An immunoglobulin-like receptor, Allergin-1, inhibits immunoglobulin E-mediated immediate hypersensitivity reactions.

Anaphylaxis is a life-threatening immediate hypersensitivity reaction triggered by antigen capture by immunoglobulin E (IgE) bound to the high-affinity IgE receptor (FcvarepsilonRI) on mast cells. However, the regulatory mechanism of mast cell activation is not completely understood. Here we identify an immunoglobulin-like receptor, Allergin-1, that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain, and show it was preferentially expressed on mast cells. Mouse Allergin-1 recruited the tyrosine phosphatases SHP-1 and SHP-2 and the inositol phosphatase SHIP. Coligation of Allergin-1 and FcvarepsilonRI suppressed IgE-mediated degranulation of bone marrow-derived cultured mast cells. Moreover, mice deficient in Allergin-1 developed enhanced passive systemic and cutaneous anaphylaxis. Thus, Allergin-1 suppresses IgE-mediated, mast cell-dependent anaphylaxis in mice.

ITAM-mediated tonic signalling through pre-BCR and BCR complexes.

Studies carried out over the past few years provide strong support for the idea that Ig alpha-Ig beta-containing complexes such as the pre-B-cell receptor and the B-cell receptor can signal independently of ligand engagement, and this has been termed tonic signalling. In this Review, I discuss recent literature that is relevant to the potential mechanisms by which tonic signals are initiated and regulated, and discuss views on how tonic and ligand-dependent (aggregation-mediated) signalling differ. These mechanisms are relevant to the possibility that tonic signals generated through immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins that are expressed by oncogenic viruses induce transformation in non-haematopoietic cells.

Organization of the resting TCR in nanoscale oligomers.

Despite the low affinity of the T-cell antigen receptor (TCR) for its peptide/major histocompatibility complex (pMHC) ligand, T cells are very sensitive to their antigens. This paradox can be resolved if we consider that the TCR may be organized into pre-existing oligomers or nanoclusters. Such structures could improve antigen recognition by increasing the functional affinity (avidity) of the TCR-pMHC interaction and by allowing cooperativity between individual TCRs. Up to approximately 20 TCRs become tightly apposed in these nanoclusters, often in a linear manner, and such structures could reflect a relatively generalized phenomenon: the non-random concentration of membrane receptors in specific areas of the plasma membrane known as protein islands. The association of TCRs into nanoclusters can explain the enhanced kinetics of the pMHC-TCR interaction in two dimensional versus three dimensional systems, but also their existence calls for a revision of the TCR triggering models based on pMHC-induced TCR clustering. Interestingly, the B-cell receptor and the FcεRI have also been shown to form nanoclusters, suggesting that the formation of pre-existing receptor oligomers could be widely used in the immune system.

Lymphocyte repertoire selection and intracellular self/non-self-discrimination: historical overview.

Immunological self/non-self-discrimination is conventionally seen as an extracellular event, involving interactions been receptors on T cells pre-educated to discriminate and peptides bound to major histocompatibility complex proteins (pMHCs). Mechanisms by which non-self peptides might first be sorted intracellularly to distinguish them from the vast excess of self-peptides have long been called for. Recent demonstrations of endogenous peptide-specific clustering of pMHCs on membrane rafts are indicative of intracellular enrichment before surface display. The clustering could follow the specific aggregation of a foreign protein that exceeded its solubility limit in the crowded intracellular environment. Predominantly entropy-driven, this homoaggregation would colocalize identical peptides, thus facilitating their collective presentation. Concentrations of self-proteins are fine-tuned over evolutionary time to avoid this. Disparate observations, such as pyrexia and female susceptibility to autoimmune disease, can be explained in terms of the need to cosegregate cognate pMHC complexes internally before extracellular display.

Mechanisms of Fc receptor and dectin-1 activation for phagocytosis.

