Inflammatory mechanisms are involved in the initiation, amplification, propagation, and resolution of Adult Respiratory Distress Syndrome (ARDS). Neutrophils accumulate in the lungs of patients with ARDS, release granular enzymes and other toxins that mediate damage to cells of the alveolar-capillary membrane in vitro, and injure lungs of experimental animals in vivo. Similarly signaling molecules, such as interleukin and biologically-active lipids, that directly activate PMNs and induced mobilization of granular contents, generation of oxygen radicals, and other functional alterations are expressed by endothelium and other cells in ARDS. The identities of many of these signaling molecules remain unknown, and the mechanisms that cause such molecules to become expressed in a dysregulated, injurious fashion, as opposed to a homeostatic, protective fashion, remain undefined. Broad goals of this project are to characterize neutrophil signaling molecules of different classes, define the mechanisms that regulate their expression, and determine mechanisms of dysregulation. We will focus here on characterization of a signaling molecule that induces neutrophil degranulation, because this event appears to be of particular importance in the pathogenesis of ARDS. In preliminary experiments, we found that endothelial cells treated with the cytokines IL-1 and TNFa or with LPS, synthesize a signaling molecule that induces PMN to secrete lactoferrin, a specific granule marker, and elastase, primary granule protease, into the fluid phase. Maximal generation of the degranulating signal is dissociated from surface expression of molecules that mediate adhesion and transmigration of PMN. These features suggest that the degranulating factor is not involved in physiologic transmigration of PMN. These features suggest that the degranulating factor is not involved in physiologic targeting events. Furthermore, endothelial cell monolayers are disrupted when incubated with PMN in the presence of the degranulating activity, suggesting that release of elastase or other PMN proteases induced by this factor mediates endothelial injury. Our hypotheses are that human endothelial cells generate and release a novel signaling molecule that stimulates PMN degranulation. PMN granular proteases amplify injury to the alveolar capillary membrane and may be key mediators of vascular destruction that occurs in ARDS. Dysregulated expression of signaling molecules by endothelial cells is a fundamental pathophysiologic mechanism in acute lung injury.
Our specific aims are: 1) To purify, characterize, sequence, and clone the new degranulating factor. 2) To characterize the biologic activities of the degranulating factor. 3) To characterize the synthesis and secretion of the factor and define its relationship to other signaling molecules. 4) To determine if the degranulating factor is found in the blood or lung tissue of patients during the acute, subacute, and reparative phases of ARDS.
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