The dynamics of interleukin‐8 and its interaction with human CXC receptor I peptide

Interleukin‐8 (CXCL8, IL‐8) is a proinflammatory chemokine important for the regulation of inflammatory and immune responses via its interaction with G‐protein coupled receptors, including CXC receptor 1 (CXCR1). CXCL8 exists as both a monomer and as a dimer at physiological concentrations, yet the molecular basis of CXCL8 interaction with its receptor as well as the importance of CXCL8 dimer formation remain poorly characterized. Although several biological studies have indicated that both the CXCL8 monomer and dimer are active, biophysical studies have reported conflicting results regarding the binding of CXCL8 to CXCR1. To clarify this problem, we expressed and purified a peptide (hCXCR1pep) corresponding to the N‐terminal region of human CXCR1 (hCXCR1) and utilized nuclear magnetic resonance (NMR) spectroscopy to interrogate the binding of wild‐type CXCL8 and a previously reported mutant (CXCL8M) that stabilizes the monomeric form. Our data reveal that the CXCL8 monomer engages hCXCR1pep with a slightly higher affinity than the CXCL8 dimer, but that the CXCL8 dimer does not dissociate upon binding hCXCR1pep. These investigations also showed that CXCL8 is dynamic on multiple timescales, which may help explain the versatility in this interleukin for engaging its target receptors.     

The expression and role of CXC chemokines in colorectal cancer

Cancer is a life-threatening disease world-wide and colorectal cancer is the second common cause of cancer mortality. The interaction between tumor cells and stromal cells plays a crucial role in tumor initiation and progression and is partially mediated by chemokines. Chemokines predominantly participate in the chemoattraction of leukocytes to inflammatory sites. Nowadays, it is clear that CXC chemokines and their receptors (CXCR) may also modulate tumor behavior by several important mechanisms: regulation of angiogenesis, activation of a tumor-specific immune response by attracting leukocytes, stimulation of tumor cell proliferation and metastasis. Here, we review the expression and complex roles of CXC chemokines (CXCL1 to CXCL16) and their receptors (CXCR1 to CXCR6) in colorectal cancer. Overall, increased expression levels of CXC chemokines correlate with poor prognosis.     

Re-evaluation of chicken CXCR1 determines the true gene structure: CXCLi1 (K60) and CXCLi2 (CAF/interleukin-8) are ligands for this receptor

The original report of chicken CXCR1 (Li, Q. J., Lu, S., Ye, R. D., and Martins-Green, M. (2000) Gene (Amst.) 257, 307-317) described it as a single exon gene, with two isoforms (differing in their start codon). In comparison with mammalian CXCR1, the reported chicken CXCR1 was longer at both the NH(2) and COOH termini, and it lacked the conserved (C/S)CXNP motif present in the last transmembrane region of all known chemokine receptors. A re-evaluation of chicken CXCR1, comparing known expressed sequence tags with the chicken genome sequence, suggested that the gene contains two exons. We isolated a cDNA corresponding to our prediction, which was significantly different in sequence to the reported CXCR1. In particular, there were three frameshifts in our sequence, compared with the reported sequence, that restored higher identity in the COOH-terminal half of the protein to mammalian CXCR1 (61% total amino acid identity compared with 52% for the reported CXCR1), restored the (C/S)CXNP motif, and gave a predicted protein of the same length as mammalian CXCR1. In human, CXCR1 is the receptor for CXCL8. In the chicken, there are two syntenic genes, CXCLi1 and CXCLi2, which look equally like orthologues of human CXCL8. We demonstrate that both of these chemokines are ligands for chicken CXCR1. We also demonstrate that heterophils express chicken CXCR1 and that the receptor is Galpha(i) protein-linked.     

Regulation of CXC chemokine receptor 4-mediated migration by the Tec family tyrosine kinase ITK

Chemokines are critical in controlling lymphocyte traffic and migration. The CXC chemokine CXCL12/SDF-1alpha interacts with its receptor CXCR4 to induce the migration of a number of different cell types. Although an understanding of the physiological functions of this chemokine is emerging, the mechanism by which it regulates T cell migration is still unclear. We show here that the Tec family kinase ITK is activated rapidly following CXCL12/SDF-1alpha stimulation, and this requires Src and phosphatidylinositol 3-kinase activities. ITK regulates the ability of CXCL12/SDF-1alpha to induce T cell migration as overexpression of wild-type ITK-enhanced migration, and T cells lacking ITK exhibit reduced migration as well as adhesion in response to CXCL12/SDF-1alpha. Further analysis suggests that ITK may regulate CXCR4-mediated migration and adhesion by altering the actin cytoskeleton, as ITK null T cells were significantly defective in CXCL12/SDF-1a-mediated actin polymerization. Our data suggest that ITK may regulate the ability of CXCR4 to induce T cell migration.     

