Comparative analysis of two types of CXCL8 from Japanese flounder (Paralichthys olivaceus).

A new type of CXCL8, named CXCL8_L1b, was identified in this research. Comparison of amino acid sequences of Japanese flounder CXCL8_L1b and CXCL8_L1a (BAB86884.1) showed only 41.2% identity. Transcripts of CXCL8_L1a were highly detected in spleen, kidney, gill and liver, while transcripts of CXCL8_L1b only were detected highly in spleen and kidney of apparently healthy fish. In fish challenged with E. tarda, transcripts of CXCL8_L1a were significantly increased at day 6, while no significant increase was detected in the mRNA level of CXCL8_L1b. On the other hand, fish infected by S. iniae significantly increased both transcripts of CXCL8_L1a and CXCL8_L1b at days 1 and 3. In VHSV-infected fish, only the transcripts of CXCL8_L1b were significantly induced at day 6. LPS and poly I:C stimulation of PBLs induced a high level of CXCL8_L1a transcripts, while CXCL8_L1b transcripts were significantly increased only post poly I:C treatment. To evaluate the chemotactic activity of CXCL8_L1a and CXCL8_L1b, Japanese flounder were intramuscularly injected with recombinant plasmids pCI-CXCL8_L1a and pCI-CXCL8_L1b. H & E staining showed that injections of both pCI-CXCL8_L1a and pCI-CXCL8_L1b caused strong immune responses in the form of intermuscular cell infiltration and capillary congestion. Injection of pCI-CXCL8_L1a and pCI-CXCL8_L1b significantly induced the expressions of genes related to inflammatory response such as IL-6 and CD8α on day 1 post-injection. The transcripts of IgM only significantly increased on day 7 post-injection of pCI-CXCL8_L1b.