Phagocytosis is a key cellular process, both during homeostasis and upon infection or tissue damage. Receptors on the surface of professional phagocytic cells bind to target particles either directly or through opsonizing ligands, and trigger actin-mediated ingestion of the particles. The process must be carefully controlled to ensure that phagocytosis is triggered efficiently and specifically, and that the antimicrobial cytotoxic responses that often accompany it are initiated only when required. In this review, we will describe and compare the molecular mechanisms that regulate phagocytosis triggered by Fcγ receptors, which mediate the uptake of immunoglobulin G-opsonized targets, and Dectin-1, which is responsible for internalization of fungi with exposed cell wall ß-glucan. We will examine how these receptors detect their ligands, how signal transduction is initiated and regulated, and how internalization is instructed to achieve rapid and yet controlled uptake of their targets.

The molecular assembly and organization of signaling active B-cell receptor oligomers.

In B cells, antigen drives the formation of B-cell receptor (BCR) clusters that initiate the formation of signaling complexes associated with the cytoplasmic domains of the BCRs. These signaling active complexes contain a number of protein and lipid kinases and phosphatases and adapter and scaffolding proteins that together function to trigger downstream signaling cascades leading to the activation of a variety of genes associated with B-cell activation. Although we are learning a considerable amount about the molecular details of the assembly of immune receptor signaling complexes, as reviewed in this volume, a fundamental question remains, namely how does antigen binding outside the cell initiate the assembly of signaling complexes inside the cell. For B cells, we do not yet understand how the information that the ectodomain of the BCR has bound to an antigen is translated across the membrane to induce changes in the cytoplasmic domains that trigger the assembly of signaling complexes. Here we describe what is known about the initiation of the antigen-driven BCR signal transduction in the newly emerging context of B-cell recognition of antigens presented by antigen-presenting cells in lymphoid tissues. We also discuss a recently proposed model for the initiation of BCR signaling termed the 'conformation-induced oligomerization model' and address the implications of this model for the mechanisms by which BCR signaling may be modulated by adapters and coreceptors.

Endocytic events in TCR signaling: focus on adapters in microclusters.

Although the critical role of T-cell receptor (TCR) microclusters in T-cell activation is now widely accepted, the mechanisms of regulation of these TCR-rich structures, which also contain enzymes, adapters, and effectors, remain poorly defined. Soon after microcluster formation, several signaling proteins rapidly dissociate from the TCR. Recent studies from our laboratory demonstrated that the movement of the adapters linker for activation of T cells (LAT) and Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) away from initial microcluster formation sites represents endocytic events. Ubiquitylation, Cbl proteins, and multiple endocytic pathways are involved in the internalization events that disassemble signaling microclusters. Several recent studies have indicated that microcluster movement and centralization plays an important role in signal termination. We suggest that microcluster movement is directly linked to endocytic events, thus implicating endocytosis of microclusters as a means to regulate signaling output of the T cell.

Apoptosis of mouse mast cells is reciprocally regulated by the IgG receptors FcγRIIB and FcγRIIIA.

BACKGROUND: Mast cells are important effector cells in allergy. They usually have a long life span and resist cell death induction. Fcγ receptor- and IgG immune complex-mediated apoptosis has been demonstrated in B-lineage cells, but not in mast cells. The aim of the current study was to investigate whether mast cells could respond to apoptosis induction by IgG immune complex aggregation of Fcγ receptors. It is known that mouse mast cells express the low-affinity Fcγ receptors FcγRIIB and FcγRIIIA, which bind IgG especially in the form of antigen-IgG immune complexes. METHODS: Mouse bone marrow-derived cultured mast cells were examined for surface expression of FcγRIIB and FcγRIIIA. Apoptosis of such cells from wild-type, FcγRIIB(-/-) or FcγRIIIA(-/-) mice was measured following receptor aggregation by IgG immune complexes. RESULTS: Our data demonstrate that aggregation of either FcγRIIB or FcγRIIIA by IgG immune complexes induced apoptosis of mouse bone marrow-derived cultured mast cells. However, mast cells expressing both FcγRIIB and FcγRIIIA were able to resist cell death induction by IgG immune complexes. CONCLUSION: Our findings reveal a fine-tuning system for regulating mast cell apoptosis through aggregating Fcγ receptors by IgG immune complexes. Such apoptosis regulation may have a substantial impact on mast cell homeostasis during allergic inflammation.

