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Hsi-Hsien Lin

Publications and source records attributed to Hsi-Hsien Lin.

15 recordsLinked to original sources
PubMed2006

CD312, the human adhesion-GPCR EMR2, is differentially expressed during differentiation, maturation, and activation of myeloid cells.

EMR2/CD312 is a member of the adhesion-GPCR family that contains extracellular EGF-like domains. Previously it has been shown to interact with chondroitin sulphate glycosaminoglycans in an isoform-specific manner. Although EMR2 expression has been found to be restricted to human myeloid cells, its expression profile has not yet been systemically characterized. In this report, we show that EMR2 receptor expression is up-regulated during differentiation and maturation of macrophages, and is conversely down-regulated during dendritic cell maturation. We also demonstrate that EMR2 receptor alternative splicing and glycosylation is regulated during myeloid differentiation. In monocytes and macrophages, EMR2 can be specifically up-regulated by LPS and IL-10 via an IL-10-mediated pathway. In inflamed tissues, EMR2 is detected in subpopulations of myeloid cells including macrophages and neutrophils. The results presented here further support the idea that EMR2 plays a role in the migration and adhesion of myeloid cells during cell differentiation, maturation, and activation.

Cell Differentiation↗
PubMed2006

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1alpha and tumour necrosis factor-alpha) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation.

Antibodies, Monoclonal↗
PubMed2005

The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)-associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80(-/-) mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80(-/-) APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80(-/-) mice generated an efferent CD8(+) T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80(-/-) mice by adoptive transfer of F4/80(+) APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8(+) T reg cells.

Adoptive Transfer↗
PubMed2005

Identification of the epidermal growth factor-TM7 receptor EMR2 and its ligand dermatan sulfate in rheumatoid synovial tissue.

OBJECTIVE: EMR2 and CD97 are closely related members of the epidermal growth factor (EGF)-TM7 family of adhesion class 7-span transmembrane (TM7) receptors. Chondroitin sulfates (CS) have recently been identified as ligands for EMR2 and CD97. CS have been implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the expression of EMR2 and the distribution of EMR2 and CD97 ligands within RA synovial tissue (ST). METHODS: ST samples were obtained by arthroscopy from 19 patients with RA, 13 patients with inflammatory osteoarthritis (OA), and 13 patients with reactive arthritis (ReA). Immunohistochemistry was performed with a monoclonal antibody against EMR2, and stained STs were analyzed by digital image analysis. Coexpression of EMR2 with cell lineage- and activation-specific markers was determined by double immunofluorescence microscopy. To evaluate the expression of EMR2 and CD97 ligands in RA synovium, binding assays were performed using EMR2- and CD97-specific multivalent fluorescent probes. RESULTS: EMR2 expression in the synovial sublining was found to be significantly higher in RA patients compared with OA and ReA control patients. Most EMR2+ cells were macrophages and dendritic cells expressing costimulatory molecules and tumor necrosis factor alpha. Dermatan sulfate was shown to be the ligand of the largest isoforms of EMR2 and CD97 in rheumatoid synovium. In addition, the smaller isoforms of CD97, but not those of EMR2, bound CD55 on fibroblast-like synoviocytes. CONCLUSION: The EGF-TM7 receptors EMR2 and CD97 are abundantly expressed on myeloid cells in ST of RA patients where their cognate ligands dermatan sulfate and CD55 are detected. These results suggest that these interactions may facilitate the retention of activated macrophages in the synovium.

Aged↗
PubMed2004

Expression of the largest CD97 and EMR2 isoforms on leukocytes facilitates a specific interaction with chondroitin sulfate on B cells.

