Identification and Expression Profiles of IL8 in

Authors: Huiying Li Yan Cai Ping Xie Guangyu Li Le Hao Qian Xiong

Publish Date: 2013/06/25

Volume: 65, Issue: 3, Pages: 537-545

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Abstract

MicrocystinLR MCLR is a widespread cyanotoxin and has immunotoxicity to animals including fish Chemokines are considered to play important roles in inflammatory response induced by MCLR In this study we cloned the fulllength cDNA of interleukin8 IL8 from bighead carp Aristichthys nobilis for the first time The fulllength IL8 cDNA was 552 bp and contained a 297bp openreading frame that encoded for a 98amino acid protein The deduced IL8 protein had a typical aspartic acid Dleucine Larginine R and a CXC motif at the Nterminal which were conserved in most fish species Phylogenetic analysis showed that bighead carp IL8 protein was grouped in the teleost IL8 lineage 2 Under normal conditions the expression of IL8 is constitutive and weak in all tested tissues However MCLR treatment could significantly increase the transcription of IL8 in bighead carp in a temporal and dosedependent pattern The present study will help us to understand more about the evolution of IL8 and its function in the MCLR induced proinflammatory response in bighead carpChemokines are a family of small cytokines secreted by cells Laing and Secombes 2004 The CXC chemokines are the principal group in the chemokine family which can be subdivided into two categories those with an glutamate Eleucine Larginine R motif ELRpositive and those without the motif ELRnegative Fernandez and Lolis 2002 Interleukin8 IL8 is a member of the CXC chemokine family that can be produced by a wide variety of cells in response to many stimulants IL8 is synthesized as a precursor with a signal peptide which is proteolytically processed to release an 8kD molecule Harun et al 2008 The active form of IL8 is a dimmer and is comprised of two identical subunits IL8 monomer contains a triplestrand antiparallel βsheet whereas the dimer comprises a sixstrand antiparallel βpleated sheet and is spanned by two long symmetryrelated αhelices which locate in the carboxy terminal of the two subunits Clore et al 1990The primary function of IL8 is the recruitment of monocytes and neutrophils the signature cells of acute inflammatory response Remick 2005 Inflammatory cells follow the increasing chemokine concentration and move toward the source of the chemokines Another pivotal function of IL8 is to activate monocytes and neutrophils The biological effects of IL8 are mediated through the binding of IL8 to two cell surface receptors CXCR1 and CXCR2 Holmes et al 1991 IL8 is also an ideal molecule for locating at acute inflammatory sites It is resistant to temperature proteolysis and some acidic environments and is relatively stable at acute inflammatory sites However IL8 is highly sensitive to oxidants and antioxidants can substantially decrease IL8 gene expression DeForge et al 1993Microcystins MCs are produced by various species of toxic cyanobacteria and are known as potent hepatotoxins MCs mainly affect the liver causing minor to widespread damage depending on the amount of toxin absorbed Butler et al 2009 Evidence has indicated that MCs also have immunotoxicity to animals or culture cells Mankiewicz et al 2002 Shen et al 2003 MCs can regulate the production of interleukin IL1 and tumornecrosis factor TNFα induce nitric oxide synthesis in macrophages Pahan et al 1998 Rocha et al 2000 and modulate the spontaneous adherence of human peripheral polymorphonuclear leukocytes PMNLs Hernández et al 2000 Moreover it has been suggested that MCs downregulate lymphocyte functions through decreasing the mRNA stability of IL2 In addition MCs depress lymphocytes response to lipopolysaccharide and produce dosedependent inhibition of polyclonal antibody response in vitro Yea et al 2001IL8 was initially purified as a neutrophil chemoattractant in human MNLs Yoshimura et al 1987 Since then many IL8 sequences have been identified Two IL8 lineages have been identified in multiple teleost species Abdelkhalek et al 2009 Both lineages were likely to have arisen from a common ancestor of mammalian IL8/ELR+CRC chemokines Van der Aa et al 2010 Lineage 1 is present in most fish such as carp Huising et al 2003 zebrafish Nomiyama et al 2008 trout Laing et al 2002 haddock CorripioMiyar et al 2007 fugu Covello et al 2009 flounder Lee et al 2001 and Atlantic cod Lineage 2 is found only in cyprinids and zebrafish Van der Aa et al 2010 However to date there has been no report about IL8 in bighead carp Aristichthys nobilisBighead carp is a commercially important fish in China Its living habits suggest that it might consume great quantities of MCproducing cyanobacteria As a freshwater phytoplanktivorous fish bighead carp is more resistant to the toxic effects of MCLR compared with mammals He et al 1997 Xie et al 2004 Therefore bighead carp has received extensive research interest as a model species for toxicology ecology physiology evolutionary genetics and etc Xie 2003In our previous study we obtained cDNA fragments of IL8 gene from bighead carp injected intraperitoneally with 200 μg MCLR/kg body weight bw using suppression subtractive hybridization SSH technique and found that it was upregulated in response to MCLR treatment unpublished data To better understand the evolution of IL8 as well as its role in the antiMCLR response in bighead carp we cloned the fulllength cDNA of IL8 using RACE method Using semiquantitative realtime PCR QPCR assay we confirmed that IL8 expression was induced in bighead carp liver by MCLR The molecular characteristics of bighead carp IL8 and gene expression profiles were further analyzed

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