RESUMEN
Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.
Asunto(s)
Fibrosis Quística/inmunología , Elastasa de Leucocito/metabolismo , Pulmón/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Animales , Células Cultivadas , Enfermedad Crónica , Fibrosis Quística/complicaciones , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Mutación/genética , Infiltración Neutrófila , Proteolisis , Infecciones por Pseudomonas/complicaciones , Inhibidor Secretorio de Peptidasas Leucocitarias/genéticaRESUMEN
The role of proteases in viral infection of the lung is poorly understood. Thus, we examined matrix metalloproteinases (MMPs) and cathepsin proteases in respiratory syncytial virus (RSV)-infected mouse lungs. RSV-induced gene expression for MMPs -2, -3, -7, -8, -9, -10, -12, -13, -14, -16, -17, -19, -20, -25, -27, and -28 and cathepsins B, C, E, G, H, K, L1, S, W, and Z in the airways of Friend leukemia virus B sensitive strain mice. Increased proteases were present in the bronchoalveolar lavage fluid (BALF) and lung tissue during infection. Mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferon-ß-deficient mice were exposed to RSV. Mavs-deficient mice had significantly lower expression of airway MMP-2, -3, -7, -8, -9, -10, -12, -13, and -28 and cathepsins C, G, K, S, W, and Z. In lung epithelial cells, retinoic acid-inducible gene-1 (RIG-I) was identified as the major RIG-I-like receptor required for RSV-induced protease expression via MAVS. Overexpression of RIG-I or treatment with interferon-ß in these cells induced MMP and cathepsin gene and protein expression. The significance of RIG-1 protease induction was demonstrated by the fact that inhibiting proteases with batimastat, E64 or ribavirin prevented airway hyperresponsiveness and enhanced viral clearance in RSV-infected mice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Catepsinas/metabolismo , ARN Helicasas DEAD-box/fisiología , Pulmón/enzimología , Metaloproteinasas de la Matriz/metabolismo , Infecciones por Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Regulación Viral de la Expresión Génica , Interferón Tipo I/inmunología , Leucina/administración & dosificación , Leucina/análogos & derivados , Pulmón/virología , Ratones , Ratones Endogámicos , Ratones Noqueados , Fenilalanina/administración & dosificación , Fenilalanina/análogos & derivados , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/efectos de los fármacos , Ribavirina/administración & dosificación , Tiofenos/administración & dosificación , Carga Viral/efectos de los fármacosRESUMEN
BACKGROUND: 1,25-Dihydroxycholecalciferol (1,25(OH)(2)D(3)) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF. METHODS: CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH)(2)D(3) or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH)(2)D(3). RESULTS: Pretreatment with 1,25(OH)(2)D(3) was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH)(2)D(3) treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH)(2)D(3) was associated with an increase in hCAP18/LL-37 mRNA and protein levels. CONCLUSIONS: Both 1,25(OH)(2)D(3) and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.
Asunto(s)
Calcitriol/farmacología , Fibrosis Quística/inmunología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Receptores de Calcitriol/agonistas , Mucosa Respiratoria/citología , Mucosa Respiratoria/inmunología , Vitaminas/farmacología , Células Cultivadas , HumanosRESUMEN
BACKGROUND: Gastro-oesophageal reflux is common in children with cystic fibrosis (CF) and is thought to be associated with pulmonary aspiration of gastric contents. The measurement of pepsin in bronchoalveolar lavage (BAL) fluid has recently been suggested to be a reliable indicator of aspiration. The prevalence of pulmonary aspiration in a group of children with CF was assessed and its association with lung inflammation investigated. METHODS: This was a cross-sectional case-control study. BAL fluid was collected from individuals with CF (n=31) and healthy controls (n=7). Interleukin-8 (IL-8), pepsin, neutrophil numbers and neutrophil elastase activity levels were measured in all samples. Clinical, microbiological and lung function data were collected from medical notes. RESULTS: The pepsin concentration in BAL fluid was higher in the CF group than in controls (mean (SD) 24.4 (27.4) ng/ml vs 4.3 (4.0) ng/ml, p=0.03). Those with CF who had raised pepsin concentrations had higher levels of IL-8 in the BAL fluid than those with a concentration comparable to controls (3.7 (2.7) ng/ml vs 1.4 (0.9) ng/ml, p=0.004). Within the CF group there was a moderate positive correlation between pepsin concentration and IL-8 in BAL fluid (r=0.48, p=0.04). There was no association between BAL fluid pepsin concentrations and age, sex, body mass index z score, forced expiratory volume in 1 s or Pseudomonas aeruginosa colonisation status. CONCLUSIONS: Many children with CF have increased levels of pepsin in the BAL fluid compared with normal controls. Increased pepsin levels were associated with higher IL-8 concentrations in BAL fluid. These data suggest that aspiration of gastric contents occurs in a subset of patients with CF and is associated with more pronounced lung inflammation.
