Selectin-dependent rolling and adhesion of leukocytes in nicotine-exposed microvessels of lung allografts

Lyudmila Sikora, Savita P. Rao, and P. Sriramarao

Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California 92121

Submitted 23 December 2002 ; accepted in final form 22 May 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The interaction of circulating leukocytes with lung microvessels is a critical event in the recruitment of effector cells into the interstitial tissue during episodes of inflammation, including smoking-induced chronic airway disease. In the present study, murine lung tissue transplanted into a dorsal skinfold window chamber in nude mice was used as a model system to study nicotine-induced leukocyte trafficking in vivo. The revascularized lung microvessels were determined to be of pulmonary origin based on their ability to constrict in response to hypoxia. We demonstrated that nicotine significantly enhanced rolling and adhesion of leukocytes within lung microvessels comprising arterioles and postcapillary venules in a dose-dependent manner, but failed to induce leukocyte emigration. Nicotine-induced rolling and adhesion was significantly higher in venules than in arterioles. Treatment of mice with monoclonal antibodies (MAbs) against L-, E-, or P-selectin after exposure of lung allografts to nicotine resulted in variable but significant inhibition of nicotine-induced rolling, whereas nicotine-induced subsequent adhesion was inhibited by MAbs against L- and P-selectin but not E-selectin. Exposure of lung allografts to nicotine along with PD-98059, a mitogen-activated protein kinase (MAPK)-specific inhibitor, resulted in significant inhibition of nicotine-induced rolling and adhesion. In vitro, exposure of murine lung endothelial cells to nicotine resulted in increased phosphorylation of mitogen-activated/extracellular signal-regulated protein kinase 1/2, which could be blocked by PD-98059. Overall, these results suggest that nicotine-induced inflammation in the airways could potentially be due to MAPK-mediated, selectin-dependent leukocyte-endothelial cell interactions in the lung microcirculation.

selectins; endothelium; leukocyte recruitment; pulmonary; adhesive interactions


THE MOST IMPORTANT ETIOLOGICAL FACTOR in chronic obstructive pulmonary disease is airway inflammation that is particularly associated with environmental tobacco smoke (ETS) and its constituents, such as nicotine. This relationship appears to strengthen with the number of cigarettes smoked. At a cellular level, lung inflammation is characterized by the recruitment of circulating leukocytes into extravascular spaces of the lungs. The molecular mechanisms mediating the initial interaction of circulating leukocytes with vascular endothelial cells, as a consequence of exposure to ETS, or constituents such as nicotine, and their subsequent accumulation in the airways, are not well understood. Because of a lack of appropriate in vivo model systems to study distinct events of human leukocyte adhesion, it has not been possible to clearly demarcate the roles of various adhesion receptors in mediating initial and subsequent events of ETS-induced leukocyte adhesion under conditions of blood flow in the airways. Earlier studies in conscious hamsters exposed to cigarette smoke (CS) have demonstrated increased rolling and subsequent adhesion of hamster leukocytes to arterioles and postcapillary venules (26, 27). Increased retention of neutrophils in the pulmonary microvessels of rabbits exposed to CS has also been demonstrated (4, 20). However, the studies thus far have not identified whether exposure to ETS or its constituents can result in increased leukocyte rolling or adhesion in lung microvessels and whether vascular selectins participate in these cellular interactions.

Nicotine has been reported to be chemotactic for human neutrophils, but not monocytes (39), and is known to prolong survival of neutrophils in vitro by suppressing apoptosis (1). In contrast, other studies demonstrated that nicotine failed to induce chemotaxis of polymorphonuclear leukocytes in vitro (32). However, the ability of nicotine to induce leukocyte trafficking and emigration in lung microvessels in vivo has not been reported, to the best of our knowledge. Earlier studies have demonstrated that CS induces the release of neutrophil-specific chemotactic factors by alveolar macrophages in smokers but not nonsmokers (16). In addition, increased expression of IL-8 (chemotactic for neutrophils and eosinophils), IL-1{beta}, IL-6, and monocyte chemoattractant protein-1 has been observed in smokers (24, 29). It has been suggested that the increased emigration of neutrophils is likely to be related to the ability of CS to induce the release of IL-8 by bronchial epithelial cells (30). Likewise, it has been demonstrated that CS stimulates the release of neutrophilic chemotactic activity from cultured bronchial epithelial cells (35). Moreover, a dose-dependent effect of CS on the inflammatory responses in human subjects has been reported (24). Although these studies demonstrate the ability of nicotine/CS to alter leukocyte survival and function, the ability of nicotine to modulate leukocyte trafficking in the lung microcirculation under conditions of flow in vivo has not been investigated.

