The receptor LMIR3 negatively regulates mast cell activation and allergic responses by binding to extracellular ceramide.

Mast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

IL-3 Triggers Chronic Rejection of Cardiac Allografts by Activation of Infiltrating Basophils.

Chronic rejection is a major problem in transplantation medicine, largely resistant to therapy, and poorly understood. We have shown previously that basophil-derived IL-4 contributes to fibrosis and vasculopathy in a model of heart transplantation with depletion of CD4+ T cells. However, it is unknown how basophils are activated in the allografts and whether they play a role when cyclosporin A (CsA) immunosuppression is applied. BALB/c donor hearts were heterotopically transplanted into fully MHC-mismatched C57BL/6 recipients and acute rejection was prevented by depletion of CD4+ T cells or treatment with CsA. We found that IL-3 is significantly upregulated in chronically rejecting allografts and is the major activator of basophils in allografts. Using IL-3-deficient mice and depletion of basophils, we show that IL-3 contributes to allograft fibrosis and organ failure in a basophil-dependent manner. Also, in the model of chronic rejection involving CsA, IL-3 and basophils substantially contribute to organ remodeling, despite the almost complete suppression of IL-4 by CsA. In this study, basophil-derived IL-6 that is resistant to suppression by CsA, was largely responsible for allograft fibrosis and limited transplant survival. Our data show that IL-3 induces allograft fibrosis and chronic rejection of heart transplants, and exerts its profibrotic effects by activation of infiltrating basophils. Blockade of IL-3 or basophil-derived cytokines may provide new strategies to prevent or delay the development of chronic allograft rejection.

Basophils trigger emphysema development in a murine model of COPD through IL-4-mediated generation of MMP-12-producing macrophages.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. It has generally been considered a non-Th2-type lung disorder, characterized by progressive airflow limitation with inflammation and emphysema, but its cellular and molecular mechanism remains ill defined, compared with that of asthma characterized by reversible airway obstruction. Here we show a previously unappreciated role for basophils at the initiation phase of emphysema formation in an elastase-induced murine model of COPD in that basophils represent less than 1% of lung-infiltrating cells. Intranasal elastase instillation elicited the recruitment of monocytes to the lung, followed by differentiation into interstitial macrophages (IMs) but rarely alveolar macrophages (AMs). Matrix metalloproteinase-12 (MMP-12) contributing to emphysema formation was highly expressed by IMs rather than AMs, in contrast to the prevailing assumption. Experiments using a series of genetically engineered mice suggested that basophil-derived IL-4, a Th2 cytokine, acted on lung-infiltrating monocytes to promote their differentiation into MMP-12-producing IMs that resulted in the destruction of alveolar walls and led to emphysema development. Indeed, mice deficient for IL-4 only in basophils failed to generate pathogenic MMP-12-producing IMs and hence develop emphysema. Thus, the basophil-derived IL-4/monocyte-derived IM/MMP-12 axis plays a crucial role in emphysema formation and therefore may be a potential target to slow down emphysema progression at the initiation phase of COPD.

Recent advances in understanding basophil-mediated Th2 immune responses.

Basophils, the least common granulocytes, represent only ~0.5% of peripheral blood leukocytes. Because of the small number and some similarity with mast cells, the functional significance of basophils remained questionable for a long time. Recent studies using newly-developed analytical tools have revealed crucial and non-redundant roles for basophils in various immune responses, particularly Th2 immunity including allergy and protective immunity against parasitic infections. In this review, we discuss the mechanisms how basophils mediate Th2 immune responses and the nature of basophil-derived factors involved in them. Activated basophils release serine proteases, mouse mast cell protease 8 (mMCP-8), and mMCP-11, that are preferentially expressed by basophils rather than mast cells in spite of their names. These proteases elicit microvascular hyperpermeability and leukocyte infiltration in affected tissues, leading to inflammation. Basophil-derived IL-4 also contributes to eosinophil infiltration while it acts on tissue-infiltrating inflammatory monocytes to promote their differentiation into M2 macrophages that in turn dampen inflammation. Although basophils produce little or no MHC class II (MHC-II) proteins, they can acquire peptide-MHC-II complexes from dendritic cells via trogocytosis and present them together with IL-4 to naive CD4 T cells, leading to Th2 cell differentiation. Thus, basophils contribute to Th2 immunity at various levels.

