Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12.

The role of angiogenesis inhibition in the antitumor activity of recombinant murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, the growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from therapeutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marker of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecules selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatment effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovascularization assays, 2 weeks of treatment were needed before severe tumor cell hypoxia was detected in K1735 tumors. The hypoxia that developed was regional and localized to tumor areas distant from blood vessels. The great majority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was sufficient to trigger K1735 apoptosis. Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vitro studies indicated that this non-hypoxia-related apoptosis was induced directly by IFN-gamma produced in response to rmIL-12 administration. These studies reveal that rmIL-12 controls K1735 tumors initially by IFN-gamma-induced apoptosis and later by hypoxia-induced apoptosis. They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediator of its therapeutic effect.

Immune suppression by recombinant interleukin (rIL)-12 involves interferon gamma induction of nitric oxide synthase 2 (iNOS) activity: inhibitors of NO generation reveal the extent of rIL-12 vaccine adjuvant effect.

Recombinant interleukin 12 (IL-12) can profoundly suppress cellular immune responses in mice. To define the underlying mechanism, recombinant murine (rm)IL-12 was given to C57BL/6 mice undergoing alloimmunization and found to transiently but profoundly suppress in vivo and in vitro allogeneic responses and in vitro splenocyte mitogenic responses. Use of neutralizing antibodies and genetically deficient mice showed that IFN-gamma (but not TNF-alpha) mediated rmIL-12-induced immune suppression. Splenocyte fractionation studies revealed that adherent cells from rmIL-12-treated mice suppressed the mitogenic response of normal nonadherent cells to concanavalin A and IL-2. Addition of an inhibitor of nitric oxide synthase (NOS) restored mitogenic responses, and inducible (i)NOS-/- mice were not immunosuppressed by rmIL-12. These results support the view that suppression of T cell responses is due to NO produced by macrophages responding to the high levels of IFN-gamma induced by rmIL-12. When a NOS inhibitor was given with rmIL-12 during vaccination of A/J mice with irradiated SCK tumor cells, immunosuppression was averted and the extent of rmIL-12's ability to enhance induction of protective antitumor immunity was revealed. This demonstrates that rmIL-12 is an effective vaccine adjuvant whose efficacy may be masked by its transient immunosuppressive effect.