Modulation of CXCR1 and CXCR3 expression on NK cells via Tim-3 in a murine model of primary biliary cholangitis.

Tim-3, which is expressed on a variety of innate immune cells including NK cells, plays a key role in many autoimmune diseases. However, the immunomodulatory actions of Tim-3 on NK cells in primary biliary cholangitis (PBC) remain uncertain. Using a murine model of PBC we evaluated the expression of Tim-3 and its ligand Gal-9 in peripheral blood, liver, and spleen. Additionally, we studied Tim-3 regulation of chemokine receptors (CXCR1 and CXCR3) in vitro. Flow cytometric analysis indicated large numbers of infiltrating NK cells in the liver which exhibited high expression of Tim-3 and CXCR3. Moreover, we found overexpression of CXCR1 in liver tissue and liver-derived NK cells in PBC mice. We also observed lower levels of soluble Tim-3 in the serum of PBC mice. In vitro experiments with liver-derived NK cells from PBC mice indicated that CXCR3 was up-regulated by treatment with recombinant mouse TIM-3 Fc (rmTim-3 Fc) to activate the Tim-3 pathway. Furthermore, stimulating normal mouse spleen NK cells with poly I:C resulted in elevated expression of CXCR1 and interferon-γ release. Nonetheless, adding rmTim-3 Fc or rmGal-9 significantly down-regulated CXCR1 expression and IFN-γ release in NK cells activated by poly I:C, proposing a means to exploit the Tim-3 pathway to reverse responses in NK cells. In conclusion, our data demonstrate that dysregulation of Tim-3/Gal-9 is involved in modulating the local immune microenvironment in PBC mice. Our findings highlight the potential of Tim-3 pathway to modulate chemokine responses in NK cells during autoimmunity.

Upregulation of interleukin (IL)-31, a cytokine producing CXCR1 peripheral immune cells, contributes to the immune abnormalities of autism spectrum disorder.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by communication deficits, impaired social interactions, and restricted stereotypical behaviors. Several immune cells are associated with immune dysfunction in ASD; however, IL-31 has not been explored in ASD. This study aims to investigate the role of inflammatory cytokines and transcription factors of the CXCR1 cells in children with ASD. In the current study, we investigated the cytokines and transcription factors produced by CXCR1+ cells (IL-31, IL-9, IL-21R, IL-21, NF-κB p65, RORγT, STAT1, and FoxP3) in peripheral blood mononuclear cells (PBMCs), from children with ASD and typically developing (TD) control children, using flow cytometric analysis. In addition, we measured mRNA and protein expression levels of IL-31 using quantitative real-time PCR and western blot analyses in PBMCs. In our study, children with ASD had increased CXCR1+IL-31+, CXCR1+IL-9+, CXCR1+IL-21R+, CXCR1+IL-21+, CXCR1+NF-κB+ p65, CXCR1+RORγT+, and CXCR1+STAT1+, and decreased CXCR1+FoxP3+ cells as compared with cells from the TD control samples. Similarly, children with ASD showed increased IL-31 mRNA and protein expression levels as compared to those of TD control samples. Our results suggest that upregulated production of inflammatory cytokines and transcription factors in CXCR1+ cells cause immunological imbalance in children with ASD. Therefore, attenuation of inflammatory cytokines/mediators and transcription factors could have a therapeutic potential in the treatment of ASD.

IL-8 Alterations in HIV-1 Infected Children With Disease Progression.

