Human macrophage C-type lectin forms a heteromeric receptor complex with Mincle but not Dectin-2.

MCL, Mincle and Dectin-2 are C-type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin-2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin-2 form a heterodimer in the mouse. In the rat, Dectin-2 is a pseudogene, but here, we examine the association of the three receptors in human. In co-transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide-linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin-2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences.

C-type lectin receptors Mcl and Mincle control development of multiple sclerosis-like neuroinflammation.

Pattern recognition receptors (PRRs) are crucial for responses to infections and tissue damage; however, their role in autoimmunity is less clear. Herein we demonstrate that 2 C-type lectin receptors (CLRs) Mcl and Mincle play an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Congenic rats expressing lower levels of Mcl and Mincle on myeloid cells exhibited a drastic reduction in EAE incidence. In vivo silencing of Mcl and Mincle or blockade of their endogenous ligand SAP130 revealed that these receptors' expression in the central nervous system is crucial for T cell recruitment and reactivation into a pathogenic Th17/GM-CSF phenotype. Consistent with this, we uncovered MCL- and MINCLE-expressing cells in brain lesions of MS patients and we further found an upregulation of the MCL/MINCLE signaling pathway and an increased response following MCL/MINCLE stimulation in peripheral blood mononuclear cells from MS patients. Together, these data support a role for CLRs in autoimmunity and implicate the MCL/MINCLE pathway as a potential therapeutic target in MS.

Dendritic Cell Activating Receptor 1 (DCAR1) Associates With FcεRIγ and Is Expressed by Myeloid Cell Subsets in the Rat.

Dendritic cell activating receptor-1 (DCAR1) is a cell-surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody against rat DCAR1, and used this to characterize receptor expression and function. Rat DCAR1 was expressed on minor subsets of myeloid cells in lymphoid tissue, but was uniformly expressed at a high level by eosinophils, and at a low level by neutrophils. It was expressed by eosinophils in the peritoneal cavity and the lamina propria of the gut, and by subsets of macrophages or dendritic cells at these sites. Polarization of peritoneal macrophages showed that DCAR1 expression was absent on M1 macrophages, and increased on M2 macrophages. DCAR1 could be expressed as a homodimer and its associated with the activating adaptor protein FcεRIγ. This association allowed efficient phagocytosis of antibody-coated beads. Additionally, cross-linking of DCAR1 on the surface of rat eosinophils lead to production of reactive oxygen species. These data show that DCAR1 is an activating receptor. Its expression on M2 macrophages and eosinophils suggests that it may play a role in the immune response to parasites.

B7H6 is a functional ligand for NKp30 in rat and cattle and determines NKp30 reactivity toward human cancer cell lines.

NK cells kill cancer cells and infected cells upon activation by cell surface receptors. Human NKp30 is an activating receptor expressed by all mature NK cells. The B7 family member B7H6 has been identified as one ligand for NKp30. Several alternative ligands have also been reported, and the field remains unsettled. To this end, we have identified full-length functional B7H6 orthologs in rat and cattle, demonstrated by phylogenetic analysis and transfection experiments. In cell-cell contact-dependent assays, chimeric NKp30 reporter cells responded strongly to B7H6 in rat and cattle. Likewise, rat NKp30 expressing target cells induced strong activation of B7H6 reporter cells. Together, these observations demonstrate that B7H6 is conserved as a functional ligand for NKp30 in mammalian species separated by more than 100 million years of evolution. B7H6 and NKp30 are pseudogenes in laboratory mice. The rat thus represents an attractive experimental animal model to study the NKp30-B7H6 interaction in vivo. B7H6 was widely expressed among human cancer cell lines, and the expression level correlated strongly with the activation of human NKp30 reporter cells. Furthermore, siRNA knockdown of B7H6 abolished NKp30 reporter responses, suggesting that B7H6 is the major functionally relevant expressed ligand for NKp30 on these cancer cell lines.

An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells.

