Masahiko Tsuboi

Tumor necrosis factor-α and interleukin-1β increase the Fas-mediated apoptosis of human osteoblasts☆☆☆★

Abstract Our recent work demonstrated functional Fas expression on human osteoblasts, and the histologic examination of the periarticular osteoporosis region in patients with rheumatoid arthritis (RA) showed apoptosis in osteoblasts. High concentrations of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6—which are thought to increase bone resorption—have been determined in RA synovium. We investigated the effect of these cytokines on the Fas-mediated apoptosis of human osteoblasts. The human osteoblastic cell line MG63 and human primary osteoblast-like cells from bone biopsy specimens were used as human osteoblasts. Fas expression on these cells was examined by flow cytometry, and Fas-mediated apoptosis induced by anti-Fas immunoglobulin M (IgM) was determined by a chromium 51 release assay, the presence of cells with hypodiploid DNA, staining with Hoechst 33258 dye, and the detection of DNA fragmentation on agarose gel electrophoresis. The proliferation of osteoblasts was analyzed by a tritiated thymidine incorporation assay. Spontaneous apoptosis was not found on cultured osteoblasts. The apoptosis of human osteoblasts was not induced by TNF-α, IL-1β, or IL-6 alone in the absence of anti-Fas IgM. In addition, proliferation of the cells was not affected by these cytokines. Fas was constitutively expressed on unstimulated osteoblasts, and treatment of these cells with IL-1β or TNF-α significantly augmented Fas expression. Human osteoblasts were committed to apoptosis with anti-Fas IgM, and the treatment of both IL-1β and TNF-α markedly increased Fas-mediated apoptosis. TNF-α augmented both Fas expression and Fas-mediated apoptosis more efficiently than did IL-1β. In addition, an additive effect on both Fas expression and Fas-mediated apoptosis was demonstrated when TNF-α and IL-1β were added to osteoblasts. IL-6 influenced neither Fas expression nor the Fas-mediated apoptosis of osteoblasts. Furthermore, no synergistic effect of IL-6 with IL-1β or TNF-α was observed. IL-1β, TNF-α, or IL-6 did not change Bcl-2 expression. Our results suggest that IL-1β and TNF-α regulate osteoblast cell number by up-regulating the Fas-mediated apoptosis of osteoblasts, one of the putative mechanisms inducing periarticular osteoporosis in patients with RA. (J Lab Clin Med 1999;134:222-31)

Fas and Fas Ligand Interaction Is Necessary for Human Osteoblast Apoptosis

We investigated the cellular and humoral interactions between peripheral blood mononuclear cells (PBMCs) and human osteoblasts, leading to apoptosis of osteoblasts. Human osteoblastic cell line MG63 and human primary osteoblast‐like cells obtained from biopsy specimens were used in this study. PBMCs were isolated from healthy donors and cultured with or without stimulation by recombinant interleukin‐2 followed by 12‐o‐tetradecanoyl‐phorbol 13‐acetate with ionomycin. Fas was functionally expressed on MG63 and primary osteoblast‐like cells. Activated PBMCs expressed Fas ligand (FasL) strongly on their surface and killed MG63 and primary osteoblast‐like cells. Cultured supernatants of activated PBMCs also induced apoptotic cell death of MG63 and primary osteoblast‐like cells. In contrast, both unstimulated PBMCs and cultured supernatants of unstimulated PBMCs did not induce apoptosis of these cells. Furthermore, the cytotoxic effect and induction of apoptosis against MG63 and primary osteoblast‐like cells by activated PBMCs and cultured supernatants were inhibited significantly by human Fas chimeric protein. Our data showed that human osteoblasts expressed Fas fuctionally and both membrane‐type and soluble form FasL from activated PBMCs induced apoptosis of these cells, providing the one possible mechanism of bone loss in inflammatory diseases such as rheumatoid arthritis.

Inhibitory effect of clarithromycin on costimulatory molecule expression and cytokine production by synovial fibroblast-like cells

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast‐like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon‐gamma (IFN‐γ)‐treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and 3H‐thymidine incorporation was counted. Intercellular adhesion molecule‐1 (ICAM‐1), LFA‐3 and vascular cell adhesion molecule‐1 (VCAM‐1) were detected on the surface of both RA and OA synoviocytes. However, ICAM‐2, B7–1 and B7–2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up‐regulated expression of ICAM‐1, LFA‐3 and VCAM‐1 by IFN‐γ, IL‐1β or 12‐o‐tetradecanoyl phorbol 13‐acetate (TPA) were markedly suppressed by clarithromycin in a dose‐dependent manner at concentrations between 0.1 and 10 μg/ml. The production of IL‐1β, IL‐6, IL‐8, granulocyte colony‐stimulating factor (G‐CSF) and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) but not IL‐1α and tumour necrosis factor‐alpha (TNF‐α) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL‐10 production. Finally, autologous T cells were stimulated by IFN‐γ‐treated synoviocytes in response to PPD. As clarithromycin suppressed HLA‐DR and costimulatory molecule expression was enhanced by IFN‐γ, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen‐specific T cell proliferation induced by synoviocytes.

