FcepsilonRI-mediated antigen endocytosis turns interferon-gamma-treated mouse mast cells from inefficient into potent antigen-presenting cells

Abstract

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-gamma (IFN-gamma) 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 FcepsilonRI-mediated antigen internalization in the regulation of the antigen-presenting function of IFN-gamma-treated mast cells. Here, we report that FcepsilonRI can reverse the IFN-gamma-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-FcepsilonRI engagement which involves tyrosines found in the immunoreceptor tyrosine-based activation motif (ITAM) embedded in the cytoplasmic tail of the FcepsilonRI beta and gamma 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 FcepsilonRI 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.

Figure 1

Surface molecule expression by mast cells. Expression of FcεRI was analysed by flow cytometry using biotinylated rat antimouse IgE (RAM-IgE) followed by phycoerythrin–streptavidin. The background fluorescence intensity was obtained with mast cells stained with biotinylated-RAM/IgE alone followed by phycoerythrin–streptavidin. Expression of MHC class II and CD117 molecules were assessed by labelling the cells with biotinylated antimouse I-A^b/d and anti-CD117 mAbs, respectively, followed by phycoerythrin–streptavidin. Negative controls were obtained by incubating cells with phycoerythrin–streptavidin alone.

Figure 2

FcεRI-mediated antigen endocytosis induces antigen presenting function in IFN-γ treated mast cells. IgE-bearing mast cells, pulsed for 1 hr with OVA–DNP (▴), were incubated at different concentrations in the presence of different T-cell hybridomas at 5×10⁴/well. Alternatively, mast cells were pulsed for 1 hr with OVA alone (▪), and cocultured with the same anti-OVA T-cell hybridomas. IL-2 content in culture supernatants (50 μl) was measured by CTLL-2 proliferation assay. Background values of mast cells cultured with different T-cell hybridomas alone were 1000±500 c.p.m. Data from one representative experiment are shown.

Figure 3

Enhancement of the lysosomal/endosomal pH compartments by NH₄Cl treatment does not increase OVA presentation by IFN-γ-treated BMMC. IFN-γ-treated BMMC (2×10⁶/ml) were left untreated (▵) or treated for 30 min at 37° in RPMI 10% FCS with NH₄Cl at 20 (□), 5 (○) or 1 (+) mM, and then pulsed with 100 μg/ml of OVA–DNP (a) or OVA (b) for 1 hr at 37°. All cells were sensitized for 3 days with anti-DNP IgE mAb to increase FcεRI expression. After washings, mast cells were diluted and cultured with the anti-OVA specific T-cell hydridoma, 3DO-54·8 (5×10⁴/well). IL-2 content in culture supernatants (50 μl) was measured by CTL-L2 proliferation assay. Background values of mast cells cultured with different T-cell hybridomas alone were 2500±500 c.p.m. The data represent the mean±SD of two different experiments.

Figure 4

IgE cross-linking by irrelevant antigen is not sufficient to induce antigen-presenting function in IFN-γ treated mast cells. Mast cells (2×10⁶/well) sensitized with anti-DNP IgE mAb were pulsed with 100 μg/ml of OVA alone (•), OVA–DNP alone (□), or a mixture of 100 ng/ml DNP–HSA and 100 μg/ml of OVA (▪) for 1 hr. After washing, the cells were cocultured at various concentrations in the presence of the T-cell hybridoma 3DO-54·8 (5×10⁴/well). As control, DNP–HSA alone did not induce any T-cell activation. IL-2 response was measured by the CTL-L2 proliferation assay. The data represent the mean±SD of two different experiments.

Figure 6

Blocking of FcεRI-mediated signalling does not prevent internalization of IgE–antigen immune complexes.IgE-sensitized mast cells (2×10⁶/ml) treated with piceatannol (30 μg/ml) or wortmannin (30 nm), were stimulated with OVA–DNP at 100 μg/ml for 2, 15 min, or 1 hr at 37°. The reaction was stopped by adding cold PBS. To measure endocytosis of the aggregated FcεRI, FcεRI expression was estimated by flow cytometry. Mast cells (4×10⁵/tube) were incubated with biotinylated rat antimouse IgE (RAM-IgE) followed by phycoerythrin–streptavidin. As control, FcεRI expression from unstimulated mast cells was also represented. The background fluorescence intensity was obtained with mast cells stained with biotinylated-RAM/IgE alone followed by phycoerythrin–streptavidin.

Figure 5

Signalling through FcεRI is required for effective antigen presentation by IFN-γ treated mast cells. To inhibit syk or PI3 kinase activation, IgE-sensitized mast cells (2×10⁶/ml) were left untreated (+) or treated with 30 (○), 10 (▵), or 3 μg/ml (□) of piceatannol for 1 hr (a), or with 30 (○), 10 (▵) or 3 nM (□) of wortmannin or alone (+) for 20 min at 37° (b). Without washing, cells were further incubated for 1 hr with 100 μg/ml OVA–DNP. After washing, the cells were cocultured, at various concentrations, with the T-cell hybridoma 3DO-54·8 (5×10⁴/well). IL-2 response was measured by the CTL-L2 proliferation assay. As control, mast cells were treated with dimethylsulphoxide (DMSO) diluted in the same range of concentrations as piceatannol or wortmannin (data not shown). To assess mast cell activation, supernatants from piceatannol- (•) or wortmannin-treated (▪) mast cells were analysed for their content in β-hexosaminidase (c). The asterisk corresponds to the amount of β-hexosaminidase released by OVA–DNP-pulsed BMMC in the absence of inhibitors. The data represent the mean±SD of three different experiments.