A major site of asbestos-induced disease is the pleura, where disease manifests itself as either inflammation, fibrosis or malignancy. Crucial to understanding asbestos-induced pleural disease is understanding the interaction of the crocidolite asbestos fiber with the mesothelial cell, since crocidolite is the fiber most associated with pleural disease and the mesothelial cell is particularly sensitive to asbestos. In recent studies, internalization of crocidolite by the mesothelial cell has been found to be increased significantly by incubating the fibers with vitronectin, the major adhesive protein of serum. Our proposed study will determine whether vitronectin-coated crocidolite asbestos is recognized and internalized via specific cell surface receptors for vitronectin on rabbit pleural mesothelial cells and whether this interaction participates in signaling the activation of gene expression. In addition, our study will address whether asbestos-induced active oxygen species contribute to fiber internalization and/or gene activation. Interleukin-8 (IL-8), a gene previously found to be induced by crocidolite asbestos in mesothelial cells, will serve as a marker of gene activation, and a novel fluorescence confocal microscopic technique will be used to count intracellular and extracellular fibers separately. First, crocidolite and control particles coated with vitronectin, other extracellular matrix proteins or serum will be incubated with mesothelial cells to determine whether vitronectin specifically increases adherence and/or internalization of crocidolite. Second, two mesothelial cell surface receptors for vitronectin, alpha-v- beta3 and alpha-v-beta5, will be blocked with specific antibodies to determine their role in mediating mesothelial cell attachment to vitronectin and in mediating mesothelial cell internalization of crocidolite. Additional vitronectin receptors will be sought using immunoprecipitation and affinity chromatography. The cellular distribution of vitronectin receptors and their possible colocalization with crocidolite will be identified using immunocytochemistry. Third, the role of vitronectin receptors in lL-8 gene expression will be determined by measuring rabbit lL-8 protein and message after vitronectin receptors are blocked or activated by integrin clustering with specific antibodies. Fourth, mesothelial-derived active oxygen species will be studied for an independent or contributory role in internalization of crocidolite by the cells and in asbestos-induced lL-8 gene expression by mesothelial cells. In this proposal, the specific interaction of crocidolite asbestos fibers with the mesothelial cell will be explored to provide new information about mechanisms of asbestos toxicity.