B cell activation triggered by the formation of the small receptor cluster: a computational study.

We proposed a spatially extended model of early events of B cell receptors (BCR) activation, which is based on mutual kinase-receptor interactions that are characteristic for the immune receptors and the Src family kinases. These interactions lead to the positive feedback which, together with two nonlinearities resulting from the double phosphorylation of receptors and Michaelis-Menten dephosphorylation kinetics, are responsible for the system bistability. We demonstrated that B cell can be activated by a formation of a tiny cluster of receptors or displacement of the nucleus. The receptors and Src kinases are activated, first locally, in the locus of the receptor cluster or the region where the cytoplasm is the thinnest. Then the traveling wave of activation propagates until activity spreads over the whole cell membrane. In the models in which we assume that the kinases are free to diffuse in the cytoplasm, we found that the fraction of aggregated receptors, capable to initiate B cell activation decreases with the decreasing thickness of cytoplasm and decreasing kinase diffusion. When kinases are restricted to the cell membrane - which is the case for most of the Src family kinases - even a cluster consisting of a tiny fraction of total receptors becomes activatory. Interestingly, the system remains insensitive to the modest changes of total receptor level. The model provides a plausible mechanism of B cells activation due to the formation of small receptors clusters collocalized by binding of polyvalent antigens or arising during the immune synapse formation.

Chemerin contributes to inflammation by promoting macrophage adhesion to VCAM-1 and fibronectin through clustering of VLA-4 and VLA-5.

Chemerin is a potent macrophage chemoattractant protein. We used murine peritoneal exudate cells (PECs) in adhesion, flow cytometry, and confocal microscopy assays to test the hypothesis that chemerin can also contribute to inflammation by promoting macrophage adhesion. Chemerin stimulated the adhesion of PECs to the extracellular matrix protein fibronectin and to the adhesion molecule VCAM-1 within a minute, with an EC(50) of 322 and 196 pM, respectively. Experiments using pertussis toxin and PECs from ChemR23(-/-) mice demonstrated that chemerin stimulated the adhesion of macrophages via the Gi protein-coupled receptor ChemR23. Blocking Abs against integrin subunits revealed that 89% of chemerin-stimulated adhesion to fibronectin was dependent on increased avidity of the integrin VLA-5 (alpha(5)beta(1)) and that 88% of adhesion to VCAM-1 was dependent on increased avidity of VLA-4 (alpha(4)beta(1)). Although chemerin was unable to induce an increase in integrin affinity as judged by the binding of soluble ligand, experiments using confocal microscopy revealed an increase in valency resulting from integrin clustering as the mechanism responsible for chemerin-stimulated macrophage adhesion. PI3K, Akt, and p38 were identified as key signaling mediators in chemerin-stimulated adhesion. The finding that chemerin can rapidly stimulate macrophage adhesion to extracellular matrix proteins and adhesion molecules, taken together with its ability to promote chemotaxis, suggests a novel role for chemerin in the recruitment and retention of macrophages at sites of inflammation.

MHC class I and TCR avidity control the CD8 T cell response to IL-15/IL-15Rα complex.

