Mechanism of vasculitis and aneurysms in Kawasaki disease: role of nitric oxide Oladapo Adewuya, * Yasuyuki Irie, Ka Bian, Edore Onigu-Otite, and Ferid Murad Department of Integrative Biology and Pharmacology, Houston Medical School, University of Texas, 6431 Fannin, Houston, TX 77030, USA Received 12 June 2002 Abstract NO in vivo has both beneficial and nonbeneficial effects depending on site and concentration. Peroxynitrite, resulting from the reaction of NO with superoxide radical, causes cellular damage. Nitrotyrosine, end product of NOÕs toxic effects on cellular proteins, is a stable compound that can be used to detect evidence of harmful quantities of NO. We sought to detect nitrotyrosine in coronary arterioles of DBA/2 mice injected intraperitoneally with Lactobacillus casei cell wall. The inflammatory response induced occurred in perivascular fashion and involved mainly macrophages. It was variable according to time points, being severe on days 10 and 14 and mild to moderate on days 3 and 7. Few basal inflammatory cells appeared in controls injected with phosphate-buffered saline. Western immunoblots of homogenized hearts on days 10 and 14 demonstrated specific nitrated proteins. Immunohistochemistry of frozen sections of diseased hearts showed positive immunoreactivity for nitrotyrosine in coronary arterioles at the same time points. These findings were absent in the controls. We also determined the expression of inducible nitric oxide synthase (iNOS) in controls on days 10 and 14. iNOS colocalized with nitrotyrosine in perivascular macrophages and coronary arterioles of treated mice. Additionally, aneurysms were found on day 10 and intracardiac hemorrhage with consequent death on day 14. These observations supply evidence that NO through its reactive product, peroxynitrite, and its antigen/tissue marker, nitrotyrosine, is directly involved in coronary arteritis and aneurysm development in mice models of Kawasaki disease (KD). This article shows that macrophages are central to this and bolsters the likelihood of L. casei being the cause of KD. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Nitrotyrosine; iNOS; Macrophages; Lactobacillus casei; Kawasaki disease; Nitric oxide NO is synthesized from L -arginine by a reaction catalyzed by nitric oxide synthase (NOS). 1 Three iso- forms of NOS have been identified. This study deals with NOS-2, also called inducible nitric oxide synthase (iNOS) or macrophage NOS (macNOS). INOS is known to produce much higher levels of NO [1] and is not expressed in inactivated cells. NO is a signaling molecule with both favorable and unfavorable effects in the human system. It has been shown to be involved in a number of inflammatory conditions such as multiple sclerosis [2], myocarditis, sepsis [3], and human cerebral infarct [4]. Its toxicity is linked to its reactive product, peroxynitrite (ONOO  ), which is formed from the reaction of NO with the su- peroxide radical (O  2 ). Peroxynitrite causes cellular damage in vitro [5]. The reaction between nitrating species and tyrosine produces a stable end product, ni- trotyrosine, which can be reliably used as a diagnostic marker to detect the presence of NO in vivo. The mouse model used was reproduced based on an established model of coronary vasculitis where immune deficient mice were studied after being injected with LCW. The coronary artery lesions that resulted indi- cated a macrophage-mediated injury [6]. In the acute and subacute phases of KD, coronary arteritis occurs and is accompanied by aneurysms [7]. Recently, mac- rophages were shown to be present primarily in the adventitial layer of coronary aneurysms in humans [8]. In our model, we injected mice intraperitonally with the sonicated cell wall of group B Lactobacillus casei, Nitric Oxide 8 (2003) 15–25 www.elsevier.com/locate/yniox NITRIC OXIDE Biology and Chemistry * Corresponding author. Fax: 409-945-9853. E-mail address: Oaadewuy@utmb.edu (O. Adewuya). 1 Abbreviations used: NOS, nitric oxide synthase; LCW, Lactoba- cillus casei cell wall; KD, Kawasaki disease; PBS, phosphate-buffered saline; HRP, horseradish peroxidase. 1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1089-8603(02)00125-8