Cytokine effects and cellular signaling pathways of grass carp HSP70 in head kidney leukocytes

In mammals, the cytokine effects of heat shock protein 70 (HSP70) have been well documented. However, the precise role and molecular mechanism of fish HSP70 in cytokine production are poorly understood. In the present study, recombinant grass carp HSP70 (rgcHSP70) was prepared by using the E. coli expression system and its effect on interleukin (IL)-1β (gcil1β) and tumor necrosis factor (TNF)-α (gctnfα) mRNA expression was examined in grass carp head kidney leukocytes (HKLs). Compared with its heated-inactivated protein, rgcHSP70 enhanced the mRNA levels of these two cytokines in a dose- and time-dependent manner. More importantly, the results of enzyme-linked immunoabsorbent assay (ELISA) showed that rgcHSP70 markedly stimulated gcIL-1β and gcTNF-α secretion from the HKLs, for the first time indicating the ability of HSP70 to regulate cytokine release in fish. Moreover, rgcHSP70 displayed more potential to increase the cytokines' release instead of their transcription in the same cell model. Subsequently, the signaling mechanisms governing the cytokine effect of rgcHSP70 were investigated, showing that rgcHSP70-induced gcil1β mRNA expression was suppressed by inhibiting p38 MAPK and NF-κB pathways, while the stimulation of rgcHSP70 on gctnfα mRNA expression were blocked by inhibiting JNK MAPK and NF-κB pathways. In parallel, rgcHSP70 was also shown to activate NF-κB pathway through regulating grass carp IκBα protein levels in a time-dependent oscillation. These findings provide further understanding of cytokine effect and regulatory mechanism of HSP70 in fish and new insights into the roles of HSP70 in teleost immunity.