In addition to neutrophilia, multiple adverse effects of cigarette smoke on mature, circulating neutrophils have been demonstrated. The consequences for neutrophils exposed to tobacco smoke, or to specific tobacco smoke components, include impaired f-actin kinetics and reduced deformability [21
], sequestration in the pulmonary microcirculation [22
], and activation in the microcirculation [23
]. The synthesis of superoxide has been reported to be both increased and decreased in tobacco smoke or nicotine-exposed neutrophils in vitro
by different research groups. However, such studies have utilized different media for neutrophil suspensions and challenge [6
]. Zappacosta et al. [24
] have reported that the phagocytic ability of neutrophils pretreated with aqueous smoke extracts is hindered. Importantly, nicotine has been shown to induce the release of elastase from human neutrophils [25
], which may play a major role in tobacco-induced destructive diseases, such as COPD. To summarize, while there are certainly conflicting data, it is clear that tobacco smoking profoundly affects multiple functions of circulating and tissue neutrophils and may shift the net balance of neutrophil activities in a destructive direction, as we have recently reviewed [6
There is a lack of data relating to the effect of tobacco smoke on neutrophil differentiation in humans. It must be acknowledged that our study model, DMSO-differentiated HL-60 cells, are not entirely similar to normal neutrophils. However, this leukemic human cell line does permit the reproducible study of differentiation while retaining many of the key effector functions of primary neutrophils, such as cytokine production, MMP release, generation of an oxidative burst, and the ability to phagocytose and kill microbes. Previously, Armstrong et al. [26
] showed that five-day exposure of undifferentiated HL-60 cells to 0.06 and 0.8 μM nicotine induced a significant increase in neutrophil elastase gene activity and protein expression over unstimulated cells, but did not initiate HL-60 cell differentiation. Otherwise, this study is the first, to the best of our knowledge, to examine the effects of nicotine on human neutrophil differentiation. Like Armstrong et al. [26
], we noted an increase in cellular protein content by infrared spectroscopy in nicotine-stimulated, undifferentiated HL-60 cells, but nicotine did not induce differentiation in these cells [26
]. In rabbits, in vivo
cigarette smoke exposure has been associated with increased numbers of both mature and band cell (immature) neutrophils liberated from the marrow into the circulation, resulting in preferential sequestration in the microvasculature [2
]. Our data shows that exposure to nicotine during differentiation promotes late phase differentiation in human HL-60 cells (Figure ).
Nicotine had no effect on cell growth or viability in undifferentiated or differentiating HL-60 cells (Figure ). Such results are in keeping with the studies of Pabst et al. [12
], who have previously observed that nicotine did not affect the viability of mature neutrophils in vitro
, as determined by trypan blue exclusion. Furthermore, our electron microscopy studies indicate that nicotine does not influence HL-60 cell ultrastructure, relative to untreated control cells.
It has been known for some time that cigarette smoking can induce leukocyte-endothelial adhesion, microvascular and macrovascular entrapment of leukocytes, and leukocyte aggregation in humans and animal models, and that key adhesion molecules including CD11b (a β2-integrin chain) are central to these processes, as we have recently reviewed [27
]. We have previously examined the influence of tobacco smoking on CD11b expression, and other adhesion molecules, on circulating human neutrophils in vivo
]. We found that tobacco smoking did not acutely influence CD11b expression levels on primary neutrophils. However, Maestrelli et al. [29
] have reported that the numbers of neutrophils expressing CD11b and CD18, but not CD11a or CD11c, are increased in chronic smoking subjects with airway obstruction, compared to smokers without airway obstruction, and hypothesized that CD11b/CD18 expression by sputum neutrophils may represent a marker for the development of chronic airway obstruction among smokers. In the current HL-60 model, we have employed CD11b as a marker of terminal differentiation. We noted no statistically significant differences in CD11b expression levels in DMSO-stimulated HL-60 cells exposed to nicotine, compared to unexposed controls. This would imply that, within the limits of this in vitro
model, any alterations in CD11b expression profiles in chronic smokers are more likely to be the result of exposure of differentiating neutrophils to components of tobacco other than nicotine, or that neutrophil CD11b expression profiles are influenced by smoke components in mature, circulating cells rather than during differentiation. Nicotine did not affect the other maturation marker examined, that is, formazan deposition.
Neutrophils enter the circulation as terminally differentiated cells. Without appropriate inflammatory stimuli, programmed cell death is initiated rapidly. Thus, circulating neutrophils normally have a short life span of less than a day. However, their longevity can be significantly increased in tissues due to the suppression of apoptosis by pro-inflammatory cytokines and other mediators [30
]. Induction of apoptosis causes loss of effector function in neutrophils, including chemotaxis, degranulation, generation of the respiratory burst, and is, thus, a key step in the resolution of inflammation. Neutrophils that die by apoptosis are predominantly phagocytosed and destroyed without releasing their proteolytic arsenal [13
]. However, neutrophils that die by necrosis release degradative proteases, including MMP-9, and other factors that can contribute to tissue degradation. Consequently, inappropriate suppression of apoptosis would be expected to facilitate or potentiate neutrophil-mediated tissue injury through prolonging neutrophil functional lifespan and abrogating the phagocytotic clearance of dead neutrophils.
