In vivo activation of tilapia nonspecific cytotoxic cells by Streptococcusiniae and amplification with apoptosis regulatory factor(s)

An important component of immediate innate responses of tilapia to stress i s the release within minutes of soluble cytokine-like substances into the p eripheral circulation. These cytokine-like stress factors bind nonspecific cytotoxic cells (NCC) and produce 3-4-fold increased cytotoxicity. In the p resent study, the in vivo responses of tilapia NCC following injection with different isolates of intact killed Streptococcus iniae was investigated. Activated cytotoxicity of NCC in the peripheral blood (PB) was produced by increased specific activity of resident cells rather than increased numbers . Tilapia injected intravenously (i.v.) with killed S. iniae produced diffe rent cytotoxicity responses compared to fish injected intraperitoneally (i. p.). In the spleen (S) and anterior kidney (AK), there was no correlation b etween S. iniae isolate and cytotoxicity response at 4, 8 or 24 h following i.p. injection. The NCC response following i.v. injection of killed bacter ia was different. Within minutes following i.v. injection, NCC cytotoxicity from the PB increased 100% compared to naive controls. The existence of su bsets of differentiated NCC in the PB was suggested because i.v, injection had no amplification effects on NCC from the AK or S. Likewise, NCC from th e PB only appeared to exhibit a degree of antigen specificity. S. iniae str ain #173 produced activation of cytotoxicity compared to isolates #164 and ATCC. Evidence for soluble factor (cytokine?) involvement in increased cyto toxicity was obtained by passive activation of NCC with serum from #173 (i. v.) injected fish. Incubation of this serum with control (naive) NCC produc ed large increases in the cytotoxicity of labelled HL-60 target cells. Simi larly obtained serum from fish injected with ATCC and #164 isolates had no amplification activity. Studies were also performed to study the mechanism( s) of passive activation. Flow cytometric analysis revealed that NCC from t he S, AK and PB constitutively expressed cytosolic (not membrane) Fast. Str ess serum treated NCC obtained from the peripheral blood produced an increa se in the expression of Fast, CAS and FADD by Western blot examination. The se data indicated that cytokine like factors in the serum of stressed tilap ia activate increased NCC cytotoxicity (possibly) by stimulating the expres sion of proteins involved in activation of programmed cell death. (C) 2000 Academic Press.