Essential role of nitric oxide and interferon-gamma for tumor immunotherapy with interleukin-10

Several laboratories have reported anti-tumor activity for high levels of interleukin-10 (IL-10) expressed as a transgene or administered as recombinant protein. The authors have reported a positive correlation for nitric oxide production and anti-tumor activity of IL-10 in a murine model of breast cancer. In the current study, they sought evidence of a mechanistic role for nitric oxide in IL-10-mediated tumor growth inhibition. They wanted to determine whether pharmacologic inhibition of nitric oxide synthase (NOS) activity reverses the therapeutic effect of IL-10. Administration of either of two NOS inhibitors, aminoguanidine (AG) or L-lysine,N6-1-iminoethyl-dihydrochloride, appears to abrogate in part the tumor growth inhibition observed when IL-10 is overexpressed as a transgene in two murine mammary tumor cell lines. Nitric oxide levels were assessed at the tumor site by measuring nitrosylated heme levels by electron spin resonance spectroscopy. Nitric oxide hemoglobin levels were lower in tumors from aminoguanidine-treated mice, indicating that effective inhibition of nitric oxide production occurred at the tumor site. Previous investigations showed that the inducible form of NOS protein (iNOS), but not constitutive NOS, was expressed at higher levels in IL-10-expressing tumors. Because iNOS is regulated at the transcriptional level, the authors compared iNOS mRNA levels in IL-10 and control tumors. Northern analysis revealed strong iNOS message expression in all six IL-10-expressing tumors examined, whereas message was faintly detected in parental or 66-neo tumors. The inducible form of NOS is responsive to induction by interferon-gamma (IFN-gamma). The role of IFN-gamma in IL-10-mediated tumor inhibition and iNOS mRNA induction was determined. When tumors were transplanted to IFN-gamma mutant mice, the tumor-inhibitory activity of IL-10 was lost. Furthermore, iNOS mRNA was no longer induced in the absence of host expression of IFN-gamma. These data indicate that nitric oxide contributes to the anti-tumor activity of IL-10 and that expression of iNOS in this context depends on IFN-gamma.