In polymyositis (PM)/dermatomyositis (DM), T cells infiltrate the muscle tissues and interact with muscle cells via cell surface molecules. Recently, myoblasts have been reported to express CD40, but little is known about the role of CD40 in myoblasts. In the present study we examined the expression and involvement of CD40 and CD40 ligand (CD40L) in the interaction between muscle cells and T cells in PM/DM. Immunohistochemical staining revealed that CD40 was expressed on muscle cells in five of five PM and four of five DM patients, and that infiltrating mononuclear cells (MNCs) expressed CD40L in all cases of PM/DM. These CD40L-expressing MNCs were primarily CD4+ T cells. IFN-γ, which is known to induce CD40 expression on various types of cells, was also expressed on the MNCs in four of the PM and four of the DM patients. Although cultured human myoblasts (SkMC 2859) did not express CD40 constitutively, IFN-γ induced CD40 expression in a dose-dependent manner. To clarify the functional roles of CD40-mediated signals, the effects of a trimeric form of recombinant human CD40L on cytokine production were studied in SkMC 2859 that were prestimulated with IFN-γ to express CD40. Recombinant human CD40L markedly increased the production of IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 of SkMC 2859. The expression of these humoral factors in muscle cells of PM and DM was demonstrated by immunohistochemistry. These results suggest that interaction between T cells and muscle cells via the CD40-CD40L system contributes to the immunopathogenesis of PM/DM by augmenting inflammation via cytokine production by the muscle cells.
Objective. Tissue fibrosis in systemic sclerosis (SSc) is attributed to excessive deposition of extracellular matrix components produced by fibroblasts in skin lesions. Angiotensin II (Ang II), a vasoconstrictive peptide, is reported to have profibrotic activity as a result of induction of the extracellular matrix. The aim of the present study was to examine the expression of Ang II and its type 1 (AT 1 ) and type 2 (AT 2 ) receptors in affected skin and dermal fibroblasts from patients with SSc and to study the role of Ang II in collagen production by SSc dermal fibroblasts.Methods. Levels of Ang II in sera from SSc patients and normal subjects were measured by a solid-phase immobilized-epitope immunoassay. Expression of angiotensinogen (Angt) in the skin was evaluated by immunohistochemistry. Expression of Angt, AT 1 , and AT 2 in cultured dermal fibroblasts was analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. Levels of type I procollagen produced by cultured dermal fibroblasts were measured by enzyme-linked immunosorbent assay.Results. Serum Ang II levels in patients with diffuse cutaneous SSc were significantly higher than those in patients with limited cutaneous SSc and in healthy donors. Immunohistochemical and immunoblotting analyses showed that Angt was present in skin from SSc patients, but not in normal skin. Angt messenger RNA (mRNA) was expressed in fibroblasts from patients with diffuse cutaneous SSc who had high levels of serum Ang II, but not in normal fibroblasts. AT 1 mRNA expression was found in both SSc and normal fibroblasts, whereas AT 2 mRNA was found only in SSc fibroblasts. Exogenous Ang II augmented the production of type I procollagen and transforming growth factor 1 by cultured fibroblasts via activation of AT 1 .
In polymyositis (PM)/dermatomyositis (DM), various cytokines, especially macrophage-derived cytokines such as IL-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha, are expressed in the inflammatory foci. We previously reported that IL-15, a novel cytokine with a biological activity similar to that of IL-2, is expressed in muscle cells in PM/DM. In the present study, we set out to investigate the regulation of IL-15 in cultured myoblasts. Myoblasts constitutively produced a low level of IL-15 and the production was augmented by stimulation with IFN-gamma, IL-1alpha, IL-1beta, TNF-alpha or lipopolysaccharide (LPS) in a dose-dependent manner. These stimuli also enhanced the expression of IL-15 mRNA. About 30-40% of IL-15 was detected intracellularly, while the rest was released into the culture supernatant. Immunohistochemical staining revealed that intracellular IL-15 was localized in the perinuclear area of the cytoplasm in the myoblasts. Despite the considerable amounts of intracellular IL-15, the myoblasts predominantly expressed authentic IL-15 mRNA isoform. This isoform generates IL-15 with long signal peptide preprotein, which is all to be secreted. The biological activity of IL-15 secreted from the myoblasts was examined using an IL-15-dependent murine T cell line, CTLL-2. Culture supernatants of the myoblasts induced a proliferative response of CTLL-2 and this was specifically inhibited by anti-IL-15 antibody. These results suggest that inflammatory stimuli induce the production of IL-15 in the muscle cells in PM/DM, and IL-15 may contribute to the immunopathogenesis by augmenting recruitment and activation of the infiltrating T cells. Blocking of IL-15 production might be of therapeutic value in PM/DM.
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that is characterized by tissue fibrosis, which may result from the activation of lesional fibroblasts exhibiting excessive production of extracellular matrix components. However, it has yet to be determined how SSc fibroblasts are activated. CD40 is a cell surface molecule expressed on various cells that is important for the response to activated T cells through CD154. CD40 mRNA was found to be constitutively expressed in both SSc and normal fibroblasts by reverse transcription PCR. Expression of CD40 protein was increased on SSc fibroblasts compared to normal fibroblasts as measured by flow cytometry. Ligation of CD40 by recombinant human CD154 (0.5–2 μg/ml) resulted in increased production of IL‐6, IL‐8, and monocyte chemoattractant protein‐1 in SSc fibroblasts in a dose‐dependent manner, whereas these phenomena were not shown in normal fibroblasts even with the addition of CD154. CD80, a costimulatory molecule, was also induced on SSc fibroblasts by CD40 ligation. In the present study, our findings suggest the possibility of a cell‐mediated response between fibroblasts and T cells in the lesional skin of SSc patients. Since it is suggested that the CD40‐CD154 system may play a crucial role in the aberrant production of immune mediators by SSc fibroblasts, blockage of CD40‐CD154 may be a novel therapeutic strategy for SSc.
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