N,N'-Diarylcyanoguanidines as antagonists of the CXCR2 and CXCR1 chemokine receptors

A series of N-(2-hydroxy-3-sulfonamidobenzene)-N'-arylcyanoguanidines was prepared. In general, these compounds proved to be potent antagonists of CXCR2 while the selectivity versus CXCR1 ranged from non-selective to >200-fold.     

Regulation of the human chemokine receptor CCR1. Cross-regulation by CXCR1 and CXCR2

To investigate the regulation of the CCR1 chemokine receptor, a rat basophilic leukemia (RBL-2H3) cell line was modified to stably express epitope-tagged receptor. These cells responded to RANTES (regulated upon activation normal T expressed and secreted), macrophage inflammatory protein-1alpha, and monocyte chemotactic protein-2 to mediate phospholipase C activation, intracellular Ca(2+) mobilization and exocytosis. Upon activation, CCR1 underwent phosphorylation and desensitization as measured by diminished GTPase stimulation and Ca(2+) mobilization. Alanine substitution of specific serine and threonine residues (S2 and S3) or truncation of the cytoplasmic tail (DeltaCCR1) of CCR1 abolished receptor phosphorylation and desensitization of G protein activation but did not abolish desensitization of Ca(2+) mobilization. S2, S3, and DeltaCCR1 were also resistant to internalization, mediated greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization, and were only partially desensitized by RANTES, relative to S1 and CCR1. To study CCR1 cross-regulation, RBL cells co-expressing CCR1 and receptors for interleukin-8 (CXCR1, CXCR2, or a phosphorylation-deficient mutant of CXCR2, 331T) were produced. Interleukin-8 stimulation of CXCR1 or CXCR2 cross-phosphorylated CCR1 and cross-desensitized its ability to stimulate GTPase activity and Ca(2+) mobilization. Interestingly, CCR1 cross-phosphorylated and cross-desensitized CXCR2, but not CXCR1. Ca(2+) mobilization by S3 and DeltaCCR1 were also cross-desensitized by CXCR1 and CXCR2 despite lack of receptor phosphorylation. In contrast to wild type CCR1, S3 and DeltaCCR1, which produced sustained signals, cross-phosphorylated and cross-desensitized responses to CXCR1 as well as CXCR2. Taken together, these results indicate that CCR1-mediated responses are regulated at several steps in the signaling pathway, by receptor phosphorylation at the level of receptor/G protein coupling and by an unknown mechanism at the level of phospholipase C activation. Moreover selective cross-regulation among chemokine receptors is, in part, a consequence of the strength of signaling (i.e. greater phosphatidylinositol hydrolysis and sustained Ca(2+) mobilization) which is inversely correlated with the receptor's susceptibility to phosphorylation. Since many chemokines activate multiple chemokine receptors, selective cross-regulation among such receptors may play a role in their immunomodulation.     

Loss of chemokine SDF-1alpha-mediated CXCR4 signalling and receptor internalization in human hepatoma cell line HepG2

Expression of the chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is absent from many carcinomas, including hepatomas. We note an early signalling defect in the hepatocellular carcinoma (HCC) cell line HepG2 that expresses the CXCR4 receptor and binds biotin-labelled SDF, but fails to stimulate downstream signalling events after engagement with SDF. In HepG2, the SDF/CXCR4 interaction did not result in calcium influx, phosphorylation and internalization of CXCR4, nor in a rapid phosphorylation of p44/42 MAP kinase. There were no CXCR4 mutations in the second chemokine binding loop or C terminal phosphorylation and internalization domains. The downstream signalling machinery in HepG2 appears to be intact since transfection of wild-type CXCR4 restored functional responsiveness. We conclude that HepG2 is unresponsive to SDF stimulation because of a defect located after receptor binding but before the activation of the signalling cascade. A hypothetical blocking molecule could hinder receptor internalization or CXCR4 signalling.     

Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells

Chemokines are secreted into the tumor microenvironment by tumor-infiltrating inflammatory cells as well as by tumor cells. Chemokine receptors mediate agonist-dependent cell responses, including migration and activation of several signaling pathways. In the present study we show that several human melanoma cell lines and melanoma cells on macroscopically infiltrated lymph nodes express the chemokine receptors CXCR3 and CXCR4. Using the highly invasive melanoma cell line BLM, we demonstrate that the chemokine Mig, a ligand for CXCR3, activates the small GTPases RhoA and Rac1, induces a reorganization of the actin cytoskeleton, and triggers cell chemotaxis and modulation of integrin VLA-5- and VLA-4-dependent cell adhesion to fibronectin. Furthermore, the chemokine SDF-1alpha, the ligand of CXCR4, triggered modulation of beta(1) integrin-dependent melanoma cell adhesion to fibronectin. Additionally, Mig and SDF-1alpha activated MAPKs p44/42 and p38 on melanoma cells. Expression of functional CXCR3 and CXCR4 receptors on melanoma cells indicates that they might contribute to cell motility during invasion as well as to regulation of cell proliferation and survival.     

