Yutaka Kawahito

15-deoxy-delta(12,14)-PGJ(2) induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats.

Peroxisome proliferator–activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and have a dominant regulatory role in adipocyte and monocyte differentiation. PPAR-γ agonists are also negative regulators of macrophage activation and have modulatory effects on tumorigenesis. In this study we demonstrate that synovial tissue localized expression of PPAR-γ in patients with rheumatoid arthritis (RA). We detected markedly enhanced expression of PPAR-γ in macrophages, as well as modestly enhanced expression in the synovial lining layer, fibroblasts, and endothelial cells. Activation of the PPAR-γ by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and the synthetic PPAR-γ ligand (troglitazone) induced RA synoviocyte apoptosis in vitro. Moreover, intraperitoneal administration of these PPAR-γ ligands ameliorated adjuvant-induced arthritis with suppression of pannus formation and mononuclear cell infiltration in female Lewis rats. Anti-inflammatory effects of 15d-PGJ2 were more potent than troglitazone. These findings suggest that PPAR-γ may be an important immunoinflammatory mediator and its ligands, especially 15d-PGJ2, may be useful in the treatment of RA.

The Severity of Ultrasonographic Findings in Nonalcoholic Fatty Liver Disease Reflects the Metabolic Syndrome and Visceral Fat Accumulation

BACKGROUND:Nonalcoholic fatty liver disease (NAFLD) is closely associated with the metabolic syndrome.AIM:We evaluated the association among the metabolic syndrome, visceral fat accumulation, and the severity of fatty liver with a new scoring system of ultrasonographic findings in apparently healthy Japanese adults.METHODS:Subjects consisted of 94 patients who received liver biopsy and 4,826 participants who were selected from the general population. Two hepatologists scored the ultrasonographic findings from 0 to 6 points. We calculated Cohens kappa of within-observer reliability and between-observer reliability. We evaluated the predictive value of the score by the area under a conventional receiver operating characteristic curve (AUC).RESULTS:Within-observer reliability was 0.95 (95% CI 0.93–0.97, P < 0.001) and between-observer reliability was 0.95 (95% CI 0.93–0.97, P < 0.001). The AUC to diagnose NAFLD was 0.980. The sensitivity was 91.7% (95% CI 87.0–95.1, P < 0.001) and the specificity was 100% (95% CI 95.4–100.0, P < 0.001). The AUC to diagnose visceral obesity was 0.821. The sensitivity was 68.3% (95% CI 51.9–81.9, P = 0.028) and the specificity was 95.1% (95% CI 86.3–99.0, P < 0.001). Adjusted odds ratio of the score for the metabolic syndrome was 1.37 (95% CI 1.26–1.49, P < 0.001).CONCLUSIONS:The scoring system with abdominal ultrasonography could provide accurate information about hepatic steatosis, visceral obesity, and the metabolic syndrome in apparently healthy people who do not consume alcohol.

Local secretion of corticotropin-releasing hormone by enterochromaffin cells in human colon

BACKGROUND/AIMS Corticotropin-releasing hormone (CRH) is a regulator of the hypothalamic-pituitary-adrenal axis and a coordinator of the gastrointestinal response to stress. In addition to its central effects, CRH has peripheral effects on the immune system. CRH is present in several human tissues, such as the brain, spinal cord, adrenal medulla, lung, liver, peripheral blood leukocytes, as well as the gastrointestinal tract. The current study examined the local production of CRH in the normal human colon. METHODS Normal human colonic tissues obtained by endoscopic biopsy were immunostained with anti-CRH and anti-5-hydroxytryptamine antibody and analyzed for CRH messenger (m)RNA by a reverse-transcribed polymerase chain reaction method and by in situ hybridization. RESULTS Immunoreactive CRH and CRH mRNA were detected in the colonic mucosal cells in the neighborhood of the base of the crypts. The mucosal cells that expressed CRH mRNA also immunostained with anti-5-hydroxytryptamine antibody. CONCLUSIONS Normal human colonic mucosal enterochromaffin cells produce CRH. CRH in the colonic mucosa may play a role in the modulation of the intestinal immune system and/or other gastrointestinal functions basally during stressful conditions.

Expression of peroxisome proliferator-activated receptors (PPARs) in human urinary bladder carcinoma and growth inhibition by its agonists.