INTRODUCTION: Reporter cells expressing a chimeric receptor that activates a reporter can be used for screening ligand-mediated signal transduction. In this study, we used reporter cells harboring an NFAT/lacZ construct that express ß-galactosidase when the chimeric receptor is stimulated. A colorimetric ß-galactosidase substrate, chlorophenol-red ß-d-galactopyranoside (CPRG), was used to detect enzymatic activity. Sub-optimal conditions have unfortunately extensively been reported with such reporter-based ß-galactosidase assays. Here, we aimed to improve the CPRG-based colorimetric assay such that receptor ligands could be effectively screened with reporter cells. METHODS: After stimulation of reporter cells, we determined ß-galactosidase activity by absorbance measurement of ß-galactosidase-dependent CPRG hydrolysis. We systematically examined each component in a standard lysis buffer most commonly reported for this type of reporter cells. Furthermore, we evaluated literature in the field. RESULTS: An increased CPRG substrate concentration combined with a different detergent, Saponin, and an optimal wavelength recording markedly increased the sensitivity for the detection of ß-galactosidase activity (≈4-fold increase). Moreover, the improved protocol resulted in increased linear time-dependent recording of enzymatic activity once cells had been lysed, and a more stable and reproducible assay to detect a ligand-stimulus with the reporter cells. The optimal time length of exposure to a stimulus was ligand-dependent. DISCUSSION: In conclusion, we provide an improved protocol with an optimized lysis buffer that gives up to a six-fold higher and more robust specific signal when NFAT/lacZ-based receptor-expressing reporter cells are exposed to a stimulus.

The Alzheimer's disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) are genetically linked to Alzheimer's disease. Here, we investigated whether human ApoE mediates signal transduction through human and murine TREM2 and sought to identify a TREM2-binding domain in human ApoE. METHODS: To investigate cell signaling through TREM2, a cell line was used which expressed an NFAT-inducible ß-galactosidase reporter and human or murine TREM2, fused to CD8 transmembrane and CD3ζ intracellular signaling domains. ELISA-based binding assays were used to determine binding affinities of human ApoE isoforms to human TREM2 and to identify a TREM2-binding domain in ApoE. RESULTS: ApoE was found to be an agonist to human TREM2 with EC50 in the low nM range, and to murine TREM2 with reduced potency. In the reporter cells, TREM2 expression was lower than in nontransgenic mouse brain. Human ApoE isoforms ε2, ε3, and ε4 bound to human TREM2 with K d in the low nM range. The binding was displaced by an ApoE-mimetic peptide (amino acids 130-149). CONCLUSIONS: An ApoE-mediated dose-dependent signal transduction through TREM2 in reporter cells was demonstrated, and a TREM2-binding region in ApoE was identified. The relevance of an ApoE-TREM2 receptor signaling pathway to Alzheimer's disease is discussed.

The Activating Human NK Cell Receptor KIR2DS2 Recognizes a ß2-Microglobulin-Independent Ligand on Cancer Cells.

The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of ß2-microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a ß2-microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after ß2-microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same ß2-microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies.

Mincle, the receptor for mycobacterial cord factor, forms a functional receptor complex with MCL and FcεRI-γ.

Upon receptor activation, the myeloid C-type lectin receptor Mincle signals via the Syk-CARD9-Bcl10-MALT1 pathway. It does so by recruiting the ITAM-bearing FcεRI-γ. The related receptor macrophage C-type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co-precipitates with FcεRI-γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI-γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI-γ increased Mincle expression and enhanced phagocytosis of Ab-coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI-γ complex is the functionally optimal form for these C-type lectin receptors on the surface of myeloid cells.

Rat macrophage C-type lectin is an activating receptor expressed by phagocytic cells.

Macrophage C-type lectin (MCL) is a membrane surface receptor encoded by the Antigen Presenting Lectin-like gene Complex (APLEC). We generated a mouse monoclonal antibody for the study of this receptor in the rat. We demonstrate that rat MCL is expressed on blood monocytes and neutrophils, as well as on several tissue macrophage populations, including alveolar and peritoneal cavity macrophages. We also demonstrate MCL expression on a subset of resident spleen macrophages. Immunohistochemistry analysis of the spleen showed staining specifically in the marginal zone and red pulp. Exposure to pro-inflammatory mediators or to yeast cell wall extract (zymosan) increased surface MCL expression on peritoneal macrophages. We characterized a rat myeloid cell line, RMW, which expresses high levels of MCL. We found that MCL co-immunoprecipitated with the activating adaptor protein FcεRIγ in these cells. Moreover, beads coated with anti-MCL antibody increased phagocytosis in the RMW cells. Together, these observations indicate that rat MCL is a receptor that activates phagocytosis in myeloid cells under inflammatory conditions.

Identification of an MHC class I ligand for the single member of a killer cell lectin-like receptor family, KLRH1.

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

The rat NK cell receptors Ly49s4 and Ly49i4 recognize nonclassical MHC-I molecules on Listeria monocytogenes-infected macrophages.

