Post-translational modification of chemokines : implications for their biological function

Abstract

Chemokines mediate a targeted immune response by orchestrating the migration of leukocytes to sites of injury. CXCL8 is a neutrophil-recruiting chemokine implicated in the pathology of many diseases. Ischaemia-reperfusion Injury (IRI) induces oxidative stress, and the reactive nitrogen species (RNS) peroxynitrite (ONOO-). Our group has shown that post-translational nitration of chemokines by peroxynitrite can affect function and detectability. These modified chemokines (if non-functional) could pose a natural mechanism for the regulation of inflammation. I have shown that nitration of CXCL8 by peroxynitrite reduces its ability to induce neutrophil migration in chemotaxis assays in vitro and murine intraperitoneal chemotaxis assays in vivo, through inhibition of both G-protein coupled receptor (GPCR) signalling and glycosaminoglycan (GAG) binding. I have used N-loop mutant and nitrated mutant versions of CXCL8 (Y13F) and CXCL1 (L15Y) to assess the importance of tyrosine residues in nitration. In both cases nitration impairs chemokine function. In collaboration with Bio-Rad, I have developed a novel antibody which has shown specificity for nitrated CXCL8 over wild type CXCL8 in initial validation studies, and a detection limit of 1-10ng/ml. This antibody has facilitated the detection of naturally occurring nitrated CXCL8 in a bronchoalveolar lavage sample from a pneumonia patient. Pending further optimisation, this antibody could be incorporated into an ELISA assay to measure nitrated CXCL8 in biological fluids. I have demonstrated that CXCL8 and 3-nitrotyrosine (a marker of ONOO- activity) production can be differentially upregulated in response to different stressing agents in breast cancer and cardiovascular cell lines in vitro. This indicates that the potential for CXCL8 to be nitrated naturally is likely to vary between health and disease states, and also between specific tissues and types of stress. Characterising the expression and function of wild type and nitrated chemokines could lead to the development of techniques measuring these molecules as biomarkers to indicate the severity of IRI, or their use as anti-inflammatory therapies.