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S.e. Escher - One of the best experts on this subject based on the ideXlab platform.
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CCR1- and CCR5-mediated inactivation of leukocytes by a nonglycosaminoglycan (non-GAG)-binding variant of n-Nonanoyl-CCL14 (NNY-CCL14)
Journal of leukocyte biology, 2010Co-Authors: Shipra Gupta, S.e. Escher, Aleksandra Heitland, Sebastian Rieder, Wolf-georg Forssmann, Sandra Schulz-maronde, Rudolf Richter, Matthias Mack, Johanna Manns, Jörn ElsnerAbstract:Intervention on chemokine receptors to prevent directional leukocyte migration is a potential therapeutic strategy. NNY-CCL14 is a CD26-resistant lead molecule, which exerts its effects on multiple chemokine receptors (CCR1, CCR2, CCR3, and CCR5). The inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation have been assigned to its interaction with CCR1 and CCR5. In this study, a non-GAG-binding variant of NNY-CCL14 was generated by mutating basic amino acids within the identified GAG-binding 49BBXB52 motif. This CD26-resistant, non-GAG binding variant, NNY-CCL14(G,A), does not promote CCR1-dependent cell arrest on modeled endothelium. Its biological activity tested on human and murine chemokine receptors revealed distinguishing properties to NNY-CCL14. As suggested by EC50 values for intracellular calcium mobilization, NNY-CCL14(G,A) demonstrated a reduced ability to activate hCCR1, but internalization and desensitization of hCCR1 were unperturbed. Surprisingly, its activity on hCCR3 was strongly reduced, and it did not internalize mCCR3. A significantly reduced chemotactic activity of eosinophils and monocytes was observed. All biological effects mediated by NNY-CCL14(G,A) via hCCR5 and mCCR5 showed no difference to NNY-CCL14. In mice treated i.v. with NNY-CCL14(G,A), a sustained in vivo down-modulation of CCR5 was achieved over 3 h. Therefore, NNY-CCL14(G,A) inactivates leukocytes by desensitizing and internalizing multiple chemokine receptors, thus rendering them unresponsive to further stimulation by natural ligands. When administered systemically, NNY-CCL14(G,A) may modulate leukocyte functions prior to their interaction with other endothelium-bound chemokines expressed under pathophysiological conditions, such as allergic inflammation.
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n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.
Allergy, 2008Co-Authors: Shipra Gupta, S.e. Escher, Wolf-georg Forssmann, Jörn Elsner, Sandra Schulz-maronde, Rudolf Richter, G. Erdmann, Ulf ForssmannAbstract:Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.
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Intravascular inactivation of CCR5 by n-Nonanoyl-CC chemokine ligand 14 and inhibition of allergic airway inflammation.
Journal of leukocyte biology, 2007Co-Authors: Shipra Gupta, Barbara Fuchs, S.e. Escher, Aleksandra Heitland, Armin Braun, Wolf-georg Forssmann, Sandra Schulz-maronde, Matthias Mack, Hanns-christian Tillmann, Jörn ElsnerAbstract:Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1 + and CCR5 + cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.
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The allergy-associated chemokine receptors CCR3 and CCR5 can be inactivated by the modified chemokine NNY-CCL11.
Allergy, 2007Co-Authors: J. Manns, S.e. Escher, S. Rieder, B. Eilers, Wolf-georg Forssmann, Jörn Elsner, Ulf ForssmannAbstract:Background: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. Methods: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. Results: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. Conclusions: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases.
