The involvement of peripheral polymorphonuclear leukocytes in the oxidative stress and inflammation among cigarette smokers.

Background: Cigarette smoking is a well-known risk factor for the development of endothelial dysfunction and the progression of atherosclerosis. Oxidative stress and inflammation have recently been implicated in endothelial dysfunction. Objectives: To assess the concomitant contribution of polymorphonuclear leukocytes to systemic oxidative stress and inflammation in cigarette smokers. Methods: The study group comprised 41 chronic cigarettesmoking, otherwise healthy males aged 45.0 + 11.5 (range 31±67 years) and 41 male non-smokers aged 42.6+ 11.3 (range 31±65) who served as the control group. The potential generation of oxidative stress was assessed by measuring the rate of superoxide release from separated, phorbol 12-myristate 13-acetate-stimulated PMNL and by plasma levels of reduced (GSH) and oxidized (GSSG) glutathione. Inflammation was estimated indirectly by: a) determining the in vitro survival of PMNL, reflecting cell necrosis; b) in vivo peripheral PMNL counts, reflecting cell recruitment; and c) plasma alkaline phosphatase levels, indicating PMNL activation and degranulation. Results: PMA-stimulated PMNL from cigarette smokers released superoxide at a faster rate than PMNL from the controls. Smokers had decreased plasma GSH and elevated GSSG levels. In vitro incubation of control and smokers' PMNL in sera of smokers caused necrosis, while control sera improved smoker PMNL survival. Smokers' PMNL counts, although in the normal range, were significantly higher than those of controls. Plasma ALP levels in smokers were significantly higher than in controls and correlated positively with superoxide release and PMNL counts. Conclusions: Our study shows that PMNL in smokers are primed in vivo, contributing concomitantly to systemic oxidative stress and inflammation that predispose smokers to endothelial dysfunction, and explains in part the accelerated atherosclerosis found in smokers. IMAJ 2002;4:1015±1019 For Editorial see page 1077 Cigarette smoking is a well-known risk factor for the development and progression of atherosclerosis [1]. The mechanism that predisposes cigarette smokers to organ injury and atherosclerosis is multifactorial and mostly unknown. Endothelial dysfunction appears to have a central role in the pathogenesis of atherosclerosis and is frequently described in smokers [2]. Oxidative stress and inflammation are among the mechanisms underlying endothelial dysfunction in clinical states associated with atherosclerosis [3]. Evidence for increased systemic oxidative stress in smokers is based mainly on decreased concentrations of endogenous antioxidants, increased oxidants and increased level of oxidation adducts [4], while evidence for systemic inflammation relies on increased levels of fibrinogen and C-reactive protein [5]. The peripheral polymorphonuclear leukocyte-mediated injury in smokers caused by inflammation and oxidative stress is known, but the majority of the reports focus on local airway pathologies [6], while only a few relate to the role of these cells in systemic oxidative stress [7]. Cigarette smoke constituents were also found to impair PMNL bactericidal activity [8]. Nicotine in vitro potentiated PMNL to release superoxide [8], while superoxide, itself derived from smoke, impaired the oxidative metabolism of PMNL [9]. We have previously established that peripheral PMNL, when primed, display enhanced superoxide release contributing to systemic oxidative stress. Simultaneously, these cells start a cascade of systemic inflammatory events, a process initiated by enhanced necrotic cell death, promoting chemotaxis and PMNL recruitment [10±12]. We have shown that primed PMNL are the common denominator in inducing systemic oxidative stress and chronic inflammation in clinical disorders known to be associated with endothelial dysfunction, accelerated atherosclerosis, and increased cardiovascular morbidity and mortality. Among these disorders are essential hypertension, diabetes mellitus type 2, chronic renal failure, and end-stage renal disease treated with hemodialysis [10±12]. Thus, the aim of the present study was to establish whether smoking could be included in the above-mentioned pathologies, predisposing smokers to endothelial dysfunction and atherosclerosis by the presence of concomitant systemic oxidative stress and chronic inflammation caused by their PMNL. Patients and Methods Two groups of subjects were enrolled in this study: 41 healthy male chronic cigarette smokers aged 45.0 + 11.5 (range 31±67 years), and 41 male non-smokers aged 42.6 + 11.3 (range 31±65) who served as the controls. The smoking load or the chronic cigarette consumption of each individual was determined by calculating the Original Articles PMNL = polymorphonuclear leukocytes PMA = phorbol 12-myristate 13-acetate GSH = reduced glutathione GSSG = oxidized glutathione ALP = alkaline phosphatase 1015 IMAJ . Vol 4 . November 2002 Neutrophils in Cigarette Smokers number of cigarette-packs smoked per day multiplied by the number of smoking years (pack-year) [13]. Smokers included in this study had to smoke no less than one pack of cigarettes (20 cigarettes) per day, had a pack-year greater than 10, and were allowed to smoke until 30 minutes before their blood withdrawal to prevent acute smoke changes as described by Blann et al. [14]. All subjects underwent a clinical examination and routine laboratory blood tests to rule out the presence of any coexisting disease. All studied subjects had normal blood pressure ( 98% pure, approximately 10 cells per isolation) were resuspended in a minimal volume (0.1±0.3 ml) of Hank's balanced salt solution, immediately counted, and diluted to a final volume of 1 ml with HBSS containing 0.5% glucose, according to different experimental needs. Sera from smokers and controls were frozen at 70C and saved for in vitro survival assays (see below).