RESUMO
The present authors report the case of an adult with chronic granulomatous disease who developed an unusual lung fibrosis associated with severe pulmonary hypertension. Histological analysis of a lung biopsy showed a diffuse infiltration with pigmented macrophages without granulomas, which particularly involved the pulmonary arterial and venular walls. Clinical and histological findings were suggestive of pulmonary veno-occlusive disease. Such a clinical association has not been previously described in the literature and might be due to the persistent expression of gp91phox at a very low level. In conclusion, the present case report illustrates a novel manifestation of chronic granulomatous disease.
Assuntos
Doença Granulomatosa Crônica/complicações , Hipertensão Pulmonar/etiologia , Doenças Pulmonares Intersticiais/etiologia , Adulto , Biópsia , Lavagem Broncoalveolar , Broncoscopia , Diagnóstico Diferencial , Diuréticos/uso terapêutico , Furosemida/uso terapêutico , Hemodinâmica , Humanos , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/fisiopatologia , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Inibidores de Fosfodiesterase/uso terapêutico , Piperazinas/uso terapêutico , Purinas/uso terapêutico , Testes de Função Respiratória , Citrato de Sildenafila , Fumar/efeitos adversos , Sulfonas/uso terapêutico , Tomografia Computadorizada por Raios XRESUMO
Cystic fibrosis (CF), is the most common life-shortening autosomal recessive disorder in Caucasians. It is caused by mutations in a single gene on the long arm of chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CF is characterized by abnormal Na+ and Cl- ion transport in several tissues, including the lungs, pancreas, gastrointestinal tract, liver, sweat glands, and male reproductive system. Progressive pulmonary disease is the dominant clinical feature of CF and accounts for morbidity and mortality. The inflammation characterized by an overabundance of activated neutrophils and macrophages on the respiratory epithelial surface is associated to a high production of reactive oxygen species (ROS) which contribute to the pathogenesis of cystic fibrosis. ROS could have different origins but the role of the NADPH oxidase system is essential. The "NADPH oxidases" (NOX/DUOX) family is an enzymatic complex formed by cytosolic and membrane subunits. Until now several homologues of the phagocytic NADPH oxidase have been identified in different tissues and it has been shown that the lungs preferentially expressed DUOX1-2. Thus, DUOX1-2 could be implicated in the anti-infectious defense system. The role of DUOX enzymes as a source of ROS in cystic fibrosis is examined as they could contribute to a better understanding of molecular mechanisms in CF. Moreover they could be a potential target for a new therapeutic approach.
Assuntos
Fibrose Cística/etiologia , NADPH Oxidases/fisiologia , Espécies Reativas de Oxigênio , Fibrose Cística/enzimologia , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Oxidases Duais , Feminino , Humanos , Masculino , Proteínas de Membrana , NADPH Oxidase 5 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Interferon-beta-1b is a valuable first-line therapy for patients with relapsing-remitting multiple sclerosis. Many non-severe cutaneous reactions to recombinant interferon beta are described at injection sites. Panniculitis after subcutaneous injection of beta interferon is a rare adverse event; we describe two such cases at beta interferon injection sites. CASE-REPORTS: Two women aged 22 years and 45 years with severe multiple sclerosis receiving immunotherapy with beta interferon were admitted to an emergency department following the appearance of extremely painful induration at injection sites rendering walking impossible after several months of interferon injections. One of the patients had fever. Histology tests showed vasculitis and capillary thrombosis in one-woman and dermal oedema in the other. MRI scanners showed extensive avascular necrosis of soft tissue without fasciitis in both patients. Interferon withdrawal and surgical debridement was carried out in one case and beta interferon was successfully reintroduced in both cases. DISCUSSION: Only two cases have been reported of panniculitis induced by subcutaneous beta interferon injection. Clinically, such cases may mimic infectious processes. The present cases show that MRI may be useful in diagnosis and that the vascular toxicity of interferon beta probably plays a role in panniculitis. Temporary withdrawal of treatment, rotation of several injection sites and alternative routes of administration may all be proposed.