Skin-infiltrating basophils promote atopic dermatitis-like inflammation via IL-4 production in mice.

BACKGROUND: Patients with atopic dermatitis (AD) often show the infiltration of basophils in the affected skin. Because basophils represent only a minor fraction among cellular infiltrates in the skin lesion, the functional significance of skin-infiltrating basophils in AD pathogenesis remains ill-defined. In this study, we aimed to clarify the role of basophils and their effector molecules triggering skin inflammation in oxazolone (OX)-induced murine model of AD. METHODS: A panel of mouse strains were sensitized and repeatedly challenged with topical applications of OX to induce AD-like skin inflammation. Both local and systemic Th2 immune responses were analyzed. RESULTS: Basophils progressively accumulated in the skin lesion but barely in draining lymph nodes (LNs). When basophils were depleted during the elicitation phase, skin inflammation was ameliorated while Th2 cell differentiation in draining LNs remained intact. The expression of IL-4 was highly upregulated in the affected skin, and basophils turned out to be the major producers of IL-4 among cellular infiltrates, suggesting the involvement of basophil-derived IL-4 in the Th2 skin inflammation. Indeed, basophil-specific IL-4-deficient mice displayed attenuated skin inflammation with a marked reduction of IL-4 in the skin lesion, even though cutaneous basophil infiltration and serum levels of IgE remained intact. CONCLUSIONS: Skin-infiltrating basophils promoted OX-induced AD-like skin inflammation through their local production of IL-4, rather than the induction of Th2 cell differentiation in draining LNs. This study suggests that the selective targeting of basophils could be a beneficial strategy in the treatment of a certain type of AD.

Aggregation makes a protein allergenic at the challenge phase of basophil-mediated allergy in mice.

A hapten is a small molecule that is not immunogenic on its own but can stimulate the production of antibodies at the sensitization phase when conjugated to carrier proteins. The hapten then reacts specifically with the antibodies generated against it to elicit an immune or allergic response at the challenge phase. Here, we compared various carrier proteins conjugated with the same hapten in their ability to induce hapten-specific IgE-mediated allergic responses in vitro and in vivo, and characterized the nature of carrier proteins that determines the magnitude of response at the challenge phase of allergic reactions. Hapten 2,4,6-trinitrophenol (TNP)-conjugated ovalbumin (TNP-OVA) and bovine serum albumin (TNP-BSA) elicited TNP-specific, mast cell-dependent, immediate-type allergic reactions at a comparable level in mice that had been passively sensitized with TNP-specific IgE. In contrast, TNP-OVA but not TNP-BSA efficiently induced a basophil-dependent, IgE-mediated chronic allergic inflammation (IgE-CAI), even though both proteins could stimulate basophils in vitro at a comparable level. By comparing different carrier proteins and structurally modifying them, we found that the formation of large aggregates is crucial for TNP-conjugated carrier proteins to efficiently elicit IgE-CAI, regardless of the type of protein. Thus, the aggregation status of carrier proteins appears to determine the magnitude of allergic response at the challenge phase of hapten-specific IgE-CAI. Our findings suggest that the allergenicity of substances is a matter of importance not only at the sensitization but also at the challenge phase in a certain type of allergy including a basophil-mediated allergic inflammation.

Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils.

Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II-expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide-MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

How do basophils contribute to Th2 cell differentiation and allergic responses?

Basophils and mast cells share some features, including basophilic granules in the cytoplasm, cell surface expression of the high-affinity IgE receptor and release of chemical mediators such as histamine. Because of this similarity and their minority status, basophils had often been erroneously considered as minor relatives or blood-circulating precursors of tissue-resident mast cells, and therefore long been neglected or underestimated in immunological studies. Taking advantage of newly developed tools, such as basophil-depleting antibodies and engineered mice deficient for only basophils, recent studies have identified previously unappreciated roles for basophils, distinct from those played by mast cells, in allergic responses, protective immunity against parasitic infections and regulation of other immune cells. In this review, we focus on two topics that we presented and discussed in the 46th Annual Meeting of the Japanese Society for Immunology held in Sendai in December 2017. The first topic is the function of basophils as antigen-presenting cells for driving Th2 cell differentiation. We demonstrated that basophils produce few or no MHC class II (MHC-II) proteins by themselves although they can acquire peptide-MHC-II complexes from dendritic cells through trogocytosis, and present them and provide IL-4 to naive CD4 T cells, promoting Th2 cell differentiation. The second topic is the basophil-specific effector molecules involved in allergic responses. Among mouse mast cell proteases (mMCPs), mMCP-8 and mMCP-11 are expressed almost exclusively by basophils. Analyses in vitro and in vivo revealed that both proteases can induce leukocyte migration through distinct mechanisms, contributing to the development of basophil-dependent allergic inflammation.