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC-I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC-I molecules on the rat macrophage cell line R2 (RT1(d)). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC-I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti-MHC-I) and AAS1 (anti-nonclassical MHC-I). IFN-γ treatment of normal R2 cells also increased the MHC-I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC-I expression on Listeria-infected cells.

The complete inventory of receptors encoded by the rat natural killer cell gene complex.

The natural killer cell gene complex (NKC) encodes receptors belonging to the C-type lectin superfamily expressed primarily by NK cells and other leukocytes. In the rat, the chromosomal region that starts with the Nkrp1a locus and ends with the Ly49i8 locus is predicted to contain 67 group V C-type lectin superfamily genes, making it one of the largest congregation of paralogous genes in vertebrates. Based on physical proximity and phylogenetic relationships between these genes, the rat NKC can be divided into four major parts. We have previously reported the cDNA cloning of the majority of the genes belonging to the centromeric Nkrp1/Clr cluster and the two telomeric groups, the Klre1-Klri2 and the Ly49 clusters. Here, we close the gap between the Nkrp1/Clr and the Klre1-Klri2 clusters by presenting the cDNA cloning and transcription patterns of eight genes spanning from Cd69 to Dectin1, including the novel Clec2m gene. The definition, organization, and evolution of the rat NKC are discussed.

Two major groups of rat NKR-P1 receptors can be distinguished based on chromosomal localization, phylogenetic analysis and Clr ligand binding.

A major subset of non-alloreactive NK cells in PVG strain rats is generally low in Ly49 receptors, but expresses the rat NKR-P1B(PVG) receptor (previously termed NKR-P1C). The NKR-P1B(+) NK subset is inhibited by a non-polymorphic target cell ligand, which we have shown here to be a C-type lectin-related molecule (Clr). Clr11 ligates two divergent NKR-P1B alleles as judged by an NFAT-driven reporter assay, and inhibits NK-cell cytotoxicity of NKR-P1B(+) NK cells. Clr11 also interacts with the prototypic NKR-P1A receptor and exerts a stimulatory influence on NK lysis. NKR-P1A and B are encoded by adjacent genes in the proximal part of the NK gene complex and show close sequence homology in their extracellular region. They diverge from another pair, NKR-P1F and -G, which is encoded by a second, distal Nkrp1 gene cluster. NKR-P1F and -G bind an overlapping panel of Clr ligands, but not Clr11. Rat Clr molecules appear to be constitutively expressed by hematopoietic cells; expression in tumor cell lines is more variable. The data show the existence of two phylogenetic groups of NKR-P1 molecules, which demonstrate conservation of ligand-binding properties independent of signaling function.

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function.

UNLABELLED: Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation. INTRODUCTION: TREM2 (triggering receptor expressed in myeloid cells-2) associates with the signaling adapter DAP12 in osteoclasts (OCs). Genetic mutation or deletion of either the TYROBP (DAP12) or TREM2 gene is associated with the human disorder of brain and bone, Nasu-Hakola disease. We and others recently showed the critical requirement for immunoreceptor tyrosine-based activation motif (ITAM) signals through DAP12 and the Fc Receptor gamma chain (FcRgamma) during OC development. Here, we further define the role of TREM2 in OC differentiation and describe a role for TREM2 in OC migration and bone resorption. MATERIALS AND METHODS: We generated monoclonal anti-mouse TREM2 antibodies (mAb), analyzed pre-osteoclasts and mature OCs for TREM2 surface expression, and determined the effect of antibody ligation on in vitro OC differentiation, resorption, and migration. TREM2 RNA interference (RNAi) was used to disrupt expression of TREM2 in pre-osteoclasts. RESULTS: Using flow cytometry, our studies reveal that TREM2 is weakly expressed on C57BL/6 bone marrow macrophages (BMMs) and is upregulated during culture with RANKL and macrophage-colony stimulating factor (M-CSF). The expression of TREM2 is unaltered in DAP12-deficient OCs. Using C57BL/6 BMMs or RAW264.7 precursors, anti-TREM2 mAb treatment with RANKL and M-CSF enhances the formation of multinuclear TRACP+ OCs compared with control mAb treatment. In contrast, these agents have no effect on DAP12-deficient precursors. Monoclonal Ab blockade of TREM2 on OCs generated from C57BL/6 BMMs results in decreased resorption of artificial calcium-phosphate substrate and dentine. Reduction of TREM2 expression in RAW264.7 cells by RNAi results in loss of OC formation in response to RANKL and M-CSF. Anti-TREM2 cross-linking enhances migration of C57BL/6 OCs and RAW246.7 OCs in response to M-CSF. CONCLUSIONS: Our studies indicate that the TREM2 receptor regulates OC multinucleation as well as resorption and migration of mature OCs. Thus, TREM2-DAP12 signals regulate both OC formation and function.