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Inhibition Of Airway Inflammation And HIV Entry By NNY-CCL14 Treatment: Role Of CCR1 And CCR5
Journal of Allergy and Clinical Immunology, 2007Co-Authors: Shipra Gupta, Barbara Fuchs, S.e. Escher, Aleksandra Heitland, Sandra Schulz-maronde, Jan Münch, Frank Kirchhoff, Matthias Mack, Hanns-christian Tillmann, Armin BraunAbstract:RATIONALE: Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive therapeutic strategy. METHODS: Efficacy and potency of NNY-CCL14, its ability to desensitize and internalize CCR1 and CCR5 on B300.19+-cell lines, human and murine leukocytes was studied by calcium release assay and flow cytometry. Airway hyper-responsiveness was measured by head-out plethysmography in NNY-CCL14-treated ovalbumin (OVA)-challenged Balb/c mice. Bronchoalveolar lavage analysis was performed on cytospins. RESULTS: NNY-CCL14 is a potent agonist on B300.19-hCCR1+ (EC50=16.4 nM), B300.19-hCCR5+ (EC50=1.7 nM) and PBMCs (EC50=3.5 nM), also activating murine CCR1 and CCR5. NNY-CCL14 completely desensitizes CCR1- and CCR5-mediated calcium release to further stimulation and effectively internalizes these receptors from cellular surface. NNY-CCL14 also potently inhibits (IC50 = 10.1 nM) CCR5 mediated HIV-1 infection. NNY-CCL14 treatment of OVA-sensitized BALB/c mice reduced influx of eosinophils (p = 0.021) and lymphocytes (p < 0.001) to bronchoalveolar lavage in association with improved airway hyperresponsiveness (p = 0.016). Above all, systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes. The rapid down-modulation is followed by a gradual CCR5 re-expression over 3 hours. Although, NNY-CCL14 internalizes CCR5 on murine lymphocytes, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. CONCLUSIONS: NNY-CCL14 is an agonist of CCR1 and CCR5 with inhibiting capacity. The inhibitory effects of NNY-CCL14 in the murine model of allergic airway inflammation can be assigned to its interaction with CCR5 and probably CCR1, but independent of CCR3. In summary, NNY-CCL14 is an interesting tool to prevent allergic and infectious diseases. Funding: DF
Shipra Gupta - One of the best experts on this subject based on the ideXlab platform.
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CCR1- and CCR5-mediated inactivation of leukocytes by a nonglycosaminoglycan (non-GAG)-binding variant of n-Nonanoyl-CCL14 (NNY-CCL14)
Journal of leukocyte biology, 2010Co-Authors: Shipra Gupta, S.e. Escher, Aleksandra Heitland, Sebastian Rieder, Wolf-georg Forssmann, Sandra Schulz-maronde, Rudolf Richter, Matthias Mack, Johanna Manns, Jörn ElsnerAbstract:Intervention on chemokine receptors to prevent directional leukocyte migration is a potential therapeutic strategy. NNY-CCL14 is a CD26-resistant lead molecule, which exerts its effects on multiple chemokine receptors (CCR1, CCR2, CCR3, and CCR5). The inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation have been assigned to its interaction with CCR1 and CCR5. In this study, a non-GAG-binding variant of NNY-CCL14 was generated by mutating basic amino acids within the identified GAG-binding 49BBXB52 motif. This CD26-resistant, non-GAG binding variant, NNY-CCL14(G,A), does not promote CCR1-dependent cell arrest on modeled endothelium. Its biological activity tested on human and murine chemokine receptors revealed distinguishing properties to NNY-CCL14. As suggested by EC50 values for intracellular calcium mobilization, NNY-CCL14(G,A) demonstrated a reduced ability to activate hCCR1, but internalization and desensitization of hCCR1 were unperturbed. Surprisingly, its activity on hCCR3 was strongly reduced, and it did not internalize mCCR3. A significantly reduced chemotactic activity of eosinophils and monocytes was observed. All biological effects mediated by NNY-CCL14(G,A) via hCCR5 and mCCR5 showed no difference to NNY-CCL14. In mice treated i.v. with NNY-CCL14(G,A), a sustained in vivo down-modulation of CCR5 was achieved over 3 h. Therefore, NNY-CCL14(G,A) inactivates leukocytes by desensitizing and internalizing multiple chemokine receptors, thus rendering them unresponsive to further stimulation by natural ligands. When administered systemically, NNY-CCL14(G,A) may modulate leukocyte functions prior to their interaction with other endothelium-bound chemokines expressed under pathophysiological conditions, such as allergic inflammation.
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n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.
Allergy, 2008Co-Authors: Shipra Gupta, S.e. Escher, Wolf-georg Forssmann, Jörn Elsner, Sandra Schulz-maronde, Rudolf Richter, G. Erdmann, Ulf ForssmannAbstract:Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.
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Intravascular inactivation of CCR5 by n-Nonanoyl-CC chemokine ligand 14 and inhibition of allergic airway inflammation.
Journal of leukocyte biology, 2007Co-Authors: Shipra Gupta, Barbara Fuchs, S.e. Escher, Aleksandra Heitland, Armin Braun, Wolf-georg Forssmann, Sandra Schulz-maronde, Matthias Mack, Hanns-christian Tillmann, Jörn ElsnerAbstract:Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1 + and CCR5 + cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.
