DISTRIBUTION OF MHC CLASS-I AND OF MHC CLASS-II MOLECULES IN MACROPHAGES INFECTED WITH LEISHMANIA-AMAZONENSIS

Macrophages, being apparently the only cells that in vivo allow the gr owth of the intracellular pathogen Leishmania, are likely candidates t o present antigens to Leishmania-specific CD4(+) and CD8(+) T lymphocy tes, known to be involved in the resolution or in the development of l esions induced by these parasites, and recognizing processed antigens bound to MHC class I and MHC class II molecules, respectively. In the present study, we analysed by confocal microscopy and by immunoelectro n microscopy the subcellular distribution of both MHC class I and clas s II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-deriv ed macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intr acellular sites where Leishmania antigens and MHC molecules meet and c an possibly interact. Double labellings with anti-MHC molecule antibod ies and with either propidium iodide or an anti-amastigote antibody al lowed localization of MHC molecules with regard to the endocytic compa rtments housing Leishmania amastigotes, organelles known as the parasi tophorous vacuoles (PV) and which most likely contain the highest conc entration of parasite antigens in the host cell. Both uninfected and i nfected macrophages from Balb/c mice expressed the MHC class I molecul es H-2K(d) and H-2D(d) on their cell surface but no significant amount of these molecules could be detected in the PV, which indicates that, if infected macrophages play a role in the induction of Leishmania-sp ecific CD8(+) T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were fo und to be associated with the PV membranes as shown previously with mi croscopic techniques at lower resolution (Antoine et al. Infect. Immun . 59, 764-775, 1991). In addition, we show here that, 48 hours after i nfection of Balb/c macrophages, in about 90% of PV containing MHC clas s II molecules, the latter were mainly or solely localized at the atta chment zone of amastigotes to PV membranes. This peculiar distribution , especially well demonstrated using confocal microscopy, was confirme d by subcellular fluorescence cytometry of infected macrophages staine d for the MHC class II molecules. The following data agree with the id ea that PV-associated MHC class II molecules establish specific intera ctions with plasma membrane components of amastigotes. First, the pola rized localization of class II appeared specific to these molecules, s ince the distribution of the lysosomal glycoproteins lgp110 and lgp120 , of the macrosialin (a macrophage-specific marker of endocytic compar tments) and of the GTP-binding protein rab7p, shown here as being PV m embrane components, was homogeneous. Second, after killing of Leishman ia with the leishmanicidal drug L-leucine methyl ester, MHC class D mo lecules remained associated for several hours with remnants of the par asites still bound to the PV membrane. Finally, polarized PV-associate d MHC class II molecules of infected Balb/c and C57BL/6 macrophages co uld be stained with the 14-4-4S and Y-3P monoclonal antibodies, respec tively; antibodies that have been described as being much more reactiv e with the compact conformers of the MHC class II molecules carrying t ightly associated peptides.