Unit: Molecular and Cellular Allergology
Director: Daëron, Marc
Our works aims 1) at investigating the molecular mechanisms which regulate, positively and negatively, cell activation in allergies; 2) at assessing the respective roles of the various human FcRs and FcR-expressing cells in murine models of allergies and inflammatory diseases, in mice whose FcRs will have been replaced by the corresponding human FcRs; and 3) at searching whether negative regulation is or not deficient in allergic patients.
The activation of hematopoietic cells is 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. As a consequence, immune responses are tightly controlled. We demonstrated previously that FcγRIIB, a family of receptors for the Fc portion (FcRs) of IgG antibodies, can inhibit activation signals triggered by immunoreceptors such as high-affinity IgE Receptors (FcεRI) expressed by mast cells and basophils. 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 phosphorylated and recruits the phosphatidylinositol 5-phosphatase SHIP1 which accounts for inhibition. These results were confirmed in vivo using FcγRIIB-/- and SHIP1-/- mice.
The research project of the unit lies on the hypothesis that allergic diseases can arise as a consequence of defects of negative regulation that prevents the outcome of allergic manifestations in normal individuals and/or of their integration. It aims at evaluating the possible contribution of negative regulation defects in allergic diseases, and at characterizing putative defects in order to correct them and/or to utilize negative regulation as a new therapeutic tool in allergies. Our work focuses on FcγRIIB-dependent negative regulation of activating FcR-dependent activation of the effector cells of allergy. This project comprises three simultaneous approaches: an in vitro analysis of the molecular interactions which negatively regulate cell activation, an in vivo analysis of interactions between human Fc Receptors in models of allergies in mice whose FcRs will have been "humanized", and an ex vivo exploration of negative regulation of cell activation in allergic patients. The main results obtained in 2005 are as follows.
Human FcγRIIB also recruit SHIP1, but not through the same mechanism as murine FcγRIIB
We showed previously that, when recruited to the ITIM of murine FcγRIIB, SHIP1 needs to be stabilized through an interaction with the cytosolic adapter Grb2, which is itself recruited to a second tyrosine-based motif (Isnardi et al. J. Biol. Chem. 2004). Human FcγRIIB lack this motif. We showed that they nevertheless recruit SHIP1, whose recruitment is stabilized by another mechanism that depends on C-terminal sequences of the human receptor (Isnardi et al., Immunol. Letters, in press).
The actin cytoskeleton, an inhibitory compartment for activation signals in mast cells
We demonstrated that, in mast cells, the recruitment of SHIP1 by FcγRIIB occurs in the actin cytoskeleton which functions as an inhibitory compartment. When FcεRI are aggregated or coaggregated with FcγRIIB, FcR aggregates associate with the cytoskeleton where the high molecular-weight isoform of SHIP1 is constitutively bound to the actin-binding protein filamin. Filamin then releases SHIP1 which remains bound to the phosphorylated ITIM of FcγRIIB. FcγRIIB thus concentrate the inhibitory phosphatase close to its substrate, in signaling complexes generated by FcεRI with which they are coaggregated (Lesourne et al. J. Immunol. 2005).
Integration of positive and negative signals by the transmembrane adapters LAT and NTAL
The transmembrane adapters LAT and NTAL are coexpressed in mast cells. Their intracytoplasmic domain contains numerous tyrosines which, when phosphorylated, concur to the organization of signaling complexes underneath aggregated FcεRI. A collaborative work with Marie and Bernard Malissen demonstrated that the dominant positive effects of LAT result from an integration of positive and negative signals which depend on distinct tyrosines (Malbec et al. J. Immunol. 2004). We also observed that NTAL has a dominant negative effect on FcεRI signaling in mast cells. Using mice deficient for one, the other or both adapters, we are analyzing the interactions between LAT and NTAL, aiming at elucidating how they these adapters generate positive and negative signals and how these signals can modulate allergic reactions.
A new model of cultured murine primary mast cells
Bone Marrow-derived Mast Cells (BMMCs) are the only available model of cultured murine primary mast cells. Their main disadvantage is that they have no physiological in vivo equivalent. We established the conditions which enable an in vitro expansion of differentiated mast cells. Under these conditions a homogeneous population of mature serosal-type mast cells can be obtained within a few weeks. These cells closely resemble peritoneal mast cells. Biological responses induced by and transduction pathways used by FcRs are not the same in these cells as in BMMCs.
A new murine receptor for IgG
We identified a new FcR, FcγRIV, encoded by a gene which maps between the genes encoding FcγRIIB and FcγRIII on mouse chromosome 1. This receptor has two glycosylated extracellular domains, a transmembrane domain and an intracytoplasmic domain which contains no activation or inhibition motif. It exists in mice only, but it is highly homologous to human FcγRIIIA. In order to be expressed at the membrane, it requires to associate with the activation motif-containing common subunit FcRγ and, when aggregated by immune complexes, it triggers activation signals. It is expressed by monocytes/macrophages and neutrophils. Using immunofluorescence on transfected cells and, in collaboration with P. England, by surface plasmon resonance on a Biacore, we showed that FcγRIV binds, with an intermediate affinity, mouse IgG2 a and IgG2b, but not IgG1 or IgG3. We demonstrated an in vivo role of FcγRIV in a model of autoimmune thrombocytopenia. When injected in normal mice, anti-platelet antibodies induce a loss of 85% blood platelets. We observed that an injection of anti-FcγRIV blocking antibodies prior to anti-platelet antibodies decreases thrombocytopenia by 60%, whereas the deletion of either one of the two other activating FcγRs (FcγRI or FcγRIII) has no effect (Bruhns et al. Immunity 2005).
In vivo roles of human FcRs in murine models of allergies in "humanized" mice
We learnt much of FcR physiology from FcR-KO mice. Due to significant differences between murine and human FcRs, results obtained in mice cannot be simply applied to humans. We have therefore undertaken the project of constructing mice whose FcRs will have been replaced by human FcRs. To express human FcRs in mice, we will use Bacterial Artificial Chromosomes (BACs) containing fragments of human chromosomes in which genes encoding human FcRs, such as, for instance, FcγRIIA, are located. When the FcγRIIA gene is isolated from other DNA sequences of the BAC, it is injected into C57BL/6 mice pronuclei. This will be done in collaboration with the Transgenese platform of the Institute directed by F. Langa-Vives. The FcγRIIATg mice thus obtained will be crossed with FcγRI/II/III-triple deficient mice (generated by S. Verbeek in the Netherlands), in order to study the role of FcγRIIA, in the absence of any endogenous FcγRs. The same strategy will be applied to other human FcγRs and FcεRs, in order to obtain mice in which all the FcRs for IgG and IgE will have been "humanized". These mice will then be injected with human IgE and IgE antibodies, and we will examine the respective roles of these receptors and of cells on which they are expressed in experimental models of inflammatory and allergic diseases.
Expression and function of FcRs on human basophils
FcRs expressed by effector cells of allergy are not well known. We have therefore started to identify these receptors, and especially, to determine the relative expression of activating and inhibitory FcRs on blood basophils from normal donors and, in collaboration with M.-T. Guinnepain and J. Laurent, at the Medical Center of the Institute, from patients suffering from various allergic diseases.
Keywords: Allergies, Immunoregulation, Antibodies, Fc Receptors, Transmembrane adapters, Intracellular signaling