3-nitrotyrosine and Myositis--Inclusion-Body

Sporadic inclusion body myositis is a severely disabling myopathy. The design of effective treatment strategies is hampered by insufficient understanding of the complex disease pathology. Particularly, the nature of interrelationships between inflammatory and degenerative pathomechanisms in sporadic inclusion body myositis has remained elusive. In Alzheimer's dementia, accumulation of β-amyloid has been shown to be associated with upregulation of nitric oxide. Using quantitative polymerase chain reaction, an overexpression of inducible nitric oxide synthase was observed in five out of ten patients with sporadic inclusion body myositis, two of eleven with dermatomyositis, three of eight with polymyositis, two of nine with muscular dystrophy and two of ten non-myopathic controls. Immunohistochemistry confirmed protein expression of inducible nitric oxide synthase and demonstrated intracellular nitration of tyrosine, an indicator for intra-fibre production of nitric oxide, in sporadic inclusion body myositis muscle samples, but much less in dermatomyositis or polymyositis, hardly in dystrophic muscle and not in non-myopathic controls. Using fluorescent double-labelling immunohistochemistry, a significant co-localization was observed in sporadic inclusion body myositis muscle between β-amyloid, thioflavine-S and nitrotyrosine. In primary cultures of human myotubes and in myoblasts, exposure to interleukin-1β in combination with interferon-γ induced a robust upregulation of inducible nitric oxide synthase messenger RNA. Using fluorescent detectors of reactive oxygen species and nitric oxide, dichlorofluorescein and diaminofluorescein, respectively, flow cytometry revealed that interleukin-1β combined with interferon-γ induced intracellular production of nitric oxide, which was associated with necrotic cell death in muscle cells. Intracellular nitration of tyrosine was noted, which partly co-localized with amyloid precursor protein, but not with desmin. Pharmacological inhibition of inducible nitric oxide synthase by 1400W reduced intracellular production of nitric oxide and prevented accumulation of β-amyloid, nitration of tyrosine as well as cell death inflicted by interleukin-1β combined with interferon-γ. Collectively, these data suggest that, in skeletal muscle, inducible nitric oxide synthase is a central component of interactions between interleukin-1β and β-amyloid, two of the most relevant molecules in sporadic inclusion body myositis. The data further

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Cell Death; Cells, Cultured; Dactinomycin; Drug Interactions; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-1beta; Muscle Cells; Myositis, Inclusion Body; Nitric Oxide Synthase Type II; RNA, Messenger; Tyrosine

Oxidative stress has been suggested as one of the pathogenetic mechanisms of inclusion body myositis (IBM). To study the role of antioxidant enzymes in myopathies with rimmed vacuoles, we examined expressions of copper, zinc superoxide dismutase (Cu, Zn-SOD) and manganese superoxide dismutase (Mn-SOD), and the relationship between SODs and other proteins localized in rimmed vacuoles in muscle biopsy specimens from three cases of sporadic IBM and two of distal myopathy with rimmed vacuoles (DMRV) as well as eight control cases of myopathies without rimmed vacuoles. Immunoblot analysis showed distinct protein bands of both SODs in IBM and DMRV using subtype-specific antibodies. Intensities of immunoreactive bands for Mn-SOD in IBM and DMRV were stronger than those in the control cases. Immunohistochemistry disclosed accumulation of both SODs in vacuolated muscle fibers in all cases of IBM and DMRV. Immunoreactivity for Mn-SOD was often colocalized with that of nitrotyrosine, cytochrome oxidase, tau, and lysosome-associated membrane proteins 2 (LAMP-2) in vacuolated fibers. Some of the Cu, Zn-SOD-positive vacuolated fibers were associated with ubiquitin. The two SODs may have different roles for cell protection, and the expression of Mn-SOD is associated with nitric oxide-induced oxidative damage in myopathies with rimmed vacuoles.

Topics: Adult; Aged; Antigens, CD; Electron Transport Complex IV; Female; Humans; Immunoblotting; Immunohistochemistry; Infant; Lysosomal Membrane Proteins; Male; Membrane Glycoproteins; Middle Aged; Mitochondria; Muscle Fibers, Skeletal; Muscle, Skeletal; Myositis, Inclusion Body; Nitric Oxide; Oxidative Stress; Superoxide Dismutase; tau Proteins; Tyrosine; Ubiquitin; Vacuoles

To investigate the possible role of nitric oxide (NO)-induced 'oxidative stress' in the pathogenesis of inclusion-body myositis (IBM), we immunostained muscle biopsies of 12 patients with IBM with isoform-specific antibodies against the neuronal and inducible forms of nitric oxide synthase and with antibodies against nitrotyrosine. Between 70 and 80% of IBM vacuolated muscle fibers contained inclusions strongly immunoreactive with all three antibodies, which by immuno-electronmicroscopy co-localized mainly to cytoplasmic paired-helical filaments, and also to amorphous structures and floccular material. Excess intracellular NO can combine with superoxide to produce highly reactive peroxynitrite which can nitrate tyrosines of proteins. The presence of nitrotyrosine is indicative of NO-induced "oxidative stress'. Our data suggest that this mechanism may play a pathogenic role in IBM.

Topics: Enzyme Induction; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Microscopy, Immunoelectron; Muscle Fibers, Skeletal; Muscular Diseases; Myositis, Inclusion Body; Neurons; Nitric Oxide Synthase; Oxidative Stress; Tyrosine