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Although alveolar epithelial type II cells (AECII) perform substantial roles in the maintenance of alveolar integrity, the extent of their contributions to immune defense is poorly understood. Here, we demonstrate that AECII activates alveolar macrophages (AM) functions, such as phagocytosis using a conditioned medium from AECII infected by P. aeruginosa. AECIIderived chemokine MCP-1, a monocyte chemoattractant protein, was identified as a main factor in enhancing AM function. We proposed that the enhanced immune potency of AECII may play a critical role in alleviation of bacterial propagation and pneumonia. The ability of phagocytosis and superoxide release by AM was reduced by MCP-1 neutralizing antibodies. Furthermore, MCP-1-/- mice showed an increased bacterial burden under PAO1 and PAK infection vs. wt littermates. AM from MCP-1-/- mice also demonstrated less superoxide and impaired phagocytosis over the controls. In addition, AECII conditioned medium increased the host defense of airway in MCP-1-/- mice through the activation of AM function. Mechanistically, we found that Lyn mediated NFkB activation led to increased gene expression and secretion of MCP-1. Consequently Lyn-/- mice had reduced MCP-1 secretion and resulted in a decrease in superoxide and phagocytosis by AM. Collectively, our data indicate that AECII may serve as an immune booster for fighting bacterial infections, particularly in severe immunocompromised conditions. [ABSTRACT FROM AUTHOR]

Introduction: Recently, proteomics approach has revealed lipid raft associated proteins such as Flotillin-1 in phagosome compartments. We have used Pseudomonas aeruginosa (PA) phagocytosis by alveolar macrophage (AM) as a model to study this mechanism. Rationale: Lyn-PI3Kinase -- Akt pathway is known to be involved in IgG receptor mediated phagosome formation. Since Lyn is also a lipid raft resident protein we propose a role for Lyn in raft mediated phagocytosis of PA by AM. Methods: Phagosome formation and localization of signaling proteins were studied by immunostaining with fluorescent antibodies followed by confocal microscopy. Lyn-YFP and PH-Akt-GFP co-transfected MHS cells (murine alveolar macrophage cell line, ATCC) were used for studying spatio-temporal association of Lyn and Akt during PA phagocytosis by live cell confocal microscopy. Lipid rafts were identified by staining with FITC labeled Cholera Toxin B subunit. Phagosomes from PA infected cells were isolated by sucrose density gradient centrifugation for biochemical characterization. Results: Lipid raft marker (green) and Lyn-YFP (yellow) were found to be co localized with PAO1 (stained red) in phagosome compartment. Both Lyn-YFP and PH-Akt-GFP were found to be co-localized in the lamellipodium and phagocytic cup. Lyn, PI3Kinase and Akt detected by immunoblotting were found to be actively recruited to phagosome fractions in PA infected cells compared to controls. Conclusions: Our data indicates that Lipid raft mediated Lyn-PI3Kinase-Akt pathway is crucial for initiating and regulating phagocytosis of PA by AM. This study will bring out newer insights into PA pathogenesis and help to understand host innate immunity. [ABSTRACT FROM AUTHOR]