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 growth of the intracellular pathogen Leishmania, are likely candidates to present antigens to Leishmania-specific CD4(+) and CD8(+) T lymphocytes, known to be involved in the resolution or in the development of lesions 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 immunoelectron microscopy the subcellular distribution of both MHC class I and class II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-derived macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intracellular sites where Leishmania antigens and MHC molecules meet and can possibly interact. Double labellings with anti-MHC molecule antibodies and with either propidium iodide or an anti-amastigote antibody allowed localization of MHC molecules with regard to the endocytic compartments housing Leishmania amastigotes, organelles known as the parasitophorous vacuoles (PV) and which most likely contain the highest concentration of parasite antigens in the host cell. Both uninfected and infected macrophages from Balb/c mice expressed the MHC class I molecules 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-specific CD8(+) T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were found to be associated with the PV membranes as shown previously with microscopic techniques at lower resolution (Antoine et al. Infect. Immun. 59, 764-775, 1991). In addition, we show here that, 48 hours after infection of Balb/c macrophages, in about 90% of PV containing MHC class II molecules, the latter were mainly or solely localized at the attachment zone of amastigotes to PV membranes. This peculiar distribution, especially well demonstrated using confocal microscopy, was confirmed by subcellular fluorescence cytometry of infected macrophages stained for the MHC class II molecules. The following data agree with the idea that PV-associated MHC class II molecules establish specific interactions with plasma membrane components of amastigotes. First, the polarized localization of class II appeared specific to these molecules, since the distribution of the lysosomal glycoproteins lgp110 and lgp120, of the macrosialin (a macrophage-specific marker of endocytic compartments) and of the GTP-binding protein rab7p, shown here as being PV membrane components, was homogeneous. Second, after killing of Leishmania with the leishmanicidal drug L-leucine methyl ester, MHC class D molecules remained associated for several hours with remnants of the parasites still bound to the PV membrane. Finally, polarized PV-associated MHC class II molecules of infected Balb/c and C57BL/6 macrophages could be stained with the 14-4-4S and Y-3P monoclonal antibodies, respectively; antibodies that have been described as being much more reactive with the compact conformers of the MHC class II molecules carrying tightly associated peptides.