Asunto(s)
Líquido del Lavado Bronquioalveolar/química , Fibrosis Quística/metabolismo , Interleucina-8/análisis , Pepsina A/análisis , Adolescente , Biomarcadores/análisis , Estudios de Casos y Controles , Niño , Preescolar , Fibrosis Quística/complicaciones , Femenino , Humanos , Lactante , Masculino , Aspiración Respiratoria/diagnóstico , Aspiración Respiratoria/etiologíaRESUMEN
alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Enfisema/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , alfa 1-Antitripsina/farmacología , Adulto , Biopsia , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Proliferación Celular , Enfisema/genética , Femenino , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Proteínas Inhibidoras de la Apoptosis/genética , Masculino , FN-kappa B/metabolismo , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Deficiencia de alfa 1-Antitripsina/metabolismo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
BACKGROUND: Neutrophil elastase (NE) activity is increased in lung diseases such as alpha(1)-antitrypsin (A1AT) deficiency and pneumonia. It has recently been shown to induce expression of cathepsin B and matrix metalloprotease 2 (MMP-2) in vitro and in a mouse model. It is postulated that increased cathepsin B and MMP-2 in acute and chronic lung diseases result from high levels of extracellular NE and that expression of these proteases could be inhibited by A1AT augmentation therapy. METHODS: Cathepsin and MMP activities were assessed in bronchoalveolar lavage (BAL) fluid from patients with A1AT deficiency, pneumonia and control subjects. Macrophages were exposed to BAL fluid rich in free NE from patients with pneumonia following pretreatment with A1AT. MMP-2, cathepsin B, secretory leucoprotease inhibitor (SLPI) and lactoferrin levels were determined in BAL fluid from A1AT-deficient patients before and after aerosolisation of A1AT. RESULTS: BAL fluid from both patients with pneumonia and those with A1AT deficiency containing free NE had increased cathepsin B and MMP-2 activities compared with BAL fluid from healthy volunteers. The addition of A1AT to BAL fluid from patients with pneumonia greatly reduced NE-induced cathepsin B and MMP-2 expression in macrophages in vitro. A1AT augmentation therapy to A1AT-deficient individuals also reduced cathepsin B and MMP-2 activity in BAL fluid in vivo. Furthermore, A1AT-deficient patients had higher levels of SLPI and lactoferrin after A1AT augmentation therapy. CONCLUSION: These findings suggest a novel role for A1AT inhibition of NE-induced upregulation of MMP and cathepsin expression both in vitro and in vivo.
Asunto(s)
Catepsina B/metabolismo , Elastasa de Leucocito/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Deficiencia de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/farmacología , Administración por Inhalación , Líquido del Lavado Bronquioalveolar/química , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Neumonía/metabolismo , Inhibidores de Serina Proteinasa/administración & dosificación , alfa 1-Antitripsina/administración & dosificaciónRESUMEN
Alpha-1 antitrypsin (A1AT) is a serine anti-protease produced chiefly by the liver. A1AT deficiency is a genetic disorder characterized by serum levels of less than 11 mumol/L and is associated with liver and lung manifestations. The liver disease, which occurs in up to 15% of A1AT-deficient individuals, is a result of toxic gain-of-function mutations in the A1AT gene, which cause the A1AT protein to fold aberrantly and accumulate in the endoplasmic reticulum of hepatocytes. The lung disease is associated with loss-of-function, specifically decreased anti-protease protection on the airway epithelial surface. The so-called 'Z' mutation in A1AT deficiency encodes a glutamic acid-to-lysine substitution at position 342 in A1AT and is the most common A1AT allele associated with disease. Here we review the current understanding of the molecular pathogenesis of A1AT deficiency and the best clinical management protocols.
