Jyothi MD, Khar A. Interleukin-2-Induced Nitric Oxide Synthase and Nuclear Factor-kB Activity in Activated NK Cells and the Production of Interferon-g. Scand J Immunol 2000;52:148±155We have previously shown that inducible nitric oxide synthase (iNOS) was up-regulated in natural killer (NK) cells when AK-5 tumour cells were transplanted subcutaneously into syngeneic Wistar rats. This study was designed to investigate the role of interleukin (IL)-2 during the induction of iNOS and to understand the subsequent events involved in NK cell activation. There was up-regulation of iNOS expression when nai È ve NK cells were cultured in the presence of recombinant IL-2. These NK cells produced a higher nitrite content and possessed cytotoxic activity against YAC-1 and AK-5 tumour cells. Induction of iNOS enhanced nuclear factor (NF)-kB binding activity in IL-2 activated NK cells, which was con®rmed using L-NAME, an NO synthesis inhibitor. Addition of L-NAME along with rIL-2 signi®cantly blocked NF-kB activity and also down-regulated the production of NO and the cytotoxic activity of NK cells. Furthermore, injection of anti-IL-2 antibody in subcutaneous tumour transplanted animals abrogated signi®cantly the expression of iNOS and NF-kB activity, leading to reduced NO production and cytotoxic activity of NK cells against YAC-1 and AK-5 cells. In addition, the expression of interferon (IFN)-g by NK cells was also inhibited in anti-IL-2 antibody injected animals compared with the control animals. Finally, there was enhanced tumour growth and delayed regression in anti-IL-2 injected animals compared with control animals.

The spontaneous regression of a rat histiocytoma, AK-5, is mediated by activated natural killer cells through antibody-dependent cellular cytotoxicity. In addition to the Fc-FcR interaction between the target and the effector cells demonstrated previously, we show the participation of costimulatory molecules B7 and CD28 in the efficient killing of the tumour cell. Blockade of the costimulatory interaction in vivo using anti-CD28 led to increased tumour growth and a suppressed cytokine response. Anti-CD28 antibody administration in vivo also diminished the cytotoxic potential of NK cells against AK-5 cells in vitro. Our studies also demonstrate the expression of B7.1 and B7.2 antigen on AK-5 tumour cells. The cytotoxic activity of natural killer cells was significantly inhibited when the effector/target cells were cultured in the presence of antibodies raised against B7.1, B7.2 and CD28. Administration of anti-CD28 in vivo also affected the efficiency of the formation of effector/target conjugates in vitro. Similarly, anti-CD28 injections affected expression of the adhesion molecules LFA 1 and ICAM 1 by splenocytes. Administration of anti-B7.1 and B7. 2 antibodies in AK-5 tumour-bearing animals showed a differential response. The cytotoxicity of natural killer cells was significantly inhibited after anti-B7.2 administration, suggesting the preferential participation of B7.2 molecules in vivo. These observations suggest an important role for B7-CD28 interaction in AK-5 tumour regression.