In this proposal we address two central questions in lung dendritic cell (DC) biology. 1. What are the mechanisms by which DC precursors enter the lung to adopt their sentinel function? 2. What role is played by DC in granulomatous inflammation? These questions build on the following two observations made recently in our laboratory. a. DC precursors, and not their mature counterparts, enter the lung in response to chemokines using mechanisms that depend, in part, on CD18 integrin and ICAM-1 expression. b. DC are the first cells to contact intravenously injected antigen-coated beads, thereby initiating granuloma formation. Origin of DC precursors. i. We establish the phenotype and function of DC precursors that migrate to the lung and provide evidence that they are a subset of monocytes. ii. Differences in the sensitivity of a. monocytes, b. interstitial lung DC precursors and c. mature lung DC to a series of chemotactic agents is examined in vitro. Parallel studies in vivo examine how the tisssue distribution of these cells is altered when a chemotactic agent is administered intra- tracheally. iii. By using mutant mice treated with blocking antibodies, we will show for the first time that DC precursor transmigration is dependent on integrins and immunoglobulin superfamily adhesion molecules. Transmigration in situ is examined at steady state, under conditions of heightened migration and after generating increased numbers of circulating DC precursors treatment with flt3 ligand. DC activity in granulomatous inflammation. Using beads coated with mycobacterial antigen, we induce granulomas in the lung and examine over time the phenotype and antigen presenting activity of the DC population in the granuloma, hilar lymph nodes and the spleen. The key role of IL-12 in the induction of the cellular immune response in granulomatous inflammation will be demonstrated by i. Immuno-localizing IL-12 in DC. ii. Quantifying IL-12 mRNA by RT-PCR-ELISA and iii. Assaying by ELISA for IL-12 cytokine secretion by DC within the first 12 h after bead inoculation. These studies contribute important new insights into the biology of pulmonary DC, their role in the immune defense of the lung and provide basic information that can be drawn upon to devise strategies to harness these cells in the defense against inhaled pathogens.
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