Leukocyte rolling in rabbit lung microvessels appears to be dependent on the engagement of selectins, whereas leukocyte adhesion has been thought to be selectin independent (22). Several studies have demonstrated leukocyte rolling and adhesion in both arterioles and postcapillary venules of the pulmonary microcirculation (21, 22); however, the ability of circulating leukocytes to respond to extravascular stimuli, such as nicotine, in the murine lung microcirculation under conditions of shear force has not been reported. In this investigation, we have used intravital microscopy to visualize leukocyte trafficking within lung allografts transplanted into the dorsal skinfold chambers implanted in nude mice. The relative importance of L-, E-, and P-selectins in mediating leukocyte rolling and adhesion in lung microvessels exposed to nicotine has been determined.

Nicotine has previously been shown to increase the activity of the mitogen-activated/extracellular signal-regulated protein kinase (ERK1/2) in neuronal cells, neuroendocrine cells, and small-cell lung carcinoma cells (5, 8, 33), which in turn are thought to influence learning and memory processes and play a potential role in lung carcinogenesis. To the best of our knowledge, there are no studies reporting the effect of nicotine on MAPK in endothelial cells. In the present study, the effect of nicotine on MAPK in murine endothelial cells in vitro and the effect of a MAPK-specific inhibitor on nicotine-induced leukocyte rolling and adhesion in murine lung microvessels in vivo have also been investigated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Lung allograft model. Dorsal skinfold chambers in nude mice were prepared as previously described (3). Adult mice (BALB/c, 8-12 wk old) were used as donors of the lung tissue to be transplanted into the skinfold window chambers of recipient nude mice. Longitudinal lung slices were fluorescently labeled and transplanted into the window of the skinfold chamber of an anesthetized recipient nude mouse aseptically in a laminar flow hood, as described in previous studies from this laboratory (19, 36, 38). The chamber containing the lung allograft was then superfused with 25-50 µl of sterile saline to keep the tissue moist and covered with a sterile siliconized coverslip kept in position by a C-ring. The recipient mouse was monitored for revascularization of the transplanted lung allografts and establishment of blood flow by intravital microscopy over a 2-wk period, as described previously (36). Briefly, the unanesthetized mouse recipient of the lung allograft was placed in a crouched position in a Plexiglas tube that was closed at one end and had a longitudinal slit to accommodate the skinfold chamber, as previously described. The tube had holes to allow the mouse to breathe. The skinfold chamber containing the lung allograft was then immobilized on a platform and placed on the stage of a Leitz Biomed intravital microscope for observation of the skinfold chamber. On the day of implantation (day 0), an overview video picture of the 5-(and 6)-{[(4-chloromethyl)-benzoyl]amino}, tetramethylrhodamine-labeled lung allograft was taken with a Leitz PL x1.6 (numerical aperture 0.05) objective. The establishment of vasculature and blood flow was observed either by intravenous administration of 50-100 µl of 2.5% FITC-dextran 500,000 (Sigma Chemical, St. Louis, MO) to obtain vessel contrast and plasma enhancement or by transillumination using a mercury or halogen lamp. Epi-illumination of FITC-labeled vessels was obtained with a silicone-intensified target camera (SIT68; DAGE MTI, Michigan City, IN) attached to the microscope and connected to a monitor (Panasonic). All images were recorded on an S-VHS videocassette recorder (HC-6600; JVC, Tokyo, Japan) for playback offline analysis. Observations were made periodically over a 2-wk period. Continued and progressive changes in establishment of blood flow were observed in the transplanted lung allograft over a 9-day period. By day 4-6 postimplantation, the lung allograft was ~50% revascularized with increased blood flow compared with day 0, and between 9 and 14 days postimplantation, all vessels in the lung allograft were completely revascularized and established blood flow with no evidence of visual thrombosis in any of the lung microvessels (36). More than 95% of the transplanted allografts were observed to revascularize and establish blood flow. Animals that were visibly healthy and had no signs of nonspecific inflammation in and around the skin chamber were included in the study. All studies involving animals were performed according to IACUC-approved protocols.

