To monitor and better understand the immunoinflammatory sequelae in sepsis and septic shock, systemic and monocyte-related cytokine responses were evaluated in baboons with experimental peritonitis induced by an E. coli-laden fibrin clot. Despite similar bacterial inocula, considerable interindividual variability in clinical manifestation and outcome of infection was observed. Because monocytes and macrophages are a key component of innate immunity, we hypothesized that early polarization of distinct activation programs in circulating monocytes that culminates in the emergence of either classically (M1) or alternatively (M2) activated monocytes may underlie the observed susceptibility or resistance to infection. To test our hypothesis, we analyzed infection-induced expression of cytokine mRNAs in monocytes isolated from surviving and dead animals. Our data show that resistance to E. coli sepsis may well be associated with a mixed M1/M2 activation state of circulating monocytes, whereas M1 phenotype appeared to be prevailing in monocytes from animals that died. Together with data on systemic cytokine responses, the latter findings indicate that morbidity and mortality of animals with gram-negative sepsis may well result from an overwhelming proinflammatory response. Collectively, our data contribute to a better understanding of cytokine networking in the immunoinflammatory response to microbial infection and suggest M1/M2 immunophenotypic profiling of readily available circulatory monocytes for early prognosis of severe infections.
Lipopolysaccharide (LPS) extracted from three strains of Salmonella typhimurium, i.e., the rough Re mutant SL1102, the rough Ra mutant TV119, and the smooth strain SH4809, was first electrodialyzed (eLPS) and then divalent cation deprived by EDTA treatment and finally made monomeric by deoxycholate solubilization. The removal of excess detergent by extensive dialysis in the absence of mineral cations resulted in the reassociation of LPS subunits into monodisperse micelles of reduced aggregation number (dLPS) as estimated by electron microscopy and gel filtration chromatography. For all LPS chemotypes tested, the developed procedure reproducibly results in stable and clear solutions of dLPS in concentrations of up to 100 mg/ml. The dLPS and eLPS preparations possessed the same reactivity with monoclonal antibodies (MAbs) raised against different LPS domains. The 100% lethal dose in galactosamine-sensitized mice of 0.01 microgram for the smooth eLPS was from 10- to 100-fold lower than that of dLPS at 0.1 to 1.0 microgram. dLPS from both the smooth strain and the Ra mutant had a significantly reduced capacity to activate the proenzyme cascade in the Limulus amoebocyte lysate assay in comparison with the slightly reduced activity of dLPS from the Re mutant. In contrast, dLPS as well as the deoxycholate-dispersed and then diluted eLPS from the smooth strain had a higher mitogenic activity on splenocytes than eLPS. The results indicate that the biological and endotoxic properties of LPS are significantly influenced by the physical state of its aggregates in aqueous solutions. The approach developed for production of a stable and dispersed form of LPS should further assist in investigation of LPS properties and interpretation of the data of endotoxic research.
We have investigated the interaction of Salmonella minnesota R595 lipopolysaccharide (ReLPS) depleted of Ca 2؉ and Mg 2؉ with both Kupffer and endothelial liver cells under serum-free conditions. Specific and saturable binding levels of 125 I-ReLPS were similar in both types of cells with respect to divalent cation independence, susceptibility to proteases, and concanavalin A inhibition. By using partial structures of ReLPS, it was demonstrated that acidic 3-deoxy-D-manno-octulosonic acid residues and phosphoryl groups on lipid A are of primary importance in ReLPS binding. The role of ionic interactions in LPS recognition by the cells was further confirmed by susceptibility of the binding to competitive inhibition by polyanions. Both ReLPS and ReLPS partial structures inhibited the specific cellular binding of acetylated low-density lipoprotein (Ac-LDL) by Kupffer cells and Ac-LDL-and formaldehyde-treated albumin by endothelial cells whose cellular accumulation is mediated by a different type(s) of scavenger receptor(s). In contrast, 125 I-ReLPS binding to Kupffer and endothelial cells was not competed by Ac-LDL or formaldehyde-treated albumin. Our results indicate the scavenger pathway of LPS uptake by Kupffer and endothelial cells and the primary role of LPS anionic properties in this process.
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