Marie-Hélène Jouvin

A Second Amplifier Function for the Allergy-Associated FcεRI-β Subunit

Genetics studies have identified the gene for the high-affinity IgE receptor (FC(epsilon)RI) beta subunit as a candidate gene for atopy. We have shown that beta is an intrinsic signaling amplifier leading to enhanced allergic responses in vivo. Here we report that beta has a second amplification function: the amplification of Fc(epsilon)RI cell surface expression. This function is due to an early association of beta with alpha, resulting in improved trafficking and maturation of alpha and receptor complexes. These data provide a possible molecular explanation for the large difference in Fc(epsilon)RI density between beta-cells such as monocytes, dendritic cells, and beta+ effector cells (mast cells, basophils). In beta+ cells, the combined signaling and expression amplification results in an estimated 12- to 30-fold amplification of downstream events.

Minimal Requirements for IgE-Mediated Regulation of Surface FcεRI

The IgE-FcεRI network plays a central role in allergic inflammation. IgE levels control cell surface levels of FcεRI and, in turn, FcεRI levels modulate the intensity of effector responses. Treatment of allergic patients with anti-IgE Abs has been shown to induce a decrease in FcεRI expression on basophils and a decrease in Ag-triggered histamine release. However, the mechanisms underlying IgE-mediated regulation of FcεRI expression remain unclear. Here, we designed an in vitro model system to establish the minimal cellular requirements for regulation of FcεRI by IgE. Using this system, we demonstrate that transcriptional regulation, hemopoietic-specific factors, and signaling are not required for IgE-mediated increases in FcεRI expression. IgE binding to the α-chain is the minimal requirement for the induction of FcεRI up-regulation. The rate of up-regulation is independent of the baseline level of expression. The mechanism of this up-regulation is the result of a combination of three factors: 1) stabilization of the receptor at the cell surface, which prevents receptor internalization and degradation; 2) use of a preformed pool of receptor comprising recycled and recently synthesized receptors; and 3) continued basal level of protein synthesis. It is possible that in vivo additional factors contribute to modulate the basic regulatory mechanism described here.

The Role of the FcεRI β-Chain in Allergic Diseases

The high affinity receptor for IgE, FcΕRI, is a multimeric surface receptor that is expressed exclusively as a tetramer on rodent cells, but exists as a tetramer or trimer on human cells. The tetrameric form is expressed on effector cells of allergic responses such as mast cells and basophils and is composed of an IgE-binding α-subunit, a β-subunit and a γ-subunit dimer. Complexes lacking the β-subunit are found on human antigen-presenting cells. On mast cells and basophils, FcΕRI is essential for IgE-mediated acute allergic reactions. Crosslinking of FcΕRI by IgE and multivalent antigen induces a signaling cascade that culminates in the release of preformed mediators and the synthesis of lipid mediators and cytokines. The β-subunit functions as an amplifier of FcΕRI expression and signaling. As a consequence, strongly enhanced mast cell effector functions and in vivo allergic reactions can be observed in the presence of FcΕRIβ. In contrast, a truncated β-isoform (βT) that is produced by alternative splicing acts as an inhibitor of FcΕRI surface expression. Thus, by producing two proteins with antagonistic functions, the FcΕRIβ gene could serve as a potent regulator of allergic responses. In addition, the genomic region encompassing the β-chain has been linked to atopy and a number of polymorphisms within the FcΕRIβ gene are associated with various atopic diseases. It remains to be elucidated how these polymorphisms might affect the allergic phenotype. These functions of the β-chain together with the described genetic linkages to atopy make it a candidate for a role in the pathophysiology of allergic diseases.

Competing Functions Encoded in the Allergy-Associated FcϵRIβ Gene

Allergic reactions are triggered via crosslinking of the high-affinity receptor for immunoglobulin E, F(c)epsilonRI. In humans, F(c)epsilonRI is expressed as a tetramer (alphabetagamma(2)) and a trimer (alphagamma(2)). The beta subunit is an amplifier of F(c)epsilonRI surface expression and signaling. Here, we show that as a consequence of alternative splicing, the F(c)epsilonRIbeta gene encodes two proteins with opposing and competing functions. One isoform is the full-length classical beta, the other a novel truncated form, beta(T). In contrast to beta, beta(T) prevents F(c)epsilonRI surface expression by inhibiting alpha chain maturation. Moreover, beta(T) competes with beta to control F(c)epsilonRI surface expression in vitro. We propose that the relative abundance of the products of the beta gene may control the level of F(c)epsilonRI surface expression and thereby influence susceptibility to allergic diseases.

Human anti-FcεRIα autoantibodies isolated from healthy donors cross-react with tetanus toxoid

Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto‐Ab) can be either pathogenic or non‐pathogenic. Auto‐Ab reacting with the α‐subunit of the high‐affinity receptor for IgE (FcϵRIα) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti‐FcϵRIα auto‐Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity‐purified the anti‐FcϵRIα Ab from an IVIg preparation using recombinant FcϵRIα. Interestingly, these anti‐FcϵRIα auto‐Ab showed no evidence of histamine release but strongly cross‐reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcϵRIα. Since the cross‐reacting Ab are non‐anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti‐FcϵRIα auto‐Ab belong to the natural Ab and serve as the parental Ab for some anti‐TTd Ab.

