Odile Malbec

The same tyrosine-based inhibition motif, in the intra-cytoplasmic domain of FcγRIIB, regulates negatively BCR-, TCR-, and FcR-dependent cell activation

The cell-triggering properties of BCR, TCR and FcR depend on structurally related immunoreceptor tyrosine-based activation motifs (ITAMs). Fc gamma RIIB have no ITAM and do not trigger cell activation. When coaggregated to BCR, they inhibit B cell activation. We show here that, when coaggregated to these receptors, Fc gamma RIIB inhibit Fc epsilon RI-, Fc gamma RIIA-, and TCR-dependent cell activation. Inhibition also affected cell activation by single ITAMs, in isolated FcR or TCR subunits. The same tyrosine-based inhibitory motif (ITIM), which is highly conserved in murine and human Fc gamma RIIB and that was previously shown to inhibit BCR-dependent B cell activation, was required to regulate TCR- and FcR-dependent cell activation. Our findings endow Fc gamma RIIB, and thus IgG antibodies, with general immunoregulatory properties susceptible to act on all ITAM-containing receptors.

Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated FcγRIIB during negative regulation of IgE-dependent mouse mast cell activation☆

We demonstrated previously that the low-affinity IgG receptors Fc gammaRIIB, which are coexpressed with the high-affinity IgE receptors Fc epsilonRI in mouse mast cells, can inhibit IgE-induced release of inflammatory mediators and cytokines by these cells. Inhibition was found to require the coaggregation of the two receptors and to depend on the presence of a tyrosine-based inhibition motif (ITIM) in the intracytoplasmic domain of Fc gammaRIIB. We report here that the coaggregation with Fc gammaRIIB does not prevent Fc epsilonRI from triggering activation signals in BMMC and induces the tyrosine phosphorylation of Fc gammaRIIB. Phosphorylated ITIM peptides bound in vitro to three SH2 domain-containing phosphatases present in BMMC lysates: the phosphotyrosine phosphatases SHP-1 and SHP-2. and the inositolphosphate phosphatase SHIP. Using BMMC generated from the SHP-1-deficient motheaten mice, SHP-1 was found to be dispensable for inhibition of mast cell activation. When analyzed for in vivo association, SHIP coprecipitated with phosphorylated Fc gammaRIIB, whereas SHP-1 or SHP-2 did not. These observations altogether indicate that Fc epsilonRI actively participates in its own regulation and that the mechanisms by which Fc gammaRIIB inhibit cell activation might be different in mast cells and in B-cells.

Peritoneal Cell-Derived Mast Cells: An In Vitro Model of Mature Serosal-Type Mouse Mast Cells

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcγRIIIA and were negatively regulated by FcγRIIB. We found that a moderate FcγRIIB-dependent negative regulation, due not to a higher FcγRIIIA/FcγRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.

The mast cell IgG receptors and their roles in tissue inflammation

Summary:  Mast cells are effector cells of the innate immune system, but because they express Fc receptors (FcRs), they can be engaged in adaptive immunity by antibodies. Mast cell FcRs include immunoglobulin E (IgE) and IgG receptors and, among these, activating and inhibitory receptors. The engagement of mast cell IgG receptors by immune complexes may or may not trigger cell activation, depending on the type of mast cell. The coengagement of IgG and IgE receptors results in inhibition of mast cell activation. The Src homology‐2 domain‐containing inositol 5‐phosphatase‐1 is a major effector of negative regulation. Biological responses of mast cells depend on the balance between positive and negative signals that are generated in FcR complexes. The contribution of human mast cell IgG receptors in allergies remains to be clarified. Increasing evidence indicates that mast cells play critical roles in IgG‐dependent tissue‐specific autoimmune diseases. Convincing evidence was obtained in murine models of multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, and glomerulonephritis. In these models, the intensity of lesions depended on the relative engagement of activating and inhibitory IgG receptors. In vitro models of mature tissue‐specific murine mast cells are needed to investigate the roles of mast cells in these diseases. One such model unraveled unique differentiation/maturation‐dependent biological responses of serosal‐type mast cells.

Signal Regulatory Proteins Negatively Regulate Immunoreceptor-dependent Cell Activation

Signal regulatory proteins of the α subtype (SIRPα) are ubiquitous molecules of the immunoglobulin superfamily that negatively regulate protein tyrosine kinase receptor-dependent cell proliferation. Their intracytoplasmic domain contains four motifs that resemble immunoreceptor tyrosine-based inhibition motifs (ITIMs) and that, when tyrosyl-phosphorylated, recruit cytoplasmic SH2 domain-bearing protein tyrosine phosphatases (SHPs). ITIMs are borne by molecules that negatively regulate cell activation induced by receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs). Because SIRPα are coexpressed with ITAM-bearing receptors in hematopoietic cells, we investigated whether SIRPα could negatively regulate ITAM-dependent cell activation. We found SIRPα transcripts in human mast cells, and we show that a chimeric molecule having the transmembrane and intracytoplasmic domains of SIRPα could inhibit IgE-induced mediator secretion and cytokine synthesis by mast cells. Inhibition required that the SIRPα chimera was coaggregated with ITAM-bearing high affinity IgE receptors (FcεRI). It was correlated with the tyrosyl phosphorylation of the SIRPα chimera and the recruitment of SHP-1 and SHP-2. The phosphorylation of FcεRI ITAMs was decreased; the mobilization of intracellular Ca2+and the influx of extracellular Ca2+ were reduced, and the activation of the mitogen-activated protein kinases Erk1 and Erk2 was abolished. SIRPα can therefore negatively regulate not only receptor tyrosine kinase-dependent cell proliferation but also ITAM-dependent cell activation.

