Alterations in ceramide concentration and pH determine the release of reactive oxygen species by Cftr-deficient macrophages on infection.
We recently demonstrated that the accumulation of ceramide in Cftr-deficient epithelial cells is important for the pathophysiology of CF. However, the role of ceramide in other lung cells, particularly lung macrophages, requires definition. In this study, we report that ceramide is accumulated in Cftr-deficient lung macrophages. Alveolar macrophages contain a vesicle population, which is stained with LysoSensor probes but not by tetramethylrhodamine dextran. These vesicles, presumably secretory lysosomes, exhibit a higher pH in Cftr-deficient macrophages than the corresponding vesicles in lung macrophages isolated from wild-type (WT) mice. Alkalinization of these vesicles in Cftr-deficient macrophages correlates with a failure of the macrophages to respond to infection with various Pseudomonas aeruginosa strains by acutely activating acid sphingomyelinase, releasing ceramide, forming ceramide-enriched membrane platforms that serve to cluster gp91(phox), and, most importantly, releasing reactive oxygen species (ROS). In contrast, these events occur rapidly in WT lung macrophages postinfection. Inhibiting ROS in WT macrophages prevents the killing of P. aeruginosa. These findings provide evidence for a novel pH-controlled pathway from acid sphingomyelinase activation via ceramide and clustering of gp91(phox) to the release of ROS in lung macrophages.
Zhang
Yang
Y
Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122, Essen, Germany.
Li
Xiang
X
Grassmé
Heike
H
Döring
Gerd
G
Gulbins
Erich
E
D000818:Animals; D002478:Cells, Cultured; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016676:Macrophages, Alveolar / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism* / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018181:Mice, Inbred CFTR; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D011550:Pseudomonas aeruginosa / Q000254:growth & development / Q000276:immunology; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D015610:Serum Bactericidal Test; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013194:Staining and Labeling
D002518:Ceramides; D003911:Dextrans; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D012235:Rhodamines; C539719:dextran tetramethylrhodamine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013108:Sphingomyelin Phosphodiesterase