Differential handling of bacterial antigens in macrophages infected with Mycobacterium leprae as studied by immunogold labeling of ultrathin sections

Abstract

Mycobacterium leprae were purified from the livers of experimentally infected armadillos, and the purity of the bacterial preparation was established by electron microscopy, immunoelectrophoresis of purified bacilli with rabbit serum raised against liver tissues from a noninfected armadillo, and gas chromatography. Such purified and intact bacilli were fixed and embedded by a gelatin-Lowicryl method for electron microscopy which preserved the mycobacterial antigens. Ultrathin sections were labeled with antisera raised in rabbits against the total antigens of the following species of mycobacteria: M. leprae, M. bovis BCG, M. avium, and a rapid-growing, nonpathogenic species, M. fallax. Bacteria were also labeled using serum raised against 2,3-diacyl-trehalose-2'-sulfate (sulfolipid-IV or SLIV) isolated and purified from M. tuberculosis. The immunolabeling was visualized under the electron microscope (EM) by using a secondary probe (goat-antirabbit IgG, H+L, coupled to 5 nm gold particles; GAR-5). EM results showed that M. leprae bacilli were highly labeled with all of the antisera used except SLIV, which was present only in discrete amounts. All of the antisera used labeled the bacterial "capsule," showing that this structure was not an artifact since it contained mycobacterial antigens. In parallel experiments, the murine J-774 macrophage cell line was infected with purified M. leprae, and fixed for EM at various time intervals for 1 week. Although the phagocytized bacteria did not multiply during the 1-week experiment, macrophages were unable to lyse them. Immunogold labeling of bacterial antigens in ultrathin sections of infected macrophages helped us to conclude: a) bacterial death and/or lysis is not a prerequisite for processing of antigens by infected macrophages; b) there was conclusive evidence for a differential antigen handling, i.e., some antigens were rapidly released (within 2 days, mostly capsular antigens) inside infected macrophages and transported to the macrophage surface, whereas others (the majority of them located in the cell-wall skeleton and in deeper bacterial structures) remained unreleased even after 4 to 7 days of infection; c) although relatively fewer epitopes reacting with anti-SLIV antibodies were found, they were rapidly released (within 2 days) inside macrophages, and exocytized to the macrophage surface. These novel findings are discussed in relation to leprosy and the current knowledge about the processing of bacterial antigens.