Time course of cigarette smoke-induced pulmonary inflammation in mice.

Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease. In the present study a murine model of tobacco smoke-induced emphysema was used to investigate the time course of airway and pulmonary inflammatory response, with a special emphasis on pulmonary dendritic cell (DC) populations. Groups of mice were exposed to either cigarette smoke or to control air for up to 24 weeks. In response to cigarette smoke, inflammatory cells (i.e. neutrophils, macrophages and lymphocytes) progressively accumulated both in the airways and lung parenchyma of mice. Furthermore, a clear infiltration of DCs was observed in airways (10-fold increase) and lung parenchyma (1.5-fold increase) of cigarette-exposed mice at 24 weeks. Flow cytometric analysis of bronchoalveolar lavage (BAL) DCs of smoke-exposed mice showed upregulation of major histocompatability complex II molecules and costimulatory molecules CD40 and CD86, compared with BAL DCs of air-exposed mice. Morphometric analysis of lung histology demonstrated a significant increase in mean linear intercept and alveolar wall destruction after 24 weeks of smoke exposure. In conclusion, the time course of the changes in inflammatory and dendritic cells in both bronchoalveolar lavage and the pulmonary compartment of cigarette smoke-exposed mice was carefully characterised.     

CD4+白细胞介素-17+辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each).Morphological changes were evaluated by mean linear intercepts and destructive index (DI).The proportion of CD4+ IL-17 + Th17, CD4+ IFN-γ+ Th1, CD4+ IL-17 +IFN-γ+ T( Th17/ Th1 ), CD8+ IFN-γ+ Tc1, CD8+ IL-21R + and CD4+ IL-17 + IL-21 + T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4)μm, (47 ±7) μm], (39.1 ± 1.6, 45.2 ±3.1 ) as compared to C12[(32 ±4) μm,28.2 ± 1.6] and C24groups [(33 ± 3 ) μm ,28.9 ± 2.1], all P ＜ O.05.The percentage of Th17 of S12 and S24 groups [(3.3 ±1.1 )%, (7.2 ±2.2)%] was significantly increased as compared with that of C12 and C24 groups [( 1.8± 0.8) %, (2.0 ± 0.6) %], all P ＜ 0.05.The mRNA levels of RORγt [( 25 ± 4), ( 35 ± 3 )] and IL-17 [(26 ± 3), (36 ± 3 )] in S12 and S24 groups were higher than in C12 [(10 ± 5 ), (13 ± 5 )] and C24 groups [( 11 ± 7 ), (8 ± 6)], all P ＜ 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ±3.7)%, (26.2 ±6.0)%], [(0.61 ±0.30)%, (1.82 ±0.52)%], [(17.0±4.5 ) %, ( 26.8 ± 8.5 ) %] was significantly increased as compared with that of C12 [( 3.8 ± 1.7 ) %,(0.27±0.17)%, (4.8 ±1.9)%] and C24 groups [(4.2±1.3)%, (0.28±0.11)%, (5.2±1.0)%], all P＜0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions ( r =0.519 - 0.797, all P ＜ 0.01 ).In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r =0.742, 0.802, all P ＜0.01 ).The percentage of CD4+ IL-17+ IL-21 +T cells was significantly increased in S12 and S24 groups [(0.19 ±0.04) %, (0.55 ± 0.24) %] compared to controls [(0.07 ± 0.03 ) %, (0.08 ± 0.03 ) %], all P ＜ 0.05.Meanwhile, as compared with that of the controls [( 1.22 ± 0.31 ), ( 1.34 ± 0.18 )], the percentage of CD8+ IL-21 R + T cells was also increased in SI 2 and S24 groups [( 2.94 ± 1.26 ), (4.12 ± 2.26 )], but there were no differences among smoke-exposure groups ( P ＞0.05 ).The frequency of CD4+ IL-17 + IL-21 + T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions (r = 0.694 -0.754, all P ＜0.05).And the frequency of CD8+ IL-21R+ T cells also had a positive correlation with emphysematous lesions ( r = 0.516, 0.725, all P ＜ 0.05).Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.     

