RESUMO
BACKGROUND: Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. OBJECTIVE: We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. METHODS: Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. RESULTS: Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. CONCLUSION AND CLINICAL RELEVANCE: These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.
Assuntos
Hipóxia Celular/fisiologia , Eosinófilos/patologia , Interleucina-8/metabolismo , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Dexametasona/farmacologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Glucocorticoides/farmacologia , Humanos , Corpos de Inclusão/patologia , Inflamação/imunologia , Inflamação/patologiaRESUMO
Common Variable Immunodeficiency (CVID) comprises a heterogeneous group of primary antibody deficiencies which lead to a range of complications, including infectious, neoplastic and inflammatory disorders. This report describes monozygotic twin brothers with CVID who developed cryptogenic liver disease and subsequently hepatopulmonary syndrome (HPS). This is the second report of the association of HPS and CVID. Its occurrence in two identical twins implicates a genetic basis.
Assuntos
Imunodeficiência de Variável Comum/diagnóstico , Síndrome Hepatopulmonar/diagnóstico , Adolescente , Imunodeficiência de Variável Comum/imunologia , Síndrome Hepatopulmonar/imunologia , Humanos , Masculino , Gêmeos MonozigóticosRESUMO
This review, based on a talk given at the RCPE Respiratory Medicine Symposium 2014, outlines the clinical spectrum of immune deficiency - antibody (B cell) deficiency, T cell defects and innate/phagocytic disorders - and discusses the relevant clinical presentations, investigations and treatments, focusing particularly on the management of adults with recurrent respiratory infections. It describes when to suspect a primary immunodeficiency, the first-line investigations to perform and the triggers that should prompt referral for further specialist opinion. The paper concludes with a look at a novel primary immunodeficiency, Activated PI3 Kinase Delta Syndrome (APDS).
Assuntos
Síndromes de Imunodeficiência/diagnóstico , Infecções Respiratórias/imunologia , Adulto , Anticorpos/sangue , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/imunologiaRESUMO
This report summarizes the establishment of the first national online registry of primary immune deficency in the United Kingdom, the United Kingdom Primary Immunodeficiency (UKPID Registry). This UKPID Registry is based on the European Society for Immune Deficiency (ESID) registry platform, hosted on servers at the Royal Free site of University College, London. It is accessible to users through the website of the United Kingdom Primary Immunodeficiency Network (www.ukpin.org.uk). Twenty-seven centres in the United Kingdom are actively contributing data, with an additional nine centres completing their ethical and governance approvals to participate. This indicates that 36 of 38 (95%) of recognized centres in the United Kingdom have engaged with this project. To date, 2229 patients have been enrolled, with a notable increasing rate of recruitment in the past 12 months. Data are presented on the range of diagnoses recorded, estimated minimum disease prevalence, geographical distribution of patients across the United Kingdom, age at presentation, diagnostic delay, treatment modalities used and evidence of their monitoring and effectiveness.
Assuntos
Síndromes de Imunodeficiência , Internet , Sistema de Registros , Feminino , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/epidemiologia , Síndromes de Imunodeficiência/terapia , Masculino , Reino Unido/epidemiologiaRESUMO
Respiratory disease is a significant cause of morbidity and mortality amongst patients with primary immunodeficiency disorders. Computed tomography (CT) plays an important role in the multidisciplinary approach to these conditions, in detecting, characterizing, and quantifying the extent of lung damage and in directing treatment. The aim of this review is to classify the primary immunodeficiency disorders and describe the thoracic complications and the associated CT findings whilst discussing the role of radiology in diagnosis and surveillance.
