Search

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli., Cell polarity is marked by re-orientation of the centrosome, but the mechanisms governing centrosome polarization are poorly understood. Here Obino et al. show that in lymphocytes centrosome-associated Arp2/3 nucleates actin that tethers the centrosome to the nucleus; activation depletes Arp2/3 from the centrosome and frees it from the nucleus.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

Allergic and autoimmune inflammation are associated with the activation of mast cells and basophils by antibodies against allergens or auto-antigens, respectively. Both cell types express several receptors for the Fc portion of antibodies, the engagement of which by antigen-antibody complexes controls their responses. When aggregated on the plasma membrane, high-affinity immunoglobulin E (IgE) receptors (FceRI) and low-affinity IgG receptors (FcγRIIIA in mice, FcγRIIA in humans) induce these cells to release and secrete proinflammatory mediators, chemokines, and cytokines that account for clinical symptoms. When coaggregated with activating receptors on the same cells, other low-affinity IgG receptors (FcγRIIB in both species) inhibit mast cell and basophil activation. We found that FcγRIIB inhibited not only signals triggered by activating receptors with which they were coengaged (cis-inhibition), but also signals triggered by receptors engaged independently (trans-inhibition). Trans-inhibition acted upon the FceRI-dependent activation of mouse mast cells, mouse basophils, and human basophils, and upon growth factor receptor (Kit)–dependent normal mouse mast cell proliferation, as well as the constitutive in vitro proliferation and the in vivo growth of oncogene (v-Abl)–transformed mastocytoma cells. Trans-inhibition was induced by receptors, whether inhibitory (FcγRIIB) or activating (FceRI), which recruited the lipid phosphatase SHIP1. By hydrolyzing PI(3,4,5)P 3 , SHIP1 induced a global unresponsiveness that affected biological responses triggered by receptors that use phosphoinositide 3-kinase to signal. These data suggest that trans-inhibition controls numerous physiological and pathological processes, and that it may be used as a therapeutic tool in inflammation, especially but not exclusively, in allergy and autoimmunity.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

When dimerized by Stem Cell Factor (SCF), the Receptor Tyrosine Kinase Kit triggers the proliferation of hematopoietic progenitors, including pro-B cells, and of some differentiated cells, including mast cells. We found previously that anti-Kit antibodies can mimic SCF and that anti-Kit-induced mast cell proliferation can be inhibited by the low-affinity IgG receptors FcγRIIB, when the two receptors are co-aggregated by IgG immune complexes. We show here that the same immune complexes inhibited anti-Kit-induced proliferation of Ba/F3 pro-B cells expressing wt Kit and FcγRIIB and that inhibition required the intracytoplasmic domain of FcγRIIB. Constitutively active Kit mutants are oncogenic. We show that Kit-dependent, ligand-independent proliferation of Ba/F3 cells expressing a constitutively dimerized Kit mutant was also inhibited by IgG immune complexes via FcγRIIB. FcγRIIB-dependent negative regulation therefore also affects Kit-dependent proliferation of transformed cells. Interestingly, the co-aggregation of Kit with FcγRIIB by immune complexes containing SCF also inhibited both growth factor-dependent and growth factor-independent proliferation of Ba/F3 cells expressing wt or mutated Kit, respectively. These results provide the basis for novel immunotherapeutical approaches of FcγRIIB-expressing tumors.

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.

Ancestral polarity protein Par3 is crucial for the polarization of B-cells toward a tethered antigen. This process is needed by these lymphocytes to uptake and process the antigen to mount the adaptive immune response., B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.

FcgammaRIIB are single-chain low-affinity receptors for the Fc portion of IgG antibodies that are widely expressed by hematopoietic cells including mast cells. We previously demonstrated that FcgammaRIIB negatively regulate cell activation triggered by receptors that possess Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) including high-affinity IgE receptors (FcepsilonRI). FcgammaRIIB possess an Immunoreceptor Tyrosine-based Inhibition Motif (ITAM) whose deletion or mutation abolishes inhibition. When coaggregated with FcepsilonRI, the FcgammaRIIB ITIM is tyrosyl-phosphorylated by the src family protein tyrosine kinase lyn, and recruits the SH2 domain-containing inositol 5-phosphatase SHIP that accounts for inhibition of cell activation. We found recently that, when coaggregated with Kit, FcgammaRIIB can also inhibit mast cell proliferation: thymidine incorporation is inhibited, cells do not enter the G1 phase of the cell cycle, the induction of cyclins D2, D3 and A is inhibited, the activation of the MAP kinases Erk1/2, JNK and p38 is decreased, Akt phosphorylation is inhibited, and SHIP coprecipitates with FcgammaRIIB. Although inhibition of Akt phosphorylation and Erk activation was abrogated in SHIP(-/-) cells, inhibition of thymidine incorporation was only partially reduced. FcgammaRIIB-dependent inhibition of Kit-mediated mast cell proliferation was however mimicked by FcgammaRIIB whose intracytoplasmic domain was replaced by the catalytic domain of SHIP. We also found that FcgammaRIIB can inhibit the proliferation of cells whose proliferation was rendered growth factor-independent because they express a mutated form of Kit that renders this RTK constitutively activated. Based on these results we developed models aiming at using FcgammaRIIB as targets for new therapeutic approaches of disease associated with mast cell activation such as allergies and diseases associated with mast cell proliferation such as mastocytosis, mastocytomas or mast cell leukemias.

