Lipopolysaccharide-Free Conditions in Primary Allow Growth and Isolation of Microglial Cells Astrocyte Cultures

Primary rat astrocyte cultures were used to isolate a macrophage population that does not adhere to the confluent glial cells. The cells multiplied vigorously in coculture with astrocytes during the 14 d culture period, provided that functionally active lipopolysaccharide (LPS) was either absent or present in very low concentrations. Based on morphological, immunocytochemical, and pharmacological data, it was concluded that the isolated cells were microglia, the resident macrophages of the brain. The findings characterized them as a distinct cell population that shares features both of peritoneal macrophages and of astroglial cells. Like peritoneal macrophages, the isolated cells were able to phagocytire as shown by their ingestion of latex beads and uptake of L-leucyl methylester. Furthermore, they were immunocytochemically stainable by a specific monoclonal antibody (ED 1) against a macrophage-specific antigen (Dijkstra et al., 1985). They also synthesized prostaglandin E, (PGE,) and secreted interleukin 1 (IL-l) upon stimulation with LPS. Upon stimulation with the ionophore A231 87, PGD,, the predominant prostaglandin of the brain, was the major PG metabolite released by these cells. In contrast to peritoneal macrophages, microglial cells were able to multiply. Proliferation of microglial cells in coculture with astrocytes was suppressed when 2 ng LPS/ml or higher concentrations were added to astroglial culture media. These astrocyte cultures, which contained approximately 1% microglia, were used to investigate the influence of LPS on prostaglandin and IL-1 secretion in order to compare astroglial and microglial features. Increasing LPS concentrations induced increased PGE, secretion, whereas PGD, secretion was essentially unaffected by LPS. The critical influence of LPS contaminations in most of the commercially available animal sera used for astrocyte cultures on cellular composition in general and on metabolism of hormones and growth factors in particular is discussed.