Inactivation of human alpha 1-proteinase inhibitor by cigarette smoke: effect of smoke phase and buffer

In order to resolve a discrepancy in the literature, we have examined the in vitro inactivation of human alpha 1-proteinase inhibitor by direct exposures either to whole cigarette smoke or to filtered (i.e., gas-phase) smoke. Wyss and coworkers (2) reported that whole smoke does not inactivate the protein, whereas we reported that gas-phase smoke does. We now find that direct exposure to gas-phase cigarette smoke causes a slightly greater inactivation of the protein than does direct exposure to whole cigarette smoke, confirming our earlier suggestion that whole smoke is less oxidizing than is gas-phase smoke. This difference, however, does not explain the dramatic difference between our previous findings and those of Wyss and coworkers (2). The explanation for the discrepancy lies in the nature of the buffers used. Wyss and coworkers used Tris buffer and the use of Tris quenches the inactivation process almost completely. Our experiments used phosphate buffer. We suggest that Tris is an unsuitable buffer for use in experiments that probe the effects of cigarette smoke.     

Inactivation of a bacterial virulence pheromone by phagocyte-derived oxidants: new role for the NADPH oxidase in host defense

Quorum sensing triggers virulence factor expression in medically important bacterial pathogens in response to a density-dependent increase in one or more autoinducing pheromones. Here, we show that phagocyte-derived oxidants target these autoinducers for inactivation as an innate defense mechanism of the host. In a skin infection model, expression of phagocyte NADPH oxidase, myeloperoxidase, or inducible nitric oxide synthase was critical for defense against a quorum-sensing pathogen, Staphylococcus aureus, but not for defense against a quorum sensing-deficient mutant. A virulence-inducing peptide of S. aureus was inactivated in vitro and in vivo by reactive oxygen and nitrogen intermediates, including HOCl and ONOO(-). Inactivation of the autoinducer prevented both the up-regulation of virulence gene expression and the downstream sequelae. MS analysis of the inactivated peptide demonstrated that oxidation of the C-terminal methionine was primarily responsible for loss of activity. Treatment of WT but not NADPH oxidase-deficient mice with N-acetyl methionine to scavenge the inhibitory oxidants increased in vivo quorum sensing independently of the bacterial burden at the site of infection. Thus, oxidant-mediated inactivation of an autoinducing peptide from S. aureus is a critical innate defense mechanism against infection with this pathogen.     

Cellular response to cigarette smoke and oxidants: adapting to survive

The gaseous and soluble phases of cigarette smoke are sources of oxidants that contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). Chronic oxidative stress of cigarette smoking induces mucus secretion and inhibits cystic fibrosis transmembrane conductance regulator function. The increased mucus viscosity renders the airways susceptible to bacterial infections, a hallmark of chronic bronchitis. Furthermore, lungs chronically exposed to the toxic mixture of oxidants in cigarette smoke show signs of endoplasmic reticulum stress, unfolded protein response, altered ceramide metabolism, and apoptosis. Fortunately, the respiratory tract has developed effective adaptive cellular mechanisms to limit oxidant damage. Numerous antioxidant enzymes and glutathione-dependent detoxification systems are increased in healthy smokers. The regulation of the antioxidant response is largely dependent on the nuclear factor erythroid 2-related factor-2 (Nrf2) pathway. However, patients with COPD have defective Nrf2 responses. Novel therapies such as 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) to correct defective Nrf2-dependent cellular response may hold promise for patients with COPD.     

Effects of cigarette smoke in mice with different levels of alpha(1)-proteinase inhibitor and sensitivity to oxidants.

