To achieve an adequate response, cells of the immune system must be tightly regulated to avoid hypo- or hyper-responsiveness. One of the mechanisms used by the immune system to avoid excessive inflammation is the modulation of the response through inhibitory receptors containing immunoreceptor tyrosine based inhibitory motifs (ITIM). Here, we show that human neutrophils from peripheral blood express the ITIM containing CD300a (also known as IRp60 and CMRF-35H) receptor. By using the HL-60 differentiation model, we show that the expression of CD300a receptor is developmentally regulated. Stimulation of human neutrophils with LPS and GM-CSF increased the cell surface expression of CD300a as a result of the rapid translocation of an intracellular pool of the receptor to the cell surface. Co-ligation of CD300a with the immunoreceptor tyrosine based activating motif (ITAM) containing CD32a (FcγRIIa) activation receptor inhibited CD32a mediated signaling, whereas it did not inhibit Toll like receptor (TLR)-4 mediated reactive oxygen species (ROS) production. Therefore, at least for human neutrophils, the inhibitory signals mediated by the CD300a receptor may be selective in their action.
neutrophils; CD300a; inhibitory receptor; reactive oxygen species; ITAM; ITIM
Signaling from immunotyrosine-based inhibitory motifs (ITIMs) neutralizes activating signals by inducing a retraction of NK cells from the surface of stimulatory cells.
Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell–activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.
Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain–containing tyrosine phosphatases SHP-1 and SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks ITIM sequences. Here we show that Fc receptor γ chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A complex results in mast cell activation such as calcium mobilization in an ITAM-dependent manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated γ chain, whereas PIR-B mediates the inhibitory signal through its ITIMs.
activation signal; Fc receptor γ chain; immunoreceptor tyrosine-based activation motif; mast cell; paired immunoglobulin-like receptor A
Receptors carrying immunoreceptor tyrosine-based inhibition motifs (ITIM) in their cytoplasmic tail control a vast array of cellular responses, ranging from autoimmunity, allergy, phagocytosis of red blood cells, graft versus host disease, to even neuronal plasticity in the brain. The inhibitory function of many receptors has been deduced on the basis of cytoplasmic ITIM sequences. Tight regulation of natural killer (NK) cell cytotoxicity and cytokine production by inhibitory receptors specific for MHC class I molecules has served as a model system to study the negative signalling pathway triggered by an ITIM-containing receptor in the physiological context of NK–target cell interactions. Advances in our understanding of the molecular details of inhibitory signalling in NK cells have provided a conceptual framework to address how ITIM-mediated regulation controls cellular reactivity in diverse cell types.
inhibition; innate immunity; natural killer cell; signalling; tyrosine phosphatase
In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM–ITAM signaling is likely to play an important role in the developmental program of NKT cells.
NKT cell development; ITAM-containing receptors; ITIM-containing receptors; TCR repertoire; NK receptor repertoire
Sialic acid binding immunoglobulin-like lectin-7 (Siglec-7) is a trans-membrane receptor carrying immunoreceptor tyrosine based inhibitory motifs (ITIMs) and delivering inhibitory signals upon ligation with sialylated glycans. This inhibitory function can be also targeted by several pathogens that have evolved to express sialic acids on their surface to escape host immune responses. Here, we demonstrate that cross-linking of Siglec-7 by a specific monoclonal antibody (mAb) induces a remarkably high production of IL-6, IL-1α, CCL4/MIP-1β, IL-8 and TNF-α. Among the three immune cell subsets known to constitutively express Siglec-7, the production of these pro-inflammatory cytokines and chemokines selectively occurs in monocytes and not in Natural Killer or T lymphocytes. This Siglec-7-mediated activating function is associated with the phosphorylation of the extracellular signal-regulated kinase (ERK) pathway. The present study also shows that sialic acid-free Zymosan yeast particles are able to bind Siglec-7 on monocytes and that this interaction mimics the ability of the anti Siglec-7 mAb to induce the production of pro-inflammatory mediators. Indeed, blocking or silencing Siglec-7 in primary monocytes greatly reduced the production of inflammatory cytokines and chemokines in response to Zymosan, thus confirming that Siglec-7 participates in generating a monocyte-mediated inflammatory outcome following pathogen recognition. The presence of an activating form of Siglec-7 in monocytes provides the host with a new and alternative mechanism to encounter pathogens not expressing sialylated glycans.
