Biomedical subjects

Gordon D Brown

Publications and source records attributed to Gordon D Brown.

At least 19 recordsLinked to original sources

PubMed2024

The Dectin-1 and Dectin-2 clusters: C-type lectin receptors with fundamental roles in immunity.

The ability of myeloid cells to recognize and differentiate endogenous or exogenous ligands rely on the presence of different transmembrane protein receptors. C-type lectin receptors (CLRs), defined by the presence of a conserved structural motif called C-type lectin-like domain (CTLD), are a crucial family of receptors involved in this process, being able to recognize a diverse range of ligands from glycans to proteins or lipids and capable of initiating an immune response. The Dectin-1 and Dectin-2 clusters involve two groups of CLRs, with genes genomically linked within the natural killer cluster of genes in both humans and mice, and all characterized by the presence of a single extracellular CTLD. Fundamental immune cell functions such as antimicrobial effector mechanisms as well as internalization and presentation of antigens are induced and/or regulated through activatory, or inhibitory signalling pathways triggered by these receptors after ligand binding. In this review, we will discuss the most recent concepts regarding expression, ligands, signaling pathways and functions of each member of the Dectin clusters of CLRs, highlighting the importance and diversity of their functions.

Lectins, C-Type↗

PubMed2006

Dectin-1 is required for beta-glucan recognition and control of fungal infection.

Beta-glucan is one of the most abundant polysaccharides in fungal pathogens, yet its importance in antifungal immunity is unclear. Here we show that deficiency of dectin-1, the myeloid receptor for beta-glucan, rendered mice susceptible to infection with Candida albicans. Dectin-1-deficient leukocytes demonstrated significantly impaired responses to fungi even in the presence of opsonins. Impaired leukocyte responses were manifested in vivo by reduced inflammatory cell recruitment after fungal infection, resulting in substantially increased fungal burdens and enhanced fungal dissemination. Our results establish a fundamental function for beta-glucan recognition by dectin-1 in antifungal immunity and demonstrate a signaling non-Toll-like pattern-recognition receptor required for the induction of protective immune responses.

Animals↗

PubMed2006

Soluble Dectin-1 as a tool to detect beta-glucans.

Beta-glucans are structural components of fungal cell walls which are involved in the immune recognition of fungal pathogens and possess beneficial immunomodulatory activities in isolated form. Here we have developed a soluble chimeric form of the major mammalian beta-glucan receptor, Dectin-1, and demonstrate its application for the detection and characterisation of soluble and insoluble beta-glucans, including fungal particles, using ELISA, flow cytometric and fluorescence-based microscopy assays.

Antibody Specificity↗

PubMed2006

Expression of functionally different dectin-1 isoforms by murine macrophages.

Dectin-1 is a specific receptor for beta-glucans and a major receptor for fungal particles on macrophages (Mphi). It is a type II membrane receptor that has a C-terminal, NK-like, C-type lectin-like domain separated from the cell membrane by a short stalk region and a cytoplasmic immunoreceptor tyrosine-based activation-like motif. We observed functional differences in dectin-1-dependent recognition of fungal particles by Mphi from different mouse strains. RT-PCR analysis revealed that mice have at least two splice forms of dectin-1, generated by differential usage of exon 3, encoding the full-length dectin-1A and a stalkless Mphi dectin-1B. Mphi from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mphi from C57BL/6 and related mice mainly expressed the smaller isoform. NIH-3T3 fibroblast and RAW264.7 macrophage cell lines stably expressing either isoform were able to bind and phagocytose zymosan at 37 degrees C. However, binding by the smaller dectin-1B isoform was significantly affected at lower temperatures. These properties were shared by the equivalent human isoforms. The relative ability of each of the isoforms to induce TNF-alpha production in RAW264.7 Mphi was also found to be different. These results are the first evidence that dectin-1 isoforms are functionally distinct and indicate that differential isoform usage may represent a mechanism of regulating cellular responses to beta-glucans.

Animals↗

PubMed2006

Prolonged reduction of leukocyte membrane-associated Dectin-1 levels following beta-glucan administration.

