title: Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors.
abstract: Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/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 and 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 microg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 microM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i. e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.
authors.affiliation: Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, Athens, Georgia 30602, USA.
authors.forename: G R
authors.initials: GR
authors.lastname: Bishop
authors.forename: L
authors.initials: L
authors.lastname: Jaso-Friedmann
authors.forename: D L
authors.initials: DL
authors.lastname: Evans
mesh_terms: D000209:Acute-Phase Proteins / Q000502:physiology; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D002413:Cations, Divalent / Q000276:immunology; D002453:Cell Cycle / Q000276:immunology; D018920:Coculture Techniques; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D004247:DNA / Q000032:analysis; D003907:Dexamethasone / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008297:Male; D013312:Stress, Physiological / Q000276:immunology; D017210:Tilapia
chemical_list: D000209:Acute-Phase Proteins; D017304:Annexin A5; D000911:Antibodies, Monoclonal; D002413:Cations, Divalent; D003907:Dexamethasone; D004247:DNA