Many cellular responses, such as autoimmunity and cytotoxicity, are controlled by receptors with cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). Here, we showed that binding of inhibitory natural killer (NK) cell receptors to human leukocyte antigen (HLA) class I on target cells induced tyrosine phosphorylation of the adaptor Crk, concomitant with dephosphorylation of the guanine exchange factor Vav1. Furthermore, Crk dissociated from the guanine exchange factor C3G and bound to the tyrosine kinase c-Abl during inhibition. Membrane targeting of a tyrosine-mutated form of Crk could overcome inhibition of NK cell cytotoxicity, providing functional evidence that Crk phosphorylation contributes to inhibition. The specific phosphorylation of Crk and its dissociation from a signaling complex, observed here with two types of inhibitory receptors, expands the signaling potential of the large ITIM-receptor family and reveals an unsuspected component of the inhibitory mechanism.
KIR3DL2/CD158k/p140 is a three domain killer cell immunoglobulin-like receptor incorporating cytoplasmic immunoreceptor tyrosine inhibitory motifs, expressed as a disulphide-bonded dimer. KIR3DL2 is a framework gene within the KIR locus and is highly polymorphic, with 62 allelic variants possibly coding for protein reported. KIR3DL2 binds to HLA-A3 and -A11 in a peptide-dependent fashion and to B27 free heavy chain forms. In addition, KIR3DL2 can also function as an innate immune receptor for delivery of CpG DNA to TLR9 in NK cells. The increased levels of expression of KIR3DL2 compared with other KIR expressed by T cell subsets in healthy individuals suggest it may function as a default KIR receptor. KIR3DL2-expressing natural killer (NK) cells and IL17 secreting CD4 T cells have been implicated in the pathogenesis of ankylosing spondylitis. Moreover, KIR3DL2 expression delineates circulating and cutaneous lymphoma T cells in Sézary’s syndrome. Here we discuss how the unique molecular attributes of KIR3DL2 impact on its function on NK and T cells and how this may relate to its role in disease.
ankylosing spondylitis; B272; CpG DNA; HLA-A11; HLA-A3; HLA-B27; KIR3DL2; Sézary’s syndrome
In this study, by the generation of a specific monoclonal antibody, we identified p75/AIRM1 (for adhesion inhibitory receptor molecule 1), a novel inhibitory receptor that is mostly confined to human natural killer cells. p75/AIRM1 is a 75-kD glycoprotein that, upon sodium pervanadate treatment, becomes tyrosine phosphorylated and associates to src homology 2 domain–bearing protein tyrosine phosphatase (SHP)-1. The p75/AIRM1 gene is located on human chromosome 19 and encodes a novel member of the sialoadhesin family characterized by three immunoglobulin-like extracellular domains (one NH2-terminal V-type and two C2-type) and a classical immunoreceptor tyrosine–based inhibitory motif (ITIM) in the cytoplasmic portion. The highest amino acid sequence similarity has been found with the myeloid-specific CD33 molecule and the placental CD33L1 protein. Similar to other sialoadhesin molecules, p75/AIRM1 appears to mediate sialic acid–dependent ligand recognition.
