Samantha J. Allen

Mast cell–expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis

Mast cells contribute importantly to both protective and pathological IgE-dependent immune responses. We show that the mast cell–expressed orphan serpentine receptor mCCRL2 is not required for expression of IgE-mediated mast cell–dependent passive cutaneous anaphylaxis but can enhance the tissue swelling and leukocyte infiltrates associated with such reactions in mice. We further identify chemerin as a natural nonsignaling protein ligand for both human and mouse CCRL2. In contrast to other “silent” or professional chemokine interreceptors, chemerin binding does not trigger ligand internalization. Rather, CCRL2 is able to bind the chemoattractant and increase local concentrations of bioactive chemerin, thus providing a link between CCRL2 expression and inflammation via the cell-signaling chemerin receptor CMKLR1.

Regulation of Chemerin Bioactivity by Plasma Carboxypeptidase N, Carboxypeptidase B (Activated Thrombin-activable Fibrinolysis Inhibitor), and Platelets

Chemerin is a potent chemoattractant for cells expressing the serpentine receptor CMKLR1 (chemokine-like receptor 1), such as plasmacytoid dendritic cells and tissue macrophages. The bioactivity of chemerin is post-translationally regulated; the attractant circulates in blood in a relatively inactive form (prochemerin) and is activated by carboxyl-terminal proteolytic cleavage. We discovered that plasma carboxypeptidase N (CPN) and B (CPB or activated thrombin-activable fibrinolysis inhibitor, TAFIa) enhanced the bioactivity of 10-mer chemerin peptide NH2-YFPGQFAFSK-COOH by removing the carboxyl-terminal lysine (K). Sequential cleavages of either a prochemerin peptide (NH2-YFPGQFAFSKALPRS-COOH) or recombinant full-length prochemerin by plasmin and CPN/CPB substantially increased their chemotactic activities. Endogenous CPN present in circulating plasma enhanced the activity of plasmin-cleaved prochemerin. In addition, we discovered that platelets store chemerin protein and release it upon stimulation. Thus circulating CPN/CPB and platelets may potentially contribute to regulating the bioactivity of leukocyte chemoattractant chemerin, and further extend the molecular link between blood coagulation/fibrinolysis and CMKLR1-mediated immune responses.

Chemokine-receptor interactions: GPCRs, glycosaminoglycans and viral chemokine binding proteins.

A key feature of the immune system is the migration of leukocytes throughout the organism in an effort to patrol for infectious pathogens, tissue damage, and other physiological insults. This remarkable surveillance system is controlled by a family of proteins called chemokines (chemoattractant cytokines), and their respective receptors. Originally discovered because of their role in cell recruitment during inflammation, it is now well recognized that chemokines are also involved in other diverse processes including lymphocyte development and homing, organogenesis, and neuronal communication. While chemokines have evolved largely for host protection, their ability to induce cell damage and inappropriate cell recruitment, can lead to disease. Thus, there is considerable interest in developing antagonists. In this review we emphasize what is known about the structural biology of chemokines, chemokine receptors, and interactions with cell surface glycosaminoglycans. We also briefly describe their role in certain diseases and strategies for interfering with chemokine function that have emerged from mechanistic and structural understanding of their function. Finally we discuss viral mechanisms for sabotaging or manipulating the chemokine system, in part to illustrate the level of molecular mimicry that viruses have achieved and the evolutionary pressure imposed on the immune system by these pathogens.

Staphylococcus aureus-Derived Staphopain B, a Potent Cysteine Protease Activator of Plasma Chemerin

Chemerin is an attractant for cells that express the serpentine receptor CMKLR1, which include immature plasmacytoid dendritic cells (pDC) and macrophages. Chemerin circulates in the blood where it exhibits low biological activity, but upon proteolytic cleavage of its C terminus, it is converted to a potent chemoattractant. Enzymes that contribute to this conversion include host serine proteases of the coagulation, fibrinolytic, and inflammatory cascades, and it has been postulated that recruitment of pDC and macrophages by chemerin may serve to balance local tissue immune and inflammatory responses. In this work, we describe a potent, pathogen-derived proteolytic activity capable of chemerin activation. This activity is mediated by staphopain B (SspB), a cysteine protease secreted by Staphylococcus aureus. Chemerin activation is triggered by growth medium of clinical isolates of SspB-positive S. aureus, but not by that of a SspBnull mutant. C-terminal processing by SspB generates a chemerin isoform identical with the active endogenous attractant isolated from human ascites fluid. Interestingly, SspB is a potent trigger of chemerin even in the presence of plasma inhibitors. SspB may help direct the recruitment of specialized host cells, including immunoregulatory pDC and/or macrophages, contributing to the ability of S. aureus to elicit and maintain a chronic inflammatory state.

Regulation of Chemerin Chemoattractant and Antibacterial Activity by Human Cysteine Cathepsins

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1

Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.

A rapid and efficient way to obtain modified chemokines for functional and biophysical studies.

Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.