The EGF-TM7 receptors CD97 and EMR2 are heptahelical molecules predominantly expressed on leukocytes. A characteristic of these receptors is their ability to interact with cellular ligands via the N-terminal epidermal growth factor (EGF)-like domains. The first two EGF domains of CD97 (but not EMR2) bind CD55 (decay-accelerating factor), while the fourth EGF domain of both CD97 and EMR2 interacts with the glycosaminoglycan chondroitin sulfate (CS). Using fluorescent beads coated with soluble recombinant CD97 and EMR2 protein, and isoform-specific monoclonal antibodies, we have determined the cellular and molecular characteristics of the interaction with CS. The fourth EGF domain of CD97 and EMR2 is expressed on activated lymphocytes and myeloid cells, whereas the ligand is specifically found on B cells within the peripheral blood. The interaction between CD97/EMR2 and CS may therefore play a role in the interaction of activated T cells, dendritic cells, and macrophages with B cells.

Antibodies, Monoclonal↗
PubMed2004

Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif.

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu downward arrow Ser(518) cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P(+1) cleavage site (Ser(518)) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P(-2) His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.

Amino Acid Motifs↗
PubMed2004

Identification and characterization of a novel human myeloid inhibitory C-type lectin-like receptor (MICL) that is predominantly expressed on granulocytes and monocytes.

Inhibitory and activatory C-type lectin-like receptors play an important role in immunity through the regulation of leukocytes. Here, we report the identification and characterization of a novel myeloid inhibitory C-type lectin-like receptor (MICL) whose expression is primarily restricted to granulocytes and monocytes. This receptor, which contains a single C-type lectin-like domain and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, is related to LOX-1 (lectin-like receptor for oxidized low density lipoprotein-1) and the beta-glucan receptor (Dectin-1) and is variably spliced and highly N-glycosylated. We demonstrate that it preferentially associates with the signaling phosphatases SHP-1 and SHP-2, but not with SHIP. Novel chimeric analyses with a construct combining MICL and the beta-glucan receptor show that MICL can inhibit cellular activation through its cytoplasmic immunoreceptor tyrosine-based inhibitory motif. These data suggest that MICL is a negative regulator of granulocyte and monocyte function.

Alternative Splicing↗
PubMed2003

Modulation of macrophage differentiation and activation by decoy receptor 3.

Decoy receptor 3 (DcR3) is a soluble receptor of the tumor necrosis factor receptor superfamily and is readily detected in certain cancer patients. Recently, we demonstrated that DcR3.Fc-treated dendritic cells skew T cell responses to a T helper cell type 2 phenotype. In this study, we further asked its ability to modulate CD14+ monocyte differentiation into macrophages induced by macrophage-colony stimulating factor in vitro. We found that DcR3.Fc was able to modulate the expression of several macrophage markers, including CD14, CD16, CD64, and human leukocyte antigen-DR. In contrast, the expression of CD11c, CD36, CD68, and CD206 (mannose receptor) was not affected in the in vitro culture system. Moreover, phagocytic activity toward immune complexes and apoptotic bodies as well as the production of free radicals and proinflammatory cytokines in response to lipopolysaccharide were impaired in DcR3.Fc-treated monocyte-derived macrophages. This suggests that DcR3.Fc might have potent, suppressive effects to down-regulate the host-immune system.

Antigens, CD↗
PubMed2003

The EGF-TM7 family: a postgenomic view.

With the human and mouse genome projects now completed, the receptor repertoire of mammalian cells has finally been elucidated. The EGF-TM7 receptors are a family of class B seven-span transmembrane (TM7) receptors predominantly expressed by cells of the immune system. Within the large TM7 superfamily, the molecular structure and ligand-binding properties of EGF-TM7 receptors are unique. Derived from the processing of a single polypeptide, they are expressed at the cell surface as heterodimers consisting of a large extracellular region associated with a TM7 moiety. Through a variable number of N-terminal epidermal growth factor (EGF)-like domains, EGF-TM7 receptors interact with cellular ligands such as CD55 and chondroitin sulfate. Recent in vivo studies demonstrate a role of the EGF-TM7 receptor CD97 in leukocyte migration. The different number of EGF-TM7 genes in man compared with mice, the chimeric nature of EMR2 and the inactivation of human EMR4 point toward a still-evolving receptor family. Here we discuss the currently available information on this intriguing receptor family.

