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In mice, interleukin-18 (IL-18) regulates Th1- or Th2-type immune responses depending on the cytokine environment and effector cells involved, and the ST2-ligand, IL-33, primarily promotes an allergic phenotype. Human basophils, major players in allergic inflammation, constitutively express IL-18 receptors, while ST2 surface expression is inducible by IL-3. Unexpectedly, freshly isolated basophils are strongly activated by IL-33, but, in contrast to mouse basophils, do not respond to IL-18. IL-33 promotes IL-4, IL-13 and IL-8 secretion in synergy with IL-3 and/or FcϵRI-activation, and enhances FcϵRI-induced mediator release. These effects are similar to that of IL-3, but the signaling pathways engaged are distinct because IL-33 strongly activates NF-κB and shows a preference for p38 MAP-kinase, while IL-3 acts through Jak/Stat and preferentially activates ERK. Eosinophils are the only other leukocyte-type directly activated by IL-33, as evidenced by screening of p38-activation in peripheral blood cells. Only upon CD3/CD28-ligation, IL-33 weakly enhances Th2 cytokine expression by in vivo polarized Th2 cells. This study on primary human cells demonstrates that basophils and eosinophils are the only direct target leukocytes for IL-33, suggesting that IL-33 promotes allergic inflammation and Th2 polarization mainly by the selective activation of these specialized cells of the innate immune system.

The systemic changes in immune mediators such as cytokine and chemokines, and their synchronized interaction that regulates the cell trafficking during Mycobacterium tuberculosis (M. tuberculosis) infection, were studied. Cytokines and chemokines were evaluated by cytometric bead array (CBA) and enzyme-linked immunosorbent assay (ELISA) in 34 pulmonary tuberculosis (PTB) patients and 30 healthy subjects. The expression of chemokine receptors was assessed by flow cytometry. A significant increase in IP-10, MIG, interleukin-8, RANTES, and interleukin-6 levels was found, whereas a decrease in interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta was observed during PTB. Significant correlation within chemokines and between cytokines was observed in PTB. All immune cells except monocytes and B cells expressed significantly higher levels of CCR1, CCR2, and CXCR2 whereas CCR7 expression was upregulated only on monocytes and neutrophils in PTB. Both T and B cells expressed significantly high levels of CXCR3 which also correlated well with the chemokine levels in PTB. Thus, it was found that chemokines function coordinately and consistently during PTB. This balanced chemokine and cytokine relationship at the periphery may aid in amplified effector immune cell trafficking and retarded monocyte migration through differential chemokine receptor expression.

T-lymphocyte trafficking is targeted to specific organs by selective molecular interactions depending on their differentiation and functional properties. Specific chemokine receptors have been associated with organ-specific trafficking of memory and effector T-cells, as well as the recirculation of naïve T-cells to secondary lymphoid organs. In addition to the acquisition of tissue-selective integrins and chemokine receptors, an additional level of specificity for T-cell trafficking into the tissue is provided by specific recognition of antigen displayed by the endothelium involving the TCRs (T-cell antigen receptors) and co-stimulatory receptors. Activation of PI3K (phosphoinositide 3-kinase) is a robust signalling event shared by most chemokine receptors as well as the TCR and co-stimulatory receptors, contributing to several aspects of T-lymphocyte homing as well as actin reorganization and other components of the general migratory machinery. Accordingly, inhibition of PI3K has been considered seriously as a potential therapeutic strategy by which to combat various T-lymphocyte-dependent pathologies, including autoimmune and inflammatory diseases, as well as to prevent transplant rejection. However, there is substantial evidence for PI3K-independent mechanisms that facilitate T-lymphocyte migration. In this regard, several other signalling-pathway components, including small GTPases, PLC (phospholipase C) and PKC (protein kinase C) isoforms, have also been implicated in T-lymphocyte migration in response to chemokine stimulation. The present review will therefore examine the PI3K-dependent and -independent signal-transduction pathways involved in T-cell migration during distinct modes of T-cell trafficking in response to either chemokines or the TCR and co-stimulatory molecules.

SummaryThe interaction of chemokines and their receptors directs lymphocyte migration, and is involved in the distribution and organization of lymphocytes within lymphoid tissues. We reasoned that abnormal chemokine receptor expression might give rise to defects of lymphocyte migration into and within lymphoid tissues, and consequently be associated with defective antibody production in primary antibody deficiencies. In this study, we have investigated the expression of chemokine receptors CXCR4, CXCR5 and CCR7 on lymphocyte subpopulations (naive and memory B cells; CD4+ and CD8+ T cells) in a cohort of patients with primary antibody deficiency (n = 23), and compared these with a group of healthy controls (n = 19). We show that there were significant differences in both the proportions of lymphocytes expressing, and the levels of expression of, specific chemokine receptors on individual lymphocyte subpopulations between patients and controls. Furthermore, these changes appeared more pronounced in patients with more severe antibody deficiency. These data support the hypothesis that abnormal lymphocyte trafficking may be involved in the pathogenesis of primary antibody deficiencies.

Chemokines are key regulators of leukocyte migration and play important roles in a number of physiological and pathological immune and inflammatory contexts. In addition to the classical signalling chemokine receptors there has emerged, recently, a new subclass of atypical chemokine receptors. This subfamily is characterised by an apparent lack of signalling and, in some cases, by an ability to internalise and degrade chemokine ligands. This review describes the family of atypical chemokine receptors with particular emphasis on the D6 receptor. The in vitro and in vivo biology of D6 is described, which indicates that D6 is active as a scavenger of inflammatory CC-chemokines and appears to play essential roles in the regulation of inflammatory responses.

