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-γ alone but bactericidal in the combined presence of IFN-γ 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 (α, β, δ) and PKC-ζ in the cytosol of C5−/− macrophages compared with C5 intact (C5+/+) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC-β and PKC-ζ 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.

TLR2-Mediated Survival of Staphylococcus aureus in Macrophages: A Novel Bacterial Strategy against Host Innate Immunity

“…This is most probably achieved through the inhibition of either the formation of the oxidase complex or the activity of particular components, with no need for newly synthesized proteins. There is an example of regulated formation of the oxidase complex in macrophages; effective association of p47 phox , a cytosolic regulator of superoxide generation by the complex, with mycobacteria-containing phagosomes seems to require the actions of C5/C5a receptor and protein kinase C (33). Further investigation is necessary to clarify the mechanism of JNK-mediated negative regulation of superoxide production in microbe-containing phagosomes.…”

Differential Contributions of C3, C5, and Decay-Accelerating Factor to Ethanol-Induced Fatty Liver in Mice

“…C5a is generally much more pro-inflammatory than C3a with respect to effects on blood flow, lymphatic drainage and vascular permeability and neutrophil activation, and consequently, with respect to local concentrations of agonists and complement components (21). C5a has also been correlated with increased expression of cytokines and chemokines (3;7) and reactive oxygen species (9). C5 has been associated with injury to lung (15) and kidney (10)(11).…”

Defect in early lung defence against Pseudomonas aeruginosa in DBA/2 mice is associated with acute inflammatory lung injury and reduced bactericidal activity in naïve macrophages

“…The increased phagocytosis could be due to increased expression of P. aeruginosa receptors on D2 naïve macrophages. Alternatively, it could be caused by attenuated activation of macrophages with the reduced production of interferon (INF)-c and/or the decreased production of reactive oxygen species (Daniel et al, 2006). Increased phagocytosis in macrophages could also evoke signals in the form of increased cytokine and chemokine production to set off a cascade of increased inflammatory host responses.…”