Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm CK-2 of rainbow trout (Oncorhynchus mykiss) has two differentially regulated alleles that encode a functional chemokine ☆ Shawna L. Semple a,1 , Shathi Eshaque a,1 , Kazuhiro Fujiki a , Cynthia Tang b , Leslie Mitchell c , Niels C. Bols a , Brian Dixon a, ⁎ a Department of Biology, University of Waterloo, 200 University Ave W., Waterloo, ON, N2L 3G1, Canada b Department of History and Classical Studies, McGill University, 845 Sherbrooke St., Montreal, QC, H3A 0G4, Canada c School of Medicine, New York University, 550 First Avenue, New York, NY, 10016, USA ARTICLE INFO Keywords: Phytohemagglutinin (PHA) Peripheral blood leukocytes Rainbow trout Chemotaxis CC chemokine ABSTRACT Rainbow trout chemokine 2 (CK-2) is currently the only known CC chemokine to have a mucin stalk. Further analysis of the mucin stalk region revealed a second, related CC chemokine sequence, denoted here as CK-2.1. This second sequence was determined to be an allele of CK-2 following genomic PCR analysis on several outbred individuals. Furthermore, in both in vivo and in vitro trials, CK-2 and CK-2.1 were both present, but appeared to have differential tissue expression in both control and PHA stimulated samples. Upon the development of a polyclonal antibody to rCK-2, CK-2 was only observed in the brain, liver and head kidney of PHA stimulated rainbow trout tissues. In comparison, when using the rainbow trout monocyte/macrophage-like cell line, RTS- 11, CK-2 protein was observed in both control and PHA stimulated conditions. When studying the function of CK-2, a chemotaxis assay revealed that both peripheral blood leukocytes and RTS-11 cells migrated towards rCK- 2 significantly at all concentrations studied when compared to truncated β 2 m. Interestingly, this migration was lowest at both the highest concentration and the lowest concentrations of CK-2. Thus, teleostean chemokine receptors may become desensitized when overstimulated as has been observed in mammalian models. The observed chemotactic function was indeed due to rCK-2 as cell migration was inhibited through pre-treatment of both the cells and the polyclonal antibody with rCK-2. As has been observed thus far with all other chemokines, CK-2 does appear to function through binding to a G-coupled protein receptor as chemotaxis could be inhibited through pre-treatment with pertussis toxin. Overall, the results of this study indicate that CK-2 is a functional chemokine that is encoded by two differentially expressed alleles in rainbow trout, CK-2 and CK-2.1. 1. Introduction The immune system of vertebrates is tremendously complex, in- volving numerous cell types, barriers and specialized systems which are all used to prevent the entry and colonization of foreign entities throughout the body. The function of this vast network is dependent upon the ability of immune cells to migrate and interact with one an- other, a role fulfilled by extracellular mediators known as chemokines. Chemotactic cytokines, or chemokines, are a large family of small cy- tokines responsible for controlling the migratory patterns and posi- tioning of immune cells (reviewed in Griffith et al., 2014). Chemokines were originally believed to have only pro-inflammatory functions but further study has revealed that these small proteins also play critical roles in both tissue homeostasis and development (Tachibana et al., 1998, Gouon-Evans et al., 2002, Virgintino et al., 2013). Regardless of their specific function, all chemokines initiate their actions through binding to appropriate receptors which can be located on a variety of cell types. Of the chemokine receptors discovered thus far, all belong to the family of pertussis toxin (PTX) sensitive, G-coupled protein re- ceptors (GCPR) (reviewed in Rossi and Zlotnik, 2000). In the highly-studied mammalian model, 46 chemokine genes are currently known (reviewed in Zlotnik et al., 2006, Nomiyama et al., 2010) and are segregated into four families based on differences in their structure and function. The largest family are the CC chemokines, so named because the first two of four cysteine residues are found adjacent to each other in members of this group. In comparison, a second family, https://doi.org/10.1016/j.vetimm.2018.02.003 Received 17 November 2017; Received in revised form 29 January 2018; Accepted 6 February 2018 ☆ The nucleotide sequence data reported in this paper has been submitted to the GenBank nucleotide sequence databases and have been assigned the accession numbers: AY372431, AY372432 and AY372433. ⁎ Corresponding author. 1 These authors should be considered co-first authors. E-mail address: bdixon@uwaterloo.ca (B. Dixon). Veterinary Immunology and Immunopathology 198 (2018) 26–36 0165-2427/ © 2018 Elsevier B.V. All rights reserved. T