The role of strain difference in the response to cigarette smoke was investigated in mice. Mice of the strains DBA/2 and C57BL/6J responded to acute cigarette smoke with a decrease of the antioxidant defenses of their bronchoalveolar lavage (BAL) fluids. On the other hand, under these conditions ICR mice increased their BAL antioxidant defenses. Mice of these three strains were then exposed to cigarette smoke (three cigarettes/d, 5 d/wk) for 7 mo. Lung elastin content was significantly decreased in C57BL/6J and DBA/2 but not in ICR mice. Also, emphysema, assessed morphometrically using three methods, was present in C57BL/6J and DBA/2 but not in ICR mice. In an additional study pallid mice, with a severe serum alpha(1)-proteinase inhibitor (alpha(1)-PI) deficiency and that develop spontaneous emphysema, were exposed to cigarette smoke for 4 mo. This resulted in an acceleration of the development of the spontaneous emphysema assessed with morphometrical and biochemical (lung elastin content) methods. All these results indicate that sensitivity to the effects of cigarette smoke is strain-dependent and cigarette smoke accelerates the effects of alpha(1)-PI deficiency.     

分泌型白细胞蛋白酶抑制剂对香烟烟雾提取物诱导人支气管上皮细胞炎症介质表达的影响

目的 检测分泌型白细胞蛋白酶抑制剂(SLPI)对香烟烟雾提取物(CSE)诱导体外培育的正常人支气管上皮细胞(NHBE)炎症介质表达水平的影响,探讨SLPI对慢性气道炎症局部保护作用的机制.方法 实验设对照组、CSE组、SLPI组和SLPI+CSE组,均采用免疫细胞化学方法检测NHBE细胞中基质金属蛋白酶-9(MMP-9)蛋白的表达,采用酶联免疫吸附试验检测各组培养上清液中白细胞介素8(IL-8)的表达.应用SPSS 11.5软件进行统计学处理,多样本均数比较采用单因素方差分析和SNK检验,若方差不齐进行校正,P<0.05为差异有统计学意义.结果 对照组能少量表达MMP-9(积分值为3.1±0.5)和IL-8[(4.9±0.6)ng/L];用CSE干预NHBE细胞能诱导MMP-9和IL-8的表达,且在一定范围内与CSE干预时间呈依赖性,24 h达峰值,MMP-9积分值和IL-8浓度分别为6.6±0.4和(17.7±1.9)ng/L,随后呈明显下降趋势;用SLPI干预NHBE细胞能明显抑制MMP-9(积分值为0.8±0.5)和IL-8[(0.7±0.6)ng/L]的表达.结论 SLPI能抑制CSE诱导的NHBE细胞的MMP-9和IL-8表达.     

Different lung responses to cigarette smoke in two strains of mice sensitive to oxidants.

The development of cigarette smoke-induced pulmonary changes in C57 Bl/6J and DBA/2 mice was investigated. Both strains are sensitive to oxidants and C57Bl/6J mice are moderately deficient in serum alpha1-proteinase inhibitor. Following chronic exposure to cigarette smoke, patchy emphysema was present in mice of both strains, but developed faster in DBA/2 mice. A positive reaction for mouse neutrophil elastase was seen on the septa of both strains. Additionally, the DBA/2 mice developed a uniform parenchymal dilation that was preceded by the appearance of apoptotic cells in areas with a low signal for vascular endothelial growth factor-receptor 2. Fibrotic areas scattered throughout the parenchyma, coupled with a positive immunohistochemical reaction for transforming growth factor-beta was seen only in DBA/2 mice. Both DBA/2 and C57Bl/6J strains showed epithelial cell injury and areas of deciliation in their airways. However, the appearance of goblet cell metaplasia was common in C57Bl/6J mice but rare in DBA/2 mice. A positive immunohistochemical reaction for interleukin (IL)-4, IL-13 and MUC5AC was seen only in the airways of C57Bl/6J mice. Strain characteristics (alpha1-proteinase inhibitor levels, sensitivity to oxidants, and constitutive levels of vascular endothelial growth factor-receptor 2) and phenotypical responses (apoptosis and cytokine distribution) may condition parenchymal and airway changes to cigarette smoke.     