CD4+白细胞介素-17+辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

目的 观察香烟烟雾暴露小鼠肺实质中CD4+白细胞介素(IL)-17+辅助性T细胞(Th17)数量及活性的表达,探讨其在香烟暴露小鼠肺部CD4+ γ-干扰素+(Th1)炎症及肺气肿中的作用及相关机制.方法 将40只雄性Balb/c小鼠按随机数字表法分为4组:对照12周(C12)组、对照24周(C24)组、烟雾暴露12周(S12)组、烟雾暴露24周(S24)组,每组10只.香烟烟雾暴露法建立小鼠肺气肿模型.HE染色观察小鼠肺气肿的改变,计算平均内衬间隔和肺泡破坏指数(DI);流式细胞术检测小鼠肺实质中CD4+IL-17+T(Th17)细胞、CD4+γ-干扰素+T(Th1)细胞、CD4+IL-17+γ-干扰素+T(Th17/Th1)细胞、CD8+γ-干扰素+T(Tc1)细胞、CD8+IL-21R+细胞及CD4+IL-17+IL-21+细胞比例;荧光定量PCR法检测小鼠肺实质中维甲酸相关孤独受体(RORγt)和IL-17的mRNA表达,并分析这些指标的相互关系.结果 S12组和S24组的平均内衬间隔[(39±4)μm和(47±7)μm]和DI(39.1±1.6和45.2±3.1)明显高于C12组[(32±4)μm和28.2±1.6]和C24组[(33±3)μm和28.9±2.1],且以S24组的增高更为明显,差异均有统计学意义(t值为4.378～15.188,均P＜0.05);S12组和S24组Th17细胞比例[(3.3±1.1)%和(7.2±2.2)%]均明显高于C12组和C24组[(1.8±0.8)%和(2.0±0.6)%];S12组和S24组RORγtmRNA表达量[(25±4)和(35±3)]及IL-17的mRNA表达量[(26±3)和(36±3)]亦明显高于C12组[(10±5)和(13±5)]和C24组[(11±7)和(8±6)],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组Th1细胞比例[(10.0±3.7)%和(26.2 ±6.0)%]、Th17/Th1细胞比例[(0.61±0.30)%和(1.82±0.52)%]及Tc1细胞比例[(17.0±4.5)%和(26.8±8.5)%]均明显高于C12组[(3.8±1.7)%、(0.27±0.17)%和(4.8±1.9)%]和C24组[(4.2±1.3)%、(0.28±0.11)%和(5.2±1.0)%],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组小鼠Th17细胞与Th1、Tc1细胞比例、平均内衬间隔、DI值均呈显著正相关(r值为0.519～0.797,均P＜0.01);Th17/Th1细胞比例与平均内衬间隔、DI值呈显著正相关(r值分别为0.742和0.802,均P＜0.01);S12组和S24组CD4+IL-17+IL-21+细胞比例[(0.19±0.04)和(0.55±0.24)]明显高于C12组和C24组[(0.07±0.03)和(0.08±0.03)],S24组增高更为明显,差异均有统计学意义(P＜0.05).S12组和S24组的CD8+IL-21R+细胞比例[(2.94±1.26)和(4.12±2.26)]高于C12组和C24组[(1.22±0.31)和(1.34±0.18)](P＞0.05);S12组及S24组小鼠CD4+IL-17+IL-21+细胞比例与Th1、Tc1细胞比例、平均内衬间隔和DI值均呈显著正相关(r值为0.694～0.754,均P＜0.05);S12及S24组小鼠CD8+IL-21R+细胞比例与平均内衬间隔和DI呈显著正相关(r值分别为0.516和0.725均P＜0.05).结论 香烟暴露导致肺气肿小鼠肺内Th17细胞数量及活性上调,并随烟雾暴露时间延长而增强;Th17细胞通过IL-21及IL-21R在肺部Th1/Tc1炎症中起重要促进作用;这对探讨COPD肺部炎症和肺气肿发生机制以及新的治疗靶点具有重要意义.

Abstract：

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each).Morphological changes were evaluated by mean linear intercepts and destructive index (DI).The proportion of CD4+ IL-17 + Th17, CD4+ IFN-γ+ Th1, CD4+ IL-17 +IFN-γ+ T( Th17/ Th1 ), CD8+ IFN-γ+ Tc1, CD8+ IL-21R + and CD4+ IL-17 + IL-21 + T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4)μm, (47 ±7) μm], (39.1 ± 1.6, 45.2 ±3.1 ) as compared to C12[(32 ±4) μm,28.2 ± 1.6] and C24groups [(33 ± 3 ) μm ,28.9 ± 2.1], all P ＜ O.05.The percentage of Th17 of S12 and S24 groups [(3.3 ±1.1 )%, (7.2 ±2.2)%] was significantly increased as compared with that of C12 and C24 groups [( 1.8± 0.8) %, (2.0 ± 0.6) %], all P ＜ 0.05.The mRNA levels of RORγt [( 25 ± 4), ( 35 ± 3 )] and IL-17 [(26 ± 3), (36 ± 3 )] in S12 and S24 groups were higher than in C12 [(10 ± 5 ), (13 ± 5 )] and C24 groups [( 11 ± 7 ), (8 ± 6)], all P ＜ 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ±3.7)%, (26.2 ±6.0)%], [(0.61 ±0.30)%, (1.82 ±0.52)%], [(17.0±4.5 ) %, ( 26.8 ± 8.5 ) %] was significantly increased as compared with that of C12 [( 3.8 ± 1.7 ) %,(0.27±0.17)%, (4.8 ±1.9)%] and C24 groups [(4.2±1.3)%, (0.28±0.11)%, (5.2±1.0)%], all P＜0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions ( r =0.519 - 0.797, all P ＜ 0.01 ).In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r =0.742, 0.802, all P ＜0.01 ).The percentage of CD4+ IL-17+ IL-21 +T cells was significantly increased in S12 and S24 groups [(0.19 ±0.04) %, (0.55 ± 0.24) %] compared to controls [(0.07 ± 0.03 ) %, (0.08 ± 0.03 ) %], all P ＜ 0.05.Meanwhile, as compared with that of the controls [( 1.22 ± 0.31 ), ( 1.34 ± 0.18 )], the percentage of CD8+ IL-21 R + T cells was also increased in SI 2 and S24 groups [( 2.94 ± 1.26 ), (4.12 ± 2.26 )], but there were no differences among smoke-exposure groups ( P ＞0.05 ).The frequency of CD4+ IL-17 + IL-21 + T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions (r = 0.694 -0.754, all P ＜0.05).And the frequency of CD8+ IL-21R+ T cells also had a positive correlation with emphysematous lesions ( r = 0.516, 0.725, all P ＜ 0.05).Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.     