IL-15 operates via a unique mechanism termed transpresentation. In this system, IL-15 produced by one cell type is bound to IL-15Rα expressed by the same cell and is presented to apposing cells expressing the IL-15Rß/γC complex. We have shown that administering soluble IL-15Rα complexed with IL-15 can greatly enhance IL-15 activity. We now show that the naive CD8 T cell response to exogenous IL-15/IL-15Rα complex is MHC class I dependent. In the absence of ß2 microglobulin, naive CD8 T cells scarcely proliferated in response to IL-15/IL-15Rα complex, whereas memory cells proliferated, although to a lesser extent, compared with levels in control mice. The loss of ß2m or FcRn slightly reduced the extended half-life of IL-15/IL-15Rα complex, whereas FcRn deficiency only partially reduced the naive CD8 T cell proliferative response to IL-15/IL-15Rα complex. In addition, we demonstrated a link between TCR avidity and the ability of a T cell to respond to IL-15/IL-15Rα complex. Thus, T cells expressing low-avidity TCR responded poorly to IL-15/IL-15Rα complex, which correlated with a poor homeostatic proliferative response to lymphopenia. The inclusion of cognate peptide along with complex resulted in enhanced proliferation, even when TCR avidity was low. IL-15/IL-15Rα complex treatment, along with peptide immunization, also enhanced activation and the migratory ability of responding T cells. These data suggest that IL-15/IL-15Rα complex has selective effects on Ag-activated CD8 T cells. Our findings have important implications for directing IL-15/IL-15Rα complex-based therapy to specific Ag targets and illustrate the possible adjuvant uses of IL-15/IL-15Rα complex.

Antigen-induced oligomerization of the B cell receptor is an early target of Fc gamma RIIB inhibition.

The FcgammaRIIB is a potent inhibitory coreceptor that blocks BCR signaling in response to immune complexes and, as such, plays a decisive role in regulating Ab responses. The recent application of high-resolution live cell imaging to B cell studies is providing new molecular details of the earliest events in the initiation BCR signaling that follow within seconds of Ag binding. In this study, we report that when colligated to the BCR through immune complexes, the FcgammaRIIB colocalizes with the BCR in microscopic clusters and blocks the earliest events that initiate BCR signaling, including the oligomerization of the BCR within these clusters, the active recruitment of BCRs to these clusters, and the resulting spreading and contraction response. Fluorescence resonance energy transfer analyses indicate that blocking these early events may not require molecular proximity of the cytoplasmic domains of the BCR and FcgammaRIIB, but relies on the rapid and sustained association of FcgammaRIIB with raft lipids in the membrane. These results may provide novel early targets for therapies aimed at regulating the FcgammaRIIB to control Ab responses in autoimmune disease.

Regulation of hematopoietic cell function by inhibitory immunoglobulin G receptors and their inositol lipid phosphatase effectors.

Numerous autoimmune and inflammatory disorders stem from the dysregulation of hematopoietic cell activation. The activity of inositol lipid and protein tyrosine phosphatases, and the receptors that recruit them, is critical for prevention of these disorders. Balanced signaling by inhibitory and activating receptors is now recognized to be an important factor in tuning cell function and inflammatory potential. In this review, we provide an overview of current knowledge of membrane proximal events in signaling by inhibitory/regulatory receptors focusing on structural and functional characteristics of receptors and their effectors Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 and SH2 domain-containing inositol 5-phosphatase-1. We review use of new strategies to identify novel regulatory receptors and effectors. Finally, we discuss complementary actions of paired inhibitory and activating receptors, using Fc gammaRIIA and Fc gammaRIIB regulation human basophil activation as a prototype.

Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses.

We report the supramolecular organization of killer Ig-like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein-tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein-tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.

Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane.

The aggregation of high affinity IgE receptors (Fcepsilon receptor I [FcepsilonRI]) on mast cells is potent stimulus for the release of inflammatory and allergic mediators from cytoplasmic granules. However, the molecular mechanism of degranulation has not yet been established. It is still unclear how FcepsilonRI-mediated signal transduction ultimately regulates the reorganization of the cytoskeleton and how these events lead to degranulation. Here, we show that FcepsilonRI stimulation triggers the formation of microtubules in a manner independent of calcium. Drugs affecting microtubule dynamics effectively suppressed the FcepsilonRI-mediated translocation of granules to the plasma membrane and degranulation. Furthermore, the translocation of granules to the plasma membrane occurred in a calcium-independent manner, but the release of mediators and granule-plasma membrane fusion were completely dependent on calcium. Thus, the degranulation process can be dissected into two events: the calcium-independent microtubule-dependent translocation of granules to the plasma membrane and calcium-dependent membrane fusion and exocytosis. Finally, we show that the Fyn/Gab2/RhoA (but not Lyn/SLP-76) signaling pathway plays a critical role in the calcium-independent microtubule-dependent pathway.