Multifaceted roles of basophils in health and disease.

Until recently, basophils had often been neglected in immunologic studies because of their minority status among immune cells or confused with tissue-resident mast cells because of some phenotypic similarities between them in spite of different anatomic localization. It is now appreciated that basophils and mast cells are distinct cell lineages and that basophils play important and nonredundant roles distinct from those played by mast cells. On the one hand, basophils contribute beneficially to protective immunity, particularly against parasitic infections. On the other hand, basophils are involved in the development of various disorders, including allergy and autoimmune disease. Basophils interact with other immune cells and nonhematopoietic cells through cell-to-cell contact or basophil-derived factors, such as cytokines and proteases, contributing to the regulation of immune and allergic responses. In this review article we highlight recent advances in our understanding of basophil pathophysiology in human subjects and animal models by consolidating research findings reported during the past 5 years. Further studies on basophils and their products will help identify suitable targets for novel therapeutics in allergy and effective vaccines against parasitic infection.

The Basophil-specific Protease mMCP-8 Provokes an Inflammatory Response in the Skin with Microvascular Hyperpermeability and Leukocyte Infiltration.

Basophils have often been erroneously considered to be minor relatives or blood-circulating precursors of tissue-resident mast cells because of some phenotypic similarity between them, including basophilic secretory granules in the cytoplasm. However, recent studies revealed that the repertoire of serine proteases stored in secretory granules is distinct in them. Particularly, mouse mast cell protease 8 (mMCP-8) is specifically expressed by basophils but not mast cells despite its name. Therefore, mMCP-8 is commonly used as a basophil-specific marker, but its functional property remains uncertain. Here we prepared recombinant mMCP-8 and examined its activity in vitro and in vivo Purified recombinant mMCP-8 showed heat-sensitive proteolytic activity when α-tubulin was used as a substrate. One intradermal shot of mMCP-8, not heat-inactivated, induced cutaneous swelling with increased microvascular permeability in a cyclooxygenase-dependent manner. Moreover, repeated intradermal injection of mMCP-8 promoted skin infiltration of leukocytes, predominantly neutrophils and, to a lesser extent, monocytes and eosinophils, in conjunction with up-regulation of chemokine expression in the skin lesion. These results suggest that mMCP-8 is an important effector molecule in basophil-elicited inflammation, providing novel insights into how basophils exert a crucial and non-redundant role, distinct from that played by mast cells, in immune responses.

MIP-1α/CCL3-expressing basophil-lineage cells drive the leukemic hematopoiesis of chronic myeloid leukemia in mice.

Basophilia is a frequently observed hematological abnormality in chronic myeloid leukemia (CML), but its pathophysiological roles are undefined. We previously demonstrated that an inflammatory chemokine, CCL3, preferentially acts on normal hematopoietic stem/progenitor cells and crucially contributes to the maintenance of leukemia initiating cells (LICs) in bone marrow (BM) during the initiation process of CML. However, the major cellular source of CCL3 in BM and the precise mechanism of CCL3-mediated maintenance of LICs remain to be investigated. To delineate the cellular process facilitating this CCL3-mediated crosstalk between normal and leukemic hematopoiesis, we precisely examined CCL3-expressing cells and their functions in both normal hematopoiesis and CML leukemogenesis. Herein, we demonstrate that basophils can constitutively express CCL3 to negatively regulate the normal hematopoietic process, especially hematopoietic reconstitution after BM transplantation. Moreover, CCL3-expressing basophil-like leukemia cells were found to accumulate in CML BM and supported the predominant expansion of LICs therein. These observations suggest that intra-BM basophil expansion can favor leukemia-tropic hematopoiesis in CML by providing CCL3, a potent inhibitor of normal hematopoiesis and that basophil-derived CCL3 may be a novel target molecule for the treatment of CML.

