Macrophages present exogenous antigens by class I major histocompatibility complex molecules via a secretory pathway as a consequence of interferon-gamma activation.

Macrophages can process and present exogenous antigens on major histocompatibility complex (MHC) class I molecules through an alternative mechanism involving the internalization of antigens and the secretion of peptides loading MHC class I molecules at the cell surface. In this paper, we found that interferon-gamma (IFN-gamma) -activated macrophages infected with Salmonella typhimurum secreted peptides able to load empty MHC Kb molecules on co-cultured TAP-2-deficient RMA-S cells, added as targets for peptide loading. The increase in class I Kb on the RMA-S cells, resulting from the macrophage-derived peptides, exhibited a comparable stability as the direct addition of an exogenous Kb-binding peptide (OVA257-264) to the RMA-S cells. In both cases, the Kb complexes were stable for at least 3 hr after separating the RMA-S cells from the macrophages. The endosomal inhibitors, leupeptin and ammonium chloride, did not inhibit the release of peptides and the increase in Kb staining on the RMA-S cells in the co-culture systems. Brefeldin A also had no effect. P815 cells previously co-cultured with Salmonella-infected macrophages became targets for cytotoxic T lymphocytes isolated from Salmonella-infected BALB/c mice. Taken together, our data suggest that IFN-gamma-activated macrophages process exogenous antigens in an intracellular compartment where serine proteases generate peptides released to the external environment for loading empty MHC class I molecules at the cell surface. This TAP-independent mechanism for the MHC class I presentation may be involved in priming cytotoxic T lymphocytes against intracellular pathogens in vivo.

Molecular characterization of the tight junction protein ZO-1 in MDCK cells.

Most of the information on the structure and function of the tight junction (TJ) has been obtained in MDCK cells. Accordingly, we have sequenced ZO-1 in this cell type, because this protein is involved in the response of the TJ to changes in Ca2+, phosphorylation, and the cytoskeleton. ZO-1 of MDCK cells comprises 6805 bp with a predicted open reading frame of 1769 amino acids. This sequence is 92 and 87% homologous to human and mouse ZO-1, respectively. Two nuclear sorting signals located at the PDZ1 and GK domains and 17 SH3 putative binding sites at the proline-rich domain were detected. We found two new splicing regions at the proline-rich region: beta had not been reported in human and mouse counterparts, and gamma, which was previously sequenced in human and mouse ZO-1, is now identified as a splicing region. The expression of different beta and gamma isoforms varies according to the tissue tested. With the information provided by the sequence, Southern blot, and PCR experiments we can predict a single genomic copy of MDCK-ZO-1 that is at least 13.16 kb long. MDCK-ZO-1 mRNA is 7.4 kb long. Its expression is regulated by calcium, while the expression of MDCK-ZO-1 protein is not.