Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

Abstract

The potential of the paired Ig-like receptors of activating (PIR-A) and inhibitory (PIR-B) types for modifying an IgE antibody-mediated allergic response was evaluated in mouse bone marrow-derived mast cells. Although mast cells produced both PIR-A and PIR-B, PIR-B was found to be preferentially expressed on the cell surface, where it was constitutively tyrosine phosphorylated and associated with intracellular SHP-1 protein tyrosine phosphatase. PIR-B coligation with the IgE receptor (FcepsilonRI) inhibited IgE-mediated mast cell activation and release of serotonin. Surprisingly, the inhibitory activity of PIR-B was unimpaired in SHP-1-deficient mast cells. A third functional tyrosine-based inhibitory motif, one that fails to bind the SHP-1, SHP-2, and SHIP phosphatases, was identified in parallel studies of FcepsilonRI-bearing rat basophilic leukemia (RBL) cells transfected with constructs having mutations in the PIR-B cytoplasmic region. These results define the preferential expression of the PIR-B molecules on mast cells and an inhibitory potential that can be mediated via a SHP-1-independent pathway.

Figure 1

Cell surface expression of PIR molecules on cultured mast cells. Nonadherent cells from the bone marrow of (a) wild-type mice (C57BL/cJ) and (b) FcεRIα^–/– mice and from the neonatal spleens of (c) wild-type mice (C3H/HeJ) and (d) me/me mice were cultured for 6 weeks with rIL-3. Cells were sequentially incubated with the PE-labeled 6C1 anti-PIR mAb, biotinylated ACK-2 anti–c-kit mAb, and APC-labeled streptavidin or with rat IgE anti-DNP mAb (dark histogram) and FITC-labeled goat anti-rat Ig antibodies before analysis by flow cytometry. Isotype-matched control rat mAb’s were used to set the quadrants for immunofluorescence analysis, and unstained background controls are indicated by open histograms.

Figure 2

PIR expression by IL-3–induced mast cells. (a) RT-PCR analysis of PIR-A, PIR-B, and actin gene expression by C3H/HeJ bone marrow–derived mast cells and the WEHI-3 myeloid cell line. The PCR products were electrophoresed in 1.5% agarose and stained with ethidium bromide. (b) Analysis of the PIR molecules on splenocytes, mast cells, and macrophages. Cell surface proteins were ¹²⁵I-labeled, solubilized in 1% NP-40, and immunoadsorbed with 6C1 anti-PIR or an isotype-matched control mAb before analysis by SDS-10% PAGE under reducing conditions as described in Methods. (c) Analysis of PIR molecules on mast cells derived from adult bone marrow of FcεRIα^–/– mice and littermate controls (upper panel) and from neonatal spleens of me/me mice and littermate controls (lower panel). Iodinated cell surface PIR proteins were resolved on SDS-10% PAGE under reducing conditions.

Figure 3

PIR-B inhibitory activity in FcεRI-mediated Ca²⁺ mobilization. Indo-1/AM dye preloaded mast cells from wild-type mice (upper panel) and motheaten mice (lower panel) were analyzed by flow cytometry for intracellular Ca²⁺ levels in the presence or absence (data not shown) of extracellular Ca²⁺. Cells were stimulated with rat IgE anti-DNP (IgE), F(ab′)₂ fragments of rat anti-PIR (anti-PIR), or both mAb’s (IgE + anti-PIR). F(ab′)₂ fragments of rabbit anti-rat Ig antibodies were used as the cross-linking reagent.

Figure 4

PIR-B inhibitory activity in FcεRI-induced serotonin release. ³H-Serotonin preloaded mast cells from wild-type neonatal spleen (left) and motheaten mutant neonatal spleen (right) were incubated with rat IgE anti-DNP mAb plus various concentrations of F(ab′)₂ fragments of rat mAb specific for PIR (filled circles) or unknown cell surface antigen (open circles). Antibody-primed cells were then triggered with F(ab′)₂ fragments of rabbit anti-rat Ig antibody as a cross-linker, and the ³H-serotonin release was measured as described in Methods. The standard deviation from the mean (circle) is indicated by bars only for the points at which this value (1 SD) is greater than the circle radius.

Figure 5

Analysis of mast cell proteins binding to PIR-B ITIM. Left panel: Metabolically labeled mast cell proteins were solubilized in 1% NP-40 lysis buffer before incubation with streptavidin-coupled beads bearing biotinylated 15-mer peptides corresponding to the mouse FcγRIIB ITIM (tyrosine-phosphorylated, pY, or tyrosine-nonphosphorylated, Y, as controls) and each of the tyrosine-phosphorylated and biotinylated PIR-B ITIM (pY713, pY742, pY770, pY794, pY824). Bound materials were resolved by SDS-8% PAGE under nonreducing conditions before exposure to x-ray films. Arrowhead indicates 120-kDa protein band precipitated by the pY742 phosphopeptide. Right panel: Proteins isolated from mast cell lysates with the same phosphopeptides were resolved by SDS-PAGE before transfer onto membranes and immunoblotting with antibodies specific for SHP-1, SHP-2, or SHIP before visualization by enhanced chemiluminescence.

Figure 6

Comparison of surface expression of FcγRIIB/PIR chimeric receptors among various stable transfectants. RBL-2H3 rat basophilic leukemia cells were transfected by electroporation with various types (YYYY, FYFF, YY, YF, and Δ712) of chimeric construct (FcγRIIB/PIR-B). These constructs encode the extracellular (EC), transmembrane (TM), and the first six amino acids of the cytoplasmic tail of the mouse FcγRIIB1 (open portions of bars) and fused with different intracytoplasmic regions (IC) of the mouse PIR-B (filled portions of bars) as depicted in the right panel. Four cytoplasmic tyrosine (Y) residues are also indicated. For the left panel, stably transfected cells were sequentially incubated with 2.4G2 rat mAb and FITC-labeled goat anti-rat Ig antibody (filled profile) or with BC4 mouse IgE mAb and FITC-labeled goat anti-mouse Ig antibody (thick line) in order to determine their cell surface levels of FcγRIIB/PIR-B chimeric receptors and endogenous FcεRI by flow cytometry. Irrelevant mouse IgG (thin line) was used as a control to obtain a background staining.

Figure 7

Inhibitory activity of FcγRIIB/PIR-B chimeric receptors expressed by RBL transfected cells. RBL-2H3 cells transfected with the indicated FcγRIIB/PIR-B constructs were incubated with various dilutions of mouse IgE in the absence (filled circles) or presence (open circles) of 2.4G2 rat anti-FcγRII/III mAb (20 μg/ml). F(ab′)₂ fragments of goat anti-murine Ig antibody (50 μg/ml) were used as a cross-linker. A representative result is shown as a mean ± 1 SD (if the SD values are greater than the radius of circles) from at least three independent experiments. Statistical analysis revealed that inhibition is significant for IgE at 1:1,000 and 1:300 dilutions in a, b, and d (data not shown).