Increasing evidence suggests that the chemokine system coordinates leukocyte migration in immunity and in- flammation, involving in the pathogenesis of many pulmonary diseases. Chemokines are small proteins which interact with specific receptors to exert their chemotactic functions for inflammatory cells and constitutive cells. Among the com- plex system, CC chemokine receptors (CCRs) represent a subfamily of chemokine receptor, which is considered to play an important role in the pathogenesis of diseases characterized by disordered inflammation and immunity, among which some respiratory diseases, such as asthma, chronic obstructive pulmonary disease, acute lung injury, comprise one impor- tant part. The present paper reviewed the research of possible relationship and progress between this system and human lung diseases. Chemokines and their receptors are essential components of Th1- and Th2-mediated responses via the re- cruitment of lymphocytes and macrophages. The local expression of homeostatic chemokines in pulmonary also plays an important role in protective immune responses. Thus, it is important to understand the potential role of CCRs in respira- tory diseases and it may provide exciting new targets for therapeutic intervention.

破骨細胞による骨吸収は, その過程において, 造血系細胞である破骨細胞前駆細胞の遊走とRANKL (Receptor activator of NF-kB ligand)による成熟した破骨細胞への分化が必要である。一方, 生理活性物質であるケモカインは造血系細胞に対して走化性を有している。本研究において我々は, RANKL刺激による破骨細胞分化におけるCCL7 (CC chemokine ligand 7; MCP-3 : monocyte chemotactic protein -3) およびCCL25 (CC chemokine ligand 25; TECK : thymus-expressed chemokine) の作用を解析した。CCL7およびCCL25は, IL-1β, ビタミンD3刺激によりマウス骨髄間質由来細胞であるST2細胞において産生が確認された。一方, CCL7の受容体CCR2 (CC chemokine receptor 2), 並びに, CCL25の受容体CCR9 (CC chemokine receptor 9)は, マウスマクロファージ系細胞株RAW264.7細胞において発現が確認された。そこで, RANKL存在下においてCCL7, CCL25リコンビナント蛋白とともにRAW264.7細胞を培養したところ, 破骨細胞形成が有意に増強された。次に, RANKL存在下において抗CCL7抗体, 抗CCL25抗体とともにRAW264.7細胞を培養したところ, 破骨細胞形成が有意に抑制された。さらに, マウスから分離した骨髄細胞を用いた破骨細胞形成系実験においても同様の結果が得られた。これらの結果より, 細胞遊走を惹起すると知られているCCL7およびCCL25は, RANKLによって誘導される破骨細胞分化, 形成においても重要な役割を担うことが示唆された。日本歯周病学会会誌(日歯周誌)51(1) : 51-61, 2009

Chemokines were originally defined as host defense proteins, however, their biological role goes well beyond this simple description of their function as immune cell chemoattractants, and they have since been shown to be involved in a number of other biological processes, including growth regulation, hematopoiesis, embryologic development, angiogenesis, and HIV-1 infection. Because of their diverse role in autoimmune diseases and AIDS, chemokines and their receptors, which belong to the G-protein-coupled receptor superfamily, have been considered good drug targets by the pharmaceutical industry.In the first part of this two-part review, we highlight recent developments in the chemokine receptor antagonist field both in the peer reviewed and in the patent literature for the CC chemokine receptors CCR1, CCR2, CCR3, and CCR4.A number of chemokine receptor antagonists have made the transition from lead compounds to clinical candidates, some of which are described here. Although there has been no clinical success yet for antagonists targeting the group of receptors discussed here, the compounds have been invaluable in generating information that should pave the way for producing successful therapeutics in the future.

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b+ myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b+ myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.

The progressive growth of Echinococcus multilocularis metacestodes and their tissue infiltration will cause organ malfunction and finally failure. In few patients, E. multilocularis metacestode proliferation will spontaneously regress, but little is known about the determinants which may restrain metacestode survival and growth. In this study, chemokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis (AE). Characteristic chemokine profiles and changes in their production were observed in AE patients and infection-free controls when their peripheral blood cells were cultured with E. multilocularis antigens. The production of CC and CXC chemokines which associate with inflammation (MIP-1 alpha/CCL3, MIP-1 beta/CCL4, RANTES/CCL5 and GRO-alpha/CXCL1) was constitutively larger in AE patients than in controls; and the elevated chemokine releases were equal in patients with progressive, stable or cured AE. Cluster analyses identified three distinct chemokine response profiles; chemokines were enhanced, depressed or produced in similar quantities in AE patients and controls. A disparate cellular responsiveness was observed in AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-alpha/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were clearly diminished, while cluster 3 chemokines (MIP-1 alpha/CCL3, MIP-1 beta/CCL4, RANTES/CCL5) augmented. The increased production of inflammatory chemokines in patients even with cured AE could be induced by residual E. multilocularis metacestode lesions which continuously stimulate production of inflammatory chemokines. E. multilocularis metacestodes also suppressed cellular chemokine production in AE patients, and this may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.

The expression of chemokine receptors and chemokine production by adult human non-transformed astrocytes, grade III astrocytoma and grade IV glioblastoma tumour cell lines were determined. Here, we show an increased expression of CXCR3 and CXCR4, and a decreased expression of CXCR1 and CCR4 by glioma cells compared to adult human astrocytes. Glioma cells showed increased production of CXCL10, whereas production of other chemokines was decreased (CXCL8, CCL2, CCL5, and CCL22). CXCL10 induced an ERK1/2-dependent increase in [(3)H] thymidine uptake. These results suggest that expression of chemokine receptor/ligand pairs such as CXCR3/CXCL10 have an important role in the proliferation of glioma cells.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1−/− mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2−/− mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3−/− mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis (EAE) induction. CCR2-deficient peripheral blood and resident peritoneal cells exhibited reduced binding capacity and biologic responses to the CCR1 ligand CCL3, suggesting that elevated levels of CCR2 ligands had down-regulated CCR1. The results indicate that signaling chemokine receptors clear chemokines from circulation and tissues. These homeostatic functions of signaling chemokine receptors need to be integrated into safety and efficacy calculations when considering therapeutic receptor blockade.