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis.

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2.

The signaling adapter protein DAP12 regulates multinucleation during osteoclast development.

UNLABELLED: Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12-associated receptors expressed by osteoclasts and examine function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts. INTRODUCTION: Osteoclasts are bone-resorbing cells derived from hematopoietic precursors in the myeloid lineage. In other myeloid cell types, the signaling adapter protein DAP12 transmits activating signals on ligation of a DAP12-associated receptor (DAR). The aim of this study was to clarify the role of DAP12 signaling during osteoclast development. MATERIALS AND METHODS: Osteoclasts from DAP12 -/- or control mice were analyzed in vitro for morphology, function, and for osteoclast markers. DARs were identified in osteoclast cultures through reverse transcriptase-polymerase chain reaction (RT-PCR). Bone density of DAP12 -/- and control mice were analyzed by microcomputed tomography. DAP12 -/- osteoclasts were retrovirally reconstituted with DAP12. RAW264.7 cells were transfected with FLAG-tagged DAP12 or TREM2 and stimulated by anti-FLAG antibody during in vitro osteoclastogenesis. RESULTS: C57BL/6 DAP12-deficient mice have higher bone mass than C57BL/6 wildtype controls. We verified the presence of DAP12 in pre-osteoclasts and osteoclasts derived from C57BL/6 or the pre-osteoclast line RAW 264.7 and identified the DARs expressed. DAP12 -/- osteoclasts developed in vitro with macrophage colony-stimulating factor (M-CSF) and RANKL formed only intensely TRACP+ mononuclear cells and failed to generate multinuclear osteoclasts. These mononuclear cells are functional osteoclast-like cells because, by RT-PCR, they express other osteoclast markers and generate resorption pits on dentine slices, although quantitative assessment of bone resorption shows decreased resorption by DAP12 -/- osteoclasts compared with C57BL/6 osteoclasts. Restoration of DAP12 expression by retroviral transduction of DAP12 -/- osteoclast precursors rescued in vitro osteoclast multinucleation. Direct stimulation of DAP12 expressed in RAW264.7 during in vitro osteoclastogenesis led to a marked increase in the number of TRACP+ multinucleated osteoclast-like cells formed. CONCLUSION: Our studies indicate that stimulation of the DAP12 adapter protein plays a significant role in formation of multinuclear osteoclasts and that DAP12 and DARs likely participate in the regulation of bony remodeling.

CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation.

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-kappaB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-beta. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and beta(3) integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.

Pattern recognition by TREM-2: binding of anionic ligands.

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

Characterization of TREM-3, an activating receptor on mouse macrophages: definition of a family of single Ig domain receptors on mouse chromosome 17.

We recently reported the cloning of two triggering receptors expressed by myeloid cells (TREM), TREM-2a and TREM-2b, which are highly homologous to each other. These receptors associate with DAP12, and ligation of TREM-2 on the surface of macrophages leads to the release of nitric oxide. Using the immunoglobulin (Ig) domain of TREM-2 to screen a mouse EST database we have isolated a novel receptor, derived from a WEHI-3 macrophage library, which shows homology to TREM-2 (20%). The DNA sequence of this receptor has been submitted to Genbank with the name TREM-3. The predicted amino acid sequence contains a single Ig domain and a transmembrane lysine residue. We found transcripts for TREM-3 in two macrophage cell lines (RAW264.7 and MT2) but not in P388D1 macrophage cells. TREM-3 transcripts could also be detected at low levels in T cell lines, but were not detectable in NK, B cell, or mast cell lines. Furthermore, in macrophage cells, transcripts for TREM-3 were up-regulated by LPS, but were down-regulated by IFN-gamma. Like TREM-1 and TREM-2, TREM-3 signals through DAP12, and when TREM-3 is transfected into an NK cell line it mediates redirected lysis. Thus, TREM-3 functions as an activating receptor. Analysis of the mouse genome reveals that the gene for TREM-3 lies adjacent to the gene for TREM-1 and in close proximity to a number of other single Ig domain receptors, including TREM-2. Thus, TREM-3 is a novel member of a family of immunoglobulin receptors that form an innate immune gene complex on chromosome 17.