Basophil tryptase mMCP-11 plays a crucial role in IgE-mediated, delayed-onset allergic inflammation in mice.

Recent studies have identified nonredundant roles for basophils in immune responses including allergy and protective immunity. It is well known that activated basophils release granule contents such as histamine and proteases as do mast cells. However, the functional significance of basophil-derived proteases remains poorly understood in contrast to those released from mast cells. For this study we generated a line of knockout (KO) mice deficient for mouse mast cell protease-11 (mMCP-11) that is preferentially expressed by basophils rather than mast cells. In spite of normal development of basophils, the mMCP-11-deficient mice showed amelioration of immunoglobulin E-mediated chronic allergic inflammation (IgE-CAI), with reduction of cutaneous swelling, microvascular permeability, and leukocyte infiltration in the skin lesion, when KO mice were compared with wild-type mice. Repeated administration of recombinant mMCP-11 in the skin induced infiltration of leukocytes, including basophils, in a tryptase activity-dependent manner. The transwell migration assay in vitro suggested that mMCP-11-mediated proteolytic products of serum protein promoted migration of basophils, eosinophils, and macrophages via 1 or more G protein-coupled receptors. Thus, basophil tryptase mMCP-11 is a crucial effector molecule for the induction of IgE-CAI. This is the first demonstration that the basophil-derived protease plays a significant role in vivo.

Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation.

OBJECTIVE: Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide-LMIR3 interaction in the development of IBD. DESIGN: The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3(-/-), mast cell-deficient Kit(W-sh/W-sh), Kit(W-sh/W-sh)LMIR3(-/-) or Kit(W-sh/W-sh) mice engrafted with WT or LMIR3(-/-) bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. RESULTS: LMIR3 deficiency exacerbated DSS-induced colitis in mice. Kit(W-sh/W-sh) mice harbouring LMIR3(-/-) mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide-LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide-LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. CONCLUSIONS: LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3(-/-) mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide-LMIR3 binding.

Real-time imaging of mast cell degranulation in vitro and in vivo.

Mast cells undergo degranulation in response to various stimuli and rapidly release pre-formed mediators present in secretory granules, leading to immediate-type allergic reactions. Mast cell degranulation is commonly detected and quantified in vitro by measuring histamine or ß-hexosaminidase released to culture medium. However, this type of assay cannot monitor degranulation of individual cells in real time, and it is not suitable for in vivo detection of degranulation. At the aim of real time imaging of mast cell degranulation at single cell level, we here developed a fluorescent protein-based indicator of degranulation, designated immuno-pHluorin (impH). When expressed in mast cells, impH is located in the membrane of secretory granules and non-fluorescent under homeostatic conditions while it turns fluorescent following degranulation, due to the pH change inside of granules during exocytosis. impH enabled us to detect polarized degranulation within one single cell when mast cells were stimulated via the small area of cell surface. Transplantation of impH-expressing mast cells into mast cell-deficient mice demonstrated that impH could function as a real-time indicator of degranulation in vivo. Thus, impH is a useful tool for imaging of mast cell activation and degranulation in vitro and in vivo, and may be applied for screening of reagents regulating mast cell degranulation.

Large particulate allergens can elicit mast cell-mediated anaphylaxis without exit from blood vessels as efficiently as do small soluble allergens.

Anaphylaxis is a rapid-onset, life-threatening allergic reaction in that IgE, mast cells and histamine are commonly involved. It can be experimentally induced in IgE-sensitized animals by intravenous injection of corresponding allergens, and the sign of anaphylactic reaction can be detected within minutes after allergen challenge. However, it remains puzzling why the anaphylactic reaction can be initiated in vivo so quickly, considering that allergens are delivered into the blood circulation while mast cells reside within peripheral tissues but not in the blood circulation. To address this issue, we compared two different forms of the same allergen, small soluble and large particulate ones, in their ability to induce anaphylaxis in IgE-sensitized mice. In contrast to our expectation, particulate allergens could induce anaphylaxis as quickly and efficiently as did soluble allergens, even though they remained inside of blood vessels. In vivo imaging analysis suggested the direct interaction of intravascular particulate allergens and perivascular mast cells across the capillary wall. Taken together with previous report that perivascular mast cells can capture IgE in the blood circulation by extending cell processes across the vessel wall, our findings imply that blood-circulating allergens, regardless of their size, can stimulate mast cells without exit from blood vessels, by means of cross-linking IgE on mast cell processes inserted into the vessel lumen, and hence initiate anaphylactic reaction so quickly.