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Inhibition Of Airway Inflammation And HIV Entry By NNY-CCL14 Treatment: Role Of CCR1 And CCR5
Journal of Allergy and Clinical Immunology, 2007Co-Authors: Shipra Gupta, Barbara Fuchs, S.e. Escher, Aleksandra Heitland, Sandra Schulz-maronde, Jan Münch, Frank Kirchhoff, Matthias Mack, Hanns-christian Tillmann, Armin BraunAbstract:RATIONALE: Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive therapeutic strategy. METHODS: Efficacy and potency of NNY-CCL14, its ability to desensitize and internalize CCR1 and CCR5 on B300.19+-cell lines, human and murine leukocytes was studied by calcium release assay and flow cytometry. Airway hyper-responsiveness was measured by head-out plethysmography in NNY-CCL14-treated ovalbumin (OVA)-challenged Balb/c mice. Bronchoalveolar lavage analysis was performed on cytospins. RESULTS: NNY-CCL14 is a potent agonist on B300.19-hCCR1+ (EC50=16.4 nM), B300.19-hCCR5+ (EC50=1.7 nM) and PBMCs (EC50=3.5 nM), also activating murine CCR1 and CCR5. NNY-CCL14 completely desensitizes CCR1- and CCR5-mediated calcium release to further stimulation and effectively internalizes these receptors from cellular surface. NNY-CCL14 also potently inhibits (IC50 = 10.1 nM) CCR5 mediated HIV-1 infection. NNY-CCL14 treatment of OVA-sensitized BALB/c mice reduced influx of eosinophils (p = 0.021) and lymphocytes (p < 0.001) to bronchoalveolar lavage in association with improved airway hyperresponsiveness (p = 0.016). Above all, systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes. The rapid down-modulation is followed by a gradual CCR5 re-expression over 3 hours. Although, NNY-CCL14 internalizes CCR5 on murine lymphocytes, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. CONCLUSIONS: NNY-CCL14 is an agonist of CCR1 and CCR5 with inhibiting capacity. The inhibitory effects of NNY-CCL14 in the murine model of allergic airway inflammation can be assigned to its interaction with CCR5 and probably CCR1, but independent of CCR3. In summary, NNY-CCL14 is an interesting tool to prevent allergic and infectious diseases. Funding: DF
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Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1
Journal of leukocyte biology, 2005Co-Authors: Shipra Gupta, Ulf Forssmann, Wolf-georg Forssmann, Sandra Schulz-maronde, Christian Kutzleb, Rudolf Richter, Alexander Kapp, Jörn ElsnerAbstract:The CC chemokine receptor 1 (CCR1) has emerged as a relevant factor contributing to inflammatory diseases such as allergic asthma. Commonly used animal models of allergic airway inflammation, especially murine models, have certain limitations. The elaborate, nonhuman, primate models of asthma display the highest comparability with the situation in humans. These models play an important role in the understanding of the pathogenesis of asthma. To improve the understanding in cynomolgus monkey models, we identified and characterized CCR1 in this nonhuman primate. Initially, we cloned the cynomolgus monkey CCR1 (cCCR1) gene, and the sequence analysis revealed high homology at the nucleotide (92%) and amino acid (88.4%) levels with its human counterpart. Human embryonic kidney 293 cells were stably transfected with cCCR1 and used in functional assays. Among those CCR1 ligands tested, CCL14(9-74) was most potent in the induction of intracellular Ca2+ fluxes as observed for human CCR1 (hCCR1). Complete cross-desensitization could be achieved between CCL14(9-74) and CCL15. However, CCL3 could not fully abrogate the response to the potent ligand CCL14(9-74). Competition-binding studies with radiolabeled CCL3 concordantly showed that CCL14(9-74) has a higher affinity to cCCR1 than hCCL3. Moreover, differential tissue-specific expression of cCCR1 was investigated by real-time quantitative polymerase chain reaction, displaying the highest levels in spleen. This study adds basic information needed for the evaluation of the role of CCR1 in the pathophysiology of asthma using the highly relevant cynomolgus monkey model and in addition, aids in the preclinical evaluation of potential novel drugs targeting CCR1.
Lois A. Salamonsen - One of the best experts on this subject based on the ideXlab platform.
