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Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism (1). However, its role in cancer immunopathology and immunotherapy is poorly understood. Using human ovarian cancers as [...]

The zebrafish lateral line primordium is a migrating collective of approximately 100 cells that deposits a series of mechanosensory organs along the main body axis (7) and whose route is [...], The directed migration of cell collectives is a driving force of embryogenesis (1-3). The predominant view in the field is that cells in embryos navigate along pre-patterned chemoattractant gradients (2). One hypothetical way to free migrating collectives from the requirement of long-range gradients would be through the self-generation of local gradients that travel with them (4,5), a strategy that potentially allows self-determined directionality. However, a lack of tools for the visualization of endogenous guidance cues has prevented the demonstration of such self-generated gradients in vivo. Here we define the invivodynamics of one key guidance molecule, the chemokine Cxcl12a, by applying a fluorescent timer approach to measure ligand-triggered receptor turnover in living animals. Using the zebrafish lateral line primordium as a model, we show that migrating cell collectives can self-generate gradients of chemokine activity across their length via polarized receptor-mediated internalization. Finally, by engineering an external source of the atypical receptor Cxcr7 that moves with the primordium, we show that a self-generated gradient mechanism is sufficient to direct robust collective migration. This study thus provides, to our knowledge, the first in vivo proof for self-directed tissue migration through local shaping of an extra-cellular cue and provides a framework for investigating self-directed migration in many other contexts including cancer invasion (6).

CXCL12 has a crucial role in maintaining HSC function, including retention in the bone marrow (8,12-14), quiescence (15,16) and repopulating activity (16). To test the hypothesis that CXCL12 production by [...], Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts (1,2) and perivascular stromal cells including endothelial cells (3), CXCL12-abundant reticular cells (4,5), leptin-receptor-positive stromal cells (6), and nestin-green fluorescent protein (GFP)-positive mesenchymal progenitors (7) have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C-X-C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations (7-11). Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterixexpressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1-cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, longterm repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

T. gondii is an opportunistic pathogen that causes encephalitis in patients with acquired defects in T-cell function (11). Several studies have established that resistance to this parasite in the central [...], Chemokines have a central role in regulating processes essential to the immune function of T cells (1-3), such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of [CD8.sup.+] T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, [CD8.sup.+] T-cell motility in the brain is well described by a generalized Levy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, [CD8.sup.+] T-cell behaviour is similar to Levy strategies reported in organisms ranging from mussels to marine predators and monkeys (4-10), and CXCL10 aids T cells in shortening the average time taken to find rare targets.

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-[kappa]B, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-[kappa]B silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-[kappa]B and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-[kappa]B inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-[kappa]B and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-[kappa]B and AP-1 in these cells. Thus dual inhibition of NF-[kappa]B and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-[kappa]B alone. keratinocyte cytokine; macrophage inflammatory protein-2; activator protein-1; nuclear factor-[kappa]B; acinar doi: 10.1152/ajpgi.00177.2010.

Muscle regeneration requires CC chemokine receptor 2 (CCR2) expression on bone marrowderived cells; macrophages are a prominent CCR2-expressing cell in this process. CCR2-/- mice have severe impairments in angiogenesis, macrophage recruitment, and skeletal muscle regeneration following cardiotoxin (CTX)-induced injury. However, multiple chemokines activate CCR2, including monocyte chemotactic proteins (MCP)-1 , -3, and -5. We hypothesized that MCP-1 is the chemokine ligand that mediates the impairments present in CCR2-/- mice. We examined muscle regeneration, capillary density, and cellular recruitment in MCP-/- and CCR2-/- mice following injury. Muscle regeneration and adipocyte accumulation, but not capillary density, were significantly impaired in MCP-1-/- compared with wild-type (WT) mice; however, muscle regeneration and adipocyte accumulation impairments were not as severe as observed in CCR2-/- mice. Although tissue levels of MCP-5 were elevated in MCP-1-/- mice compared with WT, the administration of MCP-5 neutralizing antibody did not alter muscle regeneration in MCP-1-/- mice. While neutrophil accumulation after injury was similar in all three mouse strains, macrophage recruitment was highest in WT mice, intermediate in MCP-1-/- mice, and severely impaired in CCR2-/- mice. In conclusion, while the absence of MCP-1 resulted in impaired macrophage recruitment and muscle regeneration, MCP-1-/- mice exhibit an intermediate phenotype compared with CCR2-/- mice. Intermediate macrophage recruitment in MCP-1-/- mice was associated with similar capillary density to WT, suggesting that fewer macrophages may be needed to restore angiogenesis vs. muscle regeneration. Finally, other chemokines, in addition to MCP-1 and MCP-5, may activate CCR2-dependent regenerative processes resulting in an intermediate phenotype in MCP-1-/- mice. monocyte/macrophage; angiogenesis; CC chemokine receptor 2; MCP-I/CCL2; MCP-5/CCL12 doi: 10.1152/ajpregu.00797.2009.

