Phagocytosis of particulate air pollutants by human alveolar
macrophages stimulates the bone marrow
Hiroshi
Mukae1,
James C.
Hogg1,
Dean
English1,
Renaud
Vincent2, and
Stephan F.
van
Eeden1
1 Pulmonary Research Laboratory, University of British
Columbia, St. Paul's Hospital, Vancouver, British Columbia V6Z 1Y6;
and 2 Environmental Health Directorate, Health
Canada, Ottawa, Ontario, Canada K1A OL2
 |
ABSTRACT |
Epidemiologic studies
have shown an association between the level of ambient particulate
matter < 10 µm (PM10) and cardiopulmonary mortality. We have shown that exposure of rabbits to PM10
stimulates the bone marrow. In this study, we determined whether human
alveolar macrophages (AMs) that phagocytose atmospheric
PM10 produce mediators capable of stimulating the bone
marrow. AMs incubated with PM10 for 24 h produced
tumor necrosis factor-
in a dose-dependent manner (86.8 ± 53.29 pg/ml with medium alone; 1,087.2 ± 257.3 pg/ml with 0.1 mg/ml of PM10; P < 0.02). Instillation of the
supernatants from AMs incubated with 0.1 mg/ml of PM10 into
the lungs of rabbits (n = 6) increased circulating
polymorphonuclear leukocyte (PMN) and band cell counts as well as
shortened the PMN transit time through the bone marrow (87.9 ± 3.3 h) compared with unstimulated human AMs (104.9 ± 2.4 h; P < 0.01; n = 5 rabbits). The
supernatants from rabbit AMs incubated with 0.1 mg/ml of
PM10 (n = 4 rabbits) caused a similar
shortening in the PMN transit time through the bone marrow (91.5 ± 1.6 h) compared with human AMs. We conclude that mediators
released from AMs after phagocytosis of PM10 induce a
systemic inflammatory response that includes stimulation of the bone marrow.
particulate matter less than 10 micrometers; air pollution; bone
marrow; leukocytes; cytokines
 |
INTRODUCTION |
EPIDEMIOLOGIC STUDIES
HAVE associated particulate air pollution, especially
particulate matter < 10 µm (PM10), with
increased cardiopulmonary morbidity and mortality (6, 24).
Residents of communities exposed to high levels of PM10
showed faster rates of lung function decline (19), more
hospital admissions for pneumonia, chronic obstructive lung disease,
myocardial infarctions, and heart failure after adjustment for several
individual risk factors including smoking (1, 7, 20). The
biological mechanisms responsible for this PM10-induced
pulmonary and cardiovascular morbidity and mortality are not clear
(33). Weiss et al. (36) have shown that an
increase in peripheral blood leukocyte count is a predictor of total
mortality, independent of smoking in large population-based studies,
and several independent longitudinal studies (8, 9, 12)
have linked elevations of the peripheral blood leukocyte count to
increased mortality. A leukocytosis, specifically a granulocytosis,
serves as a marker of the systemic inflammatory response
(13) and is thought to contribute to this increase in
morbidity and mortality. During an episode of acute air pollution in
Southeast Asia, Tan et al. (26) demonstrated a
leukocytosis in young military recruits that was associated with a
release of granulocytes from the bone marrow (26). This suggests that this systemic inflammatory response that includes bone
marrow stimulation is a feature of breathing polluted air and that this
leukocyte response may play a pathogenetic role in the cardiopulmonary
mortality associated with air pollution.
Our laboratory has developed a method to measure the response of the
bone marrow in rabbits with the thymidine analog
5-bromo-2'-deoxyuridine (BrdU) to label dividing polymorphonuclear
leukocytes (PMNs) in the bone marrow. This method provides the
measurement of the transit time of PMNs through the mitotic and
postmitotic pools of the bone marrow (27) and has been
used to measure the effect of inflammatory stimuli such as pneumococcal
pneumonia (27), cigarette smoking (28),
glucocorticoids (17), and interleukin (IL)-8 (30) on the transit time of PMNs through the marrow pools.
This model has also been used to show that the deposition of inert carbon particles in the lung enhanced the transit of PMNs through the
bone marrow (18). Alveolar macrophages (AMs) were
implicated in this response by showing that a similar stimulation of
the marrow by supernatants from AMs fed carbon particles
(29). Because AMs are important effector cells that
phagocytose and process inhaled particles, we suspect that they play a
key role in this bone marrow response. The present study was designed
to determine whether mediators released from human AMs after
phagocytosis of particles causes a systemic response that includes
stimulation of the bone marrow. Mediators that could be responsible for
this systemic response include granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-1, and tumor necrosis factor (TNF)-
(14, 18). In the present study,
we used TNF-
as a marker of human AM stimulation and the rabbit model to determine the collective effect of the AM-derived mediators on
the bone marrow. A major question that we addressed in this study is
whether the rabbit model we used to study bone marrow stimulation is
suitable to study the effect of mediators produced by human AMs. We
provide data showing that when human AMs phagocytose atmosphere-derived
PM10, they release mediators that stimulate the bone marrow
of rabbits. We propose that a rabbit model could be a useful tool in
identifying the AM-derived mediators responsible for the systemic
response to breathing polluted air.
 |
METHODS |
Urban air particles.