Background Inflammatory response in pulmonary fibrosis closely resembles a T-helper (Th) 2 immune response. For recruitment to an inflammatory lesion, the majority of Th1 cells express CXC chemokine receptor 3, recognizing monokine induced by interferon-gamma (Mig), interferon γ-inducible protein of 10 kD (IP-10), and interferon-inducible T-cell α chemoattractant (I-TAC). Th2 cells express CC chemokine receptor 4, recognizing thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). We investigated the Th1/Th2 chemokine production patterns by lung fibroblasts and their evaluation in bronchoalveolar lavage (BAL) fluid of interstitial lung disease. Methods The production pattern of Th1/Th2 chemokines by lung fibroblasts was examined in ELISA and quantitative reverse transcriptase polymerase chain reactions. Th1/Th2 chemokine levels in BAL fluid of idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) were examined to evaluate the clinical relevance of Th1/Th2 chemokines. Results The lung fibroblasts were polarized to produce Th1-type chemokines by the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α and the anti-fibrotic cytokine, interferon (IFN)-γ. However, the induction patterns of chemokines by these two cytokines were different, i.e., involving predominant induction of IP-10 and I-TAC by TNF-α and induction of Mig by IFN-γ. Although Mig, IP-10, and I-TAC were produced within the BAL fluid of patients, TARC and MDC were at significantly low levels. Conclusions Our results suggest that lung fibroblasts tend to induce a Th1-type immune response under normal conditions, and that a Th2-type immune response does not play a significant role in smoldering inflammation around the established lesions in IPF and NSIP.

Several studies support C-reactive protein (CRP) as a systemic cardiovascular risk factor. The recent detection of CRP in arterial intima suggests a dual activity in atherosclerosis as a circulating and tissue mediator on vascular and immune cells. In the present paper, we focused on the inflammatory effects of CRP on human monocytes, which were isolated by Ficoll-Percoll gradients and cultured in adherence to polystyrene, endothelial cell monolayer, or in suspension. Chemokine levels, adhesion molecule, and chemokine receptor expression were detected by ELISA, flow cytometry, and real-time RT-PCR. Migration assays were performed in a Boyden chamber. Stimulation with CRP induced release of CCL2, CCL3, and CCL4 in adherent monocytes through the binding to CD32a, CD32b, and CD64, whereas no effect was observed in suspension culture. This was associated with CRP-induced up-regulation of adhesion molecules membrane-activated complex 1 (Mac-1) and ICAM-1 on adherent monocytes. Blockade of Mac-1/ICAM-1 interaction inhibited the CRP-induced chemokine secretion. In addition, CRP reduced mRNA and surface expression of corresponding chemokine receptors CCR1, CCR2, and CCR5 in adherent monocytes. This effect was a result of chemokine secretion, as coincubation with neutralizing anti-CCL2, anti-CCL3, and anti-CCL4 antibodies reversed the effect of CRP. Accordingly, a reduced migration of CRP-treated monocytes to CCL2 and CCL3 was observed. In conclusion, our data suggest an in vitro model to study CRP activities in adherent and suspension human monocytes. CRP-mediated induction of adhesion molecules and a decrease of chemokine receptors on adherent monocytes might contribute to the retention of monocytes within atherosclerotic lesions and recruitment of other circulating cells.

Post-translational sulfation of tyrosines affects the affinity and binding of at least some chemokine receptors to their ligand(s) and has been hypothesized to be a feature in all chemokine receptors. This binding initiates downstream signaling cascades. By this mechanism, tyrosine sulfation can influence the cells involved in acute and chronic events of cellular immunity. These events include leukocyte trafficking and airway inflammation important in asthma and chronic obstructive pulmonary disease (COPD). We are using computational methods to convert the poorly defined hypothesis of more widespread sulfation of chemokine receptors to more specific assessments of how closely the sequence environment of each tyrosine residue resembles the sequence environment of tyrosine residues proven to be sulfated. Thus, we provide specific and readily tested hypotheses about the tyrosine residues in all of the chemokine receptors. Tyrosine sulfation was predicted with high scores in the N-terminus domain of 13 out of 18 human chemokine receptor proteins using a position-specific scoring matrix, which was determined to be 94.2% accurate based on Receiver Operating Characteristic analysis. The remaining chemokine receptors have sites exhibiting features of tyrosine sulfation. These putative sites demonstrate clustering in a manner consistent with known tyrosine sulfation sites and conservation both within the chemokine receptor family and across mammalian species. Human chemokine receptors important in asthma and COPD, such as CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8, contain at least one known or predicted tyrosine sulfation site. Recognition that tyrosine sulfation is found in most clinically relevant chemokine receptors could help the development of specific receptor-ligand antagonists to modulate events important in airway diseases.

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.

Chemokines are small, secreted proteins that orchestrate the migration of cells, which are involved in immune defence, immune surveillance and haematopoiesis. However, chemokines are also implicated in the pathology of various inflammatory diseases, cancers and HIV. The chemokine system is considerably large and has a redundancy in the repertoire of its inflammatory mediators. Therefore, strict regulation of chemokine activity is crucial. Chemokines are the substrate for various proteases including the serine protease CD26/dipeptidyl-peptidase IV and matrix metalloproteinases. Regulation by proteolytic cleavage controls and fine-tunes chemokine function by either enhancing or reducing its chemotactic activity or receptor selectivity. Often chemokines and the proteases that regulate them are produced in the same microenvironment and expression of both may be simultaneously induced by a common stimulus enabling the rapid regulation of chemokine activity. The overall impact of cleaved chemokines in cellular responses is very complex. In this review, we will give an overview on chemokine modification and the respective chemokine modifying proteases. Furthermore, we will summarize the emerging literature describing the consequences in inflammation, haematopoiesis, cancer and HIV infection upon proteolytic chemokine processing.

Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

Actions of chemokines and the interaction with specific receptors go beyond their original, defined role of recruiting leukocytes to inflamed tissues. Chemokine receptor expression in peripheral elements and resident cells of the central nervous system (CNS) represents a relevant communication system during neuroinflammatory conditions. The following examples are described in this review: Chemokine receptors play important homeostatic properties by regulating levels of specific ligands in blood and tissues during healthy and pathological conditions; chemokines and their receptors are clearly involved in leukocyte extravasation and recruitment to the CNS, and current studies are directed toward understanding the interaction between chemokine receptors and matrix metalloproteinases in the process of blood brain barrier breakdown. We also propose novel functions of chemokine receptors during demyelination/remyelination, and developmental processes.

Asthma is associated with increased numbers of T-cells in the lung. CC chemokine receptor (CCR)5 and CXC chemokine receptor (CXCR)3 have been reported to play important roles in the lung T-cell homing pathway, and may be potential targets for asthma therapy. The aim of the present study was to investigate the role of CCR5 and CXCR3 in allergen-induced acute asthma and to determine whether a novel small-molecule compound, TAK-779, targeting CCR5 and CXCR3 can attenuate allergic airway responses. Mice were sensitised with ovalbumin (OVA). mRNA expression of chemokine receptors in the lung were measured after the challenge with either aerosolised phosphate-buffered saline or OVA. OVA-sensitised mice were also treated with TAK-779. Respiratory function was measured, bronchoalveolar lavage was performed, and blood and lung samples were obtained. OVA challenge increased CCR3, CCR5 and CXCR3 expression in the lung. Treatment with TAK-779 significantly attenuated altered respiratory function and pulmonary allergic inflammation. The beneficial effect was associated with reduced expression of CCR5 and CXCR3 in the lung. These data demonstrate that blockade of CC chemokine receptor 5 and CXC chemokine receptor 3 using TAK-779, a synthetic nonpeptide compound, can prevent the development of asthma features in a mouse model. Thus, CC chemokine receptor 5 and CXC chemokine receptor 3 may be potential targets for asthma therapy.

Recently a major goal in asthma therapy is to reduce or prevent the inflammatory response of airway. Eosinophilic accumulation in the tissue is a prominent feature of allergic diseases including asthma. Production of chemokines by bronchial epithelial cells may contribute to the allergic inflammation by recruiting eosinophils. In this study we evaluated the inhibitory effect of Gamichungsangbohatang (GMCSBHT), used traditionally in treating asthma, on secretion of chemokines for eosinophils in human A549 epithelial cells. Chemokines such as eotaxin, RANTES, IL-8 were inhibited in a dose-dependent manner, but IL-16 showed no inhibition by GMCSBHT. These findings indicate that GMCSBHT might be a therapeutic value in treating asthma by suppression of chemokines secretion associated with local accumulation of eosinophils.

Infection of mice with mouse hepatitis virus (MHV) strain JHM (RJHM) induces lethal encephalitis, with high macrophage and neutrophil, but minimal T-cell, infiltration into the brain when compared to the neuroattenuated strain RA59. To determine if chemokine expression corresponds with the cellular infiltrate, chemokine protein and RNA levels from the brains of infected mice were quantified. RJHM-infected mice had lower T-cell (CXCL9, CXCL10), but higher macrophage-attracting (CCL2), chemokine proteins compared to RA59. RJHM also induced significantly higher CXCL2 (a neutrophil chemoattractant) mRNA compared to RA59. The neurovirulent spike gene chimera SJHM/RA59 induces high levels of T cells and macrophages in the brain compared to the attenuated SA59/RJHM chimera. Accordingly, SJHM/RA59 induced higher levels of CXCL9, CXCL10, and CCL2 protein compared to SA59/RJHM. Chemokine mRNA patterns were in general agreement. Thus, chemokine patterns correspond with the cellular infiltrate, and the spike protein influences levels of macrophage, but not T-cell, chemokines.

Objective: This study aimed to determine the effects of HBx on the inhibition of tumorigenesis in nude mice injected with CCL13-HBx cells. Therefore, the characteristics of the induced tumors and the phenomenon of apoptosis were assessed. Methods: The induced tumors were identified using the specific marker of hepatocellular carcinoma (HCC), anti-α-fetoprotein (AFP), and their characteristics were pathologically examined. Apoptosis was detected by DNA fragmentation, and the expression of the proapoptotic proteins p53, Bax, Bad, caspase-3, and caspase-8 and the anti-apoptotic protein Bcl-2 was detected by Western blotting. To identify possible molecules involved in the inhibition of tumorigenesis, extracts of the induced tumors were separated by 2D-PAGE, and the proteins were identified by MS. Results: The tumors of the nude mice injected with CCL13 and CCL13-HBx cells were identified as HCCs. Moreover, HBx was found to suppress tumor growth via apoptosis in the nude mice injected with CCL13-HBx cells. The MS findings revealed that phosphorylated myosin light chain was a candidate molecule involved in the inhibition of tumorigenesis. Conclusion: HBx suppressed tumorigenesis in the nude mice injected with CCL13-HBx cells, which proved to be a good animal model for the in vivo study of the effects of HBx on tumorigenesis.

Background: It is hypothesized that minimal change nephrotic syndrome (MCNS) is a consequence of immune cell dysfunction that may lead to release of glomerular permeability factors. However, the nature of such factors remains uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from 2 MCNS patients during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 24 MCNS patients and 10 patients with membranous nephropathy (MN), and from 24 healthy subjects. Results: Out of 24,446 genes screened, 171 functionally known genes were up-regulated (at least 2-fold) in PBMC from MCNS patients during the nephrosis phase. 21 genes encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretory proteins, chemokine (C-C) ligand 13 (CCL13) and a novel galectin-related protein (HSPC159). The results of quantitative RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples were higher than those in remission samples from all 24 MCNS patients examined, while these mRNA expression patterns were variable among 10 MN patients. CCL13 and HSPC159 mRNA expressions in PBMC from MCNS patients in nephrosis were significantly higher than those in nephrotic MN patients and healthy controls. Conclusion: We found that CCL13 and HSPC159 mRNA expressions in PBMC are up-regulated specifically in MCNS patients during the nephrosis phase. Further studies are necessary to clarify whether these expression changes are directly involved in the pathophysiologic processes of MCNS.