Assuntos
Interferon beta/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Paniculite/induzido quimicamente , Adjuvantes Imunológicos/toxicidade , Adulto , Feminino , Humanos , Injeções Intramusculares , Interferon beta-1a , Interferon beta/administração & dosagem , Interferon beta/toxicidade , Imageamento por Ressonância Magnética , Esclerose Múltipla/patologia , Paniculite/patologiaRESUMO
Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.
Assuntos
Aspergillus fumigatus/imunologia , Macrófagos Alveolares/imunologia , Espécies Reativas de Oxigênio , Corticosteroides/farmacologia , Animais , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , FagocitoseRESUMO
Human polymorphonuclear neutrophils play a key role in host defenses against invading microorganisms. In response to a variety of stimuli, neutrophils release large quantities of superoxide anion (O2.-) in a phenomenon known as the respiratory burst. O2.- is the precursor of potent oxidants, which are essential for bacterial killing and also potentiate inflammatory reactions. Regulation of this production is therefore critical to kill pathogens without inducing tissue injury. Neutrophil production of O2.- is dependent on the respiratory burst oxidase, or NADPH oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to O2.-. NADPH oxidase is activated and regulated by various neutrophil stimuli at infectious or inflammatory sites. Proinflammatory cytokines such as GM-CSF, TNF and IL-8 modulate NADPH oxidase activity through a priming phenomenon. These cytokines induce a very weak oxidative response by PMN but strongly enhance neutrophil release of reactive oxygen species on exposure to a secondary applied stimulus such as bacterial N-formyl peptides. Priming phenomena are involved in normal innate immune defense and in some inflammatory diseases. The mechanisms underlying the priming process are poorly understood, although some studies have suggested that priming with various agonists is regulated at the receptor and post-receptor levels. Resolution of inflammation involves desensitization phenomena and cytokines are involved in this process by various mechanisms. A better understanding of phenomena involved in the regulation of NADPH oxidase could help to develop novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide production.
Assuntos
Citocinas/farmacologia , Neutrófilos/fisiologia , Explosão Respiratória , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-8/farmacologia , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Interleukin (IL) 12 is a heterodimeric cytokine mainly produced by phagocytes-important target cells for IL-12 in particular with a chemotactic effect-and antigen-presenting cells in response to various microorganisms. Because IL-8 is a strong chemokine for polymorphonuclear neutrophils (PMNs), we investigated the effect of IL-12 on PMN IL-8 production. IL-12 alone had no significant effect, but with lipopolysaccharide (LPS) it was additive at both protein and mRNA levels. Actinomycin D at the beginning of culture inhibited IL-8 mRNA induction, whereas late addition affected IL-8 transcript stability, suggesting gene transcription involvement. Results with parthenolide and tyrphostin AG490 suggest that nuclear factor-kappaB and signal transducer and activator of transcription 4 play a role. The IL-12 additive effect was restricted to IL-8 release, with no action on cell-associated IL-8. IL-12 additive effects occurred after 18 h of culture, with no marked up-regulation of IL-12 receptor expression, and were blocked by actinomycin D added after 16 h of culture. Tumor necrosis factor (TNF) alpha and interferon (IFN) gamma had intermediate roles; their specific inhibition reduced IL-12's effect. IL-12's chemotactic mechanism seemed mediated by overproduction and release of IL-8 by human PMNs in the presence of LPS, an effect involving TNF-alpha and IFN-gamma secretion. These results point to a new role for IL-12 in inflammation, through an autocrine amplification loop.
Assuntos
Interleucina-12/farmacologia , Interleucina-8/biossíntese , Neutrófilos/imunologia , Células Cultivadas , Citocinas/farmacologia , Proteínas de Ligação a DNA/fisiologia , Dexametasona/farmacologia , Sinergismo Farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Interferon gama/fisiologia , Interleucina-10/farmacologia , Interleucina-8/genética , Cinética , Lipopolissacarídeos/farmacologia , NF-kappa B/fisiologia , Neutrófilos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Receptores de Interleucina/biossíntese , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Transativadores/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased alpha-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in alpha-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal alpha-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.