The retinoid X receptor a (RXRa) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRa regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRa in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRa plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis. nuclear hormone receptors | macrophages | innate immunity | sepsis doi/ 10.1073/pnas.0913545107

In atherosclerosis, chemokines recruit circulating mononuclear leukocytes to the vascular wall. A key factor is [CX.sub.3]CL1, a chemokine with soluble and transmembrane species that acts as both a chemoattractant and an adhesion molecule. Thromboxane [A.sub.2] and its receptor, TP, are also critical to atherogenesis by promoting vascular inflammation and consequent leukocyte recruitment. We examined the effects of TP stimulation on processing and function of [CX.sub.3]CL1, using [CX.sub.3]CL1-expressing human ECV-304 cells and primary human vascular endothelial cells. TP agonists promoted rapid shedding of cell surface [CX.sub.3]CL1, which was inhibited by pharmacological inhibitors or specific small interfering RNA targeting tumor necrosis factor-[alpha]-converting enzyme (TACE). Because it reduced cell surface [CX.sub.3]CL1, we predicted that TP stimulation would inhibit adhesion of leukocytes expressing the [CX.sub.3]CL1 cognate receptor but, paradoxically, saw enhanced adhesion. We questioned whether the enhanced ability of the remaining membrane-associated [CX.sub.3]CL1 to bind targets was caused by changes in its lateral mobility. Using fluorescence recovery after photobleaching, we found that plasmalemmal [CX.sub.3]CL1 was initially tethered but ultimately mobilized by TP agonists. TP stimulation provoked clustering of transmembrane [CX.sub.3]CL1 at sites of contact with adherent leukocytes. These data demonstrate that TP stimulation induces two distinct effects: a rapid cleavage of surface [CX.sub.3]CL1, thereby releasing the soluble chemoattractant, plus mobilization of the remaining transmembrane [CX.sub.3]CL1 to enhance the avidity of interactions with adherent leukocytes. The dual effect of TP allows [CX.sub.3]CL1 to recruit leukocytes to sites of vascular inflammation while enhancing their adhesion once recruited. leukocytes; inflammation; endothelial cells doi: 10.1152/ajpcell.00380.2009.

Acute lung injury (ALI) still poses a major challenge in critical care medicine. Neutrophils, platelets, and chemokines are all considered key components in the development of ALI. The Duffy antigen receptor for chemokines (DARC) is thought to be involved in scavenging, transendothelial transport, and presentation of neutrophil-specific chemokines. DARC is expressed on endothelial cells and erythrocytes but not on leukocytes. Here, we show that DARC is crucial for chemokine-mediated leukocyte recruitment in vivo. However, we also demonstrate that changes in chemokine and chemokine receptor homeostasis, associated with Darc gene deficiency, exert strong anti-inflammatory effects. Neutrophils from Darc gene-deficient ([Darc.sup.-/-]) mice display a more prolonged downregulation of CXCR2 during severe inflammation than neutrophils from wild-type mice. In a CXCR2-dependent model of acid-induced ALI, Dare gene deficiency prevents ALI. [Darc.sup.-/-] mice demonstrate fully preserved oxygenation, only a small increase in vascular permeability, and a complete lack of pulmonary neutrophil recruitment. Further analysis reveals that only neutrophils but neither endothelial cells nor erythrocytes from [Darc.sup.-/-] mice confer protection from ALI. The protection appears to be due to abolished pulmonary recruitment of neutrophils from [Darc.sup.-/-] mice. The generation of neutrophil-platelet aggregates, a key mechanism in both pulmonary neutrophil recruitment and thrombus formation, is also affected by altered CXCR2 homeostasis in [Darc.sup.-/-] mice. CXCR2 blockade enhances the formation of platelet-neutrophil aggregates and thereby corrects a formerly unknown bleeding defect in [Darc.sup.-/-] mice. In summary, our study suggests that chemokine/ chemokine receptor homeostasis plays a previously unrecognized and crucial role in severe ALI. endothelial cells; erythrocytes doi:10.1152/ajplung.00224.2009