Indomethacin and flurbiprofen speed recovery of rat bronchial epithelium after exposure to cigarette smoke

The cigarette smoke-induced rat model of chronic bronchitis was used to study the time course of the return of cigarette smoke-induced secretory cell hyperplasia to the normal and the capacity of two non-steroidal anti-inflammatory drugs to speed this recovery. Cigarette smoke alone significantly increased (P less than 0.05) the number of secretory cells in all of the eight airway levels studied to between 52-225% above control values. After cessation of exposure, recovery was complete by 9 days in the trachea, between 10-21 days in 'proximal' intrapulmonary airways and 43-84 days in distal bronchioli. Indomethacin and flurbiprofen, given by intraperitoneal injection at 4 mg/kg body weight for 21 days of the recovery period, significantly reduced the time taken for recovery to between 4 and 9 days in intrapulmonary airways but had no effect in the trachea.     

Bronchial leukocyte proteinase inhibitor levels in bronchial washings in asthma patients

To evaluate whether epithelial damage of airways in asthma could be related to diminished levels of the low molecular weight bronchial leukocyte proteinase inhibitor (BLPI) in airways, we determined BLPI in bronchial washings of 13 asthma patients and 13 healthy subjects, using a sensitive enzyme-linked immunoassay. The patients had asthma due to western red cedar and had bronchial washings done 24 to 48 hours after a mild to moderate asthmatic reaction induced by inhalation challenge. We did not find significant differences in BLPI concentrations in lavage fluid of asthma patients and healthy control subjects. The ratios of BLPI to albumin levels in bronchial washings appeared to be lower among asthmatic patients, but this difference was mainly due to an increase in albumin levels in lavage fluid in asthma. In addition, there were no significant differences in BLPI levels in washings obtained from main and segmental bronchi in both control subjects and asthma patients.     

Inactivation of chemotactic factor inactivator by cigarette smoke. A potential mechanism of modulating neutrophil recruitment to the lung

Activation of the complement system with generation of the potent neutrophil chemotactic factor C5a has been proposed to play a significant role in the neutrophil accumulation in the lungs of cigarette smokers. Chemotactic factor inactivator (CFI) can inhibit C5a-directed neutrophil chemotaxis by binding to the C5a cochemotaxin GcGlobulin (GcG), a vitamin-D-binding protein, and inhibiting the capacity of GcG to enhance the chemotactic activity of C5a. Because cigarette smoke can inhibit the function of some proteins, a loss of CFI functional activity induced by cigarette smoke would allow an increased capacity of GcG to augment C5a-directed neutrophil chemotaxis. In order to test this hypothesis, cigarette smoke was bubbled through a CFI solution, and the solution was evaluated for its ability to inhibit the chemotactic activity of C5a and GcG. Smoke-treated CFI inhibited only 36% of the C5a-GcG chemotactic activity. In contrast, a CFI solution treated with air inhibited 62% of the chemotactic activity (p less than 0.001). Consistent with these observations, smoke-treated CFI exhibited a decreased capacity to bind to GcG and a decreased capacity to inhibit the binding of C5a des Arg to GcG. CFI contained in the bronchial lavage fluids obtained from patients with chronic obstructive pulmonary disease secondary to cigarette smoking and asymptomatic smokers exhibited a decreased capacity to inhibit C5a-GcG neutrophil chemotaxis and to bind to GcG (p less than 0.05, both comparisons). Furthermore, smoke bubbled through normal bronchial lavage fluid decreased the capacity of CFI to bind to GcG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbic acid neutralizes reactive oxidants released by hyperactive phagocytes from cigarette smokers

During exposure to the leukoattractant FMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine) human polymorphonuclear leukocytes (PMNL) exhibit a bimodal pattern of luminol-enhanced chemiluminescence (LECL) with distinct early extracellular and later-occurring intracellular membrane-associated oxidative responses [4, 7, 14]. With the primary objective of measuring the effects of oral administration of the antioxidant ascorbate on the generation of reactive oxidants by circulating phagocytes from cigarette smokers and nonsmokers, we have developed a method for the measurement of FMLP-activated LECL in whole blood. With this method definite bimodal LECL responses, similar to those obtained with pure PMNL, were observed with FMLP-activated whole blood. No LECL responses were observed when whole blood from 3 children with chronic granulomatous disease was stimulated with FMLP, which shows that the FMLP-activated LECL responses are exclusively phagocyte-derived in blood from normal individuals. The whole blood method was used to compare the FMLP-activated LECL responses in blood from 30 asymptomatic smokers and 30 nonsmokers and to investigate the effects of co-incubation of whole blood from smokers and nonsmokers with ascorbate (2.5 × 10⁻⁵ M-2.5 × 10⁻⁴ M), as well as the effects of oral administration of the antioxidant on FMLP-activated LECL. Increased generation of both extracellular (58% mean increase,P < 0.005) and intracellular (75% mean increase,P < 0.005) phagocyte-derived oxidants was observed with FMLP-activated blood from smokers relative to nonsmokers. Co-incubation of blood with ascorbate in vitro caused a dose-dependent selective neutralization of extracellular oxidants. Similar effects were observed following the oral administration of a single dose of ascorbate (1 g). The whole blood method may be useful in identifying smokers at risk for smoking-related diseases.     