Effect of hypoxia on revascularized lung microvessels. As a functional test to ensure that the vessels in the allograft exhibited responses unique to pulmonary vessels, the effect of hypoxia on completely revascularized lung allografts (days 9-14) was studied. Recipient nude mice with revascularized lung allografts were initially observed under an intravital microscope as described earlier. Video images of the FITC-dextran-infused lung microvessels within the skinfold chamber were recorded. Thereafter, the mice were placed in a hypoxic chamber through which N2 was passed in a regulated manner such that the final O2 concentration was 10% for 1 h. The concentration of O2 was monitored with the help of an airway gas monitor (model no. 254; Datex, Madison, WI). The mice were then removed from the chamber and immediately placed on the stage of the intravital microscope for observation of the lung microvessels. All images were recorded for offline video analysis. The changes in the diameters of individual lung microvessels (n = 3, 7-10 vessels/allograft) were analyzed from recorded video images before and after exposure to hypoxia.

Effect of nicotine on leukocyte-endothelial interactions in revascularized lung microvessels and antibody blockade studies. Nude mice with completely revascularized lung allografts with well-established blood flow were selected for these studies. The coverslip from the skinfold chamber was removed and the lung allograft superfused with 50 µl of nicotine (Sigma Chemical; 10-5 to 10-9 M) or saline as a control. The ability of nicotine to induce rolling, adhesion, or transmigration of acridine orange (Sigma)-labeled circulating leukocytes (0.5 mg/mouse administered intravenously) in the lung microvessels immediately after exposure to nicotine was determined by intravital microscopy. Because the entire allograft establishes blood flow, 15-25 blood vessels representing pulmonary venules and arterioles (identified on the basis of direction of blood flow) were randomly selected for analysis of leukocyte-endothelial interactions. In additional experiments (n = 3-5), the effect of MAb against P-, E-, and L-selectin (MAbs 5H1, 9A9, and MEL-14, respectively; provided by Dr. Barry Wolitzky, MitoKor, San Diego, CA) on leukocyte rolling and adhesion in lung microvessels before as well as after exposure of lung allografts to nicotine was investigated. All antibodies were used at a concentration of 2 mg/kg body wt on the basis of previous studies (3). Initial studies carried out using either normal rat or hamster IgG as a control showed no differences in rolling and adhesion compared with saline. In all subsequent experiments, saline was used as the control. To determine whether MAPK (ERK1 and ERK2) is involved in mediating the effects of nicotine on leukocyte rolling and adhesion, in some experiments, the lung allograft was superfused with nicotine in the presence of PD-98059 (37 µM; Calbiochem, San Diego, CA), a specific inhibitor of ERK1 and ERK2, in the model described above. The concentration of PD-98059 was selected on the basis of our previous studies (2).

Image analysis. The interaction of circulating leukocytes in the lung microvessels of the skinfold chamber (i.e., rolling and adhesion) was analyzed by offline analysis of recorded video images as described in our previous studies (3). Leukocytes visibly interacting with the lung microvascular endothelium and passing at a slower rate than the main blood stream were considered as rolling cells and were quantitated by manually counting the total number of rolling cells passing through a reference point in a vessel segment. The number of rolling cells was expressed as a rolling fraction, which was a percentage of the total number of cells passing through the same reference point. Adherent cells were defined as those cells remaining stationary for at least 1 min and expressed as a percentage of total cells passing through 100-µm length of blood vessel. The mean rolling velocity of circulating, acridine orange-labeled leukocytes in nicotine- or saline-exposed lung microvessels (10 vessels/allograft) was manually determined by frame-by-frame analysis of recorded video images. Briefly, the velocity of rolling leukocytes (n = 10-30 cells/vessel) within saline- and nicotine-exposed vessels was determined by measuring the time taken for the cells to travel between two reference points, and the mean rolling velocity of leukocytes is expressed as micrometers per second.

