Nitric Oxide-Derived Species in Synovial Fluid from Patients with Juvenile Idiopathic Arthritis

ANA PAOLA N. LOTITO, MARCELO N. MUSCARÁ, MARIA HELENA B. KISS, SIMONE A. TEIXEIRA, GILBERTO S. NOVAES, IEDA MARIA M. LAURINDO, CLOVIS A. A. SILVA, and SUZANA BEATRIZ V. MELLO

ABSTRACT.

Objective. To evaluate superoxide anion (O₂–), nitrite/nitrate (NO₂–/NO₃–), and nitrotyrosine (NT) production and the contribution of myeloperoxidase (MPO) to the production of NT-containing proteins in the synovial fluid (SF) of patients with juvenile idiopathic arthritis (JIA). The affected tissues in inflammatory arthritis produce large amounts of nitric oxide (NO) or peroxynitrite (ONOO–) but there are no reports of NO or ONOO– participation in JIA. We also attempted to correlate our findings with variables of disease activity and articular damage.

Methods. We analyzed 40 patients with JIA, mean age 12.7 years, mean disease duration 7.8 years. O₂– production was measured by cytochrome C reduction after incubation of 10⁶ synovial fluid (SF) cells with or without phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan. SF and serum NO₂–/NO₃– levels were measured by Griess reaction; NT was detected by Western blot. Myeloperoxidase (MPO) activity was estimated spectrophotometrically. Clinical and laboratory variables [erythrocyte sedimentation rate, C reactive protein (CRP), and radiological score] and interleukin 6 (IL-6) levels were evaluated.

Results. NO₂–/NO₃– production was greatly enhanced in the joints of JIA patients (54.6 ± 3.2 µM) when compared with serum (13.9 ± 0.6 µM; p < 0.001). NO₂–/NO₃– levels in SF were positively correlated with the number of infiltrating lymphomononuclear cells. NT-modified proteins detected in the SF showed a high correlation with radiological score, disease duration, CRP, and IL-6.

Conclusion. Our results confirm the increased oxidative stress in children with JIA, suggesting a high in situ production of NO. The positive correlation between the expression of NT-modified proteins and variables of disease activity and damage is additional evidence that nitrogen and oxygen species may be involved in the joint destruction seen in patients with JIA. (J Rheumatol 2004; 31:992-7)

Key Indexing Terms:

JUVENILE IDIOPATHIC ARTHRITIS
NITRIC OXIDE
SUPEROXIDE
NT-MODIFIED PROTEINS
SYNOVIAL FLUID
MYELOPEROXIDASE

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From the Paediatric Division, Rheumatology Division, Department of Internal Medicine, School of Medicine, Department of Pharmacology, Institute of Biomedical Sciences, University of São Paulo; and the Rheumatology Division, Department of Medicine, School of Medicine, Catholic University of São Paulo, Brazil.

Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo FAPESP #01/01663-6 and #95/09699-7.

A.P.N. Lotito, MD, Instructor in Paediatrics; M.N. Muscará, PhD, Assistant Professor of Pharmacology; S.A. Teixeira, Research Associate; G.S. Novaes, MD, PhD, Assistant Professor of Rheumatology; I.M.M. Laurindo, MD, PhD, Assistant Professor of Rheumatology; C.A.A. Silva, MD, PhD, Assistant Professor of Paediatric Rheumatology; M.H. Kiss, MD, PhD, Professor of Paediatrics; S.B.V. Mello, PhD, Assistant Professor of Rheumatology.

Address reprint requests to Dr. S.B.V. Mello, Rheumatology Division, Department of Internal Medicine of School of Medicine, University of São Paulo, Av. Dr. Arnaldo, 455, São Paulo, 0124-6903, Brazil.

Submitted March 24, 2003; revision accepted November 5, 2003.

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