K. Migita

Osteoprotegerin (OPG) acts as an endogenous decoy receptor in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of fibroblast-like synovial cells

We examined the role of osteoprotegerin (OPG) on tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis in rheumatoid fibroblast‐like synovial cells (FLS). OPG protein concentrations in synovial fluid from patients with rheumatoid arthritis (RA) correlated with those of interleukin (IL)‐1β or IL‐6. A similar correlation was present between IL‐1β and IL‐6 concentrations. Rheumatoid FLS in vitro expressed both death domain‐containing receptors [death receptor 4 (DR4) and DR5] and decoy receptors [decoy receptor 1 (DcR1) and DcR2]. DR4 expression on FLS was weak compared with the expression of DR5, DcR1 and DcR2. Recombinant TRAIL (rTRAIL) rapidly induced apoptosis of FLS. DR5 as well as DR4 were functional with regard to TRAIL‐mediated apoptosis induction in FLS; however, DR5 appeared be more efficient than DR4. In addition to soluble DR5 (sDR5) and sDR4, OPG administration significantly inhibited TRAIL‐induced apoptogenic activity. OPG was identified in the culture supernatants of FLS, and its concentration increased significantly by the addition of IL‐1β in a time‐dependent manner. Neither IL‐6 nor tumour necrosis factor (TNF)‐α increased the production of OPG from FLS. TRAIL‐induced apoptogenic activity towards FLS was reduced when rTRAIL was added without exchanging the culture media, and this was particularly noticeable in the IL‐1β‐stimulated FLS culture; however, the sensitivity of FLS to TRAIL‐induced apoptosis itself was not changed by IL‐1β. Interestingly, neutralization of endogenous OPG by adding anti‐OPG monoclonal antibody (MoAb) to FLS culture restored TRAIL‐mediated apoptosis. Our data demonstrate that OPG is an endogenous decoy receptor for TRAIL‐induced apoptosis of FLS. In addition, IL‐1β seems to promote the growth of rheumatoid synovial tissues through stimulation of OPG production, which interferes with TRAIL death signals in a competitive manner.

Reduced blood BDCA‐2+ (lymphoid) and CD11c+ (myeloid) dendritic cells in systemic lupus erythematosus

Type 1 IFN is thought to be implicated in the autoimmune process of SLE. Plasmacytoid dendric cells (DC), which are natural IFN‐α producing cells, play a pivotal epipathogenic role in SLE. The present study was undertaken to investigate the phenotypic characteristics of peripheral blood DC in SLE patients in comparison with those of healthy controls. Samples from 20 SLE patients and 18 healthy controls were studied. Three‐colour flow cytometry was performed to identify myeloid DC, as CD11c+ lineage marker–, and HLA‐DR+ cells and plasmacytoid DC, as BDCA‐2+ linage marker–, and HLA‐DR+ cells. We used the whole blood ‘lyse/no‐wash’ procedure, which allows precise counting of peripheral blood DC. BDCA‐2+ plasmacytoid DC and CD11c+ myeloid DC were reduced in SLE patients compared with controls. Similarly, BDCA‐3+ DC were reduced in SLE patients. These results indicated that SLE patients had a reduced number of both BDCA‐2+ plasmacytoid DC and CD11c+ myeloid DC. These alternations of the DC subset may drive the autoimmune response in SLE.

Apoptosis in labial salivary glands from Sjögren's syndrome (SS) patients: comparison with human T lymphotropic virus-I (HTLV-I)-seronegative and -seropositive SS patients