白介素17抗体减轻吸烟所致慢性阻塞性肺疾病模型小鼠的气道炎症

目的 研究白介素17(interhukin-17,IL-17)抗体在吸烟所致慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型小鼠气道炎症中的作用.方法 C67/BL6雄性小鼠随机分为COPD组(8只)、COPD+IL-17抗体干预组(简称COPD+干预组,8只)和正常对照组(10只).对支气管肺泡灌洗液(BALF)进行细胞计数、染色和分类;用酶联免疫吸附试验检测小鼠肺组织匀浆IL-17水平,观察各组小鼠气道病理改变.结果 COPD组与COPD+干预组相比肺功能差异无统计学意义.COPD组、COPD+干预组小鼠与正常对照组相比BALF细胞总数显著增高[分别为(19.64±1.89)×104/ml,(15.47±2.99)×104/ml和(5.13±1.00)×104/ml,P＜0.01];COPD+干预组较COPD组细胞总数下降(P＜0.01);中性粒细胞比例[分别为(8.58+6.77)%,(22.98±8.46)%]及绝对值[(1.28±0.96)×104/ml,(4.53±1.73)×104/ml]显著下降(P值均＜0.01).肺组织HE染色病理评分COPD+干预组(73.25±18.58)较cOPD组(106.13±36.27)炎症有所减轻(P＜0.05).COPD+干预组小鼠肺组织匀浆中IL-17含量(0.084±0.041)pg/mg pro与COPD组(0.221±0.081)pg/mg pro相比显著降低(P＜0.01).结论 吸烟所致COPD小鼠模型中IL-17参与了中性粒细胞引起的气道炎症,抑制IL-17的表达,可以减少气道内中性粒细胞的数量,减轻气道炎症.     

L-NAME and L-arginine differentially ameliorate cigarette smoke-induced emphysema in mice

Nitric oxide (NO) represents one of the most important intra- and extracellular mediators and takes part in both biologic and pathologic processes. This study aimed to verify the treatment with an NO inhibitor and an NO substrate in pulmonary emphysema induced by cigarette smoke (CS) in a murine model. We compared N-acetylcysteine (NAC), a precursor of glutathione, to G-nitro-l-Arginine-Methyl Ester or l-NAME (LN), which is an NO inhibitor, and to l-arginine (LA), which is a substrate for NO formation. Mice were divided into several groups: control, CS, CS + LN, CS + LA, and CS + NAC. Control and CS groups were treated daily with a vehicle, while CS + LN, CS + LA, and CS + NAC groups were treated daily with LN (60 mg/kg), LA (120 mg/kg) and NAC (200 mg/kg), respectively. The bronchoalveolar lavage was analyzed and the lungs were removed for histological and biochemical analysis. CS increases neutrophil number. Neutrophil number was lowest in CS + LN, followed by CS + LA. The lungs of CS + LN, CS + LA and CS + NAC mice were protected compared to the lungs of CS mice, but not equal to the quality of lungs in control mice. The CS group also exhibited increased oxidative stress, which was also present in the CS + LN group and to a lesser extent in the CS + LA group. Tissue inhibitor of metalloproteinase 1 and 2 increased in the CS + LN group and to a lesser extent in the CS + LA group relative to the control group. These results suggest that LN and LA treatment protected the mouse lung from CS. However, NAC treatment was more than LN and LA. We suggest that the protection conferred by LN treatment requires a balance between proteases and antiproteases, and that protection conferred by LA treatment involves the balance between oxidants and antioxidants.     

Role of tumour necrosis factor-alpha receptor p75 in cigarette smoke-induced pulmonary inflammation and emphysema.

