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Cross talk between inflammation and coagulation plays important roles in acute or subacute progressive pulmonary fibrosis characterized by diffuse alveolar damage. Thrombomodulin is a physiological inhibitor of high-mobility group box 1 (HMGB1), and thrombin and may be effective for this condition. This study investigated the roles of HMGB1 and thrombin in the pathophysiology of bleomycin-induced pulmonary fibrosis and the efficacy of recombinant human soluble thrombomodulin (rhTM). Pulmonary fibrosis was induced in wild-type C57BL/6 mice by intratracheal instillation of bleomycin. We first assessed HMGB1, thrombin, transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) levels in bronchoalveolar lavage fluid and lung tissue sections over time. Expression of HMGB1 and thrombin was elevated before that of TGF-β1 and α-SMA and remained high during the fibrotic phase after bleomycin instillation. We next examined whether in vitro stimulation with HMGB1 and thrombin induced expression of TGF-β1 and α-SMA in cultured alveolar macrophages and lung fibroblasts, respectively, by performing quantitative PCR, enzyme-linked immunosorbent assay, Western blot, and immunofluorescence analyses. HMGB1 and thrombin stimulation induced TGF-β1 production by alveolar macrophages, and thrombin stimulation also induced α-SMA expression in lung fibroblasts. Finally, we evaluated the effect of rhTM on bleomycin-induced pulmonary fibrosis. Compared with the vehicle control, both early and late-phase administration of rhTM suppressed the fibrotic process. Our results suggest that HMGB1 and thrombin were involved in the pathophysiology of pulmonary fibrosis via production of profibrotic proteins and that rhTM attenuated bleomycin-induced pulmonary fibrosis. rhTM may be a therapeutic option for acute or subacute pulmonary fibrosis.

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries of rats with an ectopic cardiac allograft. Some studies have shown that expression of AIF-1 increased in a mouse model of trinitrobenzene sulfonic acid-induced acute colitis and in acute cellular rejection of human cardiac allografts. These results suggest that AIF-1 is related to acute inflammation. The current study used bleomycin-induced acute lung injury to analyze the expression of AIF-1 and to examine its function in acute lung injury. Results showed that AIF-1 was significantly expressed in lung macrophages and increased in bronchoalveolar lavage fluid from mice with bleomycin-induced acute lung injury in comparison to control mice. Recombinant AIF-1 increased the production of IL-6 and TNF-α from RAW264.7 (a mouse macrophage cell line) and primary lung fibroblasts, and it also increased the production of KC (CXCL1) from lung fibroblasts. These results suggest that AIF-1 plays an important role in the mechanism underlying acute lung injury.

Background Sphingosine-1-phosphate (S1P) is a biologically active phospholipid, which is derived from membrane lipid. It binds to the receptors, named S1P1–5, and regulates several signalling pathways involved in inflammation, cell survival, angiogenesis and cell migration. Concentration of S1P and expression of S1P receptors can vary according to local tissue conditions. RA is a chronic inflammatory disorder of joints and the concentration of S1P in synovial fluid is higher in RA patient than in OA patient. In vitro, S1P3 expression in RA synoviocyte is upregulated by TNFa treatment. On the other hand, it is not clarified whether S1P/S1P3 signalling pathway contributes to arthritis in RA. Objectives The objective of this study is to investigate the role of S1P/S1P3 signalling in inflammatory arthritis. Methods Collagen-induced arthritis (CIA) was induced by subcutaneous injection of bovine type II collagen emulsified in complete Freund’s adjuvant in wild-type (WT) or S1P3-knock-out (S1P3-KO) 7–9 week-old DBA/1J mice. Arthritis severity were evaluated by visual scoring and histological analysis. The severity was assessed over time by using the arthritis score, in which each paw was scored on a scale of 0–4 and the scores of all four paws were cumulated, resulting in a maximum possible score of 16 per mouse. For histopathological examination, mice were sacrificed on the 42nd day and the hindlimbs were removed and fixed in 4% buffered formaldehyde. Paraffin embedded sections of the knee joints stained with hematoxylin and eosin were systematically scanned in a microscope and scored based on cell infiltration, cartilage destruction and bone erosion parameters. S1P3 mRNA expression was examined by realtime PCR method with total RNA extracted from knee joint capsules of CIA or normal WT mice. Murine primary fibroblast like synoviocytes (FLS) were obtained from CIA mice. We examined S1P3 expression after TNFa treatment and measured cytokine produced after S1P treatment with or without TNFa pretreatment in FLS. Results S1P3 deficiency resulted in modest symptoms of arthritis and a significant reduction in synovial inflammation and bone erosions in histological analysis. S1P3 mRNA expression in knee joint capsule in CIA mice was about five times as high as that in normal mice. TNFa treatment upregulated S1P3 expression and S1P treatment enhanced IL-6 production in WT-FLS significantly. TNFa-priming enhanced S1P-induced IL-6 production, which is significantly higher in WT-FLS than in KO-FLS. This effect was not observed in MCP-1 production of WT-FLS. Conclusions S1P3-KO reduced severity of arthritis, inflammation and bone erosions in CIA. S1P3 mRNA was upregulated in inflamed joint capsule. S1P induces IL-6 production via S1P3 upregulation by TNFa in CIA-FLS. S1P3 inhibition could be a good target of the therapy for arthritis. References [1] Blaho VA and Hla T. An update on the biology of sphingosine 1-phosphate receptors. J Lipid Res55:1596–608. [2] Zhao C, Fernandes MJ, and Bourgoin SG, et al. Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-alpha. J Lipid Res49: 2323–37. Disclosure of Interest None declared