Despite the associated morbidity and mortality, underlying mechanisms leading to the development of acute lung injury (ALI) remain incompletely understood. Frequently, ALI develops in the hospital, coinciding with institution of various therapies, including the use of supplemental oxygen. Although pathological evidence of hyperoxia-induced ALI in humans has yet to be proven, animal studies involving high oxygen concentration reproducibly induce ALI. The potentially injurious role of lower and presumably safer oxygen concentrations has not been well characterized in any species. We hypothesized that in the setting of a preexisting insult to the lung, the addition of moderate-range oxygen can augment lung injury. Our model of low-dose intratracheal LPS (IT LPS) followed by 60% oxygen caused a significant increase in ALI compared with LPS or oxygen alone with increased alveolar neutrophils, histological injury, and epithelial barrier permeability. In the LPS plus oxygen group, regulatory T cell number was reduced, and macrophage activation markers were increased, compared with LPS alone. Antibody-mediated depletion of neutrophils significantly abrogated the observed lung injury for all measured factors. The enhanced presence of alveolar neutrophils in the setting of LPS and oxygen is due, at least in part, to elevated chemokine gradients signaling neutrophils to the alveolar space. We believe these results strongly support an effect of lower concentrations of oxygen to augment the severity of a mild preexisting lung injury and warrants further investigation in both animals and humans. neutrophils; supplemental oxygen; chemokine; macrophage; regulatory T cell doi:10.1152/ajplung.00308.2009

The effects of individual ELR+ CXC chemokines have been documented in experimental models of acid aspiration. However, aspiration lung injury would be influenced by the combined effects of these chemokines and other factors related to their function. Therefore, the role of the chemokine receptor CXCR2 was examined in lung injury induced by aspiration of acid and acid with gastric particulates. Anesthetized mice were given intratracheal injections of saline, acid solution, or acid containing gastric particles. Within 6 h, bronchoalveolar lavage fluid neutrophils and albumin increased relative to the severity of the insult. Immunohistochemistry and RT-PCR demonstrated striking increases in pulmonary expression of CXCR2 after aspiration. In CXCR2-deficient mice, neutrophil recruitment to airways was significantly reduced after aspiration of either acid or acid with particles. However, lung injury scores were unaffected in Ccr2-/- mice in the acid + particles group. Esterase-stained lung tissue demonstrated that focal aggregates of inflammatory cells contained neutrophils in the Ccr2-/- mice. These studies suggest CXCR2 and its ligands are dominant mediators of neutrophil recruitment to airways after aspiration. However, CXCR2-independent mechanisms recruit neutrophils into areas of cellular aggregation after aspiration of acidified gastric particulates. chemokines; receptor; inflammation; neutrophil doi:10.1152/ajplung.90635.2008

Introduction Cutaneous T-cell lymphoma (CTCL) represents a rare heterogeneous group of malignant clonal proliferations of primarily skin-directed T-helper lymphocytes. The most common subtypes of CTCL are classic mycosis fungoides (MF) [...]

Adiponectin is believed to exert hepatoprotective effects and induces CXCL8, a chemokine that functions as a survival factor, in vascular cells. In the current study, it is demonstrated that adiponectin also induces CXCL8 expression in primary human hepatocytes but not in hepatocellular carcinoma cell lines. Knock down of the adiponectin receptor (AdipoR) 1 or AdipoR2 by small-interfering RNA indicates that AdipoR1 is involved in adiponectin-stimulated CXCL8 release. Adiponectin activates nuclear factor (NF)-[kappa]B in primary hepatocytes and pharmacological inhibition of NF-[kappa]B, the p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase (ERK) 1/ERK2 reduces adiponectin-mediated CXCL8 secretion. Furthermore, adiponectin also activates STAT3 involved in interleukin (IL)-6 and leptin-mediated CXCL8 induction in primary hepatocytes. Inhibition of JAK2 by AG-490 does not abolish adiponectin-stimulated CXCL8, indicating that this kinase is not involved. Pretreatment of primary cells with 'STAT3 Inhibitor VI,' however, elevates hepatocytic CXCL8 secretion, demonstrating that STAT3 is a negative regulator of CXCL8 in these cells. In accordance with this assumption, IL-6, a well-characterized activator of STAT3, reduces hepatocytic CXCL8. Therefore, adiponectin-stimulated induction of CXCL8 seems to be tightly controlled in primary human hepatocytes, whereas neither NF-[kappa]B, STAT3, nor CXCL8 are influenced in hepatocytic cell lines. CXCL8 is a survival factor, and its upregulation by adiponectin may contribute to the hepatoprotective effects of this adipokine. adiponectin; hepatocyte; nuclear factor-[kappa]B; adiponectin receptor 1; STAT3; extracellular signal-regulated kinase; mitogen-activated protein kinase

