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 effi ciently phagocytosed by bone marrow-derived macrophages and presented to ef fector cells as part of the major histocompatibility complex (MHC) class I complex. In this report, we show that the conalbumin component of L(conalbu min) is degraded to small peptide fragments and translocated to the area of the Golgi. Golgi localization is confirmed by co-localization of L(Texas r ed-conalbumin) (L(TR-conalbumin))with both NBD-ceramide, a lipid Golgi mark er, and green fluorescent protein (GFP)-galactosyl transferase; a Golgi res ident enzyme. Incubation of the cells with brefeldin A disrupts the Golgi a nd disperses the TR-conalbumin. Furthermore, when macrophages were incubate d with another liposome-encapsulated antigen, L(ovalbumin), ovalbumin pepti des 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 macrop hages is mirrored by the in vivo uptake and Golgi localization of fluoresce nt L(conalbumin) in macrophages isolated from the spleen of a mouse injecte d 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 addi tion, when macrophages or a macrophage-derived cell line, are incubated wit h liposome-enccapsulated antigens and used as target cells in a cytotoxic T -cell (CTL) assay, the CTLs recognize the processed peptide-MHC complexes a nd kill the cells. In contrast, specific lysis of target cells by CTLs is i nhibited when the target cells are first incubated with lactacystin. These results suggest that uptake and processing of L(antigen) follows the classi cal MHC class I pathway. (C) 2000 Elsevier Science B.V. All rights reserved .