RESUMEN
Traditional enzyme kinetics provide a poor explanation for the increased risk of lung injury in alpha 1-antitrypsin (AAT) deficiency. Millimolar concentrations of leukocyte elastase, when released from single azurophil granules of activated neutrophils, lead to evanescent quantum bursts of proteolytic activity before catalysis is quenched by pericellular inhibitors. Herein, we tested the possibility that quantum proteolytic events are abnormal in AAT deficiency. We incubated neutrophils on opsonized fluoresceinated fibronectin in serum from individuals with various AAT phenotypes, and then measured and modeled quantum proteolytic events. The mean areas of the events in serum from heterozygous individuals (Pi MZ and Pi SZ) were slightly, but significantly, larger than those in serum from normal patients (Pi M). In marked contrast, mean areas of events in serum from AAT-deficient individuals were 10-fold larger than those in serum from normal patients. Diffusion modeling predicted that local elastase concentrations exceed AAT concentrations for less than 20 milliseconds and for more than 80 milliseconds in Pi M and Pi Z individuals, respectively. Thus, quantum proteolytic events are abnormally large and prolonged in AAT deficiency, leading directly to an increased risk of tissue injury in the immediate vicinity of activated neutrophils. These results have potentially important implications for the pathogenesis and prevention of lung disease in AAT deficiency.
Asunto(s)
Endopeptidasas/sangre , Neutrófilos/enzimología , Enfisema Pulmonar/enzimología , Deficiencia de alfa 1-Antitripsina/enzimología , Gránulos Citoplasmáticos/enzimología , Humanos , Hidrólisis , Mediadores de Inflamación/farmacología , Focalización Isoeléctrica , Modelos Biológicos , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fenotipo , Enfisema Pulmonar/sangre , Enfisema Pulmonar/genética , Teoría Cuántica , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/sangreRESUMEN
Membrane-bound leukocyte elastase activity on neutrophils may have potent proinflammatory effects. Herein, we report the effects of tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), N-formyl-leucyl-methionyl-phenylalanine (fMLP), and interleukin-8 (IL-8) on membrane-bound elastase expression. TNF-alpha or PAF alone induced only approximately two- to threefold increases in membrane-bound elastase but exhibited marked dose- and time-dependent priming effects for subsequent stimulation with fMLP or IL-8 (up to 20-fold increases in membrane-bound human leukocyte elastase compared with unstimulated cells). Optimally PAF-primed and fMLP-stimulated cells expressed 1.105 +/- 0.25 (SD) x 10(-17) mol [6.65 +/- 1.51 (SD) x 10(6) molecules] membrane-bound elastase activity/cell or approximately 12% of the content of unstimulated cells. Elastase binds to the cell surface by a charge-dependent mechanism since 1) incubation of cells with cationic molecules abrogated agonist-induced upregulation of membrane-bound elastase and 2) elastase was progressively eluted from the cell surface by solutions with increasing ionic strength. Thus interactions between proinflammatory mediators strikingly upregulate membrane-bound elastase on neutrophils, which may promote inflammatory responses and/or contribute to tissue injury.
Asunto(s)
Elastasa de Leucocito/metabolismo , Neutrófilos/enzimología , Humanos , Inflamación/enzimología , Interleucina-8/farmacología , Iones , N-Formilmetionina Leucil-Fenilalanina/farmacología , Factor de Activación Plaquetaria/administración & dosificación , Propiedades de Superficie , Factor de Necrosis Tumoral alfa/administración & dosificación , Regulación hacia ArribaRESUMEN
Catalytically active cathepsin G that is bound to the cell surface of human neutrophils may play a variety of roles in normal neutrophil biology and in pathobiology associated with inflammation. In this study, we describe expression of neutrophil cell surface-bound cathepsin G in response to TNF-alpha and platelet-activating factor (PAF) under conditions in which minimal free release of cathepsin G is detected. TNF-alpha and PAF alone induced modest (two- to threefold) increases in cell surface-bound cathepsin G, but exhibited a marked dose- and time-dependent priming effect for subsequent chemoattractant-induced responses (up to 15- to 25-fold increases in cell surface expression). When optimally primed (TNF-alpha, 100 U/ml, or PAF, 10(-9) M), neutrophils expressed five- to sixfold more cell surface-bound cathepsin G, in comparison with cells exposed to FMLP alone. Priming responses were more rapid with PAF (15 s to 5 min) than with TNF-alpha (1 to 60 min). Optimally primed and FMLP-stimulated neutrophils express approximately 160 ng of catalytically active cathepsin G per 10(6) cells, which represents approximately 11% of the cellular content of unstimulated cells. Cathepsin G binds to the cell surface by a charge-dependent mechanism since: 1) incubation of cells with highly positively charged molecules abrogated agonist-induced up-regulation of the cell surface expression of cathepsin G and 2) cathepsin G was eluted from the cell surface by high concentrations of NaCl. These data indicate that interactions between biologically relevant pro-inflammatory cytokines and chemoattractants serve to markedly up-regulate cell surface-bound cathepsin G. The focused catalytic activity of cell surface-bound cathepsin G may alter endothelial and epithelial barriers, promote thrombogenesis, injure extracellular matrix, and/or facilitate directed migration of neutrophils during inflammation.
