-y) to express HLA-DR molecules. Despite the fact that the IFN-y-treated HGF showed phenotypical resemblance to socalled antigen-presenting cells (APC), the IFN-y-treated HGF were ineffective stimulators of alloreactive peripheral T cells. Conversely, IFN-y-treated HGF dramatically inhibited the proliferative responses of allogeneic APC (allo-APC) or phytohaemagglutinin (PHA)-stimulated T cells. Immunosuppressive effects of culture supernatant (CS) of IFN-y-treated HGF were low and were completely abrogated by the addition of indomethacin. Moreover, the production of prostaglandin E2 (PGE2) by HGF was not affected by IFN-y. These results suggest that IFN-y-dependent immunosuppressive effects of HGF were not due to PGE2 produced by HGF. In order to investigate further the mechanism(s) of IFN-y-dependent immunosuppressive effects of HGF, activated T cells and IFN-y-treated HGF were separately cultured in the same well by collagen films which were assembled in cylindrical cells and disturbed physical interactions between T cells and HGF. The proliferative responses of T cells which directly contacted with IFN-y-treated HGF were inhibited more significantly than those of T cells which did not contact with IFN-y-treated HGF. This suggests that IFN-y-dependent immunosuppressive effects of HGF were mediated by direct interactions between T cells and activated HGF. The present results suggest that IFN-y-stimulated HGF would modulate the immune responses of locally infiltrated T cells in periodontal lesions. It is well understood that most of the periodontal diseases are characterized by chronic inflammation with dense infiltration of inflammatory cells in periodontium.' To reveal the immunopathogenesis of periodontal diseases, the locally infiltrated immunocompetent cells detected in periodontal lesions have been eagerly examined3-5 and less attention has been paid to fibroblasts which compose gingiva. Although the cells of the immune system and the fibroblasts are often considered mutually exclusive, it is becoming increasingly apparent that both are capable of significantly influencing each other. Among lymphokines affecting fibroblasts, IFN-y has been considered a unique lymphokine which induces HLA-DR expression on nonimmunocompetent cells like astrocytes,6 keratinocytes,7 thyroid cells,8 endothelial cells and fibroblasts.9 Previous reports, however, demonstrated that the interferon-gamma (IFN-y)-stimuAbbreviations: APC, antigen-presenting cells; CS, culture supernatants; HGF, human gingival fibroblasts; ICAM-1, intercellular cell adhesion molecule- 1; IFN-y, interferon-gamma; IL, interleukin; MMC, mitomycin C; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PHA, phytohaemagglutinin; PGE2, prostaglandin E2. Correspondence: Dr S. Murakami, Dept. of Periodontology and Endodontology, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565, Japan.

lated fibroblasts are less effective stimulators of allo-specific T cells. 10-12 In this study, we investigated whether IFN-y-treated human gingival fibroblasts (HGF) display immunoregulatory functions in proliferative responses of T cells induced by allogeneic antigen-presenting cells (APC) or phytohaemagglutinin (PHA). HGF were obtained from biopsies of healthy gingiva from volunteers and were maintained in vitro as described previously.'3 HGF was treated with IFN-y (250 U/ml; Chemicon International Inc., Tenecula, CA) for 6 days and then with mitomycin C (MMC) (50 pg/ml; Kyowa Hakko Kogyo Co., Tokyo, Japan) at 37' for 30 min. Peripheral T cells isolated as described previously'4 were cultured for 4 days with the IFN-ytreated HGF or MMC-treated allogeneic T-cell-depleted peripheral blood mononuclear cells (PBMC) (allo-APC) which were derived from the same donor of HGF in 200 pi of RPMI1640 medium supplemented with 10% foetal calf serum (FCS) per round-bottomed microtitre well (Corning Laboratory Sciences Co., Corning, NY). Proliferation was assayed by pulsing wells with 0 5 pCi/well (18-5 kBq/well) of [3H]TdR during the last 6 hr of the culture. At the end of the incubation, cultures were harvested by using a semiautomatic cell harvester, and [3H]TdR incorporation was measured in a liquid scincillation counter.