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvantinduced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced upregulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain. hypernociception | inflammation | prokineticin receptors

Eosinophil counts in the bronchoalveolar lavage fluid of wild-type (WT) mice increased after ovalbumin (OVA) challenge, a response that was diminished in comparably challenged low-expressing coagulation factor VII ([FVII.sup.tTA/tTA]) mice. Levels of T helper type 2 (Th2) cytokines, IL-4, IL-5, and IL-13, and eosinophil-attracting chemokines, eotaxin and RANTES, were also lower in the OVA-challenged [FVII.sup.tTA/tTA] mice. Eosinophils purified from low-FVII mice underwent apoptosis at a faster rate compared with WT eosinophils, and eosinophil migration in response to eotaxin was reduced in eosinophils obtained from [FVII.sup.tTA/tTA] mice. Airway hyperresponsiveness and mucous layer thickness were reduced in OVA-treated [FVII.sup.tTA/tTA] mice, and addition of exogenous coagulation factor X (FX) enhanced mucin production in human epithelial NCI-H292 cells. Correspondingly, incubation of FX with NCI-H292 cells resulted in activated (a) FX production, suggesting that the components required for FX activation were present on NCI-H292 cells. These results demonstrate that FVIIa functions in the asthmatic response to an allergen by stimulating lung eosinophilia, airway hyperresponsiveness, and mucin production, this latter effect through its ability to activate FX in conjunction with tissue factor. coagulation factor X; eosinophilia; T helper type 2 cytokines; chemokines; airway

Timely interaction between the egg and sperm is required for successful fertilization; however, little is known about the signaling therein. Prostaglandin (PG) E receptor EP2-deficient ([Ptger2.sup.-|-]) female mice exhibit a severe fertilization defect. We investigated the molecular events leading to this failure. We found increased gene expression for chemokines, such as Ccl2, Ccl7, and Ccl9, in [Ptger2.sup.-|-] cumulus cells (the somatic cells surrounding the egg) compared with wild-type cells. Furthermore, under physiological conditions, cumulus-derived chemokine signaling was found to have a dual action; CCL7 facilitates sperm migration to the cumulus-egg complex and integrin-mediated cumulus extracellular matrix (ECM) assembly to protect eggs. However, in the absence of [PGE.sub.2]-EP2 signaling, chronic CCL7 signaling results in excessive integrin engagement to the ECM, making the cumulus ECM resistant to sperm hyaluronidase, thereby preventing sperm penetration. Our findings indicate that [PGE.sub.2]-EP2 signaling negatively regulates the autocrine action of chemokines and prevents excessive cumulus ECM assembly. This interaction between PG and chemokine signaling is required for successful fertilization. cumulus cells | extracellular matrix | fibronectin | integrin | prostanoid receptor EP2

Given the central role of chemokines in infection, inflammation and immunity, chemokine receptors are a prime target for pharmacological intervention, and more so after the recent approval of chemokine receptor inhibitors for HIV. Allosteric inhibitors offer a largely unexploited opportunity to interfere with and modulate chemokine receptor activation and signaling. In addition to characterizing binding mode as a first step to understanding the specific mechanism underlying drug action, allosteric inhibitors pose new questions concerning different phases in drug discovery and pharmacological characterization, including the identification of appropriate screening tests, the evaluation of inhibitory effects on different signaling pathways and the implications of agonist- and signaling pathway-dependent inhibition for overall in vivo efficacy.

Exogenous carbon monoxide (CO) has anti-inflammatory and cytoprotective properties that show promise in the treatment of numerous pulmonary diseases. However, the effectiveness of CO in acute pulmonary injury associated with direct lung insult has not been shown conclusively. The purpose of this study was to determine if exogenous CO would modulate the pulmonary inflammation and lung injury that develops after acid aspiration. Groups of mice were given intratracheal (IT) injections of either saline or an acidic solution. After the IT injection, some of the mice in each group were allowed to spontaneously inhale CO (500 ppm). Mice exposed to CO for 6 h after IT acid had a significant decrease in bronchoalveolar lavage (BAL) fluid neutrophil counts and in histological evidence of lung injury. These results could not be explained by changes in BAL fluid chemokine levels or altered CXCR2 expression. The reduced neutrophil recruitment was associated with a decrease in the percentage of peripheral blood neutrophils expressing CD11b protein. However, within 24 h, the B AL neutrophil counts increased and were not different from animals without CO exposure. In addition, indices of vascular integrity were not different between animals with acid aspiration regardless of CO exposure at the later time point. These results showed that CO can modulate the early development of acute lung inflammation in this model of acid aspiration. Although these effects were eventually overwhelmed, the results suggest that CO may have efficacy during the initial treatment of aspiration lung injury. lung injury; chemokines; CXCR2; CD11b; inflammation