molecular cloning; natural killer cells; sialoadhesin; inhibitory receptor; immunoglobulin superfamily
Neonatal PMN exhibit altered inflammatory responsiveness and greater longevity compared to adult PMN; however, the involved mechanisms are incompletely defined. Receptors containing immunoreceptor tyrosine-based inhibitory motif (ITIM) domains promote apoptosis by activating inhibitory phosphatases, such as Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1), that block survival signals. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9, an immune inhibitory receptor with ITIM domains, has been shown to induce cell death in adult PMN in association with SHP-1. To test our hypothesis that neonatal PMN inflammatory function may be modulated by unique Siglec-9 and SHP-1 interactions, we compared expression of these proteins in adult and neonatal PMN. Neonatal PMN exhibited diminished cellular expression of Siglec-9, which was phosphorylated in the basal state. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment decreased Siglec-9 phosphorylation levels in neonatal PMN but promoted its phosphorylation in adult PMN, observations associated with altered survival signaling. While SHP-1 expression was also diminished in neonatal PMN, GM-CSF treatment had minimal effect on phosphorylation status. Further analysis revealed that Siglec-9 and SHP-1 physically interact, as has been observed in other immune cells. Our data suggest that age-specific interactions between Siglec-9 and SHP-1 may influence the altered inflammatory responsiveness and longevity of neonatal PMN.
Signaling by IL-4 and IL-13 via the IL-4 receptor alpha chain (IL-4Rα) plays a critical role in the pathology of allergic diseases. The IL-4Rα is endowed with an immunoreceptor tyrosine-based inhibitory motif (ITIM), centered on tyrosine 709 (Y709) in the cytoplasmic domain, that binds a number of regulatory phosphatases. The function of the ITIM in the in vivo regulation of IL-4R signaling remains unknown.
To determine the in vivo function of the IL-4Rα ITIM using mice in which the ITIM was inactivated by mutagenesis of the tyrosine Y709 residue into phenylalanine (F709).
F709 ITIM mutant mice were derived by knockin mutagenesis. Activation of intracellular signaling cascades by IL-4 and IL-13 was assessed by intracellular staining of phosphorylated signaling intermediates and by gene expression analysis. In vivo responses to allergic sensitization were assessed using models of allergic airway inflammation.
The F709 mutation increased STAT6 phosphorylation by IL-4 and, disproportionately, by IL-13. This was associated with exaggerated Th2 polarization, enhanced alternative macrophage activation by IL-13, augmented basal and antigen-induced IgE responses and intensified allergen-induced eosinophilic airway inflammation and hyperreactivity.
These results point to a physiologic negative regulatory role for the Y709 ITIM in signaling via IL-4Rα, especially by IL-13.
IL-4 receptor; IL-4; IL-13; ITIM; SHP-1; IgE; Allergic Airway Inflammation; Asthma
Elimination of extracellular aggregates and apoptotic neural membranes without inflammation is crucial for brain tissue homeostasis. In the mammalian central nervous system, essential molecules in this process are the Fc receptors and the DAP12-associated receptors which both trigger the microglial immunoreceptor tyrosine-based activation motif- (ITAM-) Syk-signaling cascade. Microglial triggering receptor expressed on myeloid cells-2 (TREM2), signal regulatory protein-β1, and complement receptor-3 (CD11b/CD18) signal via the adaptor protein DAP12 and activate phagocytic activity of microglia. Microglial ITAM-signaling receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif- (ITIM-) signaling molecules such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs can suppress the proinflammatory and phagocytic activity of microglia via ITIM signaling. Moreover, microglial neurotoxicity is alleviated via interaction of Siglec-11 with sialic acids on the neuronal glycocalyx. Thus, ITAM- and ITIM-signaling receptors modulate microglial phagocytosis and cytokine expression during neuroinflammatory processes. Their dysfunction could lead to impaired phagocytic clearance and neurodegeneration triggered by chronic inflammation.
