Ryder M I, Fujitaki R, Johnson G, Hyun W
Department of Stomatology, University of California, San Francisco, USA.
Ann Periodontol. 1998 Jul;3(1):76-87. doi: 10.1902/annals.1998.3.1.76.
Alterations of neutrophil functions by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. In the present study, we examined the in vitro effects of cigarette smoke on neutrophils at times and concentrations that may be encountered during smoke exposure. We measured the level of smoke exposure in the in vitro system by measuring the levels of nicotine and comparing these to levels in the oral cavity in smokers before and after smoking. We examined both the unstimulated and stimulated release of 2 oxidative burst products: superoxide (O-2) and hydrogen peroxide (H2O2). Salivary washings were collected from 7 smokers (> 1 pack/day) before smoking a cigarette. Immediately after they smoked a cigarette, a second set of washings was collected. In vitro exposure to smoke involved incubating aliquots of neutrophils in phosphate-buffered saline for 1 to 5 minutes. Nicotine and cotinine levels were quantitated using gas chromatography, with detection by electron impact mass spectrometry. Peripheral neutrophils were isolated from medically healthy non-smoking volunteers via a double-density gradient technique and incubated in vitro with whole cigarette smoke for 0 to 5 minutes. Phorbol myristate acetate (PMA; 10(-7) M) was used to stimulate half of the cell aliquots. Superoxide generation was assessed through the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. H2O2 production was assessed through the H2O2-dependent breakdown of dichlorofluorescin diacetate to its fluorophore and measured by flow cytometry. There was a marked elevation in salivary nicotine concentration from before smoking (mean: 80.8 ng) to after smoking (mean 1,685 ng/mL). In the in vitro smoke box system, there was a time-related elevation in nicotine from 1 to 5 minutes (50-->136 ng/mL). In PMA-stimulated cells exposed to smoke, there was a time-related inhibition of both superoxide and H2O2 production. However, in unstimulated cells exposed to smoke, there was a time-related increase in the release of superoxide and H2O2. A novel finding in unstimulated cells exposed to smoke was that there appeared to be 2 distinct populations of cells--one of "high" H2O2 producers and one of "low" H2O2 producers. The proportion of high H2O2 producers increased relative to smoke exposure. The relative production of H2O2 in the unstimulated high producers was comparable to PMA-stimulated cells at 5 minutes. This release of superoxide and H2O2 in unstimulated cells exposed to smoke may alter the pathogenic processes both in periodontal diseases and other systemic diseases.
烟草制品对中性粒细胞功能的改变可能在牙周疾病以及几种与吸烟相关的全身性疾病的发病机制中起核心作用。在本研究中，我们检测了香烟烟雾在烟雾暴露期间可能出现的时间和浓度下对中性粒细胞的体外影响。我们通过测量尼古丁水平并将其与吸烟者吸烟前后口腔中的水平进行比较，来测定体外系统中的烟雾暴露水平。我们检测了两种氧化爆发产物（超氧化物（O₂）和过氧化氢（H₂O₂））在未刺激和刺激状态下的释放情况。在7名吸烟者（每天吸烟超过1包）吸烟前收集唾液冲洗液。他们吸完烟后立即收集第二组冲洗液。体外烟雾暴露是将中性粒细胞的等分试样在磷酸盐缓冲盐水中孵育1至5分钟。使用气相色谱法定量尼古丁和可替宁水平，并通过电子轰击质谱法进行检测。通过双密度梯度技术从身体健康的非吸烟志愿者中分离外周血中性粒细胞，并在体外与全香烟烟雾孵育0至5分钟。用佛波酯（PMA；10⁻⁷ M）刺激一半的细胞等分试样。通过超氧化物歧化酶（SOD）可抑制的铁细胞色素c还原反应来评估超氧化物的产生。通过二氯荧光素二乙酸酯依赖H₂O₂分解为其荧光团并通过流式细胞术进行检测来评估H₂O₂的产生。唾液中尼古丁浓度从吸烟前的平均值80.8 ng显著升高至吸烟后的平均值1685 ng/mL。在体外烟雾箱系统中，尼古丁在1至5分钟内呈时间依赖性升高（50-->136 ng/mL）。在暴露于烟雾的PMA刺激细胞中，超氧化物和H₂O₂的产生均呈时间依赖性抑制。然而，在暴露于烟雾的未刺激细胞中，超氧化物和H₂O₂的释放呈时间依赖性增加。暴露于烟雾的未刺激细胞中的一个新发现是，似乎存在两种不同的细胞群体——一种是“H₂O₂高产生者”，另一种是“H₂O₂低产生者”。相对于烟雾暴露，H₂O₂高产生者的比例增加。未刺激的高产生者中H₂O₂的相对产生量与5分钟时PMA刺激的细胞相当。暴露于烟雾的未刺激细胞中超氧化物和H₂O₂的这种释放可能会改变牙周疾病和其他全身性疾病中的致病过程。
