H-2M molecules, like MHC class II molecules, are targeted to parasitophorous vacuoles of Leishmania-infected macrophages and internalized by amastigotes of L-amazonensis and L-mexicana

In their amastigote stage, Leishmania are obligatory intracellular parasite s of mammalian macrophages, residing and multiplying within phagolysosomal compartments called parasitophorous vacuoles (PV). These organelles have pr operties similar to those described for the MHC class II compartments of an tigen-presenting cells, sites where peptide-class ZI molecule complexes are formed before their expression at the cell surface. After infection with L eishmania amazonensis or L. mexicana, endocytosis and degradation of class II, molecules by intracellular amastigotes have also been described, sugges ting that these parasites have evolved mechanisms to escape the potentially hazardous antigen-presentation process. To determine whether these events extend to other molecules of the antigen-presentation machinery, we have no w studied the fate of the MHC molecule H-2M in mouse macrophages infected w ith Leishmania amastigotes. At least for certain class II alleles, H-2M is an essential cofactor, which catalyses the release of the invariant chain-d erived CLIP peptide from the peptide-binding groove of class II molecules a nd facilitates the binding of antigenic peptides. H-2M was detected in PV o f mouse macrophages infected with various Leishmania species including L. a mazonensis, L. mexicana, L. major and L. donovani. PV thus contain all the molecules required for the formation of peptide-class LI molecule complexes and especially of complexes with parasite peptides. The present data indic ate, however, that if this process occurs, it does not lead to a clear incr ease of SDS-stable compact ap dimers of class II. In PV that contained L. a mazonensis or L. mexicana, both class II and H-2M molecules often colocaliz ed at the level where amastigotes bind to the PV membrane, suggesting that these molecules are physically associated, directly or indirectly, and poss ibly interact with parasite components. Furthermore, as class II molecules, H-2M molecules were internalized by amastigotes of these Leishmania specie s and reached parasite compartments that also contained class II molecules. Immunostaining of H-2M within parasites was increased by treatment of infe cted macrophages with the cysteine protease inhibitors Z-Phe-AlaCHN(2) or Z -Phe-PheCHN(2) or by incubation of the parasites with the same inhibitors b efore infection. These data thus support the idea that amastigotes of certa in Leishmania species capture and degrade some of the molecules required fo r antigen presentation. To examine whether endocytosis of class II molecule s by the parasites occurs through interactions with parasite components inv olving their peptide-binding groove, we made use of the fact that a large f raction of the class LT molecules of H-2M alpha knock-out H-2(b) mice are o ccupied by the peptide CLIP and are unable to bind other peptides. We found that, in Leishmania-infected macrophages of these mutant mice, class II-CL IP complexes reached PV and were internalized by amastigotes. These results thus prove that endocytosis of class II molecules by amastigotes (1) is H- 2M-independent and (2) does not necessarily involve the peptide-binding poc ket of these molecules. Altogether, these data are compatible with an endoc ytic mechanism based on general properties shared by classical and non-clas sical class II molecules.