RESUMO
Segmental endotoxin challenge with lipopolysaccharide (LPS) can be used as a pharmacodynamic model to safely induce a transient airway inflammation in the peripheral lung of healthy subjects and to test the anti-inflammatory efficacy of investigational new drugs. In contrast to whole lung LPS challenge only a fraction of the dose is required that can be precisely administered to a specific lung region and a vehicle challenged segment as an intra-subject control can be included. The aim of this study was to assess the intra- and inter-individual variability of the response to segmental LPS challenge for the appropriate design and power calculation of future clinical trials. Two cohorts with 10 subjects each underwent two segmental LPS challenges within five weeks. The inflammatory response was evaluated in bronchoalveolar lavage (BAL) fluid at 6 (cohort 1) and 24 h (cohort 2) both in the LPS and in a vehicle challenged segment, as well as in plasma for up to 26 h post LPS challenge. While the cytokine response was more pronounced at 6 h, the influx of neutrophils and monocytes dominated at 24 h; e.g. neutrophils increased from a median (inter-quartile range, IQR) of 0.14 (0.16) and 0.09 (0.08)x10(4) cells/mL BAL fluid at baseline to 10.2 (17.1) and 19.3 (15.9)x10(4) cells/mL 24 h after the two separate challenges. The within-subject variability was higher than the between-subject variability for most of the markers. However, sample size estimations based on the variability of outcome variables found lower or equal numbers with cross-over designs compared to parallel group designs for cellular markers at 24 h and cytokine variables at 6 h. The segmental LPS challenge model was safe. Future study designs have to balance between burden to the study subjects (4 versus 2 bronchoscopies), variability (within-versus between-subject), and the desired outcome variable (cells versus chemo/cytokine).
Assuntos
Endotoxinas/toxicidade , Lipopolissacarídeos/toxicidade , Pneumonia/induzido quimicamente , Pneumonia/patologia , Adulto , Biomarcadores/sangue , Líquido da Lavagem Broncoalveolar , Broncoscopia , Quimiocinas/sangue , Estudos de Coortes , Citocinas/sangue , Feminino , Voluntários Saudáveis , Humanos , Masculino , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Reprodutibilidade dos Testes , Testes de Função RespiratóriaRESUMO
Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.
Assuntos
Benzenoacetamidas , Fatores Quimiotáticos de Eosinófilos/metabolismo , Diterpenos , Eosinófilos/fisiologia , Leucotrieno B4/biossíntese , Macrófagos Alveolares/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Eosinófilos/efeitos dos fármacos , Ginkgolídeos , Cobaias , Ácidos Hidroxâmicos/farmacologia , Indometacina/farmacologia , Lactonas/farmacologia , Leucotrieno B4/fisiologia , Inibidores de Lipoxigenase/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/biossíntese , Sensibilidade e Especificidade , Estimulação QuímicaRESUMO
Replacement therapy with exogenous surfactant has been proven successful in animal models of acute respiratory distress syndrome (ARDS). Here, we investigated the effect of seminatural surfactant Curosurf on the expression of secretory phospholipase A2 (sPLA2) in guinea-pig alveolar macrophages (AM). The latter produced an sPLA2 activity whose level was markedly reduced when culture medium was supplemented with Curosurf. This effect was concentration-dependent and was accompanied by a decrease in sPLA2 mRNA levels. By contrast, when AM were first cultured for 20 hr and then incubated with Curosurf, no significant change was observed in their sPLA2 activity. Finally, f-Met-Leu-Phe (FMLP)-induced thromboxane B2 release from AM was not altered by Curosurf, indicating that the inhibition of sPLA2 expression cannot be attributed to a nonspecific membraneous effect of Curosurf. These findings show that pulmonary surfactant modulates the expression of sPLA2 in AM and suggest that this effect may account for the clinical efficacy of surfactant replacement therapy in ARDS.