Assuntos
Imunodeficiência de Variável Comum/diagnóstico por imagem , Doenças Respiratórias/diagnóstico por imagem , Adulto , Imunodeficiência de Variável Comum/complicações , Diagnóstico Diferencial , Feminino , Humanos , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Doenças Respiratórias/complicações , Tomografia Computadorizada por Raios X/métodosRESUMO
BACKGROUND: Eosinophils are pro-inflammatory cells implicated in the pathogenesis of asthma and atopy. Apoptosis has been proposed as a potential mechanism underlying the resolution of eosinophilic inflammation and studies have indicated the ability of interventions that induce human eosinophil apoptosis to promote the resolution of eosinophilic inflammation. Recently, the cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to enhance neutrophil apoptosis and promote the resolution of neutrophilic inflammation. OBJECTIVE: The purpose of this study was to examine the expression of CDKs in human blood eosinophils, the effects of R-roscovitine on eosinophil survival in vitro and whether R-roscovitine could influence eosinophilic lung inflammation in vivo. METHODS: Eosinophils were isolated from human peripheral blood and the effects of R-roscovitine on apoptosis, degranulation and phagocytic uptake examined in vitro. The effects of R-roscovitine on eosinophilic lung inflammation in vivo were also assessed using an ovalbumin mouse model. RESULTS: Our data demonstrate that human eosinophils express five known targets for R-roscovitine: CDK1, -2, -5, -7 and -9. R-roscovitine induced eosinophil apoptosis in a time- and concentration-dependent manner but also accelerated transition to secondary necrosis as assessed by microscopy, flow cytometry and caspase activation. In addition, we show that R-roscovitine can override the anti-apoptotic signals of GM-CSF and IL-5. We report that the pro-apoptotic effect of R-roscovitine is associated with suppression of Mcl-1L expression and that this compound enhanced phagocytic clearance of eosinophils by macrophages. Finally, we show that R-roscovitine induces apoptosis in murine peripheral blood and spleen-derived eosinophils; despite this, R-roscovitine did not modulate the tissue and lumen eosinophilia characteristic of the ovalbumin mouse model of airway eosinophilia. CONCLUSION AND CLINICAL RELEVANCE: These data demonstrate that R-roscovitine is capable of inducing rapid apoptosis and secondary necrosis in eosinophils but does not affect the onset or improve the resolution of eosinophilic airway inflammation in vivo.
Assuntos
Apoptose/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Animais , Apoptose/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/imunologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eosinófilos/imunologia , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Roscovitina , Fatores de TempoRESUMO
Previous studies have identified abnormalities in the oxidative responses of the neutrophil in cystic fibrosis (CF), but it is unclear whether such changes relate to loss of membrane cystic fibrosis transmembrane conductance regulator (CFTR) or to the inflammatory environment present in this disease. The aim of the present study was to determine whether neutrophils from CF patients demonstrate an intrinsic abnormality of the respiratory burst. The respiratory burst activity of neutrophils isolated from stable DeltaF508 homozygote CF patients and matched healthy controls was quantified by both chemiluminscence and cytochrome C reduction. Expression of NADPH oxidase components and CFTR was determined by Western blotting and RT-PCR. The oxidative output from neutrophils from CF in response to receptor-linked and particulate stimuli did not differ from that of controls. Expression of NADPH oxidase components was identical in CF and non-CF neutrophils. While low levels of CFTR mRNA could be identified in the normal human neutrophil, we were unable to detect CFTR protein in human neutrophil lysates or immunoprecipitates. CFTR has no role in controlling neutrophil oxidative activity; previously reported differences in neutrophil function between CF and non-CF subjects most likely relate to the inflammatory milieu from which the cells were isolated.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/imunologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adulto , Western Blotting , Fibrose Cística/metabolismo , Feminino , Expressão Gênica/imunologia , Humanos , Masculino , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Fosfoproteínas/metabolismo , Pneumonia/imunologia , Pneumonia/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/imunologia , Explosão Respiratória/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of the current study was to investigate the prevalence and clinical associations of nontuberculous mycobacteria (NTM) in a well-characterised cohort of patients with adult-onset bronchiectasis. The sputum of all patients attending a tertiary referral bronchiectasis clinic between April 2002 and August 2003 was examined for mycobacteria as part of an extensive diagnostic work-up. NTM-positive patients subsequently had further sputa examined. A modified bronchiectasis scoring system was applied to all high-resolution computed tomography (HRCT) scans from NTM-positive patients, and a matched cohort without NTM. Out of 98 patients attending the clinic, 10 had NTM in their sputum on first culture; of those, eight provided multiple positive cultures. Three patients were treated for NTM infection. A higher proportion of NTM-positive than -negative patients were subsequently diagnosed with cystic fibrosis (two out of nine versus two out of 75). On HRCT scoring, more patients in the NTM-positive group had peripheral mucus plugging than in the NTM-negative group. In the current prospective study of a large cohort of patients with bronchiectasis, 10% cultured positive for nontuberculous mycobacteria in a random clinic sputum sample. Few clinical parameters were helpful in discriminating between groups, except for a higher prevalence of previously undiagnosed cystic fibrosis and of peripheral mucus plugging on high-resolution computed tomography in the nontuberculous mycobacteria group.
