FcεRI-mediated antigen endocytosis turns interferon-γ-treated mouse mast cells from inefficient into potent antigen-presenting cells

Previous studies in our laboratory have shown that bone-marrow-derived mast cells (BMMC) could present immunogenic peptides, from soluble antigens endocytosed through fluid phase, only if they were subjected to a 48-hr treatment with interleukin-4 (IL-4) and granulocyte–macrophage colony-stimulating factor (GM-CSF). In contrast to GM-CSF, interferon-γ (IFN-γ) which highly upregulates major histocompatibility complex (MHC) class II expression, completely inhibits the generation of immunogenic peptides. We have used this model to study the role of FcεRI-mediated antigen internalization in the regulation of the antigen-presenting function of IFN-γ-treated mast cells. Here, we report that FcεRI can reverse the IFN-γ-treated mast cells from inefficient to highly efficient antigen-presenting cells. Inhibition of the antigen presenting capacity by piceatannol, a protein tyrosine kinase (PTK) syk inhibitor, indicates that this is an active process resulting from immunoglobulin E (IgE)–antigen–FcεRI engagement which involves tyrosines found in the immunoreceptor tyrosine-based activation motif (ITAM) embedded in the cytoplasmic tail of the FcεRI β and γ chains. Antigen-presenting function was also shown to require the activation of phosphatidyl inositol 3 (PI3) kinase, downstream of PTK syk phosphorylation, since this activity was completely blocked by wortmannin, a PI3 kinase inhibitor. These data suggest that signalling generated by FcεRI provides mast cells with IgE-mediated enhanced antigen presentation to T cells and emphasize a so far unknown immunoregulatory mast-cell function that might take place in inflammatory sites

Endocytosis and recycling of the complex between CD23 and HLA-DR in human B cells

The presentation of extremely low doses of antigen to T cells is enhanced by immunoglobulin E (IgE)-dependent antigen focusing to CD23, the low-affinity receptor for IgE, expressed on activated B cells. CD23 contains a C-type lectin domain in its extracellular sequence and a targeting signal for coated pits, required for endocytosis, in its cytoplasmic sequence. CD23 is non-covalently associated with the major histocompatibility complex class II antigen, human leucocyte antigen HLA-DR, on the surface of human B cells, but the fate of this complex following endocytosis is unknown. To answer this question we have labelled these proteins on the surface of RPMI 8866 B cells and traced their route through the cytoplasm. Endocytosis mediated by anti-CD23 antibodies (BU38 and MHM6) led to the loss of CD23 from the cells. Endocytosis mediated by an antibody to HLA-DR (CR3/43) or an antigen-IgE complex (NP-BSA-anti-NP IgE), however, led to recycling of the HLA-DR-CD23 complex to the cell surface on a time scale (3-6 hr) consistent with the recycling of HLA-DR in antigen presentation. Along the latter pathway CD23 label was observed in cytoplasmic organelles that resembled the 'compartments for peptide loading' or 'class II vesicles' described by previous authors. Two features of the recycling process may contribute to the efficiency of antigen presentation. Peptide exchange may be facilitated by the proximity of HLA-DR and antigen in peptide loading compartments of the endosomal network. The return of CD23 with HLA-DR to the cell surface may then help to stabilize specific B-cell-T-cell interactions, contributing to T-cell activation