Proteomics analysis of plasma protein complex regulating leukocyte interaction.

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Sepsis and septic shock, often associated with multiple organ failure, still remain important causes of morbidity and mortality in intensive care units. Many types of therapeutic trials for the treatment of septic shock have failed. Lipopolysaccharide(LPS) is one of the major causes of septic shock. The polymyxin B-immobilized filter column (PMX) was developed for the adsorption of endotoxin by direct hemoperfusion and has been used for the treatment of LPS-induced septic shock. In this study, we demonstrated that PMX also specifically bound monocytes from the peripheral blood leukocytes of septic patients by the analysis of bound cells using immunocytochemical and electron microscopic techniques. The specific removal of monocytes from septic patients may produce beneficial effects by reducing the interaction between activated monocytes and functionally associated cells including vascular endothelial cells. We also investigated the humoral factors adsorbed on the PMX and identified the … More some of the proteins by amino acid sequencing of the purified proteins and Western blotting. These included cytokine-like proteins and macrophage migration inhibitory factor. PMX may exert beneficial effects through the adsorption of plural factors, LPS, monocytes and humoral factors in the treatment of septic shock. We also report the plural mechanisms for controlling the activity of monocytes by the regulation of the expression of adhesion molecules.Sepsis and septic shock, often associated with multiple organ failure, still remain important causes of morbidity and mortality in intensive care units. Many types of therapeutic trials for the treatment of septic shock have failed, however, recent phase III studies using recombinant activated protein C demonstrated the effectiveness of this therapy.^<1,2> Lipopolysaccharide(LPS), one of the major causes of septic shock, together with LPS binding protein binds to CD14 on the surface of monocytes/macrophages, leading to the activation of signaling molecule complex of Toll-like receptor-4 (TLR-4) and MD2. Polymyxin B can bind LPS and neutralize its biological activity, therefore, the polymyxin B-immobilized filter (PMX) column was developed for the adsorption of endotoxin by hemoperfusion.^3 There is now increasing evidence supporting the usefulness of this treatment, showing improvement of survival rate in LPS-induced circulatory disorders and systemic inflammatory response syndrome. Moreover, the effectiveness of hemoperfusion with this column for septic shock beyond LPS endotoxemia^4 prompted us to investigate additional mechanisms. Since it is well known that different populations of leukocytes are activated during septic shock and change their adhesive phenotype, we hypothesized that some population of leukocytes may be adsorbed in the column and removed from the blood circulation after treatment. To examine this hypothesis, we investigated the cellular components in the PMX columns after direct hemofiltration in four septic patients. We also investigated the humoral factors adsorbed on the PMX. For this purpose, the proteins attached to the filter were extracted and solubilized in PBS. Some of the proteins were purified chromatograpfically and their amino acid sequences were analyzed. Less