Assuntos
Bronquiectasia/microbiologia , Infecções por Mycobacterium/microbiologia , Micobactérias não Tuberculosas/isolamento & purificação , Tuberculose Pulmonar/microbiologia , Idoso , Idoso de 80 Anos ou mais , Bronquiectasia/complicações , Bronquiectasia/diagnóstico por imagem , Estudos de Coortes , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Infecções por Mycobacterium/diagnóstico por imagem , Infecções por Mycobacterium/fisiopatologia , Prevalência , Estudos Prospectivos , Escarro/microbiologia , Tomografia Computadorizada Espiral/métodos , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/patologiaRESUMO
Neutrophils play a key role in the immediate non-specific immune response, and defects in their function increase host susceptibility to a range of infective agents. However, excess activation and/or delayed clearance of these cells from an inflamed site can lead to significant tissue damage. Neutrophil priming by agents such as endotoxin, granulocyte macrophage colony stimulating factor (GM-CSF), platelet activating factor (PAF) and tumour necrosis factor-alpha (TNF alpha) may play a pivotal role in modulating the adhesive and secretory properties of these cells. Priming also appears to affect the survival of neutrophils by delaying constitutive apoptosis. The unique signal transduction events that control neutrophil priming and apoptosis, and particularly the importance of the phospholipase C and phosphoinositide 3-kinase pathways, suggest opportunities for selective pharmacological intervention.
Assuntos
Apoptose , Inflamação/fisiopatologia , Neutrófilos/fisiologia , Apoptose/fisiologia , Humanos , Necrose , Ativação de Neutrófilo , Transdução de Sinais , Fosfolipases Tipo C/fisiologiaRESUMO
Phosphoinositide 3-kinases (PI-3Ks) are enzymes that generate lipid second messenger molecules, resulting in the activation of multiple intracellular signalling cascades. These events regulate a broad array of cellular responses including survival, activation, differentiation and proliferation and are now recognised to have a key role in a number of physiological and pathophysiological processes in the lung. PI-3Ks contribute to the pathogenesis of asthma by influencing the proliferation of airways smooth muscle and the recruitment of eosinophils, and affect the balance between the harmful and protective responses in pulmonary inflammation and infection by the modulation of granulocyte recruitment, activation and apoptosis. In addition they also seem to exert a critical influence on the malignant phenotype of small cell lung cancer. PI-3K isoforms and their downstream targets thus provide novel therapeutic targets for intervention in a broad spectrum of respiratory diseases.
Assuntos
Pulmão/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Humanos , Pulmão/citologia , Músculo Liso/citologiaRESUMO
In human neutrophils, significant agonist-stimulated superoxide anion (O2-) release is observed only after exposure to a priming agent such as TNFalpha. We have investigated the potential for TNFalpha to modulate N-formyl-Met-Leu-Phe (fMLP)-triggered Ins(1,4,5)P3 and PtdIns(3,4,5)P3 accumulation. TNFalpha pretreatment did not affect basal or stimulated Ins(1,4,5)P3 levels but greatly upregulated fMLP-stimulated PtdIns(3,4,5)P3 accumulation, in a manner that matched, both temporally and in magnitude, the increase in O2- generation implying a possible role for PtdIns(3,4,5)P3 in signalling primed O2- release.
Assuntos
Inositol 1,4,5-Trifosfato/metabolismo , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Superóxidos/metabolismo , Fator de Crescimento Transformador alfa/metabolismo , Humanos , N-Formilmetionina Leucil-Fenilalanina/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Radioisótopos de Fósforo , Transdução de SinaisRESUMO
1. Neutrophil priming by agents such as tumour necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor and lipopolysaccharide causes a dramatic increase in the response of these cells to an activating agent; this process has been shown to be critical for neutrophil-mediated tissue injury both in vitro and in vivo. 2. The principle consequence of priming, aside from a direct effect on cell polarization, deformability and integrin/selectin expression, is to permit secretagogue-induced superoxide anion generation, degranulation and lipid mediator (e.g. leukotriene B4 and arachidonic acid) release. It is now recognized that most priming agents also serve an additional function of delaying apoptosis and hence increasing the functional longevity of these cells at the inflamed site. 3. The potential mechanisms underlying priming are discussed; current data suggest a dissociation between priming and changes in receptor number and/or affinity, G-protein expression, phospholipase C and phospholipase A2 activation and changes in intracellular Ca2+ concentration. However, more recent studies support a key role for protein tyrosine phosphorylation and enhanced phospholipase D and phosphoinositide 3-kinase activity in neutrophil priming. 4. Recent work has also revealed the potential for neutrophils to spontaneously and fully 'de-prime' after an initial challenge with platelet-activating factor. This ability of neutrophils to undergo a complete cycle of priming-de-priming (and re-priming) reveals a previously unrecognized flexibility in the control of neutrophil behaviour at an inflamed site.
