Cftr-independent Phagosomal Acidification in Macrophages

It was reported recently that the cystic fibrosis conductance regulator protein (CFTR) is required for acidification of phagosomes in alveolar macrophages (Di et al. Nature Cell Biol. 8:933–944, 2006). Here we determined whether the CFTR chloride channel is a generalized pathway for chloride entry into phagosomes of macrophages and whether mutations in CFTR could contribute to alveolar macrophage dysfunction. The pH of mature phagolysosomes in macrophages was measured by fluorescence ratio imaging using a zymosan conjugate containing Oregon Green 488 and tetramethylrhodamine (TMR). Acidification of phagolysosomes in J774A.1 macrophages (pH ~ 5.1 at 45 min), murine alveolar macrophages (pH ~ 5.3) and human alveolar macrophages (pH ~ 5.3) was insensitive to CFTR inhibition by the thiazol id inone CFTRinh-172. Acidification of phagolysosomes in alveolar macrophages isolated from mice homozygous for ΔF508-CFTR, the most common mutation in cystic fibrosis, was not different when compared to those isolated from wild-type mice. We also measured the kinetics of phagosomal acidification in J774A.1 and murine alveolar macrophages using a zymosan conjugate containing fluorescein and TMR. Phagosomal acidification began within 3 minutes of zymosan binding and was complete within ~15 minutes of internalization. The rate of phagosomal acidification in J774A.1 cells was not inhibited by CFTRinh-172, and was not different in alveolar macrophages from wildtype vs. ∆F508 mice. Our data indicate that phagolysosomal acidification in macrophages is not dependent on CFTR channel activity, and do not support a proposed mechanism for cystic fibrosis lung disease involving defective phagosomal acidification and bacterial killing in alveolar macrophages. Chronic lung infection and deterioration of lung function are the major causes of morbidity and death in Cystic Fibrosis (CF) (1,2). Although the genetic defect in CF was identified in 1989 mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator protein (CFTR) the mechanisms by which CFTR mutations cause lung disease remain uncertain. Various mechanisms have been proposed to link defective CFTR function to CF lung disease, such as defective airway submucosal gland secretion, abnormal airway surface liquid (ASL) composition or oxygenation, Na hyperabsorption producing ASL dehydration (reduced ASL volume), loss of CFTR regulation of other transport proteins, and intrinsic hyperinflammation (reviewed in refs. 3-6). Determination of the mechanisms linking defective CFTR function to CF lung disease is of great importance in developing rational therapies to treat CF. A recent report of defective phagolysosomal acidification in alveolar macrophages in CFTR null mice suggested a new mechanism to link defective CFTR function to CF lung disease (7). Macrophages are key protagonists of the innate immune system and patrol the body, including the alveolar surface, to engulf and destroy pathogens in their phagolysosomes (8-10). Di et al. (7) reported that phagolysosomes in alveolar macrophages acidify in a CFTR-dependent manner and that defective acidification of phagolysosomes of alveolar macrophages from CFTR null mice impaired their bactericidal activity. Interestingly, no defect in bactericidal activity was observed in peritoneal macrophages from CFTR null mice, suggesting that CFTR dependent acidification of phagolysosomes was specific to alveolar macrophages. Although the authors made no direct connection between CFTR mutations, acidification of phagosomes in alveolar macrophages, and CF disease progression, the prevailing view has emerged that defective alveolar macrophage function is important in the http://www.jbc.org/cgi/doi/10.1074/jbc.M705296200 The latest version is at JBC Papers in Press. Published on August 27, 2007 as Manuscript M705296200