Analysis of ERK phosphorylation in nicotine-exposed LEISVO murine lung endothelial cells. A murine lung endothelial cell line (kindly provided by Dr. Masanobu Kobayashi, Hokkaido School of Medicine, Sapporo, Japan) (17) of micro-vascular origin was cultured in individual petri dishes. Cultures were not tested for mycoplasma contamination. When confluent, the spent media were removed, and the cells were washed with warm serum-free RPMI (Invitrogen, Carlsbad, CA). Nicotine (10-7 M) in serum-free RPMI was added to all petri dishes except the control, to which media alone was added. All dishes were incubated at 37°C for 5 min. Nicotine was removed, and the cells were quickly rinsed once with serum-free RPMI. Fresh media (1 ml) were added to all the dishes and incubated at 37°C. At 2, 5, 10, and 15 min postnicotine exposure, the cells were lysed in cold lysis buffer [1% Triton X-100, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 µg/ml antipain, 25 µl protease inhibitor cocktail, and 100 µg/ml PMSF (all from Sigma) in PBS] by placing the petri dishes on ice on a shaker for 20 min. The control cells were harvested in a similar manner. In some experiments, the cells were exposed to nicotine in the presence of PD-98059 (37 µM) dissolved in DMSO (Sigma) for 5 min. Cells exposed to DMSO alone in serum-free RPMI without nicotine served as a control. Cell lysates were mixed with gel loading buffer (100 µM Tris · HCl, pH 7.0, 4% SDS, 0.2% bromphenol blue, 20% glycerol, and 200 mM dithiothreitol) and boiled for 2 min before SDS gel electrophoresis. An equal amount of protein was loaded from each sample in each lane. Lysates were electrophoresed with the use of 4-12% NuPage Bis-Tris gels in 2-(N-morpholino)ethanesulfonic acid SDS running buffer (Invitrogen). Resolved proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA), and the membranes were blocked with a 3% solution of blocking buffer (Upstate Biotechnology) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. This was followed by incubation with anti-phospho p44/42 MAPK in 2% blocking buffer (Cell Signaling Technology, Beverly, MA) for 1 h at room temperature or overnight at 4°C. After being washed with TBST (6 x 10 min), membranes were incubated for 1 h in horseradish peroxidase-conjugated anti-rabbit IgG (Transduction Laboratories, Lexington, KY) in 1% blocking buffer. The membranes were once again washed as described earlier, and the bound antibodies were detected with an enhanced chemiluminescence detection kit (ECL Plus; Amersham-Pharmacia Biotech, Piscataway, NJ). The blots were immediately exposed to X-Omat Blue XB-1 X-ray film (NEN, Boston, MA) for 10 s and developed.

Statistics. The significance of the interaction of nicotine-exposed vs. control circulating leukocytes and vascular endothelium in the presence and absence of anti-selectin MAb was analyzed by Student's t-test using a statistical software package (SigmaStat, Jandel Scientific). All results are expressed as means ± SE, and P values <0.05 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Nicotine increases leukocyte rolling and adhesion in lung microvessels. We examined the effect of nicotine on the interaction between circulating leukocytes and revascularized lung microvessels by intravital microscopy. Lung sections transplanted into the dorsal skinfold window chambers of nude mice were used in these studies. Complete revascularization of transplanted lung allografts with successful establishment of normal blood flow determined by injecting FITC-dextran is shown in Fig. 1, A and B. Evidence of normal blood flow within the entire lung allograft that was not sluggish was indicative of complete revascularization of the lung allograft. To determine whether vessels in the allograft exhibit responses unique to pulmonary vessels, mice bearing completely revascularized lung allografts were placed in a hypoxic chamber, and the effect on vasoconstriction was evaluated. Under these experimental conditions, lung microvessels within the allograft were found to undergo constriction (Fig. 2), suggesting that microvessels of revascularized lung allografts retain properties unique to intact pulmonary vessels, as reported previously (10).



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Fig. 1. Photomicrographs of lung sections transplanted into the dorsal skinfold chambers of mice. Lung sections from donor mice were transplanted into the dorsal skinfold window chambers of nude mice as described in MATERIALS AND METHODS. Complete revascularization of transplanted lung allografts was observed 9-14 days posttransplantation (A) with successful establishment of normal blood flow as determined by injecting FITC-dextran (B). Magnification x40 and x250, respectively.

