Monocyte chemoattractant protein 1 (MCP1), also known as monocyte chemotactic and activating factor, is the human homolog of the murine JE gene product. Purified native human MCP1 possesses potent chemotactic activity for basophils and monocytes and can augment monocyte tumoristatic activity against some tumor cell lines. MCP1 is constitutively produced by, or can be induced, in a variety of cell types. While these activities suggest that MCP1 may participate in inflammatory and immunologic processes, the biological role of MCP1 has not been studied in vivo. We hypothesize that MCP1 plays a pivotal role in the development of monocyte/macrophage-rich inflammatory lesions such as granulomas. To test this hypothesis, we will examine the role of MCP1 in the pathogenesis of a rat model of pulmonary granulomatosis. Intravenous infusion of yeast cell wall glucan results in peripheral blood monocytosis, a rise in bronchoalveolar lavage monocyte chemotactic activity, and the synchronous development of angiocentric pulmonary granulomas. Because available antibodies to human MCP1 do not neutralize rat monocyte chemotactic activity or react with rat tissue, we will clone and express rat MCP1/JE. A time course and anatomic analysis of MCP1 mRNA and protein expression during evolving pulmonary granulomatosis will be carried out by in situ hybridization and immunohistochemistry. Recruitment of mononuclear leukocytes will be quantitated by morphometry, immunohistochemistry, and in vivo radioisotopic cell labeling studies. The contribution of local cell proliferation to granuloma development will be quantitated using [3H]- thymidine pulse labeling experiments. Neutralizing anti-MCP1 antibodies, raised against rat MCP1, will be used in blocking experiments to directly and quantitatively assess the role of MCP1 in pulmonary granuloma development. The glucan induced pulmonary granulomatosis model is ideal for this study because the development of granulomas is synchronous, rapid, and anatomically discrete (i.e. angiocentric; where the relationship between MCP1 expression and mononuclear cell recruitment can be visualized). Critical to these studies is the production of rat MCP1. A cDNA library that contains MCP1 will be constructed from mRNA extracted from tumor necrosis factor- stimulated rat pulmonary artery endothelium. The cDNA library will be screened for MCP1 with degenerate oligonucleotide probes bearing highly conserved sequences from human and mouse MCP1. MCP1 cDNA clones will be amplified by polymerase chain reaction (PCR) and expressed in Spodoptera frugiperda insect cells infected with a baculovirus vector (Autographa californica nuclear polyhedrosis virus) containing MCP1 cDNA.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Lung Biology and Pathology Study Section (LBPA)
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
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