Down-regulation of complement receptor 3 and major histocompatibility complex I and II antigen-like immunoreactivity accompanies ramification in isolated rat microglia

Isolated primary microglia are highly activated in conventional culture systems. This has restricted studies to the use of late stage measures of activation rather than highly sensitive immunophenotypic and morphological criteria that mark even very early stages of microglial activation in vivo. In the present study, serum-free, serine- and glycine-free medium and poly- l-lysine coated surfaces have been used to demonstrate for the first time isolated rat microglia which (i) downregulate their immunoreactivity for antibodies recognizing complement receptor 3 and major histocompatibility complex antigens while differentiating into ramified cells, and (ii) respond to a subset of modulators with upregulation of complement receptor 3-like immunoreactivity. During 2 weeks of culturing under basal conditions, ramification was accompanied by strong downregulation of OX-42, OX-18 and OX-6 immunoreactivity (antibodies recognizing complement receptor 3 and major histocompatibility complex class I and II antigens, respectively). Ramified cells had lower level immunoreactivity for all three markers than non-ramified cells. High OX-42 immunoreactivity was also associated with morphological signs of activation previously described in vivo. Enhanced OX-42 immunoreactivity was induced by applying either serine and glycine or lipopolysaccharide (LPS) while granulocyte macrophage-colony stimulating factor increased cell number without affecting OX-42 immunoreactivity. LPS induced alterations were apparent within 24 h, were transient, and did not include changes in OX-18 or OX-6 immunoreactivity, cell number or proportion of ramified cells. The results attest to the special efficacy of this culture method for the investigation of the early microglial reaction by use of highly sensitive immunophenotypic criteria.