Asunto(s)
Hepatopatías/etiología , Enfermedades Pulmonares/etiología , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/química , Animales , Autofagia/fisiología , Humanos , Hepatopatías/genética , Hepatopatías/terapia , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/terapia , Modelos Biológicos , Conformación Proteica , Pliegue de Proteína , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/fisiopatologíaRESUMEN
Chronic lung disease is one of the most common causes of death and disability worldwide. This group of diseases is characterized by a protease burden, an infective process and a dominant pro-inflammatory profile. While SLPI (secretory leucoprotease inhibitor) was initially identified as a serine protease inhibitor, it has since been shown that SLPI possesses other properties distinct from those associated with its antiprotease capabilities that play an important role in protecting the host from infection and injury. In the course of this review, we will highlight the findings from a range of studies that illustrate the multiple functions of SLPI and its role in the resolution of the immune response.
Asunto(s)
Antiinflamatorios , Fibrosis Quística/fisiopatología , Inflamación/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Inhibidor Secretorio de Peptidasas Leucocitarias/fisiología , Antiinfecciosos , Fibrosis Quística/prevención & control , Humanos , Elastasa de Leucocito/metabolismo , Inhibidores de Proteasas , Enfermedad Pulmonar Obstructiva Crónica/prevención & control , Inhibidor Secretorio de Peptidasas Leucocitarias/química , Inhibidor Secretorio de Peptidasas Leucocitarias/inmunologíaRESUMEN
Matrix metalloproteinases 2 and 9 (MMP 2 and -9) have been implicated in the pathogenesis of atherosclerosis and aneurysm formation. The goal of the study was to establish the role of these metalloproteinases in both human atherosclerotic and non-atherosclerotic cerebral aneurysms. Eleven cerebral aneurysms (four atherosclerotic, seven non-atherosclerotic) were immunohistochemically stained for MMP 2 and -9. As controls, atherosclerotic and normal Circle of Willis arteries were similarly immunostained. All specimens were retrieved at autopsy and were paraffin-embedded. In order to evaluate the real MMP 2 and -9 activities, gelatin zymography was also performed in only two available specimens of non-atherosclerotic intracranial aneurysms, because of the relative unavailability of fresh intracranial aneurysm tissue (i.e. reluctance to excise the aneurysm fundus at surgery). Our data establish that MMP 2 and -9 were expressed minimally or not at all in normal Circle of Willis arteries but were strongly expressed in medial smooth muscle cells of atherosclerotic Circle of Willis arteries. In the aneurysm group, both MMP 2 and -9 were strongly expressed in the atherosclerotic aneurysms, but MMP 2 alone was detected in the non-atherosclerotic aneurysms. Zymography revealed a weak enzyme activity correlating to MMP 9 standard recombinant protein. MMP 2 activity was not demonstrated in either specimen. This study shows that the expression of MMP 2 and -9 is associated with atherosclerosis, be it in aneurysmal or non-aneurysmal cerebral vessels but MMP 2 appears to be specifically expressed in aneurysms devoid of atherosclerosis perhaps suggesting a pathogenic role for MMP 2 in the alteration of the extracellular matrix of cerebral arteries during aneurysm formation.
