RESUMO
Mothers giving birth to children with manifestations of neonatal lupus (NL) represent a unique population at risk for the development of clinically evident pathologic autoimmunity since many are asymptomatic and only become aware of anti-SSA/Ro positivity (anti-Ro+) based on heart block in their fetus. Accordingly, we hypothesized that the microbiome in saliva is associated with the development of autoreactivity and in some cases the progression in health status from benign to overt clinical disease including Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The study comprised a clinical spectrum of anti-Ro+ mothers, all of whom gave birth to a child with NL: 9 were asymptomatic or had an undifferentiated autoimmune disease (Asym/UAS) and 16 fulfilled criteria for SS and/or SLE. Microbial diversity was reduced across all levels from kingdom to species for the anti-Ro+ mothers vs healthy controls; however, there were no significant differences between Asym/UAS and SS/SLE mothers. Relative abundance of Proteobacteria and more specifically class Betaproteobacteria decreased with clinical severity (healthy controls <â¯Asym/UASâ¯<â¯SS/SLE). These ordered differences were maintained through the taxonomic hierarchy to three genera (Lautropia, Comamonas, and Neisseria) and species within these genera (L. mirabilis, N. flavescens and N. oralis). Biometric analysis comparing von Willebrand Factor domains present in human Ro60 with L. mirabilis proteins support the hypothesis of molecular mimicry. These data position the microbiome in the development of anti-Ro reactivity and subsequent clinical spectrum of disease.
Assuntos
Anticorpos Antinucleares/imunologia , Disbiose , Lúpus Eritematoso Sistêmico/congênito , Efeitos Tardios da Exposição Pré-Natal , Glândulas Salivares/microbiologia , Adulto , Sequência de Aminoácidos , Autoanticorpos/imunologia , Autoimunidade , Biodiversidade , Feminino , Antígenos HLA/imunologia , Humanos , Recém-Nascido , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/terapia , Masculino , Microbiota , Peptídeos/química , Peptídeos/imunologia , Gravidez , Adulto JovemRESUMO
One of the strongest associations with autoantibodies directed to components of the SSA/Ro-SSB/La ribonucleoprotein complex is the development of congenital heart block (CHB) in an offspring, an alarming prospect facing 2% of primigravid mothers with these reactivities. This risk is 10-fold higher in women who have had a previously affected child with CHB. Anti-Ro/La antibodies are necessary but insufficient to cause disease. In vitro and in vivo experiments suggest that the pathogenesis involves exaggerated apoptosis, macrophage/myfibroblast crosstalk, TGFbeta expression and extensive fibrosis in the conducting system and in some cases surrounding myocardium. A disturbing observation is the rapidity of disease progression, with advanced heart block and life-threatening cardiomyopathy observed <2 weeks from normal sinus rhythm. Once 3rd degree (complete) block is identified, reversal has never been achieved, despite dexamethasone. Current strategies include the evaluation of an early echocardiographic marker of injury, such as a prolonged PR interval and the use of IVIG as a preventative measure for pregnancies of mothers with previously affected children.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Coração Fetal/anormalidades , Bloqueio Cardíaco/congênito , Bloqueio Cardíaco/imunologia , Anticorpos Antinucleares , Doenças Autoimunes/congênito , Feminino , Idade Gestacional , Bloqueio Cardíaco/prevenção & controle , Humanos , Masculino , Gravidez , Fatores de Risco , Fatores de Tempo , Ultrassonografia Pré-NatalRESUMO
OBJECTIVE: To evaluate autoimmune disease progression in asymptomatic and pauci-symptomatic mothers of children with neonatal lupus (NL). METHODS: Clinical information on mothers enrolled in the Research Registry for NL (RRNL) was obtained from medical records. Genotyping was performed for -308A/G tumour necrosis factor (TNF)alpha, 869T/C transforming growth factor (TGF)beta and -889C/T interleukin (IL)1alpha. RESULTS: Of the 321 mothers enrolled, 229 had at least 6 months of follow-up. Of the 51 mothers who were asymptomatic at the NL child's birth, 26 progressed: 12 developed pauci-undifferentiated autoimmune syndrome (pauci-UAS), 2 poly-UAS, 7 SS, 4 SLE and 1 SLE/SS. The median time to develop any symptom was 3.15 years. Of the 37 mothers classified as pauci-UAS at the NL child's birth, 16 progressed: 5 developed poly-UAS, 6 Sjögren syndrome (SS), 4 systemic lupus erythematosus (SLE) and 1 SLE/SS. Of the pauci-UAS mothers enrolled within 1 year, the median time to progression was 6.7 years. Four mothers developed lupus nephritis (two asymptomatic, two pauci-UAS). The probability of an asymptomatic mother developing SLE by 10 years was 18.6%, and developing probable/definite SS was 27.9%. NL manifestations did not predict disease progression in an asymptomatic mother. Mothers with anti-Sjögren syndrome A antigen (SSA/)Ro and anti-Sjögren syndrome B antigen (SSB)/La were nearly twice as likely to develop an autoimmune disease as mothers with anti-SSA/Ro only. Only TGFbetaT/T was significantly higher in SLE mothers compared to asymptomatic mothers (p = 0.03). CONCLUSIONS: Continued follow-up of asymptomatic NL mothers is warranted since nearly half progress, albeit few develop SLE. While the anti-SSB/La antibodies may be a risk factor for progression, further work is needed to determine reliable biomarkers in otherwise healthy women with anti-SSA/Ro antibodies identified solely because of an NL child.
