Delyth M. Reid

Dectin-1 Is A Major β-Glucan Receptor On Macrophages

Zymosan is a β-glucan– and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti–Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan–dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

The C-type Lectin Receptor CLECSF8 (CLEC4D) Is Expressed by Myeloid Cells and Triggers Cellular Activation through Syk Kinase

Background: C-type lectins play important roles in immunity and homeostasis. Results: CLECSF8 is expressed on neutrophils and monocytes and can mediate phagocytosis, the respiratory burst and inflammatory cytokine production, in part through association with a novel adaptor. Conclusion: CLECSF8 can trigger cellular activation. Significance: This study identifies a novel C-type lectin that can control immune cell function. CLECSF8 is a poorly characterized member of the “Dectin-2 cluster” of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor.

Structure-Function Relationships and Localization of the Na/Ca-K Exchanger in Rod Photoreceptors

The structural and functional properties of the bovine rod photoreceptor Na/Ca-K exchanger and its distribution in vertebrate photoreceptor cells were studied using a panel of monoclonal antibodies. Antibodies that bind to distinct epitopes along the large hydrophilic N-terminal segment of the exchanger labeled the extracellular surface of the rod outer segment plasma membrane, whereas antibodies against a large hydrophilic loop between the two membrane domains labeled the intracellular side. Enzymatic deglycosylation studies indicated that the exchanger primarily containsO-linked sialo-oligosaccharides located within the N-terminal domain. Removal of the extracellular domain with trypsin or the large intracellular domain with kallikrein did not alter the Na+- or K+-dependent Ca2+ efflux activity of the exchanger when reconstituted into lipid vesicles. Anti-exchanger antibodies were also used to visualize the distribution of the exchanger in the retina by light and electron microscopy. The exchanger was localized to the plasma membrane of rod outer segments. No labeling was observed in the disk membranes, cone photoreceptor cells, or other retinal neurons, and only faint staining was seen in the rod inner segment. These results indicate that the O-linked glycosylated rod Na/Ca-K exchanger is specifically targeted to the plasma membrane of rod photoreceptors and has a topological organization similar to that reported for the cardiac Na/Ca exchanger. The large intracellular and extracellular domains do not directly function in the transport of ions across the rod outer segment plasma membrane, but instead may play a role in protein-protein interactions that maintain the spatial organization of the exchanger in the plasma membrane or possibly regulate transport activity of the exchanger.

Characterization and application of an in vitro detection system for studying the binding and phagocytosis of rod outer segments by retinal pigment epithelial cells

Direct and indirect radioactivity and fluorescent assays have been developed to study the interaction of rod outer segments (ROS) with retinal pigment epithelial (RPE) cells. In the direct assays ROS labelled with 125I or fluorescein isothiocyanate (FITC) have been used to measure total phagocytosis, i.e. surface binding and ingestion. In the indirect assays RPE cells were first treated with unlabelled ROS or biotinylated ROS and subsequently probed with [125I]Rho 4D2 antirhodopsin antibody or [125I]streptavidin for radioactivity measurements or with the Rho 4D2 antibody and FITC-goat anti-mouse Ig or FITC-streptavidin for fluorescent counting. In these indirect methods the number of surface bound ROS were distinguished from the number of ingested ROS by comparative labelling of non-permeabilized and permeabilized ROS-treated RPE cells. Using these assays, we have studied the binding and ingestion of bovine ROS with cultured bovine RPE cells. As in the case of newborn cultured rat RPE cells [Hall and Abrams (1987) Exp. Eye Res. 45, 907-22], binding and ingestion of bovine ROS by bovine RPE cells was saturable with respect to ROS concentration and time. At 37 degrees C ROS binding reached a saturating concentration at 1 x 10(7) ROS per well; the number of bovine ROS ingested by bovine RPE cells, however, was less than the number of rat ROS ingested by rat RPE cells. When 1 x 10(7) ROS per well was used, maximal surface binding of bovine ROS to bovine RPE cells was obtained after 2-3 hr, whereas after an initial delay, ingestion rapidly increased to a maximum at 1-2 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Recognition of DHN-melanin by a C-type lectin receptor is required for immunity to Aspergillus

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31+ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Molecular Properties of the cGMP-gated Cation Channel of Rod Photoreceptor Cells as Probed with Monoclonal Antibodies

Monoclonal antibodies have been generated against different epitopes of the cGMP-gated channel of bovine rod outer segments for use as probes to study the molecular properties of this channel protein. These monoclonal antibodies selectively labeled a 63 kDa polypeptide in both bovine rod outer segment membranes and purified channel preparations and crossreact with a 63 kDa polypeptide in rod outer segment preparations from other mammalian retinas. Immunoprecipitation studies have confirmed the identity of the 63 kDa protein as the cGMP-gated channel of rod outer segment membranes and have shown that the channel is associated with a 240 kDa protein. Solid phase radioimmune competition studies on isolated rod outer segment disks and plasma membranes have indicated that the plasma membrane has a channel density which is over 200 times greater than that in disk membranes. A monoclonal antibody against a synthetic peptide also been developed and used to study the properties of the N-terminal segment of the channel.

Detection of IL-36γ through noninvasive tape stripping reliably discriminates psoriasis from atopic eczema

This report demonstrates that sampling and detection of IL-36γ protein by non-invasive tape stripping of skin lesion provides a highly sensitive and selective diagnostic for psoriatic inflammation.