Dectin-1 is the primary pattern recognition receptor for fungal glucans. Dectin-1 mediates the internalization and biological response to glucans. We examined the effect of i.v. or i.p. glucan phosphate (GP) administration on Dectin-1 membrane expression in murine peripheral blood leukocytes, splenocytes, bone marrow, and peritoneal cells from 3 h to 10 days after injection. Circulating leukocytes were also examined for uptake and internalization of glucans from the blood. Fluorescent-labeled GP was taken up from the systemic circulation by circulating peripheral leukocytes, splenocytes, and peritoneal cells. Following internalization, glucan colocalized with Dectin-1 in an intracellular vesicle. A single parenteral injection of GP resulted in a significant reduction (approximately 33-85%) in peripheral leukocyte membrane-associated Dectin-1 positivity that lasted for up to 7 days. The loss of leukocyte membrane-associated Dectin-1 after GP administration was primarily due to decreased levels of Dectin-1 on neutrophil and monocyte membranes with no significant changes in the percentage of neutrophils or monocytes circulating in the blood. Administration of control carbohydrate polymers, i.e., mannan or pullulan, which are not ligands for Dectin-1, did not decrease Dectin-1 leukocyte positivity, indicating that the effect on Dectin-1 is specific to glucans. In fact, mannan administration increased leukocyte Dectin-1 positivity, thus demonstrating a differential effect on leukocyte Dectin-1, compared with GP. We conclude that systemic administration of GP has a specific and prolonged effect on loss of leukocyte membrane Dectin-1 positivity. These data may have important implications for developing dosing regimens for immunomodulatory carbohydrates.

Animals↗

PubMed2006

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose.

We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC(50) for mannose of approximately 20 mM compared to an IC(50) of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man(9)GlcNAc(2)). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.

Binding, Competitive↗

PubMed2006

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor.

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.

Animals↗

PubMed2006

Human MICL (CLEC12A) is differentially glycosylated and is down-regulated following cellular activation.

C-type lectins are the most diverse and prevalent lectin family in immunity. Particular interest has recently been attracted by the C-type lectin-like receptors on NK cells, which appear to regulate the activation/inhibitory balance of these cells, controlling cytotoxicity and cytokine production. We previously identified a human C-type lectin-like receptor, closely related to both the beta-glucan receptor and the lectin-like receptor for oxidized-LDL, named MICL (myeloid inhibitory C-type lectin-like receptor), which we had shown using chimeric analysis to function as an inhibitory receptor. Using a novel MICL-specific monoclonal antibody, we show here that human MICL is expressed primarily on myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Although MICL was highly N-glycosylated in primary cells, the level of glycosylation was found to vary between cell types. MICL surface expression was down-regulated during inflammatory/activation conditions in vitro, as well as during an in vivo model of acute inflammation, which we characterize here. This suggests that human MICL may be involved in the control of myeloid cell activation during inflammation.

Animals↗

PubMed2006

Dectin-1: a signalling non-TLR pattern-recognition receptor.

Dectin-1 is a natural killer (NK)-cell-receptor-like C-type lectin that is thought to be involved in innate immune responses to fungal pathogens. This transmembrane signalling receptor mediates various cellular functions, from fungal binding, uptake and killing, to inducing the production of cytokines and chemokines. These activities could influence the resultant immune response and can, in certain circumstances, lead to autoimmunity and disease. As I discuss here, understanding the molecular mechanisms behind these functions has revealed new concepts, including collaborative signalling with the Toll-like receptors (TLRs) and the use of spleen tyrosine kinase (SYK), that have implications for the role of other non-TLR pattern-recognition receptors in immunity.

Animals↗

PubMed2006

C-type lectin-like receptors on myeloid cells.

