Abstract
Administration of agonistic monoclonal antibodies or recombinant cytokines is a potential approach to enhance antitumor immunity in bone marrow transplant (BMT) recipients, but is complicated by toxicity due to proinflammatory cytokine-mediated vital organ damage. Inflammatory cytokines are known to induce free radical production which we hypothesized might be the proximate cause of toxicity in this setting. Phenyl-tert-butylnitrone (PBN) is a spin trap inhibitor, that has been used in electron paramagnetic resonance (EPR) studies to trap free radicals so they can be visualized by EPR spectroscopy. To test whether PBN could mitigate cytokine-mediated organ damage, we employed a murine syngeneic transplant model in which administration of agonistic CD40 antibody to mice early post BMT results in lethality due to production of high levels of cytokines such as IL-12 and IFN-γ, leading to fatal gut toxicity. Lethally irradiated C57BL/6J (B6) mice were transplanted with B6 BM and then treated with either IgG or anti-CD40 antibody (7 μg) on days 1–4 post-BMT. Cohorts of anti-CD40-antibody-treated mice were then administered either DMSO or PBN (50 mg/kg BID) on days 0–5 post-BMT. Administration of PBN completely protected BMT recipients from anti-CD40 antibody-induced mortality (0% survival CD40/DMSO versus 100% survival CD40/PBN). This was attributable to a significant reduction in gut toxicity as determined using a pathological scoring system. To examine the mechanism by which mice were protected, cytokine and nitrate/nitrite levels were measured to determine whether PBN inhibited production of proinflammatory mediators. Administration of anti-CD40 antibody resulted in a significant increase in IL-12, IFN-γ,TNF-α, and nitrate/nitrite levels compared to IgG/PBN-treated control mice. However, there was no significant difference in serum measurements of these cytokines in CD40/PBN compared to CD40/DMSO-treated animals. These data demonstrated that the protective effects of PBN were not attributable to a reduction in cytokine mediators and suggested that the action of PBN occurred downstream of these inflammatory cytokine signaling pathways, through inhibition of free radical production. Cytokines, such as IL-12 and IFN-γ, are also important downstream mediators of the antitumor effects induced by agonistic CD40 antibody. Therefore to directly test whether PBN compromised these antitumor effects, B6 mice were intravenously administered 107 EL4 cells and then treated on days 3-6 with either rat IgG or anti-CD40 antibody. Since EL4 cells do not express CD40, tumor regression mediated by anti-CD40 antibody is attributable to augmentation of host immunity and has been shown to dependent upon IFN-γ. B6 mice were challenged with a lethal dose EL4 tumor cells and then treated with IgG or agonistic CD40 antibody (500 μg/day) in the presence or absence of PBN. IgG-treated control mice all died within 20 days. In contrast, CD40/DMSO and CD40/PBN-treated mice all survived >80 days after tumor challenge indicating that PBN did not compromise antitumor effects. We conclude that PBN administration may represent a novel approach for reduction of toxicity without compromise of antitumor effects resulting from administration of therapeutic antibodies in BMT recipients.
Synergistic antitumor effects of celecoxib with 5-fluorouracil depend on IFN-γ