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Scleroderma is a generalized or localized disorder which leads to fibrosis of the affected organs. TGF-β has been implicated as a causal agent in its pathogenesis. In mammals, TGF-β comprises a family of three members, β1, β2 and β3. Since cutaneous wound healing is thought to result either in formation of a scar or in scar-free tissue regeneration, depending on the relative amounts of the β3 isoform, the expression of all three isoforms was studied in skin biopsies of patients with either localized or systemic scleroderma. mRNA for all three isoforms was detected in inflammatory skin areas of both disease forms, but never in sclerotic or healthy skin. Immunohistochemical analysis confirmed expression of β1 and β2 proteins in inflammatory skin of patients, whereas β3 protein appeared to be present in the subepidermal area and also found throughout the dermis of patients and healthy dermis as well.

Tumor invasion of basement membranes is a complex multi-step process. Altered adhesion, as well as increased cell locomotion contribute to tumor cell invasion and metastasis. A variety of in vitro models have been used to measure cell invasiveness. The invasion of basement membranes can be simulated in vitro in blind well Boyden Chambers using the reconstituted basement membrane matrigel or collagen type I as the invasion barrier. The aim of our study was to compare the migration and invasive capacity of epidermal tumor cells (TR 131, TR 146, SCL II, FaDu, HLaC 79) and melanoma cells derived from primary tumors (Mel Ei, Mel Ho, Mel Juso, Mel Wei) or their metastases (Mel Ju, Mel Im, Sk Mel 1, Sk Mel 28). Chemotactic response of epidermal tumor cells was increased toward fibroblast conditioned medium and fibronectin (20 micrograms/ml), while laminin (100 micrograms/ml) stimulated chemotaxis in only 3 epidermal tumor cell lines (HLaC 79, FaDu, TR 146), EGF (10 ng/ml) in only 4 cases (SCL II, FaDu, TR 131, TR 146), and interleukin-1 (IL-1) in only 1 case (FaDu). Epithelial tumor cell conditioned medium had no chemotactic influence on epithelial tumor cells. Fibroblast conditioned medium, fibronectin, EGF and PDGF were potent chemoattractants for all melanoma tumor cells, whereas IL-1 did not induce a significant chemotactic response. While two epithelial tumor cell lines (FaDu, TR 146) were able to penetrate collagen type I, matrigel was an impenetrable barrier for all epithelial tumor cells. Two cell lines from melanoma primary tumors (Mel Ho, Mel Ei) and two cell lines from melanoma metastases (Sk Mel 1, Sk Mel 28) showed no invasion through collagen type I and matrigel, whereas invasion through both barriers could be observed for the metastatic cell lines Mel Ju and Mel Im and in the primary tumor cell line Mel Wei. Therefore, the clinical observation of late and rare metastasis in epithelial tumors and early metastasis in melanoma correlate with our in vitro investigation of invasive behavior in tumor cells. No significant correlation between the invasiveness of melanoma cell lines and their clinical origin could be demonstrated suggesting the existence of subpopulations with varying invasive potential.

Direct effects of histamine on collagenous and non-collagenous protein synthesis by human skin fibroblasts were studied. Fibroblasts derived from human skin were incubated with various concentrations of histamine. Collagen and non-collagenous protein synthesis were measured by incorporation of 3H-proline. Both collagen synthesis measured as protein-bound hydroxyproline and non-collagenous protein synthesis measured as protein-bound proline increased in the presence of histamine at concentrations of 10(1)-10(2) micrograms/ml. Total RNA was extracted and m-RNA levels of various proteins were estimated by dot blot analysis, and densitometrically quantified. The levels of alpha 1(I) collagen and beta-actin m-RNA were clearly increased at the same concentrations. m-RNA levels of alpha 1(III) collagen were also increased but the rate was lower than that of alpha 1(I) collagen. No alteration of beta-tubulin m-RNA level was observed at the same concentrations. These results demonstrate that stimulation of collagen synthesis by histamine is pretranslationally controlled.

Keratinocytes and fibroblasts synthesize basement membrane proteins and even contribute to the formation of basement membrane structures following injury or tissue damage. Under these conditions many cellular functions are regulated by mediators e.g. transforming growth factor-beta, tumor necrosis factor alpha, interferon-gamma or interleukin-1 alpha. We therefore describe here their influence on synthesis of basement membrane proteins in human skin fibroblasts. A comparative analysis of mRNA steady levels coding for BM-40, nidogen, laminin B1 and B2 chains and collagen IV in fibroblasts, in primary human keratinocytes and a epidermal cell line grown in monolayer culture demonstrated that the highest amounts were present in human fibroblasts. Interferon-gamma reduces all mRNA steady state levels dose dependently in comparison to the control, while transforming growth factor-beta simultaneously induces BM-40, alpha 1 and alpha 2 (IV) collagen mRNAs. TGF-beta, however, has no effect on nidogen and laminin mRNA levels. Interleukin-1 alpha and tumor necrosis factor alpha do not affect the mRNA levels of most basement membrane proteins. However, the alpha 1 (IV) collagen mRNA is upregulated by both cytokines to 300%. These data demonstrate a specific control of the expression of several basement membrane proteins by cytokines and indicate that fibroblasts could contribute to basement membrane formation during wound healing and tissue repair.