Assuntos
Transtornos Plaquetários/patologia , Plaquetas/patologia , Neutrófilos/patologia , Fosfatase Alcalina/sangue , Fosfatase Alcalina/deficiência , Corantes Azur , Transtornos Plaquetários/sangue , Transtornos Plaquetários/genética , Plaquetas/química , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Antígenos CD18/análise , Linhagem da Célula , Tamanho Celular , Células Cultivadas , Criança , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/enzimologia , Grânulos Citoplasmáticos/metabolismo , Doenças em Gêmeos , Amarelo de Eosina-(YS) , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Isoenzimas/sangue , Isoenzimas/deficiência , Antígeno de Macrófago 1/análise , Megacariócitos/patologia , Microscopia Imunoeletrônica , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/química , Neutrófilos/enzimologia , Transporte Proteico , Receptores de Complemento 3b/análise , Coloração e Rotulagem , Síndrome , Trombopoetina/farmacologia , Fator de von Willebrand/metabolismoRESUMO
The most frequent form of chronic granulomatous disease (CGD) is caused by inactivation of the CYBB gene, which encodes the gp91-phox subunit of phagocyte NADPH oxidase. This defect prevents phagocytes from producing reactive oxygen species and thus from eradicating bacterial and fungal infections. We investigated 16 unrelated male patients with suspected X-linked CGD and gp91-phox deficiency. A mutation was found in the CYBB gene of all 16 patients, and 11 of these mutations were novel. Eleven patients (69%) had a point mutation (84G>A in two unrelated patients, and 177C>G, 217C>T, 388C>T, 676C>T, 691C>T, 868C>T, 919A>C, 1384G>T and T1514G in one case each, yielding W28X, C59W, R73X, R130X, R226X, Q231X, R290X, T307P, E462X, L505R gp-91phox). One patient had an in-frame deletion removing two amino acids (R54 and A55). Finally, insertions or duplications were found in four patients (from +1 to +31 bases). Overall, 12 (75%) of the mutations led to the production of a truncated protein. No clear correlation was found between clinical manifestations and genomic/biochemical alterations. Thirteen mothers could be tested, and all were carriers. Hum Mutat 18:163, 2001.
Assuntos
Ligação Genética/genética , Doença Granulomatosa Crônica/genética , Glicoproteínas de Membrana/genética , Mutação/genética , NADPH Oxidases , Cromossomo X/genética , Grupo dos Citocromos b/metabolismo , Análise Mutacional de DNA , Feminino , Doença Granulomatosa Crônica/metabolismo , Heterozigoto , Humanos , Masculino , NADPH Oxidase 2 , Neutrófilos/metabolismo , Polimorfismo Conformacional de Fita Simples , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência/genéticaRESUMO
IL-10 has a wide range of effects tending to control inflammatory responses. We used flow cytometry to study IL-10 binding at the polymorphonuclear neutrophil (PMN) surface and its modulation by various proinflammatory agents. Little IL-10 bound to the surface of resting PMN. However, binding was strongly increased after stimulation with LPS and proinflammatory cytokines such as TNF and GM-CSF. IL-1 and IL-8 did not significantly modify IL-10 binding. Cycloheximide had no effect on TNF-induced IL-10 binding, strongly suggesting the release of a pre-existing pool of IL-10R rather than de novo receptor synthesis by PMN. This was confirmed by the inhibitory effect of pentoxifylline, an inhibitor of degranulation. The existence of an intracellular pool of IL-10R was shown by flow cytometry, immunocytochemical staining, and Western blotting with several anti-human IL-10R Abs. In subcellular fractions of resting PMN, IL-10R was mainly located in the specific granule fraction, and was absent from azurophil granules and cytosol. We also tested the mobilization of specific granules by measuring the release of lactoferrin, their reference marker. The differential effects of the proinflammatory agents on IL-10 binding matched their effects on lactoferrin release and may therefore be related to differential mobilization of specific granules by these agents. Furthermore, the kinetics of TNF-induced up-regulation of IL-10 binding to PMN ran parallel to the kinetics of the inhibitory effect of IL-10 on the oxidative burst, suggesting a key role of IL-10R mobilization from specific granules to the membranes in optimal regulation of inflammatory responses.
