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OBJECTIVE Levels of interleukin 17A (IL 17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL 17A producing cells are present in affected SSc skin and whether IL 17A exerts a role in the transdifferentiation of myofibroblasts. METHODS Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo including IL 17A+ IL 4+ CD3+ tryptase positive a smooth muscle actin (a SMA) positive myeloperoxidase positive and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples and quantitative polymerase chain reaction Western blotting and solid phase assays were used to quantify a SMA type I collagen and matrix metalloproteinase 1 (MMP 1) production by the cultured fibroblasts. RESULTS IL 17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase positive mast cells in the production of IL 17A was observed. Fibroblasts positive for a SMA were found adjacent to IL 17A+ cells but not IL 4+ cells. However IL 17A did not induce a SMA expression in cultured fibroblasts. In the presence of IL 17A the a SMA expression induced in response to transforming growth factor ß was decreased while MMP 1 production was directly enhanced. Furthermore the frequency of IL 17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score). CONCLUSION IL 17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL 17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL 17A has a direct negative regulatory role in the development of dermal fibrosis in humans.

Objective Fibroblasts play a major role in the development of systemic sclerosis (SSc), and the occurrence of serum autoantibodies reacting with fibroblast plasma membrane antigens in SSc has been reported. This study was undertaken to investigate whether IgG from SSc sera that react with human fibroblasts modulates the fibroblasts' function. Methods Sera from 69 patients with SSc (28 with limited cutaneous SSc [lcSSc] and 41 with diffuse cutaneous SSc [dcSSc]), 30 patients with sarcoidosis, and 50 matched healthy controls were examined. We evaluated antibody binding to human skin and lung fibroblasts by cell-based enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and flow cytometry. We further investigated the ability of purified IgG to modulate 1) intercellular adhesion molecule 1 (ICAM-1) expression, 2) U937 cell adhesion to fibroblasts, and 3) fibroblast steady-state messenger RNA (mRNA) levels of interleukin-1α (IL-1α), IL-β, and IL-6, and IL-6 protein production. Results Of 69 SSc sera tested by cell-based ELISA, 58% bound to normal skin and lung fibroblasts. The prevalence of binding was significantly higher in dcSSc than in lcSSc (P < 0.05). Only IgG from SSc sera that were positive for antifibroblast antibody (AFA) induced a dose-dependent up-regulation of ICAM-1 expression and IL-6 production, enhancement of U937 cell adhesion, and increased levels of IL-1α, IL-1β, and IL-6 mRNA in fibroblasts. Up-regulation of ICAM-1 mediated by AFA IgG was inhibited by the addition of IL-1 receptor antagonist, indicating an autocrine activation loop. Conclusion Our findings confirm the presence of AFAs in SSc sera and demonstrate, for the first time, that autoantibodies reacting with fibroblast surface molecules act as an extrinsic stimulus inducing fibroblast activation in vitro.

Objective Fibroblast activation is a crucial event in the development of systemic sclerosis (SSc). Antifibroblast autoantibodies (AFAs), detectable in the sera of SSc patients, are able to induce a proinflammatory phenotype on cultured fibroblasts. This study was undertaken to investigate the mechanisms of the interaction between AFAs and living fibroblasts. Methods We coupled to fluorescein 1) IgG purified from AFA-positive and AFA-negative SSc sera (as assessed by cellular enzyme-linked immunosorbent assay) and 2) single healthy donor and pooled normal IgG. The interaction of IgG with living cultured fibroblasts from healthy individuals and from a patient with SSc was visualized by real-time confocal microscopy. Intracellular colocalization of caveolin and internalized AFA-positive IgG was assessed by immunofluorescence. Results AFA-positive IgG bound to living fibroblasts and was internalized with a cytoplasmic fibrillar pattern, in contrast to AFA-negative IgG. In the IgG tested, no correlation with antinuclear antibody activity was found. Preincubation of fibroblasts with normal IgG did not affect internalization. Internalized AFA-positive IgG colocalized with caveolin, and internalization was entirely inhibited by disassembling fibroblast caveolae with filipin. Conclusion The finding that both normal and pathologic fibroblasts specifically internalized AFA-positive, but not AFA-negative, IgG demonstrates that AFAs in SSc patient sera interact with constitutively expressed membrane molecules on fibroblasts, via an Fc-independent mechanism. The results of colocalization and inhibition experiments suggest that microdomains containing caveolin are involved in the interaction between AFAs and fibroblasts. These data, together with the reported ability of AFAs to activate fibroblasts, provide evidence for a role of AFAs in the pathogenesis of SSc.

Objective To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl–coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti–β2-glycoprotein I (anti-β2GPI) antibodies in vitro. Methods Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule 1 (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin–specific nuclear factor κB (NF-κB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-β2GPI antibodies, human recombinant IL-1β, tumor necrosis factor α, or lipopolysaccharide (LPS). Results Fluvastatin reduced, in a concentration-dependent manner (1–10 μM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-β2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, display similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-κB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-β2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 μM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. Conclusion Endothelial activation mediated by anti-β2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.