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Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b -/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b + inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

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-6C low/int peripheral blood monocytes, corresponding to CD14 dim/+ CD16 + human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115 + Ly-6C low/int monocytes. Stimulation with sphingomyelin failed to activate the CD115 + Ly-6C low/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14 dim CD16 + human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

LPS triggers inflammatory responses; however, the negative regulation of LPS responses in vivo remains poorly understood. CD300f is an inhibitory receptor among the CD300 family of paired activating and inhibitory receptors. We have previously identified ceramide as a ligand for CD300f and shown that the binding of ceramide to CD300f inhibits IgE-mediated mast cell activation and allergic responses in mouse models. Here we identify the critical role of CD300f in inhibiting LPS-induced skin inflammation. CD300f deficiency remarkably enhanced LPS-induced skin edema and neutrophil recruitment in mice. Higher levels of factors that increase vascular permeability and of factors that induce neutrophil recruitment were detected in LPS-injected skin pouch exudates of CD300f -/- mice as compared with wild-type mice. CD300f was highly expressed in mast cells and recruited neutrophils, but not in macrophages, among skin myeloid cells. CD300f deficiency failed to influence the intrinsic migratory ability of neutrophils. Ceramide-CD300f binding suppressed the release of chemical mediators from mast cells and from neutrophils in response to LPS. Adoptive transfer experiments indicated that mast cells mediated enhanced edema in LPS-stimulated skin of CD300f -/- mice, whereas mast cells together with recruited neutrophils mediated robust neutrophil accumulation. Importantly, administering a ceramide antibody or ceramide-containing vesicles enhanced or suppressed LPS-induced skin inflammation of wild-type mice, respectively. Thus, ceramide-CD300f binding inhibits LPS-induced skin inflammation, implicating CD300f as a negative regulator of Toll-like receptor 4 (TLR4) signaling in vivo ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

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.

Leukocyte mono-immunoglobulin (Ig)-like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc-induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1-LMIR5 interaction plays a physiological role in immune regulation by myeloid 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.