Staphylococcus aureus stimulates inducible nitric oxide synthase in articular cartilage

Abstract

Objective. To determine if Staphylococcus aureus stimulates the L-arginine-nitric oxide (NO) synthase pathway in articular cartilage. Methods. A heat-killed and sonicated (denatured) S aureus preparation was added to cultures of bovine articular cartilage. NO production was measured as accumulated nitrite in the culture medium and by the NO synthase-dependent conversion of 3 H-L-arginine to 3 H-L-citrulline in cartilage homogenates. Inducible NO synthase (iNOS) messenger RNA (mRNA) expression was analyzed by Northern blot. Proteoglycan synthesis was measured by 35 SO 4 incorporation into glycosamino-glycan. Results. Nitrite accumulation and 3 H-L-citrulline formation in cartilage were elevated by denatured S aureus (compared with unstimulated control cartilage) and inhibited by the NO synthase inhibitor N G-monomethyl-L-arginine. Northern blot analysis revealed increased iNOS mRNA expression in bovine chondro-cytes in response to denatured S aureus stimulation. Denatured S aureus suppressed the accumulation of 35 SO 4-labeled macromolecules representing newly synthesized proteoglycans in bovine articular cartilage. The suppressed proteoglycan synthesis was due to the presence of NO. Conclusion. These findings support the hypothesis that a component of S aureus can stimulate iNOS in articular cartilage, and that NO generated from this enzyme down-regulates cartilage matrix proteoglycan synthesis. Staphylococcus aureus is the most common microorganism recovered from patients with nongonococcal arthritis (1). Joint infection often results in a rapid and irreversible destruction of articular cartilage, leading to arthritis. Accelerated cartilage loss has been shown to be regulated by many factors that promote proteolytic degradation of matrix and inhibit matrix synthesis. Wil-liams et al (2,3) showed that a purified factor from S aureus culture medium stimulated neutral matrix metal-loproteinase activity, resulting in the increased release and degradation of proteoglycans from cartilage. Brand et al (4) and Hardy et al (5) also showed that preparations of filtered S aureus culture medium stimulated proteoglycan release in bovine and equine cartilage, respectively (compared with unstimulated controls). When produced in large amounts, nitric oxide (NO) has been implicated in conditions such as arthritis (for review, see ref. 6). Interleukin-1 (IL-1)-induced NO production has been shown to modulate matrix metal-loproteinase activity and expression in articular chon-drocytes (7-10) and to suppress the synthesis of cartilage proteoglycans in vitro and in vivo (11-13). We hypothesized that the L-arginine-NO syn-thase pathway is activated in articular cartilage in response to S aureus, and that the increased NO modulates cartilage matrix metabolism. Here we show that heat-killed and sonicated S aureus stimulated NO synthase in articular cartilage and suppressed the synthesis of pro-teoglycans, which implicates NO as a local mediator in staphylococcal septic arthritis. MATERIALS AND METHODS Materials. Tissue culture reagent...

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