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CX3CL1 and CCL14 Regulate Extracellular Matrix and Adhesion Molecules in the Trophoblast: Potential Roles in Human Embryo Implantation
Biology of reproduction, 2008Co-Authors: Natalie J. Hannan, Lois A. SalamonsenAbstract:Embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. We have previously shown that the chemokines CX3CL1 and CCL14 are abundant in endometrial vasculature, epithelial, and decidual cells at this time, and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblasts. CX3CL1 and CCL14 promote trophoblast migration. We hypothesized that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion and ECM following chemokine stimulation were examined using pathway-specific oligo-arrays and quantitative real-time RT-PCR. More than 30 genes were affected by CX3CL1 treatment, and 15 genes were found to be regulated by CCL14 treatment. Real-time RT-PCR quantitation revealed significant changes in the mRNA transcripts of alpha-catenin (CTNNA1), extracellular matrix protein 1 (ECM1), osteopontin (SPP1), integrin alpha 6 (ITGA6), matrix metalloproteinase 12 (MMP12), and integrin beta 5 (ITGB5) following chemokine treatment. Several of these genes have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin alpha 6 and SPP1 protein in first-trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors, adhesion, and ECM at the maternal-fetal interface emphasizes an important role in the controlled directional migration of trophoblasts through the maternal decidua. For the first time, this study demonstrates the direct effects of CX3CL1 and CCL14 on trophoblast adhesion molecules and ECM, suggesting mechanisms by which trophoblast cells migrate during early pregnancy.
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The Chemokines, CX3CL1, CCL14, and CCL4, Promote Human Trophoblast Migration at the Feto-Maternal Interface
Biology of Reproduction, 2006Co-Authors: Natalie J. Hannan, Rebecca L. Jones, Christine A White, Lois A. SalamonsenAbstract:Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.
Jörn Elsner - One of the best experts on this subject based on the ideXlab platform.
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CCR1- and CCR5-mediated inactivation of leukocytes by a nonglycosaminoglycan (non-GAG)-binding variant of n-Nonanoyl-CCL14 (NNY-CCL14)
Journal of leukocyte biology, 2010Co-Authors: Shipra Gupta, S.e. Escher, Aleksandra Heitland, Sebastian Rieder, Wolf-georg Forssmann, Sandra Schulz-maronde, Rudolf Richter, Matthias Mack, Johanna Manns, Jörn ElsnerAbstract:Intervention on chemokine receptors to prevent directional leukocyte migration is a potential therapeutic strategy. NNY-CCL14 is a CD26-resistant lead molecule, which exerts its effects on multiple chemokine receptors (CCR1, CCR2, CCR3, and CCR5). The inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation have been assigned to its interaction with CCR1 and CCR5. In this study, a non-GAG-binding variant of NNY-CCL14 was generated by mutating basic amino acids within the identified GAG-binding 49BBXB52 motif. This CD26-resistant, non-GAG binding variant, NNY-CCL14(G,A), does not promote CCR1-dependent cell arrest on modeled endothelium. Its biological activity tested on human and murine chemokine receptors revealed distinguishing properties to NNY-CCL14. As suggested by EC50 values for intracellular calcium mobilization, NNY-CCL14(G,A) demonstrated a reduced ability to activate hCCR1, but internalization and desensitization of hCCR1 were unperturbed. Surprisingly, its activity on hCCR3 was strongly reduced, and it did not internalize mCCR3. A significantly reduced chemotactic activity of eosinophils and monocytes was observed. All biological effects mediated by NNY-CCL14(G,A) via hCCR5 and mCCR5 showed no difference to NNY-CCL14. In mice treated i.v. with NNY-CCL14(G,A), a sustained in vivo down-modulation of CCR5 was achieved over 3 h. Therefore, NNY-CCL14(G,A) inactivates leukocytes by desensitizing and internalizing multiple chemokine receptors, thus rendering them unresponsive to further stimulation by natural ligands. When administered systemically, NNY-CCL14(G,A) may modulate leukocyte functions prior to their interaction with other endothelium-bound chemokines expressed under pathophysiological conditions, such as allergic inflammation.
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n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.
Allergy, 2008Co-Authors: Shipra Gupta, S.e. Escher, Wolf-georg Forssmann, Jörn Elsner, Sandra Schulz-maronde, Rudolf Richter, G. Erdmann, Ulf ForssmannAbstract:Background: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. Aim of the study: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. Methods: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. Results: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca2+]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. Conclusions: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.