 


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Fig. 2. Effect of hypoxia on revascularized lung allografts. Mice bearing completely revascularized lung allografts in skinfold chambers were placed in a hypoxic environment (10% O2) for 1 h and then immediately observed under an intravital microscope. All images were video recorded, and changes in the diameter of individual lung microvessels (n = 7-10 vessels/allograft) were analyzed from recorded video images before (A) and after (B) exposure to hypoxia. Arrows in A show vessels analyzed before hypoxia; arrows in B show the same vessels after exposure to hypoxic conditions. Magnification x250.

 

We next determined the ability of acridine orange-labeled circulating leukocytes to interact within the revascularized lung microvessels under conditions of flow (Fig. 3). Leukocytes were observed to minimally roll (rolling fraction <15%) and adhere (<5%) in the lung microvessels. Exposure of lung allografts to saline alone did not increase leukocyte interactions (rolling and adhesion) compared with basal levels (Fig. 3A). However, local superfusion of the lung allograft with nicotine resulted in a significant increase in leukocyteendothelial interactions (Fig. 3B). Superfusion with varying concentrations of nicotine (10-5 to 10-9 M) resulted in an immediate, dose-dependent increase in the number of rolling and adherent leukocytes, which was significantly higher than that observed with saline (Fig. 4, A and B, respectively). This nicotine-induced rolling was associated with a significant reduction in the rolling velocities of these leukocytes compared with leukocytes exposed to saline (Fig. 4C). Nicotine-induced adhesion and rolling of leukocytes was signifi-cantly higher in venules than arterioles (Fig. 5). These results suggest that nicotine exposure not only results in an increase in the number of rolling cells but also an increase in the affinity of these cells to interact with vascular endothelial cells in the lung microvessels. In addition, exposure to nicotine resulted in trapping and deformation of leukocytes during their transit through capillaries. In contrast to its effect on rolling and adhesion, exposure to nicotine did not result in emigration of the adherent leukocytes into the extravascular compartment.



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Fig. 3. Photomicrographs of nicotine-induced leukocyte rolling and adhesion in lung microvessels of revascularized lung allografts. Lung allografts transplanted into the dorsal skinfold chamber of mice were superfused with saline (A) or nicotine (B) (10-7 M). The ability of acridine orange-labeled circulating leukocytes (open arrows) to interact within revascularized lung microvessels (filled arrows) under conditions of flow was determined by intravital microscopy. Magnification x250.

 


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Fig. 4. Effect of nicotine on leukocyte rolling and adhesion. The effect of varying concentrations (Conc.) of nicotine (10-5 to 10-9 M) vs. saline (control) on rolling (A), adhesion (B), and rolling velocity (C) of circulating acridine orange-labeled leukocytes in lung microvessels was determined by offline analysis of recorded video images of revascularized lung allografts by intravital microscopy. Results are expressed as means ± SE. *P < 0.05 vs. control.

 


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Fig. 5. Nicotine-induced leukocyte rolling and adhesion in arterioles vs. venules. Revascularized lung allografts were exposed to saline (control) or nicotine (10-7 M) by local superfusion. The effect of nicotine on rolling (A), adhesion (B), and rolling velocity (C) of circulating acridine orange-labeled leukocytes in venules (converging flow) and arterioles (diverging blood flow) was analyzed by offline analysis of recorded video images. Results are expressed as means ± SE. *P < 0.05 vs. rolling and adhesion in arterioles; **P < 0.05 vs. control.

 

Nicotine-induced leukocyte rolling and adhesion is selectin dependent. Because selectins play a critical role in promoting leukocyte rolling, we examined whether nicotine-induced rolling and adhesion of leukocytes to lung microvasculature was dependent on the engagement of vascular E- and P-selectins or leukocyte-expressed L-selectin. In the first series of experiments, leukocyte rolling and adhesion was induced by superfusion of the lung allograft with nicotine after recording baseline rolling and adhesion in the lung microvessels. Nude mice were then administered saline followed by anti-L-, anti-E-, or anti-P-selectin antibodies, and their effect on leukocyte interactions was determined (Fig. 6, A and B). Administration of rat IgG, hamster IgG, or saline did not inhibit nicotine-induced leukocyte rolling and adhesion, whereas MAbs against L- and P-selectin resulted in significant inhibition of nicotine-induced rolling (>55%, P < 0.05). Anti-P-selectin antibodies inhibited nicotine-induced rolling by >90% in arterioles and venules (Fig. 6A). Anti-E-selectin antibodies, on the other hand, were the least effective in inhibiting nicotine-induced rolling (~30%). Treatment with anti-L-selectin MAbs resulted in >50% inhibition of nicotine-induced adhesion in arterioles and venules, whereas nearly complete inhibition of adhesion was observed with MAbs against P-selectin. Anti-E-selectin MAbs did not significantly inhibit nicotine-induced adhesion in both vessels (<20%; Fig. 6B). Although cell surface L-, E, and P-selectins participate in nicotine-mediated rolling within lung microvessels, blockade of L- and P-selectins alone is effective in significantly inhibiting nicotine-induced adhesion events. P-selectin appears to be the predominant adhesion molecule involved in nicotine-induced leukocyte interactions with lung microvessels since MAbs against P-selectin alone were able to inhibit leukocyte rolling and adhesion by >90%.