Arrestin 3 mediates endocytosis of CCR7 following ligation of CCL19 but not CCL21.

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 +/- 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 +/- 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2(-/-)/arrestin 3(-/-) murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.

C3a-derived peptide binds to the type I FcepsilonR and inhibits proximal-coupling signal processes and cytokine secretion by mast cells.

A peptide with the natural sequence derived from the complement component C3a, designated C3a7, and C3a9, having a modified sequence of that, was previously shown to inhibit the high-affinity IgER (FcepsilonRI)-induced secretory response of both mucosal and serosal-type mast cells. In addition, several processes that couple the FcepsilonRI stimulus to the cellular response were all suppressed in the presence of these peptides. Here, we show that peptide C3a9 binds to the FcepsilonRI on the surface of unperturbed mast cells (rat mucosal-type RBL-2H3 cell line) and remains bound even after FcepsilonRI-IgE aggregation by antigen as assessed by confocal microscopy. Moreover, that peptide interferes the initial steps of FcepsilonRI-coupling network. Namely, peptide binding to the FcepsilonRI beta-chain interrupts this chain's association with both src family protein tyrosine kinases Lyn and Fyn and enhances the internalization of the receptor. C3a9 was further found to inhibit the phosphorylation of two members of the mitogen-activated protein kinase family, extracellular signal-regulated kinase (ERK) and p38. Although ERK is usually activated via the ras-raf-mitogen-activated protein kinase/ERK kinase (MEK) pathway, our results show that C3a9 has no effect on the c-raf phosphorylation, suggesting that this complement-derived peptide inhibits ERK activation via an alternative route. C3a9 also inhibits the late-phase response to FcepsilonRI stimulus of bone marrow-derived mast cells, reducing secretion of the inflammatory cytokines IL-6 and tumor necrosis factor-alpha. Taken together, the consequence of its interference with the earliest steps of FcepsilonRI stimulus-response coupling and the C3a-derived peptide inhibits both the immediate and the late-phase responses of mast cells.

Interactions between MHC molecules and co-receptors of the TCR.

Genetic experiments indicate similarity between binding sites on MHC class I (MHCI) for CD8 and on MHCII for CD4, but the crystal structures of CD8/MHCI and CD4/MHCII complexes suggest critical differences between the interfaces in the two complexes. Biophysical analyses using ectodomains of co-receptors and MHC molecules demonstrate extremely fast kinetics and low-affinity interactions. Experiments with soluble multimeric MHC ligands suggest that CD4 and CD8 may differ in the mechanisms by which they promote the formation of ternary TCR/MHC/co-receptor complexes. Co-receptor-influenced duration of TCR signaling controls thymocyte selection. In naïve T cells, CD4/MHCII interactions may promote T-cell survival. Temporal and spatial analysis of TCR and CD4 co-clustering in the immunological synapse suggests that CD4 recruitment is regulated by the half-life of the initial TCR/MHCII complex. Diverse experimental systems have yielded conflicting data that have helped to formulate revised mechanistic models of co-receptor function.

Alterations in tyrosine protein phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes.

Accumulating data suggest that the CD4 T-cell surface antigen transduces an independent intracellular signal during antigen-mediated T-cell activation. CD4 is physically associated with the internal membrane tyrosine protein kinase p56lck and can mediate, after antibody-mediated cross-linking, the rapid enzymatic activation of Lck, implying that CD4 signalling may involve changes in tyrosine protein phosphorylation. In this report, we describe that cross-linking of CD4 results in a series of rapid changes in intracellular tyrosine protein phosphorylation. The most prominent CD4-induced tyrosine phosphorylation change involved p56lck, which became extensively phosphorylated on the carboxy-terminal tyrosine residue 505 and, to a lesser extent, lymphocytes can transduce an intracellular signal resulting in tyrosine protein phosphorylation and strongly suggest that this property of CD4 is mediated through p56lck.

Human autoantibodies against early endosome antigen-1 enhance excitatory synaptic transmission.

Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl-D-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies.