Pharmacological Stabilization of Mast Cells Abrogates
Late Thrombotic Events Induced by Diesel Exhaust
Particles in Hamsters
Abderrahim Nemmar, DVM, PhD; Peter H.M. Hoet, PhD; Jos Vermylen, MD, PhD;
Benoit Nemery, MD, PhD; Marc F. Hoylaerts, PhD
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Background—Particulate air pollution is associated with cardiovascular diseases and myocardial infarction (MI).
Methods and Results—We investigated the relationship between airway inflammation and thrombosis 24 hours after
intratracheal (IT) instillation of diesel exhaust particles (DEP; 50 ␮g/hamster). Mild thrombosis was induced in the
femoral vein by endothelial injury, and the consequences of airway inflammation on thrombogenicity were studied via
online video microscopy. Lung inflammation and histamine analysis in bronchoalveolar lavage (BAL) and plasma were
performed after pretreatment with dexamethasone (DEX) or sodium cromoglycate (SC). DEP induced airway
inflammation and histamine release in BAL and in plasma, and increased thrombosis, without elevating plasma von
Willebrand factor (vWF) levels. The IT instillation of 400-nm positively charged polystyrene particles (500
␮g/hamster), serving as particles that do not penetrate into the circulation, equally produced airway inflammation,
histamine release, and enhanced thrombosis. Histamine in plasma resulted from basophil activation. Intraperitoneal (IP)
pretreatment with DEX (5 mg/kg) abolished the DEP-induced histamine increase in BAL and plasma and abrogated
airway inflammation and thrombogenicity. The IT pretreatment with DEX (0.5 mg/kg) showed a partial but parallel
inhibition of all of these parameters. Pretreatment with SC (40 mg/kg, IP) strongly inhibited airway inflammation,
thrombogenicity, and histamine release.
Conclusions—Our results are compatible with the triggering of mast cell degranulation and histamine release by DEP.
Histamine plays an initial central role in airway inflammation, further release of histamine by circulating basophils, and
peripheral thrombotic events. Antiinflammatory pretreatment can abrogate the peripheral thrombogenicity by preventing
histamine release from mast cells. (Circulation. 2004;110:1670-1677.)
Key Words: lung 䡲 respiration 䡲 thrombosis 䡲 air pollution
H
istamine plays a major role in the pathophysiology of
inflammation. Its main source is basophils and mast
cells, which are distributed throughout the body.1 Histamine
is a powerful coronary vasoconstrictor2; it induces proinflammatory cytokine production from endothelial cells,3 upregulates P-selectin on the endothelial cell surface,4,5 and is able
to induce intimal thickening in a mouse model of thrombosis.6 Histamine also can activate platelets, and it potentiates
the aggregatory response of other agonists including adrenaline, 5-hydroxytryptamine, and thrombin.7–9 Moreover, histamine is able to modulate the activity of inflammatory cells
such as neutrophils,10 monocytes,11 and eosinophils.12
In urban environments, diesel exhaust particles (DEP) are
one of the main contributors of atmospheric particulate matter
of diameter ⬍2.5 ␮m (PM2.5). Epidemiological studies reported that exposure to PM2.5 increases susceptibility to
ischemia13 and the occurrence of myocardial infarction
(MI).14 It is currently established that acute events of atherosclerosis such as MI result from thrombosis at the site of
plaque rupture,15 but the mechanisms linking atmospheric
particulate matter to MI are not fully understood.16
Even though the elevation of histamine levels has been
correlated with the onset of MI,17–19 the evidence linking
exposure to DEP and release of histamine is more abundant in
the context of airway allergic and inflammatory processes.
Thus, DEP were reported to increase the severity of clinical
symptoms to allergen exposure by enhancing mast cell
degranulation20 and to enhance interleukin-4 and histamine
production in basophils.21 In addition, an increase in mast cell
numbers has been reported in the submucosa and elevated
histamine levels were found in nasal and bronchoalveolar
lavage (BAL) fluid after exposure to DEP in humans.22,23 We
recently showed in hamsters that DEP cause lung inflammation and enhance the occurrence of arterial and venous
Received October 31, 2003; de novo received February 26, 2004; revision received April 29, 2004; accepted May 3, 2004.