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Fig. 6. Nicotine induces selectin-dependent leukocyte rolling and adhesion in lung microvessels. The effect of anti-P- (MAb 5H1), anti-E- (MAb 9A9), and anti-L-selectin (MAb MEL-14) antibody treatment on leukocyte rolling and adhesion in lung microvessels after exposure to nicotine (10-7 M) was investigated. All antibodies were used at a concentration of 2 mg/kg body wt. Rolling (A) and adhesion (B) of circulating acridine orange-labeled leukocytes in venules and arterioles was analyzed by offline analysis of recorded video images. Results are expressed as means ± SE. *P < 0.05 vs. nicotine.

 

Pretreatment with selectin-specific antibodies prevents nicotine-induced rolling and adhesion of leukocytes. We next examined whether pretreatment with anti-selectin MAbs would prevent nicotine-induced rolling and adhesion in lung microvessels (Fig. 7, A and B). As described above, nude mice with completely revascularized lung allografts were administered with anti-selectin MAbs after recording baseline rolling and adhesion. Thereafter, lung allografts were superfused with nicotine at 10-7 M. Pretreatment with MAbs against P- and L-selectin resulted in significant inhibition of nicotine-induced leukocyte rolling in arterioles (87.5 and 82.7%, respectively) and venules (71.4 and 83.37%, respectively) compared with leukocyte rolling in mice that were not pretreated with anti-selectin MAbs before exposure to nicotine or treatment with a control antibody. These MAbs had a similar effect on nicotine-induced leukocyte adhesion in arterioles and venules, inhibiting the number of adherent cells by 65-100%, depending on the MAb (data not shown).



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Fig. 7. Pretreatment with anti-selectin antibodies inhibits nicotine-induced rolling of leukocytes. Nude mice with completely revascularized lung allografts were treated with anti-P- (MAb 5H1) or anti-L-selectin (MAb MEL-14) antibodies before superfusion with nicotine (10-7 M). Both antibodies were used at 2 mg/kg body wt. Rolling of circulating acridine orange-labeled leukocytes in arterioles (A) and venules (B) was analyzed by offline analysis of recorded video images. Results are expressed as means ± SE. *P < 0.05 vs. nicotine without anti-selectin antibody pretreatment.

 

PD-98059 inhibits nicotine-induced rolling and adhesion of leukocytes. To understand the signaling events that occur after exposure to nicotine, resulting in increased rolling and adhesion, nude mice with completely revascularized lung allografts were super-fused with nicotine and PD-98059, a specific inhibitor of MAPK, after the baseline rolling and adhesion was recorded. Exposure to PD-98059, but not DMSO (control), resulted in nearly complete inhibition of nicotine-induced leukocyte rolling in arterioles and venules (Fig. 8A). In addition, significant inhibition of nicotine-induced adhesion was observed in arterioles and venules (Fig. 8B). The reduction in rolling velocities of leukocytes induced by nicotine alone was significantly reversed by PD-98059 in arterioles and venules, with the remaining cells rolling at velocities similar to those of control cells (Fig. 8C). These findings suggest that nicotine-induced, selectin-dependent rolling and adhesion involves a MAPK signaling pathway. Further evidence for the involvement of MAPK in nicotine-induced rolling and adhesion comes from our in vitro studies with murine lung endothelial cells. Exposure of these cells to nicotine (10-7 M) for 5 min at 37°C resulted in a time-dependent increase in levels of phosphorylated ERK (Fig. 9A), which was completely inhibited when these cells were exposed to nicotine in the presence of PD-98059 (Fig. 9B).