Asunto(s)
Aterosclerosis/enzimología , Proteínas de la Matriz Extracelular/biosíntesis , Aneurisma Intracraneal/enzimología , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Adolescente , Adulto , Anciano , Aterosclerosis/patología , Círculo Arterial Cerebral/enzimología , Círculo Arterial Cerebral/patología , Femenino , Humanos , Aneurisma Intracraneal/patología , Masculino , Persona de Mediana EdadRESUMEN
Although neutrophils are a critical component of the inflammatory process, their functional regulation is incompletely understood. Of note, although pCO2 varies physiologically and pathologically in the neutrophilic milieu, its affect on neutrophil biological processes is unresolved. We demonstrate here that neutrophils respond to hypo- and hypercarbia, (0.04% and 10%) by increasing and decreasing, respectively, intracellular oxidant production (basally and in response to opsonized Escherichia coli and phorbol esters). Further, hypo- and hypercarbia increase and decrease, respectively, the release of IL-8 from LPS-stimulated cells; both effects are attenuated by the carbonic anhydrase inhibitor, acetazolamide. Anion exchange did not restore pH(i) under hypocarbic conditions, however partial restoration of pH(i) under hypercarbic conditions was achieved by Na+/H+ exchange and vacuolar ATPases. Abrogation of pCO2-induced changes in pH(i) prevented hypocarbia-induced generation of reactive oxidant species. These observations suggest that CO2 modifies neutrophil activity significantly by altering pH(i).
Asunto(s)
Dióxido de Carbono/farmacología , Interleucina-8/biosíntesis , Neutrófilos/metabolismo , Oxidantes/metabolismo , Acetazolamida/farmacología , Bicarbonatos/metabolismo , Anhidrasas Carbónicas/fisiología , Células Cultivadas , Cloruros/metabolismo , Humanos , Concentración de Iones de HidrógenoRESUMEN
Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o(-) cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor kappaB (NFkappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NFkappaB by NE was preceded by phosphorylation and degradation of IkappaB proteins, principally IkappaBbeta. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o(-) cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Delta) or TRAF-6 (TRAF-6Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Delta inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IkappaBbeta and nuclear translocation of NFkappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Bronquios/metabolismo , Interleucina-8/genética , Elastasa de Leucocito/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Receptores Inmunológicos , Regulación hacia Arriba , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Bases , Western Blotting , Bronquios/efectos de los fármacos , Línea Celular Transformada , Cartilla de ADN , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Hidrólisis , Quinasas Asociadas a Receptores de Interleucina-1 , Interleucina-8/biosíntesis , Factor 88 de Diferenciación Mieloide , FN-kappa B/metabolismo , Norepinefrina/farmacología , Factor 6 Asociado a Receptor de TNF , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A number of serine proteases, matrix metalloproteases, and cysteine proteases were evaluated for their ability to cleave and inactivate the antiprotease, secretory leucoprotease inhibitor (SLPI). None of the serine proteases or the matrix metalloproteases examined cleaved the SLPI protein. However, incubation with cathepsins B, L, and S resulted in the cleavage and inactivation of SLPI. All three cathepsins initially cleaved SLPI between residues Thr(67) and Tyr(68). The proteolytic cleavage of SLPI by all three cathepsins resulted in the loss of the active site of SLPI and the inactivation of SLPI anti-neutrophil elastase capacity. Cleavage and inactivation were catalytic with respect to the cathepsins, so that the majority of a 400-fold excess of SLPI was inactivated within 15 min by cathepsins L and S. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of cleaved SLPI in these samples whereas only intact SLPI was observed in control epithelial lining fluid samples. Active cathepsin L was shown to be present in emphysema epithelial lining fluid and inhibition of this protease prevented the cleavage of recombinant SLPI added to emphysema epithelial lining fluid. Taken together with previous data that demonstrates that cathepsin L inactivates alpha(1)-antitrypsin, these findings indicate the involvement of cathepsins in the diminution of the lung antiprotease screen possibly leading to lung destruction in emphysema.