Host defence against pathogens requires the recognition of conserved microbial molecules, or 'pathogen-associated molecular patterns' (PAMPs), by their receptors termed 'pattern recognition receptors' (PRRs), represented most notably by toll-like receptors (TLRs) and C-type lectins. The 'non-classical' C-type lectins (these that lack the residues involved in calcium binding, required for carbohydrate binding) are traditionally thought of as being restricted to natural killer (NK) or T cells, playing important roles in immune surveillance. In recent years, however, a growing number of these receptors have been identified on myeloid cells, both of human and mouse origin. In contrast to their NK counterparts that primarily control cellular activation through recognition of major histocompatibility antigen (MHC) class I and related molecules, the myeloid-expressed receptors appear to have a far more diverse range of functions and ligands, including those of exogenous origin. Some of C-type lectin-like molecules possess activating/inhibitory signalling motifs that trigger downstream signalling events, suggesting the role for these receptors as positive/negative regulators of granulocyte and monocyte functions. With the exception of a few myeloid NK-like lectins, the natural ligands for most of these receptors remain unidentified, making it difficult to define their functions in normal physiological, inflammatory or pathological conditions. Importantly, in some cases, these novel C-type lectin-like lectins, encoded by genes from the same gene cluster, can act as receptor/ligand pairs, additionally contributing to the regulation of myeloid cell functions or their interaction with other (like NK) cell types. However, the relevance and importance of such interactions still needs to be assessed. Although few of the myeloid-expressed C-type lectins have been characterized in detail, we review here each of these receptors and highlight their prospective roles in innate and adaptive immunity.

Animals↗

PubMed2005

Ligands for the beta-glucan receptor, Dectin-1, assigned using "designer" microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides.

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-alpha secretion by a Dectin-1-expressing macrophage cell line.

Animals↗

PubMed2005

The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus.

Alveolar macrophages represent a first-line innate host defense mechanism for clearing inhaled Aspergillus fumigatus from the lungs, yet contradictory data exist as to which alveolar macrophage recognition receptor is critical for innate immunity to A. fumigatus. Acknowledging that the A. fumigatus cell wall contains a high beta-1,3-glucan content, we questioned whether the beta-glucan receptor dectin-1 played a role in this recognition process. Monoclonal antibody, soluble receptor, and competitive carbohydrate blockage indicated that the alveolar macrophage inflammatory response, specifically the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, CXCL2/macrophage inflammatory protein-2 (MIP-2), CCL3/macrophage inflammatory protein-1alpha (MIP-1alpha), granulocyte-colony stimulating factor (G-CSF), and granulocyte monocyte-CSF (GM-CSF), to live A. fumigatus was dependent on recognition via the beta-glucan receptor dectin-1. The inflammatory response was triggered at the highest level by A. fumigatus swollen conidia and early germlings and correlated to the levels of surface-exposed beta glucans, indicating that dectin-1 preferentially recognizes specific morphological forms of A. fumigatus. Intratracheal administration of A. fumigatus conidia to mice in the presence of a soluble dectin-Fc fusion protein reduced both lung proinflammatory cytokine/chemokine levels and cellular recruitment while modestly increasing the A. fumigatus fungal burden, illustrating the importance of beta-glucan-initiated dectin-1 signaling in defense against this pathogen. Collectively, these data show that dectin-1 is centrally required for the generation of alveolar macrophage proinflammatory responses to A. fumigatus and to our knowledge provides the first in vivo evidence for the role of dectin-1 in fungal innate defense.

Aspergillosis↗

PubMed2005

Fish and chips: various methodologies demonstrate utility of a 16,006-gene salmonid microarray.

BACKGROUND: We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. RESULTS: We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. CONCLUSION: We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.

Animals↗

PubMed2005

Glycosylation influences the lectin activities of the macrophage mannose receptor.

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.

Animals↗

PubMed2005

Oral delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge.

Glucans are immunomodulatory carbohydrates found in the cell walls of fungi and certain bacteria. We examined the pharmacokinetics of three water-soluble glucans (glucan phosphate, laminarin, and scleroglucan) after oral administration of 1 mg/kg doses in rats. Maximum plasma concentrations for glucan phosphate occurred at 4 h. In contrast, laminarin and scleroglucan showed two plasma peaks between 0.5 and 12 h. At 24 h, 27 +/- 3% of the glucan phosphate and 20 +/- 7% of the laminarin remained in the serum. Scleroglucan was rapidly absorbed and eliminated. The liver did not significantly contribute to the clearance of plasma glucan. Biological effects were further studied in mice. Following oral administration of 1 mg, glucans were bound and internalized by intestinal epithelial cells and gut-associated lymphoid tissue (GALT) cells. Internalization of glucan by intestinal epithelial cells was not Dectin-dependent. GALT expression of Dectin-1 and toll-like receptor (TLR) 2, but not TLR4, increased following oral administration of glucan. Oral glucan increased systemic levels of interleukin (IL)-12 (151 +/- 15%) in mice. Oral glucan administration also increased survival in mice challenged with Staphylococcus aureus or Candida albicans. These data demonstrate that orally administered water-soluble glucans translocate from the gastrointestinal (GI) tract into the systemic circulation. The glucans are bound by GI epithelial and GALT cells, and they modulate the expression of pattern recognition receptors in the GALT, increase IL-12 expression, and induce protection against infectious challenge.

