PhD

dissertationWe employed knockout mice (on C57BL/6 background) to test the role of the inducible nitric oxide synthase (iNOS) in mediating Il-2 toxicity. In contrast to C3H/HeN mice, which developed hypotension and VLS after 10 does of only 180,000 IU IL-2, C57BL/6 mice were far more resistant to the effects of IL-2 requiring increased doses of 800,000 IU IL-2 (b.i.d. 5 days) to induce hypotension and VLS. Serum IFN-gamma levels were significantly more elevated by IL-2 treatment in C3H/HeN mice than in C57BL/6. Urinary excretion of NO metabolites was reduced about 70% in iNOS knockout mice (C57BL/6 iNOS-/-) following IL-2 treatment showing that this NOS isoform accounts for the majority of NO production during IL-2 treatment. A surprising finding was that these mice still developed profound hypotension and VLS. Similar findings were observed following concomitant administration of an iNOS specific inhibitors, L-NIL and 1400W with Il-2. In contrast a general nitric oxide synthase (NOS) inhibitor, N[G]-monomethly-L-arginine (MLA), prevented both hypotension and vascular lead. The superoxide dismutase mimetic, M40403, reversed only IL-2 induced hypotension, but not VLS in knockout mice. Thus, superoxide or peroxynitrite mediated mechanisms are likely responsible for hypotension, while NO-induced changed in vascular permeability results in VLS. The iNOS enzyme is not necessary for pathogenesis of IL-2 induced cardiovascular toxicity. Mice with a knockout endothelial nitric oxide synthase (eNOS), however, proved resistant to IL-2 induced hypotension and vascular lead syndrome. Urinary excretion of NO metabolites in eNOS knockout mice was reduced about 30% following IL-2 treatment compared to controls. These results imply that eNOS isoform plays a major role in the development of IL-2 induced cardiovascular toxicity. There is significant difference in the levels of cytokines expression following IL-2 treatment of C3H/HeN mice of C5BL/6 mice. We also found that LAK cell cytotoxicity is not essential for IL-2 induced hypotension and vascular leak as Beige mice (which lake NK and T-cell cytotoxicity) still show these cardiovascular responses. Beige mice retain the capability to secrete cytokines following Il-2 treatment. In contrast more severly deficient NIH-III knockout mice are partially resistant to IL-2 induced cardiovascular side effects suggesting that there are both cellular and non-cellular components to eNOS acativation