Neutrophil sequestration in the lung and pulmonary endothelial injury are central events in the pathogenesis of ARDS. The presence of both endotoxemia and complement activation in ARDS patients (see Project 6), has led us to the following hypothesis by which LPS and C5f contribute to neutrophil localization in the pulmonary microcirculation: :PS associates with a plasma component to form an altered LPS-plasma component complex that interacts with specific neutrophil receptors. That interaction diminishes neutrophil deformability and causes neutrophil retention in pulmonary capillaries. The complex also causes an up-regulation of the CD11 complex which further promotes neutrophil adherence, and 'primes' the neutrophil such that stimulation by C5f leads to prolonged adherence and enhanced secretion of elastase and toxic 02 metabolites that synergistically damage endothelial cells. Neutrophil sequestration in pulmonary capillaries will be (i) studied by first-pass techniques in rabbits, and (ii) modeled by human neutrophil retention in capillary-sized pores perfused in vitro in order to separate the effects of neutrophil formability from those of surface adhesiveness. The mechanisms by which LPS interacts with plasma components will be examined to determine if disaggregation of LPS by plasma and formation of an LPS-plasma component complex is required for activity. The availability of 3H-LPS of high specific activity will facilitate exploration of the concept that disaggregated LPS interacts with specific neutrophil surface receptors. The effect of LPS and C5f on microfilament and microtubule assembly will be used to test whether cytoskeletal assembly contributes to capillary sequestration. Antibodies to the CD11 complex will help determine whether prolonged adherence to endothelium cells in vitro is due to up-regulation of the CD11 complex, an effect possibly related to neutrophil PAF synthesis. The effect of combinations of LPS and C5f will be studied to test the hypothesis that enhancement of both initial sequestration and 'long-term' adherence will result, and that this effect can be detected in plasma samples from patients with ARDS. This project will define basic mechanisms underlying LPS and C5f- mediated neutrophil localization to the pulmonary capillary, a crucial prelude to the development of endothelial injury. This new information should improve our understanding of the basic pathogenesis of ARDS.
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