Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis

Abstract

Uric acid, the naturally occurring product of purine metabolism, is a strong peroxynitrite scavenger, as demonstrated by the capacity to bind peroxynitrite but not nitric oxide (NO) produced by lipopolysaccharide-stimulated cells of a mouse monocyte line. In this study, we used uric acid to treat experimental allergic encephalomyelitis (EAE) in the PLSJL strain of mice, which develop a chronic form of the disease with remissions and exacerbations. Uric acid administration was found to have strong therapeutic effects in a dose-dependent fashion. A regimen of four daily doses of 500 mg/kg uric acid was required to promote long-term survival regardless of whether treatment was initiated before or after the clinical symptoms of EAE had appeared. The requirement for multiple doses is likely to be caused by the rapid clearance of uric acid in mice which, unlike humans, metabolize uric acid a step further to allantoin. Uric acid treatment also was found to diminish clinical signs of a disease resembling EAE in interferon-gamma receptor knockout mice. A possible association between multiple sclerosis (MS), the disease on which EAE is modeled, and uric acid is supported by the finding that patients with MS have significantly lower levels of serum uric acid than controls. In addition, statistical evaluation of more than 20 million patient records for the incidence of MS and gout (hyperuricemic) revealed that the two diseases are almost mutually exclusive, raising the possibility that hyperuricemia may protect against MS.

Figure 1

In vitro effects of uric acid on NO production and peroxynitrite detection in cultures of RAW cells stimulated by LPS. RAW 264.7 cells were activated by LPS in the presence of various concentrations of uric acid (▪) or l-NMMA (○), and peroxynitrite (A) and NO formation (B) were assessed as described in Materials and Methods. To assess peroxynitrite, cells were washed twice with PBS after 24-hr incubation with LPS cells and 1 ml of PBS containing 5 μM DHR123 and the indicated concentrations of uric acid or l-NMMA were added. After an additional 1-hr incubation at 37°C, rhodamine fluorescence was measured as an indicator of peroxynitrite production. For measurement of NO formation, cells were stimulated for 24 hr in the presence of uric acid, and nitrite was measured in the culture medium. Data are presented as percentage of peroxynitrite or nitrite detected in replicate cultures without added uric acid or l-NMMA. (C) The peroxynitrite donor SIN-1 was titrated into RAW cells cultured in the absence (□) or presence of 1 mM uric acid (▪). Twenty-four hours later the percentage cell death was calculated by propidium iodide staining as described in Materials and Methods.

Figure 2

Effect of uric acid treatment on clinical signs of pre-existing EAE in PLSJL mice. Groups of 10 female PLSJL mice were immunized with MBP in CFA. Twelve days later, mice with clinical signs either were treated with uric acid (◊) twice daily with 10 mg per dose for 7 days (arrows) or left untreated (▴). Clinical score was assessed twice daily as described in Materials and Methods and is presented as an average.

Figure 3

Effect of uric acid treatment on survival of PLSJL mice with pre-existing EAE. Groups of 10 female PLSJL mice were immunized with MBP in CFA. Twelve days later, mice with clinical signs either were treated with uric acid (◊) twice daily at 10 mg per dose for 7 days (arrows) or left untreated (▴).

Figure 4

Effect of continued administration of uric acid on the onset of clinical signs of acute EAE in PLSJL mice. Groups of 10 female PLSJL mice were immunized with MBP in CFA as described in Materials and Methods and treated twice daily starting 5 days later with saline (▴) or 10 mg of uric acid (◊). Clinical score was assessed twice daily and is presented as an average.

Figure 5

Serum uric acid levels after single i.p. administration of uric acid. Groups of 5–10 BALB/c (▪) and SWX/J14 (⧫) mice were given a single i.p. dose of 10 mg of uric acid and bled at the indicated intervals. Uric acid levels in sera obtained before and after treatment are presented as a mean for each time point.

Figure 6

Comparison of the protective effect of one, two, and four daily doses of uric acid against EAE. Groups of 10 PLSJL mice were immunized with MBP as described in Materials and Methods. Starting 5 days later and continuing for 30 days, groups of 10 mice were treated with 10 mg of uric acid i.p. once (○), twice (◊), or four (□) times daily or with saline (▴). One group of 7/10 mice surviving without treatment to 24 days after immunization received four doses per day from day 24 (arrow) through day 35 (▪); the three mice that died before the start of treatment are excluded from the graph. Results are presented as an average of the clinical score.

Figure 7

Survival of PLSJL mice immunized with MBP in CFA and treated with 10 mg of uric acid once, twice, or four times daily starting at 5 or 24 days after immunization. Shown are the survival curves of the mice described in the legend to Fig. 6. Starting 5 days after immunization with MBP and continuing for 30 days, the groups of 10 mice were treated with 10 mg of uric acid i.p. once (○), twice (◊), or four (□) times daily or with saline (▴). A group of seven mice surviving without treatment to 24 days after immunization received four doses per day starting at that point (▪) through day 35 after immunization.

Figure 8

Effect of uric acid treatment on the clinical signs of EAE in IFN-γR KO mice. Groups of 10 female and eight male IFN-γR KO mice were immunized with MBP in CFA. Nineteen days later, half of the mice in each group began four times daily i.p. treatment with either uric acid (□), 10 mg per dose, or saline (▴). Clinical score was assessed twice daily as described in Materials and Methods, and is presented as an average in A. The percent survival of the treated and control mice is shown in B.

Figure 9

Distribution of serum uric acid levels in MS and non-MS patients. Sera were collected and uric acid levels were determined as described in Materials and Methods. For each group of 46 subjects, the majority of the values obtained fell in the range of the boxes plus bars, with outlying values denoted by ∗. The central 50% of the values fell within the boxes that also contain the median line for the group. P < 0.001 by the Wilcoxon rank sum test.