Human CD300C delivers an Fc receptor-γ-dependent activating signal in mast cells and monocytes and differs from CD300A in ligand recognition.

CD300C is highly homologous with an inhibitory receptor CD300A in an immunoglobulin-like domain among the human CD300 family of paired immune receptors. To clarify the precise expression and function of CD300C, we generated antibodies discriminating between CD300A and CD300C, which recognized a unique epitope involving amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Notably, CD300C was highly expressed in human monocytes and mast cells. Cross-linking of CD300C by its specific antibody caused cytokine/chemokine production of human monocytes and mast cells. Fc receptor γ was indispensable for both efficient surface expression and activating functions of CD300C. To identify a ligand for CD300A or CD300C, we used reporter cell lines expressing a chimera receptor harboring extracellular CD300A or CD300C and intracellular CD3ζ, in which its unknown ligand induced GFP expression. Our results indicated that phosphatidylethanolamine (PE) among the lipids tested and apoptotic cells were possible ligands for both CD300C and CD300A. PE and apoptotic cells more strongly induced GFP expression in the reporter cells through binding to extracellular CD300A as compared with CD300C. Differential recognition of PE by extracellular CD300A and CD300C depended on different amino acid residues CD300A(F56-L57) and CD300C(L63-R64). Interestingly, GFP expression induced by extracellular CD300C-PE binding in the reporter cells was dampened by co-expression of full-length CD300A, indicating the predominance of CD300A over CD300C in PE recognition/signaling. PE consistently failed to stimulate cytokine production in monocytes expressing CD300C with CD300A. In conclusion, specific engagement of CD300C led to Fc receptor γ-dependent activation of mast cells and monocytes.

A soluble form of LMIR5/CD300b amplifies lipopolysaccharide-induced lethal inflammation in sepsis.

Leukocyte mono-Ig-like receptor 5 (LMIR5, also called CD300b) is an activating receptor expressed in myeloid cells. We have previously demonstrated that T cell Ig mucin 1 works as a ligand for LMIR5 in mouse ischemia/reperfusion injury of the kidneys. In this article, we show that LMIR5 is implicated in LPS-induced sepsis in mice. Notably, neutrophils constitutively released a soluble form of LMIR5 (sLMIR5) through proteolytic cleavage of surface LMIR5. Stimulation with TLR agonists augmented the release of sLMIR5. LPS administration or peritonitis induction increased serum levels of sLMIR5 in mice, which was substantially inhibited by neutrophil depletion. Thus, neutrophils were the main source of LPS-induced sLMIR5 in vivo. On the other hand, i.p. administration of LMIR5-Fc, a surrogate of sLMIR5, bound to resident macrophages (M) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal M via its unknown ligand. Interestingly, LMIR5 deficiency profoundly reduced systemic cytokine production and septic mortality in LPS-administered mice, although it did not affect in vitro cytokine production of LPS-stimulated peritoneal M. Importantly, the resistance of LMIR5-deficient mice to LPS- or peritonitis-induced septic death was decreased by LMIR5-Fc administration, implicating sLMIR5 in LPS responses in vivo. Collectively, neutrophil-derived sLMIR5 amplifies LPS-induced lethal inflammation.

Characterization of leukocyte mono-immunoglobulin-like receptor 7 (LMIR7)/CLM-3 as an activating receptor: its similarities to and differences from LMIR4/CLM-5.

Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function.

An activating and inhibitory signal from an inhibitory receptor LMIR3/CLM-1: LMIR3 augments lipopolysaccharide response through association with FcRgamma in mast cells.

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of Fc epsilonRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by Fc epsilonRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRgamma. Analysis of FcRgamma-deficient BMMCs demonstrated that both Y276/303 and FcRgamma played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRgamma and thereby functions as an activating receptor in concert with TLR4 stimulation.