Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages.
Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-gamma, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91(phox-/-) and NO synthase 2(-/-) mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2',4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space
Myers
Jesse T
JT
Cellular and Molecular Biology Graduate Program, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Tsang
Albert W
AW
Swanson
Joel A
JA
D000818:Animals; D001854:Bone Marrow Cells / Q000201:enzymology / Q000378:metabolism / Q000382:microbiology; D002478:Cells, Cultured; D004357:Drug Synergism; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D008089:Listeria monocytogenes / Q000276:immunology / Q000378:metabolism / Q000472:pathogenicity*; D008262:Macrophage Activation / Q000235:genetics / Q000276:immunology*; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism* / Q000382:microbiology*; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D026361:Reactive Nitrogen Species / Q000378:metabolism* / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism* / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013997:Time Factors; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism* / Q000382:microbiology*
D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases