Bishop G R, Jaso-Friedmann L, Evans D L
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, Athens, Georgia 30602, USA.
Cell Immunol. 2000 Feb 1;199(2):126-37. doi: 10.1006/cimm.1999.1609.
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.
非特异性细胞毒性细胞（NCC）相当于硬骨鱼中的哺乳动物淋巴因子激活的自然杀伤细胞。罗非鱼急性期血清中存在的应激激活血清因子（SASF）可增强NCC的细胞毒性活性。在本研究中，通过流式细胞术测定，在混合物中可区分纯化的NCC与异种靶标HL-60肿瘤细胞及细胞核。如膜联蛋白-V结合和核DNA片段化结果所示，在12至16小时的杀伤试验中，被靶标HL-60细胞激活的NCC会经历激活诱导的程序性细胞死亡（AIPCD）。膜联蛋白-V结合研究还表明，NCC通过凋亡机制杀伤HL-60细胞。在50% SASF中预孵育4小时可保护NCC免受AIPCD影响。预处理还使NCC细胞毒性增加了四倍多（效应细胞/靶标细胞（E:T）比率 = 100:1）。在没有SASF预孵育的情况下，在E:T比率为1:0和1:1时，凋亡NCC的百分比分别从8%增加到91%。动力学研究（E:T = 10:1）表明，表现出HL-60依赖性AIPCD的NCC百分比在0.1至12小时之间增加，然后在接下来的60小时内随着总细胞坏死呈反比下降。用SASF对NCC进行预孵育可使NCC免受AIPCD影响超过72小时。用单克隆抗体（mab）5C6交联NCCRP-1受体，在mab浓度为1至100微克/毫升时会产生AIPCD。用SASF预孵育可完全保护NCC免受mab 5C6依赖性AIPCD影响。SASF介导的保护NCC免受AIPCD影响依赖于二价阳离子，如分别在存在10、100和1000微摩尔/升乙二胺四乙酸（EDTA）的情况下预孵育后，DNA亚二倍体增加38%、67%和88%所证明。SASF还可保护NCC免受糖皮质激素（即地塞米松）诱导的凋亡。综合这些结果表明，AIPCD会下调NCC活性。SASF释放到外周循环中可能通过增加循环能力来防止AIPCD对NCC的负调节。在急性应激源对固有免疫的影响背景下对结果进行了讨论。
