Ryder Mark I, Wu Timothy C, Kallaos Steven S, Hyun William
Division of Periodontology, Box 0650, University of California, San Francisco, San Francisco, CA 94143, USA.
J Periodontal Res. 2002 Aug;37(4):286-92. doi: 10.1034/j.1600-0765.2002.01034.x.
Tobacco smoking is a major risk factor in the incidence and severity of periodontal diseases. Alterations of neutrophil function by short-term high levels of smoke during the act of smoking (acute smoke exposure) as well as long-term exposure to lower levels of tobacco substances in the bloodstream (chronic smoke exposure) may play a role in the pathogenesis of periodontal diseases in smokers. The polymerization and depolymerization of f-actin in response to infectious agents or inflammatory mediators is a critical process in a variety of neutrophil functions. In this study, we examined the effects of in vitro smoke exposure on neutrophils from smokers and non-smokers (which may be comparable to in vivo acute smoke exposure) and neutrophils from smokers not exposed to further in vitro smoke (which may be comparable to chronic smoke exposure) on f-actin kinetics. Peripheral neutrophils were isolated from seven healthy smoking subjects and seven healthy age-matched non-smoking subjects and exposed to 1-5 min of acute smoke in a smoke box system or not exposed to further smoke (baseline controls). Selected aliquots of neutrophils from control and 5-min exposures of acute smoke were then stimulated with the chemotactic peptide F-met-leu-phe at 10(-7) M for an additional 30-360 s. Cells were fixed and permeabilized, stained for f-actin with NBD phallacidin, and analyzed by flow cytometry. From baseline to 5 min of in vitro smoke exposure, there was a 38% decline in f-actin stain in non-smokers and a 30% decline in f-actin stain in smokers (p > 0.05) with f-actin values slightly higher in smokers than-non-smokers (p > 0.05). With F-met-leu-phe stimulation, both smokers and-non-smokers demonstrated a characteristic rise in f-actin stain from 0 to 120 s with a subsequent decline to baseline at 360 s and no significant differences in f-actin levels at any time of stimulation between groups. After preincubation with 5 min of in vitro smoke, the magnitude of rise in f-actin was less in both smokers and non-smokers when compared to cells not incubated with 5 min of smoke (p < 0.05 at 120 s for both smokers and non-smokers). F-actin values in smokers were higher than-non-smokers from 30 to 360 s of F-met-leu-phe exposure (p > 0.05). These results demonstrate that in vitro smoke exposure may impair normal f-actin kinetics. These alterations in f-actin kinetics may in turn affect other neutrophil functions which may impact on the pathogenesis of periodontal diseases in smokers.
吸烟是牙周疾病发生和严重程度的主要风险因素。在吸烟过程中（急性烟雾暴露）短期高水平烟雾以及长期低水平烟草物质在血液中的暴露（慢性烟雾暴露）导致的中性粒细胞功能改变，可能在吸烟者牙周疾病的发病机制中起作用。响应感染因子或炎症介质，丝状肌动蛋白（f-肌动蛋白）的聚合和解聚是多种中性粒细胞功能中的关键过程。在本研究中，我们检测了体外烟雾暴露对吸烟者和非吸烟者的中性粒细胞（这可能与体内急性烟雾暴露相当）以及未进一步接受体外烟雾暴露的吸烟者的中性粒细胞（这可能与慢性烟雾暴露相当）的f-肌动蛋白动力学的影响。从7名健康吸烟受试者和7名年龄匹配的健康非吸烟受试者中分离外周血中性粒细胞，将其置于烟雾箱系统中暴露于急性烟雾1 - 5分钟或不进行进一步烟雾暴露（基线对照）。然后，用10⁻⁷ M的趋化肽F-甲硫-亮-苯丙氨酸刺激对照和急性烟雾暴露5分钟的中性粒细胞的选定等分试样，持续30 - 360秒。将细胞固定并通透化，用NBD鬼笔环肽对f-肌动蛋白进行染色，并通过流式细胞术进行分析。从基线到体外烟雾暴露5分钟，非吸烟者的f-肌动蛋白染色下降38%，吸烟者下降30%（p > 0.05），吸烟者的f-肌动蛋白值略高于非吸烟者（p > 0.05）。在F-甲硫-亮-苯丙氨酸刺激下，吸烟者和非吸烟者的f-肌动蛋白染色均呈现出从0到120秒特征性升高，随后在360秒降至基线，且两组在刺激的任何时间点f-肌动蛋白水平均无显著差异。在与体外烟雾预孵育5分钟后，与未与烟雾预孵育5分钟的细胞相比，吸烟者和非吸烟者的f-肌动蛋白升高幅度均较小（吸烟者和非吸烟者在120秒时p < 0.05）。在F-甲硫-亮-苯丙氨酸暴露30到360秒期间，吸烟者的f-肌动蛋白值高于非吸烟者（p > 0.05）。这些结果表明，体外烟雾暴露可能损害正常的f-肌动蛋白动力学。f-肌动蛋白动力学的这些改变可能反过来影响其他中性粒细胞功能，进而可能影响吸烟者牙周疾病的发病机制。