Assuntos
Lúpus Eritematoso Sistêmico/congênito , Mães , Anticorpos Antinucleares/imunologia , Autoanticorpos/sangue , Progressão da Doença , Feminino , Seguimentos , Genótipo , Humanos , Recém-Nascido , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Polimorfismo Genético , Sistema de Registros , Fator de Necrose Tumoral alfa/genéticaRESUMO
Nitric oxide provokes vasodilation and inhibits platelet aggregation. We examined the effect of nitric oxide on superoxide anion production by three sources: activated intact neutrophils, xanthine oxidase/hypoxanthine, and the NADPH oxidase. Nitric oxide significantly inhibited the generation of superoxide anion by neutrophils exposed to either FMLP (10(-7)M) or PMA (150 ng/ml) (IC50 = 30 microM). To determine whether the effect of nitric oxide on the respiratory burst was due to simple scavenging of O2+, kinetic studies that compared effects on neutrophils and the cell-free xanthine oxidase system were performed. Nitric oxide inhibited O2+ produced by xanthine oxidase only when added simultaneously with substrate, consistent with the short half-life of NO in oxygenated solution. In contrast, the addition of nitric oxide to neutrophils 20 min before FMLP resulted in the inhibition of O2+ production, which suggests formation of a stable intermediate. The effect of nitric oxide on the cell-free NADPH oxidase superoxide-generating system was also examined: The addition of NO before arachidonate activation (t = -6 min) significantly inhibited superoxide anion production. Nitric oxide did not inhibit O2+ when added at NADPH initiation (t = 0). Treatment of the membrane but not cytosolic component of the oxidase was sufficient to inhibit O2+ generation. The data suggest that nitric oxide inhibits neutrophil O2+ production via direct effects on membrane components of the NADPH oxidase. This action must occur before the assembly of the activated complex.
Assuntos
NADH NADPH Oxirredutases/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/farmacologia , Superóxidos/metabolismo , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , NADPH Oxidases , Neutrófilos/metabolismo , Xantina Oxidase/farmacologiaRESUMO
Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.
Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Fibronectinas/farmacologia , Óxido Nítrico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Artrite/etiologia , Cartilagem/citologia , Bovinos , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Citocinas/farmacologia , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal , Proteínas de Ligação ao GTP/metabolismo , Homeostase/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/biossíntese , Receptores de Fibronectina/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP-treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model.