Asunto(s)
Catepsinas/metabolismo , Neutrófilos/metabolismo , Catepsina G , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Inducción Enzimática , Humanos , Proteínas de la Membrana/biosíntesis , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/farmacología , Unión Proteica , Serina Endopeptidasas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface-bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.
Asunto(s)
Catepsinas/metabolismo , Elastasa de Leucocito/metabolismo , Neutrófilos/enzimología , Elastasa Pancreática/metabolismo , Serina Endopeptidasas/metabolismo , Catepsina G , Factores Quimiotácticos/farmacología , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/enzimología , Humanos , Inmunohistoquímica , Lipopolisacáridos/farmacología , Proteínas de la Membrana/fisiología , Peso Molecular , Oxidación-ReducciónRESUMEN
We have demonstrated that a discrete and naturally occurring subpopulation of human monocytes expresses a neutrophil-like proinflammatory (P) phenotype. P monocytes constitute 20-30% of the circulating monocyte pool and are characterized by 1) avid adherence to extracellular matrix through high-level cell-surface expression of alpha 5-, beta 1-, and beta 2-integrins; 2) high capacity to produce reactive oxygen species; 3) high content of serine proteinases and alpha 1-proteinase inhibitor; and 4) proteolytic activity against a soluble peptide human leukocyte elastase substrate, [3H]elastin, and solid-phase fibronectin, even in the presence of proteinase inhibitors. However, P monocytes express little or no cell-surface HLA-DR antigen, suggesting that they are unable to participate in specific immune responses. In contrast, the remainder of circulating monocytes have a low proinflammatory potential but contain the population of monocytes with high-level expression of HLA-DR antigen. P monocytes can readily be separated from the remainder of monocytes on the basis of 1) their capacity to adhere to fibronectin; and 2) their absent expression of HLA-DR antigen when flow cytometry or immunomagnetic beads are used. Our data indicate that, when recruited to sites of inflammation, P monocytes can either promote resolution of inflammation or contribute to tissue injury.
Asunto(s)
Monocitos/fisiología , Neutrófilos/fisiología , Separación Celular , Antígenos HLA-DR/análisis , Humanos , Integrinas/análisis , Monocitos/enzimología , Monocitos/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Fenotipo , Serina Endopeptidasas/análisis , alfa 1-Antitripsina/análisisRESUMEN
Only a minor proportion of monocytes responds to chemoattractants. To test the possibility that chemoattractant-responsive monocytes have distinctive functional characteristics, we enriched or depleted monocyte preparations for cells having a proinflammatory (P) phenotype and tested their responses to biologically relevant chemoattractants. We prepared monocyte subpopulations by one of three independent techniques to minimize the chances of artifacts: 1) depletion of P monocytes by adherence to fibronectin; 2) enrichment for P monocytes by negative selection for HLA-DR antigen; and 3) flow cytometric sorting. We measured responsiveness of monocyte subpopulations to N-formyl-Met-Leu-Phe, C5a, zymosan-activated serum, and monocyte chemoattractant protein-1 by three parameters: 1) polarization, 2) actin polymerization, and 3) directed migration. With each chemoattractant and each parameter, there was a striking direct relationship between the responsiveness of the monocyte preparations and their content of P monocytes. Our data indicate that the capacity of monocytes to be recruited rapidly from the vasculature into sites of inflammation is a property of a subpopulation of monocytes with a distinctive, neutrophil-like proinflammatory phenotype.