RESUMO
Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiocinas CXC/biossíntese , Epitopos de Linfócito T/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Pulmão/imunologia , Pulmão/metabolismo , Ativação Linfocitária/imunologia , Regulação para Cima/imunologia , Administração por Inalação , Administração Intranasal , Aerossóis , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Linfócitos T CD4-Positivos/citologia , Inibição de Migração Celular , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas/biossíntese , Quimiocinas/genética , Quimiocinas/imunologia , Quimiocinas CXC/antagonistas & inibidores , Fatores Quimiotáticos/antagonistas & inibidores , Fatores Quimiotáticos/biossíntese , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Substâncias de Crescimento/imunologia , Soros Imunes/administração & dosagem , Soros Imunes/farmacologia , Pulmão/citologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neutrófilos/imunologia , Ovalbumina/administração & dosagem , Ovalbumina/antagonistas & inibidores , Ovalbumina/imunologia , Ovalbumina/metabolismoRESUMO
CD4(+) T cells are thought to play a major role in the initiation and perpetuation of T helper cell, type 2 (Th2)-like allergic airway inflammation. However, it is not clear whether activation of resident antigen-specific CD4(+) T cells is in itself sufficient to induce such a phenotype. Using ovalbumin (OVA)-specific alphabeta-T cell receptor transgenic Balb/c DO11.10 mice, we were able to test this hypothesis. Nonsensitized DO11.10 mice but not wild-type mice responded to a primary OVA aerosol with a rapid and impressive bronchoalveolar lavage (BAL) neutrophilia followed by a smaller but significant eosinophilia. Responses in DO11.10 mice were mediated by OVA-specific activation of CD4(+) T cells because in vivo depletion of CD4(+) but not CD8(+) T cells abrogated inflammatory cell influx. Cytokines measured in BAL fluid (BALF) after OVA aerosol exposure of DO11.10 mice were indicative of a T helper cell, type 1 (Th1)-like immune response. Further, neutralization of interferon gamma (IFN-gamma) with antibody enhanced eosinophil influx, suggesting that IFN-gamma production was limiting the development of a Th2 response. Despite this, an increased prevalence of cells staining for mucus was seen in the bronchial epithelium, a feature more commonly associated with a Th2-immune response. Unlike what was seen in OVA-sensitized wild-type mice, multiple OVA aerosol exposures of DO11.10 mice failed to induce airway hyperresponsiveness (AHR) to inhaled methacholine. In conclusion, in vivo stimulation of resident lung CD4(+) T cells with antigen caused lung inflammation with characteristics of both a Th1- and Th2-immune response but was insufficient to directly induce AHR.
Assuntos
Hiper-Reatividade Brônquica/imunologia , Linfócitos T CD4-Positivos/imunologia , Pulmão/imunologia , Animais , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Epitopos , Feminino , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Hipersensibilidade Respiratória/imunologia , Células Th2/imunologiaRESUMO
The effects of endothelin-1 (ET-1) and sarafotoxin S6c (S6c) on cholinergic contractions elicited by electrical field stimulation (EFS) were examined in mouse tracheal preparations from healthy animals and from animals infected with parainfluenza-1 (P-1) virus. S6c (an ETB-selective agonist) and ET-1 caused marked ETA and/or ETB receptor-mediated potentiation of EFS-induced contraction in tracheal tissue from both groups. Despite the fact that such infection is known to markedly alter ET receptor density and function in mouse tracheal smooth muscle, no evidence for modulated neuronal ET receptor function was obtained. The reason for this differential sensitivity of smooth muscle and neuronal ET receptors to P-1 infection is unknown.