Disease progression in HIV-1 infected children is faster than in adults. Less than 5% of the infected children maintain stable CD4 counts beyond 7 years of infection and are termed long-term nonprogressors (LTNPs). Delineating the host immune response in antiretroviral naïve (ART) and treated HIV-1 infected children at different disease stages will help in understanding the immunopathogenesis of the disease.A total of 79 asymptomatic, perinatally HIV-1 infected children (50 ART naïve and 29 ART treated) and 8 seronegative donors were recruited in this study. T- and B-cell activation PCR arrays were performed from the cDNA, using total RNA extracted from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1 infected children at different stages of the disease. The differentially expressed genes were identified. Quantitative RT-PCR was performed for the (interleukin-8) IL-8 gene and its transcriptional mediators, that is, SHP2, GRB2, and IL-8R (IL-8 receptor/CXCR1). Plasma levels of IL-8 were measured by flow cytometry.Gene array data revealed a higher expression of IL-8 in the ART naïve HIV-1 infected progressors and in ART nonresponders than LTNPs and ART responders, respectively. Quantitative RT-PCR analysis demonstrated a significant higher expression of IL-8 (P < 0.001), its receptor CXCR1 (P = 0.03) and the upstream signaling molecule SHP2 (P = 0.04) in the progressors versus LTNPs. Plasma levels of IL-8 were significantly higher in progressors versus LTNPs (P < 0.001), and ART nonresponders versus ART responders (P < 0.001). A significant negative correlation of plasma levels of IL-8 with CD4 counts (cells/µL) was observed in HIV-1 infected ART naïve subjects (r = -0.488; P < 0.001), while the IL-8 levels positively correlated with viral load in the ART treated children (r = 0.5494; P < 0.001). ART naïve progressors on follow up demonstrated a significant reduction in the mRNA expression (P = 0.05) and plasma levels of IL-8 (P = 0.05) post 6 months of ART initiation suggesting the beneficial role of ART therapy in reducing inflammation in infected children.Our data suggest that IL-8 may serve as a potential prognostic marker in adjunct with CD4 counts to monitor disease progression in the HIV-1 infected children and the efficacy of ART.

Repertaxin, an inhibitor of the chemokine receptors CXCR1 and CXCR2, inhibits malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil.

Chemokine-mediated activation of G protein-coupled receptors CXCR1/2 promotes tumor growth, invasion, inflammation and metastasis. Repertaxin, a CXCR1/2 small-molecule inhibitor, has been shown to attenuate many of these tumor-associated processes. The present study aimed to investigate the effects of repertaxin alone and in combination with 5-fluorouracil (5-FU) on the malignant behavior of gastric cancer and the potential mechanisms. Gastric cancer MKN45 cells were treated in vitro with repertaxin and 5-FU, either alone or in combination. MTT and colony formation assay were performed to assess proliferation. Cell cycle progression and apoptosis was completed by flow cytometry. Migration and invasion were also assessed by transwell and wound-healing assay. Western blot analysis and quantitative RT-PCR were performed to determine expression of signaling molecules. MKN45 cells were also grown as xenografts in nude mice. Mice were treated with repertaxin and 5-FU, and tumor volume and weight, angiogenesis, proliferation and apoptosis were monitored. Combination of repertaxin and 5-FU inhibited MKN45 cell proliferation and increased apoptosis better than either agent alone. Similarly, enhanced effect of the combination was also observed in migration and invasion assays. The improved effect of repertaxin and 5-FU was also observed in vivo, as xenograft models treated with both compounds exhibited significantly decreased tumor volume and increased apoptosis. In conclusion, repertaxin inhibited malignant behavior of human gastric cancer MKN45 cells in vitro and in vivo and enhances efficacy of 5-fluorouracil. These data provide rationale that targeting CXCR1/2 with small molecule inhibitors may enhance chemotherapeutic efficacy for the treatment of gastric cancer.

Collagen-Derived N-Acetylated Proline-Glycine-Proline in Intervertebral Discs Modulates CXCR1/2 Expression and Activation in Cartilage Endplate Stem Cells to Induce Migration and Differentiation Toward a Pro-Inflammatory Phenotype.

The factors that regulate the migration and differentiation of cartilage endplate stem cells (CESCs) remain unknown. N-Acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine that is involved in inflammatory diseases. The purpose of this study was to detect N-Ac-PGP in degenerative intervertebral discs (IVDs) and to determine its roles in the migration and differentiation of CESCs. Enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-mass spectrometry results indicated that the levels of the proteases that generate N-Ac-PGP as well as N-Ac-PGP levels themselves increase with the progression of IVD degeneration. Immunohistochemistry and an N-Ac-PGP generation assay demonstrated that nucleus pulposus (NP) cells generate N-Ac-PGP from collagen. The effects of N-Ac-PGP on the migration and differentiation of CESCs were determined using migration assays, RT-PCR, immunoblot analysis, and ELISA. The results showed that the expression of N-Ac-PGP receptors (CXCR1 and CXCR2) in CESCs was upregulated by N-Ac-PGP. Additionally, N-Ac-PGP induced F-actin cytoskeletal rearrangement in CESCs and increased CESC chemotaxis. Furthermore, N-Ac-PGP recruited chondrocytes and spindle-shaped cells from the cartilage endplate (CEP) into the NP in vivo. These spindle-shaped cells expressed CD105 and Stro-1 (mesenchymal stem cell markers). N-Ac-PGP induced the differentiation of CESCs toward a pro-inflammatory phenotype with increased production of inflammatory cytokines rather than toward an NP-like phenotype. Our study indicated that, in the complex microenvironment of a degenerative disc, N-Ac-PGP is generated by NP cells and induces the migration of CESCs from the CEP into the NP. N-Ac-PGP induces a pro-inflammatory phenotype in CESCs, and these cells promote the inflammatory response in degenerative discs.