Human herpesvirus-6A (HHV-6A) betachemokine-receptor U51A binds inflammatory modulators CCL2, CCL5, CCL11, CCL7, and CCL13. This unique specificity overlaps that of human chemokine receptors CCC1, CCR2, CCR3, and CCR5. In model cell lines, expression leads to CCL5 down-regulation with both constitutive and inducible signaling. Here, immunomodulation pathways are investigated in human leukocytes permissive for infection. Constitutive signaling was shown using inositol phosphate assays and inducible calcium signaling by response to CCL2, CCL5 and CCL11. Constitutive signaling targets were examined using an immune response-related microarray and RT-PCR, showing down-regulation of CCL5 and FOG-2, a hematopoietic transcriptional repressor. By RT-PCR and siRNA reversion, CCL5 and FOG-2 were shown down-regulated, during peak U51A expression post infection. Two further active ligands, XCL1 and CCL19, were identified, making U51A competitor to their human receptors, XCR1 and CCR7, on T lymphocytes, NK and dendritic cells. Finally, U51A-expressing cell lines and infected ex vivo leukocytes, showed migration towards chemokine-gradients, and chemokine internalization. Consequently, U51A may affect virus dissemination or host transmission by chemotaxis of infected cells to sites of chemokine secretion specific for U51A (for example the lymph node or lung, by CCL19 or CCL11, respectively) and evade immune-effector cells by chemokine diversion and down-regulation, affecting virus spread and inflammatory pathology. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1+ and CCR5+ cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.

Chemokines are low molecular weight soluble mediators that control leukocyte trafficking during lymphocyte homeostasis and inflammation. Chemokine-mediated signaling is triggered upon chemokine binding to seven transmembrane G protein-coupled receptors. Multiple signaling pathways are activated leading to cytoskeleton rearrangements, gene transcription and receptor internalization or degradation Among the signaling molecules involved in chemokine mediated signaling, the Jak-Stat pathway has been shown to be activated very early after chemokine stimulation. There is growing evidence showing the involvement of particular Jaks and Stats, in chemokine receptor signaling both in cell lines and primary cells. Jak/Stat phosphorylation is detected soon after chemokine receptor dimerization or in response to chemokines. Also, pharmacological inhibition of Jaks, or the use of Jak deficient lymphocytes results in inhibition of chemokine-mediated responses, such as chemotaxis or integrin-mediated adhesion. This review summarizes the current data describing the involvement of the Jak-Stat pathway in chemokine-mediated signaling in T lymphocytes and discusses the potential crosstalk with other TCR and cytokine-mediated signaling pathways.

Chemokines and their receptors are important players in organism homeostasis, development and immune response to inflammatory stimuli. It has been recently confirmed that they are also involved in the development of several autoimmune diseases. In this study, we analysed the expression of two recently identified CC chemokine receptors, CCR7 and CCR8, in the central nervous system (CNS) and in peripheral tissues during chronic relapsing experimental autoimmune encephalomyelitis (ChREAE) -- an animal model of the human demyelinating disease multiple sclerosis (MS). We observed upregulation of both chemokine receptors in the CNS during the first and second attacks of ChREAE, whereas disease remission was characterized by a lower expression of those receptors. An analysis of the kinetics of CCR7 and CCR8 expression in the CNS during the first attack of the disease showed a constant increase in the first few days after the onset of clinical signs. This expression correlated with the clinical severity of ChREAE. CCR7-positive mononuclear cells were detected mostly in perivascular inflammatory cuffs in the CNS. In peripheral tissues (the spleen and kidneys) expression of both receptors was not upregulated during active ChREAE. These findings suggest that CCR7 and CCR8 may play a significant role in the pathogenesis of EAE and probably MS.

Chemokines are a superfamily of small, secreted proteins that regulate cell traffic in homeostatic and inflammatory conditions. Keratinocytes synthesize many chemokines, including members of the CC and CXC subfamilies, such as regulated on activation of normal T-cell expressed and secreted, gamma-interferon inducible protein-10, monokine induced by gamma-interferon, and thymus- and activation-regulated chemokine. They also express some chemokine receptors that mediate the inflammatory or immune response by attracting various kinds of leukocytes.

We studied the role of monocyte chemoattractant (MCP)-4/CCL13 in the pathogenesis of rheumatoid arthritis (RA). MCP-4 was highly expressed in cartilage from RA patients. Interferon-γ significantly stimulated MCP-4/CCL13 production in human chondrocytes, and this effect was enhanced in combination with interleukin-1β or tumor necrosis factor-α. MCP-4/CCL13 induces the phosphorylation of extracellular signal-regulated kinase in fibroblast-like synoviocytes and activates cell proliferation, and PD98059 completely inhibits these effects. These data suggest that interferon-γ in combination with interleukin-1β/tumor necrosis factor-α activates the production of MCP-4/CCL13 from chondrocytes in RA joints, and that secreted MCP-4/CCL13 enhances fibroblast-like synoviocyte proliferation by activating the extracellular signal-regulated kinase mitogen-activated protein kinase cascade.