Assuntos
Endotelina-1/fisiologia , Músculo Liso/fisiopatologia , Sistema Nervoso Parassimpático/fisiopatologia , Infecções Respiratórias/fisiopatologia , Infecções por Respirovirus/fisiopatologia , Respirovirus , Animais , Estimulação Elétrica , Contração Isométrica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos CBA , Músculo Liso/inervação , Receptores de Endotelina/metabolismo , Infecções Respiratórias/virologia , Infecções por Respirovirus/virologia , Traqueia/fisiopatologia , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologiaRESUMO
The aim of this study was to assess the influence of endothelin-1 (ET-1) on cholinergic nerve-mediated contractions in rat isolated tracheal smooth muscle by use of electrical-field stimulation (EFS) and [3H]choline efflux studies. EFS (80 V, 0.5 ms, 0.1-30 Hz for 10 s) evoked transient, frequency-dependent contractions of isolated tracheal preparations. Contractions were abolished in the presence of atropine or tetrodotoxin, which suggests they were mediated by acetylcholine (ACh) release from cholinergic nerves. The ETB receptor-selective agonist sarafotoxin S6c (1 nM) augmented EFS (0.6-1 Hz)-induced contractions by 179%. These effects were significantly attenuated in the presence of the ETB receptor-selective antagonist N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1- methoxycarbonyltryptophanyl-D-norleucine (BQ-788; 1 microM). ET-1 (1 nM) also markedly potentiated EFS-induced contractions (153%). This was apparently not a postjunctional effect, because ET-1 did not alter contractile responses to exogenously applied ACh. Cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu] (BQ-123;3 microM) and BQ-788 when used alone, failed to inhibit ET-1-induced potentiation of EFS-evoked contractions. However, in their combined presence, BQ-123 and BQ-788 significantly attenuated ET-1-induced potentiation of EFS responses. EFS (100 V, 0.5 ms, 3 Hz for 2 min) applied to tracheal preparations preloaded with [3H]choline, caused airway smooth muscle contraction and an efflux of radioactivity. Both sarafotoxin S6c (10 nM) and ET-1 (10 nM) significantly enhanced the EFS-induced 3H-efflux and the latter was abolished only in the combined presence of BQ-123 and BQ-788. These data indicated that ET-1 enhances cholinergic nerve-mediated contractions in rat isolated trachea via activation of prejunctional ETA and ETB receptors that were linked to increased ACh release from cholinergic nerves.
Assuntos
Acetilcolina/metabolismo , Endotelina-1/farmacologia , Músculo Liso/efeitos dos fármacos , Receptores Colinérgicos/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Animais , Estimulação Elétrica , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/metabolismo , Ratos , Ratos Wistar , Receptores Colinérgicos/fisiologia , Traqueia/inervação , Traqueia/metabolismo , TrítioRESUMO
1. In this study we have compared the effects of parainfluenza-1 respiratory tract viral infection on the density and function of ETA and ETB receptors in rat and mouse tracheal airway smooth muscle. 2. The bronchoconstrictor effect of inhaled methacholine was significantly enhanced in virus-infected rats, at both 4 and 12 days post-inoculation. That is, the concentration of methacholine causing an increase in resistance of 100% (PC100 methacholine) was significantly lower in virus-infected animals at both 4 and 12 days post-inoculation (n = 6-8; P < 0.05). 3. Total specific binding of [125I]-endothelin-1 and the relative proportions of ETA and ETB binding sites for [125I]-endothelin-1 were assessed in tracheal airway smooth muscle in parainfluenza-1-infected rats and mice at days 2, 4 and 12 post-inoculation using the ligands BQ-123 (1 microM; ETA receptor-selective) and sarafotoxin S6c (100 nM; ETB receptor-selective). Total specific binding in mice was significantly reduced at day 2 post-inoculation (n = 5; P < 0.05) but not at days 4 and 12 post-inoculation (n = 5). In control mice, the proportions of ETA and ETB binding sites were 53%:47% at day 2 and 43%:57% at day 4 and these were significantly altered by parainfluenza-1 infection such that, the ratios were 81%:19% at day 2 and 89%:11% at day 4 (P < 0.05). By day 12 post-inoculation, the proportion of ETA and ETB binding sites in tracheal smooth muscle from mice infected with parainfluenza-1 was not significantly different from control. In rat tracheal airway smooth muscle, neither total specific binding nor the ETA and ETB binding site ratio (64%:36%) were significantly altered in virus-inoculated rats at days 2, 4 or 12 post-inoculation (n = 5). 4. Parainfluenza-1 infection in mice had no effect on the sensitivity or maximal contractile effect of endothelin-1 in tracheal smooth muscle at days 2, 4 or 12 post-inoculation (n = 4). In contrast, contraction in response to the ETB receptor-selective agonist sarafotoxin S6c was attenuated by 39% at day 2 and by 93% at day 4 post-inoculation (P < 0.05). However, by day 12 post-inoculation, contractions to sarafotoxin S6c were not significantly different between control and virus-infected mice. In parainfluenza-1-infected rats, there were small but significant reductions in the sensitivity to carbachol, endothelin-1 and sarafotoxin S6c whilst the maximal responses to the highest concentrations of these agonists were not significantly altered by virus infection (n = 8). 5. BQ-123 (3 microM) had no significant effect on cumulative concentration-effect curves to endothelin-1 in tracheal preparations from control mice (n = 4) or parainfluenza-1-infected rats (n = 8). In contrast, in tissues taken from virus-infected mice at day 4 post-inoculation, BQ-123 caused a marked 9.6 fold rightward shift in the concentration-effect curve to endothelin-1 (n = 4). 6. In summary, we have demonstrated that parainfluenza-1 infection in mice transiently reduced the density of tracheal airway smooth muscle ETB receptors and this was reflected in reduced responsiveness to the ETB receptor-selective agonist sarafotoxin S6c. In contrast, whilst parainfluenza-1 infection in rats was associated with the pathological features and bronchial hyperresponsiveness common to respiratory tract viral infection, there was no selective down-regulation of ETB receptor expression or functional activity. The reasons for these species differences are not clear, but may relate to differences in the airway inflammatory response to parainfluenza-1 virus.
