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
| Autor: | Christopher R. Singh, D. Sundarsingh Daniel, Amanda K. Smith, Subramanian Dhandayuthapani, Chinnaswamy Jagannath, Robert L. Hunter, Devin R. Lindsey, Guixiang Dai |
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| Rok vydání: | 2006 |
| Předmět: |
Cytotoxicity
Immunologic Blotting Western Immunology Mice Mice Congenic Western blot Phagosomes medicine Animals Tuberculosis Immunology and Allergy Phosphorylation Protein Kinase C Protein kinase C Phagosome Complement component 5 NADPH oxidase biology medicine.diagnostic_test Activator (genetics) Macrophages Complement C5 NADPH Oxidases Mycobacterium tuberculosis Macrophage Activation Molecular biology Isoenzymes biology.protein Female Reactive Oxygen Species Intracellular |
| Zdroj: | The Journal of Immunology. 177:4688-4698 |
| ISSN: | 1550-6606 0022-1767 |
| DOI: | 10.4049/jimmunol.177.7.4688 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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