Abstract
CXCL8 (interleukin-8, IL-8) is a CXC family chemokine that recruits specific target cells and mediates inflammation and wound healing. This study reports the identification and characterization of two cxcl8 homologs from rock bream, Oplegnathus fasciatus. Investigation of molecular signature, homology, phylogeny, and gene structure suggested that they belonged to lineages 1 (L1) and 3 (L3), and designated Ofcxcl8-L1 and Ofcxcl8-L3. While Ofcxcl8-L1 and Ofcxcl8-L3 revealed quadripartite and tripartite organization, in place of the mammalian ELR (Glu-Leu-Arg) motif, their peptides harbored EMH (Glu-Met-His) and NSH (Asn-Ser-His) motifs, respectively. Transcripts of Ofcxcl8s were constitutively detected by Quantitative Real-Time PCR (qPCR) in 11 tissues examined, however, at different levels. Ofcxcl8-L1 transcript robustly responded to treatments with stimulants, such as flagellin, concanavalin A, lipopolysaccharide, and poly(I:C), and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus, when compared with Ofcxcl8-L3 mRNA. The differences in the putative promoter features may partly explain the differential transcriptional modulation of Ofcxcl8s. Purified recombinant OfCXCL8 (rOfCXCL8) proteins were used in in vitro chemotaxis and proliferation assays. Despite the lack of ELR motif, both rOfCXCL8s exhibited leukocyte chemotactic and proliferative functions, where the potency of rOfCXCL8-L1 was robust and significant compared to that of rOfCXCL8-L3. The results, taken together, are indicative of the crucial importance of Ofcxcl8s in inflammatory responses and immunoregulatory roles in rock bream immunity.
Highlights
Chemokines are a superfamily of low molecular weight (8–12 kDa) chemotactic cytokine peptides, which are multifunctional chemical messengers that can activate and orchestrate the mobilization and migration of specific subsets of leukocytes along a gradient [1]
As we described in an earlier study [40], a bacterial artificial chromosome (BAC) library was custom-constructed for rock bream, and a PCR-based method was employed in library screening to identify the positive clones containing the putative Ofcxcl8 sequences
Based on BLAST and comparative analyses, these two sequences were identified as belonging to lineages 1 (L1) and 3 (L3), and designated as Ofcxcl8-L1 and Ofcxcl8-lineage 3 (L3), respectively
Summary
Chemokines are a superfamily of low molecular weight (8–12 kDa) chemotactic cytokine peptides, which are multifunctional chemical messengers that can activate and orchestrate the mobilization and migration of specific subsets of leukocytes along a gradient [1]. Besides their leukocyte-recruiting function toward a site of infection or injury, chemokines are known to have pleiotropic biological effects in growth, differentiation, and immune regulation and angiogenesis, and appear to play a crucial mediator-role in linking innate and acquired arms of the immune system [2]. CXC chemokines that lack the ELR motif (ELR- CXC) are chemo-attractants for lymphocytes and monocytes and suppress angiogenesis [5,6]
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