Asunto(s)
Catepsina B/metabolismo , Catepsinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Proteínas/metabolismo , Animales , Sitios de Unión , Western Blotting , Lavado Broncoalveolar , Estudios de Casos y Controles , Dominio Catalítico , Catepsina B/química , Catepsina L , Catepsinas/química , Cromatografía Líquida de Alta Presión , Cisteína Endopeptidasas , Electroforesis en Gel de Poliacrilamida , Enfisema/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Epitelio/enzimología , Femenino , Humanos , Pulmón/enzimología , Masculino , Persona de Mediana Edad , Unión Proteica , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas Recombinantes/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias , Treonina/química , Factores de Tiempo , Tirosina/química , alfa 1-Antitripsina/metabolismoRESUMEN
Wegener's granulomatosis (WG) is classically associated with the presence of cytoplasmic antineutrophil cytoplasmic autoantibodies (c-ANCA). Proteinase 3 (PR3), the target antigen for c-ANCA, is inhibited by the antiprotease alpha1-antitrypsin (A1AT), and recent studies have demonstrated that WG patients who are A1AT-deficient have a worse clinical course, suggesting that a protease-antiprotease imbalance may play a role in WG. We evaluated the effect of A1AT on anti-PR3 antibody-induced activation of neutrophils. The neutrophil was chosen because of its central role in the pathogenesis of WG. Isolated neutrophils from healthy controls were incubated with tumor necrosis factor (TNF)-alpha to induce surface expression of PR3. Subsequently, they were stimulated with a monoclonal antibody to PR3, resulting in a significant increase in respiratory burst. Addition of A1AT (1 mg/ml) to the TNF-alpha- primed cells before the addition of the anti-PR3 antibody resulted in a 47% reduction in anti-PR3 antibody-induced activation. A1AT mediated this inhibitory action by preventing anti-PR3 antibody binding to PR3 on the cell, thereby preventing the PR3-FcgammaR11a cross-linkage required for cell activation. Further, anti-PR3 antibody-induced activation of neutrophils from WG patients can be reduced by 56% with A1AT. These data suggest that protease-antiprotease interactions may play a pivotal role in neutrophil activation in WG.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/farmacología , Granulomatosis con Poliangitis/metabolismo , Activación Neutrófila/efectos de los fármacos , Serina Endopeptidasas/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , alfa 1-Antitripsina/farmacología , Adulto , Anciano , Femenino , Granulomatosis con Poliangitis/etiología , Humanos , Inmunoglobulina G/metabolismo , Masculino , Persona de Mediana Edad , Mieloblastina , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas/farmacología , Estallido Respiratorio/efectos de los fármacos , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Genetic isolation by distance (IBD) has rarely been described in marine species with high potential for dispersal at both the larval and adult life-history stages. Here, we report significant relationships between inferred levels of gene flow and geographic distance in the Atlantic cod, Gadus morhua, at 10 nuclear restriction-fragment-length-polymorphism (RFLP) loci at small regional scales in the western north Atlantic region (< 1,600 km) that mirror those previously detected over its entire geographic range (up to 7,300 km). Highly significant allele frequency differences were observed among eight northwestern Atlantic populations, although the mean FST for all 10 loci was only 0.014. Despite this weak population structuring, the distance separating populations explained between 54% and 62% of the variation in gene flow depending on whether nine or 10 loci were used to estimate Nm. Across the species' entire geographic range, highly significant differences were observed among six regional populations at nine of the 10 loci (mean FST = 0.068) and seven loci exhibited significant negative relationships between gene flow and distance. At this large geographic scale, natural selection acting in the vicinity of one RFLP locus (GM798) had a significant effect on the correlation between gene flow and distance, and eliminating it from the analysis caused the coefficient of determination to increase from 17% to 62%. The role of vicariance was assessed by sequentially removing populations from the analysis and was found to play a minor role in contributing to the relationship between gene flow and distance at either geographic scale. The correlation between gene flow and distance detected in G. morhua at small and large spatial scales suggests that dispersal distances and effective population sizes are much smaller than predicted for the species and that the recent age of populations, rather than extensive gene flow, may be responsible for its weak population structure. Our results suggest that interpreting limited genetic differences among populations as reflecting high levels of ongoing gene flow should be made with caution.
Asunto(s)
Peces/genética , Genética de Población , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Femenino , Geografía , Larva/crecimiento & desarrollo , Masculino , Dinámica PoblacionalRESUMEN
Alpha1-antitrypsin (alpha1AT) deficiency is a common lethal hereditary disorder of white persons of European descent. The condition is characterized by reduced serum levels of alpha1AT, a 52-kDa glycoprotein synthesized chiefly in the liver and, to a lesser extent, by macrophages and neutrophils. Alpha1AT acts as an antiprotease and is the physiological inhibitor of neutrophil serine proteases such as neutrophil elastase cathepsin G and proteinase 3. The clinical manifestations of alpha1AT deficiency occur chiefly in the lung, with a high risk of emphysema occurring by the third or fourth decade of life. Cigarette smoking accelerates the development of emphysema in persons with alpha1AT deficiency. There is also an increased risk of liver disease in alpha1AT deficiency, which occurs mostly in childhood. In this review, we will define further the diagnosis of alpha1AT deficiency and its clinical manifestations and describe the therapeutic strategies that are currently being developed to treat the hepatic and pulmonary disease associated with this condition.