Assuntos
Grânulos Citoplasmáticos/imunologia , Grânulos Citoplasmáticos/metabolismo , Mediadores da Inflamação/fisiologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Interleucina/metabolismo , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Grânulos Citoplasmáticos/enzimologia , Gelatinases/análise , Humanos , Interfase/imunologia , Lactoferrina/metabolismo , Neutrófilos/enzimologia , Pentoxifilina/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Receptores de Interleucina-10 , Explosão Respiratória/imunologia , Frações Subcelulares/imunologia , Frações Subcelulares/metabolismo , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Polymorphonuclear neutrophils (PMN) are involved in the pathogenesis of acute lung injury (ALI), secreting numerous mediators such as proteases, reactive oxygen species, and cytokines. Because we had recently observed the ability of normal human PMN to degranulate and synthesize oncostatin M (OSM), an IL-6-family cytokine, we quantified OSM production ex vivo by highly purified blood and alveolar PMN from 24 ventilated patients with ALI, including some patients with severe pneumonia. Most of the patients had no detectable OSM in plasma, and OSM production by cultured blood PMN was similar to that of healthy controls. However, OSM was present in bronchoalveolar lavage (BAL) fluid supernatant, with significantly higher levels during pneumonia. In addition, alveolar OSM levels correlated with the number of PMN obtained by BAL, suggesting that PMN are an important source of OSM within the alveoli. Indeed, purified alveolar PMN from all of the patients, especially those with pneumonia, strongly produced OSM. Interestingly, in the latter patients, alveolar PMN always produced more OSM than autologous blood PMN. These results document the functional duality of PMN in ALI by showing the participation of PMN in the modulation of lung inflammation.
Assuntos
Pulmão/fisiopatologia , Neutrófilos/fisiologia , Peptídeos/metabolismo , Pneumonia/fisiopatologia , Alvéolos Pulmonares/fisiopatologia , Insuficiência Respiratória/fisiopatologia , Idoso , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Infecções Comunitárias Adquiridas/fisiopatologia , Citocinas/biossíntese , Citocinas/sangue , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/terapia , Humanos , Imuno-Histoquímica , Lesão Pulmonar , Pessoa de Meia-Idade , Oncostatina M , Peptídeos/análise , Peptídeos/sangue , Pneumonia/terapia , Complicações Pós-Operatórias , Respiração Artificial , Insuficiência Respiratória/terapiaRESUMO
A new megathrombocytopenic syndrome with giant platelets in peripheral blood and severe thrombocytopenia was diagnosed in a 4-month-old boy. His clinical course included repeated hemorrhagic incidents leading to death at age 37 months. Bone marrow ultrastructural analysis revealed numerous dystrophic megakaryocytes with giant membrane complexes. Although these features were similar to those described for megakaryocytes in mice lacking the gene for transcription factor p45-NF-E2, no abnormalities in the p45-NF-E2 gene could be documented. Platelet membrane analysis showed a reduction in glycoprotein (GP) Ib, but normal content of GPIIb and GPIIIa. Analysis of genes encoding for GPIb alpha and beta, GPV, and GPIX ruled out the possibility that the observed platelet abnormality is a variant of Bernard-Soulier syndrome. A moderate neutropenia was associated with a complete lack of expression of sialyl-Lewis-X on the surface of polymorphonuclear neutrophils. A common defect in posttranslational modification of glycoproteins could account for the diverse cellular abnormalities.