Chronic obstructive pulmonary disease (COPD) is characterised by a local pulmonary inflammatory response to respiratory pollutants and by systemic inflammation. Tumour necrosis factor (TNF)-alpha has been implicated in systemic effects of COPD and operates by binding the p55 (R1) and p75 (R2) TNF-alpha receptors. To investigate the contribution of each TNF-alpha receptor in the pathogenesis of COPD, the present study examined the effects of chronic air or cigarette smoke (CS) exposure in TNF-alpha R1 knockout (KO) mice, TNF-alpha R2 KO mice and wild type (WT) mice. CS was found to significantly increase the protein levels of soluble TNF-alpha R1 (by four-fold) and TNF-alpha R2 (by 10-fold) in the bronchoalveolar lavage of WT mice. After 3 months, CS induced a prominent pulmonary inflammatory cell influx in WT and TNF-alpha R1 KO mice. In TNF-alpha R2 KO mice, CS-induced pulmonary inflammation was clearly attenuated. After 6 months, no emphysema was observed in CS-exposed TNF-alpha R2 KO mice in contrast to WT and TNF-alpha R1 KO mice. CS-exposed WT and TNF-alpha R1 KO mice failed to gain weight, whereas the body mass of TNF-alpha R2 KO mice was not affected. These current findings suggest that both tumour necrosis factor-alpha receptors contribute to the pathogenesis of chronic obstructive pulmonary disease, but tumour necrosis factor-alpha receptor-2 is the most active receptor in the development of inflammation, emphysema and systemic weight loss in this murine model of chronic obstructive pulmonary disease.     

吸烟和生物燃料所致慢性阻塞性肺疾病患者临床特征差异研究

目的 观察吸烟和生物燃料所致慢性阻塞性肺疾病(COPD)患者临床特征的差异。方法 回顾性分析2011年3月至2014年3月在广州呼吸疾病研究所206例由吸烟和81例生物燃料导致的慢阻肺患者的临床资料,分别比较了两组患者的一般情况、临床症状、呼吸困难评分和合并症等方面的差异;肺功能和分级及急性加重的差异。结果 (1)一般情况的差异：吸烟所致慢阻肺患者中男性和女性的比例分别为83.5%和16.5%,生物燃料所致慢阻肺的患者中男性和女性的比例分别为14.8%和85.2%(χ2=27.2,P<0.05)。吸烟所致慢阻肺患者多见于男性,而生物燃料所致慢阻肺患者多见于女性;性别矫正后,生物燃料所致的女性COPD患者的体重指数(BMI)低于吸烟所致的女性COPD患者的BMI。其他指标如年龄两组差异无统计学意义。(2)临床症状差异：生物燃料和吸烟所致慢阻肺患者的呼吸困难指数mMRC差异无统计学意义;生物燃料所致慢阻肺患者出现喘息症状多于吸烟所致慢阻肺的患者,分别为38.3%和11.1%(χ2=17.9,P<0.05)。生物燃料所致慢阻肺患者合并过敏性疾病(如过敏性鼻炎、支气管哮喘)比例高于吸烟所致慢阻肺的患者,分别为43.2%和18%(χ2=16.1,P<0.05);而吸烟所致慢阻肺患者合并肺癌比例高于生物燃料所致慢阻肺的患者,分别为7.77%和3.7%(χ2=9.7,P<0.05)。(3)肺功能分级的差异：慢阻肺分级上,性别校正后生物燃料所致慢阻肺患者分级更多见于B级或D级,症状多。(4)急性加重的差异：生物燃料所致和吸烟所致的慢阻肺患者1年内急性加重次数无显著差异。结论 吸烟和生物燃料所致的慢阻肺在临床特征上有很多差异：生物燃料所致慢阻肺患者多见于女性,BMI低,临床症状较多,合并症以过敏性鼻炎和支气管哮喘较多,慢阻肺分级多见于B级或D级。吸烟所致慢阻肺患者多见于男性,合并症以肺癌较多。     

A murine model of cigarette smoke-induced pulmonary inflammation using intranasally administered smoke-conditioned medium

To date, few animal models of chronic obstructive pulmonary disease (COPD) exist that are ideal for the evaluation of pathophysiology, as they typically require many months of cigarette smoke exposure in inhalation facilities. Here we show that pulmonary inflammation and some of the inflammatory hallmarks of COPD can be induced in mice by cigarette smoke-conditioned media (CS) administered by the intranasal route. Balb/c mice were challenged with CS for up to 40 days. At the end of smoke treatment, mice were sacrificed and bronchoalveolar lavage (BAL) fluid collected. Total cell counts and cell differentials were performed. Enzyme-linked immunosorbent assays (ELISAs) for KC and tumor necrosis factor alpha (TNF-alpha) were performed on BAL fluid. Lungs and nasal cavities were examined histologically. Intranasal CS treatment significantly increased BAL neutrophils, lymphocytes, KC, TNF-alpha, and mucin. Changes in pulmonary reactivity to methacholine were also observed in mice challenged with CS for 40 days. The model described above demonstrates that within 1 to 8 weeks of intranasal instillation of CS, mice develop pulmonary inflammation and cellular lung changes that are characteristic of human COPD and therefore may be a good short-term in vivo model that can be utilized to monitor intervention strategies targeted for COPD.     

