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Bacterial antigen immunolabeling in macrophages after phagocytosis and degradation of Bacillus subtilis

After phagocytosis of Bacillus subtilis 168 by bone marrow-derived macrophages, the intracellular pathway followed by different antigens was studied by immunofluorescence and immunoelectron microscopy. Three different rabbit antisera were used: (i) an antiserum to B. subtilis whole cells mainly recognizing the cell wall constituents, (ii) an antiserum to teichoic acid, and (iii) an antiserum to peptidoglycan recognizing the disaccharide tetrapeptide molecules resulting from peptidoglycan degradation. During the first 3 h after phagocytosis of B. subtilis, the three antisera were confined to the same vacuolar compartments, as follows. They were first found in phagosomes gathered in the perinuclear region. Upon bacterial degradation, the three antisera colocalized in an increasing number of small dense vesicles, located in the perinuclear region, that seemed to result from the fragmentation of phagolysosomes. These vesicles correspond to an acidic compartment since they also stained for 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine, a drug known to accumulate in the acidic compartments of cells. At later time points, the antigens recognized by the three antisera followed different pathways. After 18 h, teichoic acid and peptidoglycan were no longer detectable in macrophages whereas an antigen(s) labeled with antiserum to B. subtilis whole cells remained stocked for several days in small acidic vesicles randomly distributed throughout the macrophage. This compartment appeared to be different from the one labeled during the first 3 h after ingestion of bacteria. These results suggest that the transport rate and the compartments implicated in antigen processing differ according to the antigen.