Asunto(s)
Deficiencia de alfa 1-Antitripsina/fisiopatología , alfa 1-Antitripsina/genética , Adulto , Mapeo Cromosómico , Cromosomas Humanos Par 14 , Enfisema/epidemiología , Enfisema/genética , Humanos , Elastasa de Leucocito/antagonistas & inhibidores , Hepatopatías/epidemiología , Hepatopatías/genética , Pulmón/fisiopatología , Fumar/efectos adversos , alfa 1-Antitripsina/fisiología , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/genéticaRESUMEN
Sarcoidosis is a granulomatous disease of unknown etiology associated with the expansion of IL-2-producing activated CD4(+) T lymphocytes. A number of factors including the recently described IL-18 have been implicated in IL-2 expression in vitro. We investigated the role of IL-18 in IL-2 expression in sarcoidosis. Eighteen individuals with sarcoidosis and 15 normal controls were studied. IL-18R expression and epithelial lining fluid (ELF) concentrations of IL-18 were significantly elevated in the sarcoid group (p = 0.0143 and 0.0024, respectively). Both AP1 and NF-kappaB, transcription factors that regulate IL-2 gene expression, were activated in vivo in sarcoid pulmonary CD4(+) T lymphocytes. Transcription factor activity was not detected in pulmonary CD4(+) T lymphocytes from normal controls or from peripheral blood CD4(+) T lymphocytes from individuals with sarcoidosis, further evidence of compartmentalization of the lymphoproliferative process in this condition. We examined the effects of IL-18 on AP1 and NF-kappaB in Jurkat T cells in vitro. These effects were both time and dose dependent. Examination of transcription factor activation and IL-2 gene expression in Jurkat T cells revealed that sarcoid but not normal ELF activated AP1 and NF-kappaB, induced IL-2 gene transcription, and up-regulated IL-2 protein production. Addition of IL-18 to normal ELF also induced IL-2 mRNA accumulation, whereas correspondent depletion of IL-18 from sarcoid ELF using neutralizing Abs abrogated all of the effects. These data strongly implicate IL-18 in the pathogenesis of sarcoidosis via activation of AP1 and NF-kappaB, leading to enhanced IL-2 gene expression and IL-2 protein production and concomitant T cell activation.
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Linfocitos T CD4-Positivos/inmunología , Interleucina-18/fisiología , Activación de Linfocitos/inmunología , Sarcoidosis Pulmonar/inmunología , Adulto , Líquido del Lavado Bronquioalveolar/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Citocinas/metabolismo , Epitelio/inmunología , Epitelio/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-18/metabolismo , Subunidad alfa del Receptor de Interleucina-18 , Interleucina-2/biosíntesis , Interleucina-2/genética , Células Jurkat/inmunología , Células Jurkat/metabolismo , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/sangre , Receptores de Interleucina-18 , Sarcoidosis Pulmonar/metabolismo , Sarcoidosis Pulmonar/patología , Células TH1/inmunología , Células TH1/metabolismo , Factor de Transcripción AP-1/sangre , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/inmunología , Células U937RESUMEN
Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pH(i)). We demonstrate here that pH(i) regulation is altered in neutrophils from CF patients. Although resting pH(i) is similar, pH(i) after acid loading and activation (N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pH(i). This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pH(i) as opposed to decreasing pH(i) as seen in activated CF neutrophils. These pH(i) differences in activated or acid-loaded CF neutrophils are abrogated by ZnCl(2) but not by amiloride and bafilomycin A(1), suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO(3)(-)/Cl(-) exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pH(i) regulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.
