Phylogenetic analysis of vertebrate CXC chemokines reveals novel lineage specific groups in teleost fish Jun Chen a,b , Qiaoqing Xu a,c , Tiehui Wang a , Bertrand Collet d , Yolanda Corripio-Miyar a , Steve Bird a,e , Ping Xie b , Pin Nie b , Christopher J. Secombes a,⇑ , Jun Zou a,⇑ a Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK b State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China c School of Animal Science, Yangtze University, Jingzhou, Hubei Province 434020, China d Marine Scotland Science, P.O. Box 101, 375, Victoria Road, Aberdeen AB11 9DB, UK e Department of Science and Engineering, School of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand article info Article history: Received 27 November 2012 Revised 8 May 2013 Accepted 13 May 2013 Available online 20 May 2013 Keywords: Fish Teleosts Vertebrates CXC chemokines abstract In this study, we have identified 421 molecules across the vertebrate spectrum and propose a unified nomenclature for CXC chemokines in fish, amphibians and reptiles based on phylogenetic analysis. Expanding on earlier studies in teleost fish, lineage specific CXC chemokines that have no apparent homo- logues in mammals were confirmed. Furthermore, in addition to the two subgroups of the CXCL8 homo- logues known in teleost fish, a third group was identified (termed CXCL8_L3), as was a further subgroup of the fish CXC genes related to CXCL11. Expression of the CXC chemokines found in rainbow trout, Oncorhynchus mykiss, was studied in response to stimulation with inflammatory and antiviral cytokines, and bacterial. Tissue distribution analysis revealed distinct expression profiles for these trout CXC che- mokines. Lastly three of the trout chemokines, including two novel fish specific CXC chemokines contain- ing three pairs of cysteines, were produced as recombinant proteins and their effect on trout leucocyte migration studied. These molecules increased the relative expression of CD4 and MCSFR in migrated cells in an in vitro chemotaxis assay. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Chemokines are a large family of soluble peptides directing cell migration in development and immune defence. They are small in size, typically 8–15 kDa, and in mammals are classified into four subfamilies, CC, CXC, CX 3 C and XC, based on the distinctive pat- terns of the two N terminal cysteines (Alejo and Tafalla, 2011). The CC and CXC chemokine family are the two largest groups, con- taining multiple members, whilst the CX 3 C and XC family consist of one or two members respectively. More recently a fish specific CX subfamily has been described in zebrafish (Nomiyama et al., 2008), with four members present. Chemokines can be found across the whole vertebrate spec- trum, from primitive fish to mammals, and their origin can even be found in amphioxus, an ancient chordate species that diverged approx. 520 million years ago (Sansom et al., 1992; Kuroda et al., 2003). In mammals, over 50 chemokines have been characterised to date, that interact with approximately 20 G protein-coupled receptors (Allen et al., 2007). Teleost fish appear to have an even larger repertoire of chemokines possibly due to an extra round of genome duplication, with 111 chemokines identified recently in the zebrafish genome, the majority of which (81 genes) belong to the CC family (Alejo and Tafalla, 2011; Nomiyama et al., 2008). Despite the conserved two pairs of cysteine residues seen in the CC and CXC family, the primary sequences of the chemokines are highly divergent, making it extremely difficult to determine the phylogenetic relationships among family members during evolu- tion (Nomiyama et al., 2008; DeVries et al., 2006; Peatman and Liu, 2007; Laing and Secombes, 2004). Therefore, it has been problematic to establish a common nomenclature for some chemo- kines from early vertebrates simply based on the phylogenetic classification in mammals. In this study, we have undertaken extensive analysis of the phylogeny of the CXC chemokines using the sequences that have accumulated in the nucleotide databases from various vertebrate groups, to gain an in-depth understanding of their evolutionary origin with a particular focus on fish CXC chemokines. In mammals, the CXC chemokine family consists of 17 members, divided into two main subgroups; those with an ELR sequence motif preceding the first cysteine and those without this motif (Pisabarro et al., 2006; Viola and Luster, 2008; Wang et al., 2005; Matloubian et al., 2000). The CXC chemokine ligands (CXCL) 0145-305X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dci.2013.05.006 ⇑ Corresponding authors. Tel.: +44 1224 273796. E-mail addresses: c.secombes@abdn.ac.uk (C.J. Secombes), j.j.zou@abdn.ac.uk (J. Zou). Developmental and Comparative Immunology 41 (2013) 137–152 Contents lists available at SciVerse ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci