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1: Infection and immunity, 2010 Sep 7,
KC (CXCL1)/CXCR2 Mediates Neutrophil Recruitment and is Critical for Development of Experimental Lyme Arthritis and Carditis.

[Abstract]Deletion of the chemokine receptor, CXCR2, prevents the recruitment of neutrophils into tissues and subsequent development of experimental Lyme arthritis. Following footpad inoculation of Borrelia burgdorferi, the agent of Lyme disease, expression of the CXCR2 ligand, KC (CXCL1), is highly up-regulated in the joints of arthritis-susceptible mice and is likely to play an important role in the recruitment of neutrophils to the site of infection. To test this hypothesis, we infected C3H KC(-/-) mice with B. burgdorferi and followed the development of arthritis and carditis. Ankle swelling was significantly attenuated during the peak of arthritis in the KC(-/-) mice. Arthritis severity scores were significantly lower in the KC(-/-) mice on days 11 and 21 post-infection with fewer neutrophils present in the inflammatory lesions. Cardiac lesions were also significantly decreased in KC(-/-) mice at day 21 post-infection. There were, however, no differences between C3H wild-type and KC(-/-) mice in spirochete clearance from tissues. Other CXCR2 ligands, LIX (CXCL5) and MIP-2 (CXCL2), were not increased to compensate for the loss of KC, and the production of several innate cytokines were unaltered. These results demonstrate that KC plays a critical non-redundant role in the development of experimental Lyme arthritis and carditis via CXCR2-mediated recruitment of neutrophils into the site of infection.

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2: Endocrine-related cancer, 2010 Aug 11, 58(12)
The chemokine CXCL1 induces proliferation in epithelial ovarian cancer cells by transactivation of the epidermal growth factor receptor.

[Abstract]The chemokine CXCL1 is elevated in plasma and ascites from patients with ovarian cancer. We have previously shown that CXCL1 is a marker of phosphatidylinositol 3-kinase signalling in epithelial ovarian cancer cell lines, a pathway that is commonly activated in ovarian tumours. To investigate whether CXCL1 also has functional significance in ovarian cancer, this chemokine was either downregulated using siRNAs or over-expressed by transfection of CXCL1 into the epithelial ovarian cancer cell lines SKOV3 and OVCAR-3 and proliferation assessed. Over-expression of CXCL1 increased proliferation of ovarian cancer cells over 7 days, while down-regulation was inhibitory. Treatment of cells with recombinant CXCL1 induced epidermal growth factor (EGFR) phosphorylation at Y1068, indicating cross-talk between the CXCL1 G-protein coupled receptor CXCR2 and the EGFR. CXCL1-induced proliferation was also decreased by inhibition of EGFR kinase activity and was dependent on extracellular matrix metalloprotease-mediated release of heparin-binding epidermal growth factor (HB-EGF). Involvement of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signalling was also evident since inhibition of both Ras and MEK activity decreased CXCL1-induced proliferation. CXCL1-induced ERK1/2 phosphorylation was inhibited by the MEK1 inhibitor PD98059, however EGFR phosphorylation was unaffected, indicating that CXCL1-activation of MAPK signalling is downstream of the EGFR. Taken together, these data show that CXCL1 functions through CXCR2 to transactivate the EGFR by proteolytic cleavage of HB-EGF, leading to activation of MAPK signalling and increased proliferation of epithelial ovarian cancer cells.

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3: Cell death and differentiation, 2010 Jul 2,
Ciglitazone negatively regulates CXCL1 signaling through MITF to suppress melanoma growth.