Differential modes of regulation of cxc chemokine-induced internalization and recycling of human CXCR1 and CXCR2

Studies of human neutrophil IL-8 receptors, CXCR1 and CXCR2, have shown that the two receptors are differentially regulated by ELR⁺-CXC chemokines, that they differ functionally and may have diverse roles in mediating the inflammatory process. To elucidate the role of CXCR1 and CXCR2 in inflammation and to delineate the basis for the divergent regulation of these receptors by IL-8 and NAP-2, we characterized the IL-8- and NAP-2-induced mechanisms regulating the expression of each receptor, focusing on receptor internalization and recycling. Using HEK 293 cell transfectants, IL-8 was shown to induce significantly higher levels of CXCR2 internalization than NAP-2. Moreover, although CXCR2 bound IL-8 and NAP-2 with similarly high affinity, IL-8 functionally competed with and displaced NAP-2, and prompted high levels of internalization, similar to those induced by IL-8 alone. In a system providing an identical cellular milieu for reliable comparisons between CXCR1 and CXCR2, we have shown that the mechanisms controlling the internalization of CXCR1 diverge from those regulating CXCR2 internalization. Whereas IL-8-induced internalization of CXCR1 was profoundly dependent on a region of the carboxyl terminus expressing six phosphorylation sites, internalization of CXCR2 was primarily regulated by a membrane proximal domain of the carboxyl terminus that does not express phosphorylation sites. Analysis of receptor re-expression on the plasma membrane indicated that at early time points following removal of free ligand and incubation of the cells at 37°C, receptor recycling accounted for recovery of CXCR1 and CXCR2 expression, whereas at later time points other processes may be involved in receptor re-expression. Phosphorylation-independent mechanisms were shown to direct both receptors to the recycling pathway. The differential control of CXCR1 vs CXCR2 internalization by IL-8 and NAP-2, as well as by phosphorylation-mediated mechanisms, suggests that a chemokine- and receptor-specific mode of regulation of internalization may contribute to the divergent activities of these receptors.     

Actin filaments are involved in the regulation of trafficking of two closely related chemokine receptors, CXCR1 and CXCR2

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37 degrees C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.     

Ligand-induced partitioning of human CXCR1 chemokine receptors with lipid raft microenvironments facilitates G-protein-dependent signaling

Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live-cell fluorescence resonance energy transfer imaging to detect the proximity between CXCR1-cyan fluorescent protein (CFP) and fluorescence probes that label lipid raft or non-lipid raft microdomains and using fluorescence recovery after photobleaching analysis to measure the lateral diffusion of CXCR1-CFP, we found that interleukin-8 induces association between the receptors and lipid raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling, such as Ca2+ responses and phosphatidylinositol 3-kinase activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins.     

IL-8 single-chain homodimers and heterodimers: interactions with chemokine receptors CXCR1, CXCR2, and DARC.

Covalent single-chain dimers of the chemokine interleukin-8 (IL-8) have been designed to mimic the dimeric form of IL-8 in solution and facilitate the production of heterodimer variants of IL-8. Physical studies indicated that use of a simple peptide linker to join two subunits, while allowing receptor binding and activation, led to self-association of the tethered dimers. However, addition of a single disulfide crosslink between the tethered subunits prevented this multimer from forming, yielding a species of dimer molecular weight. Crosslinked single-chain dimers bind to both IL-8 neutrophil receptors CXCR1 and CXCR2 as well as to DARC, as does a double disulfide-linked dimer with no peptide linker. In addition, neutrophil response to these dimers as measured by chemotaxis or beta-glucuronidase release is similar to that elicited by wild-type IL-8, providing evidence that the dissociation of the dimeric species is not required for these biologically relevant activities. Finally, through construction of single-chain heterodimer mutants, we show that only the first subunit's ELR motif is the single-chain variants.     

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases

Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.     