Tumor necrosis factor-alpha is central to acute cigarette smoke-induced inflammation and connective tissue breakdown

The role of tumor necrosis factor-alpha (TNF-alpha) as a mediator of cigarette smoke-induced disease is controversial. We exposed mice with knocked-out p55/p75 TNF-alpha receptors (TNF-alpha-RKO mice) to cigarette smoke and compared them with control mice. Two hours after smoke exposure, increases in gene expression of TNF-alpha, neutrophil chemoattractant, macrophage inflammatory protein-2, and macrophage chemoattractant, protein-1 were seen in control mice. By 6 hours, TNF-alpha, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 gene expression levels had returned to control values in control mice and stayed at control values through 24 hours. In TNF-alpha-RKO mice, no changes in gene expression of these mediators were seen at any time. At 24 hours, control mice demonstrated increases in lavage neutrophils, macrophages, desmosine (a measure of elastin breakdown), and hydroxyproline (a measure of collagen breakdown), whereas TNF-alpha-RKO mice did not. In separate experiments, pure strain 129 mice, which produce low levels of TNF-alpha, showed no inflammatory response to smoke at 24 hours or 7 days. We conclude that TNF-alpha is central to acute smoke-induced inflammation and resulting connective tissue breakdown, the precursor of emphysema. The findings support the idea that TNF-alpha promoter polymorphisms may be of importance in determining who develops smoke-induced chronic obstructive pulmonary disease.     

Effects of strain and treatment with inhaled aII-trans-retinoic acid on cigarette smoke-induced pulmonary emphysema in mice

Models of emphysema produced by exposing animals to cigarette smoke (CS) have potential for use in testing treatments of this disease. To better characterize development of emphysema in an animal model, male and female mice of the B6C3F1 and A/J strains were exposed to CS at 250 mg total particulate material (TPM)/m3 for 15 weeks. Emphysema was evident in both strains of mice to differing degrees of severity. The CS-induced increase in the mean linear intercept (normalized to BW) of A/J mice was 51% greater than the control value, while CS-exposed B6C3F1 had an increase of 38% in this morphometric measurement of alveolar air space enlargement. In separate experiments, female B6C3F1 mice and male A/J mice were exposed to CS for 32 weeks and 15 weeks, respectively, and were then used to test the efficacy of all trans-retinoic acid (ATRA) treatments to ameliorate emphysema lesions. Following CS exposure, the B6C3F1 mice were treated once daily for 14 days in a 3-week period by nose-only inhalation exposure to aerosols of 180 or 1,800 mg-minutes ATRA/m3. The A/J mice were treated once daily, 4 days/week, for three weeks by either intraperitoneal injection of ATRA (0.5 or 2.5 mg/kg) or inhalation exposure to ATRA (3,600 or 18,000 mg-minutes/m3). Neither the injections nor inhalation exposures of ATRA in either strain of mouse caused reversal of the emphysema. In summary, CS-induced emphysema was more severe in A/J mice than in B6C3F1 mice. Treatment with ATRA did not reverse emphysema in either strain of CS-exposed mice.     

Humic acid enhances cigarette smoke-induced lung emphysema in mice and IL-8 release of human monocytes

Tobacco smoke is the main factor in the etiology of lung emphysema. Generally prolonged, substantial exposure is required to develop the disease. Humic acid is a major component of cigarette smoke that accumulates in smokers’ lungs over time and induces tissue damage.

Objectives

To investigate whether humic acid pre-loading potentiates the development of cigarette smoke-induced lung emphysema in mice and increases IL-8 release by human monocytes.

Methods

C57BL/6J mice received humic acid or aqueous vehicle by tracheal installation on day 0 and day 7. From day 21 to day 84, the mice were exposed to cigarette smoke or clean air for 5 days/week. Twenty-four hours after the last exposure we determined leukocytes in lung lavage, heart hypertrophy and alveolar wall destruction. Human monocytes were incubated with cigarette smoke extract (CSE), humic acid or the combination overnight.

Results

Humic acid nor cigarette smoke caused alveolar wall destruction within two months. Interestingly, the combination did induce lung emphysema. Humic acid, cigarette smoke or the combination did not change leukocyte types and numbers in lung lavage fluid, but the combination caused peribronchiolar and perivascular lymphocyte infiltration. Humic acid treatment resulted in a high proportion of alveolar macrophages heavily loaded with intracellular granula. Humic acid also induces the release of IL-8 from human monocytes and enhances the CSE-induced IL-8 release.

Conclusions

Humic acid deposition in the lungs potentiates the development of cigarette smoke-induced interstitial inflammation and lung emphysema. Moreover, humic acid promotes IL-8 release from human monocytes. Since humic acid accumulates steadily in the lungs of smokers, this may provide an explanation for the natural history on late onset of this disease. The model described here offers a novel way to study emphysema and may direct the search for new therapeutic approaches.     

Cytokine and chemokine expression in cigarette smoke-induced lung injury in guinea pigs.