Allergy-associated polymorphisms of the FcεRIβ subunit do not impact its two amplification functions

Two variants of the β-chain of the high affinity IgE receptor FcεRI, I181L-V183L and E237G, have been found associated with allergy. We have previously shown that the β-chain plays at least two distinct amplifier functions. It amplifies FcεRI surface expression and signaling, resulting in an estimated 12- to 30-fold amplification of downstream events. To test the hypothesis that the I181L-V183L and E237G β variants may be functionally relevant and could directly contribute to an allergic phenotype, we have evaluated the functional impact of the β variants on the two amplifier functions of β. We found that these variants have no direct effect on the β amplifier functions. However, the possibility remains that these variants are in linkage disequilibrium with other more relevant polymorphisms or are affecting unknown β-chain functions.

The TRPM4 Channel Controls Monocyte and Macrophage, but Not Neutrophil, Function for Survival in Sepsis

A favorable outcome following acute bacterial infection depends on the ability of phagocytic cells to be recruited and properly activated within injured tissues. Calcium (Ca2+) is a ubiquitous second messenger implicated in the functions of many cells, but the mechanisms involved in the regulation of Ca2+ mobilization in hematopoietic cells are largely unknown. The monovalent cation channel transient receptor potential melastatin (TRPM) 4 is involved in the control of Ca2+ signaling in some hematopoietic cell types, but the role of this channel in phagocytes and its relevance in the control of inflammation remain unexplored. In this study, we report that the ablation of the Trpm4 gene dramatically increased mouse mortality in a model of sepsis induced by cecal ligation and puncture. The lack of the TRPM4 channel affected macrophage population within bacteria-infected peritoneal cavities and increased the systemic level of Ly6C+ monocytes and proinflammatory cytokine production. Impaired Ca2+ mobilization in Trpm4−/− macrophages downregulated the AKT signaling pathway and the subsequent phagocytic activity, resulting in bacterial overgrowth and translocation to the bloodstream. In contrast, no alteration in the distribution, function, or Ca2+ mobilization of Trpm4−/− neutrophils was observed, indicating that the mechanism controlling Ca2+ signaling differs among phagocytes. Our results thus show that the tight control of Ca2+ influx by the TRPM4 channel is critical for the proper functioning of monocytes/macrophages and the efficiency of the subsequent response to infection.

The Tetraspanin CD63 Is Required for Efficient IgE-Mediated Mast Cell Degranulation and Anaphylaxis

Mast cell (MC) activation through the high-affinity IgE receptor FcεRI leads to the release of mediators involved in immediate-type allergic reactions. Although Abs against the tetraspanins CD63 and CD81 inhibit FcεRI-induced MC degranulation, the intrinsic role of these molecules in FcεRI-induced MC activation is unknown. In MCs, CD63 is expressed at the cell surface and in lysosomes (particularly secretory lysosomes that contain allergic mediators). In this study, we investigated the role of CD63 in MC using a CD63 knockout mouse model. CD63-deficiency did not affect in vivo MC numbers and tissue distribution. Bone marrow–derived MC developed normally in the absence of CD63 protein. However, CD63-deficient bone marrow–derived MC showed a significant decrease in FcεRI-mediated degranulation, but not PMA/ionomycin-induced degranulation, as shown by β-hexosaminidase release assays. The secretion of TNF-α, which is both released from granules and synthesized de novo upon MC activation, was also decreased. IL-6 secretion and production of the lipid mediator leukotriene C4 were unaffected. There were no ultrastructural differences in granule content and morphology, late endosomal/lysosomal marker expression, FcεRI-induced global tyrosine phosphorylation, and Akt phosphorylation. Finally, local reconstitution in genetically MC-deficient Kitw/w-v mice was unaffected by the absence of CD63. However, the sites reconstituted with CD63-deficient MC developed significantly attenuated cutaneous anaphylactic reactions. These findings demonstrate that the absence of CD63 results in a significant decrease of MC degranulation, which translates into a reduction of acute allergic reactions in vivo, thus identifying CD63 as an important component of allergic inflammation.

Role of the extracellular domain of FcɛRIα in intracellular processing and surface expression of the high affinity receptor for IgE FcɛRI

The high affinity receptor for immunoglobulin E, Fc epsilon RI, is a critical component of IgE-mediated allergic reactions. It is expressed as a tetramer (alphabetagamma(2)) made of an IgE-binding alpha chain and a signaling module formed by the beta chain and a dimer of gamma chains. It is expressed in humans and rodents on basophils and mast cells at a high level, and, upon activation, it induces the liberation of allergy mediators. In humans a trimeric form lacking the beta chain also exists (alphagamma(2)). This trimeric form is expressed on antigen presenting cells where it acts to facilitate antigen presentation via IgE. Both the expression and the signaling capacity of the trimer are lower than those of the tetramer. The differences between human (tetrameric and trimeric) and murine (tetrameric only) expression is explained in part by the fact that mouse alpha cannot be expressed at the cell surface in the absence of beta, while human alpha can. Here we demonstrate that the capacity of human alpha to be expressed at the cell surface in the absence of beta is encoded entirely in its extracellular domain. These findings show that the extracellular domain of the type I transmembrane protein Fc epsilon RI alpha plays a role in Fc epsilon RI intracellular processing and expression at the cell surface.