Assuntos
Megacariócitos/ultraestrutura , Neutropenia/diagnóstico , Oligossacarídeos/metabolismo , Trombocitopenia/diagnóstico , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Humanos , Lactente , Contagem de Leucócitos , Masculino , Neutropenia/patologia , Neutrófilos/metabolismo , Contagem de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Antígeno Sialil Lewis X , Síndrome , Trombocitopenia/metabolismo , Trombocitopenia/patologiaRESUMO
BACKGROUND: in response to a variety of stimuli, phagocytes release large quantities of reactive oxygen species (ROS), which are essential for bacterial killing. However, excessive ROS production not appropriately compensated by antioxidant molecules can lead to oxidative stress, which may also play an important role in pathogenesis of HIV infection. In fact, ROS participate in chronic inflammation, HIV replication and the apoptosis of cells of the immune system. OBJECTIVE AND STUDY DESIGN: we used flow cytometry to study, in whole blood, the activation and redox status of polymorphonuclear neutrophils (PMN) and monocytes at different stages of the disease. RESULTS: we showed that neutrophils and monocytes from HIV-infected patients spontaneously produced increased amounts of H2O2. This increased H2O2 production was associated with alterations of adhesion molecules expression at the cell surface, which also reflected basal activation of phagocytes from the HIV-infected patients. In monocytes, basal H2O2 production correlated with viral load. This increased ROS production was associated with changes in the expression of the antiapoptotic/antioxidant compounds Bcl-2 and thioredoxin along the course of the disease. This modulation could result from a dual regulation by oxidative stress and could explain at least in part why monocyte numbers remain relatively stable throughout the disease. Monocytes expressed a normal maximal capacity to produce ROS in optimal conditions of stimulation. In contrast, after ex vivo priming with TNFalpha or IL-8, neutrophils showed a decreased H2O2 production in response to bacterial N-formyl peptides. This latter impairment correlated with the decrease in CD4+ lymphocyte numbers and with IL-8 and IL-6 plasma levels. CONCLUSIONS: the increased basal ROS production by phagocytes could participate to the oxidative injury which has been implicated in the pathophysiology of HIV infection. In addition, the decreased priming of H2O2 production by neutrophils could contribute to the increased susceptibility of HIV-infected patients to bacterial infections.
Assuntos
Soropositividade para HIV/imunologia , HIV-1 , Monócitos/metabolismo , Neutrófilos/metabolismo , Estresse Oxidativo , Actinas/sangue , Adulto , Citocinas/sangue , Citometria de Fluxo , Soropositividade para HIV/sangue , Humanos , Peróxido de Hidrogênio/sangue , Selectina L/sangue , Receptores de Lipopolissacarídeos/sangue , Antígeno de Macrófago 1/sangue , Ativação de Neutrófilo , Oxirredução , Proteínas Proto-Oncogênicas c-bcl-2/sangue , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/sangueRESUMO
OBJECTIVE: We assessed the clinical and histologic features of angiogenesis inhibition in a transgenic mouse model of arthritis that closely resembles rheumatoid arthritis (RA) in humans. METHODS: KRN/NOD mice, which spontaneously develop arthritis, were treated with TNP-470, an angiogenesis inhibitor. Disease was monitored by use of clinical indices and histologic examinations; circulating blood levels of vascular endothelial growth factor were determined by enzyme-linked immunosorbent assay. RESULTS: In the preventive protocol, with TNP-470 administration at a dosage of 60 mg/kg of body weight, the onset of arthritis was delayed and its clinical intensity was rather mild; 100% of placebo-treated transgenic mice developed arthritis that led to severe articular destruction. At a dosage of 90 mg/kg of TNP-470, the appearance of clinical signs was delayed for a longer period of time and disease was almost abolished. The therapeutic regimen alleviated clinical signs only when given during the very early stage of disease. Reductions in cartilage and bone destruction by TNP-470 treatment were observed histologically, a feature that was still evident at 30 and 80 days after injections were withdrawn. CONCLUSION: Our demonstration that in vivo administration of an angiogenesis inhibitor suppresses arthritis and protects from bone destruction provides new insight into the pathogenesis of the disease and opens new possibilities in the treatment of RA in humans.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Reabsorção Óssea/prevenção & controle , Sesquiterpenos/uso terapêutico , Animais , Artrite Reumatoide/patologia , Artrite Reumatoide/prevenção & controle , Cicloexanos , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/sangue , Linfocinas/sangue , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , O-(Cloroacetilcarbamoil)fumagilol , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.