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K_i > 1 μm). Further, CC-CXCL8 failed to mobilize Ca²⁺ in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca²⁺ in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.     

The role of CXC chemokine ligand 4/CXC chemokine receptor 3-B in breast cancer progression

Chemokines and their receptors participate in the development of cancers by enhancing tumor cell proliferation, angiogenesis, invasion, metastasis and penetration of tumor immune cells. It remains unclear whether CXC chemokine ligand 4 (CXCL4)/CXC chemokine receptor 3-B (CXCR3-B) can be used as an independent molecular marker for establishing prognosis for breast cancer patients. We evaluated CXCL4 and CXCR3-B expression in 114 breast cancer tissues and 30 matched noncancerous tissues using immunohistochemistry and western blot, and determined the correlation between their expression and clinicopathologic findings. We observed that breast cancer tissues express CXCL4 strongly and CXCR3-B weakly compared to noncancerous tissues. Strong CXCL4 expression was detected in 94.7% and weak CXCR3-B expression was detected in 78.9% of the tissues. Therefore, CXCL4/CXCR3-B might play a crucial role in breast cancer progression. We found no significant correlation between CXCL4 and age, tumor stage, tumor grade or TNM stage. CXCR3-B was associated significantly with tumor grade. Moreover, the Chi-square test of association showed that the expression of CXCL4/CXCR3-B might be an independent prognostic marker for breast cancer. Therefore, we suggest that CXCR3-B is an indicator of poor prognosis and may also be a chemotherapeutic target.     

CXCL12 induces tyrosine phosphorylation of cortactin, which plays a role in CXC chemokine receptor 4-mediated extracellular signal-regulated kinase activation and chemotaxis

CXC chemokine receptor 4 (CXCR4) plays a role in the development of immune and central nervous systems as well as in cancer growth and metastasis. CXCR4-initiated signaling cascades leading to cell proliferation and chemotaxis are critical for these functions. The present study demonstrated that stimulation of CXCR4 by its ligand, CXCL12, induced transient translocation of cortactin from endosomal compartments to the cell periphery where it colocalized with CXCR4 followed by internalization of CXCR4 together with cortactin into endosomes. Cortactin was co-immunoprecipitated with CXCR4 in response to CXCL12 treatment in a time-dependent manner. Ligand stimulation induced phosphorylation of cortactin at tyrosine 421, and the phosphorylation was both c-Src- and dynamin-dependent. Cortactin overexpression promoted CXCR4 internalization and recycling. However, overexpression of a cortactin mutant in which tyrosine 421 was replaced with alanine (cortactin-Y421A) or knockdown of cortactin with RNA interference (RNAi) reduced CXCR4 internalization in response to CXCL12. CXCR4-mediated activation of extracellular signal-regulated kinases 1 and 2 was significantly prolonged by overexpression of wild-type cortactin but not by the cortactin-Y421A mutant and was inhibited by cortactin knockdown with RNAi. Moreover, CXCL12-induced chemotaxis was enhanced by cortactin overexpression, reduced by overexpression of the cortactin-Y421A mutant, and blocked by cortactin knockdown with RNAi. These data provide strong evidence for an important role of cortactin in CXCR4 signaling and trafficking as well in the receptor-mediated cell migration.     

