3-nitrotyrosine and Autoimmune-Diseases

The free radical-mediated damage to proteins results in the modification of amino acid residues, cross-linking of side chains and fragmentation. l-Tyrosine and protein bound tyrosine are prone to attack by various mediators and reactive nitrogen intermediates to form 3-nitrotyrosine (3-NT). Activated macrophages produce superoxide (O2(·-)) and NO, which are converted to peroxynitrite ONO2(-). 3-NT formation is also catalyzed by a class of peroxidases utilizing nitrite and hydrogen peroxide as substrates. Evidence supports the formation of 3-NT in vivo in diverse pathologic conditions and 3-NT is thought to be a relatively specific marker of oxidative damage mediated by peroxynitrite. Free/protein-bound tyrosines are attacked by various RNS, including peroxynitrite, to form free/protein-bound 3-NT, which may provide insight into the etiopathogenesis of autoimmune conditions. The formation of nitrotyrosine represents a specific peroxynitrite-mediated protein modification; thus, detection of nitrotyrosine in proteins is considered as a biomarker for endogenous peroxynitrite activity. The peroxynitrite-driven oxidation and nitration of biomolecules may lead to autoimmune diseases such as systemic lupus. The subsequent release of altered proteins may enable them to act as antigen-inducing antibodies against self-proteins. Hence, tyrosine nitrated proteins can act as neoantigens and lead to the generation of autoantibodies against self proteins in various autoimmune disorders.

Topics: Animals; Autoimmune Diseases; Autoimmunity; Biomarkers; Free Radicals; Humans; Nitrogen; Oxidation-Reduction; Proteins; Tyrosine

Glucocorticoid (GC) excess often elicits serious adverse effects on the vascular system, such as hypertension and atherosclerosis, and effective prophylaxis for these complications is limited. We sought to reveal the mechanism underlying GC-induced vascular complications. Responses in forearm blood flow to reactive hyperemia in 20 GC-treated patients were significantly decreased to 43+/-8.9% (mean+/-SEM) from the values obtained before GC therapy (130+/-14%). An administration of vitamin C almost normalized blood flow responses. In human umbilical vein endothelial cells (HUVECs), production of hydrogen peroxide was increased up to 166.5+/-3.3% of control values by 10(-7) mol/L dexamethasone (DEX) treatment (P<0.01). Concomitant with DEX-induced hydrogen peroxide production, intracellular amounts of peroxynitrite significantly increased and those of nitric oxide (NO) decreased, respectively (P<0.01). Immunoblotting analysis using anti-nitrotyrosine antibody showed that peroxynitrite formation was increased in DEX-treated HUVECs. Using inhibitors against metabolic pathways for generation of reactive oxygen species (ROS), we identified that the major production sources of ROS by DEX treatment were mitochondrial electron transport chain, NAD(P)H oxidase, and xanthine oxidase. These findings suggest that GC excess causes overproduction of ROS and thereby perturbs NO availability in the vascular endothelium, leading to vascular complications in patients with GC excess.

Topics: Adolescent; Adult; Aged; Antioxidants; Autoimmune Diseases; Cells, Cultured; Dexamethasone; Electron Transport; Endothelium, Vascular; Enzyme Inhibitors; Female; Forearm; Glucocorticoids; Humans; Hydrogen Peroxide; Male; Middle Aged; Muscle, Skeletal; NADH, NADPH Oxidoreductases; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Reactive Oxygen Species; Regional Blood Flow; Superoxides; Tyrosine; Vasodilation; Xanthine Oxidase

To estimate oxidative stress and antioxidant components during different stages of autoimmune liver diseases and assess their possible implication on disease progression.. We determined several markers of oxidative injury (isoprostane, aldehydes, protein carbonyls, 3-nitrotyrosine, and myeloperoxidase) and antioxidant components (glutathione, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase) in whole blood, serum, and urine in 49 patients with autoimmune cholestatic liver diseases (AC) and 36 patients with autoimmune hepatitis (AIH) and healthy subjects matched for sex and age.. Both AC and AIH patients had increased levels of all lipid and protein oxidative injury products and significantly decreased whole blood glutathione levels compared to controls. AIH patients had significantly higher levels of aldehydes and glutathione peroxidase activity and significantly lower protein carbonyl levels compared to AC patients. Protein carbonyl and isoprostane levels increased and glutathione levels decreased gradually with progression from mild fibrosis to severe fibrosis and cirrhosis in both AC and AIH patients. In addition, both cirrhotic AC and AIH patients had significantly higher protein carbonyls compared to non-cirrhotics.. We provide novel findings in support of a major contribution of oxidant/antioxidant imbalance in the progression of liver injury in AC and AIH.