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Intravascular inactivation of CCR5 by n-Nonanoyl-CC chemokine ligand 14 and inhibition of allergic airway inflammation.
Journal of leukocyte biology, 2007Co-Authors: Shipra Gupta, Barbara Fuchs, S.e. Escher, Aleksandra Heitland, Armin Braun, Wolf-georg Forssmann, Sandra Schulz-maronde, Matthias Mack, Hanns-christian Tillmann, Jörn ElsnerAbstract:Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1 + and CCR5 + cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.
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The allergy-associated chemokine receptors CCR3 and CCR5 can be inactivated by the modified chemokine NNY-CCL11.
Allergy, 2007Co-Authors: J. Manns, S.e. Escher, S. Rieder, B. Eilers, Wolf-georg Forssmann, Jörn Elsner, Ulf ForssmannAbstract:Background: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. Methods: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. Results: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. Conclusions: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases.
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Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1
Journal of leukocyte biology, 2005Co-Authors: Shipra Gupta, Ulf Forssmann, Wolf-georg Forssmann, Sandra Schulz-maronde, Christian Kutzleb, Rudolf Richter, Alexander Kapp, Jörn ElsnerAbstract:The CC chemokine receptor 1 (CCR1) has emerged as a relevant factor contributing to inflammatory diseases such as allergic asthma. Commonly used animal models of allergic airway inflammation, especially murine models, have certain limitations. The elaborate, nonhuman, primate models of asthma display the highest comparability with the situation in humans. These models play an important role in the understanding of the pathogenesis of asthma. To improve the understanding in cynomolgus monkey models, we identified and characterized CCR1 in this nonhuman primate. Initially, we cloned the cynomolgus monkey CCR1 (cCCR1) gene, and the sequence analysis revealed high homology at the nucleotide (92%) and amino acid (88.4%) levels with its human counterpart. Human embryonic kidney 293 cells were stably transfected with cCCR1 and used in functional assays. Among those CCR1 ligands tested, CCL14(9-74) was most potent in the induction of intracellular Ca2+ fluxes as observed for human CCR1 (hCCR1). Complete cross-desensitization could be achieved between CCL14(9-74) and CCL15. However, CCL3 could not fully abrogate the response to the potent ligand CCL14(9-74). Competition-binding studies with radiolabeled CCL3 concordantly showed that CCL14(9-74) has a higher affinity to cCCR1 than hCCL3. Moreover, differential tissue-specific expression of cCCR1 was investigated by real-time quantitative polymerase chain reaction, displaying the highest levels in spleen. This study adds basic information needed for the evaluation of the role of CCR1 in the pathophysiology of asthma using the highly relevant cynomolgus monkey model and in addition, aids in the preclinical evaluation of potential novel drugs targeting CCR1.
Natalie J. Hannan - One of the best experts on this subject based on the ideXlab platform.
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CX3CL1 and CCL14 Regulate Extracellular Matrix and Adhesion Molecules in the Trophoblast: Potential Roles in Human Embryo Implantation
Biology of reproduction, 2008Co-Authors: Natalie J. Hannan, Lois A. SalamonsenAbstract:Embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. We have previously shown that the chemokines CX3CL1 and CCL14 are abundant in endometrial vasculature, epithelial, and decidual cells at this time, and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblasts. CX3CL1 and CCL14 promote trophoblast migration. We hypothesized that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion and ECM following chemokine stimulation were examined using pathway-specific oligo-arrays and quantitative real-time RT-PCR. More than 30 genes were affected by CX3CL1 treatment, and 15 genes were found to be regulated by CCL14 treatment. Real-time RT-PCR quantitation revealed significant changes in the mRNA transcripts of alpha-catenin (CTNNA1), extracellular matrix protein 1 (ECM1), osteopontin (SPP1), integrin alpha 6 (ITGA6), matrix metalloproteinase 12 (MMP12), and integrin beta 5 (ITGB5) following chemokine treatment. Several of these genes have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin alpha 6 and SPP1 protein in first-trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors, adhesion, and ECM at the maternal-fetal interface emphasizes an important role in the controlled directional migration of trophoblasts through the maternal decidua. For the first time, this study demonstrates the direct effects of CX3CL1 and CCL14 on trophoblast adhesion molecules and ECM, suggesting mechanisms by which trophoblast cells migrate during early pregnancy.