Administration, Oral↗

PubMed2005

A role for fungal {beta}-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice.

A combination of genetic and environmental factors can cause autoimmune disease in animals. SKG mice, which are genetically prone to develop autoimmune arthritis, fail to develop the disease under a microbially clean condition, despite active thymic production of arthritogenic autoimmune T cells and their persistence in the periphery. However, in the clean environment, a single intraperitoneal injection of zymosan, a crude fungal beta-glucan, or purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice, but only transient arthritis in normal mice. Blockade of Dectin-1, a major beta-glucan receptor, can prevent SKG arthritis triggered by beta-glucans, which strongly activate dendritic cells in vitro in a Dectin-1-dependent but Toll-like receptor-independent manner. Furthermore, antibiotic treatment against fungi can prevent SKG arthritis in an arthritis-prone microbial environment. Multiple injections of polyinosinic-polycytidylic acid double-stranded RNA also elicit mild arthritis in SKG mice. Thus, specific microbes, including fungi and viruses, may evoke autoimmune arthritis such as rheumatoid arthritis by stimulating innate immunity in individuals who harbor potentially arthritogenic autoimmune T cells as a result of genetic anomalies or variations.

Animals↗

PubMed2005

The human beta-glucan receptor is widely expressed and functionally equivalent to murine Dectin-1 on primary cells.

We identified the C-type-lectin-like receptor, Dectin-1, as the major receptor for fungal beta-glucans on murine macrophages and have demonstrated that it plays a significant role in the cellular response to these carbohydrates. Using two novel, isoform-specific mAb, we show here that human Dectin-1, the beta-glucan receptor (betaGR), is widely expressed and present on all monocyte populations as well as macrophages, DC, neutrophils and eosinophils. This receptor is also expressed on B cells and a subpopulation of T cells, demonstrating that human Dectin-1 is not myeloid restricted. Both major functional betaGR isoforms - betaGR-A and betaGR-B - were expressed by these cell populations in peripheral blood; however, only betaGR-B was significantly expressed on mature monocyte-derived macrophages and immature DC, suggesting cell-specific control of isoform expression. Inflammatory cells, recruited in vivo using a new skin-window technique, demonstrated that Dectin-1 expression was not significantly modulated on macrophages during inflammation, but is decreased on recruited granulocytes. Despite previous reports detailing the involvement of other beta-glucan receptors on mature human macrophages, we have demonstrated that Dectin-1 acted as the major beta-glucan receptor on these cells and contributed to the inflammatory response to these carbohydrates.

Animals↗

PubMed2005

Dectin-2 is predominantly myeloid restricted and exhibits unique activation-dependent expression on maturing inflammatory monocytes elicited in vivo.

Dectin-2 is a recently described dendritic-cell-associated receptor, suggested to be involved in the initiation and maintenance of UV-induced tolerance. To understand the physiological relevance of the proposed functions of this C-type lectin-like receptor, we have generated monoclonal antibodies against its extracellular domain and performed a detailed study of its expression. In naive mice, Dectin-2 has a novel distribution pattern compared with other myeloid markers, but is predominantly expressed by a wide variety of tissue macrophages. Its expression was limited on dendritic cells and notably absent from brain microglia and choroid plexus or meningeal macrophages. On peripheral blood monocytes, Dectin-2 expression was very low on the surface but was transiently and markedly up-regulated on induction of inflammation in vivo using a variety of stimuli. This change in Dectin-2 expression occurs on 'inflammatory' monocytes after arrival at the inflammatory lesion as demonstrated by adoptive cell-transfer studies, and is independent of whether the macrophages elicited by the stimuli ultimately expressed Dectin-2. These observations show Dectin-2 expression to be characteristic of monocyte activation/maturation at an inflammatory lesion and provide a new perspective on the interpretation of Dectin-2 function in vivo.

Adoptive Transfer↗