Abstract
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a proapoptotic member of the TNF family of type II membrane proteins, which constitutes one component of T cell cytotoxicity. TRAIL has been reported to induce apoptosis in several tumor cell lines including kidney carcinomas. However, it is not known whether TRAIL could mediate killing of hepatocellular carcinomas. Recent studies demonstrated that TRAIL is expressed on human T cell clones in response to interferons (IFNs). This study was therefore conducted to determine if IFNs could elicit tumoricidal effects on a hepatoma cell line through regulation of TRAIL expression on peripheral blood T (PBT) cells. Fresh human PBT cells, obtained from healthy volunteers, were cultured in RPMI1640 medium (3.5×106cells/ml) containing 10%FCS, 100 (g/ml streptomycin and penicillin. PBT cells were prestimulated for 12 hr on plates coated with anti-CD3/TCR monoclonal antibody (10μg/ml) in the presence or absence of IFNs (α or β;200IU/ml). After washing with culture medium, the cells were used as effector cells. Surface TRAIL expression on human PBT was analyzed with flow cytometry. The human hepatoma cell line Hep G2 was cultured in Dulbecco's modified Eagle Medium plus 10%FCS, 100μg/ml streptomycin and penicillin. Tumoricidal effect of PBT cells was determined by release of LDH into the culture media. Freshly isolated PBT cells did not express a detectable level of TRAIL on their surface. However, after stimulation with IFN-α or β for 12-48 hr in the presence of anti-CD3 monoclonal antibody, a rapid and remarkable TRAIL expression was induced. Co-culture with PBT cells that were not stimulated with anti-CD3 or IFNs resulted in moderate increases in LDH release. In marked contrast, IFNs augmented the cytotoxic activity of anti-CD3-stimulated PBT cells against Hep G2 cells, as evidenced by 3-4 fold increases in LDH release after co-culture for 24 hr with anti-CD3 plus IFN- stimulated PBT cells. These increases were significantly (-70%) suppressed by inclusion of a neutralizing anti-TRAIL antibody (10μg/ml) in culture media, indicating that the IFN-enhanced cytotoxicity was predominantly mediated by TRAIL. The results indicate that IFN can augment the cytotoxic activity of CD3-stimulated PBT cells against Hep G2 cells by enhancing TRAIL-mediated cytotoxicity. This finding may lead to identifying a new mechanism of the anti-tumor effect of IFN against hepatocellular carcinoma.
