RESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre. METHOD: Healthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR. RESULTS: Negative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD. CONCLUSION: Saliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.
Assuntos
Mediadores da Inflamação/metabolismo , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Saliva/metabolismo , Escarro/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar , Feminino , Humanos , Inflamação/diagnóstico , Inflamação/metabolismo , Pessoa de Meia-IdadeRESUMO
Endotoxin, tumor necrosis factor (TNF), and organic dust constitute proinflammatory stimuli involved in the initiation of inflammation. The major receptor for endotoxin (lipopolysaccharide [LPS]) is Toll-like receptor 4 (TLR4), whereas TLR2 binds to agents from gram-positive bacteria. The aim of the study was to elucidate whether TLR2 and TLR4, expressed on primary bronchial epithelial cells (PBECs), are influenced by exogenous (organic dust and LPS) and endogenous (TNF) stimuli and whether this interaction is influenced by a glucocorticosteroid. The cells were exposed to LPS (10 microg/ml), TNF (10 ng/ml), or dust (100 microg/ml) 1.5 and 6 h, in the presence or absence of budesonide (10(-6) M) in vitro. The mRNA expression of interleukin (IL)-6, IL-8, TLR2, and TLR4 were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and IL-6 and IL-8 release was assessed with enzyme-linked immunosorbent assay (ELISA). To elucidate the importance of TLR-signaling for IL-6 and IL-8 secretion, the effect of TLR-blockers was studied. Endotoxin, TNF, and dust stimulated the release of IL-6 and IL-8 in a time-dependent manner. Budesonide significantly attenuated the release and expression of IL-6 and IL-8 after exposure. Budesonide did not influence TLR expression, but costimulation with LPS, TNF, or dust together with budesonide increased TLR2 expression synergistically. Blocking of TLR2 and TLR4 reduced cytokine secretion in stimulated cells. Budesonide reduced IL-6 and IL-8 production and enhanced expression of TLR2 in PBECs only in the presence of a proinflammatory stimulus. These findings contribute to our understanding of the beneficial effects of glucocorticosteroids during chronic obstructive pulmonary disease (COPD) exacerbations and asthma, which are frequently caused by microorganisms.
Assuntos
Brônquios/efeitos dos fármacos , Budesonida/farmacologia , Células Epiteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Receptor 2 Toll-Like/biossíntese , Brônquios/citologia , Brônquios/imunologia , Brônquios/metabolismo , Células Cultivadas , Poeira , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Citometria de Fluxo , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-8/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
OBJECTIVE: Exposure in connection with cleaning piggeries induces airway inflammation. The aim of this study was to compare the health effects related to two different cleaning processes in a piggery. METHODS: In a cross-over study design, 12 subjects were randomly exposed for three hours during the cleaning of a piggery with a high pressure water jet, with and without pre-cleaning using a robot. We assessed lung function, bronchial responsiveness, symptoms, body temperature, and exhaled nitric oxide, and performed blood sampling and nasal lavage before and after both exposures. RESULTS: Compared with ordinary cleaning without the use of a robot, pre-cleaning with a robot significantly reduced the increase in bronchial responsiveness (P=0.049), total cell number (P=0.0029), and pro-inflammatory cytokines (IL-8 level [P=0.016]) in nasal lavage, and diminished the increase in neutrophils (P=0.0029) in the blood. CONCLUSION: Pre-cleaning of a piggery with a robot reduced exposure to dust and endotoxin, and resulted in an attenuation of the increase in bronchial responsiveness and the airway inflammatory response compared pre-cleaning without a robot.
