In their amastigote stage, Leishmania live in mammalian macrophages within parasitophorous vacuoles (PV), organelles of phagolysosomal origin that, in macrophages activated with IFN-gamma, contain major histocompatibility complex (MHC) class II molecules apparently devoid of invariant chains. We have now studied the fate of PV-associated class II molecules in mouse bone marrow-derived macrophages infected with L. amazonensis amastigotes using immunocytochemical and biochemical approaches. We have found that at least a part of these class II molecules was internalized by amastigotes and reached structures very often located in their posterior poles. This process was much more obvious if infected macrophages were incubated with protease inhibitors like antipain, chymostatin, Z-Phe-AlaCHN2 and Z-Phe-PheCHN2, or if amastigotes were pre-treated with the irreversible cysteine protease inhibitor Z-Phe-AlaCHN2 before infection, clearly indicating that amastigotes also degraded the internalized class II molecules. Study of infected macrophage cryosections by immuno-electron microscopy allowed the identification of the class II-positive structures in amastigotes as the lysosome-like organelles known as megasomes. Other PV membrane components like the prelysosomal/lysosomal glycoproteins Igp110, Igp120 and macrosialin could not be detected in megasomes of amastigotes even after treatment of macrophages with protease inhibitors, suggesting the involvement of some specific mechanism(s) for the internalization of class II molecules. Interestingly, after treatment of infected macrophages with various protease inhibitors (antipain, leupeptin, E-64, Z-Phe-AlaCHN2, Z-Phe-PheCHN2), PV membrane as well as megasomes of amastigotes become positive for invariant chains. A quantitative analysis of amastigote-associated class II molecules based on enzyme immunoassays showed that: (a) amastigotes extracted from macrophages treated with both IFN-gamma and antipain or Z-Phe-AlaCHN2 contained a much greater amount of class II than amastigotes extracted from macrophages treated with IFN-gamma alone; (b) class II molecules associated with the former were mainly intracellular and, at least some of them, were complexed with invariant chains or fragments of invariant chains; (c) amastigotes pre-incubated with Z-Phe-AlaCHN2 before infection accumulated a greater amount of intracellular class II than amastigotes pre-incubated without inhibitor, clearly indicating that the blockade of parasite cysteine proteases was sufficient to enhance the pool of these molecules within megasomes. On the whole, these data are consistent with the idea that class II molecules reaching PV are newly synthesized and still complexed with intact invariant chains or with partially degraded invariant chains.(ABSTRACT TRUNCATED AT 400 WORDS)

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