The long term objective of this project is a better understanding of the mechanisms and control of human lung hypersentivity disorders and asthma. This project will focus on the central cell of lung hypersentivity, the human lung mast cell (HLMC). The HLMC by virtue of its anatomic localization, elaboration of potent chemical mediators upon stimulation and the effects of these mediators on cellular and tissue targets can produce the pathophysiologic manifestations of these disorders: airway constriction, edema and hypersecretion. The studies involve enzymatically dispersing human lung to a single cell suspension, separating lung cells (and HLMC) by elutriation into morphologically and functionally distinct subpopulations based on their diameter, and further separating these fractions on the basis of density by flotation through step-gradients. The density-step also results in purification of HLMC. One isolated, HLMC subpopulations will be examined with respect to ultrastructure, mediator content and capacity to release mediators. Selective responsiveness to relevant triggering stimuli will be explored. These stimuli include hypoxia, anaphylatoxins, bacterial fragments (f-met peptide), and a newly described mediator, an alveolar macrophage (AM) - derived releasing factor. Also, selective responsiveness of subpopulations to pharmacologic inhibition of release to the above stimuli will be examined. The agents to be tested include B-adrenergic agonists, PGE, membrane cholesterol binders and cholesterol synthesis inhibitors. The crucial role of membrane cholesterol in release will be explored through quantitative measurements of cholesterol synthesis during mast cell activation-secretion. Novel mechanisms of HLMC release inhibition through manipulation of the membrane lipid micro-environment will be studied.
