RESUMO
Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are relatively resistant to apoptosis. Transforming growth factor-beta (TGF)-beta is strongly implicated in the pathogenesis of SSc and we and others have shown that TGF-beta can activate Akt, a kinase with potent anti-apoptotic effects. To determine whether Akt was activated in SSc, we quantified phospho-Akt expression in skin fibroblasts in vitro by western blot analysis and a functional kinase assay. In addition, the relative proportion of fibroblasts containing activated Akt in was quantified by immunohistochemistry on skin sections insitu. Analysis of Akt phosphorylation of skin fibroblasts in vitro suggested increased phosphorylation of Akt, and evaluation of skin sections by immunohistochemistry revealed significantly higher percentages of fibroblasts that stained for phospho-Akt compared with controls (78% +/- 14.0% vs 13% +/- 9%, p < 0.001). In addition, co-incident staining of phospho-Akt and alpha-smooth muscle actin was observed in some fibroblasts. These findings indicate that Akt is activated insitu in skin fibroblasts from patients with SSc. Akt activation may contribute to resistance to apoptosis, selection of disease-inducing fibroblasts, and, possibly, myofibroblasts.
Assuntos
Fibroblastos/enzimologia , Fibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Apoptose/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/fisiologia , Pele/enzimologia , Pele/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
A variety of complement components have been detected on apoptotic cells and proposed to facilitate recognition and/or ingestion by phagocytes. The triggers for complement activation remain uncertain. To determine the role of IgM in classical pathway activation and clearance of apoptotic cells in vitro and in vivo, we quantified these parameters in mice deficient in serum IgM (sIgM). Phagocytosis by bone marrow-derived macrophages of apoptotic cells incubated with serum deficient in sIgM was markedly reduced, similar to apoptotic cells incubated with C1q deficient serum in vitro. Similarly, intraperitoneal clearance of apoptotic cells and cellular C3 deposition were significantly reduced in mice deficient in sIgM compared to wild-type mice. Clearance and C3 deposition were reconstituted by addback of IgM. In mice deficient in both sIgM and Clq, addback of both serum factors was required for restoration of clearance. These findings indicate that, on a quantitative basis, sIgM is a potent factor required for intraperitoneal phagocytosis of apoptotic cells, and further demonstrate that IgM and C1q work in concert to activate complement, resulting in C3 deposition on the apoptotic cell surface and ultimately, efficient clearance of the apoptotic cell by macrophages.