title: Humanized forms of the CXCR1/CXCR2 antagonist, bovine CXCL8((3-74))K11R/G31P, effectively block ELR-CXC chemokine activity and airway endotoxemia pathology.
abstract: Glu-Leu-Arg (ELR)-CXC chemokines are important in acute responses to bacterial infections, wherein neutrophils are often critical to pathogen clearance. However, excessive neutrophil recruitment augments the pathology of many diseases. We have shown that bovine CXCL8((3-74))K11R/G31P (bG31P) is a highly effective ELR-CXC chemokine and neutrophil antagonist in cattle, but herein we wished to determine whether humanized forms of this antagonist would be similarly effective. We thus examined the independent contributions of the bovine-human-discrepant amino acids within CXCL8 to the biological activity of bG31P. We first examined the effect of wholesale ligation of the carboxy half of hCXCL8 onto the amino half of bG31P, and found that this human-bovine chaemeric G31P (hbG31P; i.e., bCXCL8((3-44))K11R/G31P-hCXCL8((45-72))) fully retained the ELR-CXC chemokine antagonist activity of bG31P. Thus, hbG31P blocked the abilities of human CXCL8 to chemoattract human neutrophil or induce reactive oxygen intermediate (ROI) release. It was also a highly effective antagonist in vivo in a guinea pig model of airway endotoxemia. We next methodically moved through the 5' half of the hbG31P cDNA, using site-directed mutagenesis to one-by-one make substitutions at each remaining discrepant amino acid (i.e., T3K, H13Y, T15K, E35A, and S37T). We generated and tested the agonist and antagonist activities of each analogue using human neutrophils and human CXCL8. We found that none of these possessed better antagonist activities than hbG31P. Our data thus suggests that partially humanized analogues of bG31P display significant potential as antagonists of human ELR-CXC chemokines.
authors.affiliation: Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.
authors.forename: Xixing
authors.initials: X
authors.lastname: Zhao
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Town
authors.forename: Xiaobei
authors.initials: X
authors.lastname: Zhang
authors.forename: Wenjun
authors.initials: W
authors.lastname: Wang
authors.forename: John R
authors.initials: JR
authors.lastname: Gordon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D054360:Chemokine CXCL1 / Q000037:antagonists & inhibitors*; D002633:Chemotaxis / Q000187:drug effects; D019446:Endotoxemia / Q000188:drug therapy*; D006168:Guinea Pigs; D006801:Humans; D016209:Interleukin-8 / Q000037:antagonists & inhibitors* / Q000494:pharmacology / Q000627:therapeutic use; D008168:Lung / Q000473:pathology*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000187:drug effects; D010446:Peptide Fragments / Q000494:pharmacology* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000378:metabolism; D023062:Receptors, Interleukin-8A / Q000037:antagonists & inhibitors*; D023063:Receptors, Interleukin-8B / Q000037:antagonists & inhibitors*
chemical_list: C457773:CXCL8(3-73)K11R,G31P, bovine; D054360:Chemokine CXCL1; D016209:Interleukin-8; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D002118:Calcium