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Fig. 8. Nicotine-induced leukocyte rolling and adhesion involves a mitogen-activated protein kinase (MAPK)-dependent mechanism. Nude mice with completely revascularized lung allografts were superfused with nicotine (10-7 M) alone or with nicotine and PD-98059 (37 µM). The effect of PD-98059 on rolling (A), adhesion (B), and rolling velocity (C) of circulating acridine orange-labeled leukocytes was determined by offline analysis of recorded video images. Results are expressed as means ± SE. *P < 0.01 vs. nicotine.

 


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Fig. 9. Analysis of extracellular signal-regulated kinase phosphorylation in nicotine-exposed murine lung endothelial cells. Murine lung endothelial cells (LEISVO) cultured in individual petri dishes were exposed to media containing nicotine (10-7 M) or RPMI alone (control) at 37°C for 5 min. At 2, 5, 10, and 15 min, cells exposed to nicotine were lysed. Control (C) cells were lysed at 15 min. Supernatants from the cell lysates were subjected to Western blotting with anti-phospho p44/42 MAPK (A). In some experiments, cells were incubated with nicotine in the presence or absence of PD-98059 (37 µM) for 5 min and lysed at 2 and 5 min after exposure to nicotine for Western blot analysis. Control cells were incubated with RPMI alone or RPMI containing DMSO (B).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Adherence of circulating neutrophils and eosinophils to the vascular endothelium and their accumulation in inflamed tissues is the hallmark of pulmonary inflammation. ETS has been identified as a major risk factor for chronic obstructive pulmonary disease. Furthermore, smoking not only induces wheezing in patients with asthma (6) but also contributes to childhood asthma in children exposed to passive smoke (7, 11). The molecular and cellular mechanisms mediating the sequestration of circulating leukocytes into the airways of smokers during ETS-induced airway inflammation are not well recognized. In the present study, we used a unique mouse lung allograft model (36) to study the effect of nicotine on circulating leukocytes in revascularized lung microvessels by intravital microscopy. The revascularized lung microvessels were confirmed to be of pulmonary origin based on previous histology studies (36) as well as current studies that demonstrate their ability to vasoconstrict in response to hypoxia, a property known to be unique to pulmonary vessels (10). This model allowed direct visualization of leukocytes in the lung microcirculation continuously and was not limited by the movement or changes in the shape of the lungs during respiration or ventilation. We demonstrated that nicotine, a major constituent of ETS, induced increased intravascular rolling and adhesion of circulating leukocytes in a murine model of revascularized lung microcirculation but failed to induce any emigration of adherent leukocytes in vivo. Nicotine-induced leukocyte rolling and adhesion was significantly higher in venules compared with arterioles. Although shear rates were higher in venules vs. arterioles of revascularized lung allografts (36) similar to intact lungs (21, 23), it is likely that the strength of the adhesive interactions mediated by venular adhesion molecules was significantly greater than that of adhesion molecules expressed by the arteriolar endothelium.

A key feature of smoking-induced airway inflammation is the specific interaction of circulating leukocytes with adhesion molecules expressed by the pulmonary vasculature. Thus the predominant recruitment of leukocytes in lungs during episodes of chronic bronchitis or asthmatic bronchitis is suggestive of trafficking through the lung microcirculation. For instance, the transit of polymorphonuclear neutrophils (PMN) through the lung microvasculature is slowed due to increased retention in the capillaries during inhalation of CS in rabbits and humans (4, 20, 28). These studies also suggested that CS might cause lung damage by activation of PMN in the capillary bed. Likewise, the increase in local concentration of neutrophils in airways of smokers has been related to the presence of CS in the lungs, and the lesions that characterize emphysema are thought to result from the destruction of lung tissue by neutrophils that remain within the pulmonary vessels (28). Moreover, the presence of CS in the alveoli has also been suggested to activate PMN in the lung microvessels of rabbits (20). The recruitment of neutrophils into the pulmonary microvessels involves the engagement of both CD11/CD18-dependent and -independent mechanisms (9). Likewise, exposure of rabbit neutrophils to CS results in the upregulation of CD18 integrin and decreased expression (shedding) of L-selectin. Increased adhesion of peripheral blood monocytes isolated from smokers was mediated by CD11b/CD18, which was prevented by intake of vitamin C by smokers (40). Although vitamin C appears to prevent CS-induced leukocyte aggregation and adhesion to endothelium in vivo (25), the ability of anti-selectin molecules to block nicotine/CS-induced leukocyte rolling and adhesion in the lung microvasculature has not been investigated.