Balance of signals generated from the engaged activating and inhibitory surface receptors regulates mature NK cell activities. The inhibitory receptors signal through immunoreceptor tyrosine based inhibitory motifs (ITIM), and recruit phosphatases such as SHP-1 to inhibit NK cell activation. To directly examine the importance of SHP-1 in regulating activities and cell fate of mature NK cells, we used our established lentiviral-based engineering protocol to knock down the SHP-1 protein expression in primary C57BL/6NCrl cells. Gene silencing of the SHP-1 in primary NK cells abrogated the ability of ITIM-containing NK inhibitory receptors to suppress the activation signals induced by NK1.1 activating receptors. We followed the fates of stably transduced SHP-1 silenced primary NK cells over a longer period of time in IL-2 containing cultures. We observed an impaired IL-2 induced proliferation in the SHP-1 knockdown NK cells. More interestingly, these “de-regulated” SHP-1 knockdown NK cells mediated specific self-killing in a real-time live cell microscopic imaging system we developed to study NK cell cytotoxicity in vitro. Selective target recognition of the SHP-1 knockdown NK cells revealed also possible involvement of the SHP-1 phosphatase in regulating other NK functions in mature NK cells.
Novel immune-type receptor (NITR) genes are members of diversified multigene families that are found in bony fish and encode type I transmembrane proteins containing one or two extracellular immunoglobulin (Ig) domains. The majority of NITRs can be classified as inhibitory receptors that possess cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs). A much smaller number of NITRs can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain, which permits partnering with an activating adaptor protein.
Forty-four NITR genes in medaka (Oryzias latipes) are located in three gene clusters on chromosomes 10, 18 and 21 and can be organized into 24 families including inhibitory and activating forms. The particularly large dataset acquired in medaka makes direct comparison possible to another complete dataset acquired in zebrafish in which NITRs are localized in two clusters on different chromosomes. The two largest medaka NITR gene clusters share conserved synteny with the two zebrafish NITR gene clusters. Shared synteny between NITRs and CD8A/CD8B is limited but consistent with a potential common ancestry.
Comprehensive phylogenetic analyses between the complete datasets of NITRs from medaka and zebrafish indicate multiple species-specific expansions of different families of NITRs. The patterns of sequence variation among gene family members are consistent with recent birth-and-death events. Similar effects have been observed with mammalian immunoglobulin (Ig), T cell antigen receptor (TCR) and killer cell immunoglobulin-like receptor (KIR) genes. NITRs likely diverged along an independent pathway from that of the somatically rearranging antigen binding receptors but have undergone parallel evolution of V family diversity.
We report the cloning and functional characterization in the mouse and the rat of a novel natural killer (NK) cell receptor termed KLRE1. The receptor is a type II transmembrane protein with a COOH-terminal lectin-like domain, and constitutes a novel KLR family. Rat Klre1 was mapped to the NK gene complex. By Northern blot and flow cytometry using newly generated monoclonal antibodies, KLRE1 was shown to be expressed by NK cells and a subpopulation of CD3+ cells, with pronounced interstrain variation. Western blot analysis indicated that KLRE1 can be expressed on the NK cell surface as a disulphide-linked dimer. The predicted proteins do not contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or a positively charged amino acid in the transmembrane domain. However, in a redirected lysis assay, the presence of whole IgG, but not of F(ab′)2 fragments of a monoclonal anti-KLRE1 antibody inhibited lysis of Fc-receptor bearing tumor target cells. Moreover, the tyrosine phosphatase SHP-1 was coimmunoprecipitated with KLRE1 from pervanadate-treated interleukin 2–activated NK cells. Together, our results indicate that KLRE1 may form a functional heterodimer with an as yet unidentified ITIM-bearing partner that recruits SHP-1 to generate an inhibitory receptor complex.
natural immunity; lymphocytes; immunological receptors; molecular sequence data; protein-tyrosine-phosphatase
Most coinhibitory receptors regulate T cell responses through an immunoreceptor tyrosine-based inhibitory motif (ITIM) that recruits protein tyrosine phosphatases (PTP) to mediate inhibitory function. Because syndecan-4 (SD-4), the coinhibitor for DC-HIL, lacks such an ITIM, we posited that SD-4 links with a PTP in an ITIM-independent manner. We showed SD-4 to associate constitutively with the intracellular protein syntenin but not with the receptor-like PTP CD148 on human CD4+ T cells. Binding to DC-HIL allowed SD-4 to assemble with CD148 through the help of syntenin as a bridge, and this process upregulated the PTP activity of CD148, which is required for SD-4 to mediate DC-HIL’s inhibitory function. Using a mouse model, we found SD-4 to reside away from the immunological synapse formed between T cells and APC during activation of T cells. These findings indicate that SD-4 is unique among known T cell coinhibitors, in employing CD148 to inhibit T cell activation at a site distal from the synapse.
