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 α4ß7 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 α4ß7-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 α4ß7-mediated cell adhesion in vitro, and these effects could contribute to the efficacy of GCs in treating intestinal inflammation in vivo.

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 promotes human monocyte survival via a reduction in oxidative stress.

OBJECTIVE: The CX3C chemokine fractalkine (CX3CL1) has a critical role in the development of atherogenesis because apolipoprotein-E-deficient mice lacking CX3CL1 or its receptor CX3CR1 develop smaller plaques and polymorphisms in CX3CR1 are associated with altered risk of cardiovascular disease. CX3CR1 is found on numerous cell types involved in atherogenesis but seems to have a key role in monocyte function. We aimed to elucidate the role of CX3CL1 in human monocyte survival and determine the mechanism by which CX3CL1 spares monocytes from apoptosis. APPROACH AND RESULTS: Primary human monocytes were prepared from healthy donors and subjected to serum-starvation to induce spontaneous apoptosis. The addition of CX3CL1, but not other chemokines tested, promoted monocyte survival in a dose-dependent manner with full-length CX3CL1 (including the mucin stalk) having a more potent antiapoptotic effect than chemokine-domain CX3CL1. The prosurvival effect of CX3CL1 was evident in both monocyte subsets although nonclassical monocytes were more prone to spontaneous apoptosis. In addition, we found that the effect of CX3CL1 was independent of CX3CR1 genotype. Serum-starvation increased the level of intracellular reactive oxygen species, and this was reduced by the addition of CX3CL1. Inhibition of oxidative stress with an antioxidant prevented monocyte apoptosis, indicating that this is the dominant mechanism of cell death targeted by CX3CL1. CONCLUSIONS: CX3CL1 has a substantial and highly reproducible antiapoptotic effect on human monocytes, via a mechanism involving a reduction in oxidative stress. This suggests that CX3CL1 is likely to play a key role in human atherogenesis and may provide a novel therapeutic target in cardiovascular disease.

Variation in Brain Morphology of Intertidal Gobies: A Comparison of Methodologies Used to Quantitatively Assess Brain Volumes in Fish.

When correlating brain size and structure with behavioural and environmental characteristics, a range of techniques can be utilised. This study used gobiid fishes to quantitatively compare brain volumes obtained via three different methods; these included the commonly used techniques of histology and approximating brain volume to an idealised ellipsoid, and the recently established technique of X-ray micro-computed tomography (micro-CT). It was found that all three methods differed significantly from one another in their volume estimates for most brain lobes. The ellipsoid method was prone to over- or under-estimation of lobe size, histology caused shrinkage in the telencephalon, and although micro-CT methods generated the most reliable results, they were also the most expensive. Despite these differences, all methods depicted quantitatively similar relationships among the four different species for each brain lobe. Thus, all methods support the same conclusions that fishes inhabiting rock pool and sandy habitats have different patterns of brain organisation. In particular, fishes from spatially complex rock pool habitats were found to have larger telencephalons, while those from simple homogenous sandy shores had a larger optic tectum. Where possible we recommend that micro-CT be used in brain volume analyses, as it allows for measurements without destruction of the brain and fast identification and quantification of individual brain lobes, and minimises many of the biases resulting from the histology and ellipsoid methods.

Microhabitat use affects brain size and structure in intertidal gobies.

The ecological cognition hypothesis poses that the brains and behaviours of individuals are largely shaped by the environments in which they live and the associated challenges they must overcome during their lives. Here we examine the effect of environmental complexity on relative brain size in 4 species of intertidal gobies from differing habitats. Two species were rock pool specialists that lived on spatially complex rocky shores, while the remainder lived on dynamic, but structurally simple, sandy shores. We found that rock pool-dwelling species had relatively larger brains and telencephalons in particular, while sand-dwelling species had a larger optic tectum and hypothalamus. In general, it appears that various fish species trade off neural investment in specific brain lobes depending on the environment in which they live. Our previous research suggests that rock pool species have greater spatial learning abilities, enabling them to navigate their spatially complex environment, which may account for their enlarged telencephalon, while sand-dwelling species likely have a reduced need for spatial learning, due to their spatially simple habitat, and a greater need for visual acuity. The dorsal medulla and cerebellum size was unaffected by the habitat in which the fish lived, but there were differences between species indicative of species-specific trade-offs in neural investment.

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.

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 ß-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 ß-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.

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.

Contrasting in vitro vs. in vivo effects of a cell membrane-specific CC-chemokine binding protein on macrophage chemotaxis.

UNLABELLED: Chemokines (CK) provide directional cues that mediate the recruitment of leukocytes to sites of inflammation. Broad-spectrum blockade of the CC-CK family, using the vaccinia virus 35K protein, has been shown to cause a potent reduction of systemic inflammation in models of atherosclerosis, vein graft disease and arthritis. We have used a cell membrane-targeted form of 35K, Mem35K, to probe whether cell-associated blockade of chemokine response is sufficient to reduce cell recruitment in inflammation. In Tie2cre mice, activation of a flox-stop Mem35K transgene directed conditional expression of Mem35K in leukocytes and endothelial cells, confirmed by Western blotting, flow cytometry and immunofluorescence microscopy. This conditional Mem35K expression was sufficient to increase cell surface CCL5 binding and reduce chemotaxis in vitro to CCL5, CCL2 and CCL3 but not to non-CC-CK chemoattractants, LTB4, C5a or chemerin. However, in vivo monocyte recruitment into the peritoneum driven by zymosan or CC-chemokine injection, which was demonstrated to be CC-CK dependent using CCR2-/- mice, was not reduced by Mem35K expression, despite the expression of functional Mem35K protein. These findings highlight differing requirements for cell-associated anti-inflammatory activity in in vitro and in vivo models. KEY MESSAGE: Mem35K is a cell-associated CC-chemokine binding protein. Conditional Mem35K transgenic mice show expression Mem35K in leukocytes. Mem35K blocks in vitro primary macrophage chemotaxis specifically towards CC-chemokines. Mem35K expression is not sufficient to reduce inflammation in vivo. The requirements for anti-inflammatory activity in vitro and in vivo are different.

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.

Suppressor of cytokine signalling protein SOCS3 expression is increased at sites of acute and chronic inflammation.

Treatment of cells with cytokines and growth factors leads to the synthesis of Suppressor of Cytokine Signalling (SOCS) proteins that act as potent negative regulators of signalling via the Jak/STAT pathway. We used immunohistochemistry to identify cells and pathologies where SOCS3 expression might influence acute and chronic inflammatory responses in human tissues. Epitope and GFP tagged SOCS3 fusion proteins were localised predominantly in the nucleus of transfected cells and a validated anti SOCS3 antiserum revealed the expression of SOCS3 in the nucleus and cytoplasm of macrophages, endothelial and epithelial cells in a wide range of normal tissues in tissue microarrays (n = 31 different tissues). Nuclear SOCS3 was only seen in cells expressing a high level of the protein. Comparative immunostaining of acute, chronically and granulomatously inflamed human tissues revealed higher levels of nuclear and cytoplasmic SOCS3 expression in inflamed than in corresponding normal tissues, particularly in recruited leukocyte populations, but also in epithelia. The staining appeared more intense, suggesting higher expression levels, in areas where inflammation was more acute, consistent with the time course of SOCS3 induction described in vitro. Expression of SOCS3 protein by leucocytes and other cell types in tissue sections could be a useful marker of cells undergoing acute or chronic stimulation by cytokines in vivo.

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.

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.