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin-8 (IL-8) and leukotriene B4 (LTB4 ) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll-like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down-regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL-8-induced and LTB4 -induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down-regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL-8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Expression of CXCR1 (interleukin-8 receptor) in murine macrophages after staphylococcus aureus infection and its possible implication on intracellular survival correlating with cytokines and bacterial anti-oxidant enzymes.

Interaction with the live Staphylococcus aureus promotes secretion of interleukin-8 (IL-8), although the expressions of functional CXCR1 (IL-8RA) in murine macrophages have not been identified. Expression of CXCR1 was induced in S. aureus-infected macrophages, whereas, CXCR1 was undetectable in control macrophages. CXCR1 blocking significantly reduced the phagocytosis of S. aureus and TNF-α, IL-6, IL-1ß, IFN-γ, IL-12, and IL-8 production and increased release of MIP-2 and soluble TNF-R1. Increased bacterial catalase and decreased superoxide dismutase (SOD) activities by S. aureus with concomitant decrease in hydrogen peroxide (H2O2), superoxide anion, and nitric oxide (NO) release were observed in case of prior CXCR1 blocking. In the presence of cytochalasin D, S. aureus-mediated induction of IL-8 was inhibited concomitant with decreased bacterial count suggesting that internalization of S. aureus was necessary for induction of IL-8. Shedding of TNF-R1 due to CXCR1 blocking after S. aureus inoculation was critical for neutralization of TNF-α signaling and arrests the inflammation.

High expression of GPER1, EGFR and CXCR1 is associated with lymph node metastasis in papillary thyroid carcinoma.

Clinical and epidemiological studies have shown that estrogen may be involved in the development and progression of papillary thyroid carcinoma (PTC). G protein-coupled estrogen receptor 1 (GPER1) is a novel seven-transmembrane estrogen receptor that functions alongside traditional nuclear estrogen receptors (ERs) to regulate the cellular responses to estrogen. The purpose of this study was to examine GPER1, EGFR and CXCR1 expression in PTC and to assess the association of their expression with clinicopathological indicators. GPER1, EGFR and CXCR1 protein expression in 129 PTCs, 61 nodular hyperplasia and 118 normal thyroid tissue specimens were analyzed using immunohistochemistry. The protein expression levels of these three molecules were up-regulated in PTCs. High protein expression of GPER1, EGFR and CXCR1 was significantly correlated with lymph node metastasis (LNM) (P ≤ 0.001). Furthermore, GPER1, EGFR and CXCR1 protein expression were correlated with one another. Concomitant high expression of these molecules had stronger correlation with LNM than did each alone (P = 0.002 for GPER1/EGFR, P = 0.013 for GPER1/CXCR1, P = 0.018 for EGFR/CXCR1 and P < 0.001 for GPER1/EGFR/CXCR1). Additionally, GPER1, EGFR and CXCR1 mRNA expression was assessed in 30 PTCs, 10 nodular hyperplasia and 10 normal thyroid tissue specimens using real-time RT-PCR. GPER1, EGFR and CXCR1 mRNA expression levels were up-regulated in PTCs, and high mRNA expression of GPER1, EGFR and CXCR1 was significantly correlated with LNM (P < 0.001 for all these three molecules). These results demonstrated that the evaluation of GPER1, EGFR and CXCR1 expression in PTC may be useful in predicting the risk of LNM.

Association of CXCR1 and 2 expressions with gastric cancer metastasis in ex vivo and tumor cell invasion in vitro.