Assuntos
Mediadores da Inflamação/fisiologia , Inflamação/imunologia , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Humanos , Neutrófilos/fisiologia , Fosfolipase D/metabolismo , Explosão Respiratória , Fosfolipases Tipo C/metabolismoRESUMO
Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-alpha (TNF-alpha), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-alpha inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4, and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-alpha was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-alpha neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-alpha to accelerate apoptosis was lost if neutrophils were primed with 1 micromol/L PAF or aged for 6 hours before TNF-alpha addition. The TNFR55-selective TNF-alpha mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-alpha. Although the TNFR75-selective mutant (D143F) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-alpha-stimulated apoptosis. Hence, TNF-alpha has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.
Assuntos
Antígenos CD/fisiologia , Apoptose/fisiologia , Neutrófilos/citologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Fragmentação do DNA , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Cinética , Leucotrieno B4/farmacologia , Lipopolissacarídeos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Ácido Fítico/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Exposure of neutrophils to agents such as lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major upregulation of subsequent agonist-induced NADPH oxidase activation. This priming effect is a prerequisite for neutrophil-mediated tissue damage and has been widely considered to be an irreversible process. We have investigated the potential for neutrophils to recover from a priming stimulus by studying the effects of platelet-activating factor (PAF). PAF did not stimulate respiratory burst activity directly, but caused a rapid (maximal at 10 minutes) and concentration-dependent (EC50 50.2 nmol/L) increase in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide anion release. At time-points > 10 minutes, this priming effect spontaneously declined, with return to basal levels of fMLP-stimulated superoxide anion generation by 120 minutes. An identical priming time-course was observed with N-methyl carbamyl PAF, a nonmetabolizable analogue of PAF, indicating that the transient nature of PAF-induced priming was not secondary to PAF metabolism. Two structurally diverse PAF receptor antagonists (UK-74,505 and WEB 2086), added 10 minutes after PAF addition, increased the rate of decay of the priming effect. In contrast, TNF-alpha-induced priming, which was of a similar magnitude to that observed for PAF, was slower to evolve (maximal at 30 minutes) and remained constant for at least 120 minutes. The reversible nature of PAF-induced priming was confirmed by demonstrating that PAF-, but not TNF-alpha-, induced cell polarization (shape change) and CD11b-dependent neutrophil binding of albumin-coated latex beads was also transient, with return to basal, unstimulated levels by 120 minutes. Furthermore, cells that had spontaneously deprimed following PAF exposure retained their capacity to be fully reprimed by a subsequent addition of either PAF or TNF-alpha. These data imply that neutrophil priming is not an irreversible event: the demonstration of a cycle of complete priming, depriming, and repriming offers the potential for functional recycling of neutrophils at sites of inflammation.
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Adulto , Azepinas/farmacologia , Adesão Celular , Polaridade Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Di-Hidropiridinas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Antígeno de Macrófago 1/fisiologia , Microesferas , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Éteres Fosfolipídicos/farmacologia , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Explosão Respiratória , Superóxidos/metabolismo , Fatores de Tempo , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Lung injury in a variety of disease states is critically dependent on neutrophil-mediated inflammatory responses. Neutrophil recruitment to sites of infection or tissue damage requires co-ordinated regulation of neutrophil adhesion and activation status. We have examined the effects of treatment of human peripheral blood neutrophils with priming agents [lipopolysaccharide (LPS). tumor necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF)] upon expression of CD11a. CD11b. CD11c. CD35 and CD62-1 and CD11b function to assess whether subtle regulation of neutrophil adhesion potential accompanies augmented formyl-methionyl-leucyl-phenylalanine-stimulated superoxide production. We have found that there are differential effects of priming concentrations of these agents. For LPS. CD62L loss occurs in the absence of changes in CD11b, whereas for PAF. CD11b up-regulation occurs in the absence of detectable loss of CD62-L. However, for TNF-2, decreased expression of CD62-L occurs concomitantly with increased expression of CD11b. In addition, we have shown that priming agents augment CD11b functional activity in a manner that parallels the priming of the respiratory burst. Thus, priming agents may differentially regulate neutrophil adhesive capacity and data presented in this manuscript suggest that the increased effector cell function observed in primed cells may be associated with a distinct repertoire of potential adhesive interactions.
