Takahiko Aoyagi

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)

TNF-alpha-mediated expression of membrane-type matrix metalloproteinase in rheumatoid synovial fibroblasts.

Degradation of the extracellular matrix plays an important role in rheumatoid articular destruction. Rheumatoid synovial fibroblasts secrete a large amount of matrix‐degrading metalloproteinases (MMPs), which initiate tissue damage by proteolytic degradation of collagens and proteoglycans. Cytokines, such as interleukin‐1α, ‐1β or tumour necrosis factor (TNF)‐α, are potent inducers of MMPs in rheumatoid synovial fibroblasts. MMPs are synthesized and secreted as latent pro‐enzymes and their activation is achieved by proteolytic cleavage of the propeptide domain at the N‐terminus of the molecule. Thus, the interaction of the pro‐enzymes with specific activators determines the enzymatic activity in the extracellular space. In the present study, we identified a novel mechanism for the activation of pro‐MMP‐2, which can be achieved through the interaction of the inflammatory cytokine, TNF‐α, with synovial fibroblasts. Although MMP‐2 is constitutively secreted by synovial fibroblasts as a pro‐enzyme, stimulation of fibroblasts by TNF‐α‐induced secretion of MMP‐2 in an active form. In support of this result, TNF‐α stimulation‐induced membrane‐type matrix metalloproteinase (MT‐MMP), a newly identified MMP‐2‐specific activator, on synovial fibroblasts. Cycloheximide analysis demonstrated that protein synthesis may be required for TNF‐α‐mediated MT‐MMP expression on synovial fibroblasts. Our results suggest that TNF‐α induces MMP‐2 activation in part by up‐regulating MT‐MMP expression, thus representing a new mechanism for cytokine‐mediated articular destruction in rheumatoid arthritis (RA).

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.

Importance of NF-κB in rheumatoid synovial tissues: in situ NF-κB expression and in vitro study using cultured synovial cells

OBJECTIVES To examine whether inhibition of NF-κB induces apoptosis of human synovial cells stimulated by tumour necrosis factor α (TNFα), interleukin 1β (IL1β), and anti-Fas monoclonal antibody (mAb). METHODS The expression of proliferating cell nuclear antigen (PCNA), NF-κB, and the presence of apoptotic synovial cells were determined in synovial tissues. Apoptosis of cultured synovial cells was induced by inhibition of NF-κB nuclear translocation by Z-Leu-Leu-Leu-aldehyde (LLL-CHO). The activation of caspase-3 and expression of XIAP and cIAP2 in synovial cells in LLL-CHO induced apoptosis was also examined. RESULTS Abundant PCNA+ synovial cells were found in rheumatoid arthritis (RA) synovial tissue, though a few apoptotic synovial cells were also detected in the RA synovial tissues. Nuclear NF-κB was expressed in RA synovial cells. Electrophoretic mobility shift assay showed that treatment of cells with TNFα or IL1β significantly stimulated nuclear NF-κB activity. A small number of apoptotic synovial cells expressing intracellular active caspase-3 were found after treatment of cells with LLL-CHO. Although treatment of RA synovial cells with TNFα or IL1β alone did not induce apoptosis, apoptosis induced by LLL-CHO and caspase-3 activation were clearly enhanced in TNFα or IL1β stimulated synovial cells compared with unstimulated synovial cells. Furthermore, induction of apoptosis of synovial cells with caspase-3 activation by anti-Fas mAb was clearly increased by LLL-CHO. The expression of cIAP2 and XIAP in synovial cells may not directly influence the sensitivity of synovial cells to apoptosis induced by LLL-CHO. CONCLUSION The results suggest that NF-κB inhibition may be a potentially important therapeutic approach for RA by correcting the imbalance between apoptosis and proliferation of synovial cells in RA synovial tissue.

Interferon-alpha and dexamethasone inhibit adhesion of T cells to endothelial cells and synovial cells

We investigated whether interferon‐gamma (IFN‐γ), interferon‐alpha (IFN‐α) and glucocorticoids affected the adhesion of T cells to human umbilical endothelial cells or human synovial cells. About 30% of peripheral blood T cells could bind to unstimulated endothelial cells, but only a few T cells could bind to unstimulaled synovial cells. When both endothelial cells and synovial cells were cultured with recombinant IFN‐γ (rlFN‐γ), the percentage of T cell binding to both types of cells increased in a dose‐dependent manner. rIFN‐α and dexamethasone blocked the T cell binding to unstimulated endothelial cells. Furthermore, rIFN‐α and dexamethasone suppressed T cell binding to both endothelial cells and synovial cells stimulated by IKN‐γ, and also inhibited intercellular adhesion molecule‐ l (ICAM‐1) expression on both endothelial cells and synovial cells stimulated by 1 FN‐γ. These results suggest that IFN‐α and glucocorticoids may inhibit T cell binding to endothelial cells or synovial cells by modulating adhesion molecule expression on these cells.

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.

Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis‐inducing signal pathway, we investigated the effects of NO on rheumatoid synovial‐cell apoptosis induced by Fas ligation with anti‐Fas antibody. Pretreatment of synovial cells with the NO donor S‐nitro‐N‐acetylpenicillamine (SNAP) prevented the Fas‐mediated induction of apoptosis. The activation of caspase‐3 was required to mediate Fas‐induced synovial cell apoptosis. The NO donor SNAP inhibited Fas‐induced caspase‐3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas‐induced caspase‐8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase‐3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.

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.

Infection of human synovial cells by human T cell lymphotropic virus type I. Proliferation and granulocyte/macrophage colony-stimulating factor production by synovial cells.

The present study was performed to clarify the relationship between human T cell lymphotropic virus type I (HTLV-I) infection and chronic inflammatory arthropathy. To determine the ability of HTLV-I to infect synovial cells and the effect on synovial cell proliferation, synovial cells were cocultured with the HTLV-I-producing T cell lines (MT-2 or HCT-1). After coculture with HTLV-I-infected T cells, the synovial cells expressed HTLV-I-specific core antigens, and HTLV-I proviral DNA was detected from the synovial cells by polymerase chain reaction. These cocultured synovial cells with HTLV-I-infected T cells proliferated more actively than the synovial cells cocultured with uninfected T cells. This stimulatory effect of HTLV-I-infected T cells on synovial cell proliferation seems necessary to contact each other. After being cocultured with MT-2 cells, synovial cells proliferated more actively than control cells even after several passages. Furthermore, HTLV-I-infected synovial cells produced significant amounts of granulocyte/macrophage colony-stimulating factor. These results suggest that HTLV-I can infect synovial cells, resulting their active proliferation and may be involved in the pathogenesis of proliferative synovitis similar to that found in rheumatoid arthritis.

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.