Endotoxin tolerance alters macrophage membrane regulatory G proteins

Administration of sublethal doses of endotoxin (LPS) or tumor necrosis factor-alpha (TNF alpha) renders rats tolerant to supralethal doses of LPS. Peritoneal macrophages from tolerant rats are refractory to LPS induced arachidonic acid (AA) metabolism and cytokine production in vivo, and exhibit reduced membrane GTPase activity and GTP gamma S binding. Since LPS stimulated AA metabolism is mediated by Gi alpha proteins, we sought to determine whether Gi alpha and/or other G proteins are reduced in LPS tolerance. Rats were rendered tolerant by two daily sublethal doses of Salmonella enteritidis LPS, 100 micrograms/kg and 500 micrograms/kg administered intraperitoneally. Animals were allowed to rest for 72 hours. Alternatively, tolerance to LPS was induced by sublethal administration of human recombinant TNF alpha (10 micrograms/kg) intraperitoneally 24 hrs before the experiments. Macrophage membrane G protein content was determined by immunoblot analysis with specific antisera to Gi1,2 alpha, Gi3 alpha, Gs alpha and the G protein beta subunits (G beta). Membrane G proteins were differentially decreased in tolerant macrophages. In macrophages from rats rendered tolerant by sublethal doses of LPS, Gi3 alpha was reduced the most to 48 +/- 8% of control (n = 3, P < 0.05) and this reduction was significant compared to those of other G proteins. Gi1,2 alpha and G beta were reduced to 73 +/- 5% (n = 3, P < 0.05) and 65 +/- 4% (n = 3, P < 0.05) of control respectively. Gs alpha(L) and Gs alpha(H) were also reduced to 61 +/- 5% (n = 3, P < 0.05) and 68 +/- 3% (n = 3, P < 0.05) of control, respectively. In contrast, only Gi3 alpha was reduced in macrophage membranes from rats pretreated with TNF alpha. Gi3 alpha was reduced to 57 +/- 11% of control (n = 4, P < 0.05) whereas Gi1,2 alpha and G beta were not significantly affected. These results demonstrate selective changes in tolerant macrophage membrane G proteins and suggest a potential role for Gi3 alpha in mediating LPS tolerance. The molecular mechanisms underlying these changes and their significance in LPS tolerance merit further investigation.