CD148; Co-inhibitory receptors; DC-HIL; Syndecan-4; T cells
Human and mouse B cells lacking functional DAP12 are hyperresponsive, and DAP12 works with MAIR-II (CD300d) to negatively regulate B cell activity.
DAP12, an immunoreceptor tyrosine-based activation motif–bearing adapter protein, is involved in innate immunity mediated by natural killer cells and myeloid cells. We show that DAP12-deficient mouse B cells and B cells from a patient with Nasu-Hakola disease, a recessive genetic disorder resulting from loss of DAP12, showed enhanced proliferation after stimulation with anti-IgM or CpG. Myeloid-associated immunoglobulin-like receptor (MAIR) II (Cd300d) is a DAP12-associated immune receptor. Like DAP12-deficient B cells, MAIR-II–deficient B cells were hyperresponsive. Expression of a chimeric receptor composed of the MAIR-II extracellular domain directly coupled to DAP12 into the DAP12-deficient or MAIR-II–deficient B cells suppressed B cell receptor (BCR)–mediated proliferation. The chimeric MAIR-II–DAP12 receptor recruited the SH2 domain–containing protein tyrosine phosphatase 1 (SHP-1) after BCR stimulation. DAP12-deficient mice showed elevated serum antibodies against self-antigens and enhanced humoral immune responses against T cell–dependent and T cell–independent antigens. Thus, DAP12-coupled MAIR-II negatively regulates B cell–mediated adaptive immune responses.
Human paired Ig-like type 2 receptor α (PILRα) has been expressed, purified and crystallized. A diffraction data set has been collected to 1.3 Å resolution.
Human paired immunoglobulin-like (Ig-like) type 2 receptor α (PILRα) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILRα can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) to inhibit phophorylations induced by activation signals. The extracellular region of human PILRα comprises one immunoglobulin superfamily V-set domain and a stalk region. The V-set domain (residues 13–131) of human PILRα was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILRα protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 Å resolution at SPring-8 BL41XU; they belong to space group P212121, with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 Å, and contain one molecule per asymmetric unit.
paired Ig-like type 2 receptor α; membrane proteins
Siglecs (sialic acid-binding, immunoglobulin [Ig]-like lectins) are a family of single-pass transmembrane cell surface proteins found predominantly on leukocytes. Their unique structural characteristics include an N-terminal carbohydrate-binding (“lectin”) domain that binds sialic acid, followed by a variable number of Ig-like domains, hence these structures are a subset of the Ig gene superfamily. Another unique feature of Siglecs is that most, but not all, possess so-called immunoreceptor tyrosine-based inhibitory motifs (“ITIMs”) in their cytoplasmic domains, suggesting that these molecules function in an inhibitory capacity. Siglec-8, the eighth member identified at the time, was discovered as part of an effort initiated almost a decade ago to identify novel human eosinophil and mast cell proteins. Since that time, its selective expression on human eosinophils and mast cells has been confirmed. On eosinophils, Siglec-8 engagement results in apoptosis, whereas on mast cells, inhibition of FcεRI-dependent mediator release, without apoptosis, is seen. It has subsequently been determined that the closest functional paralog in the mouse is Siglec-F, selectively expressed by eosinophils but not expressed on mast cells. Despite only modest homology, both Siglec-8 and Siglec-F preferentially recognize a sulfated glycan ligand closely related to sialyl Lewis X, a common ligand for the selectin family of adhesion molecules. Murine experiments in normal, Siglec-F-deficient mice and hypereosinophilic mice have resulted in similar conclusions that Siglec-F, like Siglec-8, plays a distinctive and important role in regulating eosinophil accumulation and survival in vivo. Given the resurgent interest in eosinophil-directed therapies for a variety of disorders, plus its unique additional ability to also target the mast cell, therapies focusing on Siglec-8 could some day prove to be a useful adjunct to our current armamentarium for the treatment of asthma, allergies and related disorders where overproduction and overactivity of eosinophils and mast cells is occurring.