Objective Synovial fluid pH is decreased in patients with rheumatoid arthritis (RA); however, the underlying mechanisms are unclear. We undertook this study to examine the mechanism by which synovial fluid pH is regulated and to explore the possibility of a therapeutic strategy by manipulating this mechanism. Methods We determined the pH and lactate concentration in synovial fluid from 16 RA patients. Cultured synovial fibroblasts (SFs) from the inflamed joints of 9 RA patients (RASFs) were examined for the expression of ion transporters that regulate intracellular and extracellular pH. The ion transporter up-regulated in RASF lines was then suppressed in RASFs by small interfering RNA (siRNA), and the effect of transfection on viability and proliferation was investigated. Finally, we examined the therapeutic effect of electrotransfer of monocarboxylate transporter 4 (MCT4)–specific siRNA into the articular synovium of mice with collagen-induced arthritis (CIA). Results Synovial fluid pH correlated inversely with both the Disease Activity Score in 28 joints using the C-reactive protein level and the synovial fluid lactate levels. RASFs exhibited up-regulated transcription of MCT4 messenger RNA. MCT4 exported intracellular lactate into the extracellular space. RASFs had significantly higher MCT4 protein levels than did SFs from patients with osteoarthritis. Knockdown of MCT4 induced intrinsic apoptosis of RASFs, thereby inhibiting their proliferation. Moreover, electrotransfer of MCT4-specific siRNA into the articular synovium of mice with CIA significantly reduced the severity of arthritis. Conclusion RA activity correlated with decreased synovial fluid pH. This may be due to increased MCT4 expression in RASFs. Silencing MCT4 induced apoptosis in RASFs and reduced the severity of CIA, suggesting that MCT4 is a potential therapeutic target for inflammatory arthritis.

Background Patients with glucocorticoid-induced osteoporosis (GIOP) are at very high risk of fracture, and patients with severe GIOP often experience fractures during treatment with bisphosphonates. Teriparatide (TPTD) is the only currently available anabolic agent expected to be effective for GIOP. Once-weekly TPTD decreased bone resorption marker with primary osteoporosis different from daily TPTD, but it has not yet been tested with GIOP. Objectives To evaluate the efficacy of once-weekly TPTD for patients with GIOP and inadequate response to bisphosphonates. Methods Patients with GIOP and collagen diseases treated with prednisolone for at least 6 months with inadequate responses to bisphosphonates were administered once-weekly TPTD. Bone density of the lumbar spine and femoral neck, measured as percent young adult mean (YAM); serum concentrations of cross-linked N-terminal telopeptides of type I collagen (NTx), bone alkaline phosphatase (BAP), and calcium; and FRAX were measured at baseline and 6, 12 and 18 months after starting TPTD. Results Of the 12 GIOP patients with collagen diseases enrolled, nine (seven females, two males; mean age 57.4 ± 11.1 years) completed treatment, including six with systemic lupus erythematosus, two with rheumatoid arthritis, and one with adult onset still disease. Only one new fracture event, a lumbar compression fracture, occurred during the study period, although seven patients experienced eight fracture events within 18 months before starting TPTD (p = 0.04). Lumbar spine YAM significantly improved at 18 months (p = 0.04), whereas femoral neck YAM did not (p = 0.477). Serum NTx, BAP, Ca, and FRAX were not significantly affected by TPTD treatment. Conclusions Once-weekly TPTD reduces fracture events and increases bone density of the lumbar spine of GIOP patients with inadequate response to bisphosphonates.