The chemokine receptors CXCR1 and CXCR2 couple to distinct g protein-coupled receptor kinases to mediate and regulate leukocyte functions

The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized, and internalized. In this study, we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, short hairpin RNA was used to inhibit GRK2, 3, 5, and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6 increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization, and internalization, respectively, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect on CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6(-/-)) displayed an increase in CXCR2-mediated G protein activation but in vitro exhibited a decrease in chemotaxis, receptor desensitization, and internalization relative to wild-type (GRK6(+/+)) cells. In contrast, neutrophil recruitment in vivo in GRK6(-/-) mice was increased in response to delivery of CXCL1 through the air pouch model. In a wound-closure assay, GRK6(-/-) mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure compared with GRK6(+/+) animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis.     

Distinct mechanisms of agonist-induced endocytosis for human chemokine receptors CCR5 and CXCR4

Desensitization of the chemokine receptors, a large class of G protein-coupled receptors, is mediated in part by agonist-driven receptor endocytosis. However, the exact pathways have not been fully defined. Here we demonstrate that the rate of ligand-induced endocytosis of CCR5 in leukocytes and expression systems is significantly slower than that of CXCR4 and requires prolonged agonist treatment, suggesting that these two receptors use distinct mechanisms. We show that the C-terminal domain of CCR5 is the determinant of its slow endocytosis phenotype. When the C-tail of CXCR4 was exchanged for that of CCR5, the resulting CXCR4-CCR5 (X4-R5) chimera displayed a CCR5-like trafficking phenotype. We found that the palmitoylated cysteine residues in this domain anchor CCR5 to plasma membrane rafts. CXCR4 and a C-terminally truncated CCR5 mutant (CCR5-KRFX) lacking these cysteines are not raft associated and are endocytosed by a clathrin-dependent pathway. Genetic inhibition of clathrin-mediated endocytosis demonstrated that a significant fraction of ligand-occupied CCR5 trafficked by clathrin-independent routes into caveolin-containing vesicular structures. Thus, the palmitoylated C-tail of CCR5 is the major determinant of its raft association and endocytic itineraries, differentiating it from CXCR4 and other chemokine receptors. This novel feature of CCR5 may modulate its signaling potential and could explain its preferential use by HIV for person-to-person transmission of disease.     

Expression and function of the SDF-1 chemokine receptors CXCR4 and CXCR7 during mouse limb muscle development and regeneration

The chemokine, SDF-1/CXCL12, and its receptor, CXCR4, have been implied to play major roles during limb myogenesis. This concept was recently challenged by the identification of CXCR7 as an alternative SDF-1 receptor, which can either act as a scavenger receptor, a modulator of CXCR4, or an active chemokine receptor. We have now re-examined this issue by determining whether SDF-1 would signal to C2C12 myoblasts and subsequently influence their differentiation via CXCR4 and/or CXCR7. In addition, we have analyzed CXCR7, CXCR4, and SDF-1 expression in developing and injured mouse limb muscles. We demonstrate that in undifferentiated C2C12 cells, SDF-1-dependent cell signaling and resulting inhibitory effects on myogenic differentiation are entirely mediated by CXCR4. We further demonstrate that CXCR7 expression increases in differentiating C2C12 cells, which in turn abrogates CXCR4 signaling. Moreover, consistent with the view that CXCR4 and CXCR7 control limb myogenesis in vivo by similar mechanisms, we found that CXCR4 expression is the highest in late embryonic hindlimb muscles and drops shortly after birth when secondary muscle growth terminates. Vice versa, CXCR7 expression increased perinatally and persisted into adult life. Finally, underscoring the role of the SDF-1 system in muscle regeneration, we observed that SDF-1 is continuously expressed by endomysial cells of postnatal and adult muscle fibers. Analysis of dystrophin-deficient mdx mice additionally revealed that muscle regeneration is associated with muscular re-expression of CXCR4. The apparent tight control of limb muscle development and regeneration by CXCR4 and CXCR7 points to these chemokine receptors as promising therapeutic targets for certain muscle disorders.     

Expression of functional CCR and CXCR chemokine receptors in podocytes

Chemokines and their receptors play an important role in the pathogenesis of acute and chronic glomerular inflammation. However, their expression pattern and function in glomerular podocytes, the primary target cells in a variety of glomerulopathies, have not been investigated as of yet. Using RT-PCR, we now demonstrate the expression of CCR4, CCR8, CCR9, CCR10, CXCR1, CXCR3, CXCR4, and CXCR5 in cultured human podocytes. Stimulation of these receptors induced a concentration-dependent biphasic increase of the free cytosolic calcium concentration in podocytes in culture. In addition, we demonstrate that podocytes release IL-8 in the presence of FCS and that IL-8 down-regulates cell surface CXCR1. Chemokine stimulation of the detected CCRs and CXCRs increased activity of NADPH-oxidase, the primary source of superoxide anions in podocytes. Immunohistochemistry studies revealed only diffuse and weak CXCR expression in healthy human glomerula. In contrast, in membranous nephropathy, a characteristic podocyte disorder, the expression of CXCR1, CXCR3, and CXCR5 is up-regulated in podocytes. In conclusion, podocytes in culture and podocytes in human kidney sections express a set of chemokine receptors. The release of oxygen radicals that accompanies the activation of CCRs and CXCRs may contribute to podocyte injury and the development of proteinuria during membranous nephropathy.     