The available evidence for an effect of tobacco smoke on neutrophil longevity is controversial. Acrolein, a tobacco smoke component, has been shown to suppress pro-apoptotic signals in systemic neutrophils in an in vitro
model. Aoshiba et al. [31
] reported that nicotine can suppress neutrophil apoptosis. A more recent report concluded high nicotine doses did not influence apoptosis in freshly isolated peripheral neutrophils [32
]. Mariggio et al. [33
] have presented the alternate message, showing nicotine to be a potent inducer of apoptosis in systemic neutrophils, and Yoshida et al. [32
] have reported that high nicotine doses induce DNA cleavage in HL-60 cells. Therefore, an urgent need to clarify the influence of physiologically relevant nicotine exposure on apoptosis in neutrophils has been identified.
We hypothesized that the neutrophilia noted in smokers may partly be due to suppression of apoptosis and that pro-apoptotic signaling may be suppressed even during differentiation. Yet, contradicting this hypothesis, infrared spectroscopy analysis of the molecular profiles of nicotine-stimulated HL-60 cells revealed apoptosis-associated cellular alterations, including increases in the cellular lipid content and in the DNA-protein ratio (Figure ). However, such pro-apoptotic influences of nicotine on HL-60 cells during differentiation did not result in committed apoptosis, as would have been confirmed by propidium iodide staining in the flow cytometry experiments. Furthermore, nicotine exerted no statistically significant positive or negative effect on camptothecin-induced apoptosis. Clearly, definitive conclusions on the influence of nicotine on apoptosis in differentiating neutrophils would require the detection of biomarkers of cell apoptosis, such as Annexin V staining, Caspase 3 activation, and DNA fragmentation. However, within the limits of our study, nicotine exposure during differentiation neither promoted nor inhibited apoptosis in our in vitro assays.
Thus, HL-60 cells are highly resistant to physiologically relevant doses of nicotine during differentiation, with respect to alterations to cell proliferation, viability, cell cycle, ultrastructural characteristics and the development of terminal differentiation markers. However, nicotine exposure during differentiation adversely affects key effector functions in HL-60 cells. It is known that smokers exhibit increased risk of multiple infectious diseases, as discussed earlier. We show that the oxidative burst in response to PMA is quantitatively diminished in HL-60 cells differentiated under the influence of nicotine (Figure ), which is reflected in an impaired ability to kill the Gram-negative periodontal pathogen, P. gingivalis
(Figure ). Interestingly, smokers are known to have increased risk of P. gingivalis
], to harbour greater numbers of P. gingivalis
] and to be more susceptible to periondontitis than non-smokers [6
]. It should be noted that the use of a single agent to stimulate ROS production is a study limitation. Nevertheless, further studies into the molecular mechanisms of nicotine-induced suppression of the oxidative burst and bactericidal capabilities in neutrophils, are warranted.
Finally, matrix metalloproteinases are a family of at least eighteen secreted and membrane-bound zinc-endopeptidases. Collectively, these enzymes can degrade most, if not all, the components of the extracellular matrix. Neutrophil-derived, MMP-9-mediated tissue degradation and the instructional role of MMP-9 in directing the inflammatory response are key, and perhaps critical, events in the etiology of chronic obstructive pulmonary disease (COPD) [38
]; systemic and cerebral vascular diseases [39
], asthma [2
], and periodontitis[6
]. Tobacco smoke exposure is the major environmental risk factor each of these diseases, and herein we show that exposure to the major tobacco constituent, nicotine, during HL-60 differentiation exacerbates the rapid, LPS-induced MMP-9 release in a dose- and α7nAChR-dependent manner (Figure ). MMP-2 was not similarly influenced. Engagement of α7nAChR on other innate immune cells by nicotine, or the endogenous α7nAChR agonist – acetylcholine, is known to have a profound influence on the inflammatory response to Gram negative stimuli, as we have recently discussed elsewhere [40
]. Therefore, these data are in keeping with, and extend, previous reports of increased MMP-9 concentrations in the bronchoalveolar lavage fluids of smoke exposed mice [41
], increased neutrophil gelatinase-associated lipocalin [NGAL: a neutrophil-derived protein that associates with MMP-9 [42
] concentrations in blood and bronchoalveolar lavage fluid of smokers, compared with non-smokers [43
]; and those of Nakamura et al. [44
], who were the first to suggest an increased systemic MMP-9 load in smokers. It is not yet clear whether this increase in the circulating MMP-9 burden arises from activation of circulating neutrophils or from translocation from inflammatory cells activated to produce MMP-9 in the pulmonary environment. Further studies into the mechanisms underlying nicotine-induced, α7nAChR-dependent augmentation of LPS-induced MMP-9 release could allow the development of refined and specific therapeutic strategies for the treatment of a number of important tobacco-associated inflammatory diseases and conditions.