GM-CSF autoantibodies and neutrophil dysfunction in pulmonary alveolar proteinosis.

BACKGROUND: Increased mortality from infection in patients with pulmonary alveolar proteinosis occurs in association with high levels of autoantibodies against granulocyte-macrophage colony-stimulating factor (GM-CSF). We tested the hypothesis that neutrophil functions are impaired in patients with pulmonary alveolar proteinosis and that GM-CSF autoantibodies cause the dysfunction. METHODS: We studied 12 subjects with pulmonary alveolar proteinosis, 61 healthy control subjects, and 12 control subjects with either cystic fibrosis or end-stage liver disease. We also studied GM-CSF-/- mice and wild-type mice. We evaluated basal neutrophil functions, neutrophil functions after priming by GM-CSF to augment antimicrobial functions, and the effects of highly purified GM-CSF autoantibodies on neutrophil functions in vitro and in vivo. RESULTS: Neutrophils from subjects with pulmonary alveolar proteinosis had normal ultrastructure and differentiation markers but impaired basal functions and antimicrobial functions after GM-CSF priming. GM-CSF-/- mice also had reduced basal neutrophil functions, but functions after GM-CSF priming were unimpaired. The neutrophil dysfunction characteristic of pulmonary alveolar proteinosis was reproduced in a dose-dependent fashion in blood specimens from healthy control subjects after incubation with affinity-purified GM-CSF autoantibodies isolated from patients with pulmonary alveolar proteinosis. The injection of mouse GM-CSF antibodies into wild-type mice also caused neutrophil dysfunction. CONCLUSIONS: The antimicrobial functions of neutrophils are impaired in patients with pulmonary alveolar proteinosis, owing to the presence of GM-CSF autoantibodies. The effects of these autoantibodies show that GM-CSF is an essential regulator of neutrophil functions.

PU.1 redirects adenovirus to lysosomes in alveolar macrophages, uncoupling internalization from infection.

Adenovirus is endocytosed and efficiently destroyed by human and murine alveolar macrophages (AMs) and rapidly cleared from the lungs of wild-type but not GM-CSF(-/-) mice. We hypothesized that GM-CSF may regulate adenovirus clearance in AMs via the transcription factor PU.1 by redirecting virion trafficking from the nucleus to lysosomes. This hypothesis was tested in murine AM cell lines with altered GM-CSF and/or PU.1 expression including MH-S (GM-CSF(+/+)PU.1(Pos)), mAM (GM-CSF(-/-)/PU.1(Neg)), and mAM(PU.1+) (GM-CSF(-/-)/PU.1(Pos); PU.1-transduced mAM cells) and A549 (an epithelial-like cell line) using a human adenovirus expressing a beta-galactosidase reporter. In PU.1(Neg) mAM and A549 cells, adenovirus efficiently escaped from endosomes, translocated to the nucleus, and expressed the viral reporter in most cells. In marked contrast, in PU.1(Pos) mAM(PU.1+) and MH-S cells, adenovirus failed to escape from endosomes, colocalized exclusively with endosome/lysosome markers (Rab5, Rab7, and Lamp1), and rarely expressed the reporter. Retroviral expression of PU.1 in A549 cells blocked endosomal escape, nuclear translocation and reporter expression. Inhibition of endosome acidification also blocked escape, nuclear translocation, and reporter expression in PU.1(Neg) cells. The effect of PU.1 on viral trafficking and transduction could not be explained by an effect on endosome acidification or on differences in viral load. PU.1 reduced expression of integrin beta(5), a host factor important for endosomal escape of adenovirus, suggesting that PU.1 redirects adenoviral trafficking by modulating integrin signaling. These results demonstrate that PU.1 uncouples infection from internalization in AMs, providing a mechanism for AMs to avoid infection by adenovirus during clearance.