The MAb studies described here demonstrate that vascular P-selectin and leukocyte-expressed L-selectin play an important role in the recruitment of leukocytes to both arterioles and venules exposed to nicotine in the lung microcirculation. Nicotine has previously been shown to enhance leukocyte rolling via P-selectin in the cerebral microcirculation of mice (41). Interestingly, treatment with anti-L- and anti-P-selectin inhibited nicotine-induced leukocyte adhesion. Although selectins, in general, are not known to support adhesion, it is likely that inhibition of the rolling step mediated by leukocyte-expressed L-selectin and/or P-selectin is sufficient to block subsequent stable arrest to nicotine-activated endothelial cells. E-selectin appears to be absent on unstimulated vascular endothelium and is upregulated 1-5 h in most tissues when stimulated (12). However, it is not known whether nicotine has any effect on stimulation of early E-selectin expression that may account for the marginal inhibition of leukocyte rolling observed with anti-E-selectin antibodies (Fig. 6). In the same study, it was also shown that significant expression of P-selectin was present even in the absence of stimulation, with a rapid time-dependent upregulation as early as 5 min when stimulated, and exhibited the largest responses in the mesentery and lung. Interestingly, in our in vivo studies of leukocyte trafficking in lung microcirculation, we failed to discern any potential effect of nicotine exposure on leukocyte chemotaxis. This is in sharp contrast to earlier studies (39) wherein nicotine induced chemotaxis of neutrophils in vitro. However, other investigators (32, 34) have failed to observe any nicotine-induced chemotaxis of polymorphonuclear leukocytes in vitro, supporting our observation. Nicotine, on the other hand, has been shown to enhance chemotactic responses of human polymorphonuclear leukocytes to selected stimuli such as formylmethionylleucylphenylalanine (31, 15, 13). Our studies in vivo thus shed further evidence on the function of nicotine as a proinflammatory mediator that supports rolling and adhesion of leukocytes but fails to induce chemotaxis in vivo under conditions of flow in lung microvessels.

Although overall, our data demonstrate that nicotine is likely to induce the upregulation of not only vascular P-selectin but also the expression of L-selectin ligands, the mechanism by which these molecules are upregulated at a cellular level and how they influence the rolling behavior of circulating leukocytes is not understood. Previous studies from our laboratory demonstrate that rolling of human eosinophils in inflamed postcapillary venules at physiological shear stress is blocked by inhibitors of MAPK (2). In addition, other investigators have demonstrated that nicotine increases the activity of MAPK or ERK1/2 in various cells, affecting critical cellular events (5, 8, 33). However, the effect of nicotine on modulating phosphorylation of MAPK in lung endothelial cells and the effect of this modulation on leukocyte rolling and adhesion has not been delineated. In the present study, nicotine was found to increase levels of phosphorylated ERK1/2 in murine lung-derived endothelial cells (Fig. 9). In the presence of a MAPK-specific inhibitor, not only was the increase in phosphorylated ERK1/2 abolished, but the nicotine-induced leukocyte rolling and adhesion was also significantly inhibited (Fig. 8). Other investigators have demonstrated that expression of P- and L-selectin is dependent on phosphorylation of ERK1/2 (14, 18). Together, these data further substantiate the anti-selectin MAb studies in that exposure to nicotine results in the upregulation of vascular endothelium-expressed selectins and L-selectin ligands, a process that appears to be dependent on the MAPK signaling pathway. Our studies, for the first time, elucidate the proinflammatory role of nicotine in mediating the recruitment of leukocytes in both arterioles and venules of the lung microvasculature.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by California Tobacco-Related Disease Research Program Grants 7RT-0197 and 10RT-0171 and the National Institute of Allergy and Infectious Diseases Grant AI-35796 (to P. Sriramarao).


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Sriramarao, Division of Vascular Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Dr., San Diego, CA 92121 (E-mail: rao{at}ljimm.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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