Apoptosis is a type of cell death that occurs during morphogenesis and development of the immune system. One of the mechanisms is mediated through the Fas and Fas ligand (FasL) pathway. To determine the possible involvement of Fas and its ligand in salivary gland destruction, we analysed the appearance of nuclei with DNA fragmentation by using nick end labelling (TUNEL) and the expression of Fas and FasL by immunohistochemistry in labial salivary glands. Furthermore, we compared the features of apoptosis in labial salivary glands between HTLV‐I− and HTLV‐I+ SS. When the frozen sections of 10 primary SS patients in the absence of anti‐HTLV‐I antibody were examined, several apoptotic cells were found in the acinar and ductal epithelial cells as well as infiltrated mononuclear cells. Both Fas and FasL were detected in the infiltrated mononuclear cells. Acinar epithelial cells, which are surrounded by FasL+ mononuclear cells, were also double‐positive with Fas and FasL, although the expression of FasL was localized at their apical border, suggesting that apoptosis of mononuclear cells was achieved by activation‐induced mechanisms through Fas/FasL pathways, and that of acinar epithelial cells was mediated by FasL derived from either acinar epithelial cells themselves or infiltrated mononuclear cells. Interestingly, Fas expression in ductal epithelial cells was localized around the lumen side of the ducts, indicating that FasL secreted from acinar epithelial cells may induce Fas‐mediated apoptosis of ductal epithelial cells. We also studied the labial salivary glands from nine SS patients with anti‐HTLV‐I antibodies. There was no significant difference in the occurrence of apoptotic cells or in the expression of Fas and FasL between HTLV‐I+ and HTLV‐I− SS patients. It was of note that neither the expression of Fas and FasL nor the presence of apoptotic cells were determined in labial salivary glands from subjects without SS. These findings indicate that Fas‐mediated apoptosis in salivary glands could be involved in the pathological manifestations of SS, irrespective of HTLV‐I seropositivity.

Suppressive effect of leflunomide metabolite (A77 1726) on metalloproteinase production in IL-1β stimulated rheumatoid synovial fibroblasts

Leflunomide, an isoxazol derivative structurally unrelated to other immunomodulatory drugs, has proven to be efficacious in the treatment of rheumatoid arthritis (RA). This study was conducted to elucidate the mechanism by which leflunomide mediated antirheumatic effects. We investigated the effects of A77 1726, leflunomides active metabolite, on mitogen‐activated protein kinase (MAPK) activation in IL‐1β‐stimulated rheumatoid synovial fibroblasts. The effects of A77 1726 on the secretion of matrix metalloproteinases (MMPs) from rheumatoid synovial fibroblasts were also examined. A77 1726 partially suppressed IL‐1β‐induced ERK1/2 and p38 kinase activation. In contrast, A77 1726 efficiently suppressed IL‐1β‐stimulated JNK1/2 kinase activation. Although no suppressive effect was demonstrated on MMP‐2, A77 1726 markedly inhibited MMP‐1, 3, and 13 secretions from IL‐1β‐stimulated rheumatoid synovial fibroblasts. Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) was constitutively produced from rheumatoid synovial fibroblasts and the suppressive effects of A77 1726 on TIMP‐1 production were minimal. Our results suggest that the suppression of the MAPK signalling pathway and MMP synthesis in rheumatoid synovial fibroblasts is a possible mechanism for the inhibitory activity of leflunomide against rheumatoid arthritis.

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.

Cytokine regulation of HLA on thyroid epithelial cells

The regulation of class I and class II HLA expression in human thyroid follicular cells was studied in vitro. Tumour necrosis factor‐alpha (TNF‐α) enhanced the expression of class I antigen on thyrocytes, but these cytokines had little effect on the expression of class II antigen. Interleukin‐lβ (IL‐ lβ) and interleukin‐6(IL‐6) did not affect class I and class II antigen expression. The combination of interferon‐gamma (IFN‐γ) with TNF‐α or IL‐1β enhanced the induction of class I and class II antigens, compared with the effect of IFN‐β alone. Neither class I nor class II expression was induced by lL‐6 alone or in combination with IFN‐γ. These findings suggest that TNF‐α and IL‐1β may have an important role in inappropriate expression of HLA antigens on thyrocytes in thyroid gland.

