Role of Th1-stimulating cytokines in bacillus Calmette–Guérin (BCG)-induced macrophage cytotoxicity against mouse bladder cancer MBT-2 cells

Abstract

Previously, we have demonstrated that macrophages exhibited cytotoxicity toward mouse bladder cancer MBT-2 cells upon bacille Calmette-Guérin (BCG) stimulation. In this study, we have investigated the role of Th1-stimulating cytokines in BCG-induced macrophage cytotoxicity. Thioglycollate-elicited peritoneal exudate cells (PECs) were used as a conventional source for macrophages and the induction of PEC effector functions (cytolytic activity and cytokine production) by BCG was evaluated in vitro. The BCG-activated PECs showed potent cytotoxicity and killed MBT-2 cells in a dose-dependent manner. Depletion of T cells, natural killer (NK) cells, or both, in PEC preparations exhibited a marginal or small reduction of MBT-2 cell killing, suggesting that macrophages played a primary role in PEC cytotoxicity. Transwell assays indicated that the maximal PEC cytotoxicity required both direct cell-cell contact and soluble factors such as interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. Neutralizing endogenous cytokines interleukin (IL)-12, IL-18, IFN-gamma or TNF-alpha reduced PEC cytotoxicity by 38%, 22%, 15% and 94%, respectively. Supplementation of BCG with recombinant (r)IL-2, rIL-12 or rIL-18 increased PEC cytotoxicity by approximately twofold. Compared with control BCG for PEC stimulation, rBCGs expressing IL-2 or IL-18 showed enhanced MBT-2 cell killing by PECs. Increased cytokine production (IFN-gamma, TNF-alpha and IL-6) was also observed in rBCG-stimulated PEC cultures. Taken together, these results suggest that Th1-stimulating cytokines play an important role in BCG-induced macrophage cytotoxicity and that combination of BCG with selected Th1-stimulating cytokines, either supplemented or expressed by BCG, may enhance the effect of BCG in the treatment of bladder cancer patients.