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* Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, TX 77030; and
Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78229
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-
alone but bactericidal in the combined presence of IFN- 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 p47phox 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 p47phox onto bacterial phagosomes. Western blot analysis demonstrated a defective phosphorylation of PKC (
, 
, 
) and PKC-
in the cytosol of C5–/– macrophages compared with C5 intact (C5+/+) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC- and PKC- 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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Heart, Lung, and Blood Institute Grant HL68520.
2 D.S.D. and G.D. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Chinnaswamy Jagannath, MSB2200, Department of Pathology and Laboratory Medicine, University of Texas Health Sciences Center, Houston, TX 77030. E-mail address: Chinnaswamy.Jagannath{at}uth.tmc.edu
4 Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; C5a-R, C5a-receptor; ROS, reactive oxygen species; PKC, protein kinase C; BM, bone marrow; DCFDA, dihydro-dichloro-fluorescein diacetate; iNOS, inducible NO synthase; RLU, relative light unit; AFU, average fluorescence unit; NT, nitrotyrosine; ONOO, peroxynitrite.
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