Human osteoclasts express different CXC chemokines depending on cell culture substrate: molecular and immunocytochemical evidence of high levels of CXCL10 and CXCL12

Chemokines are important mediators of chemotaxis, cell adherence, and proliferation and exert specific functions in bone remodeling. Despite the potential intriguing role of chemokines in the regulation of osteoclast (OC) functions, little is known about the expression of chemokines and their receptors in human OCs at different stages of differentiation. Therefore, we analyzed the expression of CXC chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5) and ligands (CXCL8, CXCL10, CXCL12 and CXCL13) both at molecular and protein levels, in human OCs grown on plastic or calcium phosphate-coated slides at different stages of differentiation. Real-time PCR showed that CXCR1, CXCR2, CXCR3, CXCR4, CXCR5 and CXCL8 were expressed in undifferentiated cells and significantly decreased during OC differentiation. By contrast, CXCL10 and CXCL12 were strongly upregulated from day 0 to day 8 in cells grown on calcium phosphate-coated slides. Immunocytochemistry showed that OCs grown on plastic expressed CXCR3, CXCR4, CXCR5, CXCL8 and CXCL12, while they were negative for CXCR1, CXCR2 and CXCL10. Interestingly, both at molecular and protein levels CXCL10 and CXCL12 significantly increased only when cells were differentiated on calcium phosphate-coated slides. These data suggest that the selection of a substrate that better mimics the tridimensional structure of bone tissue, thus favoring OC maturation and differentiation, may be necessary when studying osteoclastogenesis in vitro.     

The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer

Chronic inflammation may increase the risk to develop cancer, for instance esophagitis or gastritis may lead to development of esophageal or gastric cancer, respectively. The key molecules attracting leukocytes to local inflammatory sites are chemokines. We here provide a systematic review on the impact of CXC chemokines (binding the receptors CXCR1, CXCR2, CXCR3 and CXCR4) on the transition of chronic inflammation in the upper gastrointestinal tract to neoplasia. CXCR2 ligands, including GRO-α,β,γ/CXCL1,2,3, ENA-78/CXCL5 and IL-8/CXCL8 chemoattract pro-tumoral neutrophils. In addition, angiogenic CXCR2 ligands stimulate the formation of new blood vessels, facilitating tumor progression. The CXCR4 ligand SDF-1/CXCL12 also promotes tumor development by stimulating angiogenesis and by favoring metastasis of CXCR4-positive tumor cells to distant organs producing SDF-1/CXCL12. Furthermore, these angiogenic chemokines also directly enhance tumor cell survival and proliferation. In contrast, the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 are angiostatic and attract anti-tumoral T lymphocytes and may therefore mediate tumor growth retardation and regression. Thus, chemokines exert diverging, sometimes dual roles in tumor biology as described for esophageal and gastric cancer. Therefore extensive research is needed to completely unravel the complex chemokine code in specific cancers. Possibly, chemokine-targeted cancer therapy will have to be adapted to the individual's chemokine profile.     

The role of CXC chemokines in the transition of chronic inflammation to esophageal and gastric cancer

Chronic inflammation may increase the risk to develop cancer, for instance esophagitis or gastritis may lead to development of esophageal or gastric cancer, respectively. The key molecules attracting leukocytes to local inflammatory sites are chemokines. We here provide a systematic review on the impact of CXC chemokines (binding the receptors CXCR1, CXCR2, CXCR3 and CXCR4) on the transition of chronic inflammation in the upper gastrointestinal tract to neoplasia. CXCR2 ligands, including GRO-α,β,γ/CXCL1,2,3, ENA-78/CXCL5 and IL-8/CXCL8 chemoattract pro-tumoral neutrophils. In addition, angiogenic CXCR2 ligands stimulate the formation of new blood vessels, facilitating tumor progression. The CXCR4 ligand SDF-1/CXCL12 also promotes tumor development by stimulating angiogenesis and by favoring metastasis of CXCR4-positive tumor cells to distant organs producing SDF-1/CXCL12. Furthermore, these angiogenic chemokines also directly enhance tumor cell survival and proliferation. In contrast, the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 are angiostatic and attract anti-tumoral T lymphocytes and may therefore mediate tumor growth retardation and regression. Thus, chemokines exert diverging, sometimes dual roles in tumor biology as described for esophageal and gastric cancer. Therefore extensive research is needed to completely unravel the complex chemokine code in specific cancers. Possibly, chemokine-targeted cancer therapy will have to be adapted to the individual's chemokine profile.     

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.     

Diverging mechanisms of activation of chemokine receptors revealed by novel chemokine agonists

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions.     