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.     

趋化因子SDF-1及受体CXCR4研究进展

趋化因子(chemokine)是一类一级结构相似,以对白细胞等多种细胞具有趋化定向运动作用为特征的小分子蛋白。功能研究表明,趋化因子在胚胎发育、血管生成、炎症、肿瘤、艾滋病等机体多种生理和病理过程中发挥重要作用,部分趋化因子的衍生物或抑制物具有潜在的临床应用前景。不久的将来,趋化因子及其受体可能成为疾病治疗的分子靶点。     

G protein-coupled receptor kinase 5 phosphorylation of hip regulates internalization of the chemokine receptor CXCR4

Regulation of the magnitude, duration, and localization of G protein-coupled receptor (GPCR) signaling responses is controlled by desensitization, internalization, and downregulation of the activated receptor. Desensitization is initiated by the phosphorylation of the activated receptor by GPCR kinases (GRKs) and the binding of the adaptor protein arrestin. In addition to phosphorylating activated GPCRs, GRKs have been shown to phosphorylate a variety of additional substrates. An in vitro screen for novel GRK substrates revealed Hsp70 interacting protein (Hip) as a substrate. GRK5, but not GRK2, bound to and stoichiometrically phosphorylated Hip in vitro. The primary binding domain of GRK5 was mapped to residues 303-319 on Hip, while the major site of phosphorylation was identified to be Ser-346. GRK5 also bound to and phosphorylated Hip on Ser-346 in cells. While Hip was previously implicated in chemokine receptor trafficking, we found that the phosphorylation of Ser-346 was required for proper agonist-induced internalization of the chemokine receptor CXCR4. Taken together, Hip has been identified as a novel substrate of GRK5 in vitro and in cells, and phosphorylation of Hip by GRK5 plays a role in modulating CXCR4 internalization.     

Computer‐aided assessment of the chemokine receptors CXCR3, CXCR4 and CXCR7 expression in gallbladder carcinoma

Gallbladder carcinoma (GBC) is a vicious and invasive disease. The major challenge in the clinical treatment of GBC is the lack of a suitable prognosis method. Chemokine receptors such as CXCR3, CXCR4 and CXCR7 play vital roles in the process of tumour progression and metastasis. Their expression levels and distribution are proven to be indicative of the progression of GBC, but are hard to be decoded by conventional pathological methods, and therefore, not commonly used in the prognosis of GBC. In this study, we developed a computer‐aided image analysis method, which we used to quantitatively measure the expression levels of CXCR3, CXCR4 and CXCR7 in the nuclei and cytoplasm of glandular and interstitial cells from a cohort of 55 GBC patients. We found that CXCR3, CXCR4 and CXCR7 expressions are associated with the clinicopathological variables of GBC. Cytoplasmic CXCR3, nuclear CXCR7 and cytoplasmic CXCR7 were significant predictive factors of histology invasion, whereas cytoplasmic CXCR4 and nuclear CXCR4 were significantly correlated with T and N stage and were associated with the overall survival and disease‐free survival. These results suggest that the quantification and localisation of CXCR3, CXCR4 and CXCR7 expressions in different cell types should be considered using computer‐aided assessment to improve the accuracy of prognosis in GBC.     

Blocking HIV-1 infection via CCR5 and CXCR4 receptors by acting in trans on the CCR2 chemokine receptor

The identification of chemokine receptors as HIV-1 coreceptors has focused research on developing strategies to prevent HIV-1 infection. We generated CCR2-01, a CCR2 receptor-specific monoclonal antibody that neither competes with the chemokine CCL2 for binding nor triggers signaling, but nonetheless blocks replication of monotropic (R5) and T-tropic (X4) HIV-1 strains. This effect is explained by the ability of CCR2-01 to induce oligomerization of CCR2 with the CCR5 or CXCR4 viral coreceptors. HIV-1 infection through CCR5 and CXCR4 receptors can thus be prevented in the absence of steric hindrance or receptor downregulation by acting in trans on a receptor that is rarely used by the virus to infect cells.