Kumi Izawa

TIM1 is an endogenous ligand for LMIR5/CD300b: LMIR5 deficiency ameliorates mouse kidney ischemia/reperfusion injury

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.

Myelodysplastic syndromes are induced by histone methylation–altering ASXL1 mutations

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal–truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2–mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT–induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

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.

An Activating and Inhibitory Signal from an Inhibitory Receptor LMIR3/CLM-1: LMIR3 Augments Lipopolysaccharide Response through Association with FcRγ in Mast Cells

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of FcεRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by FcεRI 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 FcRγ. Analysis of FcRγ-deficient BMMCs demonstrated that both Y276/303 and FcRγ 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 FcRγ and thereby functions as an activating receptor in concert with TLR4 stimulation.

Functional analysis of activating receptor LMIR4 as a counterpart of inhibitory receptor LMIR3

The leukocyte mono-Ig-like receptor (LMIR) belongs to a new family of paired immunoreceptors. In this study, we analyzed activating receptor LMIR4/CLM-5 as a counterpart of inhibitory receptor LMIR3/CLM-1. LMIR4 is expressed in myeloid cells, including granulocytes, macrophages, and mast cells, whereas LMIR3 is more broadly expressed. The association of LMIR4 with Fc receptor-γ among immunoreceptor tyrosine-based activation motif-bearing molecules was indispensable for LMIR4-mediated functions of bone marrow-derived mast cells, but dispensable for its surface expression. Cross-linking of LMIR4 led to Lyn- and Syk-dependent activation of bone marrow-derived mast cells, resulting in cytokine production and degranulation, whereas that of LMIR3 did not. The triggering of LMIR4 and TLR4 synergistically caused robust cytokine production in accordance with enhanced activation of ERK, whereas the co-ligation of LMIR4 and LMIR3 dramatically abrogated cytokine production. Notably, intraperitoneal administration of lipopolysaccharide strikingly up-regulated LMIR3 and down-regulated LMIR4, whereas that of granulocyte colony-stimulating factor up-regulated both LMIR3 and LMIR4 in granulocytes. Cross-linking of LMIR4 in bone marrow granulocytes also resulted in their activation, which was enhanced by lipopolysaccharide. Collectively, these results suggest that the innate immune system is at least in part regulated by the qualitative and quantitative balance of the paired receptors LMIR3 and LMIR4.

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K−) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K−, we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K−-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K− probe could be useful in investigating stem cells as well as quiescent cells.

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 KitW-sh/W-sh, KitW-sh/W-shLMIR3−/− or KitW-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. KitW-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.

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 (Mϕ) and stimulated transient inflammation in mice. Consistently, LMIR5-Fc induced in vitro cytokine production of peritoneal Mϕ 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 Mϕ. 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.

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

Background: Human CD300C is not fully characterized because of the unavailability of its specific antibody. Results: Stimulation with a specific CD300C antibody activates human monocytes and mast cells that express high levels of CD300C. Conclusion: Specific engagement of CD300C, but not its co-engagement with CD300A, delivers an Fc receptor-γ-dependent activating signal. Significance: The activating function of CD300C is associated with its ligand specificity. 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.

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.