Linker for activation of T cells integrates positive and negative signaling in mast cells

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcεRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like αβ or γδ T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Negative regulation of hematopoietic cell activation and proliferation by FcγRIIB

FcγRIIB are single-chain, low-affinity receptors for the Fc portion of IgG which belong to the immunoglobulin superfamily (Daeron 1997a). They bind IgG antibodies either complexed to multivalent soluble antigens or bound to cell membrane antigens. The FcγRIIB gene possesses three exons that encode the intracytoplasmic domain. By alternative splicing, it generates two (in humans) or three (in mice) isoforms of membrane FcγRIIB that differ only in their intracytoplasmic sequences (Ravetch et al. 1986; Hibbs et al. 1986; Lewis et al. 1986;Brooks et al. 1989; Latour et al. 1996). FcγRIIB are involved in the internalization of IgG immune complexes; and FcγRIIB isoforms were shown to differentially mediate the phagocytosis of particulate immune complexes and/or the endocytosis of soluble immune complexes (Daeron et al. 1993). No FcγRIIB isoform is able to trigger cell activation (Daeron et al. 1992). Instead, all isoforms were found to negatively regulate cell activation.

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation.

OBJECTIVE Mast cells are tissue-resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis. METHODS In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR(-/-) and control mast cells were engrafted into mast cell-deficient WBB6 F1-Kit(w) /Kit(W-v) (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR(-/-) animals and in murine and human mast cell cultures. RESULTS Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR(-/-) mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR(-/-) mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin-8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR(-/-) mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis. CONCLUSION Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a-modulated expression of FcγR.

A novel druglike spleen tyrosine kinase binder prevents anaphylactic shock when administered orally

BACKGROUND The spleen tyrosine kinase (Syk) is recognized as a potential pharmaceutical target for the treatment of type I hypersensitivity reactions including allergic rhinitis, urticaria, asthma, and anaphylaxis because of its critical position upstream of immunoreceptor signaling complexes that regulate inflammatory responses in leukocytes. OBJECTIVE Our aim was to improve the selectivity of anti-Syk therapies by impeding the interaction of Syk with its cellular partners, instead of targeting its catalytic site. METHODS We have previously studied the inhibitory effects of the anti-Syk intracellular antibody G4G11 on Fc epsilonRI-induced release of allergic mediators. A compound collection was screened by using an antibody displacement assay to identify functional mimics of G4G11 that act as potential inhibitors of the allergic response. The effects of the selected druglike compounds on mast cell activation were evaluated in vitro and in vivo. RESULTS We discovered compound 13, a small molecule that inhibits Fc epsilonRI-induced mast cell degranulation in vitro and anaphylactic shock in vivo. Importantly, compound 13 was efficient when administered orally to mice. Structural analysis, docking, and site-directed mutagenesis allowed us to identify the binding cavity of this compound, located at the interface between the 2 Src homology 2 domains and the interdomain A of Syk. CONCLUSION We have isolated a new class of druglike compounds that modulate the interaction of Syk with some of its macromolecular substrates implicated in the degranulation pathway in mast cells.

The Engagement of Activating FcγRs Inhibits Primate Lentivirus Replication in Human Macrophages

We previously reported that the stimulation of monocyte-derived macrophages (MDM) by plate-bound i.v. Igs inhibits HIV-1 replication. In this study, we show that IgG immune complexes also suppress HIV-1 replication in MDMs and that activating receptors for the Fc portion of IgG–FcγRI, FcγRIIA, and FcγRIII–are responsible for the inhibition. MDM stimulation through FcγRs induces activation signals and the secretion of HIV-1 modulatory cytokines, such as M-CSF, TNF-α, and macrophage-derived chemokine. However, none of these cytokines contribute to HIV-1 suppression. HIV-1 entry and postintegration steps of viral replication are not affected, whereas reduced levels of reverse transcription products and of integrated proviruses, as determined by real-time PCR analysis, account for the suppression of HIV-1 gene expression in FcγR-activated MDMs. We found that FcγR-dependent activation of MDMs also inhibits the replication of HIV-2, SIVmac, and SIVagm, suggesting a common control mechanism for primate immunodeficiency lentiviruses in activated macrophages.