[Abstract]We have previously demonstrated that the thiazolidinedione ciglitazone inhibited, independently of PPARgamma activation, melanoma cell growth. Further investigations now show that ciglitazone effects are mediated through the regulation of secreted factors. Q-PCR screening of several genes involved in melanoma biology reveals that ciglitazone inhibits expression of the CXCL1 chemokine gene. CXCL1 is overexpressed in melanoma and contributes to tumorigenicity. We show that ciglitazone induces a diminution of CXCL1 level in different human melanoma cell lines. This effect is mediated by the downregulation of microphthalmia-associated transcription factor, MITF, the master gene in melanocyte differentiation and involved in melanoma development. Further, recombinant CXCL1 protein is sufficient to abrogate thiazolidinedione effects such as apoptosis induction, whereas extinction of the CXCL1 pathway mimics phenotypic changes observed in response to ciglitazone. Finally, inhibition of human melanoma tumor development in nude mice treated with ciglitazone is associated with a strong decrease in MITF and CXCL1 levels. Our results show that anti-melanoma effects of thiazolidinediones involve an inhibition of the MITF/CXCL1 axis and highlight the key role of this specific pathway in melanoma malignancy.Cell Death and Differentiation advance online publication, 2 July 2010; doi:10.1038/cdd.2010.75.

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4: Parasite immunology, 2010 May, 32(5)
Parasite excretory-secretory proteins elicit TRIF dependent CXCL1 and IL-6 mediated allergic inflammation.

[Abstract]Summary Currently, little information is available regarding innate immunity to helminthic parasite infection. In this study, we isolated the excretory-secretory (ES) proteins from Anisakis simplex (sea mammal intestinal parasite) third stage larva. We determined that the levels of IL-17 in the lung and lung draining lymph node of mice were increased sixfold as a result of intranasal treatment with ES proteins. The ES protein treatment elicited pro-inflammatory cytokine and chemokine secretion (especially IL-6 and CXCL1) from mouse lung epithelial cell line and primary lung epithelial cells. In addition, the expression of IL-6 and CXCL1 in mouse embryonic fibroblast (MEF) cells was significantly increased by the ES protein treatment, but we did not detect these effects in the TRIF(-/-) MEF cells. These elevations of IL-6 and CXCL1 expression were also not diminished by RNase treatment. In conclusion, the ES proteins of helminthic parasite larva may elicit TRIF dependent pro-inflammatory cytokines, and this is not double-stranded RNA.

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5: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 2010 May 21,
Aberrant expression of selectin E, CXCL1, and CXCL13 in chronic endometritis.

[Abstract]Chronic endometritis is often identified in the patients with unexplained infertility, and is histopathologically characterized by infiltration of plasmacytes within the endometrial stroma. In parallel with stromal plasmacyte infiltration, the endometrial functional layer in chronic endometritis is invaded by B cells, which are a rare leukocyte subset residing within the basal layer in the nonpathological endometrium. In this study, we investigated the molecular expression underlying this unusual increase of B cells in chronic endometritis. Twenty-two out of 76 infertile patients were diagnosed with chronic endometritis from the stromal plasmacyte infiltration, and the endometrium contained numerous stromal B-cell aggregates and glandular single B cells. However, the other major leukocyte subsets, including T cells, natural killer cells, macrophages, and neutrophils were comparable in densities in chronic endometritis and nonpathological endometrium. The microvascular endothelium showed immunoreactivity to adhesion molecule selectin E and chemokine CXCL13 along with immunoreactivity to CXCL1 in the glandular epithelium in chronic endometritis, but not in the nonpathological endometrium. Lipopolysaccharide significantly induced surface selectin E expression and CXCL13 secretion in uterine microvascular endothelial cells, and CXCL1 secretion in endometrial epithelial cells in vitro. These findings indicated that the aberrant local microenvironment triggered possibly by bacterial infection has a role in selective extravasation of circulating B cells in chronic endometritis.Modern Pathology advance online publication, 21 May 2010; doi:10.1038/modpathol.2010.98.

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6: Critical care medicine, 2010 Jan 14,
Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury.

[Abstract]OBJECTIVE:: To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. DESIGN:: C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01-1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. SETTING:: University hospital research laboratory. SUBJECTS:: Anesthetized male mice and murine cardiomyocytes. MEASUREMENTS AND MAIN RESULTS:: Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. CONCLUSIONS:: Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

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7: Endocrinology, 2009 Jan 15,
The chemokine Cxcl1 is a novel target gene of PTH/PTHrP in committed osteoblasts.