Assuntos
Antígenos CD/imunologia , Adesão Celular , Ativação de Neutrófilo , Neutrófilos/imunologia , Adesão Celular/efeitos dos fármacos , Humanos , Integrina alfaXbeta2/imunologia , Selectina L/imunologia , Lipopolissacarídeos/farmacologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno de Macrófago 1/imunologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Receptores de Complemento 3b/imunologia , Explosão Respiratória/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Inositol hexakisphosphate (InsP6) is a ubiquitous and abundant cytosolic inositol phosphate that has been reported to prime human neutrophils for enhanced agonist-stimulated superoxide anion generation. This led to the proposal that the release of InsP6 from necrotic cells may augment the functional responsiveness of neutrophils at an inflammatory focus. The aim of this study was to examine whether the functional effects of InsP6 in neutrophils are receptor-mediated and establish the magnitude of this priming effect relative to other better characterized priming agents. 2. Analysis of [3H]-InsP6 binding to human neutrophil membranes in 20 mM Tris, 20 mM NaCl, 100 mM KCl, 5 mM EDTA (pH 7.7) buffer using 0.1 mg ml-1 membrane protein and 2.5 nM [3H]-InsP6 (90 min, 4 degrees C), demonstrated specific low affinity [3H]-InsP6 binding that was non-saturable up to a radioligand concentration of 10 nM. 3. [3H]-InsP6 displacement by InsP6 gave a Hill coefficient of 0.55 and best fitted a two-site logistic model (53% KD 150 nM, 47% KD 5 microM). [3H]-InsP6 binding also displayed low (3 fold) selectivity for InsP6 over Ins(1,3,4,5,6)P5. 4. The specific [3H]-InsP6 binding displayed a pH optimum of 8, was abolished by pre-boiling the membranes, and was enhanced by Ca2+, Mg2+ and Na+. 5. In incubations with intact neutrophils, where high levels of specific [3H]-LTB4 binding was observed, no [3H]-InsP6 binding could be identified. 6. Preincubation of neutrophils with 100 microM InsP6 had no effect on resting cell morphology, but caused a minor and transient (maximal at 30 s) enhancement of (0.1 nM) fMLP-induced shape change (% cells shape changed: fMLP 53 +/- 3%, fMLP+InsP6 66 +/- 4%). Similarly, InsP6 (100 microM, 30 s) had no effect on basal superoxide anion generation and, compared to lipopolysaccharide (LPS, 100 ng ml-1, 60 min), tumour necrosis factor-alpha (TNF alpha, 200 u ml-1, 30 min) or platelet-activating factor (PAF, 100 nM, 5 min) caused only a small enhancement of 100 nM fMLP-stimulated superoxide anion generation (fold-increase in superoxide anion generation over fMLP alone: InsP6 1.8 +/- 0.3, LPS 6.8 +/- 0.6, TNF alpha 5.2 +/- 0.7, PAF 5.8 +/- 0.6). 7. While these data support the presence of a specific, albeit low affinity, [3H]-InsP6 binding site in human neutrophil membrane preparations, the lack of binding to intact cells implies that the functional effects of InsP6 (ie. enhanced fMLP-stimulated superoxide anion generation and shape change) are not receptor-mediated.
Assuntos
N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Ácido Fítico/farmacologia , Receptores Citoplasmáticos e Nucleares/análise , Explosão Respiratória/efeitos dos fármacos , Superóxidos/metabolismo , Adulto , Humanos , Lipopolissacarídeos/farmacologia , Neutrófilos/metabolismo , Ácido Fítico/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Androstadienos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neutrófilos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Ceramidas/farmacologia , Sinergismo Farmacológico , Humanos , Cinética , Neutrófilos/citologia , Neutrófilos/fisiologia , Fosfatidilinositol 3-Quinases , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Transdução de Sinais , Esfingomielina Fosfodiesterase/farmacologia , WortmaninaRESUMO
Platelet-derived growth factor (PDGF) has been reported to induce chemotaxis, degranulation, and superoxide anion generation, and to increase the expression of CD11b/CD18 in human neutrophils; hence, it has been proposed as an important regulator of neutrophil function. Most of the studies on PDGF, however, have been complicated by the use of nonrecombinant PDGF or the use of mixed leukocyte cell preparations. Assessment of the effects of recombinant human PDGF-AB or -BB which display agonist activity against both PDGF receptor subtypes failed to demonstrate any effect of this peptide on neutrophil shape change, respiratory burst activity, CD11/CD18, or CD62-L expression, inositol 1,4,5-trisphosphate accumulation, or phosphorylation of mitogen-activated protein kinase. This apparent lack of effect of PDGF was consistent with our findings that neutrophils display no specific 125I-PDGF-AB or -BB binding and lack detectable mRNA for PDGF alpha-receptor and beta-receptors. These data indicate that human neutrophils do not possess functional PDGF receptors and question previous reports of a functional effect of this peptide in these cells.