The Activation Effect Of S100A9on Fibroblasts In Scleroderma

BackgroundScleroderma is an autoimmune disease, characterized by progressive fibrosis of skin and internal organs. There are two different clinical subsets of SSc. The diffuse cutaneous form is characterized by rapidly progressive fibrosis of the skin and visceral organs, while in the limited cutaneous form, skin and organ fibrosis is generally limited and the progression is slow. Fibrosis is the most remarkable clinic character in the process of scleroderma, and it is also the major threaten of influencing on the life quality of patients, even the patients’life. Although the pathogenesis underlying scleroderma has been widely studied, the exact molecular mechanisms remain obscure.S100A9, one member of the S100proteins, is constitutively expressed in cells of the myelomonocytic lineage. It is confirmed that S100A9is chemotactic for neutrophils, and increase their transendothelial migration. It also induces inflammatory cells secretion of cytokines and chemokines and thus, initiation and amplification of inflammation. Recently, S100A9is found to be associated with many inflammatory and autoimmune conditions. Our laboratory found that the skin tissues from bleomycin treated mice had significantly increased expression of S100A9. We hypothesized that the cytokine may be involved in scleroderma. However, there is no precise data about its mechanism in scleroderma, which prompted us to explore its potential involvement in the disorder.ObjectiveWe performed this study to determine the protein and gene expression levels of S100A9in patients with scleroderma, and to identify any associations in expression of S100A9with clinical or laboratory parameters in SSc patients, and to understand the role of S100A9in mice models of scleroderma established by local injections of bleomyein (BLM), and to investigate the effect of S100A9on human skin fibroblast proliferation and production of cytokines and collagen, and the pathways involved, giving new insights into the possible mechanism.MethodsFirstly,26localized scleroderma (LS),31limited cutaneous SSc (lcSSc) and57diffuse cutaneous SSc (dcSSc) patients were recruited in the present study. The expression of S100A9in plasma was measured using ELISA and mRNA levels in peripheral blood were assessed using RT-PCR. The expression and distribution of S100A9and its receptor RAGE in skin tissues was analyzed by immunohistochemistry. Secondly, the expression of S100A9in skin of scleroderma mice model was measured by RT-PCR and Western blot. The changes of the skin appearance, histopathy of skin lesion and the levels of inflammatory cytokines and collagen of skin were observed after local injection of S100A9with or without bleomycin in the mouse skin. Thirdly, human skin fibroblasts were cultured in vitro, and exposed to S100A9with or without blocking antibody. Proliferation of fibroblasts was measured using CCK8and BrdU assay, and mRNA levels of inflammatory cytokines induced by S100A9were analyzed by real-time PCR. RAGE expression and MAPK pathways phosphorylation and NF-κB activation was determined in fibroblasts after S100A9exposure by Western blot.ResultsPart11. The plasma concentrations of S100A9were significantly higher in dcSSc patients than normal controls. Increased plasma levels of S100A9in dcSSc patients were associated with organ involvements and several autoantibodies. 2. Transcription levels of S100A9in peripheral blood were found elevated in three kinds of patients than normal controls. The mRNA expression of S100A9in dcSSc patients was much higher than lcSSc and LS patients.3. Immunohistochemistry demonstrated higher S100A9expression in sclerotic skin than in normal skin. The number of S100A9or RAGE positive fibroblasts was also significantly increased.Part21. High dose of bleomycin (0.3mg/ml) induced scleroderma mice model characterized by increased fiber bundles and up-regulated collagen deposition which was examined by HE staining and Masson’s trichrome staining. While, low dose of bleomycin (0.2mg/ml) did not cause obvious fibrotic skin.2. Compared to the control group, the gene and protein levels of S100A9were increased gradually in the mice skin after local injection of bleomycin (0.3mg/ml) for1,2, or3weeks.3. After local injections of S100A9and low dose of bleomycin, an intense dermal sclerosis was observed in the mice model. The dermal thickness in model mice was remarkably increased and the amount of inflammatory cytokines, a-SMA, and hydroxyproline in skin was also obviously enhaned, compared with high dose of bleomycin treated mice.4. Compared to the control group, high dose of S100A9caused amounts of inflammatory cells infiltration and increased the dermal thickness in the mice skin after3week injection.Part31. S100A9promoted proliferation of fibroblasts and up-regulated expression of pro-inflammatory cytokines IL-6, IL-8, IL-1β and HMGB1. S100A9also induced fibroblasts to produce collagen type I, a-SMA and RAGE. In addition, S100A9caused a significant increase in ERK1/2phosphorylation while the status of p38and JNK MAPK phosphorylation remained unchanged. Treatment of cells with S100A9also enhanced NF-κB activation.2. RAGE blocking antibody pretreatment inhibited the S100A9-induced cell proliferation, cytokine production and pathway phosphorylation. Meanwhile, S100A9-mediated cell activation was suppressed significantly by ERK1/2MAPK inhibitor and NF-κB inhibitor.3. Comparing to control group, local injections of S100A9indued a significant increase in ERK1/2MAPK and NF-κB phosphorylation in the mice skin. S100A9enhanced RAGE expression and ERK1/2MAPK and NF-κB phosphorylation in low dose of bleomycin injection group.Conclusion1. Highly elevated expression of S100A9was found in dcSSc patients and there was close correlation with disease severity and serological abnormalities, suggesting that the protein may play important roles in the development of systemic sclerosis.2. S100A9aggravated bleomycin induction of sclerotic changes of the mice skin and S100A9lonely could cause inflammation and fibrosis in skin, indicating that S100A9has participated in the development of scleroderma.3. S100A9activated fibroblasts via receptor RAGE and ERK1/2MAPK and NF-κB pathways mediated, and showed pro-inflammation and pro-fibrosis property. Therefore, S100A9played an important role in the pathogensis of scleroderma.