Recent studies have demonstrated that peroxisome proliferator activator‐receptors(PPAR)‐γ is expressed in various cancer tissues and its ligand induces growth arrest of these cancer cells through apoptosis. In our study, we investigated the expression of PPAR‐α, β and γ in human bladder tumor (BT) and normal bladder (NB) tissues as well as the effects of PPAR‐γ ligands. Specimens were obtained from 170 patients with BT and 20 with NB. The expressions were investigated using RT‐PCR and immunohistochemical methods. We also investigated the inhibitory effect of PPAR‐γ ligands on BT‐derived cell line. Immunoreactive PPAR‐α and ‐β were significantly apparent in both BT and NB tissues. Although no marked expression of PPAR‐γ was observed in NB tissue, significant expression was found in BT tissue. The extent and intensity of immunoreactive PPAR‐γ polypeptides in BT cells were statistically much greater than those of NB cells. Correlation between PPAR‐γ expression and tissue type or progression of bladder cancer was observed; PPAR‐γ expression was higher in G3 of bladder cancer than in G1 and was higher in advanced than in early cancer. PPAR‐γ agonists, troglitazone and 15‐deoxy‐Δ12, 14‐prostaglandin J2 inhibited the growth of the BT cells. PPAR‐γ is expressed in bladder tumor, and results suggest that PPAR‐γ ligands may mediate potent antiproliferative effects against BT cells. Thus, PPAR‐γ has the ability to become a new target in treatment of bladder tumor.

IDENTIFICATION OF FOUR NEW QUANTITATIVE TRAIT LOCI REGULATING ARTHRITIS SEVERITY AND ONE NEW QUANTITATIVE TRAIT LOCUS REGULATING AUTOANTIBODY PRODUCTION IN RATS WITH COLLAGEN-INDUCED ARTHRITIS

OBJECTIVE Collagen-induced arthritis (CIA) is a polygenic model of experimentally induced autoimmunity and chronic joint inflammation. This study maps genetic loci that regulate CIA susceptibility in DA/Bkl (DA) and BN/SsNHsd (BN) rats. METHODS Genome scans covering chromosomes 1-20 and interval mapping techniques using 159 simple sequence-length polymorphism markers were used to identify quantitative trait loci (QTLs) that regulate CIA in (DA x BN)F2 hybrids. Serum antibody titers to type II collagen were determined by enzyme-linked immunosorbent assay. RESULTS DA rats were high responders to porcine type II collagen (PII) and developed severe CIA (100%). BN rats were low responders to PII and resistant to CIA (0%). BN genes strongly repressed PII-induced CIA. Only 12% of (DA x BN)F1 rats (7 of 60) and 31% of (DA x BN)F2 rats (307 of 1,004) developed CIA. Three new QTLs (Cia11, Cia12, and Cia13) with significant logarithm of odds (LOD) scores of 5.6, 4.6, and 4.5, respectively, plus a suggestive QTL (Cia14*, LOD 3.0) regulating arthritis severity were identified on chromosomes 3, 12, 4, and 19. A new QTL, Ciaa3, associating with anticollagen antibody titer (antibody to PII LOD 6.5; antibody to rat type II collagen LOD 5.2) mapped to chromosome 9. Of 10 CIA QTLs previously identified in (DA x F344) and (DA x ACI) rats, only Cia1 in the major histocompatibility complex and a region coincident to Cia5 on chromosome 10 (LOD >8.0) influenced CIA severity in (DA x BN)F2 rats. CONCLUSION Since CIA exhibits many of the pathologic features of rheumatoid arthritis, the data indicate that the variety of genetic elements regulating human autoimmune and rheumatic diseases may be much larger and more varied than originally envisioned.

Myeloid-Derived Suppressor Cells Play Crucial Roles in the Regulation of Mouse Collagen-Induced Arthritis

Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. The role of MDSCs in autoimmune diseases remains controversial, and little is known about the function of MDSCs in autoimmune arthritis. In this study, we clarify that MDSCs play crucial roles in the regulation of proinflammatory immune response in a collagen-induced arthritis (CIA) mouse model. MDSCs accumulated in the spleens of mice with CIA when arthritis severity peaked. These MDSCs inhibited the proliferation of CD4+ T cells and their differentiation into Th17 cells in vitro. Moreover, MDSCs inhibited the production of IFN-γ, IL-2, TNF-α, and IL-6 by CD4+ T cells in vitro, whereas they promoted the production of IL-10. Adoptive transfer of MDSCs reduced the severity of CIA in vivo, which was accompanied by a decrease in the number of CD4+ T cells and Th17 cells in the draining lymph nodes. However, depletion of MDSCs abrogated the spontaneous improvement of CIA. In conclusion, MDSCs in CIA suppress the progression of CIA by inhibiting the proinflammatory immune response of CD4+ T cells. These observations suggest that MDSCs play crucial roles in the regulation of autoimmune arthritis, which could be exploited in new cell-based therapies for human rheumatoid arthritis.

Induction of cyclooxygenase-2 in monocyte/macrophage by mucins secreted from colon cancer cells

Up-regulation of cyclooxygenase-2 (COX-2) and overproduction of prostaglandins have been implicated in the initiation and/or progression of colon cancer. However, it is uncertain in which cells and how COX-2 is induced initially in the tumor microenvironment. We found that a conditioned medium of the colon cancer cell line, LS 180, contained a factor to induce COX-2 in human peripheral blood mononuclear cells. This factor was purified biochemically and revealed to be mucins. A small amount of mucins (≈100 ng of protein per ml) could elevate prostaglandin E2 production by monocytes. The mucins induced COX-2 mRNA and protein levels of monocytes in a dose- and time-dependent manner, indicating a COX-2-mediated pathway. We also have examined immunohistochemically the localization of COX-2 protein and mucins in human colorectal cancer tissues. It is noteworthy that COX-2-expressing macrophages were located around the region in which mucins were detectable, suggesting that COX-2 also was induced by mucins in vivo. These results suggest that mucins produced by colon cancer cells play a critical role in the initial induction of COX-2 in the tumor microenvironment.

