title: Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing.
abstract: Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Cleveland, OH, 44106, USA. lxr2@po.cwru.edu
authors.forename: L
authors.initials: L
authors.lastname: Ramachandra
authors.forename: C V
authors.initials: CV
authors.lastname: Harding
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000187:drug effects; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D004351:Drug Resistance; D005434:Flow Cytometry; D006681:HLA-D Antigens / Q000096:biosynthesis / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007840:Latex / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010047:Ovalbumin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010587:Phagocytosis / Q000187:drug effects; D010588:Phagosomes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*
chemical_list: C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D000949:Histocompatibility Antigens Class II; C111389:I-Ad antigen; D007166:Immunosuppressive Agents; D007840:Latex; C111390:OVA 323-339; D010446:Peptide Fragments; D020126:Brefeldin A; D010047:Ovalbumin