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

Complement C5-deficient (C5^-/-) macrophages derived from B.10 congenic mice ware found to be defective to 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 colocalizatlon of p47^phox protein of the NADPH oxidase (phox) complex with GFP-espressing 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 (α, β, δ) and PKC-ζ in the cytosol of C5^-/- macrophages compared with C5 intact (C5 ^+/+) macrophages. Furthermore, in site 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.