From the Laboratory of Pneumology, Lung Toxicology Unit (A.N., P.H.M.H., B.N.), and the Center for Molecular and Vascular Biology (J.V.,
M.F.H.), Katholieke Universiteit Leuven, Leuven, Belgium.
Correspondence to Prof B. Nemery, Katholieke Universiteit Leuven, Laboratory of Pneumology, Lung Toxicology Unit, Herestraat, 49, B-3000
Leuven, Belgium. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/01.CIR.0000142053.13921.21
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Nemmar et al
thrombosis24 and that these effects persisted up to 24 hours.25
An instrumental role of histamine in these processes was
evidenced by pretreating hamsters with a histamine H1
receptor antagonist, which inhibited both lung inflammation
and thrombotic events without affecting the histamine production itself.25
The aim of this study was to further investigate the links
among histamine release, pulmonary inflammation, peripheral thrombogenicity, and vascular inflammation. To this end,
the consequences of systemic and local pulmonary antiinflammatory pretreatments by glucocorticoids and sodium
cromoglycate (SC), a well-established stabilizer of mast cell
and basophil degranulation, were investigated on a number of
inflammatory parameters. These studies revealed a primary
role for mast cell degranulation in lung inflammation, which
in turn triggered inflammation-related peripheral thrombotic
events.
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Methods
The experiments were performed in accordance with protocols
approved by the Institutional Animal Care and Research Advisory
Committee of the Katholieke Universiteit Leuven.
Particles
DEP (SRM 1650) obtained from the National Institute of Standards
and Technology were suspended in sterile pyrogen-free saline (NaCl
0.9%) containing Tween 80 (0.1%). Polystyrene particles with a
uniform diameter of 400 nm amine modified (Bangs Laboratories)
were suspended in saline. These particles are positively charged at
physiological pH.26 To minimize their aggregation, particle suspensions were always sonicated (Branson 1200, VEL) for 15 minutes
and vortexed immediately (⬍1 minute) before dilution and before
intratracheal (IT) administration. Control animals received saline
alone or saline containing Tween 80 (0.1%), as appropriate.
Diesel-Induced Thrombosis and Mast Cells
1671
Determination of Histamine in BAL and Plasma
The histamine content was determined by a radioimmunoassay kit
(Immunotech) in BAL and plasma obtained after centrifuging
(1000g⫻10 min, 4°C) venous blood collected from the abdominal
vena cava on ethylenediaminetetraacetic acid. The BAL and plasma
samples were stored at ⫺20°C.
Determination of Plasma von Willebrand
Antigen Levels
Plasma von Willebrand factor (vWF) antigen levels were measured
by an enzyme-linked immunosorbent assay (ELISA) technique, as
previously described,28 and expressed as a percentage of the absorbance value for a normal plasma pool obtained from 10 untreated
hamsters.
Determination of Total Histamine After Cell Lysis
To assess the contribution of basophils to the appearance of
histamine in plasma, we performed histamine release experiments
separately in whole blood collected from hamsters IT instilled with
vehicle or DEP. The total histamine in cell suspensions was
quantified after cell lysis in distilled water and twice freeze-thawing,
according to the manufacturer’s recommendation (Immunotech).
Histamine was measured as described above and compared with
histamine levels in the corresponding plasma.
Pretreatment With Dexamethasone or
Sodium Cromoglycate
Hamsters were pretreated with dexamethasone (DEX; Sigma), injected either IP (5 mg/kg) or IT (0.1 or 0.5 mg/kg), or with SC
(Sigma) given IP (40 mg/kg29), 1 hour before DEP or vehicle
instillation. Animals were then allowed to recover. They were
subjected to either experimental thrombosis induction or the analysis
of BAL and plasma parameters 24 h later, as outlined above. In
addition, in the experiments involving pretreatment with SC, platelet
function was assessed ex vivo in the platelet function analyzer
PFA-100 (Dade Behring),30 with venous blood collected from the
abdominal vena cava on hirudin (20 ␮g/mL) and supplemented with
0.4% citrate. The closure time reflects platelet aggregate formation in
a shear stress– dependent manner.30
Intratracheal Instillation of Particles
Male and female hamsters (Pfd Gold, University of Leuven, Belgium) weighing 100 to 110 g were used. The animals were
anesthetized intraperitoneally (IP) with sodium pentobarbital (60
mg/kg). The tracheal zone was shaved and disinfected with ethanol
(70%), and the trachea was exposed for the IT administration of 120
␮L of vehicle or DEP (50 ␮g/animal) or 400-nm polystyrene
particles (500 ␮g/animal).