Assuntos
Pneumopatias/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Pneumopatias/induzido quimicamente , Pneumopatias/patologia , Macaca mulatta , Oligopeptídeos , Peptídeo Hidrolases/metabolismo , Peptidil Dipeptidase A/sangue , Inibidores de Proteases/metabolismo , Alvéolos Pulmonares/enzimologia , SRS-A/metabolismo , Acetato de Tetradecanoilforbol , Irrigação Terapêutica , alfa 1-AntitripsinaRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms that are independent of their effects on prostaglandin biosynthesis. We examined the effects of sodium salicylate and piroxicam on GTP/GDP exchange by a regulatory G protein (G alpha i). Plasma membrane and cytosol of human neutrophils were prepared by nitrogen cavitation and discontinuous sucrose density centrifugation. Salicylate (3 mM) and piroxicam (50 microM) reduced [35S]GTP gamma S binding to purified plasma membranes [65 +/- 3.7 and 75 +/- 5.3% (P < 0.003) of control, respectively]. Membrane-associated G alpha/beta gamma was solubilized by treatment of plasma membranes with sodium cholate. NSAIDs did not inhibit binding of GTP to solubilized G alpha/beta gamma derived from detergent-treated plasma membranes. Lipid reconstitution was achieved by detergent dialysis followed by the addition of bilayer liposomes (phosphatidylcholine). Salicylate and piroxicam inhibited GTP gamma S binding to G alpha/beta gamma derived from solubilized plasma membranes reconstituted in phosphatidylcholine vesicles (bilayer structures) but had no effect when phosphatidylethanolamine (hexagonal phase II structure) was used for reconstitution. Salicylate and piroxicam had no effect on GTP binding to cytosolic fractions in which soluble G alpha i exists as a free subunit, suggesting that the effect required either assembly of G alpha i/beta gamma heterotrimer or the presence of a lipid bilayer. Although the addition of purified bovine beta gamma subunits to dialyzed cytosol increased both the total GIP binding capacity and the pertussis toxin-dependent ADP-ribosylation of G alpha i, consistent with assembly of a G protein heterotrimer, NSAIDs had no effect on GTP binding. In contrast, NSAIDs inhibited GTP binding to heterotrimeric G alpha cytosol/beta gamma bovine when the complex was inserted into bilayer liposomes. The data indicate that salicylate and piroxicam disrupt neutrophil function via their capacity to interfere with GTP/GDP exchange at an alpha subunit of a regulatory G protein, an effect which requires assembly of the active heterotrimer G alpha i/beta gamma in a phospholipid bilayer.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Ligação ao GTP/química , Bicamadas Lipídicas/química , Neutrófilos/efeitos dos fármacos , Fosfolipídeos/química , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Neutrófilos/química , Neutrófilos/metabolismo , Piroxicam/farmacologia , Salicilatos/farmacologia , Ácido Salicílico , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Radioisótopos de EnxofreRESUMO
It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.
Assuntos
Leucócitos Mononucleares/enzimologia , Neutrófilos/enzimologia , Óxido Nítrico Sintase/sangue , Adenosina Difosfato Ribose/sangue , Animais , Sequência de Bases , Linhagem Celular , Separação Celular/métodos , Primers do DNA/genética , DNA Complementar/sangue , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/sangue , Humanos , Técnicas In Vitro , Inflamação/enzimologia , Leucócitos Mononucleares/metabolismo , Camundongos , Dados de Sequência Molecular , Neutrófilos/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/sangue , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Nitric oxide (NO) is synthesized via the oxidation of arginine by a family of nitric oxide synthases (NOS), which are either constitutive (ie. endothelial (ec)NOS and neuronal (nc)NOS) or inducible (iNOS). The production of nitric oxide plays a vital role in the regulation of physiological processes, host defence, inflammation and immunity. Pro-inflammatory effects include vasodilation, oedema, cytotoxicity and the mediation of cytokine-dependent processes that can lead to tissue destruction. Nitric oxide-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis and osteoarthritis. Conversely, the production of NO by endothelial cell NOS may serve a protective, or anti-inflammatory, function by preventing the adhesion and release of oxidants by activated neutrophils in the microvasculature. In this chapter we describe the multifaceted role of nitric oxide in inflammation and address the potential therapeutic implications of NOS inhibition.
Assuntos
Inflamação/metabolismo , Óxido Nítrico/biossíntese , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Humanos , Inflamação/patologia , Articulações/metabolismo , Articulações/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Óxido Nítrico Sintase/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologiaRESUMO
The association of isolated congenital heart block (CHB) with maternal autoantibodies to SSA/Ro and SSB/La ribonucleoproteins is approaching the predictable, even in mothers who are completely asymptomatic. Indeed, this model of passively acquired autoimmunity offers an exceptional opportunity to examine the effector arm of immunity and define the pathogenicity of an autoantibody in mediating tissue injury. The study of CHB exemplifies not only translational research, which inherently draws upon clinical observations and explores them in the laboratory, but "integrational" research which attempts to fit critical clinical and basic observations together, even those seemingly at odds. The spectrum of conduction abnormalities includes second and third-degree block, but injury can extend to the myocardium and endocardium, in rare cases without AV nodal dysfunction. The rarity of disease continues to drive the search for factors (fetal and environmental) that might amplify the effects of the maternal autoantibodies. The identification of exaggerated apoptosis, macrophage/myfibroblast crosstalk, TGF beta expression, and extensive fibrosis in the conducting system and in some cases surrounding myocardium in fetuses dying with CHB, provide in vivo support for several parallel lines of in vitro investigation. Specifically, the consideration of exaggerated apoptosis as the initial link between maternal antibody and tissue injury led to the observation that cardiocytes are capable of phagocytosing autologous apoptotic cardiocytes and anti-Ro/La antibodies inhibit this function. Recognizing that this perturbation of physiologic efferocytosis might divert uptake to professional Fc gamma R-bearing phagocytes fits well with experiments demonstrating macrophage secretion of pro-inflammatory and fibrosing cytokines when coincubated with apoptotic cardiocytes bound by Ro/La antibodies. While CHB is rare, its study should set precedent for defining the role of autoantibodies in driving end organ disease.
