A. Neil Barclay

Lymphoid/Neuronal Cell Surface OX2 Glycoprotein Recognizes a Novel Receptor on Macrophages Implicated in the Control of Their Function

The OX2 membrane glycoprotein (CD200) is expressed on a broad range of tissues including lymphoid cells, neurons, and endothelium. We report the characterization of an OX2 receptor (OX2R) that is a novel protein restricted to cells of the myeloid lineage. OX2 and its receptor are both cell surface glycoproteins containing two immunoglobulin-like domains and interact with a dissociation constant of 2.5 microM and koff 0.8 s(-1), typical of many leukocyte protein membrane interactions. Pervanandate treatment of macrophages showed that OX2R could be phosphorylated on tyrosine residues. Blockade of the OX2-OX2R interaction with an OX2R mAb exacerbated the disease model experimental allergic encephalomyelitis. These data, together with data from an OX2-deficient mouse (R. M. Hoek et al., submitted), suggest that myeloid function can be controlled in a tissue-specific manner by the OX2-OX2R interaction.

CD200 and membrane protein interactions in the control of myeloid cells.

OX2 (now designated CD200) is a membrane protein expressed by a broad range of cell types. It is the ligand for a receptor restricted to myeloid cells, with the potential to deliver inhibitory signals. This is indicated by the CD200-deficient mouse model, in which myeloid cells are more activated when stimulated immunologically than cells from normal mice. The unusual tissue distribution of CD200 indicates where myeloid cells can be restrictively controlled through cell-cell contact. Recent data on CD200 will be reviewed in the context of other proteins that might have similar roles, in particular, the interaction between CD47 and SIRPalpha (CD172a).

Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.

A new superfamily of cell surface proteins related to the nerve growth factor receptor

In this article Susan Mallett and Neil Barclay discuss the molecular and functional features of a new superfamily of membrane proteins defined by the presence of cysteine-rich motifs originally identified in the low-affinity nerve growth factor receptor. This superfamily includes two lymphocyte proteins of unknown function and two receptors for tumor necrosis factor.

Transient intercellular adhesion: the importance of weak protein-protein interactions

Intercellular adhesion is a complex phenomenon central to the development, structure and functioning of all multicellular organisms. Adhesion is mediated by distinct families of cell-adhesion molecules (CAMs), and recent studies have identified key characteristics of CAMs that influence their function. Affinity and kinetic analyses using a novel technique based on surface plasmon resonance have shown that CAM interactions that mediate transient cell adhesion may have surprisingly low affinities and extremely fast dissociation rate constants.

Signal Regulatory Proteins in the Immune System

Signal regulatory proteins (SIRPs) constitute a family of transmembrane glycoproteins with extracellular Ig-like domains. Several SIRP family members have thus far been identified on myeloid and other cells in man, mouse, rat, and cattle. In the present study, we provide a description of the SIRP multigene family, including a number of previously undescribed SIRP genes, based on the complete genome sequences of various mammalian and bird species. We discuss this information in the context of the known immunological properties of the individual SIRP family members. Our analysis reveals SIRPs as a diverse multigene family of immune receptors, which includes inhibitory SIRPα, activating SIRPβ, nonsignaling SIRPγ, and soluble SIRPδ members. For each species, there appears to be a single inhibitory SIRPα member that, upon interaction with the “self” ligand CD47, controls “homeostatic” innate immune effector functions, such as host cell phagocytosis. The activating SIRPβ proteins show considerable variability in structure and number across species and do not bind CD47. Thus the SIRP family is a rapidly evolving gene family with important roles in immune regulation.

Localisation of the MRC OX‐2 Glycoprotein on the Surfaces of Neurones

Abstract: The MRC OX‐2 monoclonal antibody recognises membrane glycoproteins of Mr 41,000 in rat brain and 47,000 on thymocytes. It also reacts with follicular dendritic cells in lymphoid organs, endothelium, smooth muscle, and B‐lymphocytes. Indirect immunoperoxidase staining of cryostat sections showed that OX‐2 antigen was present throughout the cerebellum, with staining of both grey and white matter. Blood vessels were also stained. The Purkinje cell layer appeared to be unlabelled. Double‐immunofluorescence staining of cerebellar interneurone cultures with MRC OX‐2 antibody and tetanus toxin showed that all tetanus‐positive cells (neurones) were MRC OX‐2‐positive. Glial fibrillary acidic protein‐positive astrocytes were not labelled by MRC OX‐2 antibody. Thus OX‐2 antigen is one of the few biochemically characterised components of neuronal membranes and its properties are compared with those of the neuronal membrane glycoprotein Thy‐1.

Frontline: Optimal T cell activation requires the engagement of CD6 and CD166

The T cell surface glycoprotein, CD6 binds CD166 in the first example of an interaction between a scavenger receptor cysteine‐rich domain and an immunoglobulin‐like domain. We report that in human these proteins interact with a KD =0.4–1.0 μM and Koff ≥0.4–0.63 s–1, typical of many leukocyte membrane protein interactions. CD166 also interacts in a homophilic manner but with around 100‐fold lower affinity (KD =29–48 μM and Koff ≥ 5.3 s–1). At concentrations, that will block the CD6/CD166 interaction, soluble monomeric CD6 and CD166 inhibit antigen‐specific human T cell responses. This is consistent with extracellular engagement between CD6 and CD166 being required for an optimal immune response.

Paired Receptor Specificity Explained by Structures of Signal Regulatory Proteins Alone and Complexed with Cd47.

CD47 is a widely distributed cell-surface protein that acts a marker of self through interactions of myeloid and neural cells. We describe the high-resolution X-ray crystallographic structures of the immunoglobulin superfamily domain of CD47 alone and in complex with the N-terminal ligand-binding domain of signal regulatory protein alpha (SIRPalpha). The unusual and convoluted interacting face of CD47, comprising the N terminus and loops at the end of the domain, intercalates with the corresponding regions in SIRPalpha. We have also determined structures of the N-terminal domains of SIRPbeta, SIRPbeta(2), and SIRPgamma; proteins that are closely related to SIRPalpha but bind CD47 with negligible or reduced affinity. These results explain the specificity of CD47 for the SIRP family of paired receptors in atomic detail. Analysis of SIRPalpha polymorphisms suggests that these, as well as the activating SIRPs, may have evolved to counteract pathogen binding to the inhibitory SIRPalpha receptor.

Analysis of cell-adhesion molecule interactions using surface plasmon resonance

The molecular interactions that mediate cell adhesion are often very weak, making them difficult to study. However, real-time optical biosensors based on surface plasmon resonance (SPR) are greatly facilitating the biochemical analysis of these interactions. Analysis of the T cell surface molecule CD2 has shown that adhesion molecules can interact with very low affinities (Kd approximately 100 microM) and dissociate with half lives of approximately 0.2 seconds or less. SPR has been combined with site-directed mutagenesis to delineate the interacting surfaces of CD2 and its ligand, CD48, quantify the contribution of individual residues to the binding energy, and determine the binding orientation of these surfaces in the CD2-CD48 complex. Furthermore, SPR has been combined with in situ modification of carbohydrates on purified glycoproteins to analyze the binding specificity of lectins such as CD22. Researchers have discovered the potential pitfalls of SPR, which can lead to inaccurate affinity and kinetic measurements.