Polymorphonuclear leukocyte and monocyte chemoattractants produced by human fibroblasts

Abstract

The elaboration of leukocyte chemotactic factors by human fibroblasts was studied. 12 lines of normal fibroblasts obtained by skin biopsy and then cultured in vitro produced chemoattractants (assessed by modified Boyden-chamber techniques) for both peripheral blood polymorphonuclear leukocytes and monocytes (obtained by Hypaque-Ficoll and dextran sedimentation). Chemotactic activity was not present performed in fibroblasts, and cycloheximide blocked its elaboration. The chemotactic activity of crude-culture supernate was heat stable (56 degrees C for 30 min), trypsin- and pronase-sensitive, and neuraminidase resistant. Characterization of the chemotactic activity by gel filtration (Sephadex G-75) showed two active fractions, one with mol wt greater than 100,000 and the other less than 10,000. In studies designed to relate these chemotactic factors to collagen, we have confirmed that type I collagen and alpha 1-chain; are chemotactically active for monocytes but not polymorphonuclear leukocytes. However, the chemotactic activity in fibroblast-culture media was media was distinct from collagen in that it attracted neutrophils, it was not precipitated by 25% ammonium sulfate, and it was resistant to collagenase treatment; ascorbic acid, in concentrations known to stimulate fibroblast collagen synthesis, had no effect on the elaboration of the chemotactic factors. Furthermore, amino acid analysis of Sephadex G-75 fractions with chemotactic activity failed to reveal amino acids such as hydroxyproline characteristic of collagen. In addition to the chemotactic factors secreted by fibroblasts, a heat-resistant factor (30 min at 56 degrees C) which generated the chemotactically active fragment of C5 (C5a) from human serum was also secreted. The elaboration of mediators of the inflammatory and immune responses by fibroblasts may initiate and(or) modulate local skin inflammatory reactions and play a protective role in vivo.