Assuntos
MAP Quinase Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Fosfoproteínas/metabolismo , Butadienos/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinases/antagonistas & inibidores , Maleimidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NADPH Desidrogenase/metabolismo , NADPH Oxidases , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Nitrilas/farmacologia , Mapeamento de Peptídeos , Fosfopeptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Explosão Respiratória/efeitos dos fármacosRESUMO
BACKGROUND/AIMS: Several observations point to an important role of interactions between polymorphonuclear neutrophils and cytokines in severe alcoholic hepatitis. The polymorphonuclear neutrophil activation status and the local and systemic pro- and anti-inflammatory cytokine responses were quantified. The effect of corticosteroids, widely used in this setting, was evaluated using these parameters. METHODS: We studied blood polymorphonuclear neutrophil functions in terms of L-selectin and beta2-integrin expression, H2O2 production and IL-8 and tumor necrosis factor alpha synthesis capacity. We also measured IL-8, tumor necrosis factor alpha and IL-10 plasma and liver tissue levels. Fifteen patients with alcoholic hepatitis were compared to 15 patients with alcoholic cirrhosis without alcoholic hepatitis, and to 10 healthy volunteers. The impact of a 28-day course of corticosteroids on blood neutrophils activation status and cytokine levels was evaluated in patients with alcoholic hepatitis. RESULTS: Blood polymorphonuclear neutrophils were activated, as shown by increased H2O2 production (48+/-6 vs 29+/-6 MFI in healthy controls), and decreased L-selectin expression (300+/-61 vs 449+/-59 in healthy controls). Upon stimulation, polymorphonuclear neutrophils synthesized large amounts of IL-8 (21.7+/-9.2 ng/ml vs 8.8+/-10 ng/ml in healthy controls) and tumor necrosis factor alpha (524+/-132 pg/ml vs 79+/-144 pg/ml in healthy controls). Tumor necrosis factor alpha and IL-8 plasma and tissue levels were markedly increased as IL-10 was barely detectable in alcoholic hepatitis patients, compared to cirrhotic patients and healthy controls. During steroid therapy, plasma levels of the pro-inflammatory cytokine IL-8 fell as early as day 14, while levels of the anti-inflammatory cytokine IL-10 increased on day 21. Finally, polymorphonuclear neutrophil functions returned to normal after treatment. CONCLUSION: Severe alcoholic hepatitis appears to be associated with polymorphonuclear neutrophil activation and an imbalance between pro- and anti-inflammatory cytokines; during steroid therapy a normalization of these parameters was observed.
Assuntos
Corticosteroides/administração & dosagem , Citocinas/sangue , Hepatite Alcoólica/sangue , Hepatite Alcoólica/tratamento farmacológico , Neutrófilos/metabolismo , Adulto , Idoso , Feminino , Humanos , Peróxido de Hidrogênio/sangue , Selectina L/sangue , Masculino , Pessoa de Meia-Idade , Ativação de NeutrófiloRESUMO
Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.
Assuntos
Doença Granulomatosa Crônica/enzimologia , NADPH Oxidases/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/metabolismo , Linfócitos B/citologia , Linfócitos B/enzimologia , Western Blotting , Linhagem Celular Transformada , Membrana Celular/enzimologia , Grupo dos Citocromos b/isolamento & purificação , Grupo dos Citocromos b/metabolismo , Citosol/química , Citosol/enzimologia , Ativação Enzimática , Estabilidade Enzimática , Teste de Complementação Genética , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/patologia , Humanos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Neutrófilos/citologia , Neutrófilos/enzimologia , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Rare hereditary deficiencies have been described which affect each functional stage of polymorphonuclear neutrophils. They almost invariably lead to recurrent acute infection. Among the abnormalities involving adhesion and motility, the following can be noted: the Buckley syndrome; and leucocyte type 1 and 2 adhesion deficiencies, respectively caused by a deficiency in membrane expression of beta 2 integrin CD11/CD18, and sialyl lewis X. Granulation system abnormalities include relatively non-symptomatic myeloperoxidase deficiency, specific granulation deficiency or the Chediak-Higashi syndrome with the presence of giant lysosomal granulations. Chronic or familial septic granulomatosis constitutes the main disease described due to the oxidative PMN burst connected with the functional impairment of one of the constituents of NADPH oxidase (with an incidence of one in 5.10(6) to one in 10(6) births) The transmission is X-linked, or autosomal recessive depending on the mutation. The antenatal detection of the X-linked component, gp91 phox, can be made in suspected carrier mothers. In addition to the standard treatment (Bactrim and Itraconazole), bone marrow transplantation may also be carried out, and in future gene therapy may be introduced.
