CCL2 nitration is a negative regulator of chemokine-mediated inflammation

Catriona E Barker,Hugues Lortat-Jacob,Graeme O’Boyle,Simi Ali,John A Kirby,Neil S Sheerin,Sarah Thompson

doi:10.1038/srep44384

Abstract

Chemokines promote leukocyte recruitment during inflammation. The oxidative burst is an important effector mechanism, this leads to the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO). The current study was performed to determine the potential for nitration to alter the chemical and biological properties of the prototypical CC chemokine, CCL2. Immunofluorescence was performed to assess the presence of RNS in kidney biopsies. Co-localisation was observed between RNS-modified tyrosine residues and the chemokine CCL2 in diseased kidneys. Nitration reduced the potential of CCL2 to stimulate monocyte migration in diffusion gradient chemotaxis assays (p < 0.05). This was consistent with a trend towards reduced affinity of the nitrated chemokine for its cognate receptor CCR2b. The nitrated chemokine was unable to induce transendothelial monocyte migration in vitro and failed to promote leukocyte recruitment when added to murine air pouches (p < 0.05). This could potentially be attributed to reduced glycosaminoglycan binding ability, as surface plasmon resonance spectroscopy showed that nitration reduced heparan sulphate binding by CCL2. Importantly, intravenous administration of nitrated CCL2 also inhibited the normal recruitment of leukocytes to murine air pouches filled with unmodified CCL2. Together these data suggest that nitration of CCL2 during inflammation provides a mechanism to limit and resolve acute inflammation.

Highlights

The mechanisms by which ongoing inflammation is resolved are unclear
In order to examine the effects of oxidative stress in vivo, we used biopsy sections taken from patients who had been diagnosed with acute tubular necrosis (ATN) caused by ischaemia reperfusion injury (IRI) sustained during kidney transplantation
This was compared to staining in kidney sections from the unaffected pole of tumour nephrectomies. 3-nitrotyrosine appeared to be present constitutively, in the tubules, in both normal and ATN kidneys (Fig. 1); a staining pattern which has been previously documented[24]

Summary

Introduction

The mechanisms by which ongoing inflammation is resolved are unclear. Limiting the production of pro-inflammatory factors, including chemokines, is one potential mechanism. In order to form the chemokine gradients needed for in vivo function, chemokines bind to GAGs such as heparan sulphate[15,16] Endothelial expression of these cell surface GAGs increases during the stresses induced by transplantation, resulting in increased endothelial potential to bind and present chemokines[17]. It is thought that modification of proinflammatory cytokines by peroxynitrite abrogates function, but this is dependent on numerous factors, including cell type, anti-oxidant levels (scavenger glutathione) etc. Molon et al.[23] reported that nitrated CCL2 cannot be detected by commercially available antibodies as the modification causes epitope loss This has important implications for studies using antibody based techniques to study chemokine levels in inflammatory environments

Methods

Results

Conclusion