Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand‐binding studies, functional responses, and RNA analysis by RT‐PCR. Uptake of 125I‐C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High‐affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated KD of 1–2 nM. Low‐affinity binding sites with a KD of 209 nM were largely expressed (n≥ 4 × 106 sites per cell) and probably did not reflect a receptor‐mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT‐PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti‐C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca2+] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C‐terminus Arg considerably decreased the [Ca2+] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.

SUMMARYConstitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinfiammatory cytokine IL-1Q has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory eflfect on factor H secretion. In this study, we examined the combined eflFectof IL-1 Q and glucocorticoids on C3 and factor B expression by the endotheiial cell. When dexamethasone or hydrocortisone were added to IL-la, significant potentialization of ILl a-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10"' to 10"^M for dexamethasone and 50-200U for IL-lo. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of 1L-1« on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-lo was not observed for another marker of endothelial activation, IL-lainduced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-la therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endolhelium stimulation by glucocorticoids and IL-la, C3a is generated in the vicinity ofthe endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-la and endogenous glucocorticoids coexist, such as in septic shock.

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