Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian l ymphokine-activated natural killer cells. The cytotoxic activities of NCC a re enhanced by stress-activated serum factors (SASF) present in tilapia acu te-phase serum. In the present study purified NCC and xenogeneic target HL- 60 tumor cells and nuclei were distinguishable in mixtures determined by fl ow cytometry, NCC activated by target HL-60 cells undergo activation-induce d programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V bindi ng studies also demonstrated that NCC kill. HL-60 cells by an apoptotic mec hanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF, Pret reatment also produced more than a fourfold increase in NCC cytotoxicity (e ffector/target (E:T) ratio = 100:1), In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1: 0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 a nd 12 h and then decreased inversely with total cell necrosis over the next 60 h, Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h, Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 mu g/mL mab concentrations. Preincubation with SASF completely protected NCC from mab BCG-dependent AIPCD, SASF-media ted protection of NCC from AIPCD was dependent upon divalent cations, as de monstrated by increases in DNA hypoploidy of 38, 67, and 88% following prei ncubation in the presence of 10, 100, and 1000 mu M EDTA, respectively. SAS F also protected NCC from glucocorticoid- (i.e., dexamethasone) induced apo ptosis, Combined, these results demonstrated that NCC activity is down-regu lated by AIPCD, Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Resu lts are discussed in the context of the effects of acute stressors on innat e immunity. (C) 2000 Academic Press.