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr[7.sup.-/-] mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr[7.sup.-/-] mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr[7.sup.-/-] heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4. chemokines | heart | heterodimerization | immunology | endothelium

Neuropeptides play an important role in the active communication between the nervous and immune systems. Substance P (SP) is a prominent neuropeptide involved in neurogenic inflammation and has been reported to exert various proinflammatory actions on inflammatory leukocytes including neutrophils. The present study further investigated the modulatory effect of SP (1 [micro]M) on chemokine production and chemokine receptor expression in primary mouse neutrophils. Our results showed that SP primed neutrophils for chemotactic responses not only to the CXC chemokine macrophage inflammatory protein (MIP)-2/CXCL2 but also to the CC chemokine MIP-I[alpha]CCL3. The activating effect of SP on neutrophils was further evidenced by upregulation of the CD11b integrin, the activation marker of neutrophils. SP induced both the mRNA and protein expression of the chemokines MIP-1[alpha]/CCL3 and MIP-2/CXCL2 in neutrophils and upregulated the chemokine receptors CC chemokine receptor (CCR)-1 and CXC chemokine receptor (CXCR)-2. This stimulatory effect on chemokine and chemokine receptor expression in neutrophils was further found to be neurokinin-1 receptor (NK-1R) specific. Pretreatment with selective NK-IR antagonists inhibited SP-triggered activation of neutrophils and chemokine and chemokine receptor upregulation. Moreover, SP-induced chemokine upregulation was NF-[kappa]B dependent. SP time dependently induced NF-[kappa]B p65 binding activity, I[kappa]Bp[alpaha] degradation, and NF-[kappa]B p65 nuclear translocation in neutrophils. Inhibition of NF-[kappa]B activation with its inhibitor Bay11-7082 (10 [micro]M) abolished SP-induced NF-[kappa]B binding activity and upregulation of MIP1[alpha]/CCL3 and MIP-2/CXCL2 in neutrophils. Together, these results suggest that SP exerts a direct stimulatory effect on the expression of chemokines and chemokine receptors in mouse neutrophils. The effect is NK-1R mediated, involving NF-[kappa]B activation. chemokines and receptors; neuro-immune interaction; neurokinin-1 receptor; primary leukocytes; NF-KB activation

The transcription factor nuclear factor-[kappa]B (NF-[kappa]B) is activated by oxidative stress and pro-inflammatory stimuli and controls the expression of numerous genes involved in the inflammatory response. Dampening NF-[kappa]B activation and thereby limiting the inflammatory response have been suggested as a potential strategy to prevent chronic inflammatory diseases. In cultured monocytes, anthocyanins isolated from bilberries and black currants (Medox) efficiently suppressed LPS-induced activation of NF-[kappa]B. Furthermore, we studied the effect of anthocyanin supplementation (Medox, 300 mg/d for 3 wk) in a parallel-designed, placebo-controlled clinical trial (n = 120 men and women aged 40-74 y). Differences were observed in several NF-[kappa]B related inflammatory mediators in the Medox group compared to placebo. The changes in the NF-[kappa]B-controlled pro-inflammatory chemokines IL-8, 'regulated upon activation, normal T cell expressed and secreted,' (RANTES) and IFN[alpha] (an inducer of NF-[kappa]B activation) in the Medox group (45, 15, and 40% decreases from baseline, respectively) differed from those in the placebo group (20, 0, and 15% decreases from baseline, respectively) (P < 0.050). Similarly, changes in IL-4 and IL-13, 2 cytokines that mediate pro-inflammatory responses and induce NF-[kappa]B activation, in the Medox group (60 and 38% decreases from baseline, respectively) tended to differ from those in the placebo group (4 and 6% decreases) (P = 0.056 and, P = 0.089, respectively).These data suggest that anthocyanin supplementation may have a role in the prevention or treatment of chronic inflammatory diseases by inhibition of N F-[kappa]B transactivation and deceased plasma concentrations of pro-inflammatory chemokines, cytokines, and inflammatory mediators.

