Ayumi Hida

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.

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.

Detection of apoptosis-specific autoantibodies directed against granzyme B-induced cleavage fragments of the SS-B (La) autoantigen in sera from patients with primary Sjögren's syndrome

The objective of this study was to detect autoantibodies against granzyme B cleavage products in sera from patients with primary Sjögrens syndrome (SS). Cell lysates derived from human salivary gland (HSG) cell lines were incubated with granzyme B. The susceptibility to the generation of cleavage fragments of SS autoantigens was assayed by immunoblotting using sera from 57 primary SS patients, 17 primary SS patients with malignant lymphoma (ML), 28 systemic lupus erythematosus (SLE) patients, and 20 healthy controls. A 27 kD protein was recognized by serum autoantibodies in 8 (14·0%) of 57 primary SS patients, 5 (29·4%) of 17 SS patients with ML, 2 (7·1%) of 28 SLE patients, but not in 20 normal subjects. This protein was recognized by anti‐SSB (La) monoclonal antibodies. Granzyme B‐treated recombinant La protein was also shown to migrate as a discrete 27 kD protein by SDS PAGE. Blocking studies demonstrated the existence of an apoptosis‐specific B cell epitope present in sera from 2 of 8 primary SS patients and in 2 of 5 primary SS patients with ML which recognized the 27 kD protein. Granzyme B‐induced La fragments are generated during cytotoxicity in vitro. This is the first report describing autoantibodies in sera from primary SS patients that specifically recognize fragments of the La protein that are produced by the granzyme B protease.

SUCCESSFUL TREATMENT OF RAPIDLY PROGRESSIVE LUPUS NEPHRITIS ASSOCIATED WITH ANTI-MPO ANTIBODIES BY INTRAVENOUS IMMUNOGLOBULINS

Abstract: We report a case of systemic lupus erythematosus (SLE) associated with crescentic glomerulonephritis and myeloperoxidase-specific anti-neutrophil cytoplasmic antibodies (MPO-ANCA). A 34-year-old Japanese female patient diagnosed with SLE developed rapidly progressive renal failure and nephrotic syndrome. Haemodialysis was required to restore renal function. Methylprednisolone pulse therapy followed by plasmapheresis did not suppress the progression of renal failure, so she was treated with high-dose intravenous immunoglobulin (IV-IG) therapy, which was well tolerated and effectively prevented renal failure. A renal biopsy showed diffuse proliferative lupus nephritis (WHO classification IVc) with predominant crescent formation and scant subendothelial immune deposits. These findings indicate that, in addition to lupus nephritis, which usually results from the deposition of circulating or locally formed immune complexes, MPO-ANCA may be involved in the pathogenesis of crescentic glomerulonephritis. Furthermore, we propose that IV-IG is an effective therapy for MPO-ANCA-related renal crisis in lupus nephritis.

Anti-TIF1-γ antibody and cancer-associated myositis A clinicohistopathologic study

Objective:We aimed to analyze the clinical and histopathologic features of cancer-associated myositis (CAM) in relation to anti–transcriptional intermediary factor 1 &ggr; antibody (anti-TIF1-&ggr;-Ab), a marker of cancer association. Methods:We retrospectively studied 349 patients with idiopathic inflammatory myopathies (IIMs), including 284 patients with pretreatment biopsy samples available. For the classification of IIMs, the European Neuromuscular Center criteria were applied. Patients with CAM with (anti-TIF1-&ggr;-Ab[+] CAM) and without anti-TIF1-&ggr;-Ab (anti-TIF1-&ggr;-Ab[−] CAM) were compared with patients with IIM without cancers within and beyond 3 years of myositis diagnosis. Results:Cancer was detected in 75 patients, of whom 36 (48%) were positive for anti-TIF1-&ggr;-Ab. In anti-TIF1-&ggr;-Ab(+) patients with CAM, cancers were detected within 1 year of myositis diagnosis in 35 (97%) and before 1 year of myositis diagnosis in 1. All the anti-TIF1-&ggr;-Ab(+) patients with CAM satisfied the dermatomyositis (DM) criteria, including 2 possible DM sine dermatitis cases, and were characterized histologically by the presence of perifascicular atrophy, vacuolated fibers (VFs), and dense C5b-9 deposits on capillaries (dC5b-9). In contrast, 39 anti-TIF1-&ggr;-Ab(−) patients with CAM were classified into various subgroups, and characterized by a higher frequency of necrotizing autoimmune myopathy (NAM). Notably, all 7 patients with CAM classified into the NAM subgroup were anti-TIF1-&ggr;-Ab(−) and exhibited no dC5b-9 or VFs. Conclusions:CAM includes clinicohistopathologically heterogeneous disease entities. Among CAM entities, anti-TIF1-&ggr;-Ab(+) CAM has characteristically shown a close temporal association with cancer detection and the histopathologic findings of dC5b-9 and VFs, and CAM with NAM is a subset of anti-TIF1-&ggr;-Ab(−) CAM.

Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cells

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP) is thought to be a putative regulator of pro‐gelatinase A (MMP‐2) in the rheumatoid synovium. In this study, we examined the effects of IL‐1β, one of the inflammatory cytokines, on the expression of MT1‐MMP and the activation of pro‐MMP‐2 using rheumatoid synovial cells. We also studied the effects of KE‐298 (2‐acetylthiomethyl‐4‐(4‐methylphenyl)‐4‐oxobutanoic acid), a new disease‐modifying anti‐rheumatic drug (DMARD), on MT1‐MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast‐like synovial cells) were cultured with KE‐298 (25–100 µg/ml) in the presence of IL‐1β for 48 h. Activation of pro‐MMP‐2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription–polymerase chain reaction (RT–PCR) methods were used to detect MT1‐MMP mRNA. MT1‐MMP protein expression on synovial cells was examined by anti‐MT1‐MMP immunoblot. An active form of MMP‐2 was demonstrated in the culture media conditioned by IL‐1β‐stimulated synovial cells. In addition, MT1‐MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL‐1β treatment. KE‐298 blocked this IL‐1β‐induced pro‐MMP‐2 activation and MT1‐MMP expression, but did not affect IL‐1β‐induced tissue inhibitor of metalloproteinase‐2 (TIMP‐2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL‐1β results in the induction of MT1‐MMP expression. Given that MT1‐MMP promotes matrix degradation by activating pro‐MMP‐2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE‐298 may prevent this process by down‐regulating MT1‐MMP expression.

Protection of mitochondrial perturbation by human T-lymphotropic virus type 1 tax through induction of Bcl-xL expression.

This study was designed to determine the inhibitory role of human T-lymphotropic virus type 1 (HTLV-1) tax against apoptotic cell death. We used JPX-9 cells, a Jurkat subclone generated by the stable introduction of a tax expression-plasmid vector, and induced tax expression in JPX-9 cells with CdCl2. Expression of Bcl-2, Bcl-xL, and Bax in JPX-9 cells was assessed with Western blot analysis. Both tax-negative and tax-positive JPX-9 cells were incubated in the presence of several apoptogenic stimuli, and sensitivity to apoptogenic stimuli was also evaluated. Compared with tax-negative JPX-9 cells, Bcl-xL expression was clearly augmented in tax-positive JPX-9 cells. These cells were resistant to both receptor-mediated apoptosis (induced by anti-Fas IgM and tumor necrosis factor-related apoptosis-inducing ligand) and chemical-induced apoptosis (induced by pyrrolidine dithiocarbamate, etoposide, and staurosporine), as evidenced by the presence of hypodiploid DNA-positive cells, activation of caspase-3 and caspase-9, disruption of mitochondrial transmembrane potential (DeltaPsim) and inhibition of cytochrome c release in tax-positive JPX-9 cells compared with tax-negative JPX-9 cells. Our results suggest that tax-mediated Bcl-xL expression inhibits apoptosis of activated T-cells in HTLV-1-seropositive subjects, which consequently promotes the onset of autoimmune disorders such as Sjögrens syndrome.

Clinicopathologic features of myositis patients with CD8-MHC-1 complex pathology.

Objective: To determine the clinical features of myositis patients with the histopathologic finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class 1 (CD8-MHC-1 complex), which is shared by polymyositis (PM) and inclusion body myositis (IBM), in relation to the p62 immunostaining pattern of muscle fibers. Methods: All 93 myositis patients with CD8-MHC-1 complex who were referred to our hospital from 1993 to 2015 were classified on the basis of the European Neuromuscular Center (ENMC) diagnostic criteria for IBM (Rose, 2013) or PM (Hoogendijk, 2004) and analyzed. Results: The 93 patients included were 17 patients with PM, 70 patients with IBM, and 6 patients who neither met the criteria for PM nor IBM in terms of muscle weakness distribution (unclassifiable group). For these PM, IBM, and unclassifiable patients, their mean ages at diagnosis were 63, 70, and 64 years; autoimmune disease was present in 7 (41%), 13 (19%), and 4 (67%); hepatitis C virus infection was detected in 0%, 13 (20%), and 2 (33%); and p62 was immunopositive in 0%, 66 (94%), and 2 (33%), respectively. Of the treated patients, 11 of 16 PM patients and 4 of 6 p62-immunonegative patients in the unclassifiable group showed responses to immunotherapy, whereas all 44 patients with IBM and 2 p62-immunopositive patients in the unclassifiable group were unresponsive to immunotherapy. Conclusions: CD8-MHC-1 complex is present in patients with PM, IBM, or unclassifiable group. The data may serve as an argument for a trial of immunosuppressive treatment in p62-immunonegative patients with unclassifiable myositis.