Endothelial cell (EC) surface platelet-activating factor (PAF), together with other cell adhesion molecules including P-selectin, plays an important role in the neutrophil (PMN)-EC adhesion cascade. Human PMN have high and low affinity state PAF receptors, each coupled to separate signal transduction pathways through which different PMN (patho)physiological responses are modulated. Alkyl-PAF is a complete PMN agonist because it interacts with both PAF receptors, triggering full-blown, potentially detrimental PMN activation. Acyl-PAF, on the other hand, is only a partial PMN agonist, preferentially interacting with only the high affinity PAF receptor. On the basis of both thermodynamic and (patho)physiological considerations, it would be expected that modulation of the PMN-EC adhesion cascade by EC surface-associated alkyl-PAF will lead to a significantly different outcome than when acyl-PAF is the predominate species. After EC stimulation with inflammatory mediators, cytokines, enzymes or reactive oxygen species, human EC synthesize both acyl-PAF and alkyl-PAF via the remodelling pathway. Two cytokines implicated in the pathogenesis of endotoxic shock (IL-1alpha and TNFalpha) strongly favor EC synthesis of alkyl-PAF while other inflammatory mediators (ATP, bradykinin and histamine) preferentially induce EC synthesis of acyl-PAF. In view of the preceding, we propose the following two hypotheses: l) EC surface expression of acyl-PAF plays an important role in the PMN-EC adhesion cascade by fostering PMN emigration without fully activating the PMN or causing EC injury; 2) EC surface expression of alkyl-PAF predisposes the vascular endothelium to PMN mediated injury through a greatly amplified PMN-EC adhesion cascade that results in maximal juxtacrine/paracrine priming and activation of EC-adherent PMN. We are now in a unique position to examine these hypotheses because we have recently identified conditions which allow for the preferential synthesis of either acyl-PAF or alkyl-PAF by stimulated human EC. Therefore, we can definitively characterize the normal and detrimental consequences that EC expression of either surface- associated acyl-PAF or alkyl-PAF have on the PMN-EC adhesion cascade. In the proposed studies we will examine the following (patho)physiological responses that are inhibitable by PAF receptor antagonists and occur after adhesion of PMN to EC expressing acyl-PAF or alkyl-PAF on their cell surface: l) PMN-EC adherence; 2) PMN activation; and 3) EC injury (detachment, necrosis, apoptosis). ThroUgh the logical design and straightforward nature of the proposed studies, we will gain a wealth of novel and important information about the (patho)physiological roles that surface-associated EC acyl-PAF and alkyl-PAF play during the interaction of PMN with EC. We are confident that this investigation will greatly expand our understanding of the cell biology of the PAF-dependent mechanisms that modulate both normal PMN trafficking as well as PMN mediated injury of the vascular endothelium, such as in ischemia/reperfusion injury and the microvascular in associated with endotoxic shock.
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