cytochalasin-d and 5-dimethylamiloride

We studied MHC class II (MHC-II)-restricted antigen processing of viable Streptococcus pyogenes by murine macrophages for presentation of two CD4 T cell epitopes of the surface M5 protein. We show that presentation of both epitopes was prevented if actin polymerization was inhibited by cytochalasin D, but not if clathrin-dependent receptor-mediated endocytosis was prevented, suggesting uptake of streptococci by phagocytosis or macropinocytosis was required for presentation of the surface M protein. However, treatment of macrophages with amiloride, which selectively blocks membrane ruffling and subsequent macropinocytosis, inhibited the response to one epitope (M5(308-319)), but had no effect on presentation of the other (M5(17-31)). The effect of the inhibitors on uptake of streptococci was analyzed by electron microscopy. Cytochalasin D completely blocked uptake of streptococci, while dimethyl-amiloride only inhibited uptake into spacious compartments. Neither of the inhibitors altered the cell-surface expression of MHC-II and costimulatory molecules analyzed by flow cytometry. The data suggest that distinct epitopes of a protein associated with viable bacteria may be presented optimally following different uptake mechanisms in the same antigen-presenting cells.

Topics: Amiloride; Amino Acid Sequence; Animals; Antigen Presentation; Antigens, Bacterial; Antigens, CD; B7-1 Antigen; B7-2 Antigen; Cadaverine; CD4-Positive T-Lymphocytes; CD40 Antigens; Cell Line; Cytochalasin D; Epitopes; Histocompatibility Antigens Class II; Macrophages; Membrane Glycoproteins; Mice; Microscopy, Electron; Molecular Sequence Data; Streptococcus pyogenes