Satoshi Urayama

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.

Nuclear factor-κB and caspases co-operatively regulate the activation and apoptosis of human macrophages

Accumulating evidence suggests that macrophages function as major effector cells in the pathological process of various human diseases. We examined here the role of nuclear factor‐κB (NF‐κB) and caspases in the regulation of activation and apoptosis of macrophages. Activation of the human monoblastic leukaemia cell line, U937, by phorbol 12‐myristate 13‐acetate (PMA) increased the expression of CD14/CD86, and cytokine production. PMA stimulation also increased the expression of both pro‐caspase‐8 and pro‐caspase‐3 in U937, but not apoptosis or intracellular caspase‐3 activity. PMA also increased the expression of X‐chromosome‐linked inhibitor of apoptosis protein (XIAP) in U937, suggesting an inhibitory action for XIAP on the caspase cascade in PMA‐stimulated U937. Electrophoretic mobility shift assay (EMSA) showed a significant increase of nuclear NF‐κB activity in PMA‐stimulated U937. When a potent NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was added to U937 cell culture in the presence of PMA, apoptosis was triggered by activation of caspase‐3, which was induced by caspase‐8 activation. XIAP expression was markedly suppressed in PMA‐treated U937 in the presence of PDTC. The inhibitors of caspase‐8 and caspase‐3 mostly inhibited apoptosis of U937 treated with PMA in the presence of PDTC. Furthermore, a phenotype of U937 treated with PMA and PDTC in the presence of caspase inhibitor was almost identical to that of unstimulated U937. Our results suggest that the signalling pathways involved in the activation and apoptosis of human macrophages could be co‐operatively regulated by the use of NF‐κB and caspase inhibitors, thus enabling the control of macrophage function and number.

Regulation of synovial cell apoptosis by proteasome inhibitor.

OBJECTIVE Recent studies have shown the importance of proteasome function in the regulation of apoptosis. This study examined whether inhibition of proteasome function mediates apoptosis of synovial cells, and whether cytokines modulate this process. METHODS Type B synovial cells (fibroblast-like synovial cells) were cultured with tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta1 (TGFbeta1), and further incubated in the presence of variable concentrations of Z-Leu-Leu-Leu-aldehyde (LLL-CHO), a proteasome inhibitor. During this process, apoptosis of synovial cells was determined by Hoechst 33258 dye staining and 51Cr release assay. The involvement of caspase cascade was examined using enzyme activity assay and blocking experiments by peptide inhibitors. The expression of pro-caspases, Bcl-2-related proteins, and X chromosome-linked inhibitor of apoptosis (XIAP) in synovial cells was examined by Western blot analysis. RESULTS Apoptosis of cultured synovial cells was induced in a dose-dependent manner by LLL-CHO. Activation of caspase cascade through caspase-8 to caspase-3 was essential during this process. Pretreatment of synovial cells with TNF alpha significantly augmented both the activation of caspases and the proportion of apoptosis in synovial cells induced by LLL-CHO, whereas TGFbeta1 pretreatment markedly suppressed these phenomena. The ratio of the expression of Bcl-2 to Bax or Bcl-xL to Bax, and XIAP expression in synovial cells may not be directly associated with the susceptibility of synovial cells to apoptosis by LLL-CHO. CONCLUSION Apoptosis of synovial cells was induced by inhibition of proteasome function through the activation of caspase cascade, and this process was clearly modulated by cytokines. These data provide new insight into the regulatory mechanisms controlling synovial cells in rheumatoid synovitis by proteasome inhibitors, and might be useful for the design of new therapeutic strategies in rheumatoid arthritis.

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.

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.

FK506 Augments Glucocorticoid-Mediated Cyclooxygenase–2 Down-Regulation in Human Rheumatoid Synovial Fibroblasts

Prostaglandins (PG) formed by cyclooxygenase (COX) enzymes are important mediators of inflammation in rheumatoid arthritis. The contribution of the inducible COX-2 to inflammation in the rheumatoid synovium is well documented. We examined the regulation of COX-2 mRNA and protein expression in response to both glucocorticoids (GC) and FK506 using rheumatoid synovial fibroblasts. Combined treatment of FK506 and a low concentration of dexamethasone (DEX) (10−9 M) down-regulated synovial COX-2 mRNA and protein expression. In contrast, neither FK506 nor DEX (10−9 M) alone influenced COX-2 expression. Immunocytochemical studies showed that pretreatment with FK506 enhanced the nuclear translocation of the glucocorticoid receptor (GR) in synovial fibroblasts in the presence of low concentrations of DEX (10−9 M). Transient transfection experiments showed that treatment of cells with FK506 enhanced the expression of glucocorticoid-responsive gene reporter in the presence of DEX (10−9 M). NF-κB is known to mediate the transcriptional activation of the COX-2 gene. Electrophoretic mobility shift assay demonstrated that DNA-binding activity of NF-κB was suppressed more profoundly by FK506 plus DEX (10−9 M) treatment with those of DEX (10−9 M) alone in IL-1β-stimulated synovial cells. Our results indicated that FK506-induced potentiation of GR-mediated repression of synovial COX-2 gene transcription is the result of increased translocation of GR to the nucleus and subsequent repression of NF-κB transactivation. Our results also suggest that FK506 may exert anti-inflammatory effects in the rheumatoid synovium by potentiating GR-mediated signal transduction.

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.

CD4+ T-cell-mediated cytotoxicity against staphylococcal enterotoxin B-pulsed synovial cells.

Apoptosis of synovial cells in rheumatoid arthritis (RA) synovium determined in vivo is suggested to counteract the overgrowth of synovium. Immunohistological examination has revealed the infiltration of activated CD4+ T cells, which express Fas ligand (FasL), in RA synovium. The presence of a putative antigen (Ag) of autoimmune disorders in a target organ may induce the activation of specific T cells in the inflammatory region such as RA synovium. We examined the possible role of CD4+ T cells activated by synovial cells in a staphylococcal enterotoxin B (SEB)‐dependent manner, inducing synovial cell apoptosis. Synovial cells were cultured with or without interferon‐γ (IFN‐γ) and further incubated with CD4+ T cells in the presence of SEB. After the cocultivation, both the cytotoxicity and FasL expression of CD4+ T cells were investigated. Constitutive Fas expression was detected on both unstimulated and IFN‐γ‐stimulated synovial cells. CD4+ T cells did not kill SEB‐pulsed unstimulated synovial cells efficiently. In contrast, when CD4+ T cells were incubated with IFN‐γ‐stimulated synovial cells with SEB whose human leucocyte antigen (HLA)‐DR and ‐DQ expression was markedly induced, significant cytotoxicity by these cells against synovial cells was detected. The addition of anti‐HLA‐DR and ‐DQ monoclonal antibodies (mAbs) or human Fas chimeric protein (hFas‐Fc) reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN‐γ‐stimulated synovial cells with SEB was significantly induced. Furthermore, the addition of mAbs against CD54, CD58 and CD106 inhibited both the cytotoxicity and FasL expression of CD4+ T cells induced by IFN‐γ‐stimulated synovial cells in the presence of SEB, indicating the importance of costimulatory molecules on synovial cells in activating CD4+ T cells. Our results suggest that CD4+ T cells are activated by synovial cells by an SEB‐dependent manner and express FasL, inducing Fas‐mediated apoptosis of the latter cells. These phenomena may regulate the overgrowth of synovial cells in RA synovium.