| BackgroundToll-like receptors (TLRs) are involved in recognition of a broad range of pathogens. Their activation triggers the innate immune response, and is crucial to the successful induction of adaptive immunity. Respiratory infections and environmental stimuli influence the risk of developing COPD and the disease progression. As the key receptors of the innate immune system, TLRs are the important link between environmental exposure and regulation of adaptive immunity. Recent studies suggest that TLRs activation is likely to contribute to the establishment and maintenance of inflammation in COPD. But most of our knowledge of TLRs has emerged from studies of mice. The contribution of TLRs function to human COPD is less advanced.ObjectiveTo investigate the role of TLRs in the pathogenesis of COPD, we first compared the expression of TLR2 and TLR4 in circulating CD14+ monocytes and neutrophils harvested from clinically stable COPD patients, smokers with normal lung function and never smokers. Then the surface expression of TLR2 and TLR4 was phenotypically characterized on circulating CD14+ monocytes and neutrophils in COPD patients and patients with chronic persistent asthma. In addition, the effects of Th1- type cytokines and corticosteroid on the expression of TLRs in human respiratory epithelial cells were also examined.MethodsThe study population consisted of 30 COPD patients without evidence for acute exacerbation, 21 smokers with normal lung function, 25 healthy non-smokers and 30 patients with persistent asthma. The expression of TLR2, TLR4 surface molecules on human CD14+ monocytes and neutrophils was assessed using FACS analysis by flow cytometry. Spirometries were performed in all participants. Sputum bacterial isolation and serum C-reactive protein (CRP) measurement were performed in COPD patients. Serum tumour necrosis factor-α(TNF-α) and interleukin-8 (IL-8) levels of COPD and asthma patients were measured by means of ELISA. In addition, the human epithelial lung cancer cell line, A549, were stimulated with Interferon-γ(IFN-γ) , TNF-αand dexamethasone singly, or in a combination. Real-time PCR were used to assess TLRs mRNA expression.ResultsClinical study1. CD14+ monocytes expressed significantly higher levels of TLR2 and TLR4 than meutrophils. The expression of TLR2 was positive correlated with the expression of TLR4 on monocytes. The expression of TLR2, and TLR4 as well, in monocytes were positive correlated with that in neutrophils.2. There were 16 clinically stable COPD patients with sputum pathogenic bacterial quantity more than 10~5 CFU/mL among 24 patients whose sputum sample was valid. All the COPD patients had a normal C-reactive protein level.3. Analysis among COPD patients, smokers with normal lung function, and nonsmokers:a. The expression of TLR2 and TLR4 on monocytes in healthy smokers was reduced as compared to nonsmokers ( P<0.05), while in COPD patients the expression was down-regulated more than healthy smokers( P<0.05).b. The expression of TLR2 on neutrophils in healthy smokers was reduced as compared to nonsmokers( P<0.05).The expression of TLR2 on neutrophils showed a tendency to be lower in COPD patients than healthy smokers. There was no significant difference among 3 groups in terms of the expression of TLP4 on neutrophils (P<0.05).c. The expression of TLR2 and TLR4 on monocytes was positively correlated with lung function parameters, including FEV1% predicted and FEV1/FVC( P<0.05). The expression of TLR2 on neutrophils was also positively correlated with the lung function parameters above( P<0.05).4. The TLR2 positive and TLR4 positive cell percent of CD14 monocytes was significantly higher in patients with chronic persistent asthma, than that in healthy controls( P<0.05), while the expression of TLR2 and TLR4 on neutrophils were down regulated in asthma patients. The expression intensities of TLR2 and TLR4 on monocytes showed a tendency to be correlated with serume IgE level in asthma patients.5. Analysis between patients with COPD and patients with asthma:a. Compared with patients with asthma, the expression of TLR2 anf TLR4 on circulating neutrophils was significantly increased in patients with COPD ( P<0.05), while the expression of TLR2 and TLR4 on monocytes was reduced in patients with COPD (P<0.01).b. Compared with patients with asthma, the serum IL-8 level was increased in COPD patients, while the serum TNF-αlevel was reduced in COPD patients.c. The expression of TLR2 and TLR4 on neutrophils were positively associated with serum IL-8 level ( P<0.01). The TLR2 positive and TLR4 positive percent of monocytes were positively associated with serum TNF-αlevel (P<0.01).Laboratory study1. IFN-γsignificantly up regulated the expression of TLR3 on A549 cells (3.78 folds compared with control, P<0.05).2. TNF-αenhanced the expression of TLR2 and TLR4 on A549 cells (1.62 and 1.22 folds compared with control, respectively, P<0.05).3. Dexamethasone significantly up regulated the expression of TLR2 and TLR4 on A549 cells (3.27 and 3.43 folds compared with control, respectively, P<0.05).4. Dexamethasone potentiated TLR2 expression induced by IFN-γor TNF-α(6.25 and 6.09 folds, respectively, P<0.05).5. TLR2 expression was significantly enhanced after stimulation with a combination of TNF-αand IFN-γ(3.66 fold compared with control, P<0.05). Dexamethasone synergistically enhanced TLR2 expression in combination with TNF-αand IFN-γ(29.81 fold compared with control, P<0.05).Conclusion1. CD14+ monocytes expressed significantly higher levels of TLR2 and TLR4 than meutrophils in healthy non-smokers. 2. Smoke depressed the the expression of TLRs on monocytes and neutropils.3. The innate immune response might be depressed in patients with COPD, and the down regulation of TLRs was associated with the disease progression.4. The character of TLRs expression in patients with COPD might play a role in neutrophic inflammation.5. Th-1 type cytokines and corticosteroid regulated the expression of respiratory epithelial cells. Corticosteroid in combination with TNF-αand IFN-γcould synergistically enhance TLR2 expression on respiratory epithelial cells.
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