title: Processing and presentation of a mycobacterial antigen 85B epitope by murine macrophages is dependent on the phagosomal acquisition of vacuolar proton ATPase and in situ activation of cathepsin D.
abstract: Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston 77030, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Singh
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Moulton
authors.forename: Lisa Y
authors.initials: LY
authors.lastname: Armitige
authors.forename: Akhil
authors.initials: A
authors.lastname: Bidani
authors.forename: Mark
authors.initials: M
authors.lastname: Snuggs
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: D000143:Acids; D000217:Acyltransferases / Q000276:immunology*; D000818:Animals; D017951:Antigen Presentation / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D000939:Epitopes / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D007371:Interferon-gamma / Q000096:biosynthesis; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000166:cytology / Q000276:immunology; D010588:Phagosomes / Q000201:enzymology* / Q000276:immunology*; D034741:RNA, Small Interfering / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D000143:Acids; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D000939:Epitopes; D034741:RNA, Small Interfering; D007371:Interferon-gamma; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis; D002402:Cathepsin D; D025262:Vacuolar Proton-Translocating ATPases