Metabolism of fibroblasts plays a key role in wound healing, fibrosis, rheumatoid arthritis, and similar physiological and pathological processes. The regulatory influence of eicosanoids, an important class of inflammatory mediators, on fibroblast metabolism, in these processes is, to date, unclear. The aim of this study was to investigate the effect of some eicosanoids on chemotaxis and protein synthesis of fibroblasts in vitro. Of twelve eicosanoids tested, only 5(S)-HETE, LTB4, and 12(S)-HETE were active as chemo-attractants for fibroblasts. 5(S)-HETE was the most potent attractant. It exerted its maximal activity at 10−10 mol/l. 12(S)-HETE and LTB4 caused similar dose dependent fibroblast chemotaxis with a maximum of activity at 10−7 M and 5 × 10−8 M, respectively. Hydroxylation of LTB4 on C20 or methylation of the carboxy group of 12(S)-HETE decreased reactivity of the parent compounds only slightly. Eicosanoid induced chemotaxis could be antagonized by 12(S)-HETE but not by the proteinaceous chemoattractants fibronectin, PDGF, or EGF. Receptors for peptide and eicosanoid mediated chemotaxis are thus different. Inhibition of collagen synthesis was observed in the presence of 5(S)-HETE and 12(S)-HETE while total protein synthesis was unaffected by 12(S)-HETE and augmented by 5(S)-HETE. These data suggest that certain eicosanoids specifically regulate fibroblast activities in wound healing and similar events of connective tissue reorganization.

Mast cell infiltration and accumulation is known to occur in tissue fibrosis. Increased numbers of mast cells are detected in scleroderma or hypertrophic scar skin, however, neither the role of mast cells nor the interaction of fibroblasts and mast cells in fibrosis are fully understood. A growing body of evidence indicate that mast cells are rich source of cytokines, growth factors or chemokines, which are suggested to play an important role in the induction of fibrosis. Recent in vivo and in vitro studies suggest the involvement of monocyte chemoattractant protein-1 (MCP-1), a member of the C-C chemokine family, in fibrosis. Here, we examined the effect of stem cell factor (SCF), a mast cell growth factor, on MCP-1 gene expression in a human mast cell line, HMC-1, and as well as the effect of MCP-1 on alpha1(I) collagen gene expression in human skin fibroblasts. HMC-1 cells spontaneously expressed MCP-1 mRNA transcripts, which was detectable by in situ hybridization and Northern blot analysis. Stimulation with SCF further upregulated MCP-1 mRNA expression in a time- and dose-dependent manner, and stimulation with 100 ng/ml SCF for 24 h induced a 3-fold increase of MCP-1 mRNA expression in HMC-1 cells as compared with unstimulated cells. The concentration of MCP-1 protein in the culture supernatants of 50 ng/ml SCF-stimulated HMC-1 cells (3816+/-70 pg/ml) was significantly elevated compared to unstimulated cells (2588+/-130 pg/ml) (P < 0.01), as assessed by ELISA. Adversely, MCP-1 induced alpha1(I) collagen mRNA expression in normal skin fibroblasts dose-dependently. Finally, comparative study revealed that the concentration of SCF in the culture supernatants of scleroderma fibroblasts at primary passages was significantly increased (344.6+/-182.4 pg/ml), as compared with normal skin fibroblasts (72.4+/-20.2 pg/ml) (P

Systemic sclerosis (SSc) is a connective tissue disease with unknown etiology characterized by excessive deposition of collagen in the skin as well as various internal organs. One of the characteristic histological features is the presence of infiltrating mononuclear cells in the dermis in its early stage. As well as T cells, macrophages are implicated to play an important role in the initial pathologic changes associated with SSc by releasing fibrogenic cytokines, including transforming growth factor-beta or platelet-derived growth factor. However, the precise mechanism for increased monocyte/macrophage recruitment in the lesional skin of SSc is still not completely elucidated. Monocyte chemoattractant protein-1 (MCP-1) is a predominant monocyte chemoattractant secreted by various cells types including mononuclear cells, fibroblasts, smooth muscle cells, endothelial cells, or keratinocytes. In this study, we examined the expression of MCP-1 protein and mRNA in the lesional skin of seven patients with SSc by immunohistochemistry and in situ hybridization. Results of immunohistochemistry showed that MCP-1 was detected on infiltrating mononuclear cells and fibroblastic cells in scleroderma skin, whereas normal skin showed only minimal MCP-1 expression. We demonstrated the expression of MCP-1 mRNA in infiltrating mononuclear cells and keratinocytes in scleroderma and contact dermatitis skin. In addition, signals were also detected in fibroblasts in the lesional skin of scleroderma, whereas fibroblasts in normal skin and contact dermatitis skin did not express MCP-1 mRNA. These findings suggest that MCP-1 plays a role in recruiting monocyte/macrophages in the lesional skin of scleroderma and that activated fibroblasts in scleroderma are involved in this process.