Proteasome inhibitors block the entry of liposome-encapsulated antigens into the classical MHC class I pathway.

Liposome-encapsulated conalbumin (L(conalbumin)) is an antigen that is efficiently phagocytosed by bone marrow-derived macrophages and presented to effector cells as part of the major histocompatibility complex (MHC) class I complex. In this report, we show that the conalbumin component of L(conalbumin) is degraded to small peptide fragments and translocated to the area of the Golgi. Golgi localization is confirmed by co-localization of L(Texas red-conalbumin) (L(TR-conalbumin))with both NBD-ceramide, a lipid Golgi marker, and green fluorescent protein (GFP)-galactosyl transferase, a Golgi resident enzyme. Incubation of the cells with brefeldin A disrupts the Golgi and disperses the TR-conalbumin. Furthermore, when macrophages were incubated with another liposome-encapsulated antigen, L(ovalbumin), ovalbumin peptides were observed in the Golgi area and MHC class I-peptide complexes could be detected on the cell surface by both immunofluorescence microscopy and flow cytometry. The Golgi localization observed in vitro in cultured macrophages is mirrored by the in vivo uptake and Golgi localization of fluorescent L(conalbumin) in macrophages isolated from the spleen of a mouse injected with L(TR-conalbumin). The accumulation of peptide fragments in the Golgi is inhibited by the addition of the proteasome inhibitors, lactacystin and MG-132, demonstrating the role of the proteasome in this activity. In addition, when macrophages or a macrophage-derived cell line, are incubated with liposome-enccapsulated antigens and used as target cells in a cytotoxic T-cell (CTL) assay, the CTLs recognize the processed peptide-MHC complexes and kill the cells. In contrast, specific lysis of target cells by CTLs is inhibited when the target cells are first incubated with lactacystin. These results suggest that uptake and processing of L(antigen) follows the classical MHC class I pathway.