In this report, the direct effect of cigarette smoke on neutrophil migration and on β2-integrin activation and function in the endothelial transmigration of neutrophils was investigated. Exposing mice to cigarette smoke for 5 days resulted in the presence of CD11b expressing neutrophils in the lungs, showing that cigarette smoke attracts neutrophils. For a more in depth study, we exposed freshly isolated human neutrophils to CSE to study possible migratory effects and involvement of Mac-1. To our knowledge, this is the first study to show that CSE can induce the migration of neutrophils in vitro. Moreover, CSE activated the β2-integrin Mac-1 on the neutrophil leading to firm adhesion to fibrinogen. Furthermore, neutrophils transmigrate through endothelium in response to CSE via the activation of β2-integrins, since functionally blocking CD11b and CD18 decreased this transmigration. Taken together, our data provide evidence for a critical role of β2-integrins in the firm adhesion and transmigration of neutrophils in response to cigarette smoke in vitro and in vivo.
Cigarette smoke consists of more than 4000 compounds, known to be mutagenic, carcinogenic, antigenic and cytotoxic [27, 28] and it induces a peripheral inflammatory response in all smokers . This is reflected in increased macrophage and neutrophil numbers, changes in expression surface markers, elevated levels of chemokines (amongst which are CXCL1 and CXCL8) and cytokines (such as TNF-α and IL-1β) and increased production of proteolytic enzymes by different immune cells, such as macrophages and neutrophils [18, 28, 30].
Exposing mice for five days to cigarette smoke results in an influx of CD11b expressing cells. We concluded that these cells were neutrophils due to the characteristic segmented nuclei. Other studies also observed increased numbers of neutrophils in BALF and lung tissue after one week [26, 31] and five months cigarette smoke exposure in mice . These in vivo findings led to the hypothesis that cigarette smoke may has as a direct migratory effect. Interestingly, it has recently been suggested by Barnes that smoke may induce neutrophil movement . In the present study, we confirm this hypothesis and demonstrate that CSE induces the migration of neutrophils. Although it has been reported that nicotine, a major constituent of cigarette smoke, can have a chemotactic effect on neutrophils , other groups found no effect or even an inhibitory effect [35–37] or describe this effect as very weak and state that nicotine enhances neutrophil responsiveness to other chemo-attractants . Another major constituent of smoke, acrolein, inhibits chemotaxis [36, 39]. Since CSE consist of more than 4000 components, it is likely that other chemical compounds or combinations of soluble compounds are responsible for the chemotactic effect on neutrophils.
To our knowledge, we are the first to show a direct migratory effect of CSE. Other groups have investigated the priming effect of cigarette smoke. Bridges and Hsieh demonstrated that water-soluble fractions (WSF) and cigarette smoke condensates (CSC) were able to inhibit endotoxin-activated serum-induced chemotaxis of neutrophils . Selby et al. described that exposure of human neutrophils in vitro to cigarette smoke resulted in impaired cell spreading and chemokinesis, but did not influence zymosan-activated serum-induced chemotaxis . However, both groups did not investigate the direct migratory effect of cigarette smoke on neutrophils. The inhibitory effects of cigarette smoke on activated serum-induced chemotaxis of neutrophils, as described in the studies of Bridges and Hsieh and Selby et al. [40, 41], may be explained by the fact that adding the cigarette smoke to the neutrophils leads to immobilization of the cells; the cells are attracted by the cigarette smoke in the upper chamber and do not actively need to go to the lower chamber.
Neutrophils use β2-integrins, such as Mac-1, for nearly every step in transmigration . During inflammation, β2-integrin on the neutrophil are activated by chemokines such as CXCL8, leading to neutrophilic firm adhesion to endothelial cells and subsequently to transmigration. This activation is essential, since it leads to a conformational change in structure, going from an inactive, low affinity state to an active, high-affinity state . We show that CSE activates β2-integrins on the neutrophil leading to firm adhesion to fibrinogen and transmigration through an endothelial cell layer, since functionally blocking CD11b and CD18 decreased these effects of cigarette smoke. Consistent with previous studies describing β2-integrin expression patterns of smokers [29, 42, 43], we found that CSE-induced adhesion and transmigration of neutrophils are the result of β2-integrin activation, and more specifically Mac-1 activation. It may be possible that cigarette smoke exposure leads to an up-regulation of β2-integrin expression on the neutrophil. Koethe et al. described that pre-incubating PMNs with CSC resulted in a 2.5 fold increase in CD11b/CD18 expression as compared to the control . However, Selby et al. reported that acute in vivo cigarette smoking of up to 4 cigarettes did not change the expression patterns of CD18 on the neutrophils . From these results it can be suggested that besides the activation of β2-integrins, CSE incubation may result in an up-regulation of the CD11b/CD18 expression.
Cigarette smoke enhances the expression of E- and P-selectin  and ICAM-1 and VCAM-1  on HUVECs, thereby increasing rolling interactions and subsequent firm adhesion of neutrophils to HUVECs via β2-integrins Mac-1 and LFA-1 and β1-integrin VLA-4 . In our experiments, neutrophils migrated through an endothelial cell layer in response to CSE. Administration of cigarette smoke did not affect the permeability of the HUVEC layer in the transmigration assay since the electrical resistance of the confluent HUVEC layer did not change before and after CSE exposure (Table 1). This leads to the conclusion that neutrophils actively migrate through the layer in response to cigarette smoke.
Although CXCL8 has a stronger migratory effect and fMLP is a stronger activator of β2-integrins, cigarette smoke exposure leads to a 1.5 fold increase in migration and to a 1.5 to 3 fold increase in adhesion as compared to control. Mortaz and colleagues have recently shown that CSE exposure to human neutrophils results in the release of CXCL8 . Furthermore, earlier studies have demonstrated that exposure of neutrophils to CXCL8 leads to the phosphorylation of focal adhesion kinase (FAK) and CXCL8 induces FAK cellular redistribution . FAK activation is involved in cellular adhesion and spreading processes of neutrophils . Thus in the in vivo situation, we cannot rule out the additional chemotactic effects of cigarette smoke-induced released inflammatory mediators from for example epithelial cells, pulmonary neutrophils and resident macrophages.
Taken together, this is the first study that describes CSE as a potent inducer of neutrophil movement. Besides this migratory effect, CSE plays a critical role in β2-integrin activation, which leads to an increase in firm adhesion and transmigration through HUVECs. These observations may contribute to a better understanding of neutrophilic transmigration in smokers and COPD patients and potentially offer a new target in disease management.