Assuntos
Anticorpos Antinucleares/fisiologia , Apoptose/imunologia , Bloqueio Cardíaco/congênito , Bloqueio Cardíaco/imunologia , Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Autoanticorpos/fisiologia , Feminino , Bloqueio Cardíaco/patologia , Humanos , Troca Materno-Fetal/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , GravidezRESUMO
Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.
Assuntos
Apoptose/imunologia , Autoantígenos/metabolismo , Bloqueio Cardíaco/congênito , Epitopos Imunodominantes/metabolismo , Ribonucleoproteínas/metabolismo , Autoantígenos/imunologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Epitopos/métodos , Feminino , Bloqueio Cardíaco/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Imunoglobulina G/imunologia , Troca Materno-Fetal/imunologia , Gravidez , Ribonucleoproteínas/imunologia , Síndrome de Sjogren/imunologia , Antígeno SS-BRESUMO
In flares of systemic lupus erythematosus (SLE), endothelial cells activated by immune stimuli are potential participants in the inflammatory processes, which contribute to injury. Elevated levels of circulating endothelial cells (CEC) may be a proxy for vascular injury, as demonstrated in patients with sickle cell anemia during acute crises. In active SLE, CEC levels in peripheral blood are elevated (vs healthy controls and correlate with plasma C3a). CEC may reflect widespread unrecognized, ongoing injury despite the absence of clinical stigmata of vasculitis in patients with SLE.
Assuntos
Endotélio Vascular/imunologia , Molécula 1 de Adesão Intercelular/análise , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Óxido Nítrico/metabolismo , Biomarcadores/análise , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Masculino , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Regulação para Cima , Doenças Vasculares/imunologiaRESUMO
S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was greater than 60 min. The addition of resting PMN did not affect the T1/2 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10(-7) M) reduced measurable SNO-GSH (15 microM) at 5 min (48 +/- 5.0% control, P less than 0.05). Incubation (5 min, 37 degrees C) of PMN with 10 microM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 +/- 3 vs 78 +/- 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.
Assuntos
Glutationa/análogos & derivados , Neutrófilos/metabolismo , Compostos Nitrosos/síntese química , Cisteína/química , Glutationa/síntese química , Glutationa/química , Glutationa/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Indóis/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Compostos Nitrosos/metabolismo , Oxindóis , Piroxicam/farmacologia , S-Nitrosoglutationa , Salicilatos/farmacologia , Ácido SalicílicoRESUMO
OBJECTIVE: Nitric oxide (NO) is induced by exposure of endothelial cells (EC) to acetylcholine, where it acts in a paracrine manner to relax smooth muscle and as a defensive molecule to inhibit the adhesion of leukocytes to EC. The mechanism(s) of the antiadhesive properties of constitutive NO are poorly understood. In these studies, we found that NO induced by acetylcholine exerts autocrine effects, which interfere with normal adhesion mechanisms. METHODS: The function of the adhesion molecule intercellular adhesion molecule 1 (CD54) of EC was measured using latex beads coated with antibody to CD54 as a model for CD54 ligation by the leukocyte beta2 integrin. Recruitment of filamentous actin (F-actin) and of the signaling molecule vasodilator-stimulated phosphoprotein (VASP) was measured by immunofluorescence microscopy. RESULTS: Exposure of EC to anti-CD54 beads induced the subplasmalemmal assembly of F-actin and VASP. Acetylcholine blocked the anti-CD54 bead-induced translocation of F-actin and VASP; this effect was reversed by inhibition of NO production. The NO action did not interfere with binding, but completely inhibited the assembly of the focal activation complex, which we believe is necessary for firm heterotypic adhesion between leukocyte and EC. Further studies indicated that the NO effect was due to its capacity to raise cGMP. Platelet endothelial cell adhesion molecule 1 (CD31, also implicated in leukocyte adhesion) did not mimic CD54 responses. CONCLUSION: These results indicate that the ligation of endothelial cell CD54 induces the assembly of subplasmalemmal F-actin and the recruitment of VASP. NO derived from constitutive nitric oxide synthase acts to disrupt these CD54-elicited endothelial cell responses. This action may protect vascular endothelium from leukocyte-mediated injury.
