The Experts below are selected from a list of 22875 Experts worldwide ranked by ideXlab platform
Krishna Rajarathnam - One of the best experts on this subject based on the ideXlab platform.
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Chemokine CXCL1 mediated neutrophil recruitment role of glycosaminoglycan interactions
Scientific Reports, 2016Co-Authors: Kirti V Sawant, Krishna Mohan Sepuru, Roberto P Garofalo, Krishna Mohan Poluri, Amit K Dutta, Anna Troshkina, Krishna RajarathnamAbstract:The Chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury.
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CXCL1 mgsa is a novel glycosaminoglycan gag binding Chemokine structural evidence for two distinct non overlapping binding domains
Journal of Biological Chemistry, 2016Co-Authors: Krishna Mohan Sepuru, Krishna RajarathnamAbstract:Abstract In humans, the Chemokine CXCL1/MGSA (hCXCL1), plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We have addressed this missing knowledge by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using nuclear magnetic resonance (NMR) spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding, and are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) was also previously identified as the GAG-binding domain for the related Chemokine CXCL8/IL-8. The second domain, consisting of residues from the N-terminus, 40s turn, and 3rd β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that GAG-bound Chemokine completely occludes receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating Chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.
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CXCL1 mgsa is a novel glycosaminoglycan gag binding Chemokine structural evidence for two distinct non overlapping binding domains
Journal of Biological Chemistry, 2016Co-Authors: Krishna Mohan Sepuru, Krishna RajarathnamAbstract:In humans, the Chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related Chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating Chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.
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Chemokine CXCL1 mediated neutrophil trafficking in the lung role of cxcr2 activation
Journal of Innate Immunity, 2015Co-Authors: Kirti V Sawant, Renling Xu, Hal K Hawkins, Elena Sbrana, Deepthi Kolli, Roberto P Garofalo, Krishna RajarathnamAbstract:The Chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and the monomer-dimer equilibrium regulate the neutrophil function. However, the molecular mechanisms linking the CXCL1/CXCR2 axis and the neutrophil ‘beneficial' and ‘destructive' phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by the CXCL1 wild type (WT), which exists as monomers and dimers, and by a nondissociating dimer. Whereas the WT, compared to the dimer, was more active at low doses, both the WT and the dimer elicited a large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by the WT or dimer was not detrimental to lung tissue integrity and, further, could not be correlated to surface CXCR2 levels. We conclude that the CXCL1 monomer-dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment.
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molecular basis of glycosaminoglycan heparin binding to the Chemokine CXCL1 dimer
Journal of Biological Chemistry, 2013Co-Authors: Krishna Mohan Poluri, Kirti V Sawant, Prem Raj B Joseph, Krishna RajarathnamAbstract:Glycosaminoglycan (GAG)-bound and soluble Chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which Chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the Chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating Chemokine and exists as both monomers and dimers (Kd = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” Chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt Chemokine-GAG interactions and neutrophil homing to the target tissue.
Robert G Webster - One of the best experts on this subject based on the ideXlab platform.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G WebsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92→Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50 %) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1α, IL1β, IL6, IFN-γ and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G Webster, Mark Y SangsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
Peter C Doherty - One of the best experts on this subject based on the ideXlab platform.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G WebsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92→Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50 %) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1α, IL1β, IL6, IFN-γ and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G Webster, Mark Y SangsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
Aleksandr S Lipatov - One of the best experts on this subject based on the ideXlab platform.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G WebsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92→Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50 %) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1α, IL1β, IL6, IFN-γ and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
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pathogenesis of hong kong h5n1 influenza virus ns gene reassortants in mice the role of cytokines and b and t cell responses
Journal of General Virology, 2005Co-Authors: Aleksandr S Lipatov, Samita Andreansky, Richard J Webby, Diane J Hulse, Jerold E Rehg, Scott Krauss, Daniel R Perez, Peter C Doherty, Robert G Webster, Mark Y SangsterAbstract:The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92-->Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and Chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50%) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1alpha, IL1beta, IL6, IFN-gamma and Chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
John T Martin - One of the best experts on this subject based on the ideXlab platform.
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the Chemokine CXCL1 is a novel target gene of parathyroid hormone pth pth related protein in committed osteoblasts
Endocrinology, 2009Co-Authors: Done Onan, E H Allan, Julian M W Quinn, Jonathan H Gooi, S Pompolo, Natalie A Sims, Matthew T Gillespie, John T MartinAbstract:The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the cysteine-X-cysteine (CXC) family Chemokine ligand 1 (CXCL1) as a novel immediate PTH/PTHrP-responsive gene. CXCL1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. CXCL1 mRNA levels were up-regulated 1 h after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold) and in rat metaphyseal bone (5-fold) 1 h after a single sc injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted CXCL1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for CXCL1, is highly expressed in osteoclast precursors (hemopoietic cells) ...
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the Chemokine CXCL1 is a novel target gene of parathyroid hormone pth pth related protein in committed osteoblasts
Endocrinology, 2009Co-Authors: Done Onan, E H Allan, Julian M W Quinn, Jonathan H Gooi, S Pompolo, Natalie A Sims, Matthew T Gillespie, John T MartinAbstract:The PTH receptor (PTHR1) is expressed on osteoblasts and responds to PTH or PTHrP in an endocrine or autocrine/paracrine manner, respectively. A microarray study carried out on PTHR1-positive osteoblasts (Kusa 4b10 cells) identified the cysteine-X-cysteine (CXC) family Chemokine ligand 1 (CXCL1) as a novel immediate PTH/PTHrP-responsive gene. CXCL1 is a potent neutrophil chemoattractant with recognized roles in angiogenesis and inflammation, but a role in bone biology has not been described. CXCL1 mRNA levels were up-regulated 1 h after either PTH or PTHrP treatment of differentiated Kusa 4b10 osteoblasts (15-fold) and mouse calvarial osteoblasts (160-fold) and in rat metaphyseal bone (5-fold) 1 h after a single sc injection of PTH. Furthermore, PTH treatment stimulated a 10-fold increase in secreted CXCL1 protein by both Kusa 4b10 cells and calvarial osteoblasts. Immunohistochemistry and PCR demonstrated that CXCR2, the receptor for CXCL1, is highly expressed in osteoclast precursors (hemopoietic cells) but is predominantly undetectable in the osteoblast lineage, suggesting that osteoblast-derived CXCL1 may act as a chemoattractant for osteoclast precursors. Confirming this hypothesis, recombinant CXCL1 dose-dependently stimulated migration of osteoclast precursors in cell culture studies, as did conditioned media from Kusa 4b10 cells treated with PTH. These data indicate that local action through the PTHR1 could stimulate cells of the osteoblast lineage to release a Chemokine capable of attracting osteoclast precursors to the bone environment.