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
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ABSTRACT
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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
, 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.
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MATERIALS AND METHODS
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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.
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RESULTS
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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.
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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.
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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.
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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.
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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).
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DISCUSSION
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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
|
---|
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.
 |
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