Assuntos
Produtos Biológicos , Inibidores Enzimáticos/farmacologia , Macrófagos Alveolares/enzimologia , Fosfolipases A/antagonistas & inibidores , Fosfolipídeos , Surfactantes Pulmonares/farmacologia , Animais , Cobaias , Masculino , Fosfolipases A2 , Síndrome do Desconforto Respiratório/tratamento farmacológicoRESUMO
Extra-cellular and cell-associated Ca(2+)-dependent phospholipases A2 and released thromboxane B2 were correlated to exudation and cell migration during rat pleurisy induced by carrageenan or zymosan. Extra-cellular phospholipase A2 was delayed with respect to acute inflammation, while cell-associated phospholipase A2 closely correlated with cell migration and thromboxane B2 levels. This confirms that the subcellular localization of phospholipases A2 is linked to their physiological action and, in particular, suggests that the cell-associated, rather than the extracellular enzyme, accounts for the production of eicosanoids.
Assuntos
Fosfolipases A/metabolismo , Pleurisia/enzimologia , Animais , Biomarcadores , Carragenina , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Exsudatos e Transudatos/enzimologia , Cinética , Masculino , Fosfolipases A2 , Pleurisia/induzido quimicamente , Prostaglandina-Endoperóxido Sintases/metabolismo , Radioimunoensaio , Ratos , Ratos Wistar , Tromboxano B2/metabolismo , ZimosanRESUMO
The occurrence of a 14-kDa secretory phospholipase A2 (PLA2) in guinea pig alveolar macrophages (AM) and its relationship with the release of arachidonic acid (AA) were investigated. Freshly collected AM showed no detectable PLA2 activity as measured by the in vitro hydrolysis of phosphatidic acid. However, the PLA2 activity increased progressively when AM were maintained in culture to reach a level 60- to 100-fold greater than basal values within 20 h, with a parallel secretion into the incubation medium. By contrast, the activities of other phospholipid-hydrolyzing enzymes (platelet-activating factor acetylhydrolase and lysophospholipase) were modified only marginally. Both intra- and extracellular increases of PLA2 activity were abrogated with actinomycin D or cycloheximide. The enhanced PLA2 activity preferentially hydrolyzed negatively charged phospholipids in the order phosphatidic acid > phosphatidylglycerol > phosphatidylethanolamine > phosphatidylcholine, had an optimum pH of 7.5, and required a millimolar Ca2+ concentration for optimal activity and an apparent molecular mass of 14 kDa. Taken together, these results suggest that cultured AM elaborate an enzyme similar to the group II PLA2. On the other hand, our results show that AM hydrolyzed exogenous 2-arachidonoyl phosphatidylcholine and released AA and metabolites on FMLP stimulation. However, in contrast to the increase observed in the activity of the 14-kDa PLA2, the enzymatic activity involved in the hydrolysis of 2-arachidonoyl phosphatidylcholine and AA release remained constant with the culture duration of AM. Finally, dexamethasone markedly inhibited the increase of PLA2 activity, but only marginally inhibited the release of AA and metabolites from FMLP-stimulated AM. We conclude that guinea pig AM elaborate a 14-kDa PLA2 similar to the group II PLA2 through RNA- and protein synthesis-dependent processes. This elaboration appears to be induced by the adhesion of AM and is clearly dissociated from the liberation of AA.