Assuntos
Músculo Liso/ultraestrutura , Vírus da Parainfluenza 1 Humana , Receptores de Endotelina/fisiologia , Infecções Respiratórias/fisiopatologia , Infecções por Respirovirus/fisiopatologia , Traqueia/ultraestrutura , Animais , Antagonistas dos Receptores de Endotelina , Endotelina-1/farmacologia , Técnicas In Vitro , Masculino , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos CBA , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Peptídeos Cíclicos/farmacologia , Ratos , Ratos Wistar , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/metabolismo , Infecções Respiratórias/virologia , Infecções por Respirovirus/virologia , Fatores de Tempo , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologiaRESUMO
Although initial research interest in the ETs was focused on their cardiovascular effects, it is now clearly established that these peptides have wide-ranging activities in the respiratory track. Importantly, ET-1 is synthesized, stored, released and metabolized in the lung, suggesting that these activities may be relevant to both physiological function and pathophysiological processes in the lung. To the present time, only two ET receptor subtypes have been definitively characterized in the lung, namely ETA and ETB receptors, both of which have been shown to mediate contraction and mitogenesis in airway smooth muscle from humans and animals. However, the possibility that further receptor subtypes might be defined from functional, biochemical, and molecular biological studies cannot be dismissed. It is apparent that the ETs may have a role to play in the pathogenesis of several pulmonary diseases. However, most of the current evidence for this falls short of establishing convincing causal associations. Perhaps the most convincing data relate to pulmonary hypertension. Although it is too early to ascribe a role for the ETs in the pathophysiology of asthma, the preliminary data are intriguing. Thus far, research has centered largely on the bronchoconstrictor effects of ET-1 in vitro and in vivo. In relation to asthma, it is important that future studies should focus increasingly on the effects of the ETs in nerves and inflammatory cells. In addition, the effects of chronic airway exposure to ET-1 on smooth muscle and fibroblast proliferation is an important area for future research. The unequivocal testing of the pathophysiological role of the ETs in asthma requires the clinical evaluation of potent and selective receptor antagonists for the various ET receptor subtypes. It seems likely that these studies will be conducted in the not-too-distant future, as compounds which possess the appropriate pharmacological profile become available for clinical evaluation. Additionally, ECE-1 and ECE-2 present as potentially important targets for therapeutic intervention, although the development of selective nonpeptide inhibitors may be some years away.
Assuntos
Endotelinas/fisiologia , Fenômenos Fisiológicos Respiratórios , Sequência de Aminoácidos , Animais , Brônquios/efeitos dos fármacos , Brônquios/fisiologia , Endotelinas/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/fisiologia , Dados de Sequência Molecular , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiologia , Veias Pulmonares/metabolismo , Veias Pulmonares/fisiologia , Sistema Respiratório/metabolismoRESUMO
The potent bronchoconstrictor and mitogenic actions of the peptide endothelin-1 (ET-1) on airway smooth muscle may contribute significantly to the bronchial obstruction observed in asthma. However, the status of the receptor-effector systems that mediate these actions of ET-1 in asthmatic airways is currently unknown. Thus, we have used quantitative autoradiographic and isometric-tension recording techniques to evaluate the density, distribution, and function of the specific receptors that mediate the actions of ET-1 in both asthmatic and nonasthmatic airways. Here, we report that similar numbers of specific binding sites for [125I]-ET-1 exist in asthmatic and nonasthmatic airways, with the greatest densities located in airway smooth muscle in both tissue types. The ETB-receptor subtype constituted approximately 82% and 88% of these receptors for ET-1 in asthmatic and nonasthmatic human bronchial smooth muscle, respectively, and mediated contraction in response to this peptide. In addition, a component of ET-1-induced contraction appeared to be mediated by a non-ETB, BQ-123-resistant mechanism. Furthermore, a small population of ETA sites was identified that did not mediate contraction, but which may have a role in ET-1-induced prostanoid release and airway smooth-muscle proliferation. Interestingly, bronchial smooth muscle from asthmatic lung was significantly less sensitive to the contractile effects of ETB receptor activation, consistent with desensitization of this receptor subtype in response to the increased production and release of ET-1 that occurs in this disease.