Assuntos
Acetilcolina/farmacologia , GMP Cíclico/farmacologia , GMP Cíclico/fisiologia , Endotélio Vascular/citologia , Adesões Focais/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/fisiologia , Óxido Nítrico/fisiologia , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Endotélio Vascular/ultraestrutura , Humanos , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Veias UmbilicaisRESUMO
The metabolites of arachidonic acid known as the leukotrienes are a class of lipid mediators which have potent and diverse biological effects in pulmonary tissue. Leukotrienes C, D, and E (LTC4, LTD4, and LTE4) are known to be principal mediators of immunoglobulin E (IgE)-mediated hypersensitivity reactions in lung tissue. It is therefore important to develop reliable and quantitative isolation techniques for estimating levels of these mediators in tissue. In this study, LTC4, LTD4, and LTE4 were separated from other arachidonate metabolites by organic extraction procedures. 5-Hydroxyeicosatetraeonic acid and leukotriene B4 extract efficiently into the organic layer of aqueous:ether or aqueous:chloroform extractions, whereas arachidonate metabolites containing conjugated peptides (e.g., LTC4, LTD4, and LTE4) failed to extract into these organic solvents. An extraction step was therefore developed that affords quantitative extraction of LTC4, LTD4, and LTE4 into the organic phase of an isopropanol:ether:H2O mixture. This step is the key for a two-step extraction method that isolates histamine, LTC4, LTD4, and LTE4 with a recovery of 100, 85, 75, and 57%, respectively. One advantage of this separation procedure for obtaining these mediators by organic extraction is an ability to expediently process many samples. Furthermore, the leukotriene content of extracted samples can be analyzed using the guinea pig ileum bioassay without interference from vasoamines or platelet-activating factor. These later substances are eliminated from leukotriene-enriched fractions by this extraction process. When histamine and LTC4 were added to supernatant fluids recovered from isolated lung tissue, they were quantitatively recovered using this extraction method.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Líquidos Corporais/análise , Hipersensibilidade/metabolismo , Pulmão/metabolismo , SRS-A/análogos & derivados , SRS-A/metabolismo , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cobaias , Histamina/metabolismo , Imunoglobulina E/fisiologia , Leucotrieno E4 , Metabolismo dos LipídeosRESUMO
OBJECTIVE: In the current studies we have examined the effects of nitric oxide, and its redox derivatives peroxynitrite and S-nitrosothiol, S-nitrosocysteine, on nuclear factor kappaB (NF-kappaB) activation in cytokine-stimulated bovine chondrocytes. METHODS: The kinetics of NF-kappaB activation (p65 nuclear translocation) were assessed by immunofluorescence and immunoblot assays. RESULTS: We observed that the two nitric oxide redox species, peroxynitrite and S-nitrosocysteine, exert opposing effects on NF-kappaB activation. However, in lipopolysaccharide (LPS)/cytokine-stimulated chondrocytes (LPS, IL-1beta and TNF-alpha (LIT)) in the presence or absence of the NOS inhibitor L-NG-monomethyl arginine citrate (L-NMMA), the results indicate that nitric oxide causes persistent activation of NF-kappaB, most likely via generation of the free radical derivative peroxynitrite. CONCLUSION: The studies indicate that while nitric oxide is not required for immediate NF-kappaB activation in cytokine-stimulated chondrocytes, its effect is to sustain nuclear translocation of p65 and thereby provide a persistent "on signal" to NF-kappaB dependent gene transcription. Persistent activation of NF-kappaB may represent a mechanism by which nitric oxide sustains catabolic processes and promotes cartilage degeneration in osteoarthritis.
