Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.

Abstract

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) We have reported (1) that Borna disease virus, rabies virus, and herpes simplex virus induce the increased expression of inducible nitric oxide synthase (iNOS) mRNA in the brains of intrathecally infected mice and rats. We and others (2) also found that the induction of experimental allergic encephalomyelitis resulted in a similar increase in iNOS mRNA expression, suggesting that a similar phenomenon may occur in the brains of patients with multiple sclerosis (MS). We investigated this hypothesis by detecting iNOS mRNA in extracts of brains from patients who died with MS and from extracts of brain tissues from patients who died from nonneurological diseases. We have also colocalized the iNOS expressing cells by using immunocytochemical techniques combined with reverse transcriptase (RT) driven-in situ PCR and demonstrated the presence of iNOS protein by the same double labeling procedures. Finally, as a surrogate marker for nitric oxide (NO) presence, we have immunocytochemically localized nitroty-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.rosine adducts in histological sections of brain from MS patients. MATERIALS AND METHODSSources and Selection of Specimens. The samples used in these studies were collected from patients with an antemortem diagnosis of MS based on clinical presentation and reconfirmed by histopathology after death. All MS samples were found to contain pathognomonic MS plaques; control brains were examined and found to be pathologically unremarkable. In most cases, autopsy samples were of frontal lobes, which provided mixed white and gray matter.In two of the MS cases, special efforts were made to preserve the tissue morphology. Small portions of the brain specimens were frozen in liquid nitrogen immediately after sectioning and then stored at -70°C. This procedure preserved the architecture of the brain significantly better than direct freezing of fresh brain specimens at -80°C, which results in ice crystal formation in the tissue and distorts its architecture.Cell Culture and Cytokine Activation. A549 human pulmonary epithelial cells were purchased from the American Type Culture Collection. Cells were grown in Ham's F-12 medium supplemented with 10% (vol/vol) fetal calf serum, penicillin (50 units/ml), streptomycin (50 ,ug/ml), and 5 ,tM L-glutamine. Cells were seeded into 6-well tissue culture clusters (Falcon) for Northern blot analysis or seeded into 8-well Lab-Tek culture slides (Nunc) for RT-in situ PCR. For those cultures stimulated for the expression of iNOS, interleukin 1(3 (IL-1,B at 100 units/ml), tumor necrosis factor a (TNF-a at 10 ng/ml), and interferon y (IFN-,y at 500 units/ml) were added for 8 h.For use in Northern blot analysis, the cells were scraped from the culture substrate, washed three ti...

Cerebrospinal fluid and serum uric acid levels in patients with multiple sclerosis

“…In the wide variety of immune-mediated mechanisms and molecules that could potentially cause CNS tissue damage in MS, many results point to the pathogenetic significance of nitric oxide (NO) (2)(3)(4). Several studies have shown that NO is produced in excess in the CNS (5)(6)(7) and NO metabolites are increased in body fluids of patients with MS (8)(9)(10). The toxicity of NO is significantly enhanced when it combines with superoxide to rapidly form the peroxynitrite anion (11).…”

Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions

“…Two early reports that examined the expression of NADPH diaphorase staining as an indicator of NO production supported a role for astrocytes as a source of NO in MS lesions, 14,15 however, two subsequent studies using a reverse transcriptase in situ polymerase chain reaction (PCR) hybridization and/or immunocytochemical approach failed to detect iNOS in astrocytes, and instead implicated cells of the monocyte/macrophage lineage. 16,17 More recently, a study of brain biopsies from two acute cases of MS in young adults detected signal for iNOS in both reactive astrocytes and perivascular monocytes/macrophages, whereas no signal was found in more chronic MS cases. 18 These data suggest that the extent of lesion activity may critically affect which cell types express iNOS in the lesion.…”

Nitric Oxide Trapping of the Tyrosyl Radical of Prostaglandin H Synthase-2 Leads to Tyrosine Iminoxyl Radical and Nitrotyrosine Formation

“…Nitrotyrosine has been detected in samples from a wide variety of disease states, including acute lung injury (1, 2), atherosclerosis (3,4), neurodegenerative diseases (5)(6)(7)(8), bacterial and viral infections (9 -11), aging (12), chronic inflammation (13), and exposure to cigarette smoke (14) or carbon monoxide (15). In most of the above cases, the nitrotyrosine detection was presumed to be the result of the reaction of tyrosine residues with peroxynitrite/ peroxynitrous acid.…”