Heparin-bound chemokine CXCL8 monomer and dimer are impaired for CXCR1 and CXCR2 activation: implications for gradients and neutrophil trafficking.

Prem Raj B Joseph,Kirti V Sawant,Krishna Rajarathnam

doi:10.1098/rsob.170168

Prem Raj B Joseph, Kirti V Sawant + Show 1 more

Open Access

https://doi.org/10.1098/rsob.170168

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Abstract

Chemokine CXCL8 plays a pivotal role in host immune response by recruiting neutrophils to the infection site. CXCL8 exists as monomers and dimers, and mediates recruitment by interacting with glycosaminoglycans (GAGs) and activating CXCR1 and CXCR2 receptors. How CXCL8 monomer and dimer interactions with both receptors and GAGs mediate trafficking is poorly understood. In particular, both haptotactic (mediated by GAG-bound chemokine) and chemotactic (mediated by soluble chemokine) gradients have been implicated, and whether it is the free or the GAG-bound CXCL8 monomer and/or dimer that activates the receptor remains unknown. Using solution NMR spectroscopy, we have now characterized the binding of heparin-bound CXCL8 monomer and dimer to CXCR1 and CXCR2 receptor N-domains. Our data provide compelling evidence that heparin-bound monomers and dimers are unable to bind either of the receptors. Cellular assays also indicate that heparin-bound CXCL8 is impaired for receptor activity. Considering dimer binds GAGs with higher affinity, dimers will exist predominantly in the GAG-bound form and the monomer in the free form. We conclude that GAG interactions determine the levels of free CXCL8, and that it is the free, and not GAG-bound, CXCL8 that activates the receptors and mediates recruitment of blood neutrophils to the infected tissue.

Highlights

A hallmark of infection is the immediate and robust recruitment of circulating neutrophils to the target tissue [1,2,3,4]
We used binding-induced chemical shift changes as structural probes to characterize whether CXCR1 or CXCR2 binding to heparin-bound CXCL8 monomers or dimers results in a ternary complex
CXCL8 exists as monomers and dimers, and animal model studies have shown that GAG interactions and monomer – dimer equilibrium regulate recruitment, and that the recruitment profiles of the monomers and dimers are distinctly different [37,38]

Summary

Introduction

A hallmark of infection is the immediate and robust recruitment of circulating neutrophils to the target tissue [1,2,3,4]. Chemokines mediate trafficking of neutrophils and other cell types to distal and remote locations in various tissues and organs [5,6,7,8]. Humans express around 50 chemokines, and all share a similar structural fold, exist as monomers and dimers (and some as higher order oligomers), and exert their function by binding G-protein-coupled receptors and sulfated glycosaminoglycans (GAGs) [9,10,11,12,13]. A subset of seven chemokines characterized by the highly conserved N-terminal ELR motif recruit neutrophils by activating CXCR1 and CXCR2 receptors [14,15]. Neutrophil-activating chemokines (NACs), released at the site of infection by resident cells, form concentration gradients that serve as beacons and guide the blood neutrophils to the infected site. Functional studies for NAC CXCL1 and CXCL8 have established that both monomers and dimers function as high-affinity CXCR2 agonists and that the CXCL8 monomer alone functions as a high-affinity CXCR1 agonist [16,17,18,19,20]

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