Taiichiro Miyashita

Granzyme B leakage-induced cell death: a new type of activation-induced natural killer cell death

Activation‐induced natural killer (NK) cell death is very rapid compared to activation‐induced T or B cell death. Here we show that NK cell activation is accompanied by the leakage of granzymeB from intracellular granules into the cytoplasm. Evidence for granzyme B leakage includes the formation of granzyme B/serine proteinase inhibitor 9 (PI‐9) complexes that are detected by immunoprecipitation as well as colocalization of granzyme B and PI‐9 detected by immunocytochemistry. The pro‐apoptotic molecule Bid, a specific substrate for granzyme B, was cleaved within 2 min following CD2‐induced NK cell activation, suggesting that granzyme B triggers apoptosis by directing Bid to mitochondrial membranes. The granzyme B/PI‐9 protein ratio was found to mirror the percentage of CD2‐induced NK cell death, suggesting that an excess of leaked granzyme B over its inhibitor is a major determinant of cell death. We suggest that granzyme B leakage‐induced cell death is an important determinant of activation‐induced NK cell death and that this process may be important for the fate of NK cells which encounter malignant or virus‐infected cells.

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.

Functional changes in rheumatoid fibroblast‐like synovial cells through activation of peroxisome proliferator‐activated receptor γ‐mediated signalling pathway

Peroxisome proliferator‐activated receptor γ (PPARγ) is a ligand dependent transcriptional factor known to be a regulator of adipogenesis. Recent studies have also shown that stimulation of PPARγ inhibits the transcriptional activities of other nuclear factors and down‐regulates proinflammatory cytokine synthesis in T cells and monocytes. We examined, in the present study, the functional significance of PPARγ expressed in fibroblast‐like synovial cells (FLS) isolated from patients with rheumatoid arthritis (RA). Incubation of FLS with a synthetic PPARγ ligand, troglitazone, inhibited endogenous production of TNF‐α, IL‐6 and IL‐8, as well as matrix metalloprotease‐3 (MMP‐3), without inducing apoptosis of the cells. The gelatinase activity of FLS culture media was also inhibited by troglitazone. Electrophoretic mobility shift assay (EMSA) showed a significant reduction in the DNA binding activity of NF‐κB in troglitazone‐treated FLS in response to TNF‐α or IL‐1β. Moreover, long‐term cultivation of FLS with troglitazone resulted in morphological changes with marked lipid accumulation in these cells. Our results show a negative regulatory function for PPARγ on cytokine and MMP production together with inhibition of cytokine‐mediated inflammatory responses in rheumatoid synovial cells. Our results also suggest that FLS could differentiate into adipocyte‐like cells in the presence of proper stimulatory signals including PPARγ.

CP690,550 inhibits oncostatin M-induced JAK/STAT signaling pathway in rheumatoid synoviocytes

IntroductionInterleukin (IL)-6-type cytokines exert their effects through activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade. The JAK/STAT pathways play an important role in rheumatoid arthritis, since JAK inhibitors have exhibited dramatic effects on rheumatoid arthritis (RA) in clinical trials. In this study, we investigated the molecular effects of a small molecule JAK inhibitor, CP690,550 on the JAK/STAT signaling pathways and examined the role of JAK kinases in rheumatoid synovitis.MethodsFibroblast-like synoviocytes (FLS) were isolated from RA patients and stimulated with recombinant oncostatin M (OSM). The cellular supernatants were analyzed using cytokine protein chips. IL-6 mRNA and protein expression were analyzed by real-time PCR method and ELISA, respectively. Protein phosphorylation of rheumatoid synoviocytes was assessed by Western blot using phospho-specific antibodies.ResultsOSM was found to be a potent inducer of IL-6 in FLS. OSM stimulation elicited rapid phosphorylation of STATs suggesting activation of the JAK/STAT pathway in FLS. CP690,550 pretreatment completely abrogated the OSM-induced production of IL-6, as well as OSM-induced JAK/STAT, and activation of mitogen-activated kinases (MAPKs) in FLS.ConclusionsThese findings suggest that IL-6-type cytokines contribute to rheumatoid synovitis through activation of the JAK/STAT pathway in rheumatoid synoviocytes. Inhibition of these pro-inflammatory signaling pathways by CP690,550 could be important in the treatment of RA.

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.

