Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen-derived peptides.

Abstract

The chemotactic response of human dermal fibroblasts to type I, II, and III human collagens and collagenderived peptides was quantitated by an in vitro assay. All three native human collagens and constituent a chains can serve as chemoattractants for fibroblasts in vitro. When type I, II, and III collagens were digested by bacterial collagenase, the resulting peptides were also chemotactic. In addition, synthetic di-and tripeptides containing hydroxyproline were also chemotactic for fibroblasts. Since (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Type IV collagen, isolated initially from basement membranes, has been more difficult than the other three collagens to characterize biochemically and may be heterogeneous (13)(14)(15).We have recently developed a method by which fibroblast chemotaxis to various stimuli can be quantitated in vitro (16). By using this technique, we have isolated and characterized a human lymphocyte-derived chemotactic factor for fibroblasts, LDCF-F (16). In the present study we have found that type I, II, and III collagens and collagen-derived peptides can effect chemotactic migration of fibroblasts. MATERIALS AND METHODS Preparation of Collagens and Constituent a Chains.Human type I and II collagens were obtained from the bone and cartilage of extremities previously amputated to treat peripheral vascular disease. Type III collagen was purified from human livers obtained at autopsy. Collagens were solubilized from these tissues by limited proteolysis with pepsin under nondenaturing conditions and purified by methods previously described (3, 9). Type I collagen was also obtained from the skin of 3-week-old lathyritic chickens (17).Heat-denatured collagen solutions were fractionated on carboxymethyl-cellulose to isolate purified a chains as described (1,3,9).The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 871Fibroblast Chemotaxis Assay. Fibroblast chemotaxis was measured in mtro by methods previously described in detail (16). The assay uses blindwell modified Boyden chemotaxis chambers equipped with gelatin-treated polycarbonate filters having 8-,m pores. Normal human dermal fibroblasts maintained as continuous cell lines were used as indicator cells in the assay. Fibroblasts were harvested from monolayers by either trypsinization or EDTA treatment. Fibroblasts chemotactic activity was determined after chambers were incubated for 150 min by counting the fibroblasts migrating to the lower surface of the polycarbonate filters in 20 oil immersion fields. Substances were assayed in triplicate, and the final chemotactic activity of a sample was expressed as the mean + SEM.Lyophilized collagen and a chain preparations were solubilized for use in the chemotaxis studies by dissolving them in 0.5 M acetic acid, stirring them overnight (40), and then dialyzing them against large volumes of 10 mM phosphate/0.14 M NaCl at p...

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