Wright S C, Bonavida B
J Immunol. 1987 Mar 15;138(6):1791-8.
The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.
本研究旨在评估其他细胞因子对含NKCF上清液裂解活性的可能贡献。我们比较了人NKCF与纯化的重组人rLT和rTNF的一些功能特性。发现rLT的靶细胞特异性与NKCF有很大不同，因为rLT既无种属特异性也无NK特异性。此外，抗rLT抗体不影响NKCF的裂解活性。这些结果表明LT对NKCF介导的裂解活性没有显著贡献。发现rTNF的靶细胞特异性与NKCF相关，但有一种NK抗性细胞系在20小时的51Cr释放试验中被rTNF裂解除外。然而，在20小时的试验中，通过台盼蓝排斥法评估，rTNF对所测试的任何靶细胞均无毒性，除非靶细胞用51Cr标记。相反，如通过台盼蓝排斥法检测到的，NKCF确实能杀死未用51Cr标记的靶细胞。对rTNF和NKCF裂解活性的这种机制差异的进一步分析表明，rTNF与放线菌酮或丝裂霉素C联合使用，但不与IFN-γ联合使用，能够裂解未标记的U937靶细胞。此外，用与用于51Cr标记细胞相同浓度的非放射性Na2CrO4预处理U937靶细胞，通过台盼蓝排斥法评估，其对rTNF介导的裂解变得敏感。这些发现表明，rTNF在20小时内对几种敏感靶细胞的裂解需要存在其他可能具有亚致死毒性和/或抑制大分子合成的因子。抗体抑制研究表明，抗TNF可部分或完全抑制含NKCF的未分级上清液对U937的裂解。然而，在色谱聚焦柱上对这种上清液进行分级分离，产生了两个不同的活性峰，在pH 5至6和7至8范围内洗脱。抗TNF可抑制NKCF的酸性形式，但不能抑制中性形式。得出的结论是，NKCF活性部分由TNF或抗原相关分子以及其他一些不同因子介导。抗TNF对NK CMC缺乏一致的抑制作用表明，单独的TNF不足以介导NK活性，或者它无法与添加的抗体结合。
