Culture and functional studies of mouse macrophages on native-like fibrillar type I collagen

Freshly isolated, starch-elicited mouse peritoneal macrophages (Mø) attached very efficiently to type I collagen in vitro, if collagen molecules were arranged in ordered supra-molecular assemblies corresponding to the precursor native fibrils. After 6-20h of incubation, the collagen-bound cells were observed to secrete fibronectin, which presumably enhanced cell-collagen interaction associated with cellular differentiation. Mø attachment to collagen could be temporarily inhibited by addition of the linear tri-peptide Arg-Gly-Asp (RGD) to the culture media. This inhibition was much more pronounced when using the cyclic RGD-containing peptide cGRGDSPA. Similarly, cells could be easily detached from the fibrillar collagen layers within 20 min at 37 degrees C by RGDS, GRGDS or cGRGDSPA but not by the glutamate-containing RGES peptide. Using antibodies to known collagen receptors, attachment of Mø to type I collagen fibers was best inhibited by antibodies directed against the alpha2 and beta1 integrin subunits. The presence of these integrins on Mø was confirmed by immunofluorescence. Binding of the alpha2beta1 integrin on collagen was divalent cation-dependent and was supported by magnesium but not by calcium. Cells recovered by RGD-mediated detachment from collagen were highly phagocytic and synthesized DNA when exposed to growth factors. These cells could be activated for cytotoxicity by treatment with interferon-gamma and lipopolysaccharide. Comparative in vitro assays performed on macrophages cultured on plastic and on collagen allowed the detection of NO production by activated macrophages followed by spontaneous deactivation for cells cultivated on collagen. These findings suggest that Mø can recognize native collagen of type I through functional interactions with their specific triple helix-binding integrin receptors indicating that integrins other than those directed to fibronectin may also occupy active focal points on the cell at the initial phase of attachment.