Gemma E. White

CC Chemokine Receptors and Chronic Inflammation—Therapeutic Opportunities and Pharmacological Challenges

Chemokines are a family of low molecular weight proteins with an essential role in leukocyte trafficking during both homeostasis and inflammation. The CC class of chemokines consists of at least 28 members (CCL1-28) that signal through 10 known chemokine receptors (CCR1-10). CC chemokine receptors are expressed predominantly by T cells and monocyte-macrophages, cell types associated predominantly with chronic inflammation occurring over weeks or years. Chronic inflammatory diseases including rheumatoid arthritis, atherosclerosis, and metabolic syndrome are characterized by continued leukocyte infiltration into the inflammatory site, driven in large part by excessive chemokine production. Over years or decades, persistent inflammation may lead to loss of tissue architecture and function, causing severe disability or, in the case of atherosclerosis, fatal outcomes such as myocardial infarction or stroke. Despite the existence of several clinical strategies for targeting chronic inflammation, these diseases remain significant causes of morbidity and mortality globally, with a concomitant economic impact. Thus, the development of novel therapeutic agents for the treatment of chronic inflammatory disease continues to be a priority. In this review we introduce CC chemokine receptors as critical mediators of chronic inflammatory responses and explore their potential role as pharmacological targets. We discuss functions of individual CC chemokine receptors based on in vitro pharmacological data as well as transgenic animal studies. Focusing on three key forms of chronic inflammation—rheumatoid arthritis, atherosclerosis, and metabolic syndrome—we describe the pathologic function of CC chemokine receptors and their possible relevance as therapeutic targets.

Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling.

Aims Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. Methods and results CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. Conclusion We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Fractalkine: A Survivor's Guide: Chemokines as Antiapoptotic Mediators

Chemokines are a family of low-molecular-weight proteins essential to the directed migration of cells under homeostatic and pathological conditions. Fractalkine (CX3CL1) is an unusual chemokine that can act as either a soluble or membrane-bound mediator and signals through the G protein-coupled chemokine receptor CX3CR1, expressed on monocytes, natural killer cells, T cells, and smooth muscle cells. Accumulating evidence suggests that fractalkine, in addition to its role in chemotaxis and adhesion of leukocytes, supports the survival of multiple cell types during homeostasis and inflammation. This review presents the evidence obtained from several disease models implying an antiapoptotic function for fractalkine and shows how this is relevant to the pathology of atherosclerosis and other vascular diseases. We discuss whether the key role of fractalkine, unlike other chemokines, is the promotion of cell survival and whether this has implications for vascular disease.

Chapter 17 Zymosan‐Induced Peritonitis as a Simple Experimental System for the Study of Inflammation

The acute inflammatory response occurs as a result of tissue injury or infection and is characterized by the coordinated recruitment of leukocytes in response to inflammatory mediators including chemokines. This process generally resolves within a matter of days, and normal tissue architecture is restored by a process of wound healing. Failure to resolve the injury can result in chronic inflammation. Much of our understanding of the specific mediators and cell types involved in acute inflammation has come from sterile peritonitis models. The injection of a wide range of irritants into the peritoneal cavity induces the hallmarks of inflammation, including pain, leukocyte infiltration, and synthesis of inflammatory mediators. Intraperitoneal injection of zymosan, a polysaccharide cell wall component derived from Saccharomyces cerevisiae, has been widely used as a self-resolving model of acute inflammation that peaks within a few hours and is cleared within 48 to 72 h. We have used the zymosan-induced peritonitis model extensively to quantify the recruitment of monocytes and neutrophils into the peritoneal cavity and to study the effects of existing and novel antiinflammatory drugs. We discuss some of the applications and advantages of the zymosan-induced peritonitis model and describe the method for analysis of leukocyte recruitment and inflammatory mediator production in response to zymosan.

A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Fractalkine: one chemokine, many functions.