Inhibitory effect of a new anti-rheumatic drug T-614 on costimulatory molecule expression, cytokine production, and antigen presentation by synovial cells.

The present study was undertaken to investigate the immunoregulatory effects of T-614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) on synovial cells in vitro. Synovial cells were cultured with T-614 in the presence or absence of various cytokines. After incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analyzed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of T-614 on the function of synovial cells as antigen-presenting cells (APCs). The costimulatory molecules including CD54, CD58, and CD106 were constitutionally expressed on the surface of synovial cells. However, neither CD80 nor CD86 nor CD102 was found on the surface, and these costimulatory molecules could not be induced by any cytokines. T-614 itself did not affect the costimulatory molecule expression and cytokine production of unstimulated synovial cells. The stimulation of synovial cells with interferon-gamma (IFN-gamma), interleukin-1beta, or 12-O-tetradecanoyl phorbol 13-acetate enhanced the expression of costimulatory molecules and the proinflammatory cytokine production of these cells. Both the up-regulated expression of these costimulatory molecules and the enhanced production of proinflammatory cytokines were significantly inhibited by T-614. Autologous T cell proliferation in response to purified protein derivative by IFN-gamma-treated synovial cells was significantly suppressed by T-614. T-614 has considerable immunosuppressive effects on synovial cells by inhibiting the costimulatory molecule expression and cytokine production of these cells and the antigen-specific T cell proliferation mediated by the synovial cells. These results suggest that T-614 plays an important immunoregulatory role in rheumatoid synovial tissues.

Inhibitory effects of interleukin-10 on synovial cells of rheumatoid arthritis.

This paper describes the immunoregulatory effects of interleukin‐10 (IL‐10) on synovial cells in vitro. Synovial cells were cultured with IL‐10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme‐linked immunosorbent assay, respectively. We also examined the effect of IL‐10 on the function of synovial cells as antigen‐presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon‐γ (IFN‐γ), IL‐1β, or 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up‐regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL‐10. Autologous T‐cell proliferation was stimulated by purified protein derivative (PPD) in IFN‐γ‐treated synovial cells and treatment of these synovial cells with IL‐10 also suppressed T‐cell proliferation. Our results suggest that IL‐10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis.

The effects of the immunosuppressant rapamycin on the growth of rheumatoid arthritis (RA) synovial fibroblast.

RA is a chronic inflammatory disease characterized by mononuclear cell infiltration and the overgrowth of synovial fibroblast. This invasive growth of synovial tissues corresponds with the progressive destruction of articular carilage and bone. Several immunosuppresive agents, such as cyclophosphamide, cyclosporin A and mizoribine, have been clinically used to control disease progresssion, though relatively little is known of their effects on rheumatoid synovium. Rapamycin exhibits a strong immunosuppressive activity by acting on T cell signalling pathways. In the present study we examined the effects of rapamycin on the growth of synovial fibroblast isolated from RA patients. Platelet‐derived growth factor (PDGF) is a potent growth factor in synovial fibroblasts isolated from RA patients. PDGF and serum stimulation resulted in a rapid phosphorylation of tyrosine and activation of mitogen‐activated protein kinase (MAP kinase), 70‐kD‐S6 kinase (P70S6K) and 90‐kD‐S6 kinase (P90rsk). Rapamycin, a macrolide immunosuppressant, inhibited completely growth factor‐induced synovial fibroblast proliferation and P70S6K activation. In contrast, tyrosine phosphorylation and activation of MAP kinases and P90rsk were not influenced by rapamycin treatment. Our data demonstrate that growth factor‐mediated P70S6K activation is closely related to the growth of synovial fibroblast, and suggest the efficacy of rapamycin for controlling synovial hyperplasia in RA.

Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25‐week‐old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506‐treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl‐2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone.

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves’ disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves’ disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vβ elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-γ (IFN-γ)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-γ-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-γ-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-γ-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-γ in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.

Expression and function of Fas and Fas ligand on peripheral blood lymphocytes in normal subjects

We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1beta (IL-1beta), IL-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas+ target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Bax was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas+ target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.

Two elderly cases of familial mediterranean fever with rheumatoid arthritis

Familial Mediterranean fever (FMF) is a genetic autoinflammatory disorder that usually develops before 20 years of age and is characterized by periodic fever with serositis and arthritis. Both FMF and rheumatoid arthritis (RA) involve arthritis; however, their coexistence is rare. We describe two RA patients with an MEFV mutation in exon 2, who were diagnosed with FMF at an age of over 50 years. We also discuss the possibility that MEFV mutations could modulate RA disease activity.