Effects of immunosuppressive agents on nuclear factor NF-kB activation in jurkat cells

Abstract

Effects of immunosuppressive agents on nuclear transcription factor NF-κB activation were investigated as an approach to elucidate the mechanisms of immunosuppression. Three copies of NF-κB binding site were inserted into test plasmid containing the chloramphenicol acetyltransferase gene. When Jurkat cells were transiently transfected with the recombinant construct, NF-κB activation could be induced by a combination of PMA and PHA and inhibited by pyrrolidine dithiocarbamate as a well-known potent inhibitor. Based on the results, immunosuppressive agents such as 2-acetylaminofluorene (AAF), 2-aminofluorene (AF), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 7,12-Dimethylbenz[a]anthracene (DMBA), cychlophosphamide (CP) and aflatoxin B1 ($AFB_1$) were tested for their ability to affect the NF-κB activation by both PHA and PMA. AF produced the most potent inhibition. Complete inhibition of NF-κB by AF appeared at concentration of 5 μM. The effect of AAF was also severe even if the higher concentration of AAF was required to make the same inhibition as that of AF. About 25 μM of AAF reduced the NF-κB activity by 60% and 100 μM by 100% to the background level. TCDD also produced an inhibition but a little. CP and DMBA had no effect on NF-κB activation in mixed culture of transfected cells and primary rat hepatocytes as well as in the transfectants only. On the other hand, 100 μM; $AFB_1$ strongly inhibited the NF-κB activation in transfected cells cocultured with hepatocytes while direct treatment of $AFB_1$ had no effect on NF-κB activity in transfected cells in the absence of hepatocytes. Conclusively, these data suggest that the inhibition of NF-κB activation may be involved in the immunosuppression provoked by AAF, AF, TCDD (partially) and $AFB_1$ (cocultured with rat hepatocytes), whereas the immunosuppression of CP and DMBA in the presence of hepatocytes may not be mediated by the regulation of NF-κB activity.