Assuntos
Condrócitos/efeitos dos fármacos , Cisteína/análogos & derivados , Sequestradores de Radicais Livres/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico/farmacologia , Animais , Bovinos , Células Cultivadas , Condrócitos/metabolismo , Cisteína/farmacologia , Citocinas/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Inibidores Enzimáticos/farmacologia , Imunofluorescência/métodos , Immunoblotting/métodos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase Tipo II , Compostos Nitrosos/farmacologia , Ácido Peroxinitroso/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , ômega-N-Metilarginina/farmacologiaRESUMO
Human neutrophils biosynthesize the chemoattractant leukotriene B4 (LTB4) and metabolize LTB4 to omega oxidative products 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). In this study, we prepared the C-1 methyl ester and N-methyl amide of LTB4 and then examined neutrophil chemotaxis and metabolism of these derivatives of LTB4. The results show that chemical modification of LTB4 at carbon atom 1 dramatically affects metabolism of the lipid molecule. The free acid form of LTB4 was taken up and metabolized by human neutrophils, while the methyl ester and N-methyl amide derivatives were poor substrates for omega oxidation. Although human neutrophils were poorly attracted to the methyl ester of LTB4, the amide derivative was a complete agonist of the neutrophil chemotactic response and displayed an ED50 for chemotaxis identical to that of LTB4. Therefore, we concluded that omega oxidation is not a requirement for the neutrophil chemotactic response induced by LTB4. These results also indicate that the N-methyl amide of LTB4 may be a useful ligand for the elucidation of molecular mechanisms operative in neutrophil chemotaxis to LTB4, since the C-1 derivative is not further metabolized. Two separate responses of human neutrophils are elicited by LTB4, resulting in both cellular activation and generation of omega oxidation products. It appears that putative receptors on the neutrophils can distinguish between LTB4 and certain derivatives that are structurally identical except for modification at the C-1 position (i.e., the methyl ester). LTB4 derivatives modified at the C-1 position do not undergo conversion to omega oxidation products by the neutrophil.
Assuntos
Quimiotaxia de Leucócito/efeitos dos fármacos , Leucotrieno B4/farmacologia , Neutrófilos/efeitos dos fármacos , Humanos , Leucotrieno B4/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Oxirredução , Relação Estrutura-AtividadeRESUMO
The neonatal lupus syndromes (NLS), while quite rare, carry significant mortality and morbidity in cases of cardiac manifestations. Although anti-SSA/Ro-SSB/La antibodies are detected in > 85% of mothers whose fetuses are identified with congenital heart block (CHB) in a structurally normal heart, when clinicians applied this testing to their pregnant patients, the risk for a woman with the candidate antibodies to have a child with CHB was at or below 1 in 50. While the precise pathogenic mechanism of antibody-mediated injury remains unknown, it is clear that the antibodies alone are insufficient to cause disease and fetal factors are likely contributory. In vivo and in vitro evidence supports a pathologic cascade involving apoptosis of cardiocytes, surface translocation of Ro and La antigens, binding of maternal autoantibodies, secretion of profibrosing factors (e.g., TGFbeta) from the scavenging macrophages and modulation of cardiac fibroblasts to a myofibroflast scarring phenotype. The spectrum of cardiac abnormalities continues to expand, with varying degrees of block identified in utero and reports of late onset cardiomyopathy (some of which display endocardial fibroelastosis). Moreover, there is now clear documentation that incomplete blocks (including those improving in utero with dexamethasone) can progress postnatally, despite the clearance of the maternal antibodies from the neonatal circulation. Better echocardiographic measurements which identify first degree block in utero may be the optimal means of approaching pregnant women at risk. Prophylactic therapies, including treatment with intravenous immunoglobulin, await larger trials. In order to achieve advances at both the bench and bedside, national research registries established in the US and Canada are critical.
Assuntos
Bloqueio Cardíaco/congênito , Lúpus Vulgar/congênito , Autoanticorpos/análise , Feminino , Bloqueio Cardíaco/diagnóstico , Bloqueio Cardíaco/imunologia , Humanos , Recém-Nascido , Lúpus Vulgar/diagnóstico , Gravidez , Diagnóstico Pré-Natal , Prognóstico , SíndromeRESUMO
Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells. However, it is highly unstable, which is an obstacle to detection and quantitation. We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB). The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions. The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated. These studies show the following properties of nitric oxide in aqueous solutions. (i) Nitric oxide is highly reactive to oxygen. (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product. (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution. We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable. From these studies, we conclude that deoxygenated solutions stabilize nitric oxide. An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.