The hypothesis that the neutrophil chemoattractant CXC chemokines KC and macrophage inflammatory protein-2 (MIP-2) are involved in neutrophil transmigration and liver injury was tested in C3Heb/FeJ mice treated with galactosamine (Gal, 700 mg/kg), endotoxin (ET, 100 [micro]g/kg), or Gal + ET (Gal/ET). Hepatic KC and MIP-2 mRNA levels and plasma CXC chemokine concentrations were dramatically increased 1.5 h after Gal/ET or ET alone and gradually declined up to 7 h. Murine recombinant cytokines (TNF-[alpha], IL-1[alpha], and IL-1[beta]), but not Gal/ET, induced CXC chemokine formation in the ET-resistant C3H/HeJ strain. To assess the functional importance of KC and MIP-2, C3Heb/FeJ mice were treated with Gal/ET and control IgG or a combination of anti-KC and anti-MIP-2 antibodies. Anti-CXC chemokine antibodies did not attenuate hepatocellular apoptosis, sinusoidal neutrophil sequestration and extravasation, or liver injury at 7 h. Furthermore, there was no difference in liver injury between BALB/cJ wild-type and CXC receptor-2 gene knockout (CXCR[2.sup.-/-]) mice treated with Gal/ET. The higher neutrophil count in livers of CXCR[2.sup.-/-] than in wild-type mice after Gal/ET was caused by the elevated number of neutrophils located in sinusoids of untreated CXCR[2.sup.-/-] animals. The pancaspase inhibitor Z-Val-Ala-Asp-fluoromethylketone eliminated Gal/ET-induced apoptosis and neutrophil extravasation and injury but not CXC chemokine formation. Thus Gal/ET induced massive, cytokine-dependent CXC chemokine formation in the liver. However, neutrophil extravasation and injury occurred in response to apoptotic cell injury at 6-7 h and was independent of CXC chemokine formation. inflammatory liver injury; neutrophil cytotoxicity; apoptosis; caspases; CXC receptor 2

An immense number of bacteria reside within the intestinal lumen. The task of appropriately identifying and responding to microbial threats lies primarily with the single layer of cells that line the intestinal tract. Intestinal epithelial cells have developed a number of strategies aimed at identifying microorganisms and eliciting the appropriate inflammatory response. The pathogen recognition mechanisms and the signaling and inflammatory events that ensue within the intestine are the focus of this review. chemokines; eicosanoids; defensins; pathogen-associated molecular patterns; enteric bacteria

Pain, a critical component of host defense, is one hallmark of the inflammatory response. We therefore hypothesized that pain might be exacerbated by proinflammatory chemokines. To test this hypothesis, CCR1 was cotransfected into human embryonic kidney (HEK)293 cells together with transient receptor potential vanilloid 1 (TRPV1), a cation channel required for certain types of thermal hyperalgesia. In these cells, capsaicin and anandamide induced [Ca.sup.2+] influx mediated by TRPV1. When CCR1:TRPV1/HEK293 cells were pretreated with CCL3, the sensitivity of TRPV1, as indicated by the [Ca.sup.2+] influx, was increased [approximately equal to] 3-fold. RT-PCR analysis showed that a spectrum of chemokine and cytokine receptors is expressed in rat dorsal root ganglia (DRG). Immunohistochemical staining of DRG showed that CCR1 is coexpressed with TRPV1 in >85% of small-diameter neurons. CCR1 on DRG neurons was functional, as demonstrated by CCL3-induced [Ca.sup.2+] ion influx and PKC activation. Pretreatment with CCL3 enhanced the response of DRG neurons to capsaicin or anandamide. This sensitization was inhibited by pertussis toxin, U73122, or chelerythrine chloride, inhibitors of Gi-protein, phospholipase C, and protein kinase C, respectively. Intraplantar injection of mice with CCL3 decreased their hot-plate response latency. That a proinflammatory chemokine, by interacting with its receptor on small-diameter neurons, sensitizes TRPV1 reveals a previously undescribed mechanism of receptor cross-sensitization that may contribute to hyperalgesia during inflammation. CCRI | chemokine receptor | inflammation | pain

Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX3C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, C[X.sub.3]CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1[beta], TNF-[alpha], and IFN-[gamma], the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-[beta]. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-[gamma] and TNF-[alpha] induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-[beta] had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone ([10..sup.-8]-[10.sup.-6] M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun N[H.sub.2]terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 [micro]M) and SB-203580 (20 [micro]M), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-[gamma]- and TNF-[alpha]-induced JNK phosphorylation remained unaltered in the presence of TGF-[beta] but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-[beta]- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors. chemokine; airway inflammation; asthma; chronic obstructive pulmonary disease