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Figure 1. IFN--;-treated HGF suppressed allo-APC- or PHA-induced T-cell proliferative responses. Peripheral T cells (I x 105/well) cultured in the presence of non-treated or IFN-y-treated HGF (2 x 104/well) with MMC-treated allo-APC (1 x 105/well) (a) or PHA (1:4000 dilution) (b). In (b). indomethacin (1I pg/ml) was added to the culture as indicated. Proliferation assay was carried out by pulsing wells with 0 5 ,iCi well (18 5 kBq, well) of [3H]TdR during the last 6 hr of the culture. Results are expressed as arithmetic means and standard errors of triplicate cultures. were

Although the T cells were strongly stimulated to proliferate by allogeneic T-cell-depleted PBMC (allo-APC) as expected, IFN-,-treated allogeneic HGF which were HLA-DR+ and intercellular cell adhesion molecule-I (ICAM-1) (data not shown) bright did not stimulate the T cells despite the phenotypical resemblance of IFN-,-treated HGF to APC (Fig. la). Previous reports demonstrated that IFN-;1-treated fibroblasts were not effective in inducing primary allogeneic mixed lymphocyte reaction (allo-MLR), but were effective in inducing secondary allo-MLR.'° " However, purified CD4+ T cells, which had been primed with allo-APC in vitro, were not effectively stimulated to proliferate with the IFN-;'-treated HGF (data not shown). Moreover, IFN--,j-treated HGF failed to exhibit the APC function even when exogeneous interleukin-l (IL-I), IL-2 or indomethacin was added to the culture (data not shown). Interestingly, when T cells were stimulated with allo-APC in the presence of IFN-R'-treated HGF, the allo-APC-induced proliferative response was significantly blocked (Fig. la). The effect of IFN-;-treated HGF on PHA (Difco Laboratories, Detroit, MI)-induced proliferative responses was next examined. When peripheral T cells were stimulated with PHA in the presence of non-treated HGF, proliferative responses of T cells were partially inhibited (Fig. Ib). Moreover, the immunosuppressive effects were clearly augmented by pretreating HGF with IFN-y (250 U/ml) (Fig. lb). We also found that IFN-;'-treated HGF suppressed the proliferative responses of T cells induced by antiCD3 antibody (data not shown). Both autologous and allogeneic HGF exhibited the suppressive effects on T-cell responses to the same extent and no significant difference in the suppressive effect was observed. These results clearly demonstrate that HGF exhibit immunosuppressive function in an IFN-s'-dependent manner. To examine the possible involvement of prostaglandin E2 (PGE2) in the immunosuppressive function of HGF, indomethacin (Wako Pure Chemical Industries, Tokyo, Japan) was added to the culture when PHA-stimulated T cells were cultured with non-treated or IFN- '-treated HGF. As shown in Fig. I b, indomethacin did not completely restore the suppressed proliferative responses induced by IFN-'-treated HGF. It was also demonstrated that culture supernatant (CS) which had

been prepared in the presence of indomethacin did not exhibit suppressive effects on proliferative responses of T cells (data not shown). Moreover, we revealed by radioimmunoassay (RIA) that PGE2 production by HGF was not increased by the treatment of IFN-y (data not shown). These results suggest that the main factor in CS of HGF which suppresses the proliferative responses of T cells is PGE, but that the IFN--'-dependent immunosuppression is not due to PGE2 In order to investigate the mechanism(s) by which IFN-)'dependent immunosuppression of HGF occurs, the chamber system was introduced. As shown in Fig. 2, the requirement of direct interactions between T cells and HGF was examined by using cylindrical cells in which collagen films are assembled (Cellgen CM24, Koken, Tokyo, Japan). IFN-,'-treated or nontreated HGF (4 x 104 cells) were inoculated into the upper side of transparent collagen films of the equipment in the well of 24well plate (Corning Laboratory Sciences Co.), and T cells (2 x 105 cells) were added to the basal compartment (Fig. 2a). The T cells were cultured in the presence of PHA (1:4000 dilution) for 4 days and proliferation of the T cells was assayed as described above. When T cells were cultured in the different chamber from that in which HGF were cultured, T cells would be affected only by the soluble factor(s) secreted by HGF. As shown in Fig. 2b, slight suppression was observed when PHAstimulated T cells and HGF were cultured separately in the same well. This suppression was not affected by IFN-y treatment of HGF (Fig. 2b) and was completely abrogated by addition of indomethacin (data not shown). Conversely, when PHAstimulated T cells and HGF were cultured with contacting each other, the immunosuppressive effects were increased. Moreover, the effects were significantly augmented by treating HGF with IFN--y. These results demonstrate that HGF suppressed T-cell proliferation by contacting with the T cells and that IFN-y treatment augmented the physical-contact-dependent immunosuppression. Until now, Korn'5 reported that dermal fibroblasts demonstrated immunosuppressive function and that the immunosuppression could be explained solely by PGE, secreted from the fibroblasts. In this report, we first clarified that immunosuppression by HGF requires physical contact between T cells and any