Statistics
Experimental Thrombosis Model
The total cell count in BAL increased to 189.7⫾28.9⫻104/mL
as compared with control (2.5⫾0.4⫻104/mL, P⬍0.0001) 24
hours after the IT instillation of DEP (50 ␮g/animal). The IT
instillation of 400-nm amine polystyrene particles (500 ␮g/
animal) resulted in a less pronounced but nevertheless marked
cellular influx (25.0⫾4.4⫻104/mL versus 2.3⫾0.6⫻104/mL in
controls, P⬍0.005). Figure 1a shows that the IT instillation of
DEP or 400-nm particles led to significant increases in polymorphonuclear neutrophil (PMN) numbers in BAL, reaching
similar proportions in both treatments (34% and 39% of the total
cell number, respectively). The rest of the cells were mainly
macrophages, the proportion of lymphocytes being below 2%.
Histamine concentrations in BAL increased significantly in
the DEP and 400-nm particle groups as compared with their
respective controls, and the average increase (7-fold) was
similar for both types of particles, despite differences in
infiltrating cell numbers (Figure 1b).
In vivo thrombogenesis was assessed 24 hours after the IT instillation of DEP or vehicle, as recently described.26,27 Briefly, a surgically exposed segment of the femoral vein was illuminated with
green (540 nm) light for 2 minutes after the IV injection of Rose
Bengal (20 mg/kg; Sigma), thus producing a free radical–mediated
endothelial lesion with subsequent platelet-rich thrombosis. The
kinetics of thrombus generation were monitored for 40 minutes with
the use of an online microscope video camera, and thrombosis was
quantified via image analysis. The size of the thrombus is measured
as the total area under the curve during plots of the light intensity
(expressed in arbitrary units, AU) versus time.
Analysis of BAL Fluid
Animals were killed with an overdose of sodium pentobarbital 24
hours after the IT instillation of DEP or vehicle. BAL was performed
by cannulating the trachea and lavaging the lungs with 4.5 mL (3
times 1.5 mL) of sterile NaCl 0.9%. BAL cells were counted and
identified with Diff-Quik stain.
Data are mean⫾SEM. Comparisons between groups were performed
by an unpaired Student t test or a 1-way analysis of variance and then
by the Newman-Keuls test.
Results
Histamine Release and Lung Inflammation
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September 21, 2004
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Figure 1. PMN (a) and histamine (b) in BAL fluid. Data obtained
24 h after IT instillation of vehicle or DEP (50 ␮g/animal) or
400-nm amine-modified polystyrene particles (500 ␮g/animal),
as indicated. PMN expressed as % total cell numbers.
Mean⫾SEM (each group, n⫽4 to 7). Statistical analysis by
unpaired Student t test.
Experimental thrombus formation in vivo was strongly enhanced by the IT instillation of DEP or 400-nm particles, and the
magnitude was similar for both types of particles (Figure 2a).
The concentrations of histamine in plasma also increased (2fold) after DEP or 400-nm particle instillation (Figure 2b). No
effect of DEP or 400-nm particle administration was observed
on plasma vWF concentrations (Figure 2c).
Origin of Plasma Histamine
Table 1 compares the concentrations of histamine after cell
lysis in vitro in whole blood and plasma histamine in vivo
after the IT administration of vehicle, DEP, or 400-nm
polystyrene particles. In the vehicle-instilled groups, the
difference between the total blood histamine concentration
(blood cells and plasma) after cell lysis and plasma histamine
concentration was 35⫾6 nmol/L (saline-treated animals) or
36⫾5 nmol/L (animals treated with saline containing Tween
80). The corresponding values found in the groups treated
with 400-nm amine particles and DEP were 12.2⫾1.8 nmol/L
(P⫽0.01) and 17.8⫾1.5 nmol/L (P⬍0.05), respectively.
