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A4728 - Surfactant Protein A1: Effects on the Toponome of the Mouse Alveolar Macrophage
Author Block: D. S. Phelps1, X. Zhang1, D. Shearer2, J. Weisz2, J. Floros1; 1Pediatrics, Penn State Univ, Hershey, PA, United States, 2Obstetrics and Gynecology, Penn State Univ, Hershey, PA, United States.
The innate host defense molecules, human surfactant protein-A1 (hSP-A1) and hSP-A2, differentially affect the proteomic expression profile and function of alveolar macrophages (AM). We studied, male SP-A knockout (KO) mice rescued with a treatment of in vitro expressed hSP-A1. The macrophages were harvested by bronchoalveolar lavage after 18h, plated onto slides, fixed, and frozen for later analysis. KO mice treated with vehicle served as controls. We analyzed the slides using the Toponome Imaging System (TIS) that enables studying the AM toponome (combination of the “proteome” and “interactome”) within intact cells. TIS is a robotically controlled serial immunostainer that enables sequential immunostaining and imaging of a single slide with multiple FITC-conjugated antibodies. The resulting images are analyzed pixel-by-pixel to identify the proteins present in each pixel. The group of proteins present in each pixel is designated as a combinatorial molecular phenotype (CMP), which represents organized protein clusters that are postulated to contribute to a specific cell function. We analyzed 19 cells from each mouse immunostained with 13 antibodies and three mice per condition. Focusing on the 20 most abundant CMPs for each cell we tabulated the incidence of each of the 13 markers studied and we prepared graphs that allowed us to more easily compare the relative amounts of the markers. Phenotypes were defined as cells exhibiting similar patterns of expression of markers. We found that the AM populations were diverse and that each sample contained cells with multiple phenotypes. Of the 114 cells analyzed, no two cells were identical. However, we did find groups of cells with similar phenotypes. Some phenotypes were found predominantly in vehicle-treated KO mice, and others found mostly in SP-A1-treated mice. There were also phenotypes that were equally abundant in both groups. These patterns suggested that SP-A had both positive and negative regulatory influences on the expression of some markers constituting various phenotypes, whereas others were unaffected by the presence or absence of SP-A. In summary, using the data from this novel microscopic technology we are able to analyze individual cells within populations of AM: a) we have confirmed that AM from a given individual are very diverse, consisting of several subpopulations, and that no two cells are identical; b) we have also demonstrated that AM phenotype is dependent, at least in part, on the presence of SP-A. These findings indicate that SP-A treatment may be useful for the therapeutic manipulation of AM function in vivo.