Assuntos
Ácido Araquidônico/metabolismo , Dexametasona/farmacologia , Macrófagos Alveolares/enzimologia , Fosfolipases A/biossíntese , Animais , Cálcio/farmacologia , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Cobaias , Concentração de Íons de Hidrogênio , Leucotrieno B4/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Masculino , Peso Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Fosfatidilcolinas/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Tromboxano B2/metabolismoRESUMO
The effect of human rIL-1 beta on the release of arachidonic acid (AA) and on the phospholipase A2 (PLA2) activity in guinea pig eosinophils was investigated. Stimulation of [3H]AA-labeled eosinophils with the ionophore A23187 resulted in a time and concentration-dependent release of AA in parallel to hydrolysis of endogenous phosphatidylcholine (PC). Both events were abrogated by the chelation of intracellular free calcium, but not by its depletion from the medium, suggesting that the ionophore-induced AA release involves a PLA2 activity dependent on the mobilization of intracellular calcium. Addition of human rIL-1 beta (0.01 to 100 ng/ml) to eosinophils for 15 min had no effect on the release of AA induced by the ionophore. However, prolonged incubation with human rIL-1 beta (30 to 180 min) inhibited in a concentration- and time-dependent manner the release of AA and the hydrolysis of phosphatidylcholine in ionophore-stimulated eosinophils. Our results also showed that eosinophil homogenates contain a calcium-dependent PLA2 whose activity was markedly reduced when eosinophils were pretreated with human rIL-1 beta. The inhibition was time and concentration dependent and was observed in the presence of calcium and phospholipid excess. Finally, studies with Fura-2-loaded eosinophils showed that the ionophore A23187 stimulated an increase in intracellular calcium concentration that was not altered by pretreating the eosinophils with human rIL-1 beta. These results suggest that human rIL-1 beta inhibits the release of AA by eosinophils via the inhibition of a PLA2 activity and through a calcium-independent mechanism. Inhibition by human rIL-1 beta required a prolonged incubation (30 to 180 min) and was observed after its removal from the medium, suggesting that human rIL-1 beta did not interact directly with the PLA2 itself, but with a metabolic process involved with the regulation of its activity in eosinophils.
Assuntos
Eosinófilos/enzimologia , Interleucina-1/farmacologia , Fosfolipases A/antagonistas & inibidores , Animais , Ácido Araquidônico/metabolismo , Calcimicina/farmacologia , Cálcio/metabolismo , Ácido Egtázico/farmacologia , Cobaias , Técnicas In Vitro , Fosfatidilcolinas/metabolismo , Fosfolipases A2 , Proteínas Recombinantes/farmacologiaRESUMO
We have applied the technique of sputum induction by hypertonic saline in asthmatics and nonatopic control subjects to study an array of indices of airway inflammation believed to be relevant to asthma pathogenesis. Compatible with a central role for eosinophils and mast cells in asthma, sputum of asthmatic subjects contained increased numbers of eosinophils and levels of eosinophil cationic protein (ECP) and mast cell tryptase. Eosinophil numbers, and ECP and histamine levels correlated with the degree of methacholine airways responsiveness, and ECP, tryptase, and histamine correlated with raised concentrations of albumin. Using the micro-Boyden chamber technique eosinophil chemotactic activity was identified only in the sputum from asthmatics. The correlation between the raised levels of total IgA, IL-8/IgA complexes, and tryptase and the degree of sputum eosinophilia and ECP levels, suggests possible mechanisms for eosinophil chemotaxis and activation in asthma. Row cytometric analysis of sputum lymphocytes showed an increase in CD4+ T cells and T cells expressing intercellular adhesion molecule-1 (ICAM-1) in asthma which, together with the finding of raised levels of soluble ICAM-1 in the sputum, indicates upregulation of this adhesion molecule. Finally, the proportion of CD16+ natural killer (NK) cells was reduced in the sputum of asthmatics. These observations highlight the importance of the airway inflammation in causing asthma and further confirm the usefulness of sputum induction as a tool in asthma research.