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Fibrosis Quística/metabolismo , Hidrógeno/metabolismo , Membranas Intracelulares/metabolismo , Neutrófilos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácidos/farmacología , Adolescente , Adulto , Álcalis/metabolismo , Bronquiectasia/metabolismo , Bronquiectasia/patología , Líquido del Lavado Bronquioalveolar/citología , Cloruros/farmacología , Fibrosis Quística/patología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Monocitos/fisiología , Neutrófilos/efectos de los fármacos , Protones , Valores de Referencia , Compuestos de Zinc/farmacologíaRESUMEN
Hydrogen peroxide is a component of cigarette smoke known to be essential for inactivation of alpha(1)-antitrypsin, the primary inhibitor of neutrophil elastase. To establish the molecular basis of the inactivation of alpha(1)-antitrypsin, we determined the sites oxidized by hydrogen peroxide. Two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation was known to cause loss of anti-elastase activity. The other, methionine 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-elastase activity, an effect not previously recognized. The equal susceptibility of methionine 358 and methionine 351 to oxidation was confirmed by mass spectrometry. To verify this finding, we produced recombinant alpha(1)-antitrypsins in which one or both of the susceptible methionines were mutated to valine. M351V and M358V were not as rapidly inactivated as wild-type alpha1-antitrypsin, but only the double mutant M351V/M358V was markedly resistant to oxidative inactivation. We suggest that inactivation of alpha(1)-antitrypsin by oxidation of either methionine 351 or 358 provides a mechanism for regulation of its activity at sites of inflammation.
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Elastasa de Leucocito/antagonistas & inhibidores , Metionina/metabolismo , alfa 1-Antitripsina/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genéticaRESUMEN
Cystic fibrosis (CF) is a lethal, hereditary disorder characterized by a neutrophil-dominated inflammation of the lung. We sought to determine whether neutrophils from individuals with CF release more neutrophil elastase (NE) than neutrophils from normal subjects. Our results showed that peripheral blood neutrophils (PBNs) from normal subjects and individuals with CF contained similar amounts of NE, but after preincubation with CF bronchoalveolar lavage (BAL) fluid, significantly more NE was released by CF PBNs, a release that was amplified further by incubation with opsonized Escherichia coli. To determine which components of CF BAL fluid stimulated this excessive NE release from CF PBNs, we repeated the experiments after neutralization or immunoprecipitation of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha and IL-8 were used as activating stimuli, CF PBNs released significantly greater amounts of NE compared with PBNs from control subjects and individuals with bronchiectasis. These results indicate that CF PBNs respond abnormally to TNF-alpha and IL-8 in CF BAL fluid and react to opsonized bacteria by releasing more NE. This may help explain the increased NE burden seen in this condition.
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Fibrosis Quística/metabolismo , Interleucina-8/fisiología , Elastasa de Leucocito/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Antígenos CD/metabolismo , Líquido del Lavado Bronquioalveolar/química , Niño , Preescolar , Humanos , Interleucina-8/análisis , Interleucina-8/farmacología , Antígeno de Macrófago-1/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Norepinefrina/metabolismo , Pruebas de Precipitina , Isoformas de Proteínas/metabolismo , Receptores Fc/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8A , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The yeast transcription factor Ace2p regulates expression of the chitinase gene CTS1 in a cell cycle-dependent manner. Nuclear localisation of Ace2p is restricted to late M and early G phases of the mitotic cell cycle. We show here that this nuclear localisation is directly associated with regulation of CTS1 expression. Using a version of Ace2p tagged with a c-myc epitope, we show that the protein is excluded from the nucleus of cells during most phases of the mitotic cell cycle. A mutant derivative in which one threonine and two serine residues, which are candidate phosphorylation sites, were replaced by alanine (to mimic constitutive dephosphorylation) is localised in the nucleus throughout the cell cycle. The mechanism of localisation of Ace2p therefore involves regulation of its phosphorylation state, and closely resembles that used by the homologous transcription factor Swi5p. The wild-type Ace2 protein associates with Cdc28p in vivo, suggesting this may be the kinase that mediates the phosphorylation event. The stability of the protein is greatly reduced in a mutant that is constitutively localised to the nucleus, but is restored in a deletion derivative which remains in the cytoplasm. Ace2p is therefore controlled throughout the cell cycle at three levels: transcription, nuclear localisation, and proteolysis.