BACKGROUND: CXCR1 and CXCR2, cell surface receptors of interleukin-8, regulate cell migration and alteration of their expression has been associated with poor prognosis of various cancers. The aim of this study was to detect their expression in gastric cancer to identify associations with another cell adhesion molecule, matrix metalloproteinase-9 (MMP9), and with clinicopathological data ex vivo, and then explore their potential role in gastric cancer cells in vitro. MATERIALS AND METHODS: A total of 172 cases of gastric cancer tissue specimens were collected for immunohistochemical analysis of CXCR1, CXCR2, and MMP9 expression. Expression of CXCR1 and CXCR2 proteins was knocked in or down using their cDNA and shRNA, respectively, in gastric cancer cell lines to assess the changed cell phenotypes and gene expression. RESULTS: CXCR1, CXCR2, and MMP9 were expressed in 61.0%, 77.9%, and 75.6% of gastric cancer tissues, respectively. Moreover, CXCR1 and CXCR2 expression was associated with tumor differentiations, advanced clinical stages, lymph node, and distant metastasis of gastric cancer. Similarly, MMP9 expression was associated with CXCR1 and CXCR2. Expression of these three proteins was interrelated. In vitro study showed that levels of CXCR1 and CXCR2 proteins were associated with the capacity of gastric cancer cell migration, while knockdown of their expression inhibited gastric cancer cell migration and invasion abilities in vitro. In contrast, overexpression of CXCR1 and CXCR2 proteins promoted tumor cell migration and invasion. At the gene levels, knockdown of CXCR1 or CXCR2 expression suppressed expression of Ets-1, SRC-1, and JNK proteins and phosphorylated c-Jun and Erk1/2. Conversely, upregulation of CXCR1 or CXCR2 promoted expression of Ets-1, SRC-1, JNK, and c-Jun proteins and phosphorylated JNK, c-Jun and Erk1/2. CONCLUSIONS: These findings suggest that CXCR1 and CXCR2 play an important role in gastric cancer progression. Further study will be performed to investigate whether target of their expression can be used as a novel strategy in clinical control of gastric cancer metastasis.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

Neuroblastoma-derived TGF-ß1 modulates the chemokine receptor repertoire of human resting NK cells.

In this study, we show that neuroblastoma (NB) cell conditioning affects the chemokine receptor repertoire of human resting NK cells. In particular, NB cells upregulated the expression of CXCR4 and CXCR3 in all NK cells and downregulated CX3CR1 in the CD56(dim) subset. On the contrary, the expression of CXCR1 and CCR7 remained unaltered. The phenomenon was dependent on the release by NB cells of TGF-ß1, and rTGF-ß1 induced a chemokine receptor repertoire identical to that of NB-conditioned NK cells. The immune modulatory role of TGF-ß1 appears to be dose dependent because low amounts of the cytokine were sufficient to modulate CXCR4 and CX3CR1 expression, intermediate amounts modified that of CXCR3, and high amounts were necessary to downregulate the expression of the NKp30 activating receptor. Notably, a similar receptor modulation was observed in rTGF-ß2-conditioned NK cells. Finally, the analysis of NK cells from patients with stage 4 NB suggests that NB conditioning could exert in vivo an immune modulatory effect resembling that emerged from in vitro experiments. Altogether our data propose a novel tumor escape-mechanism based on the modulation of chemokine receptors that play pivotal roles in NK cells bone marrow homing, egress, or recruitment into peripheral tissues.

Human labour is associated with a decline in myometrial chemokine receptor expression: the role of prostaglandins, oxytocin and cytokines.

PROBLEM: Human labour is an inflammatory process with a heavy infiltration of immune cells into the myometrium and cervix induced by local chemokine production. Myometrial cells also express chemokine receptors, but there is little information about their behaviour or function during pregnancy and labour. METHOD OF STUDY: We studied the behaviour of the receptors (CCR2, CXCR1 and CXCR2) for the CCL2 and CXCL8 in human myometrium, because both have been shown to be important in labour. RESULTS: We found that there was a significant decline in the mRNA expression of all three receptors in the upper segment and a similar trend in the lower segment with the onset of term labour (TL). Chemokine receptor mRNA expression was increased by stretch, reduced by oxytocin and PGF(2α) acting via phospholipase C (PLC). CXCR2 declined with exposure to CXCL8, consistent with the negative relationship observed in labouring myometrial tissue. The mRNA changes were confirmed by western analysis and flow cytometry. CONCLUSION: These data show that myometrial chemokine receptor expression is reduced with the onset of term labour probably in response to the increased activity of chemokines, oxytocin and PGF(2α) .

Abnormal spontaneous interleukin 8 receptor expression: a brief report of two cases.