Assuntos
Criação de Animais Domésticos , Doenças Profissionais/prevenção & controle , Exposição Ocupacional/prevenção & controle , Doenças Respiratórias/prevenção & controle , Robótica , Adulto , Animais , Temperatura Corporal , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/prevenção & controle , Estudos Cross-Over , Feminino , Humanos , Masculino , Líquido da Lavagem Nasal/imunologia , Óxido Nítrico/análise , Saúde Ocupacional , Testes de Função Respiratória , Doenças Respiratórias/imunologia , Doenças Respiratórias/fisiopatologia , Suínos , Adulto JovemRESUMO
INTRODUCTION: Alveolar macrophages (AMs) are equipped with innate immune receptors such as toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). In primary bronchial epithelial cells, exposure of toll-like receptor (TLR) ligands or tumor necrosis factor-alpha (TNF-α) increased TLR2 mRNA expression and reduced interleukin-8 (IL-8) release when coincubated with glucocorticosteroids. The aim of this study was to compare TLR2 and TLR4 expression levels and the effect of a glucocorticosteroid after stimulation with TLR ligands on AMs from smokers with and without COPD compared with the healthy controls. SUBJECTS AND METHODS: Bronchoalveolar lavage was performed, and AMs were isolated from smokers with (n=10) and without COPD (n=11) and healthy controls (n=10) and stimulated ex vivo with peptidoglycan (PGN), lipopolysaccharide (LPS), or TNF-α ± budesonide (Bud). Blocking antibodies to TLR2 or TLR4 were added before stimulation with LPS or PGN ± Bud, respectively. The release of proinflammatory cytokine (TNF-α), chemoattractant (CXCL8), and TLR expression was analyzed by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. RESULTS: LPS, PGN, and TNF-α induced an increased release of IL-8 and TNF-α in the AMs in all the groups independent of smoking or disease. These responses were inhibited by a glucocorticosteroid (Bud) in all the three groups, except PGN-induced IL-8 secretion in smokers without COPD. Bud increased TLR2 expression in the healthy controls and smokers without COPD. Costimulation of TLR ligands and Bud significantly enhanced TLR2 mRNA expression in both groups of smokers compared with TLR ligands alone. In smokers, costimulation with PGN and Bud significantly increased TLR2 expression when compared with Bud alone. On stimulation with the TLR4 agonist, LPS downregulated TLR4 mRNA expression in all the three groups. CONCLUSION: The combination of glucocorticosteroids with TLR ligands can increase TLR2 expression, thereby improving host defense in smokers. Also this combination can decrease the secretion of proinflammatory cytokines and chemokines as an anti-inflammatory response. Our findings indicate that glucocorticosteroid therapy strengthens immune defense pathways, which may have implication during exacerbation caused by microorganisms.
Assuntos
Anti-Inflamatórios/farmacologia , Budesonida/farmacologia , Glucocorticoides/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Fumar/imunologia , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacos , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Imunidade Inata/efeitos dos fármacos , Interleucina-8/imunologia , Interleucina-8/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Peptidoglicano/farmacologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fumar/efeitos adversos , Fumar/genética , Fumar/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Intercellular communication is essential for defense and survival of the organism. The aim of the study was to find out whether there is an active crosstalk between airway cells constituting the first line of defense, alveolar epithelial cells (A549) and neutrophils, following activation with pro-inflammatory stimuli in vitro and to explore whether this communication is altered in chronic obstructive pulmonary disease (COPD), a condition characterized by chronic airway and lung inflammation. METHODS: Blood neutrophils from healthy subjects and COPD patients were co-cultured with A549 cells in pure medium and in medium containing lipopolysaccharide (LPS), peptidoglycan (PGN), or tumor necrosis factor. The expression of Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), and CD14 on the cell surface of neutrophils was assessed by flow cytometry, and release of CXCL8 (IL-8) and the soluble CD14 (sCD14) was measured in the supernatant with enzyme-linked immunosorbent assay (ELISA). RESULTS: On neutrophils, the surface expression of TLR2 was diminished following activation with all three pro-inflammatory stimuli, and membrane bound (mCD14) and TLR4 expression were significantly increased in co-cultures compared to single cell cultures, irrespective of pro-inflammatory stimulation. There was a correlation between CXCL8 and sCD14 in LPS-stimulated co-cultured cells (r=0.82; p<0.01). CONCLUSION: An active crosstalk between A549 cells and blood neutrophils was clearly demonstrated, both in unstimulated cells and following activation with pro-inflammatory stimuli, in vitro. Co-culturing implied synergy and correlation between LPS-induced release of sCD14 and CXCL8, which indicates that sCD14 may be donated by neutrophils to epithelial cells facilitating TLR4-signaling. Furthermore, TLR2 on neutrophils was found to be down-regulated by pro-inflammatory stimuli.
RESUMO
INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is characterized by non-reversible airflow limitation and systemic engagement. Bacterial colonization in the lungs is common in COPD-patients and may be associated with frequent acute exacerbations. Pattern-recognition receptors (PRRs), like Toll-like receptor 2 (TLR2), TLR4 and CD14 are expressed on most immunologic active cell types and are most likely of importance in COPD patho-physiology. MATERIAL AND METHODS: Twenty smokers with and 20 without COPD and 20 healthy non-smokers participated in the study. At two visits, induced sputum was collected after spirometry, blood was sampled and bronchoscopy with bronchoalveolar lavage was performed. Expression of TLR2, TLR4 and CD14 on different cell types and soluble receptors were assessed in the different compartments. RESULTS: Expression of TLR2 was lower on sputum neutrophils and soluble TLR2 (sTLR2) was higher in the supernatant in the COPD group, indicating a down regulation of TLR2 at the transit from blood to sputum. Expression of CD14 on sputum neutrophils and gene expression of CD14 on alveolar macrophages was up-regulated in the two smoking groups compared with non-smokers. No differences between the groups were found regarding TLR4 expression. CONCLUSIONS: Pattern-recognition receptors (PRRs), that are expected to make a first line of defense against invading micro-organisms, are differently regulated in smokers with COPD compared with smokers without airflow limitation and non-smokers. This is likely of importance in COPD patho-physiology, in particular for exacerbations, which often are caused by micro-organisms.