These lower values compared with the vehicle groups show
that blood basophils had been at least partially degranulated
in both cases. This result pleads in favor of a role for
basophils in the release of histamine in the circulation.
Figure 2. Thrombosis (a), histamine in plasma (b), and plasma
vWF (c). Data obtained 24 h after IT instillation of vehicle or DEP
(50 ␮g/animal) or 400-nm amine-modified polystyrene particles
(500 ␮g/animal), as indicated. Cumulative thrombus size
expressed in AU as total light intensity over 40 min after mild
photochemical damage to femoral vein; vWF expressed as %
normal value in reference animals (n⫽10). Mean⫾SEM (each
group, n⫽4 to 10). Statistical analysis by unpaired Student t
test.
Intraperitoneal DEX Prevents Inflammation,
Thrombotic Events, and Histamine Release
The IP pretreatment of control hamsters with DEX (5 mg/kg)
did not significantly affect the total cell numbers in BAL
(1.8⫾0.5⫻104/mL) or the proportion of PMN as compared
with nontreated controls (Figure 3a). The pretreatment did
strongly reduce the DEP-induced rise of total BAL cells to
control numbers (2.3⫾0.8⫻104/mL, P⬍0.001) and the proportion of PMN (8%, Figure 3a). The histamine concentra-
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Diesel-Induced Thrombosis and Mast Cells
1673
TABLE 1. Histamine Concentrations in Plasma and Whole
Blood After Cell Lysis
Histamine, nmol/L
Vehicle
400-nm Particles
Vehicle
DEP
Plasma
21.6⫾3.3
37.6⫾2.9*
23.6⫾3.7
43.5⫾3.9*
Whole blood
57.0⫾7.0
49.8⫾3.1
59.5⫾9.2
61.3⫾3.4
In distilled water and twice freeze-thawing, after IT instillation of vehicle or
400-nm polystyrene amine-modified particles (500 ␮g/animal) or DEP (50
␮g/animal).
*Pⱕ0.01, values significantly different from vehicle group. Data are
mean⫾SEM (each group, n⫽4).
tions in BAL also were strongly lowered (Figure 3b). Similarly, the IP administration of DEX neutralized the DEPinduced thrombus formation in vivo almost completely
(Figure 4a); likewise, histamine levels in plasma were normalized (Figure 4b).
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Dose-Dependent Inhibition by Intratracheal DEX
on Lung Inflammation, Thrombotic Events, and
Histamine Release
The IT pretreatment of control hamsters with DEX did not affect
the total cell numbers in BAL. The total number of cells found
Figure 4. Thrombosis (a) and histamine release in plasma (b) IP
injection of DEX. Data obtained 24 h after IT instillation of vehicle or DEP (50 ␮g/animal) with or without IP pretreatment with
DEX (5 mg/kg), as indicated. Mean⫾SEM (each group, n⫽4 to
8). Cumulative thrombus size expressed in AU as total light
intensity over 40 min after mild photochemical damage to femoral vein. Statistical analysis by Newman-Keuls test. Data for
non–DEX-pretreated animals same as Figure 2.
Figure 3. PMN influx (a) and histamine release (b) in BAL after
IP DEX. Data obtained 24 h after IT instillation of vehicle or DEP
(50 ␮g/animal) with or without IP pretreatment with DEX (5
mg/kg), as indicated. PMN expressed as % total cell numbers.
Mean⫾SEM (each group, n⫽4 to 7). Statistical analysis by
Newman-Keuls test. Data for non–DEX-pretreated animals same
as Figure 1.
in BAL were 2.0⫾0.2⫻104/mL and 1.7⫾0.4⫻104/mL after the
IT administration of DEX at 0.1 and 0.5 mg/kg, respectively.