Interleukin 8 (CXCL8) is an autocrine chemokine specific for the chemoattraction and activation of granulocytes, NKT cells and T lymphocytes. Patients with tuberculosis and latent Mycobacterium tuberculosis infection were assessed for the spontaneous expression of CXCR1 (CD128) and CXCR2 on lymphocytes and monocytes. Compared with ex vivo profiles, increased spontaneous CXCR2 expression and normal CXCR1 expression were found on lymphocytes in two out of 59 individuals. Monocytes showed normal ex vivo profiles for both receptors. After stimulation with purified protein derivative, the in vitro levels of CXCL8 were below the median levels of all patients with prior tuberculosis. Spontaneous CXCR2 modulation did not cause notable variation in the in vitro levels of CXCL8.

Endothelial cells overexpressing interleukin-8 receptors reduce inflammatory and neointimal responses to arterial injury.

BACKGROUND: Interleukin-8 (IL8) receptors IL8RA and IL8RB on neutrophil membranes bind to IL8 and direct neutrophil recruitment to sites of inflammation, including acutely injured arteries. This study tested whether administration of IL8RA- and/or IL8RB-transduced rat aortic endothelial cells (ECs) accelerates adhesion of ECs to the injured surface, thus suppressing inflammation and neointima formation in balloon-injured rat carotid arteries. We tested the hypothesis that targeted delivery of ECs by overexpressing IL8RA and IL8RB receptors prevents inflammatory responses and promotes structural recovery of arteries after endoluminal injury. METHODS AND RESULTS: Young adult male rats received balloon injury of the right carotid artery and were transfused intravenously with ECs (total, 1.5×10(6) cells at 1, 3, and 5 hours after injury) transduced with adenoviral vectors carrying IL8RA, IL8RB, and IL8RA/RB (dual transduction) genes, AdNull (empty vector), or vehicle (no EC transfusion). ECs overexpressing IL8Rs inhibited proinflammatory mediators expression significantly (by 60% to 85%) and reduced infiltration of neutrophils and monocytes/macrophages into injured arteries at 1 day after injury, as well as stimulating a 2-fold increase in reendothelialization at 14 days after injury. IL8RA-EC, IL8RB-EC, and IL8RA/RB-EC treatment reduced neointima formation dramatically (by 80%, 74%, and 95%) at 28 days after injury. CONCLUSIONS: ECs with overexpression of IL8RA and/or IL8RB mimic the behavior of neutrophils that target and adhere to injured tissues, preventing inflammation and neointima formation. Targeted delivery of ECs to arteries with endoluminal injury provides a novel strategy for the prevention and treatment of cardiovascular disease.

Enhancement of interleukin-8-induced chemotactic response and reactive oxygen species production in HL-60 cells expressing CXCR1.

Neutrophils play a pivotal role in immunity against infection by ingesting and killing invading microbes. Neutrophils isolated from human peripheral blood have been used for a number of studies conducted for evaluation of immunomodulating drugs, cytokines, and microbe products. Human promyelocytic leukemia cells, HL-60, have been extensively studied because they can differentiate into neutrophil-like cells by addition of all-trans retinoic acid or dimethyl sulfoxide. For a system that would always allow experimental use of granulocytic cells in a uniformly activated state, we have established HL-60 cell lines with increased migratory activity by transducing the CXC chemokine receptor 1 (CXCR1) gene. When these cell lines were primed with CXC chemokine ligand 8 (IL-8), a slight increase in reactive oxygen species production induced by phorbol myristate acetate (PMA) or zymosan A stimuli was observed. A significance increase in migratory activity was noticed when the HL-60 cells transduced CXCR1 were stimulated with IL-8 in the Boyden chamber method. The gene-transduced HL-60 cell lines may be used as a substitute for neutrophils in screening the effects of various immunomodulating drugs on the migratory activity induced by IL-8.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.

Optimization of purification and refolding of the human chemokine receptor CXCR1 improves the stability of proteoliposomes for structure determination.