Topics: Adult; Aged; Aldehydes; Antioxidants; Autoimmune Diseases; Carbon; Catalase; Disease Progression; Female; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Hepatitis, Autoimmune; Humans; Isoprostanes; Liver Diseases; Male; Middle Aged; Oxidative Stress; Peroxidase; Superoxide Dismutase; Tyrosine

Vitiligo is a common pigmentary skin disorder of unknown etiology. Many studies show the defective mitochondrial functionality in vitiligo patients, but the potential role of mitochondrial DNA (mtDNA) in the pathogenesis of vitiligo remains to be investigated. Recent evidences demonstrate that mitochondria possess their own nitric-oxide-synthase and can produce endogenous peroxynitrite (ONOO(-)). This study was undertaken to investigate the role of ONOO(-)-modified-mitochondrial-DNA (ONOO(-)-mtDNA) in vitiligo autoimmunity. Our data revealed that ONOO(-)-induced modifications in mtDNA caused structural alterations. Specificity of immunoglobulin G (IgG) from vitiligo patients (n=26) and controls (n=25) were analysed towards ONOO(-)-mtDNA. Vitligo-IgG samples (Vt-IgG) show preferential binding to ONOO(-)-mtDNA in comparison with native mtDNA (p<0.01). Anti-ONOO(-)-mtDNA-IgG show cross-reactivity with isolated DNA from vitiligo patients. Furthermore, levels of anti-ONOO(-)-mtDNA-IgG, inducible-nitric-oxide-synthase (iNOS), nitric oxide (NO) and nitrotyrosine were higher among vitiligo patients whose disease durations (DD) were ⩾5 years as compared to patients with lower DD (DD<5 years). In conclusion, this is the first study to demonstrate the role of ONOO(-)-modified mtDNA in vitiligo patients. Our data provide an important insight into the immunological mechanisms occur in vitiligo. The ONOO(-)-mtDNA may be useful in elucidating the mechanisms of disease pathogenesis.

Topics: Adolescent; Adult; Autoantibodies; Autoimmune Diseases; Autoimmunity; Child; DNA, Mitochondrial; Female; Humans; Immunoglobulin G; Male; Mitochondria; Nitric Oxide; Nitric Oxide Synthase; Nitrosation; Peroxynitrous Acid; Tyrosine; Vitiligo; Young Adult

Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4(th) day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases ( approximately 3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-gamma) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

Topics: Animals; Autoimmune Diseases; Cells, Cultured; Cytokines; Drug Administration Schedule; Female; Gene Expression Regulation; Kidney; Liver; Mice; Mice, Inbred MRL lpr; Nitric Oxide Synthase Type II; Oxidation-Reduction; Oxidative Stress; Protein Carbonylation; Spleen; Trichloroethylene; Tyrosine

Excessive production of nitric oxide (NO) by inducible NO synthase (iNOS) contributes to the progression of myocardial damage in myocarditis. Some dihydropyridine calcium channel blockers reportedly inhibit NO production and proinflammatory cytokines and the present study sought to clarify if a low dose of benidipine, a novel dihydropyridine calcium channel blocker, would ameliorate experimental autoimmune myocarditis (EAM). Rats with or without myocarditis were administered oral benidipine at a dose of 3 mg. kg(-1). day(-1) for 3 weeks. Low-dose benidipine did not decrease blood pressure significantly compared with the untreated group, but markedly reduced the severity of myocarditis. Myocardial interleukin-1beta (IL-1beta) expression and IL-1beta-positive cells were significantly less in rats with EAM that were treated with low-dose benidipine compared with untreated rats. Also, myocardial iNOS expression and iNOS-positive cells were markedly reduced in in the treated rats compared with the untreated group. Furthermore, myocardial NO production and nitrotyrosine expression were suppressed by the treatment in rats with EAM. The cardioprotection of low-dose benidipine may be caused by suppression of inflammatory cytokines and inhibition of NO production.

Topics: Acute Disease; Animals; Autoimmune Diseases; Blood Pressure; Calcium Channel Blockers; Calcium Channels, L-Type; Cytokines; Dihydropyridines; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Heart Rate; Myocarditis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Rats; Rats, Inbred Lew; Tyrosine

