Role Of Inteleukin-8in The Inflammatory Response In Grass Carp (Ctenopharyngodon Idella)

Objective: Interleukin-8(IL-8) is a member of the CXC chemokine family, andplays an important role in inflammation, cancer and other diseases by activatingneutrophils, basophils and lymphocytes, and regulating their chemotaxis. Grass carp(Ctenopharyngodon idella) is one of the major aquaculture species in China, but isprone to infection by pathogenic microorganisms, presents severe inflammation andeven death. Based on the cloning of IL-8gene from grass carp in a previous study in ourlaboratory, we expressed recombinant grass carp interleukin-8(rgcIL-8) by using aprokaryotic expression system, and identified the function of rgcIL-8in grass carp. Withan objective to explore the role of fish IL-8in the inflammatory response in grass carp,we prepared anti-rgcIL-8polyclonal antibody, and then measured the production of IL-8in immune-related tissues of grass carp under normal and pathological conditions.Methods: Firstly, the rgcIL-8expressed in E.coli BL21cells was purified throughNi-NTA column affinity chromatography, and its biological effects were analyzed bytranswell migration assay and by in vivo stimulation test. Secondly, the purified rgcIL-8was used as the immunogen to generate an anti-rgcIL-8polyclonal antibody, thus thespecifcity and titer of the antibody were determined by Western blot and indirectELISA methods. The optimal working concentration of antigen-antibody in indirectELISA for detecting IL-8protein in grass carp were determined by using chessboardtitration. Finally, the native IL-8production and distribution in normal immune-relatedtissues from normal and/or LPS-induced grass carp were detected byimmunofluorescence assay with the anti-rgcIL-8antibody.Results: Transwell migration assay showed rgcIL-8exerted obvious chemotacticactivity on neutrophils in head kidney of grass carp, in a concentration dependentmanner. Results from in vivo stimulation test indicated that rgcIL-8could induce theleukocytes to accumulate at the stimulated sites, resulted in histopathological changessimilar to an acute inflammatory response. The polyclonal antibody assays showed theanti-rgcIL-8antibody could specifically recognize native gcIL-8, and its titer was determined to be1:40000. The expression pattern of gcIL-8in immune-related tissuesin grass carp was analyzed by using the indirect ELISA optimized with anti-rgcIL-8polyclonal antibody, the results revealed that the gcIL-8was constitutively expressed inall immune-related tissues. LPS could significantly upregulate the expression of gcIL-8in each immune-related tissue. The expression of gcIL-8in the thymus gland, liver andintestine rapidly increased and peaked4h after LPS stimulation, whereas that in thehead kidney, gill, trunk kidney and spleen peaked at12h. Thus, these results confirmedthe importance of gcIL-8in immunomodulatory effects in grass carp.Conclusion: It was validated that grass carp IL-8had significant chemotacticactivity at the cellular level and in vivo proinflammatory effect. We successfullygenerated the anti-rgcIL-8polyclonal antibody with higher specificity and titer, andestablished indirect ELISA suitable for detecting grass carp IL-8, by which theproduction and distribution of IL-8protein in each immune-related tissue from normaland/or LPS-induced grass carp were investigated for the first time. The role of IL-8inthe inflammatory response in grass carp was primarily confirmed. This provides a basisfor further exploring the functional mechanism of IL-8in fish and discovering noveltargeted agents for inhibiting fish inflammation.