Constitutive expression of tumor necrosis factor-alpha in cytotoxic cells of teleosts and its role in regulation of cell-mediated cytotoxicity

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are the main killer cell populations of the immune system. The mechanisms by which these cells recognize target cells vary considerably, while the effector molecules used to facilitate target cell death are highly conserved. The main pathways utilized by killer cells consist of granule exocytosis and those mediated by members of the TNF superfamily. Nonspecific cytotoxic cells (NCC) are the first identified cytotoxic cell population in teleosts. We have previously demonstrated the expression of granzymes and Fas ligand in these cells. This is the first report of the expression of tumor necrosis factor-alpha in these killer cells. A cDNA coding for TNF was cloned and sequenced from NCC purified from Nile tilapia (Oreochromis niloticus). Factors regulating the transcriptional modulation of TNF in these cells were identified by RT-PCR analysis. The mature form of tilapia TNF was expressed as a recombinant protein and biological activities were analyzed. Using a cross-reacting anti-TNF polyclonal antibody, analysis of TNF expression suggested that tilapia NCC constitutively express the membrane-bound as well as secreted forms of TNF. Recombinant tilapia TNF effectively induced cytotoxicity in the mammalian cell line WEHI, although to a lesser extent compared to the murine TNF. Treatment with recombinant TNF protected NCC from activation-induced cell death. Recombinant tilapia TNF was also effective in upregulation of granzyme transcription in tilapia NCC. These data suggest that teleost TNF may play a role in diverse effector functions of cytotoxic cells from ectotherms, similar to the biological functions described for mammalian TNF.