KIR2DL4 is an unusual killer cell immunoglobulin-like receptor (KIR) family member in terms of its structure, expression, cellular localization, and signaling properties. The most conserved KIR in evolution, it is referred to as a framework KIR gene and is expressed by all natural killer (NK) cells and a subset of T cells. Although it has a long cytoplasmic tail that is typical of inhibitory KIR, engagement of this receptor results in the activation of NK cells, not for cytotoxicity, but for cytokine and chemokine secretion. Unlike all other KIRs, which are expressed on the surface of NK cells, KIR2DL4 resides in endosomes. It signals from this intracellular site for a proinflammatory and proangiogenic response, using a novel endosomal signaling pathway that involves the serine/threonine kinases DNA-PKcs and Akt. The only known ligand of KIR2DL4 is HLA-G. Soluble HLA-G accumulates in KIR2DL4+ endosomes. Unlike classical HLA molecules that serve as ligands for other KIR family members, in healthy individuals, HLA-G expression is restricted to the fetal trophoblast cells that invade the maternal decidua during early pregnancy. Since NK cells constitute the predominant lymphocyte subset at this site, the proinflammatory/proangiogenic outcome of the interaction between KIR2DL4 and soluble HLA-G supports a role for KIR2DL4 in the extensive remodeling of the maternal vasculature during the early weeks of pregnancy.
NK; KIR; HLA-G; pregnancy
Osteoclasts (OC) are bone-resorbing, multinucleated cells that are generated via fusion of OC precursors (OCP). The frequency of OCP is elevated in patients with erosive inflammatory arthritis and metabolic bone diseases. Although many cytokines and cell surface receptors are known to participate in osteoclastogenesis, the molecular mechanisms underlying the regulation of this cellular transformation are poorly understood. Herein, we focused our studies on the dendritic cell-specific transmembrane protein (DC-STAMP), a seven-pass-transmembrane receptor-like protein known to be essential for cell-to-cell fusion during osteoclastogenesis. We identified an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail of DC-STAMP, and developed an anti-DC-STAMP monoclonal antibody 1A2 that detected DC-STAMP expression on human tumor giant cells, blocked OC formation in vitro, and distinguished four patterns of human PBMC with a positive correlation to OC potential. In freshly isolated monocytes, DC-STAMPhigh cells produced a higher number of OC in culture than DC-STAMPlow cells and the surface expression of DC-STAMP gradually declined during osteoclastogenesis. Importantly, we showed that DC-STAMP is phosphorylated on its tyrosine residues and physically interacts with SHP-1 and CD16, an SH2-domain-containing tyrosine phosphatase and an ITAM-associated protein, respectively. Taken together, these data show that DC-STAMP is a potential OCP biomarker in inflammatory arthritis. Moreover, in addition to its effect on cell fusion, DC-STAMP dynamically regulates cell signaling during osteoclastogenesis.
DC-STAMP; osteoclast; signaling; ITIM; ITAM; SHP-1; OCP; biomarker; Ps; PsA; CD16
Immune responses are regulated by opposing positive and negative signals triggered by the interaction of activating and inhibitory cell surface receptors with their ligands. Here, we describe novel paired activating and inhibitory immunoglobulin-like receptors, designated myeloid-associated immunoglobulin-like receptor (MAIR) I and MAIR-II, whose extracellular domains are highly conserved by each other. MAIR-I, expressed on the majority of myeloid cells, including macrophages, granulocytes, mast cells, and dendritic cells, contains the tyrosine-based sorting motif and the immunoreceptor tyrosine-based inhibitory motif-like sequences in the cytoplasmic domain and mediates endocytosis of the receptor and inhibition of IgE-mediated degranulation from mast cells. On the other hand, MAIR-II, expressed on subsets of peritoneal macrophages and B cells, associates with the immunoreceptor tyrosine-based activation motif-bearing adaptor DAP12 and stimulates proinflammatory cytokines and chemokine secretions from macrophages. Thus, MAIR-I and MAIR-II play important regulatory roles in cell signaling and immune responses.
