Expression patterns of IL-8 protein during bacterial LPS-induced inflammatory response in grass carp (Ctenopharyngodon idella)

In order to investigate the expression characteristics of IL-8 during the lipopolysaccharide (LPS)-induced inflammatory process in grass carp (Ctenopharyngodon idella), recombinant grass carp IL-8 (rgcIL-8) was generated in prokaryotic expression system, and an anti-rgcIL-8 polyclonal antibody was prepare by immunizing mice with the purified rgcIL-8 alternately via intraperitoneal and subcutaneous injections. The polyclonal antibody was validated for specificity and titer by Western blotting and enzyme-linked immunosorbent assay (ELISA), and was used to detect IL-8 in immune-related tissues from healthy and/or LPS-stimulated grass carp by flow cytometry analysis. Results showed that the antibody titer peaked at 40 000. Flow cytometry analysis confirmed that IL-8 protein was expressed constitutively in all tested tissues of healthy fish, with higher levels in head kidney and gill. Moreover, the expression of IL-8 protein was found to be significantly up-regulated by LPS stimulation in all tested fish tissues, the protein levels peaked in intestine, liver and trunk kidney at 4 h, in head kidney and spleen at 12 h, and in thymus at 24 h following LPS stimulation, respectively. Our results provide further evidence that IL-8 is a key player in host inflammatory responses. Therefore, it is proposed that an elevated expression level of IL-8 protein could be used as a marker for monitoring the early inflammation, and be considered as a risk indicator for inflammatory diseases in aquaculture.