[Abstract]The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the CXC family chemokine ligand 1 (Cxcl1) as a novel immediate PTH/PTHrP responsive gene. Cxcl1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. Cxcl1 mRNA levels were up-regulated one hour after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold), and in rat metaphyseal bone (5-fold) one hour after a single subcutaneous injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted Cxcl1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for Cxcl1, is highly expressed in osteoclast precursors (haemopoietic cells) but is predominantly undetectable in the osteoblast lineage, suggesting that osteoblast-derived Cxcl1 may act as a chemoattractant for osteoclast precursors. Confirming this hypothesis, recombinant Cxcl1 dose-dependently stimulated migration of osteoclast precursors in cell culture studies, as did conditioned media from Kusa 4b10 cells treated with PTH. This data indicate that local action through the PTHR1 could stimulate cells of the osteoblast lineage to release a chemokine capable of attracting osteoclast precursors to the bone environment.

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8: European cytokine network, 2008 Dec, 19(4)
Prokineticin 1 induces CCL4, CXCL1 and CXCL8 in human monocytes but not in macrophages and dendritic cells.

[Abstract]Prokineticin 1 and 2 (PROK1 and PROK2) are two small proteins largely expressed in inflammatory tissues and involved in monocyte activation and differentiation. The focus of this study was to evaluate whether PROK1 was able to induce chemokine secretion in human monocytes, in monocyte-derived macrophages and in monocyte-derived dendritic cells, an aspect not addressed thus far. Here, we show for the first time, using flow cytometry, that PROK receptors 1 and 2 are present on the surface of human monocytes. Subsequently, monocytes were selected to investigate the chemokine response after stimulation by PROK1. Our results show that only three chemokines (CCL4, CXCL1 and CXCL8) were significantly induced at both the transcript and protein level, and that PROK1 induces most potently CXCL8, in a dose-dependent manner. From a mechanistic point of view, by blocking independently Galphai protein or intracellular calcium, monocytes lose the ability to secrete CXCL8 in response to PROK1. Finally, we observed that CCL4, CXCL1 and CXCL8 secretion, following PROK1 induction, is only observed in monocytes and not in monocyte-derived macrophages and dendritic cells. Our results demonstrate that, in vitro, the differentiation status of monocytes influences chemokine production after stimulation by PROK1, and that this chemokine production is geared toward a pro-inflammatory response. This could represent a novel amplification loop of leukocyte recruitment, extravasation and tissue invasion.

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9: The American journal of pathology, 2009 Jan, 174(1)
Neuroprotection and remyelination after autoimmune demyelination in mice that inducibly overexpress CXCL1.

[Abstract]In rodents, the chemokine CXCL1 both induces the proliferation and inhibits the migration of oligodendrocyte precursor cells. We previously reported that in multiple sclerosis, the same chemokine is expressed by hypertrophic astrocytes, which associate with oligodendrocytes that express the receptor CXCR2. To investigate whether chemokines influence repair after autoimmune demyelination, we generated GFAP-rtTA x beta-Gal-TRE-CXCL1 double-transgenic (Tg) mice that inducibly overexpress CXCL1 under the control of the astrocyte-specific gene, glial fibrillary acidic protein. Experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, was induced in these animals (and controls) by the subcutaneous injection of myelin oligodendrocyte glycoprotein, and after disease onset, CXCL1 production was initiated by the intraperitoneal injection of doxycycline. Double-Tg animals displayed a milder course of disease compared with both single (CXCL1 or glial fibrillary acidic protein)-Tg and wild-type controls. Pathologies were similar in all groups during the acute stage of disease. During the chronic disease phase, both inflammation and demyelination were diminished in double-Tg mice and Wallerian degeneration was markedly decreased. Remyelination was strikingly more prominent in double-Tg mice, together with an apparent increased number of oligodendrocytes. Moreover, cell proliferation, indicated by BrdU incorporation within the central nervous system, was more widespread in the white matter of double-Tg animals. These findings suggest a neuroprotective role for CXCL1 during the course of autoimmune demyelination.

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10: Molecular pain, 2008, 4(1)
The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons.

[Abstract]BACKGROUND: Altered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons. RESULTS: We report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 - 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS. CONCLUSION: Many studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.

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11: Innate immunity, 2008 Apr, 14(2)
Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis.

[Abstract]The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC(- /-) and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.