Molecular cloning, characterization and expression analysis of a miiuy croaker (Miichthys miiuy) CXC chemokine gene resembling the CXCL9/CXCL10/CXCL11

Chemokines are a large family of chemotactic cytokines playing crucial roles in the innate immune response. In the present study, we report the cloning of a CXC chemokine gene resembling the closely related CXCL9/CXCL10/CXCL11 from the miiuy croaker Miichthys miiuy (MimiCXC). Both 5'-RACE and 3'-RACE were carried out in order to obtain the complete cDNA, which consists of a 73 bp 5'-UTR, a 369 bp open reading frame encoding 122 amino acids and a 715 bp 3'-UTR. The deduced MimiCXC contains a 19-aa signal peptide and a 103-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 4.8%-65.6% sequence identities to mammalian CXC chemokines and the highest sequence identity of 65.6% is between MimiCXC and CXCL10 chemokine. Three exons and two introns were identified in MimiCXC gene. The MimiCXC gene was constitutively expressed in all tissues tested, although at different levels. Upon induction with Vibrio anguillarum, MimiCXC gene expression was up-regulated in kidney and spleen, however, down-regulated in liver. These results indicate that MimiCXC may be involved in immune responses as well as homeostatic processes in miiuy croaker.     

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay.     

The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR+ CXC chemokine-induced angiogenic activity

We have previously shown that members of the ELR(+) CXC chemokine family, including IL-8; growth-related oncogenes alpha, beta, and gamma; granulocyte chemotactic protein 2; and epithelial neutrophil-activating protein-78, can mediate angiogenesis in the absence of preceding inflammation. To date, the receptor on endothelial cells responsible for chemotaxis and neovascularization mediated by these ELR(+) CXC chemokines has not been determined. Because all ELR(+) CXC chemokines bind to CXC chemokine receptor 2 (CXCR2), we hypothesized that CXCR2 is the putative receptor for ELR(+) CXC chemokine-mediated angiogenesis. To test this postulate, we first determined whether cultured human microvascular endothelial cells expressed CXCR2. CXCR2 was detected in human microvascular endothelial cells at the protein level by both Western blot analysis and immunohistochemistry using polyclonal Abs specific for human CXCR2. To determine whether CXCR2 played a functional role in angiogenesis, we determined whether this receptor was involved in endothelial cell chemotaxis. We found that microvascular endothelial cell chemotaxis in response to ELR(+) CXC chemokines was inhibited by anti-CXCR2 Abs. In addition, endothelial cell chemotaxis in response to ELR(+) CXC chemokines was sensitive to pertussis toxin, suggesting a role for G protein-linked receptor mechanisms in this biological response. The importance of CXCR2 in mediating ELR(+) CXC chemokine-induced angiogenesis in vivo was also demonstrated by the lack of angiogenic activity induced by ELR(+) CXC chemokines in the presence of neutralizing Abs to CXCR2 in the rat corneal micropocket assay, or in the corneas of CXCR2(-/-) mice. We thus conclude that CXCR2 is the receptor responsible for ELR(+) CXC chemokine-mediated angiogenesis.     

Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2

The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.     

Epigenetics underpinning the regulation of the CXC (ELR+) chemokines in non-small cell lung cancer

Background

Angiogenesis may play a role in the pathogenesis of Non-Small Cell Lung cancer (NSCLC). The CXC (ELR⁺) chemokine family are powerful promoters of the angiogenic response.

Methods

The expression of the CXC (ELR⁺) family members (CXCL1-3/GROα-γ, CXCL8/IL-8, CXCR1/2) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of these chemokines was examined in normal bronchial epithelial and NSCLC cell lines.

Results

Overall, expression of the chemokine ligands (CXCL1, 2, 8) and their receptors (CXCR1/2) were down regulated in tumour samples compared with normal, with the exception of CXCL3. CXCL8 and CXCR1/2 were found to be epigenetically regulated by histone post-translational modifications. Recombinant CXCL8 did not stimulate cell growth in either a normal bronchial epithelial or a squamous carcinoma cell line (SKMES-1). However, an increase was observed at 72 hours post treatment in an adenocarcinoma cell line.

Conclusions

CXC (ELR⁺) chemokines are dysregulated in NSCLC. The balance of these chemokines may be critical in the tumour microenvironment and requires further elucidation. It remains to be seen if epigenetic targeting of these pathways is a viable therapeutic option in lung cancer treatment.