CX3CL1 (Fractalkine) is an atypical chemokine with a documented role in the development of numerous inflammatory diseases including atherosclerosis. In this issue of Blood , Landsman and colleagues demonstrate that interactions between CX3CL1 and its receptor CX3CR1 are involved in monocyte survival

Site-directed Mutagenesis of the CC Chemokine Binding Protein 35K-Fc Reveals Residues Essential for Activity and Mutations That Increase the Potency of CC Chemokine Blockade

Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and β-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced β-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.

Acute exposure to apolipoprotein a1 inhibits macrophage chemotaxis in vitro and monocyte recruitment in vivo

Apolipoprotein A1 (apoA1) is the major protein component of high-density lipoprotein (HDL) and has well documented anti-inflammatory properties. To better understand the cellular and molecular basis of the anti-inflammatory actions of apoA1, we explored the effect of acute human apoA1 exposure on the migratory capacity of monocyte-derived cells in vitro and in vivo. Acute (20–60 min) apoA1 treatment induced a substantial (50–90%) reduction in macrophage chemotaxis to a range of chemoattractants. This acute treatment was anti-inflammatory in vivo as shown by pre-treatment of monocytes prior to adoptive transfer into an on-going murine peritonitis model. We find that apoA1 rapidly disrupts membrane lipid rafts, and as a consequence, dampens the PI3K/Akt signalling pathway that coordinates reorganization of the actin cytoskeleton and cell migration. Our data strengthen the evidence base for therapeutic apoA1 infusions in situations where reduced monocyte recruitment to sites of inflammation could have beneficial outcomes. DOI: http://dx.doi.org/10.7554/eLife.15190.001

Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells

CCR9 expressed on T lymphocytes mediates migration to the small intestine in response to a gradient of CCL25. CCL25-stimulated activation of α4β7 integrin promotes cell adherence to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by vascular endothelial cells of the intestine, further mediating gut-specific homing. Inflammatory bowel disease is a chronic inflammatory condition that primarily affects the gastrointestinal tract and is characterized by leukocyte infiltration. Glucocorticoids (GCs) are widely used to treat inflammatory bowel disease but their effect on intestinal leukocyte homing is not well understood. We investigated the effect of GCs on the gut-specific chemokine receptor pair, CCR9 and CCL25. Using human peripheral blood-derived T lymphocytes enriched for CCR9 by cell sorting or culturing with all-trans retinoic acid, we measured chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion to plate-bound MAdCAM-1. Dexamethasone (DEX), a specific GC receptor agonist, significantly reduced CCR9-mediated chemotaxis and adhesion to MAdCAM-1 without affecting CCR9 surface expression. In contrast, in the same cells, DEX increased CXCR4 surface expression and CXCL12-mediated signaling and downstream functions. The effects of DEX on human primary T cells were reversed by the GC receptor antagonist mifepristone. These results demonstrate that GCs suppress CCR9-mediated chemotaxis, intracellular calcium flux, and α4β7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.

Hydrodynamic Gene Delivery of CC Chemokine Binding Fc Fusion Proteins to Target Acute Vascular Inflammation In Vivo

Blockade of CC chemokines is an attractive yet under utilized therapeutic strategy. We report the in vivo pharmacokinetics of a broad-spectrum vaccinia virus CC chemokine binding protein (35 K) fused to human IgG1 Fc. We demonstrate that the in vivo efficacy of the protein can be interrogated using hydrodynamic gene delivery of a standard mammalian expression plasmid. High plasma levels of the 35 K-Fc protein are maintained for at least 14 days post gene transfer, with the protein still detectable at 5 weeks. We confirm that the protein has biological activity in acute inflammation, causing a significant reduction in monocyte recruitment during zymosan induced peritonitis. The ability of 35 K-Fc to block more complex pathologies is demonstrated using aortic digests to assess angiotensin II mediated leukocyte recruitment to the aorta. Angiotensin II causes upregulation of mCCL2 in the aorta causing the accumulation of CCR2+ cells. Peak monocyte recruitment to the aorta occurs within 3 days and this process is CC chemokine dependent, being significantly reduced by hydrodynamic delivery of 35 K-Fc.