Macrophages infiltrating an inflamed or injured tissue undergo development of coordinated sets of properties that contribute to tissue damage, repair, and remodeling. The purpose of this study was to determine whether macrophages isolated from normal or inflamed retina are programmed to a distinct set of properties and to examine whether the development of experimental autoimmune uveoretinitis (EAU) affects macrophage function.. EAU was induced in Lewis rats, and a retina-derived macrophage-enriched population was generated by density centrifugation during the prepeak, peak, and resolution phases of the disease. Cell surface phenotype was assessed by two- and three-color flow cytometry, and function was determined in vitro by nitric oxide (NO) production, with or without further cytokine stimulation or by immunohistochemistry to determine expression of beta-glucuronidase, nitric oxide synthase (NOS)-2, and nitrotyrosine.. Myeloid-derived cells from normal retina were programmed similar to TGF-beta-stimulated uncommitted bone-marrow-derived macrophages (BMDMs). Contrary to BMDM behavior, retina-isolated macrophages displayed distinct properties and phenotype at different phases of the disease course and remained resistant throughout, to further cytokine challenge in vitro. During peak disease, retina-isolated macrophages had characteristics of IFN-gamma/TNF-alpha primed cells (nitrotyrosine positive and NO producing). Despite equivalent numbers of macrophages during resolution, their function reverted to characteristics of TGF-beta primed cells (beta-glucuronidase positive).. Resident retinal myeloid-derived cells are primed and are resistant to further cytokine stimulation, and, similar to macrophages derived during EAU recovery, behave operationally as though TGF-beta primed. During peak inflammation, infiltrating macrophages adapt to concurrent hierarchical Th1 T-cell response (IFN-gamma/TNF-alpha), generating NO. The results provide evidence of in vivo programming of macrophages within the retina.

Topics: Animals; Autoimmune Diseases; Cytokines; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Glucuronidase; Immunoenzyme Techniques; Immunophenotyping; Macrophage Activation; Macrophages; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Lew; Retina; Retinitis; Tyrosine; Uveitis

Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death. In certain mouse major histocompatibility complex (MHC) backgrounds, myocarditis and inflammatory cardiomyopathy can be triggered by immunization with heart muscle-specific proteins. Similarly, chronic heart disease in humans has been linked to certain HLA alleles, such as HLA-DQ6. However, there is no experimental evidence showing that human MHC class II molecules and peptides derived from human proteins are involved in the pathogenesis of myocarditis and DCM.. We generated double CD4- and CD8-deficient mice transgenic for human CD4 (hCD4) and human HLA-DQ6 to specifically reconstitute the human CD4/DQ6 arm of the immune system in mice. Transgenic hCD4 and HLA-DQ6 expression rendered genetically resistant C57BL/6 mice susceptible to the induction of autoimmune myocarditis induced by immunization with cardiac myosin. Moreover, we identified heart-specific peptides derived from both mouse and human alpha-myosin heavy chains capable of inducing inflammatory heart disease in hCD4 and HLA-DQ6 double transgenic mice but not in hCD4 single transgenic littermates. The autoimmune inflammatory heart disease induced by the human heart muscle-specific peptide in hCD4 and HLA-DQ6 double transgenic mice shared functional and phenotypic features with the disease occurring in disease-susceptible nontransgenic mice.. Our data provide the first genetic and functional evidence that human MHC class II molecules and a human alpha-myosin heavy chain-derived peptide can cause inflammatory heart disease and suggest that human inflammatory cardiomyopathy can be caused by organ-specific autoimmunity. The humanized mice generated in this study will be an ideal animal model to further elucidate the pathogenesis of inflammatory heart disease and facilitate the development of rational treatment strategies.

Topics: Animals; Autoimmune Diseases; CD4 Antigens; Disease Susceptibility; HLA-DQ Antigens; Humans; Immunization; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocarditis; Myocardium; Myosin Heavy Chains; Myosins; Peptide Fragments; Phenotype; Tyrosine

We studied the effect of nitric oxide (NO) on experimental autoimmune myocarditis (EAC) in rats. We examined the role of inducible nitric oxide synthase (iNOS), an enzyme that produces NO, on hearts affected with EAC, by testing the effects of aminoguanidine (AG), a selective iNOS inhibitor, on the course of EAC. Western blotting detected iNOS in the affected cardiac tissues, but not in CFA immunized cases. Immunohistochemically, the majority of ED1+ macrophages in the EAC lesions were positive for iNOS and nitrotyrosine. A high dose of AG (200 mg/kg/day) significantly reduced the incidence of EAC (p < 0.05) and ameliorated the histological score for the cardiac inflammation (p < 0.01) compared with the low dose AG (100 mg/kg/day) and vehicle treated groups. The immunoblot analysis showed that a high dose of AG effectively suppressed iNOS in hearts affected with EAC. An iNOS band was barely detected in the high dose AG (200 mg/kg) treated group, while it was distinctively visualized in the vehicle and low dose AG (100 mg/kg) treated groups. These results suggest that iNOS is upregulated in EAC lesions and increased NO production plays an important role in the development of EAC. In addition, selective iNOS inhibitors may have a therapeutic role in treating certain autoimmune diseases including EAC.

Topics: Animals; Autoimmune Diseases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidines; Myocarditis; Myocardium; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Inbred Lew; Tyrosine