The IT pretreatment with 0.1 mg/kg of DEX was ineffective in
reducing the DEP-induced increase of total cells in BAL
(230.0⫾47.0⫻104/mL) and the corresponding PMN influx
(38%; Figure 5a), whereas 0.5 mg/kg of DEX did reduce the
DEP-induced increase of total cells in BAL (to 19.5⫾3.3⫻104/
mL, P⬍0.001) as well as the proportion of PMN (to 17%;
Figure 5a). Similarly, the histamine accumulation in BAL
induced by DEP was not prevented by the IT instillation of 0.1
mg/kg of DEX, but it was partially prevented by IT pretreatment
with 0.5 mg/kg of DEX (Figure 5b).
No inhibition of thrombus formation was recorded after the
IT instillation of 0.1 mg/kg of DEX, but 0.5 mg/kg of DEX
partially inhibited in vivo thrombus formation (Figure 6a). No
effect on plasma histamine concentrations was observed after
pretreatment with 0.1 mg/kg of DEX (Figure 6b); however,
after the administration of 0.5 mg/kg of DEX, the DEPinduced histamine increase in plasma was reduced, although
the drop failed to reach statistical significance (Figure 6b).
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Figure 5. PMN influx (a) and histamine release (b) in BAL after
IT DEX. Data obtained 24 h after IT instillation of vehicle or DEP
(50 ␮g/animal) with or without IT pretreatment with DEX (0.5 or
0.1 mg/kg), as indicated. PMN expressed as % total cell numbers. Mean⫾SEM (each group, n⫽4 to 7). Statistical analysis by
Newman-Keuls test. Data for non–DEX-pretreated animals same
as Figure 1.
SC Inhibition of Histamine Release, Lung
Inflammation, and Platelet Activation
The IP pretreatment of control hamsters with SC (40 mg/kg)
did not significantly affect the total cell numbers in BAL
(2.5⫾0.4⫻104/mL) or the proportion of PMN as compared
with nontreated controls (Figure 7a); however, this pretreatment strongly reduced the DEP-induced increase of total
BAL cell numbers to 4.0⫾0.4⫻104/mL (P⬍0.001) and the
proportion of PMN to 12% (Figure 7a). Likewise, SC
pretreatment reduced the histamine concentrations in BAL to
control values (Figure 7b). The IP administration of SC
strongly reduced histamine release in plasma (Figure 7c).
Platelet activation, as analyzed ex vivo, also was potently
inhibited (Table 2).
Discussion
Short-term increases in particulate air pollution are associated
with acute cardiovascular events in people with preexisting
vascular disease and diabetes mellitus and in older
adults.14,31–33
Ultrafine particles (UFPs; ie, particles of diameter ⬍0.1
␮m) can translocate rapidly from the lungs into the systemic
circulation in humans34 and hamsters.35 We previously demonstrated that the IV or IT administration of positively
charged ultrafine (60 nm) polystyrene particles not only
causes rapid lung inflammation but also enhances thrombotic
events.26,36 We also provided evidence that DEP cause
marked pulmonary inflammation and could aggravate thrombosis up to 24 hours after their deposition in the lungs.25
Pretreating hamsters with a histamine H1 receptor antagonist
strongly reduced lung inflammation at all time points investigated (ie, 1 h, 6 h, 24 h). Such pretreatment reduced the
thrombotic events at 6 and 24 hours but not at 1 hour after
DEP administration. These findings are compatible with
platelet activation being caused by direct passage of the
ultrafine fraction of DEP (or their associated constituents) at
1 hour and by lung inflammation at later time points.25
In contrast to DEP that contain UFP (or their associated
components) capable of translocation into the blood34,35,37 and
capable of directly affecting the vascular end points, large
particles such as 400-nm polystyrene particles are unlikely to
cross the lung– blood barrier to any significant extent38,39; neither
do they possess associated components that can leak and pass
into the blood. We have already shown that 1 hour after their IT
instillation, positively charged amine-modified 400-nm particles
(500 ␮g/hamster) did not cause any thrombotic events, although
they did trigger lung inflammation. In contrast, positively
charged amine particles of 60 nm (UFP) were prothrombotic,
this effect being presumably related to their passage into the
blood.36 In the present study, we wanted to assess the link
between lung inflammation and systemic prothrombotic effects
independent of any direct passage from the lung into the
bloodstream. Therefore, we IT instilled positively charged
400-nm polystyrene particles and DEP and studied their late
effects on lung inflammation, histamine release, thrombotic
events, and endothelial activation in hamsters.
