Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2 Federica Casilli a,1 , Andrea Bianchini a,1 , Isabelle Gloaguen b , Leda Biordi c , Edoardo Alesse c , Claudio Festuccia c , Barbara Cavalieri d , Raffaele Strippoli e , Maria Neve Cervellera a , Rosa Di Bitondo a , Elisabetta Ferretti e , Fabrizio Mainiero e , Cinzia Bizzarri a , Francesco Colotta a , Riccardo Bertini a, * a Dompe ´ S.p.A. Research Center, L’Aquila, Italy b Consorzio Biolaq, L’Aquila, Italy c Department of Experimental Medicine, University of L’Aquila, Italy d Department of Clinical and Biological Sciences, University of Turin, Italy e Department of Experimental Medicine and Pathology, University of ‘La Sapienza’, Rome, Italy Received 6 August 2004; accepted 20 October 2004 Abstract Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites. # 2004 Elsevier Inc. All rights reserved. Keywords: Interleukin-8; Polymorphonuclear leukocytes; Adhesion; Degranulation; Phagocytosis; Repertaxin 1. Introduction The recruitment of leukocytes into sites of tissue damage is a normal physiological response designed to fight infection, remove damaged cells and stimulate healing. However, the uncontrolled recruitment of leukocytes often exacerbates tissue damage, slows healing and in some cases may lead to host death. Among chemotactic factors generated at the site of inflammation, interleukin-8 (CXCL8/IL-8) is a key mediator of human polymorphonuclear leukocyte (PMN) recruitment and activation [1,2]. CXCL8 is also involved in T lymphocyte and natural killer (NK) cell recruitment [3,4]. CXCL8 belongs to the C–X–C branch of chemokine family, which also includes growth-related oncogene (CXCL1/GRO), neutrophil-activating peptide-2 (CXCL7/NAP-2), epithelial neutrophil-activating peptide (CXCL5/ENA-78) and granulocyte chemotactic protein-2 (CXCL6/GCP-2) [5,6]. Interleukin-8 receptor 1 (CXCR1) www.elsevier.com/locate/biochempharm Biochemical Pharmacology 69 (2005) 385–394 Abbreviations: PMN, human polymorphonuclear leukocytes; CXCL8/ IL-8, interleukin-8; CXCR1, interleukin-8 receptor 1; CXCR2, interleukin- 8 receptor 2; GPCR, G protein-coupled receptor; NK, natural killer; LPS, bacterial endotoxin; C5a, fifth component of complement; fMLP, N-formyl- methionyl-leucyl-phenylalanine; CXCL12/SDF-1, stromal cell-derived fac- tor-1; CXCL6/GCP-2, granulocyte chemotactic protein-2; PBS, phosphate buffered saline; FBS, fetal bovine serum; MMP-9, matrix metalloproteinase 9; IL-1b, interleukin-1b; EDTA, ethylenediaminetetracetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase * Corresponding author. Tel.: +39 0862 338375; fax: +39 0862 338219. E-mail address: bertini@dompe.it (R. Bertini). 1 They contributed equally to this work. 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.10.007