Ascorbic acid neutralizes reactive oxidants released by hyperactive phagocytes from cigarette smokers

During exposure to the leukoattractant FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) human polymorphonuclear leukocytes (PMNL) exhibit a bimodal pattern of luminol-enhanced chemiluminescence (LECL) with distinct early extracellular and later-occurring intracellular membrane-associated oxidative responses [4, 7, 14]. With the primary objective of measuring the effects of oral administration of the antioxidant ascorbate on the generation of reactive oxidants by circulating phagocytes from cigarette smokers and nonsmokers, we have developed a method for the measurement of FMLP-activated LECL in whole blood. With this method definite bimodal LECL responses, similar to those obtained with pure PMNL, were observed with FMLP-activated whole blood. No LECL responses were observed when whole blood from 3 children with chronic granulomatous disease was stimulated with FMLP, which shows that the FMLP-activated LECL responses are exclusively phagocyte-derived in blood from normal individuals. The whole blood method was used to compare the FMLP-activated LECL responses in blood from 30 asymptomatic smokers and 30 nonsmokers and to investigate the effects of co-incubation of whole blood from smokers and nonsmokers with ascorbate (2.5 X 10(-5) M-2.5 X 10(-4)M), as well as the effects of oral administration of the antioxidant on FMLP-activated LECL. Increased generation of both extracellular (58% mean increase, P less than 0.005) and intracellular (75% mean increase, P less than 0.005) phagocyte-derived oxidants was observed with FMLP-activated blood from smokers relative to nonsmokers. Co-incubation of blood with ascorbate in vitro caused a dose-dependent selective neutralization of extracellular oxidants. Similar effects were observed following the oral administration of a single dose of ascorbate (1 g). The whole blood method may be useful in identifying smokers at risk for smoking-related diseases.     

Erythrocytes prevent inactivation of alpha 1-antitrypsin by cigarette smoke

Red blood cells (RBC) possess strong antioxidant activity. We tested whether this activity was sufficient to prevent the oxidative inactivation of alpha 1-antitrypsin (alpha 1-AT) by cigarette smoke. We found that RBC in physiological concentrations completely prevented the inactivation of alpha 1-AT. The major erythrocytic antioxidants, catalase, superoxide dismutase and glutathione were then selectively inhibited and the RBC retested. Only the inhibition of catalase significantly impaired the protective ability of added erythrocytes. We suggest that RBC antioxidants may be an important variable in determining the degree of protection of alpha 1-AT against oxidation.     

A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells.

Using a xenotransplantation system in which immortalized nontumorigenic human bronchial epithelial cells (BEAS-2B cells) are grown in deepithelialized rat tracheas that are subcutaneously transplanted into athymic nude mice, we exposed BEAS-2B cells either to cigarette smoke condensate or to the tobacco-specific N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1- butanone. After 6 mo the carcinogen-exposed BEAS-2B cells were neoplastically transformed to invasive adenocarcinomas. Cell lines obtained from xenografts exposed in vivo to chemicals exhibited several features typical of malignant lung cancer cells, such as increased in vivo invasiveness that correlated well with enhanced type IV collagenolytic activity, resistance to serum-induced growth inhibition, and increased expression of transforming growth factor alpha and its cellular-membrane receptor. Invasiveness, similar to that seen after exposure to phorbol esters, was also detected after in vitro exposure of BEAS-2B cells to cigarette smoke condensate. Collectively, these data indicate that cigarette smoke condensate and N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone induce in vivo phenotypic changes in BEAS-2B cells similar to the progressive changes that occur during human lung carcinogenesis.     

