Nitric oxide (NO) is a biologically pluripotent compound that is induced during immune responses and overproduced in murine models of lupus. We have found in murine lupus: 1) pharmacologic blockade of NO production ameliorates lupus-like disease, 2) nitration of tyrosine residues of several proteins including catalase with alternations in catalase function in the kidney, and 3) increased levels of NO in the spleens modulate splenocyte apoptosis. In our retrospective studies of human lupus, serum measures of NO (serum nitrate) correlated with clinical disease activity, although there was overlap between normals and lupus patients which may reflect effects of dietary nitrate intake. To definitively determine NO production in human lupus compared to controls and the effects of NO in disease pathogenesis, we propose a prospective study of lupus patients and murine studies to provide insight into immune factors promoting NO production in disease. These studies are incorporated in the following specific aims: 1. Prospectively follow 70 lupus patients with primarily early disease monitoring disease activity and systemic NO production via serum nitrate/nitrite and 3nitrated proteins quarterly for 3 years. 2. A. Measure apoptosis of PBMCs in lupus patients and controls both in vivo and in vitro, correlating apoptosis with disease activity and NO production. B. Determine NOS2 expression in human lupus kidneys and correlate NOS2 expression with disease class, activity, and chronicity. C. Identify serum factors in lupus sera that stimulate NO production by PBMCs. D. Identify human lupus serum and kidney proteins that are nitrated using mass spectrometry analysis. 3. A. Using genetically deficient mice, we will determine the role of immunoglobulin, C3, complement factor B, and CD40 in macrophage activation and NO production in MRL-lpr mice. B. We will assess if L-NIL, a specific inhibitor of NOS2, is effective in preventing lupus-like disease in MRL-lpr mice. C. We will determine if estrogen modulates NO production in MRL-lpr mice using ovariectomy studies and estrogen receptor knockout mice.
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