[alpha]-Naphthylisothiocyanate (ANIT) is a hepatotoxin that causes severe neutrophilic inflammation around portal tracts and bile ducts. The chemotactic signals that provoke this inflammatory response are unknown. In this study, we addressed the possibility that ANIT upregulates CXC chemokines in the liver and that these compounds mediate hepatic inflammation and tissue injury after ANIT treatment. Mice treated with a single dose of ANIT (50 mg/kg) exhibited rapid hepatic induction of the CXC chemokine macrophage inflammatory protein-2 (MIP-2). MIP-2 derived primarily from hepatocytes, with no apparent contribution by biliary cells. In ANIT-treated mice, the induction of MIP-2 coincided with an influx of neutrophils to portal zones; this hepatic neutrophil recruitment was suppressed by 50% in mice that lack the receptor for MIP-2 (CXCR[2.sup.-/-]). Interestingly, despite their markedly reduced degree of hepatic inflammation, CXCR[2.sup.-/-] mice displayed just as much hepatocellular injury and cholestasis after ANIT treatment as wild-type mice. Moreover, after long-term exposure, ANIT CXCR[2.sup.-/-] mice developed liver fibrosis that was indistinguishable from that in wild-type mice. In summary, our data show that CXC chemokines are responsible for some of the hepatic inflammation that occurs in response to ANIT but that these compounds are not essential to the pathogenesis of either acute or chronic ANIT hepatotoxicity. hepatotoxicity; cholangiocyte; neutrophil; fibrosis; macrophage inflammatory protein-2

Macrophage-derived cytokines may provoke the inflammatory response in lung injury. Because macrophage influx is a prominent feature of the cellular inflammatory response accompanying the development of bronchopulmonary dysplasia, we hypothesized that blocking macrophage influx would reduce overall cellular influx and oxidative damage. Newborn rats were exposed at birth to 95% [O.sub.2] or air for 1 wk, and hyperoxia-exposed pups were injected with anti-monocyte chemoattractant protein-1 (MCP-1) or IgG control on days 3-5. MCP-1 was increased in bronchoalveolar lavage fluid and in histological sections from the 95% [O.sub.2]-exposed, IgG-injected pups compared with air-exposed controls. At 1 wk, anti-MCP-1-treated pups had reduced leukocyte numbers, both macrophages and neutrophils, in bronchoalveolar lavage fluid compared with IgG-treated controls. Cytokine-induced neutrophil chemoattractant-1, the rat analog of IL-8, was not significantly decreased in lavage fluid but was reduced in lung cells in anti-MCP-1-treated pups. Tissue carbonyls, a measure of protein oxidation, were decreased in anti-MCP-1-treated pups. Anti-MCP-1 treatment prevented neutrophil influx and reduced protein oxidation in hyperoxia-exposed newborn rats. bronchopulmonary dysplasia; oxidative stress; white blood cell

Pulmonary and activation-regulated chemokine (PARC/CCL18) belongs to the family of CC chemokines and shares 61% sequence identity with monocyte inflammatory protein (MIP)-1[alpha]. Produced by dendritic ceils and macrophages primarily in the lung, PARC is known to be chemotaetic for T cells. Because PARC's biological function is largely unknown, we screened various leukocyte populations for PARC expression and for response to PARC, with the idea that the cellular source may link PARC to disease states in which it may be involved. Here we report that eosinophils obtained from individuals with mild eosinophilia express PARC as assessed by RT-PCR on eosinophil RNA. The eosinophil preparations were free of monocytes, a known source of PARC, and no RT-PCR product was obtained from neutrophils. Furthermore, PARC protein was detected by ELISA in the supernatants of eosinophils from seven of nine donors and in higher concentration in the supernatants of monocytes on day 1 of culture. Purified recombinant PARC activated human monoeytes/macrophages kept in culture for 3-4 days but not freshly isolated monocytes. The threshold dose for [Ca.sup.2+] mobilization as determined fluorometrically in indo 1-AM-labeled monocytes was 5 nM; maximal response was reached with ~50 nM PARC. PARC was chemotactic for these cultured monocytes and caused actin polymerization determined by FITC-phalloidin binding and fluorescence-activated cell sorting analysis. In contrast, PARC activated neither neutrophils nor eosinophils. Eosinophil production of PARC, its chemotactic effect on monocytes and lymphocytes, and PARC's previously described localization to the lung suggest that this chemokine might play a role in pulmonary leukocyte trafficking. novel source; novel target cell