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.

Allergic symptoms result from the release of granular and lipidic mediators and of cytokines by inflammatory cells. The whole process is initiated by the aggregation of mast cell and basophil high-affinity IgE receptors (Fc epsilon RI) by IgE and antigen. We report here that IgE-induced release of mediator and cytokine can be inhibited by cross-linking Fc epsilon RI to low-affinity IgG receptors (Fc gamma RII) which are constitutively expressed on mast cells and basophils. Using a model of stable transfectants in RBL-2H3 cells expressing endogeneous rat Fc epsilon RI and recombinant murine Fc gamma RII, we showed that inhibition requires that Fc epsilon RI be crosslinked to Fc gamma RII by the same multivalent ligand. Inhibition of cross-linked receptors left non-cross-linked Fc epsilon RI capable of triggering mediator release and was reversible upon disengagement. Both isoforms of wild-type Fc gamma RII were equally capable of inhibiting Fc epsilon RI-mediated mast cell activation provided they had an intact intracytoplasmic domain. Our results demonstrate that mast cell secretory responses triggered by high-affinity receptors for IgE may be controlled by low-affinity receptors for IgG. This regulation of Fc epsilon RI-mediated mast cell activation is of potential interest in mast cell physiology and in allergic pathology.

Some nonpathogenic bacteria were found to have protective effects in mouse models of allergic and autoimmune diseases. These “probiotics” are thought to interact with dendritic cells during Ag presentation, at the initiation of adaptive immune responses. Many other myeloid cells are the effector cells of immune responses. They are responsible for inflammation that accounts for symptoms in allergic and autoimmune diseases. We investigated in this study whether probiotics might affect allergic and autoimmune inflammation by acting at the effector phase of adaptive immune responses. The effects of one strain of Lactobacillus casei were investigated in vivo on IgE-induced passive systemic anaphylaxis and IgG-induced passive arthritis, two murine models of acute allergic and autoimmune inflammation, respectively, which bypass the induction phase of immune responses, in vitro on IgE- and IgG-induced mouse mast cell activation and ex vivo on IgE-dependent human basophil activation. L. casei protected from anaphylaxis and arthritis, and inhibited mouse mast cell and human basophil activation. Inhibition required contact between mast cells and bacteria, was reversible, and selectively affected the Lyn/Syk/linker for activation of T cells pathway induced on engagement of IgE receptors, leading to decreased MAPK activation, Ca2+ mobilization, degranulation, and cytokine secretion. Also, adoptive anaphylaxis induced on Ag challenge in mice injected with IgE-sensitized mast cells was abrogated in mice injected with IgE-sensitized mast cells exposed to bacteria. These results demonstrate that probiotics can influence the effector phase of adaptive immunity in allergic and autoimmune diseases. They might, therefore, prevent inflammation in patients who have already synthesized specific IgE or autoantibodies.

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-Kitw/KitW-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 Fc? receptors (Fc?R) 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 Fc?R.

Mast cells are cells of the innate immune system whose biological responses are markedly modulated by effector molecules of adaptive immunity, i.e., antibodies. They thus contribute to anti-infectious defense but also to antibody-dependent inflammatory responses. They are especially well known as inducers of allergic reactions. They are widely distributed in most tissues, but in low numbers. They are not readily purified, and with a poor yield. For these reasons, means to generate large numbers of homogenous non-transformed mast cells have been developed. We describe here (1) fractionation methods suitable for purifying mouse or rat peritoneal mast cells and for purifying human mast cells of various origins, and (2) conditions for generating pure cultured mast cell populations from mouse, rat, and human tissues.

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.

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.

