Signaling through the high affinity IgE receptor Fc⑀RI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor Fc␥RII. We used a recently described fusion protein, GE2, which is composed of key portions of the human ␥1 and the human ⑀ heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of Fc␥RII and Fc⑀RI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor Fc␥RII but not Fc␥RI or Fc␥RIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating Fc⑀RI and Fc␥RII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca 2؉ mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62 dok ; Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fc⑀-Fc␥ co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human Fc⑀RI-mediated responses can be inhibited by co-aggregation with Fc␥RIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.Mast cells generate pro-inflammatory mediators that initiate and propagate allergic inflammation. IgE-mediated mediator release is initiated through the high affinity IgE receptor Fc⑀RI, 1 in which the ␣ subunit has IgE binding activity and the ␤ and ␥ subunits have signaling activity. Signaling is primarily mediated by immunoreceptor tyrosine-based activation motifs (ITAMs) located in the carboxyl-terminal cytoplasmic tails of each of the ␤ and ␥ subunits. ITAMs are typically 26-or 27-amino acid stretches including two YXXL motifs separated by 9 or 10 amino acids. Cross-linking IgE-primed receptors with a multivalent antigen results in the activation of Lyn, which in turn phosphorylates ITAMs, creating docking sites for Syk kinase family members and permitting signal propagation (for review, see Ref. 1).Many immune cells that express ITAM-containing antigen receptors also express receptors with a related immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ITIMs are 13-amino acid sequences containing a single YXXL motif (2). They were first recognized in the cytoplasmic tails of the Fc␥RIIB isoform (Fc␥RIIB1) of rodent B cells. Their negative signaling activity was revealed by studies showing ...

We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, Fc epsilon RI. The structure of the second domain of CD2 was used as a modelling template for the second alpha-chain domain of Fc epsilon RI, the C-C' loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C' region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C' peptide mimics act as competitive inhibitors of IgE binding. The cyclic L- and retro D-peptides exhibited KDs of approximately 3 microM and 11 microM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response.

The MS4A (membrane-spanning 4-domain family, subfamily A) family of proteins contains some well-known members including MS4A1 (CD20), MS4A2 (FcɛRIβ) and MS4A3 (HTm4). These three MS4A family members are expressed on the cell surface of specific leukocyte subsets and have been well characterized as having key roles in regulating cell activation, growth and development. However, beyond MS4A1-3 there are a large number of related molecules (18 to date in humans) where our understanding of their biological roles is at a relatively nascent stage. This review examines the larger MS4A family focusing on their structure, expression, regulation and characterized and/or emerging biological roles. Our own work on one family member MS4A8B, and its possible role in epithelial cell regulation, is also highlighted.

The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcepsilonRI) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of IgE-Fc (Cepsilon3) as the site of the interaction with FcepsilonRI. We have prepared a series of site-directed mutants of human IgE-Fc, particularly focusing on the N-terminal "linker" region and AB loop of Cepsilon3. The kinetics of binding IgE and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of IgE-Fc for FcepsilonRI by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in Fcepsilon3-4, a truncated form of IgE-Fc which lacks the Cepsilon2 domain pair, and thus it does not exert its effect through altering the quaternary structure of IgE-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of FcepsilonRII (CD23), the low-affinity receptor for IgE, demonstrating the structural integrity of the mutated IgE-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of IgE-Fc or Fcepsilon3-4. These results demonstrate the importance of the N-terminal linker region of Cepsilon3 in the interaction of IgE with FcepsilonRI.

Previous investigators have reported that deletion of the protein tyrosine kinase Lyn alters mast cell (MC) signaling responses but does not affect or reduces the cytokine-mediated proliferation of mouse bone marrow-derived MC (BMMC) precursors and of mature MC. We observed that Lyn-deficient mice have more peritoneal MC than wild-type (WT) mice. Studies to explore this unexpected result showed that Lyn(-/-) BM cells expand faster than WT cells in response to interleukin (IL)-3 and stem-cell factor over the 4-5 weeks required to produce a >95% pure population of granular, receptor with high affinity for immunoglobulin E-positive BMMC. Furthermore, differentiated Lyn(-/-) BMMC continue to proliferate more rapidly than WT BMMC and undergo less apoptosis in response to cytokine withdrawal. Additionally, Lyn(-/-) BMMC support greater IL-3-mediated phosphorylation of the prosurvival kinase, Akt, and the proliferative kinase, extracellular-regulated kinase 1/2. These results identify Lyn as a negative regulator of murine MC survival and proliferation.