Assuntos
Asma/metabolismo , Fatores Quimiotáticos de Eosinófilos/análise , Molécula 1 de Adesão Intercelular/análise , Escarro/química , Adulto , Quimases , Humanos , Mediadores da Inflamação/análise , Pessoa de Meia-Idade , Testes de Função Respiratória , Serina Endopeptidases/análise , Escarro/citologia , TriptasesRESUMO
There is abundant evidence for T-lymphocyte recruitment into the airways in allergic inflammatory responses. This study has tested the hypothesis that T-cell chemotaxis induced by platelet-activating factor (PAF) and human recombinant interleukin-8 (hrIL-8) can be attenuated by inhibition of phosphodiesterase activity and raised intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels. This study used theophylline, a nonselective phosphodiesterase (PDE) inhibitor, and rolipram, a selective PDE4 inhibitor, to study the effect of PDE inhibition on T-cell chemotaxis. The beta2-adrenoceptor agonist, salbutamol, the adenylyl cyclase activator, forskolin, and the cAMP analogue, dibutyryl cAMP (db-cAMP), were used to demonstrate a role for raised cAMP levels. T-cells were obtained from 10 atopic asthmatics, and the phenotype of migrating cells was examined by flow cytometry. Theophylline caused an inhibition of both PAF-and hrIL-8-induced chemotaxis (mean+/-SEM maximum inhibition at 1 mM: 73+/-4% and 48+/-8% for hrIL-8 and PAF, respectively) that was not specific for the CD4+, CD8+, CD45RO+ or CD45RA+ T-cell subsets. T-cell chemotaxis was more sensitive to treatment with rolipram whose effect was already significant from 0.1 microM on hrIL-8-induced chemotaxis. Both a low concentration of salbutamol (0.1 mM) and forskolin (10 microM) potentiated the inhibitory effect of a low concentration of theophylline (25 microM) on responses to PAF but not to hrIL-8. Finally, T-cell chemotaxis was also inhibited by db-cAMP. It is concluded that attenuation of T-cell chemotaxis to two chemoattractants of relevance to asthma pathogenesis can be achieved via phosphodiesterase inhibition and increased intracellular 3', 5'-cyclic monophosphate using drugs active on cyclic nucleotide phosphodiesterase. This action may explain the anti-inflammatory effects of theophylline and related drugs in asthma.
Assuntos
Quimiotaxia de Leucócito , AMP Cíclico/fisiologia , Diester Fosfórico Hidrolases/fisiologia , Linfócitos T , Albuterol/farmacologia , Asma/imunologia , Bucladesina/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Colforsina/farmacologia , Humanos , Interleucina-8/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fator de Ativação de Plaquetas/farmacologia , Rolipram/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Teofilina/farmacologiaRESUMO
The mechanisms that cause T cell recruitment into inflamed airways of asthmatic individuals are poorly understood. It has been shown previously that both natural exposure to allergen and challenge in the laboratory induce T cell accumulation in the bronchial mucosa of sensitized asthmatics. To study the mechanisms involved in this process, we have used an explant model in which bronchial biopsies taken from mild atopic asthmatic volunteers during fiberoptic bronchoscopy were stimulated in culture for 24 h by the common aeroallergen house dust mite (Dermatophagoides pteronyssinus (Der p)). Analysis of culture supernatants showed that stimulation with Der p significantly enhanced both the generation of T cell chemotactic activity by the mucosal tissue, as assayed in microchemotaxis chambers, and the production of IL-16 and RANTES. Neutralization experiments showed that IL-16 contributed more to the chemotactic activity than RANTES. The fusion protein CTLA-4-Ig, blocking B7:CD28 costimulation, and dexamethasone both significantly reduced the ex vivo production of chemotactic activity and release of IL-16 and RANTES. The proteasome inhibitor Cbz-Ile-Glu(OtBu)-Ala-leucinal also had a significant inhibitory effect on T cell chemotactic activity and IL-16 but not RANTES generation, indicating a role for nuclear factor NF kappa B activation. These results indicate that allergen stimulates cells within the bronchial mucosa to increase IL-16 and RANTES release, both of which contribute to T cell accumulation in asthmatic airways. The allergen-induced chemotactic activity is dependent on cell activation via CD28 and involves, at least partly, NF-kappa B.