Influence of Janus Kinase Inhibition on Interleukin 6-mediated Induction of Acute-phase Serum Amyloid A in Rheumatoid Synovium

Objective. Inhibition of intracellular signal transduction is considered to be a therapeutic target for chronic inflammation. The new Janus kinase (JAK)3 inhibitor CP690,550 has shown efficacy in the treatment of rheumatoid arthritis (RA). We investigated the influence of JAK/STAT inhibition using CP690,550 on the induction of acute-phase serum amyloid A (SAA), which is triggered by interleukin 6 (IL-6) stimulation in rheumatoid fibroblast-like synoviocytes (RA-FLS). Methods. IL-6-stimulated gene expression of the acute-phase serum amyloid A genes (A-SAA; encoded by SAA1+SAA2) and SAA4 was analyzed by reverse transcriptase-polymerase chain reaction. The intracellular signaling pathway mediating the effects of CP690,550 on IL-6-stimulated JAK/STAT activation was assessed by measuring the phosphorylation levels using Western blots. Results. IL-6 trans-signaling induced A-SAA messenger RNA (mRNA) expression in RA-FLS. By contrast IL-6 stimulation did not affect SAA4 mRNA expression, which is expressed constitutively in RA-FLS. IL-6 stimulation elicited rapid phosphorylation of JAK2 and STAT3, which was blunted by CP690,550. CP690,550 abrogated IL-6-mediated A-SAA mRNA expression in RA-FLS. Similarly, CP690,550 inhibited IL-6-mediated A-SAA mRNA expression in human hepatocytes. Conclusion. Our data indicated that CP690,550 blocked IL-6-induced JAK2/STAT3 activation, as well as the induction of A-SAA. Inhibition of IL-6-mediated proinflammatory signaling pathways by CP690,550 may represent a new antiinflammatory therapeutic strategy for RA and AA amyloidosis.

Association of Increased Frequencies of HLA-DPB1*05∶01 with the Presence of Anti-Ro/SS-A and Anti-La/SS-B Antibodies in Japanese Rheumatoid Arthritis and Systemic Lupus Erythematosus Patients

Introduction Autoantibodies to ribonucleoprotein are associated with a variety of autoimmune diseases, including rheumatoid arthritis (RA). Many studies on associations between human leukocyte antigen (HLA) alleles and RA have been reported, but few have been validated in RA subpopulations with anti-La/SS-B or anti-Ro/SS-A antibodies. Here, we investigated associations of HLA class II alleles with the presence of anti-Ro/SS-A or anti-La/SS-B antibodies in RA. Methods An association study was conducted for HLA-DRB1, DQB1, and DPB1 in Japanese RA and systemic lupus erythematosus (SLE) patients that were positive or negative for anti-Ro/SS-A and/or anti-La/SS-B antibodies. Results An increased prevalence of certain class II alleles was associated with the presence of anti-Ro/SS-A antibodies as follows: DRB1*08∶03 (Pc = 3.79×10−5, odds ratio [OR] 3.06, 95% confidence interval [CI] 1.98–4.73), DQB1*06∶01 (Pc = 0.0106, OR 1.70, 95%CI 1.26–2.31), and DPB1*05∶01 (Pc = 0.0040, OR 1.55, 95%CI 1.23–1.96). On the other hand, DRB1*15∶01 (Pc = 0.0470, OR 3.14, 95%CI 1.63–6.05), DQB1*06∶02 (Pc = 0.0252, OR 3.14, 95%CI 1.63–6.05), and DPB1*05∶01 (Pc = 0.0069, OR 2.27, 95% CI 1.44–3.57) were associated with anti-La/SS-B antibodies. The DPB1*05∶01 allele was associated with anti-Ro/SS-A (Pc = 0.0408, OR 1.69, 95% CI 1.19–2.41) and anti-La/SS-B antibodies (Pc = 2.48×10−5, OR 3.31, 95%CI 2.02–5.43) in SLE patients. Conclusion HLA-DPB1*05∶01 was the only allele associated with the presence of both anti-Ro/SS-A and anti-La/SS-B antibodies in Japanese RA and SLE patients.

Thyroid papillary carcinoma with solid sclerosing change in IgG4-related sclerosing disease

IgG4‐related sclerosing disease (IgG4‐RSD) is an inflammatory and fibrosing disorder characterized by lymphoplasmacytic inflammation with infiltration of various organs, including the pancreas, bile ducts, lung, kidney, and retroperitoneum. As for malignancy in IgG4‐RSD, only limited literature is available. We report here a case of thyroid papillary carcinoma showing unique morphology in IgG4‐RSD. Solid tumor nests were surrounded by dense IgG4‐positive plasma cells and fibrosis at both the primary site and metastatic lymph nodes. In contrast the background thyroid showed focal lymphocytic thyroiditis. IgG4‐related sclerosing sialadenitis and autoimmune pancreatitis were also diagnosed, and prednisolone treatment improved symptoms and serum IgG4 abnormality. To the best of our knowledge, this is the first documentation of a malignancy of the thyroid gland occurring in a background of IgG4‐RSD. A brief review of the literature on the relationship between IgG4 and malignancy is included.