Endocytic Internalization of Adenovirus, Nonspecific Phagocytosis, and Cytoskeletal Organization Are Coordinately Regulated in Alveolar Macrophages by GM-CSF and PU.1

Abstract

GM-CSF gene-targeted (GM−/−) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM−/− mice compared to wild-type (GM+/+) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM−/− mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM−/− mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM+/+ AM cell line. Phagocytosis of 4-μm latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM−/− AMs restored phagocytosis of 4-μm beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin αV and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110γ isoform, important in macrophage phagocytic function, was absent in GM−/− AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small (∼100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.