The guinea pig model of cigarette smoke (CS)-induced lung injury is known to exhibit many pathophysiological similarities to chronic obstructive pulmonary disease (COPD), but the expression profiles of inflammatory mediators in the lung are poorly understood. Quantitative real-time RT-PCR was used in this study to investigate the pulmonary expression profiles of cytokine and chemokine mRNA in response to single or repeated CS exposure in guinea pigs. A single CS exposure did not induce obvious inflammatory cell infiltration into the lungs, but it led to significant increases in the mRNA expression of tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-8, and monocyte chemoattractant protein (MCP)-1, and decreases in IL-5 and granulocyte-macrophage colony-stimulating factor. Repeated CS exposure induced many features of COPD, such as marked accumulation of macrophages and neutrophils, augmented protease activities, lung structural alterations and increased airway resistance, accompanied by significant increases in the mRNA expression of IL-1beta and MCP-1 and decreases in IL-2, IL-5, transforming growth factor-beta, and eotaxin. In conclusion, in guinea pigs, inflammatory mediator changes in the lungs following cigarette smoke exposure are largely similar to those reported for smokers and/or chronic obstructive pulmonary disease patients. This model will therefore be useful to further understand the pathogenesis of chronic obstructive pulmonary disease.     

Short-term cigarette smoke exposure enhances allergic airway inflammation in mice

RATIONALE: Epidemiologic studies suggest that tobacco smoke contributes to the prevalence and occurrence of exacerbations in asthma. The effect of active smoking in adolescents with atopy is poorly understood. OBJECTIVES: We developed an experimental model to investigate the influence of smoking on antigen-induced airway inflammation and airway responsiveness in mice that were previously sensitized. METHODS: Ovalbumin (OVA)-sensitized BALB/c mice were exposed to air or mainstream smoke (5 days/week) and to phosphate-buffered saline (PBS) or OVA aerosol (3 times/week) for 2 weeks (n = 8 for each group). RESULTS: Airway responsiveness to intravenously injected carbachol was increased (p < 0.05) in smoke- and OVA-exposed mice compared with all other groups. There was an additive effect of smoke and OVA exposure on total cell numbers, macrophages, and dendritic cells in bronchoalveolar lavage fluid and on CD4+ and CD8+ T lymphocytes and dendritic cells in lung tissue (p < 0.05 compared with mice exposed to smoke and PBS and to mice exposed to air and OVA). Concurrent smoke and OVA exposure augmented OVA-specific IgE in serum compared with air and OVA exposure. In lavage fluid supernatant, eotaxin was increased in air- and OVA-exposed mice. The further increase observed in the group exposed to both OVA and cigarette smoke came close to formal significance (p = 0.06). Thymus- and activation-regulated chemokine was augmented in mice exposed to either smoke or OVA, without additional effect. CONCLUSIONS: Our data indicate that acute concurrent exposure to allergen and mainstream cigarette smoke enhances airway inflammation and airway responsiveness in previously sensitized mice.     

Mechanisms of cigarette smoke-induced stimulation of rapidly adapting receptors in canine lungs.

The stimulation of rapidly adapting receptors (RARs) in the lungs evoked by cigarette smoke consists of an initial and either a type I or a type II delayed response (Kou and Lee, J. Appl. Physiol. 68: 1203, 1990). In the type I response, receptor activity increased during expirations and exhibited a prominent cardiac modulation, whereas in the type II response, receptor discharge reached its peak during inspiration at peak transpulmonary pressure. To investigate the mechanisms of this stimulation, we recorded the vagal afferent activity arising from 39 RARs and delivered a single breath (120 ml) of cigarette smoke in 15 anesthetized, open-chest and artificially ventilated dogs. Studies were repeated after a pretreatment with aerosolized hexamethonium (3-8 breaths, 10%), aerosolized isoproterenol (12-15 breaths, 2%), or after the cardiac impact on the lung had been minimized by elevating the apex of the heart. The initial response of RARs was totally abolished by hexamethonium but was not affected by isoproterenol. The increase of total lung resistance induced by cigarette smoke and the concomitant type II delayed response of RARs were both abolished by isoproterenol. The type I delayed response of RARs was eliminated or largely attenuated after the apex of the heart had been elevated. Based upon these results, we conclude that a direct effect of nicotine on these receptors may be responsible for the immediate stimulation while the systemic effects of absorbed nicotine may play a part in evoking the delayed stimulation.     

Vitamin C prevents cigarette smoke-induced leukocyte aggregation and adhesion to endothelium in vivo.