The CXC chemokines cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2) are potent neutrophil chemoattractants in rats. We have previously shown that CINC, unlike MIP-2 and most other proinflammatory cytokines, is elevated in the systemic circulation in response to an intratracheal (IT) challenge. Therefore, we hypothesized that CINC generated within the lung selectively enters the vascular compartment to facilitate pulmonary neutrophil recruitment. Rats were administered IT LPS, and plasma CINC and MIP-2 levels were measured 90 min and 4 h after injection, along with mRNA expression in lung, spleen, liver, and kidney. Ninety minutes and 4 h after IT LPS, CINC and MIP-2 mRNA expression were largely confined to lung homogenate, but of the two chemokines, only CINC was present in plasma. In separate experiments, rats received IT injections of recombinant CINC and/or MIP-2. Here, plasma levels of CINC, but not MIP-2, were significantly increased throughout the 4-h observation period. This finding was verified by individually administering [sup.125]I-labeled forms of each chemokine. Instillation of recombinant MIP-2 or CINC into the lung increased the number of neutrophils recovered in bronchoalveolar lavage fluid at 4 h, and this effect was enhanced when both chemokines were administered together. In addition, intravenous (IV) CINC, but not IV MIP-2, increased pulmonary neutrophil recruitment in response to IT MIP-2. Our results show that CINC, in contrast to MIP-2, is selectively transported from the lung to the systemic circulation, where it promotes neutrophil migration into the lung in response to a chemotactic stimulus. compartmentalization; macrophage inflammatory protein-2; intratracheal chemokines; mRNA; intratracheal lipopolysaccharide

Asthma is characterized by an increased production of eosinophil-active C-C chemokines by the airway epithelium. Recent studies have identified the presence of important quantities of labile zinc in the conducting airways. We hypothesized that modulation of this labile zinc could influence the production of proinflammatory chemokines in respiratory epithelial cells. The zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the heavy metal chelator 2,3-dimercapto-l-propanesulfonic acid (DMPS) were used to reduce the labile zinc content of A549, BEAS-2B, and HFL-1 cells. Northern blot analysis and RNase protection assay were used to study the effects of the zinc chelators on mRNA expression. DMPS and TPEN specifically inhibited the production of eotaxin, regulated on activation, normal T-cell expressed, and presumably secreted, and monocyte chemotactic protein-1 in TNF-[alpha]-stimulated respiratory epithelial cells and fibroblasts through labile zinc chelation. The inhibitory effects of DMPS and TPEN were associated with the decreased binding of the zinc-finger transcription factor GATA-1, whereas no change in NF-[kappa]B activation was observed. Together these results demonstrate that modulation of the labile pool of zinc can regulate gene expression and protein synthesis of C-C chemokines in lung epithelial cells and fibroblasts. CC chemokines; inflammation; asthma; regulated on activation, normal T-cell expressed, and presumably secreted; monocyte chemotactic protein

Angiogenesis occurs in skeletal muscle in response to exercise training. To gain insight into the regulation of this process, we evaluated the mRNA expression of factors implicated in angiogenesis over the course of a training program. We studied sedentary control (n = 17) rats and both sedentary (n = 18) and exercise-trained (n = 48) rats with bilateral femoral artery ligation. Training consisted of treadmill exercise (4 times/day, 1-24 days). Basal mRNA expression in sedentary control muscle was inversely related to muscle vascularity. Angiogenesis was histologically evident in trained white gastrocnemius muscle by day 12. Training produced initial three- to sixfold increases in VEGF, VEGF receptors (KDR and Flt), the angiopoietin receptor (Tie-2), and endothelial nitric oxide synthase mRNA, which dissipated before the increase in capillarity, and a substantial (30- to 50-fold) but transient upregulation of monocyte chemoattractant protein 1 mRNA. These results emphasize the importance of early events in regulating angiogenesis. However, we observed a sustained elevation of the angiopoietin 2-to-angiopoietin 1 ratio, suggesting continued vascular destabilization. The response to exercise was (in general) tempered in high-oxidative muscles. These findings place importance on cellular events coupled to the onset of angiogenesis. capillaries; peripheral vascular disease; endothelium; cytokines; chemokines

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1[beta] and tumor necrosis factor (TNF)-[alpha]. An imbalance between proteases and anti-proteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, [[alpha].sub.1]-antitrypsin, or N[alpha]-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1[beta] and TNF-[alpha]. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1[beta] and TNF-[alpha] may stimulate lung fibroblasts to release NCA and MCA by a proteasedependent mechanism and that serine protease inhibitors may attenuate the release. neutrophil; monocyte; interleukin-8; monocyte chemoattractant protein-1; granulocyte/macrophage colony-stimulating factor