TNF-alpha inhibits
collagen synthesis and at high concentrations stimulates
collagenase synthesis in fibroblasts. As fluid from chronic inflammatory lesions contains significant levels of
TNF-alpha, it is puzzling why these lesions exhibit dense accumulations of disorganized
collagen. In this study we determined if low concentrations of
TNF-alpha may inhibit the
collagen phagocytic pathway in fibroblasts and thereby contribute to
fibrosis.
Collagen phagocytosis was measured by flow cytometric assessment of internalized, fluorescent
collagen beads.
TNF-alpha induced a dose-dependent reduction (optimal dose: 40%
at 10 ng/ml; p<0.001) in the proportion of phagocytic cells and a twofold reduction of the number of internalized beads per cell but did not alter the total number of vital cells.
TNF-alpha reduced by twofold the degradation of
collagen films. Fluid flow shear-force assays demonstrated that
TNF-alpha caused a 72% reduction (p < 0.05) in strong binding of
collagen-coated beads to cells indicating that
TNF-alpha may inactivate receptors and inhibit
collagen binding. Furthermore,
TNF-alpha reduced cell contact area with
collagen substrates by threefold and inhibited reattachment of trypsinized cells by fourfold. Although levels of
collagen receptors were increased by
TNF-alpha (53% increase in alpha(2) (beta)1
integrin; p<0.001, 20% increase in alpha(1)beta(1)), the receptors were inactivated by the
cytokine. The reduced phagocytic activity of
TNF-alpha-treated cells was restored to control levels by treatment with the
integrin-activating Abs A16G6 and JBS2.
TNF-alpha inhibited focal adhesion formation and
phosphotyrosine staining in focal adhesions. These effects were replicated by the
tyrosine kinase inhibitor genistein, which also inhibited phagocytosis. Collectively, these data indicate that
TNF-alpha inhibits adherence and phagocytosis of
collagen. These effects are mediated by a reduction in the strength of alpha(2)beta(1)
integrin binding to
collagen, possibly through
tyrosine kinases in focal adhesions. At low concentrations of
TNF-alpha (10 ng/ml) that are found in the periphery of chronic inflammatory lesions, we suggest that inhibition of the
collagen phagocytic pathway may contribute to
fibrosis.