Basophils represent less than 1% of peripheral blood leukocytes and have often been considered as minor and possibly redundant circulating mast cells. The recent finding that basophils readily generate large quantities of T helper 2 (Th2) cytokines such as IL-4 provided new insights into the possible role of basophils in allergic disorders and immunity to pathogens. However, in-depth studies on basophils, particularly their functions in vivo, have been hampered by the lack of appropriate animal models, such as mutant animals deficient only in basophils. Here, we established a mAb that reacted with mouse basophils and depleted them when administered in vivo. The mAb treatment of mice did not show any significant effect on classical allergic reactions such as passive cutaneous anaphylaxis and contact hypersensitivity. In contrast, it completely abolished the development of IgE-mediated chronic allergic dermatitis that is characterized by massive eosinophil infiltration, even though basophils accounted for only approximately 2% of the infiltrates. The treatment during the progression of the dermatitis resulted in drastic reduction in numbers of infiltrating eosinophils and neutrophils, concomitantly with elimination of basophils from the skin lesions. Thus, basophils play a pivotal role in the development of IgE-mediated chronic allergic inflammation, as an initiator rather than as an effector.

Expression of chemokine receptors by tumors, specifically CCR4 on cutaneous T cell lymphomas, is often associated with a poor disease outcome. To test the hypothesis that chemokine receptor-expressing tumors can be successfully controlled by delivering toxins through their chemokine receptors, we have generated fusion proteins designated chemotoxins: chemokines fused with toxic moieties that are nontoxic unless delivered into the cell cytosol. We demonstrate that chemokines fused with human RNase eosinophil-derived neurotoxin or with a truncated fragment of Pseudomonas exotoxin 38 are able to specifically kill tumors in vitro upon internalization through their respective chemokine receptors. Moreover, treatment with the thymus and activation-regulated chemokine (CCL17)-expressing chemotoxin efficiently eradicated CCR4-expressing cutaneous T cell lymphoma/leukemia established in NOD-SCID mice. Taken together, this work represents a novel concept that may allow control of growth and dissemination of tumors that use chemokine receptors to metastasize and circumvent immunosurveillance.

A subset of CC chemokines, acting through CC chemokine receptors (CCRs) 1 to 5, is instrumental in shaping inflammatory responses. Recently, we and others have demonstrated that the atypical chemokine receptor D6 actively sequesters and destroys many of these proinflammatory CC chemokines. This is critical for effective resolution of inflammation in vivo. Inflammation can be protumorigenic, and proinflammatory CC chemokines have been linked with various aspects of cancer biology, yet there is scant evidence supporting a critical role for these molecules in de novo tumor formation. Here, we show that D6-deficient mice have increased susceptibility to cutaneous tumor development in response to chemical carcinogenesis protocols and, remarkably, that D6 deletion is sufficient to make resistant mouse strains susceptible to invasive squamous cell carcinoma. Conversely, transgenic D6 expression in keratinocytes dampens cutaneous inflammation and can confer considerable protection from tumor formation in susceptible backgrounds. Tumor susceptibility consistently correlated with the level of recruitment of T cells and mast cells, cell types known to support the development of skin tumors in mice. These data demonstrate the importance of proinflammatory CC chemokines in de novo tumorigenesis and reveal chemokine sequestration by D6 to be a novel and effective method of tumor suppression.

Helicobacter pylori -infected gastric mucosa is characterized by infiltration of inflammatory cells such as neutrophils and eosinophils. However, little information is available on the relationship between H. pylori virulence factors and chemokine expression in eosinophils. This study investigates the role of vacuolating cytotoxin (VacA) in chemokine expression from human eosinophils. Eosinophils were isolated from the peripheral blood of healthy volunteers using a magnetic cell separation system. VacA + H. pylori water-soluble proteins (WSP) induced higher expression of interleukin-8, growth-related oncogene alpha, monocyte chemotactic protein 1, and RANTES (regulated on activation, normal, T-cell expressed and secreted) than Vac − WSP in human eosinophils, as assessed by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay. Purified VacA not only increased chemokine expression but also activated p65/p50 NF-κB heterodimers and phosphorylated IκB kinase (IKK) α/β signals in human eosinophils. Inhibition of NF-κB and IKK significantly decreased the chemokine expression in VacA-stimulated eosinophils. Furthermore, VacA-induced NF-κB activation and chemokine release from eosinophils were dependent on Ca 2+ influx and mitochondrial generation of reactive oxygen intermediates (ROI). These results suggest that NF-κB and IKK signals via Ca 2+ influx and mitochondrial ROI play a role in the up-regulation of chemokine expression in eosinophils stimulated with H. pylori VacA.

Chemokines play a critical role in shaping innate and adaptive immunity. These molecules also participate in maintaining the immune balance in the body. Apart from their regulatory role, these mediators are involved in several inflammatory and autoimmune diseases including viral infection such as HIV-1/AIDS. Chemokine co-receptor CCR5 and CXCR4 and their ligands significantly contribute to HIV-1 disease progression. C-C chemokines such CCL3, CCL4 and CCL5 have been shown to possess antiviral effects by binding to HIV-1 co-receptors. CCL2, a member of the C-C chemokine family, displays a different feature instead. It is a potential enhancer rather than inhibitor of viral replication, a property exhibited by most of the C-C chemokine members. In addition, the role of CCL2 is well established in forming a Th2 type of response by directing differentiation of Th0 cells towards Th2 type, a unique feature of HIV-1 disease. We propose a hypothesis in which the chemotactic nature of CCL2 drives recruitment of target cells to the site of infection as one of the mechanisms operating in vivo that favours viral replication and eventually a high viral load in infected individuals.

The fields of regulatory T (Treg) cells and chemokines/chemokine receptors have progressed rapidly in the last few years. Treg cells, especially CD4(+)CD25(+) Treg cells, play a critical role in maintaining self-tolerance and immune homeostasis. Chemokines and chemokine receptors are crucial for lymphoid development, homing and immunological regulation. This review will discuss the biological effects of chemokines and chemokine receptors on regulating the migration and development of CD4(+)CD25(+) Treg cells, and the potential clinical implications of these findings when considering chemokine receptors as therapeutic targets.