A common feature of cigarette-smoke (CS)-associated diseases such as atherosclerosis and pulmonary emphysema is the activation, aggregation, and adhesion of leukocytes to micro- and macrovascular endothelium. A previous study, using a skinfold chamber model for intravital fluorescence microscopy in awake hamsters, has shown that exposure of hamsters to the smoke generated by one research cigarette elicits the adhesion of fluorescently labeled leukocytes to the endothelium of arterioles and small venules. By the combined use of intravital microscopy and scanning electron microscopy, we now demonstrate in the same animal model that (i) CS-induced leukocyte adhesion is not confined to the microcirculation, but that leukocytes also adhere singly and in clusters to the aortic endothelium; (ii) CS induces the formation in the bloodstream of aggregates between leukocytes and platelets; and (iii) CS-induced leukocyte adhesion to micro- and macrovascular endothelium and leukocyte-platelet aggregate formation are almost entirely prevented by dietary or intravenous pretreatment with the water-soluble antioxidant vitamin C (venules, 21.4 +/- 11.0 vs. 149.6 +/- 38.7 leukocytes per mm2, P < 0.01; arterioles, 8.5 +/- 4.2 vs. 54.3 +/- 21.6 leukocytes per mm2, P < 0.01; aortas, 0.8 +/- 0.4 vs. 12.4 +/- 5.6 leukocytes per mm2, P < 0.01; means +/- SD of n = 7 animals, 15 min after CS exposure). No inhibitory effect was observed by pretreatment of the animals with the lipid-soluble antioxidants vitamin E or probucol. The protective effects of vitamin C on CS-induced leukocyte adhesion and aggregation were seen at vitamin C plasma levels (55.6 +/- 22.2 microM, n = 7) that can easily be reached in humans by dietary means or supplementation, suggesting that vitamin C effectively contributes to protection from CS-associated cardiovascular and pulmonary diseases in humans.     

A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels.

Cigarette smoking produces pulmonary hypertension (PHT) through unknown mechanisms. In animal models acute smoke exposure induces cell proliferation in the small arteries adjacent to the alveolar ducts, and chronic exposure results in muscularisation of these vessels, with changes related to the development of PHT. Studies indicate that serine-elastase inhibitors can prevent experimental monocrotaline-induced PHT. This study examined whether they could also prevent cigarette smoke-induced pulmonary vascular disease. Guinea-pigs were exposed to cigarette smoke or air for 6 months. Some animals also received ZD0892, an orally active, synthetic, selective, serine-elastase inhibitor. The percentage of muscularised, small, pulmonary arteries was determined by morphometric analysis of histological sections and vascular cell proliferation by proliferating cell nuclear antigen staining. Vascular cell proliferation was markedly increased in the smoke-exposed animals and the percentage of completely muscularised small vessels was increased four-fold. Cell proliferation indices correlated with muscularisation indices. In the animals treated with ZD0892, the number of completely muscularised vessels was reduced by 50% and cell proliferation was reduced by 61%. These data suggest that smoke-induced cell proliferation leads to pulmonary arterial muscularisation. Serine-elastase inhibitors appear to be able to reduce cell proliferation and vascular remodelling.     

Exposure to neonatal cigarette smoke causes durable lung changes but does not potentiate cigarette smoke-induced chronic obstructive pulmonary disease in adult mice

The impact of early childhood cigarette smoke (CS) exposure on CS-induced chronic obstructive pulmonary disease (COPD) is unknown. This study was performed to evaluate the individual and combined effects of neonatal and adult CS exposure on lung structure, function, and gene expression in adult mice. To model a childhood CS exposure, neonatal C57/B6 mice were exposed to 14 days of CS (Neo CS). At 10 weeks of age, Neo CS and control mice were exposed to 4 months of CS. Pulmonary function tests, bronchoalveolar lavage, and lung morphometry were measured and gene expression profiling was performed on lung tissue. Mean chord lengths and lung volumes were increased in neonatal and/or adult CS-exposed mice. Differences in immune, cornified envelope protein, muscle, and erythrocyte genes were found in CS-exposed lung. Neonatal CS exposure caused durable structural and functional changes in the adult lung but did not potentiate CS-induced COPD changes. Cornified envelope protein gene expression was decreased in all CS-exposed mice, whereas myosin and erythrocyte gene expression was increased in mice exposed to both neonatal and adult CS, suggesting an adaptive response. Additional studies may be warranted to determine the utility of these genes as biomarkers of respiratory outcomes.     

肺气肿小鼠肺组织CD8+ IL-17+T细胞变化及作用机制

目的 观察香烟烟雾暴露肺气肿小鼠肺组织中CD8+IL-17+T细胞(Tc17)的变化,探讨Tc17在香烟烟雾暴露肺气肿小鼠中的作用及上调的可能机制.方法 将40只雄性Balb/c小鼠按随机数字表随机分为A(12周健康对照组)、B(24周健康对照组)、C(12周香烟烟雾暴露肺气肿组)、D(24周香烟烟雾暴露肺气肿组)组,每组10只.香烟烟雾暴露法建立小鼠肺气肿模型,观察小鼠肺气肿的改变,计算平均内衬间隔(Lm)和肺泡破坏指数(DI);流式细胞术检测小鼠肺组织中Tc17、CD8+IL-17+趋化因子受体6(CCR6)+细胞及CD8+IL-17+CCR6+细胞;荧光定量PCR法检测小鼠肺组织中维甲酸相关孤独受体(RORγt)、IL-17 mRNA表达;ELISA法检测小鼠肺组织匀浆中IL-1β、IL-6、IL-23、转化生长因子(TGF)β及趋化因子CCL20水平,并分析这些指标间的相关性.结果 (1)C、D组Lm、DI、Tc17/CD8+ T细胞比例、CD8+IL-17+CCR6+细胞/CD8+ T细胞比例、CD8+IL-17+CCR6+细胞/Tc17比例、RORγt和IL-17 mRNA相对表达量校正值、趋化因子CCL20浓度、IL-1β、IL-6、IL-23、TGFβ水平均明显高于A、B组,以D组增高更为显著,差异均有统计学意义(P值均小于0.05).(2)C、D组RORγt mRNA与IL-17 mRNA相对表达量校正值呈正相关(r=0.651,P＜0.05).C、D组Tc17/CD8+ T细胞比例与Lm、DI、趋化因子CCL20、IL-1β、IL-6、IL-23、TGFβ呈正相关(r值分别为0.734、0.884、0.899、0.531、0.643、0.799、0.637,P＜0.01).结论 香烟烟雾暴露肺气肿小鼠肺内Tc17上调;IL-1β、IL-6、IL-23、TGFβ升高及CCR6、趋化因子CCL20趋化作用促使Tc17上调.