DEP and 400-nm Polystyrene Particles Cause
Lung Inflammation, Thrombotic Events, and
Histamine Release
Our present results show that 24 hours after IT instillation of
DEP, histamine was elevated in BAL and plasma in association
with airway inflammation and thrombosis. Compared with DEP,
the 400-nm amine polystyrene particles produced less cellular
influx, which presumably is related to the different size and
surface chemistry for these particles. The concentrations of
histamine released in BAL or plasma were comparable for both
types of particles, suggestive of a maximal degranulation of mast
cells by both types of particles. No changes in plasma vWF
concentration were observed. vWF is synthesized by endothelial
cells, stored in the Weibel-Palade bodies, and secreted on
endothelial activation.40 Increased plasma vWF levels have been
associated with cardiovascular disease.41 The lack of endothelial
vWF release 24 hours after DEP or 400-nm particle administration in the present study suggests that the prothrombotic tendency developed without endothelial activation. In addition, we
showed previously that no activation of the intravascular coagulation occurred.26
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Figure 6. Thrombosis (a) and histamine release in
plasma (b) after IT DEX. Data obtained 24 h after
IT instillation of vehicle or DEP (50 ␮g/animal) with
or without IT pretreatment with DEX (0.5 or 0.1
mg/kg), as indicated. Cumulative thrombus size
expressed in AU as total light intensity over 40 min
after mild photochemical damage to femoral vein.
Mean⫾SEM (each group, n⫽4 to 8). Statistical
analysis by Newman-Keuls test. Data for non–DEXpretreated animals same as Figure 2.
Histamine H1 receptor antagonism strongly reduced DEPinduced inflammation as well as systemic platelet activation
and prothrombotic tendency.25 In the present study, we
investigated further the relative role of histamine levels in the
lung and blood by targeting its cellular sources (ie, mast cells
and basophils), and we assessed its role in the inflammatory
and thrombotic processes accompanying DEP exposure.
Origin of Plasma Histamine
We verified whether histamine in plasma originated from basophil degranulation or whether it resulted from diffusion, after
pulmonary mast cell degranulation, from the lungs into the
bloodstream. To this end, we compared histamine concentrations before and after cell lysis in whole blood in vitro, which
reflects the in vivo release from basophils into the circulation.
We found lower histamine concentrations contained within
basophils after cell lysis in both 400-nm particle– and DEPexposed groups, as compared with vehicle groups. In other
words, this analysis revealed that the increased plasma histamine
concentrations after the IT instillation of DEP or 400-nm
polystyrene particles resulted from the activation of blood
basophils. This finding is in agreement with the study of
Devouassoux et al,21 who showed that DEP enhance histamine
production from basophils in vitro. It also follows from this
analysis that the peripheral events leading to basophil degranulation are secondary to lung inflammation because they also
occurred with the 400-nm particles, which stay in the lung. A
possible mechanism for this activity is the release of platelet
activating factor42,43 into the circulation, thus causing basophil
activation, but this possibility still needs to be verified.
Effect of DEX on Lung Inflammation and
Thrombotic Events
The prothrombotic “enhancement” of peripheral vascular
thrombosis after endothelial damage constitutes a realistic
experimental approach for human thrombosis underlying MI.
The dose of DEP used here to trigger such thrombotic events
is within realistic exposure during peak levels of air pollution.24 We pretreated hamsters with DEX, a synthetic glucocorticoid (GC), which is a potent antiinflammatory
agent44,45 known to exert cardiovascular protective effects.46
The pharmacological actions of GC often are species specific
or cell-type specific. In rodents, it has been reported that in
vitro47 and in vivo48 pretreatment with GC inhibits the release
of mast cell mediators, including histamine. In humans, DEX
exerts direct inhibitory effects on human mast cell maturation49 and inhibits histamine release from human basophils.50
DEX at 5 mg/kg is effective in inhibiting silica-induced
pulmonary inflammation in rats.51 Here, IP administration of
DEX strongly inhibited histamine release in BAL and plasma
and reduced the DEP-induced PMN influx by 76% and thrombosis in vivo by 60%. These reductions are comparable to those
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September 21, 2004
TABLE 2.
