Functional characterization of grass carp (Ctenopharyngodon idella) interleukin-2 in head kidney leukocytes

Interleukin (IL)-2 belongs to the four-helix bundle cytokine family and plays key roles in growth, survival, activation-induced cell death and differentiation of the immune cells. In cyprinid fish, only common carp interleukin-2 (il2) has been cloned because of relatively low sequence homology between carp Il-2 and its homologs in other fish species. In the present study, the coding sequence of grass carp Il-2 (gcIl-2) was cloned and its identity was verified via bioinformatic analysis. Tissue distribution study showed that grass carp il2 (gcil2) mRNA was expressed in thymus, head kidney and gill with relatively high levels. Recombinant gcIl-2 (rgcIl-2) protein was subsequently prepared by using a prokaryotic expression system followed by a refolding method. The purified rgcIl-2 displayed an ability to stimulate the cell proliferation along with an increased mRNA expression of cd4l but not cd8a, igm or mcsfr in grass carp head kidney leukocytes (HKLs), suggesting the possible involvement of gcIl-2 in T helper (Th) cell proliferation. In the same cell model, rgcIl-2 significantly enhanced mRNA expression of some cytotoxic molecules including perforin-like protein 2, granzyme B-like and Fas ligand, indicating the modulation of cytotoxic cells by gcIl-2 in grass carp HKLs. Besides, gene expression of regulatory T (Treg) cell- and Th1/2 cell-related cytokines or transcription factors was detected in grass carp HKLs treated by rgcIl-2. Results showed that rgcIL2 treatment increased the mRNA expression of foxp3, cd25l, ifng2, il12p35, tbet, tnfa, il2, il4/13a, il4/13b and gata3l in HKLs, implying the regulatory roles of Il-2 in the expression of these immune genes and its possible involvement in differentiation of Treg and Th1/2 cells. These observations together with the related studies in other fishes suggest the existence of cytotoxic cells, Treg and Th1/2 subpopulations in fish species and the functional roles of Il-2 in these cells.