Dysregulation of gastric H,K-ATPase by cigarette smoke extract

AIM： To test whether the expression and activity of H,K-ATPase in parietal cells would be affected by cigarette smoke extract.
METHODS： Extracts of cigarette smoke were administered into mice by gastric gavage （5 mg/kg body weight/day） for 3 d or in drinking water for 7 or 14 d. For the latter, each day a mouse consumed 5 mL water containing extracts of two cigarettes, on average. Control littermate mice received only vehicle. To compare the amount of H,K-ATPase in control and smoke-treated mice, the stomach was processed for Western blotting and immunohistochemical analysis using monoclonal antibodies specific for α- or β-subunits of H,K-ATPase. The p-nitrophenylphospatase activity assay was used as a measurement for K-dependent H,K-ATPase activity.
RESULTS： Probed transblots showed an increase in the amount of H,K-ATPase in smoke-treated mice which was confirmed by immunohistochemistry and was found to be due to increased amounts of protein per parietal cell rather than an increased parietal cell number. The increase in the amount of H,K-ATPase was associated with an enhancement of its enzymatic activity. K-dependent activity in control and smoketreated mice was significantly different （respectively, 0.12 μmol/mg vs 0.27μmol/mg per minute, P 〈 0.05）.
CONCLUSION： Administration of cigarette smoke extract is associated with an increase in the amount and activity of H,K-ATPase and hence, smokers are susceptible to development of peptic ulcer.     

香烟烟雾对细胞生物学功能的影响

香烟烟雾中含有大量的化学有毒物质,其颗粒物和挥发性有机物可不同程度地造成炎症的产生、细胞毒性增加、染色体损伤和DNA链断裂,从而引起慢性阻塞性肺疾病、肺癌等疾病的发生.氧化应激机制是目前研究的热点.本文主要从吸烟与呼吸系统疾病、香烟烟雾对细胞的损伤机制、香烟烟雾对气道上皮细胞的作用三大方面对香烟烟雾对细胞生物学功能的影响进行综述,有助于呼吸道疾病的诊断和治疗.     

Bronchoconstriction and apnea induced by cigarette smoke: Nicotine dose dependence

Recently, evidence was presented to suggest that nicotine absorbed from cigarette smoke was the main cause of smoke-induced bronchoconstriction (Hartiala et al., J Appl Physiol (1985) 59(1): 64–71). However, due to the qualitative nature of the data, it remains unclear whether cigarette-smoke-induced bronchoconstriction is related to the nicotine content of the smoke in a dose-dependent manner. Experiments were performed on intact anesthetized dogs (n = 6). Each animal spontaneously inhaled 300 cc smoke containing low (0.37 mg), medium (1.46 mg), or high (1.80 mg) levels of nicotine. Isometric tension was measured in an isolated tracheal segment not exposed to the smoke as an index of bronchoconstriction. In all dogs there was a nicotine dose-dependent increase in tracheal tension. The time in expiration (T_e) in the breath following smoke inhalation was prolonged, the magnitude of prolongation being dependent upon the nicotine content of the smoke. These results suggest that bronchoconstriction and changes in T_e induced by cigarette smoke are nicotine-dependent.     

香烟烟雾暴露对支气管哮喘影响机制的研究进展

支气管哮喘(简称哮喘)是一种以可逆性气流受限为特征的慢性气道炎症性疾病,在世界范围内的发病率有明显增加.流行病学研究结果显示,环境污染可能是其发病原因之一.香烟烟雾是一种重要的环境污染物,近年来研究结果显示,母亲孕期吸烟、儿童被动吸烟以及主动吸烟均可增加哮喘发病率,长期吸烟能够增加哮喘发作严重度及病死率,且减弱对糖皮质激素(简称激素)治疗的反应性.本文对香烟烟雾暴露与哮喘发病和病情发展的关系及其影响机制进行综述.