Abstract：

Objective To evaluate the expression of Tc17 in a cigarette smoke-induced mice model of emphysema.To explore the probable mechanisms about how Tc17 cells to elevate in lungs of mice.Methods Forty male Balb/c mice were randomly divided into four groups,including control group ( 12 weeks,C12),control group (24 weeks,C24),smoke-exposure group (12 weeks,S12) and smoke-exposure group (24 weeks,S24 ),10 mice each group,Emphysema of mice was observed by HE pigmentation.Morphological changes were evaluated by mean linear intercepts (Lm) and destructive index (DI).The proportion of CD8+ IL-17 + Tc17,CD8+ IL-17 + CC chemokine receptor type 6 ( CCR6 ) + and 6CCR6 + Tc17 cells in lungs of mice was determined by flow cytometry.The mRNA expressions of retinoidrelated orphan nuclear receptor(RORγt) and IL-17 were evaluated by real-time PCR.The levels of IL-1 β,IL-6,IL-23,transforming growth factor β (TGFβ) and CC chemokine ligand 20 (CCL20) were tested by ELISA.Correlations among these indexes were analyzed.Results Lm and DI were significantly higher in S12 and S24 than in C12 and C24,S24 in particular (t value 4.378-15.188,all P ＜ 0.05).The percentages of Tc17 in S12 and S24[(9.28 ± 1.12)%,( 13.13 ±3.56)%]was significantly increased as compared with that in C12 and C24[(2.40 ±0.60 )%,(2.64 ±0.96 )%],S24 in particular.The mRNA levels of RORγt and IL-17 in S12 and S24 were higher than in C12 and C24,S12 and S24 in particular.There was significant difference (all P ＜0.05 ).The frequency of Tc17 cells had a positive correlation with Lm and DI ( r value were 0.734 and 0.884 respectively,P ＜ 0.01 ).The percentages of CD8+ IL-17 + CCR6 +T cells and CCR6 + Tc17 were significantly elevated in S12 and S24 compared to C12 and C24,S24 in particular (all P ＜ 0.05 ).There was positive correlation between Tc17 cell ratio and CCL20 levels( r =0.899,P ＜0.01 ).The levels of IL-1 β,IL-6,IL-23 and TGFβ in S12 and S24 were significantly increased as compared with that in C12 and C24.There was significant difference (all P ＜0.05).Meanwhile,the frequency of Tc17 cells had a positive correlation with IL-1β,IL-6,IL-23,and TGFβ.Conclusions An up-regulation of proportions Tc17 in lungs of cigarette smoke-induced emphysema mice were detected.The CCR6/CCL20 axis and the increased IL-1β,IL-6,IL-23 and TGFβ probably contributed to this up-regulation.     

Platinum nanoparticle antioxidants inhibit pulmonary inflammation in mice exposed to cigarette smoke

Recent evidence implicates increased oxidative stress as an important mechanism of the pulmonary inflammation that occurs in cigarette smokers. Since cigarette smoke (CS) contains and generates a large amount of reactive oxygen species (ROS) that elicit pulmonary inflammation, antioxidants may become effective therapeutic agents for CS-related inflammatory lung diseases, such as chronic obstructive pulmonary disease. Platinum nanoparticles stabilized with polyacrylate to form a stable colloid solution (PAA-Pt) are a new class of antioxidants that has been shown to efficiently quench ROS. In the present study we investigated the therapeutic effects of PAA-Pt on pulmonary inflammation in smoking mice. PAA-Pt or saline was administered intranasally to DBA/2 mice, which were then exposed to CS or control air daily for 3 days. Mice were sacrificed 4 h after their final exposure to CS or control air. CS exposure caused depletion of antioxidant capacity, NFκB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt prior to CS exposure was found to inhibit these changes. Intranasal administration of PAA-Pt alone did not elicit pulmonary inflammation or toxicity. In in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a CS extract. These results suggest that platinum nanoparticles act as antioxidants that inhibit pulmonary inflammation induced by acute cigarette smoking.