Assuntos
Asma/metabolismo , Brônquios/química , Receptores de Endotelina/análise , Adolescente , Adulto , Asma/fisiopatologia , Autorradiografia , Brônquios/efeitos dos fármacos , Brônquios/fisiopatologia , Carbacol/farmacologia , Relação Dose-Resposta a Droga , Endotelinas/farmacologia , Feminino , Humanos , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/química , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/agonistas , Receptores de Endotelina/efeitos dos fármacos , Receptores de Endotelina/fisiologia , Análise de Regressão , Venenos de Víboras/farmacologiaRESUMO
[125I]-endothelin-1 ([125I]-ET-1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non-asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non-asthmatic and asthmatic lung. The use of the receptor subtype-selective ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non-asthmatic alveolar wall tissue demonstrated that 29-32% of specific [125I]-ET-1 binding was to ETA sites and 68-71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.
Assuntos
Asma/metabolismo , Pulmão/química , Receptores de Endotelina/análise , Adolescente , Adulto , Autorradiografia , Antagonistas dos Receptores de Endotelina , Feminino , Humanos , Pulmão/metabolismo , Masculino , Peptídeos Cíclicos/farmacologia , Receptores de Endotelina/metabolismoRESUMO
1. Autoradiographic studies were conducted to investigate the receptor subtypes for endothelin-1 (ET-1) that were present in the ovine respiratory tract. In addition, the receptor subtypes mediating contraction of airway smooth muscle and the possible involvement of extracellular Ca2+ and inositol phosphate generation in intracellular signal transduction were assessed. 2. Specific [125I]-ET-1 binding in ovine trachea increased in a time- and concentration-dependent manner. Autoradiographic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding were associated with submucosal glands and with cells immediately below the epithelial basement membrane (lamina propria). The ETA receptor-selective antagonist, BQ 123 (1 microM), virtually abolished specific binding to airway smooth muscle. Quantitative analyses of autoradiographic data describing the time-dependence of specific [125I]-ET-1 binding in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ETA receptors. BQ 123 (1 microM) also abolished specific binding to structures associated with submucosal glands, whereas the ETB receptor selective agonist, sarafotoxin S6c (100 nM) had little effect on this binding, indicating the predominance of ETA receptors at these sites. In contrast, ETB receptors predominated in the lamina propria, since sarafotoxin S6c abolished specific binding in this tissue. 3. High levels of specific [125I]-ET-1 binding were also detected in the alveoli and in the walls of blood vessels and small airways in ovine peripheral lung. Specific binding associated with alveoli was reduced to similar extents by BQ 123 (1 MicroM; 54%) and sarafotoxin S6c (100 nM; 40%), suggesting the coexistence of both ETA and ETB receptors in approximately equal proportions in this tissue. In contrast,specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in the presence of BQ 123 (1 MicroM), but was unaffected by sarafotoxin S6c, indicating the presence of only ETA receptors at these sites.4. ET-1 caused concentration-dependent contractions of ovine tracheal smooth muscle which were inhibited in the presence of BQ 123 (1 MicroM). ET-1 also caused concentration-dependent contraction of ovine lung parenchyma strips. In contrast, the ETB receptor-selective agonists, sarafotoxin S6c and BQ 3020, were virtually inactive as spasmogens in both tracheal smooth muscle and lung strip preparations.Thus contraction was mediated by ETA receptors in ovine tracheal smooth muscle and this is consistent with binding and autoradiographic data demonstrating a homogeneous population of these binding sites in this tissue. Contraction of parenchymal lung strip preparations to ET-1 was mediated via non-ETB receptors, presumably ETA receptors, with contributions to this response perhaps coming from airway and vascular smooth muscle and from alveolar wall contractile cells.5. ET-1-induced contraction of tracheal smooth muscle was not significantly altered in the presence of indomethacin (5 MicroM), indicating that cyclo-oxygenase metabolites of arachidonic acid were not involved in this response. Contraction induced by ET-1 was virtually abolished in Ca2+-free medium containing 0.1 mM EGTA, indicating that this response was dependent upon the influx of extracellular Ca2 .Contraction was inhibited by about 50% in the presence of nicardipine (1 MicroM), indicating that a significant component of this response was mediated via the activation of L-type Ca2+ channels.6. ET-1 caused poorly defined increases in the accumulation of intracellular inositol phosphates in ovine tracheal smooth muscle. The maximal response to ET-1 was less than 20% of that to the cholinoceptor agonist, carbachol. Furthermore, sarafotoxin S6c was inactive. These data, when taken together with the results of autoradiographic and contraction studies, indicate that ovine airway smooth muscle contraction in response to ET-1 is mediated via ETA receptors which are linked to the influx of extracellular Ca2+, partly through voltage-dependent channels. ETB receptors also exist in the lamina propria of ovine trachea and in peripheral alveoli, perhaps residing in vascular endothelial cells.