Increased numbers of pulmonary dendritic cells (DCs) are recruited to the lungs during allergic airway inflammation and contribute to the maintenance of the inflammatory immune response. The chemokine receptors that directly control DC accumulation into the lungs are largely unknown. To explore this issue, we generated mixed bone marrow chimeric mice containing both wild-type and knockout cells for a given chemokine receptor. After induction of allergic airway inflammation, we specifically tracked and compared chemokine receptor knockout vs wild-type DC populations through various lung compartments. Using this approach, we show that CCR2, but not CCR5 or CCR6, directly controls the accumulation of DCs into allergic lungs. Furthermore, the size of inflammatory monocyte populations in peripheral blood was strikingly CCR2 dependent, suggesting that CCR2 primarily mediates the release of monocytic DC precursors into the bloodstream.

Streptococcus pneumoniae (the pneumococcus) is estimated to cause approximately 1 million deaths per year in children in the developing world.1 The pneumococcus is the primary cause of pneumonia and meningitis in children in the developing world1 and is the leading cause of community-acquired bacteremia in children in sub-Saharan Africa.2 The burden of invasive pneumococcal disease (IPD) has increased in areas with a high prevalence of HIV infection.3 In South Africa, the disease incidence in children younger than 2 years of age has been shown to be 73 per 100,000 children per year in HIV-uninfected children compared with 3036 per 100,000 children per year in HIV-infected children.4 Chemokines (or chemoattractant cytokines) are subdivided into 4 distinct subfamilies (CC, CXC, CX3C, and C) depending on the arrangement and number of the conserved N-terminal cysteine molecules. The CC chemokines (also called β chemokines) have the first 2 cysteine molecules adjacent, include regulated on activation normal T cells expressed and secreted (RANTES or CCL5), monocyte chemotactic protein-1 (MCP-1 or CCL2), macrophage inflammatory protein-1α (MIP-1α or CCL3) and MIP-1β (CCL4), and they preferentially attract monocytes and/or macrophages and eosinophils. The CXC chemokines (also called α chemokines) have the first 2 cysteine molecules separated by an amino acid, which includes interleukin (IL)-8 (or CXCL8), growth-related oncogene (GROα or CXCL1), and epithelial cell–derived neutrophil activating peptide (ENA-78 or CXCL5), and they preferentially attract neutrophils.5,6 Interferon-γ (IFNγ) has been demonstrated to modulate the release of chemokines in pneumococcal cell wall–stimulated mouse microglia differentially, supporting the view that activated microglia are a major intrinsic source of central nervous system (CNS) chemokine production and that IFNγ potentially regulates this microglial chemokine response pattern.7 Because chemokines are specific for particular cell types, the ratio and concentration of different chemokines in different body compartments determine the composition of the different cell types within the particular compartment. To understand the host response, IPD, more fully, it is important to gain insight into the production and regulation of chemokines at local sites of inflammation. The HIV coreceptor CCR5 also acts as a receptor for CCL3, CCL4, and CCL5, and receptor binding and CCR5-dependent inhibition of HIV infection are induced by CCL8.8 CCR2b, the receptor for CCL2 (and CCL7, CCL8, and CCL13) is also a coreceptor for HIV and is expressed on peripheral blood lymphocytes and monocytes and/or macrophages.9 In HIV infection, CC chemokines induce an inflammatory response attracting noninfected target cells to the site of active viral replication and also induce intracellular signaling that enhances viral replication.10 The aims of this study were to investigate the chemokine response to IPD in children with pneumonia and meningitis and to examine the influence of HIV infection on the chemokine response, pneumococcal bacterial loads, and outcome.

Background and Objectives Chemokines are soluble mediators involved in angiogenesis, cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells.Design and Methods Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation, colony formation), chemokine receptor expression, constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells.Results Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors, but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression, but, compared to CD34− AML cells, CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore, a broad constitutive chemokine release profile was detected for most patients, and the following chemokine clusters could be identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFκB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased.Interpretation and Conclusions Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile.

ATP is abundantly released from stressed or damaged cells in response to mechanical stimulation, bacteria, or noxious agents. In this study, we have investigated the possible involvement of P2 receptors (receptor for extracellular nucleotides) in the expression and release of inflammatory mediators by human keratinocytes. Notably, extracellular ATP displayed a complex regulation of IFN-gamma-stimulated chemokine expression, with upregulation of CC chemokine ligand 2 (CCL2), CCL5 and CXC chemokine ligand 8 (CXCL8), and suppression of the receptor CXC chemokine receptor 3 (CXCR3), CXCL9, CXCL10, and CXCL11. The effect of ATP was mimicked by ADP and adenosine-5'-O-3-thiotriphosphate, whereas 2',3'-O-(4-benzoylbenzoyl) ATP (BzATP) downmodulated all chemokines investigated. UTP had no effect on IFN-gamma-stimulated chemokine secretion. The broad-spectrum P2 receptor antagonist suramin and the selective P2Y1 inhibitor adenosine 3'-phosphate 5'-phosphosulfate counteracted the effect of ATP on secretion of all the chemokines examined, whereas pyridoxal phosphate 6-azophenyl 2',4'-disulfonic acid and KN62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl] 4 phenylpiperazine) partially prevented the inhibitory effect of ATP on CXCL10 secretion, but on the other hand potentiated the ATP-stimulatory effect on CCL5, CCL2, and CXCL8 release. In lesional skin of psoriasis and atopic dermatitis patients, intense P2X7 reactivity was confined to the cell membrane of the basal layer, whereas diffuse P2Y1 immunostaining was found throughout the epidermis. Collectively, our data suggest that the orchestrated activation of distinct P2Y and P2X receptors modulates skin inflammation.

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.

