The overall goal of this proposal is to investigate how sustained increases in intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) suppress neutrophil function and attenuate neutrophil-mediated acute lung injury. Agents that cause sustained increases in cAMP have been shown to inhibit the activation of neutrophils. We have previously shown that methylxanthines, which inhibit phosphodiesterases (PDEs), can prevent neutrophils from damaging the lungs and other organs in animal models of acute lung injury. Further preliminary studies with isolated human neutrophils have shown the efficacy of PDE inhibitors and a cAMP analog in suppressing neutrophil function following activation by endotoxin and tumor necrosis factor; however, more extensive studies are needed to determine the role of changes in cAMP levels in the intracellular signalling pathways that mediate neutrophil function. We now propose to: (1) quantitatively correlate the time course of changes in intracellular cAMP levels with the activation and suppression of neutrophil functions including chemiluminescence, degranulation, and expression of CD11/CD18 (surface glycoproteins associated with cell adhesion in neutrophils); (2) determine the effects of neutrophil activators and suppressors on the intermediaries of intracellular signal transduction pathways of neutrophils, including inositol triphosphate, diacylglycerol, the translocation of protein kinase C from cytosol to membrane, and calcium flux; (3) employ whole-cell and patch-clamp recording to characterize the effects of activators and suppressors on neutrophil ion channels and to intracellularly administer key intermediates and inhibitors of signal transduction; and (4) determine, in vivo, the effectiveness of agents that alter cAMP levels in preventing neutrophil-mediated acute lung injury. These studies should reveal how sustained increases in intracellular cAMP suppress the activation of neutrophils, and may eventually contribute to new pharmacologic approaches to the prevention and treatment of the adult respiratory distress syndrome (ARDS).