PFA-100
Closure Time in the Platelet Function Analyzer
Closure time, s
Vehicle
DEP
SC⫹Vehicle
SC⫹DEP
248⫾18
136⫾5*
228⫾25
209⫾34†
Blood samples taken from hamsters 24 h after IT DEP instillation (50
␮g/animal) or vehicle with or without pretreatment with SC (40 mg/kg IP). Data
are mean⫾SEM (each group, n⫽4).
*P⬍0.05 vs vehicle-treated group.
†P⬍0.05 vs DEP-treated group.
partially reduced the airway inflammation and subsequent
thrombosis after DEP instillation to a comparable degree.
DEP-Triggered Mast Cell Degranulation and
Histamine Release Initiate Lung Inflammation and
Subsequent Thrombotic Events
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Figure 7. PMN influx (a) and histamine release in BAL (b) and in
plasma (c) after IP SC. Data obtained 24 h after IT instillation of
vehicle or DEP (50 ␮g/animal) with or without IP pretreatment
with SC (40 mg/kg), as indicated. PMN expressed as % total
cell numbers. Mean⫾SEM (each group, n⫽4 to 7). Statistical
analysis by Newman-Keuls test. Data for non–SC-pretreated
animals same as Figures 1, 2.
observed after pretreatment with the H1 receptor antagonist
diphenhydramine25 during the DEP-induced PMN influx. This
result suggests a key role for histamine in triggering cellular
inflammation in the lung and associated thrombotic events.
Inspired by the observations that the IT instillation of DEX
decreases inflammatory cell influx and protein levels in BAL
in oleic acid–induced lung injury52 and because pulmonary
inflammation precedes the systemic effects triggered by DEP,
we used this approach to modulate the thrombotic response to
DEP exposure via antiinflammatory treatment. Although 0.1
mg/kg IT of DEX failed to affect histamine levels and
thrombotic and inflammatory events, 0.5 mg/kg of DEX
DEX affects the release of cytokines and other inflammatory
mediators from various types of cells.53 Mast cell degranulation releases not only histamine but also serotonin, proteases,
and various cytokines. Even though we already documented a
major role for histamine in the observed effects,25 it remained
to be shown whether the effects of DEX resulted from
inhibiting histamine release or whether they resulted from
any other antiinflammatory action. We used SC, a wellknown stabilizer of mast cells and basophil degranulation.54
Using the platelet function analyzer ex vivo, we recently
confirmed the relationship between platelet activation measured ex vivo and the development of thrombotic events in
vivo after DEP exposure.24,25
Our results show that pretreatment with SC29 strongly
inhibited the DEP-mediated histamine release in BAL and
plasma and the neutrophil influx in BAL and peripheral
platelet activation. The parallelism between antiinflammatory
pretreatment and SC on histamine levels, in association with
our earlier findings using the H1 receptor antagonist diphenhydramine,25 pleads in favor of a key role for mast cells’
degranulation and histamine release in initiating inflammatory effects in the lung and subsequent peripheral prothrombotic events. We can conclude therefore that the prevention of
inflammatory and prothrombotic events by DEX probably
stems from the stabilization of mast cells.
Conclusion
Our data provide evidence for a direct link between the
DEP-induced release of histamine and pulmonary inflammation. Lung inflammation in turn causes peripheral systemic
responses and platelet activation. Mast cell stabilization and
antiinflammatory pulmonary treatment abrogate the histamine production in the lung and are capable of reducing the
inflammation-induced thrombotic events in the circulation.
Acknowledgments
This work was supported by the funds of the Katholieke Universiteit
Leuven (OT/02/45) and the Fund for Scientific Research Flanders
(G.0165.03). The help of P. Vandervoort in performing the vWF
ELISA is highly appreciated.
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Pharmacological Stabilization of Mast Cells Abrogates Late Thrombotic Events Induced
by Diesel Exhaust Particles in Hamsters
Abderrahim Nemmar, Peter H.M. Hoet, Jos Vermylen, Benoit Nemery and Marc F. Hoylaerts
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Circulation. 2004;110:1670-1677; originally published online September 13, 2004;
doi: 10.1161/01.CIR.0000142053.13921.21
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