ENDOTOXIN TOLERANCE IS ASSOCIATED WITH ALTERED GTP-BINDING PROTEIN FUNCTION

Previous studies ave suggested that guanine nucleotide regulatory (G) proteins modulate endotoxin-stimulated peritoneal macrophage arachidonic acid (AA) metabolism. Endotoxin-stimulated metabolism of AA by peritoneal macrophages is decreased in endotoxin tolerance (Rogers et al. Prosta-glandins 31: 639-650, 1986). These observations led to a study of G protein function and AA metabolism by peritoneal macrophages in endotoxin tolerance. Endotoxin tolerance was induced by the administration of sublethal doses of endotoxin. AA metabolism was assessed by measurement of thromboxane B2 (TxB2), a cyclooxygenase metabolite. NaF (5 mM), an activator of G proteins, significantly stimulated TxB2 synthesis in control macrophages from 7.7 +/- 0.2 to 19.1 +/- 0.6 (SE) ng/m (P < 0.05) at 2 h and was partially inhibited by pertussis toxin, suggesting a G protein-dependent mechanism. Salmonella enteritidis endotoxin (50 mug/ml) stimulated a similar increase in TxB2 levels (23 +/- 0.4 ng/ml, P < 0.05). In contrast to control macrophages, macrophages from endotoxin-tolerant rats stimulated with either NaF or S. enteritidis endotoxin had TxB2 levels that were only 30 and 2% of the respective stimulated control cells. Basal guanosinetriphosphatase (GTPase) activity (33 +/- 6 pmol.mg-1.min-1) in endotoxin-tolerant macrophage membranes was significantly lower (P < 0.05) than control basal activity (158 +/- 5 pmol.mg-1.min-1). This suppression of macrophage GTPase activity was apparent 48 h after the first in vivo sublethal endotoxin injection (100 mug/kg ip). The reduced GTPase activity paralleled in vitro cellular hyporesponsiveness to endotoxin-stimulated TxB2 production. NaF significantly increased control macrophage membrane GTPase activity, but not in membranes from endotoxin-tolerant macrophages. Neither lipid A, the active component of endotoxin, nor endotoxin directly stimulated macrophage membrane GTPase activity. However, a 2-h preincubation of macrophages with endotoxin significantly increased basal GTPase activity in control macrophage membranes, but not in endotoxin-tolerant macrophage membranes. These data suggest that 1) endotoxin-induced AA metabolism in macrophages is modulated by proteins and 2) desensitization to endotoxin produced by repeated sublethal injections results in decreased G protein function.