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AimsFractalkine (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 resultsCX3CL1 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.ConclusionWe 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.

The theory of ecological cognition poses that the brains and behaviour of animals are shaped by the environmental challenges they face in their everyday lives. Site fidelity and homing ability was tested for five species of intertidal rock pool fish by tagging and displacing them to new rock pools at various distances from their 'home' rock pools. Three of the species were rock pool specialists whilst the remaining two spend a small proportion of their life in rock pools during early ontogeny. The three specialists showed strong site fidelity with [50 % of individuals found in the same pool 42 days after tagging. In contrast, the non-specialist species showed low fidelity and poor homing abilities. Homing success in the rock pool specialists remained relatively stable as displacement distance increased. The effect of body size on homing ability was species dependent, with only one species showing a significantly greater tendency to home with increasing size.

Abstract-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. (Arterioscler Thromb Vasc Biol. 2012;32:589-594.)Key Words: apoptosis Ⅲ atherosclerosis Ⅲ macrophages Ⅲ vascular biology Ⅲ chemokines F irst recognized in 1987 with the discovery of interleukin-8, the chemokine family today numbers at least 46 members divided into 4 families on the basis of structure. 1,2 As their name-chemotactic cytokinessuggests, all members of the family share the ability to chemoattract cells expressing their cognate G protein-coupled receptors. The variety of cellular functions ascribed to chemokines is vast and growing, including adhesion, proliferation, survival, angiogenesis, and regulation of proinflammatory gene expression. In addition, chemokine receptor expression has been described for virtually every cell type studied, under both homeostatic and inflammatory conditions. Dysregulation of chemokine and chemokine receptor expression is associated with multiple disease states, including cardiovascular disease, cancer, neurodegenerative disease, and systemic inflammatory disease, such as rheumatoid arthritis and systemic lupus erythematosus (reviewed in 3 ).Fractalkine (CX3CL1) is the only member of the CX3C chemokine family and is expressed as a membrane-bound molecule with the chemokine domain attached via a mucinlike stalk to the cell surface. 4 A recent analysis of fractalkine expression using a Cx3cl1 cherry :Cx3cr1 gfp knock-in mouse identified neurons and epithelial cells in the lung, kidney, and intestine as the major sites of fractalkine expression, confirming previous reports. 5,6 Fractalkine can also be expressed by endothelial and smooth muscle cells under inflammatory conditions. 7,8 Cleavage at the base of the mucin stalk is mediated by at least 2 enzymes, ADAM10 and ADAM17, which function under homeostatic and inflammatory conditions, respectively. 9 -11 The structure of membrane-bound and soluble fractalkine is presented in Figure 1. Fractalkine is the unique ligand for the chemokine receptor CX3CR1, which is expressed on monocytes, natural killer cells, T cells, and smooth muscle cells, 12,13 where...

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.

Rock pools can be found in inter-tidal marine environments worldwide; however, there have been few studies exploring what drives their, fish species composition, especially in Australia. The rock-pool environment is highly dynamic and offers a unique natural laboratory to study the habitat choices, physiological limitations and adaptations of inter-tidal fish species. In this study rock pools of the Sydney region were sampled to determine how the physical (volume, depth, rock cover and vertical position) and biological (algal cover and predator presence) parameters of pools influence fish distribution and abundance. A total of 27 fish species representing 14 families was observed in tide pools at the four study locations. The five most abundant species were Bathygobius cocosensis, Centropogon australis, Enneapterygius atrogulare, Lepidoblennius haplodactylus and Microcanthus strigatus, which together represented 71% of the total number of fish recorded. Larger rock pools containing more algal and rock ledge cover hosted a larger and more diverse population of fish. Furthermore, certain species were only found in pools with specific characteristics, such as the presence of loose shells, a variety algae or rock cover, suggesting a high degree of habitat specificity. By contrast, some species were ubiquitous and thus can likely tolerate a wide variety of physical conditions.

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.