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213. Chemokines: key players at the maternal–fetal interface
Reproduction Fertility and Development, 2008Co-Authors: Natalie J. Hannan, L. A. SalamonsenAbstract:Establishment of pregnancy requires extensive communication at the maternal-fetal interface and involves a plethora of locally acting molecules, including the chemokines. Chemokines are multifunctional molecules initially described for roles in leukocyte trafficking, but since found to participate in many other processes such as differentiation and directed migration. Previously we have shown that the chemokines, CX3CL1 and CCL14, are abundant in human endometrial vasculature, leukocytes, epithelial and decidual cells at the time of implantation and that their receptors, CX3CR1 and CCR1, are present on invading human trophoblast. CX3CL1 and CCL14 directly promote human trophoblast migration. We hypothesised that these endometrial chemokines promote trophoblast migration by regulating adhesion molecules and extracellular matrix (ECM) components on the trophoblast, similar to mechanisms used in leukocyte trafficking. Trophoblast cells (AC1M-88) used previously, showed a marked increase in adhesion to fibronectin following treatment with CX3CL1 and CCL14. Alterations in trophoblast adhesion associated and ECM genes following chemokine stimulation were examined using pathway specific oligo-arrays and quantitative real-time RT–PCR. Over 30 transcripts were affected by CX3CL1 treatment and 15 were regulated by CCL14 treatment. Real-time RT–PCR confirmed significant changes in the mRNA transcripts of α-catenin (CTNNA1), extracellular matrix protein-1 (ECM1), osteopontin (SPP1), integrin α6 (ITGA6), matrix metalloproteinase-12 (MMP12) and integrin β5 (ITGB5) following chemokine treatment. Several of these molecules have previously been implicated in implantation. Immunohistochemistry confirmed the presence of integrin α6, SPP1 and ECM1 protein in first trimester human implantation sites. The temporal and spatial expression of chemokines, their receptors and adhesion related molecules at the maternal-fetal interface emphasises an important role in the controlled directional migration of trophoblast through the maternal decidua. For the first time this study demonstrates direct effects of CX3CL1 and CCL14 on trophoblast adhesion and ECM molecules suggesting mechanisms by which trophoblast cells migrate during early pregnancy.
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The Chemokines, CX3CL1, CCL14, and CCL4, Promote Human Trophoblast Migration at the Feto-Maternal Interface
Biology of Reproduction, 2006Co-Authors: Natalie J. Hannan, Rebecca L. Jones, Christine A White, Lois A. SalamonsenAbstract:Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and subsequent trophoblast invasion of the decidua. Chemokines, critical regulators of leukocyte migration, are abundant in endometrial epithelial and decidual cells at this time. We hypothesized that endometrial chemokines stimulate trophoblast invasion. Chemokine receptors CX3CR1 and CCR1 were immunolocalized in human first-trimester implantation sites, specifically to endovascular extravillous trophoblasts, but not to the invading interstitial EVTs (iEVTs), with weak staining also on syncytium. CCR3 was localized to invading iEVTs and to microvilli on the syncytial surface. Expression of CX3CL1 (fractalkine), CCL7 (MCP-3), and their receptors (CX3CR1, CCR1, CCR2, CCR3, and CCR5) mRNA was examined in cellular components of the maternal-embryonic interface by RT-PCR. Both chemokines were abundant in entire endometrium and placenta, endometrial cells (primary cultures and HES, a human endometrial epithelial cell line) and trophoblast cell lines (JEG-3, ACIM-88, and ACIM-32). Chemokine receptor mRNA was expressed by placenta and trophoblast cell lines: CCR1 by all trophoblast cell types, whereas CCR2, CCR3, and CX3CR1 were more variable. CX3CR1, CCR1, CCR2, and CCR5 were also expressed by endometrial cells. Migration assays used the trophoblast cell line most closely resembling extravillous cytotrophoblast (AC1M-88). Trophoblast migration occurred in response to CX3CL1, CCL14, and CCL4, but not CCL7. Endometrial cell-conditioned media also stimulated trophoblast migration; this was attenuated by neutralizing antibodies to CX3CL1 and CCL4. Thus, chemokines are expressed by maternal and embryonic cells during implantation, whereas corresponding receptors are on trophoblast cells. Promotion of trophoblast migration by chemokines and endometrial cell conditioned medium indicates an important involvement of chemokines in maternal-fetal communication.