Unit: Molecular and Cellular Allergology
Director: Daëron, Marc
Cell activation results from the transient displacement of a balance between positive and negative signals delivered by activating and inhibitory receptors. Both types of receptors are co-expressed on cells involved in allergic reactions. Signals are integrated by transmembrane adapter molecules which organize intracellular signaling complexes. Our working hypothesis is that allergic diseases result from defects in the mechanisms that maintain this balance and prevent the outcome of clinical symtoms in normal individuals. During the last year, we have focused our work on negative signaling generated by FcγRIIB, the low-affinity IgG receptors which we demonstrated previously that they can inhibit cell activation, and by two transmembrane adapters, LAT and NTAL. We found 1) that the intracytoplasmic domain of FcγRIIB contains two tyrosine-based motifs which cooperate to recruit SHIP1, the phosphatase that is responsible for inhibition; 2) that inhibition depends on the F-actin skeleton where SHIP1 is located and to which FcγRIIB associate upon engagement ; 3) that LAT integrates not only positive but also negative signals generated by high-affinity IgE receptors in mast cells; and 4) that NTAL functions as an inhibitor of mast cell activation. Our projects include: 1) further in vitro analyses of the intracellular molecular interactions that regulate cell activation; 2) an in vivo study of interactions between human FcRs in murine allergy models using "humanized" mice; 3) an ex vivo exploration of negative regulation of cell activation in allergic patients.
The activation, proliferation and differentiation of cells involved in the immune response are under the control of membrane receptors that trigger positive and negative signals. These signals are integrated through the sequential interactions of intracellular molecules recruited within signaling complexes that build up underneath co-engaged receptors and whose composition varies with time. As a consequence, the immune response is tightly controlled and, although potentially deleterious, immune effectors are non pathogenic in normal individuals. The research project of the unité lies on the hypothesis that allergic diseases can arise as a consequence of defects - that might possibly affect any step during the allergic reaction - in negative regulation that prevents the outcome of allergic manifestations in normal individuals. It will aim at evaluating the possible contribution of negative regulation defects in allergic diseases, and at characterizing the putative defects in order to correct them and/or to utilize negative regulation as a new therapeutic tool in allergies.
We demonstrated previously that FcγRIIB, a family of receptors for the Fc portion (FcRs) of IgG antibodies expressed by most cells of hematopoietic origin, can inhibit activation and proliferation signals triggered by immunoreceptors and growth factor receptors, respectively. Inhibition depends on an Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) that we first identified in the intracytoplasmic domain of murine and human FcγRIIB and that was subsequently found in a large number of other inhibitory receptors. When FcγRIIB are co-aggregated with activating receptors on the same cell by a common extracellular ligand, such as immune complexes, their ITIM is phopshorylated and recruits the phopshatidylinositol 5-phosphatase SHIP1 which accounts for inhibition. These results were confirmed in vivo using FcγRIIB-/- mice. These mice exhibited enhanced anaphylactic reactions and developed autoimmune diseases. During the last year, we further studied mechanisms by which FcγRIIB generate negative signals and we undertook an analysis of mechanisms by which positive and negative signals generated by high-affinity IgE receptors (FcεRI) are integrated in mast cells. These works led to the following results.
The FcγRIIB intracytoplasmic domain contains a second tyrosine-based inhibitory motif
We identified a second tyrosine-based motif, in the intracytoplasmic domain of murine FcγRIIB, and demonstrated its role in the recruitment of SHIP1. In order for this phosphatase to be recruited by FcγRIIB, the binding of its SH2 domain to the phosphorylated FcγRIIB ITIM indeed needs to be stabilized by Grb2 and/or Grap. These cytosolic adapter molecules bind both to this second motif, in FcγRIIB, via their SH2 domain and, via their C-terminal SH3 domain, to proline-rich sequences in the C-terminal region of SHIP1. These simultaneous interactions enable the formation of a stable tri-molecular complex (Isnardi et al., J. Biol. Chem. 2004).
The F-actin skeleton is critical for FcγRIIB-dependent negative regulation
We have found that, in mast cells, the recruitment of SHIP1 by murine FcγRIIB occurs in the cytoskeleton. Having observed that FcγRIIB and SHIP1 are located in different subcellular compartments in resting mast cells, we investigated where these molecules could meet when FcγRIIB are co-aggregated with FcεRI. We found that receptor complexes associate with the cytoskeleton where the high-molecular isoform of SHIP1 is constitutively bound to an actin-binding protein, filamin. SHIP1, subsequently dissociates from filamin, and remains bound onto FcγRIIB. This mechanism enables phosphorylated FcγRIIB to concentrate the phosphatase at proximity of signaling complexes that form underneath FcεRI with which they are co-aggregated (Lesourne et al., J. Immunol., 2005, in press).
LAT integrates positive and negative signals delivered into mast cells by FcεRI
LAT is a transmembrane adapter the intracytoplasmic domain of which contains 9 tyrosine residues. When phopshorylated, these tyrosines recruit and organize the signaling complex induced upon FcεRI engagement. We investigated the roles played by LAT tyrosines in mast cells derived from knock-in mice bearing point mutations of one, three or the four C-terminal LAT tyrosines and generated by Marie and Bernard Malissen at the Centre d'Immunologie de Marseille-Luminy. We could assign distinct roles to these tyrosines in mast cell activation. These tyrosines indeed differentially contribute to biological responses (release of granular mediators and cytokine secretion) and to signals leading to the generation of intracellular effctors of cell activation (intracellular Ca2+ and MAP kinases). Interestingly, we found that the dominant positive effects of LAT result from an integration of positive and negative signals that depend on distinct tyrosines and that differentially regulate the metabolic pathways leading to exocytosis and to cytokine secretion (Malbec et al., J. Immunol. 2004)
LAT and NTAL are a pair of antaogonistic molecules in mast cells
NTAL is another transmembrane adapter which closely resembles LAT. This molecule was proposed to play a role similar to the role played by LAT, in B cells and macrophages which do not express LAT. Interestingly, mast cells express both LAT and NTAL. Because we had observed, in the above study, that although markedly reduced, IgE-induced activation was not abolished in LAT-/- mast cells, we hypothesized that LAT and NTAl might have complementary roles in mast cells. We therefore examined mast cells derived from LAT-deficient, NTAL-deficient or LAT and NTAL-deficient mice, generated by Marie and Bernard Malissen. Surprisingly, we found that LAT and NTAL play antagonistic roles in mast cells. Whereas LAT functions as an amplifier, NTAL indeed functions as an inhibitor of IgE-induced mast cell activation. NTAL therefore appears as a novel inhibitory molecule of allergic reaction (Roget et al., article en preparation).
Our projects comprise three complementary approaches that will be conducted simultaneously: 1) an in vitro analysis that will further dissect the intracellular molecular interactions which negatively regulate cell activation, 2) an in vivo analysis of interactions between human Fc Receptors in models of allergies in mice in which all endogenous FcRs will have been replaced by human FcRs and 3) an ex vivo exploration of negative regulation of cell activation in allergic patients.
Keywords: Allergies, Immunoregulation, Antibodies, Fc Receptors, Transmembrane adapters, Intracellular signaling