Assuntos
Síndrome da Aderência Leucocítica Deficitária/genética , Neutrófilos/fisiologia , Antígenos CD/sangue , Antígenos CD18/sangue , Humanos , Síndrome da Aderência Leucocítica Deficitária/sangue , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/diagnóstico , Antígeno de Macrófago 1/sangue , Cromossomo XRESUMO
Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms. Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation. This process is known as priming. We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms (Mycobacterium bovis, BCG, and Salmonella typhimurium). No T- or B-cell quantitative or qualitative defects were found. Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease. Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H(2)O(2) production after pretreatment with LPS (5 microg/mL for 30 min), TNFalpha (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10(-6) M for 5 min). In these conditions, H(2)O(2) production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H(2)O(2). Moreover, monocytes from the patient showed an impaired capacity to kill S. typhimurium in vitro. Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst. This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens.
Assuntos
Monócitos/metabolismo , Neutrófilos/metabolismo , Explosão Respiratória , Adulto , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Criança , Consanguinidade , Grupo dos Citocromos c/metabolismo , Citocinas/farmacologia , Feminino , Genes Recessivos , Humanos , Peróxido de Hidrogênio/sangue , Peróxido de Hidrogênio/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Monócitos/efeitos dos fármacos , Monócitos/microbiologia , Monócitos/patologia , Mycobacterium bovis/imunologia , Mycobacterium bovis/fisiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/microbiologia , Neutrófilos/patologia , Disfunção de Fagócito Bactericida/enzimologia , Disfunção de Fagócito Bactericida/imunologia , Disfunção de Fagócito Bactericida/metabolismo , Disfunção de Fagócito Bactericida/patologia , Recidiva , Explosão Respiratória/efeitos dos fármacos , Infecções por Salmonella/enzimologia , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologiaRESUMO
NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.
Assuntos
Citoesqueleto/metabolismo , Ativação de Neutrófilo/fisiologia , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Actinas/metabolismo , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Citoesqueleto/enzimologia , Detergentes/química , Humanos , NADPH Oxidases/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/ultraestrutura , Octoxinol/química , Fosfoproteínas/deficiência , Testes de Precipitina , Solubilidade , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
We used flow cytometry to analyze the expression of adhesion molecules and the oxidative burst of whole-blood polymorphonuclear neutrophils (PMN) from 26 patients with periodontitis. Three different clinical entities were studied: adult periodontitis (AP), localized juvenile periodontitis (LJP), and rapidly progressive periodontitis (RPP). Unstimulated PMN from the patients showed reduced Lewis x, sialyl-Lewis x, and L-selectin expression relative to those from healthy control subjects. These alterations were present whatever the severity of periodontal disease. However, PMN from RPP patients showed increased basal H2O2 production and decreased L-selectin shedding. These latter impairments, which correlated with increased IL-8 plasma levels, could contribute to initial vascular damage. In addition, decreased IL-8 priming of H2O2 production by PMN from RPP patients could account for a lower bactericidal capacity of PMN, leading to the large number of bacteria in the subgingival region of RPP patients. Soluble L-selectin plasma levels were also decreased in the RPP group, indicating more severe or diffuse endothelial damage. These abnormalities were not found in the patients with less destructive forms of periodontitis (AP and LJP). Porphyromonas gingivalis, a bacterial pathogen known to increase IL-8 production by PMN, was found in the periodontal pockets of RPP patients only. These results show links among PMN abnormalities, the clinical form of periodontitis, and the gingival bacterial flora.