The human chemokine receptor CXCR1 is a G-protein coupled receptor that has been successfully expressed in E. coli as inclusion bodies, and purified and refolded in multi-milligram quantities required for structural studies. Expression in E. coli enables selective and uniform isotopic labeling with (13)C and (15)N for NMR studies. Long-term chemical and conformational stability and oligomeric homogeneity of CXCR1 in phospholipid bilayers are crucial for structural studies under physiological conditions. Here we describe substantial refinements in our previously described purification and reconstitution procedures for CXCR1 in phospholipid bilayers. These refinements have led to the preparation of highly purified, completely monomeric, proteoliposome samples that are stable for months at 35°C while subject to the high power radiofrequency irradiations of solid-state NMR experiments. The principal changes from the previously described methods include: 1) ensure that CXCR1 is pure and homogeneously monomeric within the limits of detection (>98%); 2) monitor and control the pH at all times especially following the addition of TCEP, which serves as a reducing agent but also changes the pH; 3) slowly refold CXCR1 with the complete removal of all traces of SDS using a KCl precipitation/dialysis method; and 4) ensure that the molar ratio between the CXCR1 and the phospholipids does not change during refolding and detergent removal. NMR samples prepared with these protocols yield reproducible results over a period of many months at 35°C. This purification and refolding protocol is likely to be applicable with minimal changes to other GPCRs as well as other membrane proteins.

CXC chemokine receptor-1 is expressed by hepatocytes and regulates liver recovery after hepatic ischemia/reperfusion injury.

UNLABELLED: CXC chemokines mediate hepatic inflammation and injury following ischemia/reperfusion (I/R). More recently, signaling through CXC chemokine receptor-2 (CXCR2) was shown to delay liver recovery and repair after I/R injury. The chemokine receptor CXCR1 shares ligands with CXCR2, yet nothing is known about its potential role in liver pathology. In the present study, we examined the role of CXCR1 in the injury and recovery responses to I/R using a murine model. CXCR1 expression was undetectable in livers of sham-operated mice. However, after ischemia CXCR1 expression increased 24 hours after reperfusion and was maximal after 96 hours of reperfusion. CXCR1 expression was localized largely to hepatocytes. In order to assess the function of CXCR1, CXCR2(-/-) mice were treated with the CXCR1/CXCR2 antagonist, repertaxin. Prophylactic treatment with repertaxin had no effect on acute inflammation or liver injury. However, when repertaxin was administered 24 hours postreperfusion there was a significant increase in hepatocellular injury and a delay in recovery compared to control-treated mice. CXCR1(-/-) mice also demonstrated delayed recovery and regeneration after I/R when compared to wild-type mice. In vitro, hepatocytes from CXCR2(-/-) mice that were stimulated to express CXCR1 showed increased proliferation in response to ligand. Hepatocyte proliferation was decreased in CXCR1(-/-) mice in vivo. CONCLUSION: This is the first report to show that CXCR1 expression is induced in hepatocytes after injury. Furthermore, the data suggest that CXCR1 has divergent effects from CXCR2 and appears to facilitate repair and regenerative responses after I/R injury.

Enrichment of Foxp3+ CD4 regulatory T cells in migrated T cells to IL-6- and IL-8-expressing tumors through predominant induction of CXCR1 by IL-6.

Analysis of cytokine and chemokine production by tumor cell lines including five lung cancers, a malignant mesothelioma, and a malignant melanoma recently established in our laboratory showed rather high production of IL-8 in all tumors and IL-6 in one lung cancer, the malignant mesothelioma, and the malignant melanoma. We investigated the migration of PBMCs to these tumor cells using Transwell plates and showed enrichment of Foxp3(+) CD4 regulatory T cells (Tregs) in migrated T cells to both IL-6- and IL-8-producing tumors. Marked induction of CXCR1 expression on Foxp3(+) CD4 Tregs by IL-6 followed by IL-8-mediated migration appeared to be responsible for enriched migration. Frequent production of IL-8 by the tumors and Treg migration to those tumors through induction of IL-8R expression by IL-6 is one of the mechanisms for tumor escape.

Expression of zebrafish cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation.

Cxcl8 is a pro-inflammatory chemokine, best known for its role in neutrophil chemotaxis. Signalling through its receptors, Cxcr1 and Cxcr2, is induced by inflammatory stimuli evoked by microbial, chemical or environmental stress, and hormonal signals. While it is recognised that Cxcl8 signalling is active in the gut mucosa, this is not as well understood as its role in leukocyte trafficking. Here, we report the characterisation of genes encoding the zebrafish Cxcl8, Cxcr1 and Cxcr2. By a combination of genomic, expression and functional analyses, we show that the Cxcl8 signalling pathway is conserved in zebrafish. As in humans, cxcl8 is expressed in zebrafish leukocytes. Transcripts were also detected in intestinal epithelial cells, and this expression is upregulated under inflammatory conditions caused by bacterial or chemical insult. Expression of cxcr1 and cxcr2 is robust within the developing gut. This work provides a model for the study of Cxcl8 signalling during gut inflammation.