Real-time microscopy of interactions between alveolar macrophages and the alveolar epithelium in situ

Background: Alveolar macrophages (AMs) are the first line of defense in the lung. The mechanisms of how alveolar macrophages interact with the alveolar epithelium to modulate lung inflammation are not well defined. Methods: To visualize AMs in situ , we expressed Enhanced Yellow Fluorescent Protein in CD11c+ AMs (AM YFP ). We isolated and blood-perfused lungs of AM YFP mice at constant pulmonary artery, left atrial and alveolar pressures of 10, 3 and 5 cmH 2 O, respectively. We imaged the lungs by confocal microscopy. We micropunctured single alveoli and microinfused dyes and reagents. To define the role of gap junctional channels (GJCs) in lung injury, we crossed AM YFP mice with Cx43 floxed/floxed mice (AM YFP Cx43 -/- ). We treated mice with intra-nasal instillations of 1 mg/kg LPS 24 h before we excised the lungs. Results: Calcium uncaging experiments in wild type mice revealed that 38±10% of all AMs formed functional GJCs with the alveolar epithelium. As compared to wild type mice, AM-specific knockout of Cx43 in AMCx43 -/- mice resulted in a 2-fold higher neutrophil recruitment to the lung 24 h after LPS and reduced survival after 25 mg/kg LPS (P<0.05). Cytokines of macrophages origin (MIP-1alpha) but also cytokines that were predominantly of epithelial origin (CXCL1 and 5) were increased in the bronchoalveolar lavage, suggesting that AM-epithelial communication may serve as a feedback loop to regulate lung inflammation. Conclusions: For the first time, we report that AMs form GJCs with the alveolar epithelium. The interaction between AMs and the epithelium through GJCs was crucial in that knockout of GJCs in AMs aggravated lung injury (HL78645, HL64896).