Lung CD11chigh dendritic cells (DC) are comprised of two major phenotypically distinct populations, the CD11bhigh DC and the integrin αEβ7+ DC (CD103+ DC). To examine whether they are functionally distinguishable, global microarray studies and real-time PCR analysis were performed. Significant differences between the two major CD11chigh DC types in chemokine mRNA expression were found. CD11bhigh DC is a major secretory cell type and highly expressed at least 16 chemokine mRNA in the homeostatic state, whereas CD103+ DC highly expressed only 6. Intracellular chemokine staining of CD11chigh lung cells including macrophages, and ELISA determination of sort-purified CD11chigh cell culture supernatants, further showed that CD11bhigh DC produced the highest levels of 9 of 14 and 5 of 7 chemokines studied, respectively. Upon LPS stimulation in vitro and in vivo, CD11bhigh DC remained the highest producer of 7 of 10 of the most highly produced chemokines. Induction of airway hyperreactivity and lung inflammation increased lung CD11bhigh DC numbers markedly, and they produced comparable or higher amounts of 11 of 12 major chemokines when compared with macrophages. Although not a major producer, CD103+ DC produced the highest amounts of the Th2-stimulating chemokines CCL17/thymus and activation-related chemokine and CCL22/monocyte-derived chemokine in both homeostasis and inflammation. Significantly, CCL22/monocyte-derived chemokine exhibited regulatory effects on CD4+ T cell proliferation. Further functional analysis showed that both DC types induced comparable Th subset development. These studies showed that lung CD11bhigh DC is one of the most important leukocyte types in chemokine production and it is readily distinguishable from CD103+ DC in this secretory function.

The D6 chemokine receptor can bind and scavenge several chemokines, including the T-helper 2 (Th2)-associated chemokines CCL17 and CCL22. Although D6 is constitutively expressed in the lung, its pulmonary function is unknown.This study tested whether D6 regulates pulmonary chemokine levels, inflammation, or airway responsiveness during allergen-induced airway disease.D6-deficient and genetically matched C57BL/6 mice were sensitized and challenged with ovalbumin. ELISA and flow cytometry were used to measure levels of cytokines and leukocytes, respectively. Mechanical ventilation was used to measure airway reactivity.The ability of D6 to diminish chemokine levels in the lung was chemokine concentration dependent. CCL17 and CCL22 were abundant in the airway, and their levels were attenuated by D6 when they were within a defined concentration range. By contrast, airway concentrations of CCL3, CCL5, and CCL11 were low and unaffected by D6. Allergen-challenged D6-deficient mice had more dendritic cells, T cells, and eosinophils in the lung parenchyma and more eosinophils in the airway than similarly challenged C57BL/6 mice. By contrast, D6-deficient mice had reduced airway responses to methacholine compared with C57BL/6 mice. Thus, D6 has opposing effects on inflammation and airway reactivity.The ability of D6 to scavenge chemokines in the lung is dependent on chemokine concentration. The absence of D6 increases inflammation, but reduces airway reactivity. These findings suggest that inhibiting D6 function might be a novel means to attenuate airway responses in individuals with allergic asthma.

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Recruitment of leukocytes is a characteristic feature of tissue injury in systemic lupus erythematosus, including lupus nephritis. Locally secreted chemokines and their receptors are important mediators of leukocyte recruitment to the specific sites of immune complex injury, and contribute to renal inflammatory disease in the initiation and progression phase. Therefore, chemokines and chemokine receptors represent potential therapeutic targets in lupus nephritis. In this review we summarize available experimental and human data supporting their functional role in lupus nephritis. Moreover, interventional studies with chemokine and chemokine receptor antagonists that show the therapeutic potential of chemokine antagonists in experimental models of lupus nephritis and potentially in human renal disease are discussed.

SummaryAtherosclerosis is a chronic inflammatory disease that represents the primary cause of heart disease and stroke.The recruitment of inflammatory cells in the intima is an essential step in the development and progression of atherosclerosis.This process is triggered by local production of chemokines and chemokine receptors from activated endothelial cells and inflammatory cells. Various members of the CC chemokine family (e.g. MCP- 1/CCL2) as well as CXC family (e.g. IL-8/CCL8, IP-10/CXCL10, SDF-1/CXCL12) and, more recently, fractalkine/CX3CL1 have been implicated in atherosclerosis development. Latest findings in animal models suggest that blocking chemokine/chemokine receptor interactions may serve as a suitable approach to treat atherosclerosis. Likewise, chemokine antagonists that inhibit leukocyte recruitment could particularly be interesting to treat inflammation in response to myocardial infarction, the major consequence of atherosclerosis.

Purpose We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. Methods Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors. Results Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly. Conclusions We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.

Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4+T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.

The hepatitis B virus X protein (HBx), which is encoded by hepatitis B virus (HBV), plays crucial roles in the tumorigenesis of HBV associated hepatocellular carcinoma (HCC). Recent studies suggest that the HBx is involved in regulation of host immune cytokines and chemokines in HBV-associated HCC patients. However, effects of the HBx on autocrine chemokine expression profiles of hepatoma cells, which were shown in modulation of tumor-immune cell interactions, have not been investigated comprehensively. In the present study, human hepatoma cell lines SMMC-7721 and HepG2 were transfected with HBx-expressing plasmid. Human chemokine antibody array 1 (RayBio®), which simultaneously detects 38 chemokine factors, was used to determine chemokine expression profiles. Real-time polymerase chain reaction (real-time PCR) was used to further confirm the differential expression of chemokines. Chemokine antibody array revealed that all 38 chomekines were found to be expressed by SMMC-7721 and HepG2 cell lines. Interleukin-8 (IL-8) was obviously up-regulated, and epithelial neutrophil-activating protein 78 (ENA78), eosinophil chemotactic protein-1 (Eotaxin-1), monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3 and macrophage inflammatory protein-3β (MIP-3β) were significantly declined in both cell lines following transfection of HBx-expressing plasmid. Other chemokines showed little or no significant changes. HBx-induced differential chemokine expression levels were validated by real-time PCR. Hierarchical cluster analysis identified a distinction of chomekine expression profiles between HBX-expressing hepatoma cell lines and controls. Our findings provide new evidence that HBx is able to selectively regulate chomekines in hepatoma cells that may be involved in the regulation of tumor-immune cell interactions.