Amino Acid Sequence↗
PubMed2003

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide.

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.

Adjuvants, Immunologic↗
PubMed2003

Proteolytic cleavage of the EMR2 receptor requires both the extracellular stalk and the GPS motif.

EMR2 is a human myeloid-restricted member of the EGF-TM7 receptor family that contains a highly conserved G protein-coupled receptor proteolysis site (GPS) in the membrane-proximal region. Here the post-translational proteolytic cleavage of EMR2 at GPS was investigated. We show the cleavage occurs at Leu517-Ser518 and is independent of the transmembrane domains. The non-covalent association of the resulting extracellular alpha-subunit and transmembrane beta-subunit requires a minimum of eight amino acids in the beta-subunit. The GPS motif is necessary, but not sufficient for receptor cleavage, which requires the entire extracellular stalk. Thus, an alternatively spliced EMR2 isoform with a truncated stalk fails to undergo proteolytic cleavage. Alternative splicing therefore provides a means to regulate GPS cleavage, producing receptors with two distinct structures.

Alternative Splicing↗
PubMed2003

The epidermal growth factor-like domains of the human EMR2 receptor mediate cell attachment through chondroitin sulfate glycosaminoglycans.

Using multivalent protein probes, an evolutionarily conserved endogenous ligand for EMR2, a human myeloid cell-restricted EGF-TM7 receptor, was identified on the surface of a number of adherent cell lines. In addition, in situ staining of the ligand has revealed specific in vivo patterns consistent with a connective tissue distribution. The interaction is conserved across species and mediated exclusively by the largest EMR2 isoform containing 5 epidermal growth factor (EGF)-like modules. Antibody-blocking studies subsequently revealed that the fourth EGF-like module constitutes the major ligand-binding site. The largest isoform of CD97, a related EGF-TM7 molecule containing an identical EGF-like module, also binds to the putative EMR2 ligand. Through the use of mutant Chinese hamster ovary (CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as the enzymatic removal of specific cell surface GAGs, the molecular identity of the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS GAGs blocked the EMR2-ligand interaction in a dose-dependent manner. EMR2-CS interaction is Ca2+- and sulphation-dependent and results in cell attachment. This is the first report of a GAG ligand for the TM7 receptors extending the already vast repertoire of stimuli of the GPCR superfamily.

Alternative Splicing↗
PubMed2002

EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line A20.

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.

Amino Acid Sequence↗
PubMed2002

The human EGF-TM7 family member EMR2 is a heterodimeric receptor expressed on myeloid cells.

The EGF-TM7 family is a group of class B seven-span transmembrane (TM7) receptors expressed predominantly by cells of the immune system. Family members CD97, EMR1, EMR2, EMR3, and ETL are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain by a stalk. The EGF domain region of the recently identified EMR2 differs from that of CD97 in only 6 out of 236 amino acids. Although small, this difference has been shown to alter ligand specificity. To analyze the structure and cellular distribution of EMR2, a specific monoclonal antibody (2A1) was generated. Use of 2A1 has demonstrated EMR2, like CD97, to be expressed as a heterodimeric receptor consisting of an extracellular alpha part and a TM7/cytoplasmic beta part. Analysis of EMR2 expression on primary blood leukocytes, on hematopoietic cells lines, and in situ revealed a myeloid-restricted profile. Highest expression levels were detected on the more mature CD16(+) blood monocytes, on macrophages, and on BDCA-3(+) myeloid DC, whereas little if any expression was found on granulocytes. Unlike CD97, no expression was observed on resting or activated lymphocytes. Different expression patterns and the inability of EMR2 to interact with the CD97 ligand CD55 indicate that the molecular twins EMR2 and CD97 likely have nonredundant functions.

Antibodies, Monoclonal↗