Progress in clinical and biological research最新文献

Roles for LBP and CD14 in the LPS dependent activation of a wide variety of cells have been established. In the work described here, we describe roles for these proteins in the binding and uptake of LPS by cells which express membrane CD14 and those which do not. Surprisingly, cell activation and LPS uptake appear to be independent phenomena with different protein requirements.

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.

Since the earliest days of antibiotic chemotherapy to treat infection with Gram-negative microbes, investigators have recognized that such treatments may result in the release of microbial constituents that might, in turn, exacerbate the pathophysiological manifestations of disease. Both in vitro studies and in vivo animal experiments have over the years provided evidence in support of this concept; however, the actual clinical importance of this phenomenon to patients with Gram-negative sepsis is unclear. Recently published reports from a number of laboratories have shown that cell wall-active antibiotics that differ in their fundamental mechanisms of action in disrupting microbial growth (via selective interactions with various penicillin binding proteins) also differ in their relative ability to induce the release of biologically active endotoxin both in vitro and in vivo. Further, quantitative differences in total endotoxin release correlate well with antibiotic-initiated morphological changes in the microbe. Of potential significance is the finding that these differences are also reflected in differential production of cytokines from endotoxin-stimulated mononuclear phagocytes and other host target cells, including 11-6 and TNF. Since these immunologic hormones have been strongly implicated as contributing factors to the pathogenesis of Gram-negative sepsis, interest in the potential use of this chemotherapeutic approach as a means of controlling the host immunopathologic response has increased. Carefully controlled clinical trials in which different antibiotic treatments are correlated with production of cytokines will be of significant potential value in evaluating the actual significance of this phenomenon in the Gram-negative septic patient.