Y. Shimahukuro, S. Murakaini & H. Okada

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this paper, we would speculate that IFN-, may induce or increase expression of such molecule(s) on HGF. The fact that IFN-;-treated HGF suppressed proliferative responses of both autologous and allogeneic T cells to the same extent (data not shown) suggests that the compatibility of HLA phenotype between T cells and HGF is not essential for the induction of IFN-,-dependent immunosuppression of HGF. To date, we have not obtained any evidence that anti-HLA-DR antibody abrogates the IFN- j-dependent immunosuppression of HGF. Recently it has been demonstrated that various cell adhesion molecules are expressed on a variety of cells. These molecules play a maijor role in cell cell interactions and are involved in Tcell activation.1 67 So far, some of those molecules, such as LFA3. ICAM-I. have been identified on fibroblasts'" (S. Murakarmi. unpublished observations). ICAM- 1, which is one of the ligands of LFA-1, is expressed on HGF and the expression is upregulated by IFN-; .I However, anti-ICAM-I antibody, 84H 10, did not reverse the inhibition by IFN-,-treaIted HGF to T-cell responses (data not shown). Therefore it is not yet clear if or how ICAM-1 and LFA-1 participate in the induction of IFN--,depenident immunosuppression of HGF. Further study needs to be carried out to determine the essential molecule(s) on T cells and HGF for the induction of the immunosuppression as described in this paper. Present findings demonstrate that IFN-,-treated HGF exhibit suppressive effects in a direct-interaction-dependent manner a-nd suggest that it is possible for HGF to modulate local immune responses in inflammatory pcriodontal lesions by regulating the responses of locally infiltrated T cells.

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Figure 2. IF N-,-depenldenit irrmilunosuppressive effeccts of HJGF required direct intertictioens between T cells anld HGF. (I-) Schernllitic illustratititn of the chtiarnber systernl. Cylinidricail cells in whlich collagven filmlls wsere a~ssen-bled we rc introduced. I n this culturc sNystern, T cells a~nd HG F werc cultuled ill tile samei \vwcll of thc 24-well Culttire plaite without conltactill~y C~Ich other. The upper side of colkalgen films and the bastIl compolelit \xere natifiecd A a~nd B. respectivcly. (b) HGF hcid becil cultured ill the preselice or tabsence of 250) U nil1 I FN--, anld theil trecited w-ith 5() lig 1111 M MC.The HGF (4 x 10' \Aell ) tind periplhrctl T ccils (2'x 10' wicl )w\Nere cultured ais indicaited wlith PHA ( I :4000 dilution) for 4 d,-tNs. Proliferaition of T cclls wacs alssayed als described in Fig. I The results represcilt airithmiletic l1lC~tr1S ar1id staindalrd errors of triplicalte Cultulcs. The bacvkground c p mn + SE of T cells was I '() (0'1).

HGF and that I FN-,,I treatment clearly augmnetts this physicalcontact-dependent immunosuppression w ithout affecting the production of PGE,. It can be speculated that the immunosuppressive effects of HGF described in this paper meay partiallyv explalin the inability of IFN--i-treated HGF as APC. We also

found thalt IL-1 increases the inimunosuppressive functions of HGF. However, the immunosuppressive effects induced by IL-1I were completely inhibited by indomethacin, suggesting that ILI dependent i'mmunosuppression of HGF was caused by increased production of PGE, without affecting the physicalcontact-dependent mechanism (data not shown). The imnportant question still remnaining is what cell surface mnolecule(s) mediate T- cell-HGF interalctions and act clS negative signal transducers. Based on the results described in

We are grateful to Drs Y. Takeda. T. Saho and R. Isoda for their excellent technical assistance and helpful discussion. We allso thank Ms Kirsten Malpas for help in preparing this manuscript. This work was supported in part hb Grant-in-Aid for Scientific Research from the Ministry of Education. Science and Culture of Japan (no. (03771356 and (277 1 -(5).

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