Effects of Janus kinase inhibitor tofacitinib on circulating serum amyloid A and interleukin‐6 during treatment for rheumatoid arthritis

The Janus kinase inhibitor tofacitinib is currently being investigated as a disease‐modifying agent in rheumatoid arthritis (RA). We investigated the in‐vivo effects of tofacitinib treatment for 4 weeks on elevated circulating acute‐phase serum amyloid (SAA) levels in 14 Japanese patients with RA. SAA levels fell from 110·5 ± 118·5 μg/ml (mean ± standard deviation) at treatment initiation to 15·3 ± 13·3 μg/ml after 4 weeks treatment with tofacitinib. The reduction in SAA levels was greater in patients receiving tofacitinib plus methotrexate compared with those receiving tofacitinib monotherapy. Tofacitinib was also associated with reduced serum interleukin (IL)‐6, but had no effect on serum levels of soluble IL‐6 receptor. Patients were divided into groups with adequate (normalization) and inadequate SAA responses (without normalization). Serum IL‐6 levels were reduced more in the group with adequate SAA response compared with those with inadequate SAA response. These results suggest that tofacitinib down‐regulates the proinflammatory cytokine, IL‐6, accompanied by reduced serum SAA levels in patients with active RA. The ability to regulate elevated serum IL‐6 and SAA levels may explain the anti‐inflammatory activity of tofacitinib.

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.

Inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway in rheumatoid synovial fibroblasts using small molecule compounds

Janus kinase (JAK) inhibitors have been developed as anti‐inflammatory agents and have demonstrated clinical efficacy in rheumatoid arthritis (RA). We investigated if JAK‐3‐selective inhibition alone could disrupt cytokine signalling in rheumatoid synovial fibroblasts. In‐vitro studies were performed using synovial fibroblasts isolated from patients with RA. Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target‐gene expression levels were measured by reverse transcription–polymerase chain reaction (RT–PCR) or real‐time PCR. The JAK inhibitors CP‐690,550 and INCB028050 both suppressed activation of JAK‐1/‐2/‐3 and downstream STAT‐1/‐3/‐5, as well as the expression levels of target proinflammatory genes (MCP‐I, SAA1/2) in oncostatin‐M (OSM)‐stimulated rheumatoid synovial fibroblasts. In contrast, the JAK‐3‐selective inhibitor, PF‐956980, suppressed STAT‐1/‐5 activation but did not affect STAT‐3 activation in OSM‐stimulated rheumatoid synovial fibroblasts. In addition, PF‐956980 significantly suppressed MCP‐1 gene expression, but did not block SAA1/2 gene expression in OSM‐stimulated rheumatoid synovial fibroblasts. These data suggest that JAK‐3‐selective inhibition alone is insufficient to control STAT‐3‐dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK‐1/‐2, is needed to control the proinflammatory cascade in RA.

Induction of interleukin-23 p19 by serum amyloid A (SAA) in rheumatoid synoviocytes.

In this study, we investigated the roles of serum amyloid A (SAA) in T helper 17 (Th17)‐related cytokine induction in rheumatoid arthritis (RA) synoviocytes. Synoviocytes isolated from rheumatoid arthritis (RA) patients were stimulated with recombinant SAA and IL‐23 expression was investigated using reverse transcriptase‐polymerase chain reaction and Western blot. The involvement of mitogen‐activated protein kineases (MAPKs) and nuclear factor (NF)‐κB in SAA‐induced interleukin (IL)‐23 p19 expression was investigated using pharmacological inhibitors. In RA synoviocytes, SAA induced the expression of IL‐23 p19 and p40 mRNA expression. The SAA‐stimulated expression of p19 was rapid (< 3 h), and insensitive to polymyxin B treatment. This SAA‐stimulated expression of IL‐23 p19 was inhibited completely by inhibitors of NF‐κB, p38MAPK and dexamethasone. Interestingly, the SAA‐induced IL‐23, p19 and p40 production was accompanied by enhanced expression of IL‐1β, but not transforming growth factor‐β. These results indicate that SAA is a significant inducer of IL‐23 and IL‐1β in RA synoviocytes and potentially activates the IL‐23/IL‐17 pathway in the RA synovium. Our data present a novel interaction between inflammation and autoimmunity by an acute‐phase protein.