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.

International audience; We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.

The intracytoplasmic domain of FcγRIIB, a family of widely expressed IgG receptors, contains an Immunoreceptor Tyrosine-based Inhibition Motif (ITIM) that accounts for the ability of these receptors to negatively regulate cell activation and cell proliferation induced by receptors containing Immunoreceptor Tyrosine-based Activation Motif (ITAMs) and by receptors with an intrinsic protein tyrosine kinase activity. These properties lie in the affinity of the FcγRIIB ITIM for SH2 domain-containing inositol 5-phosphatases (SHIPs). FcγRIIB are the only known ITIM-bearing receptors capable of recruiting SHIPs in vivo. The present review discusses recent findings that established the molecular bases for the affinity of the FcγRIIB ITIM for SHIPs, that explained why SHIPs, rather than SH2 domain-containing protein tyrosine phosphatases, are recruited by FcγRIIB in vivo, that unraveled a dual mechanism used by FcγRIIB to negatively regulate B cell activation via SHIP1, and that demonstrated the role of SHIP1 in FcγRIIB-dependent negative regulation of mast cell proliferation induced by the growth factor receptor Kit.

International audience; Signal regulatory proteins of the alpha subtype (SIRPalpha) 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 SIRPalpha are coexpressed with ITAM-bearing receptors in hematopoietic cells, we investigated whether SIRPalpha could negatively regulate ITAM-dependent cell activation. We found SIRPalpha transcripts in human mast cells, and we show that a chimeric molecule having the transmembrane and intracytoplasmic domains of SIRPalpha could inhibit IgE-induced mediator secretion and cytokine synthesis by mast cells. Inhibition required that the SIRPalpha chimera was coaggregated with ITAM-bearing high affinity IgE receptors (FcepsilonRI). It was correlated with the tyrosyl phosphorylation of the SIRPalpha chimera and the recruitment of SHP-1 and SHP-2. The phosphorylation of FcepsilonRI ITAMs was decreased; the mobilization of intracellular Ca(2+) and the influx of extracellular Ca(2+) were reduced, and the activation of the mitogen-activated protein kinases Erk1 and Erk2 was abolished. SIRPalpha can therefore negatively regulate not only receptor tyrosine kinase-dependent cell proliferation but also ITAM-dependent cell activation.

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.

Crosslinking of the B-cell receptor (BCR) for antigen to low-affinity receptors for IgG (Fc gamma RII) inhibits B-cell activation induced by BCR aggregation. The cell-triggering properties of the BCR depend on tyrosine-containing activation motifs (TAM), in the intracytoplasmic domain of its Ig alpha and Ig beta subunits. TAMs also account for the cell-triggering capabilities of the T-cell receptor (TCR) for antigen, in T lymphocytes, and of the high-affinity receptor for IgE (Fc epsilon RI), in mast cells. Using as a model, rat basophilic leukemia cells (RBL-2H3) stably transfected with cDNA encoding wild-type or mutated murine or human Fc gamma RIIB and chimeric molecules having the intracytoplasmic domain of the FcR gamma subunit or of TCR-CD3 zeta subunit, we found that the inhibitory properties of Fc gamma RII are neither restricted to B cells nor to BCR-dependent cell activation, but can be extended to other cells and, as a general rule, to TAM-dependent cell activation.

In the present work, we studied the phagocytic and endocytic properties of murine Fc gamma RII in mast cells. Mouse mast cells express high-affinity receptors for monomeric IgE and three low-affinity receptors for complexed IgG: Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RIII. In previous studies we showed that, when aggregated by multivalent ligands, murine Fc gamma RIII, but not Fc gamma RII, triggers the release of inflammatory mediators and cytokines by mast cells. Upon Fc gamma R aggregation, mast cells not only release intracellular materials, they also internalize particulate and soluble immune complexes. We compared the ability of the two Fc gamma RII isoforms to trigger phagocytosis and endocytosis in RBL-2H3 cells stably transfected with cDNAs encoding wild-type, deleted, and tyrosine mutant Fc gamma RIIb1 or Fc gamma RIIb2. We found that Fc gamma RIIb2, but not Fc gamma RIIb1, triggered both phagocytosis and endocytosis. We identified distinct intracytoplasmic sequences necessary for Fc gamma RIIb2-mediated endocytosis and phagocytosis respectively, and we observed that two tyrosine residues, located in each of these sequences, are critical for endocytosis and/or phagocytosis. Our data indicate that the two internalization pathways diverge as early as signal transduction.

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.

International audience; The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function.