A Critical Role for Allograft Inflammatory Factor-1 in the Pathogenesis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is characterized by massive synovial proliferation, angiogenesis, subintimal infiltration of inflammatory cells and the production of cytokines such as TNF-α and IL-6. Allograft inflammatory factor-1 (AIF-1) has been identified in chronic rejection of rat cardiac allografts as well as tissue inflammation in various autoimmune diseases. AIF-1 is thought to play an important role in chronic immune inflammatory processes, especially those involving macrophages. In the current work, we examined the expression of AIF-1 in synovial tissues and measured AIF-1 in synovial fluid (SF) derived from patients with either RA or osteoarthritis (OA). We also examined the proliferation of synovial cells and induction of IL-6 following AIF-1 stimulation. Immunohistochemical staining showed that AIF-1 was strongly expressed in infiltrating mononuclear cells and synovial fibroblasts in RA compared with OA. Western blot analysis and semiquantitative RT-PCR analysis demonstrated that synovial expression of AIF-1 in RA was significantly greater than the expression in OA. AIF-1 induced the proliferation of cultured synovial cells in a dose-dependent manner and increased the IL-6 production of synovial fibroblasts and PBMC. The levels of AIF-1 protein were higher in synovial fluid from patients with RA compared with patients with OA (p < 0.05). Furthermore, the concentration of AIF-1 significantly correlated with the IL-6 concentration (r = 0.618, p < 0.01). These findings suggest that AIF-1 is closely associated with the pathogenesis of RA and is a novel member of the cytokine network involved in the immunological processes underlying RA.

C‐MYC antisense oligodeoxynucleotides can induce apoptosis and down‐regulate Fas expression in rheumatoid synoviocytes

OBJECTIVE To investigate the role of c-myc in the pathogenesis of rheumatoid arthritis (RA) and the mechanism of synovial apoptosis. METHODS Using cultured human synoviocytes from patients with RA and c-myc antisense oligodeoxynucleotides (AS ODN), we examined the inhibition of cell proliferation by the MTT assay and the induction of apoptosis with TUNEL staining and fluorescence microscopy. In addition, the effect of c-myc on down-regulation of Fas expression was analyzed by flow cytometry, cytotoxicity assay, and reverse transcriptase-polymerase chain reaction. RESULTS Treatment with c-myc AS ODN induced inhibition of cell proliferation, along with down-regulation of c-Myc protein and c-myc messenger RNA (mRNA) expression. The morphologic changes of synovial cell death were typical of apoptosis. In addition, c-myc AS ODN treatment down-regulated expression of Fas mRNA but not Fas antigen. Analysis of the involvement of the caspase cascade revealed that the cytotoxic activity of c-myc AS ODN was completely blocked by inhibitors of both caspase 1 (YVAD-FMK) and caspase 3 (DEVD-FMK). CONCLUSION Our results strongly suggest that c-myc AS ODN might be a useful therapeutic tool in RA and clarify that cell death by c-myc AS ODN is induced through the caspase cascade, similar to Fas-induced apoptosis. In addition, combination therapy with anti-Fas antibody and c-myc AS ODN reduced Fas-dependent cytotoxicity.

Reciprocal expression of MRTF-A and myocardin is crucial for pathological vascular remodelling in mice

Myocardin‐related transcription factor (MRTF)‐A is a Rho signalling‐responsive co‐activator of serum response factor (SRF). Here, we show that induction of MRTF‐A expression is key to pathological vascular remodelling. MRTF‐A expression was significantly higher in the wire‐injured femoral arteries of wild‐type mice and in the atherosclerotic aortic tissues of ApoE−/− mice than in healthy control tissues, whereas myocardin expression was significantly lower. Both neointima formation in wire‐injured femoral arteries in MRTF‐A knockout (Mkl1−/−) mice and atherosclerotic lesions in Mkl1−/−; ApoE−/− mice were significantly attenuated. Expression of vinculin, matrix metallopeptidase 9 (MMP‐9) and integrin β1, three SRF targets and key regulators of cell migration, in injured arteries was significantly weaker in Mkl1−/− mice than in wild‐type mice. In cultured vascular smooth muscle cells (VSMCs), knocking down MRTF‐A reduced expression of these genes and significantly impaired cell migration. Underlying the increased MRTF‐A expression in dedifferentiated VSMCs was the downregulation of microRNA‐1. Moreover, the MRTF‐A inhibitor CCG1423 significantly reduced neointima formation following wire injury in mice. MRTF‐A could thus be a novel therapeutic target for the treatment of vascular diseases.