ITAM; ITIM; innate immunity; DAP12; myeloid cells
NKB1 is one member of a growing family of killer cell inhibitory receptors (KIR). It is expressed on natural killer (NK) cells and T cells, and has been shown to inhibit cytolytic functions of these cells upon interacting with its ligand, HLA-B (Bw4). We demonstrate here that the cytoplasmic region of NKB1 is capable of inhibiting T cell activation in Jurkat cells. The tyrosine phosphorylation of the NKB1 KIR consensus motif, YxxL(x)26 YxxL, induces an association with the protein tyrosine phosphatase 1C (PTP1C). Importantly, mutation of both tyrosines in the motif abolished the inhibitory functions of NKB1 and abrogated PTP1C association. Mutational analysis of the individual tyrosines suggest that the membrane proximal tyrosine may play a crucial role in mediating the inhibitory signal. These results demonstrate that KIR can not only inhibit cytolytic activity, but can also negatively regulate T cell receptor activation events that lead to downstream gene activation, and further supports a model that implicates PTP1C as a mediator in the KIR inhibitory signal.
Natural Killer (NK) cells play an important role in the early immune response to cancer. The NKp44 activating receptor is the only Natural Cytotoxicity Receptor that is expressed exclusively by primate NK cells; yet, its cellular ligands remain largely unknown. Proliferating Cell Nuclear Antigen (PCNA) is overexpressed in cancer cells. We show that the NKp44 receptor recognizes PCNA. Their interaction inhibits NK cell function through the NKp44-Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM). The physical interaction of NKp44 and PCNA is enabled by recruitment of target cell PCNA to the NK immunological synapse. We demonstrate that PCNA promotes cancer survival by immune evasion through inhibition of NKp44-mediated NK cell attack.
PECAM-1 (CD31) is an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing surface glycoprotein expressed on various hematopoietic cells as well as on endothelial cells. PECAM-1 has been shown to play roles in regulation of adhesion, migration and apoptosis. The BCR/ABL fusion tyrosine kinase is expressed in chronic myeloid leukemia and Philadelphia-positive (Ph+) acute lymphoblastic leukemia cells, and its inhibition by the clinically used tyrosine kinase inhibitors imatinib or dasatinib induces apoptosis of these cells. In the present study, we demonstrate that PECAM-1 is tyrosine phospho rylated in its ITIM motifs in various BCR/ABL-expressing cells including primary leukemia cells. Studies using imatinib and dasatinib as well as transient expression experiments in 293T cells revealed that PECAM-1 was phosphorylated directly by BCR/ABL, which was enhanced by the imatinib-resistant E255K and T315I mutations, or partly by the Src family tyrosine kinases, including Lyn, which were activated dependently or independently on BCR/ABL. We also demonstrate by using a substrate trapping mutant of SHP2 that tyrosine phosphorylated PECAM-1 binds SHP2 and is a major substrate for this tyrosine phosphatase in BCR/ABL-expressing cells. Overexpression of PECAM-1 in BCR/ABL-expressing cells, including K562 human leukemia cells, enhanced cell adhesion and partially inhibited imatinib-induced apoptosis involving mitochondria depolarization and caspase-3 cleavage, at least partly, in an ITIM-independent manner. These data suggest that PECAM-1 may play a role in regulation of apoptosis as well as adhesion of BCR/ABL-expressing cells to modulate their imatinib sensitivity and would be a possible candidate for therapeutic target in Ph+ leukemias.
PECAM-1; BCR/ABL; imatinib; apoptosis; leukemia