title: The reduced bactericidal function of complement C5-deficient murine macrophages is associated with defects in the synthesis and delivery of reactive oxygen radicals to mycobacterial phagosomes.
abstract: Complement C5-deficient (C5(-/-)) macrophages derived from B.10 congenic mice were found to be defective in killing intracellular Mycobacterium tuberculosis (MTB). They were bacteriostatic after activation with IFN-gamma alone but bactericidal in the combined presence of IFN-gamma and C5-derived C5a anaphylatoxin that was deficient among these macrophages. Reduced killing correlated with a decreased production of reactive oxygen species (ROS) in the C5(-/-) macrophages measured using fluorescent probes. Furthermore, a lack of colocalization of p47(phox) protein of the NADPH oxidase (phox) complex with GFP-expressing MTB (gfpMTB) indicated a defective assembly of the phox complex on phagosomes. Reconstitution with C5a, a known ROS activator, enhanced the assembly of phox complex on the phagosomes as well as the production of ROS that inhibited the growth of MTB. Protein kinase C (PKC) isoforms are involved in the phosphorylation and translocation of p47(phox) onto bacterial phagosomes. Western blot analysis demonstrated a defective phosphorylation of PKC (alpha, beta, delta) and PKC-zeta in the cytosol of C5(-/-) macrophages compared with C5 intact (C5(+/+)) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC-beta and PKC-zeta were the isoforms that are not phosphorylated in C5(-/-) macrophages. Because Fc receptor-mediated phox assembly was normal in both C5(-/-) and C5(+/+) macrophages, the defect in phox assembly around MTB phagosomes was specific to C5 deficiency. Reduced bactericidal function of C5(-/-) macrophages thus appears to be due to a defective assembly and production of ROS that prevents effective killing of intracellular MTB.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: D Sundarsingh
authors.initials: DS
authors.lastname: Daniel
authors.forename: Guixiang
authors.initials: G
authors.lastname: Dai
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Singh
authors.forename: Devin R
authors.initials: DR
authors.lastname: Lindsey
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Smith
authors.forename: Subramanian
authors.initials: S
authors.lastname: Dhandayuthapani
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hunter
authors.forename: Chinnaswamy
authors.initials: C
authors.lastname: Jagannath
mesh_terms: D000818:Animals; D015153:Blotting, Western; D003182:Complement C5 / Q000172:deficiency*; D003602:Cytotoxicity, Immunologic; D005260:Female; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism; D008262:Macrophage Activation / Q000276:immunology; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D020297:Mice, Congenic; D009169:Mycobacterium tuberculosis / Q000276:immunology; D019255:NADPH Oxidases / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism*; D014376:Tuberculosis / Q000276:immunology*
chemical_list: D003182:Complement C5; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C