The urban air dust preparation EHC-93 was obtained from Environmental
Health Directorate (Health Canada, Ottawa, ON) (34, 35).
The particles were recovered from bag-house filters of the single-pass
air filtration system of the Environmental Health Center in Ottawa
(100% outdoor air), and their elemental and organic contents have been
reported elsewhere (35). The dispersed particles have a
mass median diameter of 4-5 µm, and 20% of the mass is associated with the fraction of particulate matter < 2.5 µm
based on chemical profile and size distribution (34). The
particles have a low direct cytotoxicity to lung macrophages
(2) and contain endotoxin levels well below those detected
in similar preparations such as SRM-1649 (3).
Stimulation of AMs in vitro.
Human AMs were harvested from bronchoalveolar lavage (BAL) fluid from
lungs resected for small peripheral tumors (n = 6 patients). All patients were smokers aged 62 ± 5 yr (3 women and
3 men). The BAL fluid was obtained from a noninvolved segment or lobe of the resected lung. Human AMs harvested from BAL fluid were >90%
viable (trypan blue exclusion method) and consisted of 98% AMs and 2%
lymphocytes and neutrophils (as determined by morphological identification of the cells by a qualified pathologist). Rabbit AMs
were harvested by BAL from the lungs of normal 6-wk-old rabbits. After
the rabbit was killed with pentobarbital sodium, a polyethylene tube
was inserted into the trachea, and the lungs were washed with 50 ml of
saline. More than 98% of cells recovered were AMs, and the cells were
>90% viable. All specimens were tested for possible endotoxin
contamination (positive specimens were excluded) with the
Limulus amebocyte lysate test (detection level of 0.1 endotoxin unit/ml; E-TOXATE, Sigma, St. Louis, MO). Human and rabbit AMs were incubated with different concentrations of EHC-93. EHC-93 (0.01-0.1 mg/ml) particles were suspended in RPMI 1640 medium and 10% fetal calf serum. Cells (0.5 × 106/ml) were incubated with the PM10 suspension
at 37°C in 5% CO2 for 24 h in a 24-well plate. This
time point was selected after a time-series experiment in which
supernatants were harvested 2, 4, 8, 12, and 24 h after EHC-93
exposure. AMs only at the 24-h time point were significantly different
from control AMs incubated only with RPMI 1640 medium. The
supernatants were harvested, and to remove all particulate matter from
the supernatants, they were filtered through 0.2-µm filters and
centrifuged at 180 g for 10 min before storage at
70°C.
TNF-
levels in the supernatants were determined by ELISA with a
commercially available kit (Genzyme Diagnostics, Cambridge, MA). The
supernatants used for the in vivo studies (see Effect of
mediators from AMs on the bone marrow: experimental
protocol) were generated by incubating AMs with either RPMI 1640 medium (control group) or 0.1 mg/ml of PM10.
Effect of mediators from AMs on the bone marrow: experimental
protocol.
Female New Zealand White rabbits (weight 2.3-2.8 kg;
n = 19) were used, and all studies were approved by the
Animal Experimentations Committee of the University of British Columbia
(Vancouver, BC).
BrdU (100 mg/kg; Sigma) was administered to rabbits 24 h before
the intrabronchial instillation of the supernatants by infusion through
the marginal ear vein at a concentration of 15 mg/ml in normal sterile
saline over a period of 5 min (29). For bronchial instillation of the supernatants, the rabbits were anesthetized with
ketamine hydrochloride (80-100 mg/kg intramuscularly) and xylazine
(10-15 mg/kg intramuscularly) and challenged with intrabronchial instillation of supernatants (0.6 ml/kg) from either human AMs stimulated with PM10 or rabbit AMs stimulated with
PM10. The supernatant was instilled in the lower lobe of
the lung under fluoroscopic control by inserting a pediatric
nasogastric tube through the trachea (29). The peripheral
blood samples were obtained from the central ear artery just before the
BrdU injection and 3, 6, 9, 12, 24, 36, 48, 72, 96, 120, 144, and
168 h after the intrabronchial instillation. One milliliter of
blood was collected in standard Vacutainer tubes containing EDTA
(Becton Dickinson, Rutherford, NJ) for leukocyte counts, and an
additional 1 ml was collected in tubes containing acid citrate-dextrose
for the detection of BrdU-labeled PMNs (PMNBrdU). Fentanyl
(0.02 mg/kg) and droperidol (1.0 mg/kg) were used subcutaneously as
sedation at each time point of blood collection.
The animals were killed with an overdose of pentobarbital
sodium, the chests were opened rapidly, the base of the heart was ligated, and the lungs were removed and inflated at 25 cmH2O by intratracheal instillation of 10%
phosphate-buffered Formalin for histological evaluation. The tissue
blocks were sectioned (3 µm thick) and stained with hematoxylin and
eosin. The tissue sections were evaluated at ×400 magnification in
computer-generated random fields of view with a light microscope
(Nikon, Japan, Tokyo).
Total white blood cell counts were determined on a model SS80 Coulter
Counter (Coulter Electronics, Hialeah, FL). Differential counts were
obtained by counting 100 leukocytes in randomly selected fields of view
on Wright-stained blood smears, and 100 PMNs were evaluated in randomly
selected fields of view to determine the changes in the number of band
cells. Blood collected in acid citrate-dextrose was used to obtain
leukocyte-rich plasma as previously described in detail
(27). Leukocyte-rich plasma was cytospun onto
3-aminopropyltriethoxysilane-coated slides by cytocentrifugation
at 180 g with a Cytospin 2 (Shandon Lab Products, Cheshire,
UK) for 5 min. The cytospin specimens were air-dried and stained with
the alkaline phosphatase-anti-alkaline phosphatase method to determine
the fraction of PMNBrdU in each specimen as previously
described (27).
Evaluation of PMNBrdU.
PMNBrdU were evaluated as previously described
(27). Briefly, PMNBrdU were divided into three
groups according to the intensity of nuclear staining with an
arbitrarily designated grading system. This grading system was designed
to evaluate the transit time of the myeloid cells that were in their
last division in the mitotic pool when exposed to BrdU (G3), those that
were in the middle (G2), and those that were in their first division
(G1). These slides were coded and examined without knowledge of the
group or sampling time. Fields were selected in a systematic randomized fashion, and 200 cells were evaluated per specimen.
Transit time of PMNBrdU through the bone marrow was
calculated as previously described (27). Briefly, the
number of PMNBrdU was corrected for the disappearance
(half-life) of cells in the circulation. In a previous study, Bicknell
et al. (5), using a whole blood transfusion
method, reported that the half-life of PMNBrdU in rabbits
was 270 min or 4.5 h. We have applied this rate of exponential
loss of PMNBrdU from the circulation to calculate the
number of PMNBrdU released from the bone marrow and the
transit time through the different pools (27).
Statistical analysis.
All values are expressed as means ± SE. Differences in TNF-
values and PMN transit times were evaluated with a one-way ANOVA. In
vivo data were analyzed with a two-way ANOVA for repeated measures, and
Bonferroni's correction was done for multiple comparisons. A value of
P < 0.05 was accepted as significant.
 |
RESULTS |
Composition of particles.
Table 1 summarizes the elemental
composition of the EHC-93 particles as total content and soluble
content as adapted from Vincent et al. (35). The
calculated dose of soluble elements instilled into the lungs of rabbits
is comparable to that in rats exposed for 4 h to 5 mg/m3 of EHC-93 and is considered environmentally relevant
(34). This dose does not cause histological changes
indicative of injury in the lung. Similarly, qualitative evaluation of
lung tissue sections in our study showed no signs of inflammation in
regions of supernatant instillation compared with contralateral control regions of the lung (Fig. 1B).

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Fig. 1.
A: photomicrograph of human alveolar
macrophages (AMs) stained with Wright's solution after incubation for
24 h with the urban air particles (EHC-93) in vitro. The majority
of AMs ingested 1 or more particles (arrow). Bar, 10 µm.
B: region of lung tissue where the supernatants were
instilled. There were no inflammatory changes in either the smaller
bronchioles, alveolar walls, or air spaces. Bar, 20 µm.
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TNF-
produced by human AMs stimulated with PM10.
When human AMs were incubated with 0.1 mg/ml of PM10 for
24 h, they phagocytosed the particles (Fig. 1A).
Incubation with different concentrations of PM10 resulted
in a dose-dependent increase in TNF-
secretion by human AMs (Fig.
2), and the 0.1 mg/ml concentration of
PM10 resulted in a response similar to that produced
by 1 µg/ml of lipopolysaccharide (Sigma).

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Fig. 2.
Tumor necrosis factor (TNF)- production by human AMs
exposed to urban air particles and lipopolysaccharide (LPS; 1 µg/ml).
PM10, particulate matter < 10 µm. Data are
means ± SE of 5 (urban air particles) and 3 (LPS) experiments
performed with AMs from different donors. The urban air particles
caused a dose-dependent increase in TNF- production, and the results
with highest dose of particles were similar to those with
LPS.* P < 0.05 vs. medium.
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Supernatants from AMs and the release of PMNs from the bone marrow.
Instillation of the supernatants from human AMs (6 h; P < 0.05) and rabbit AMs (6 h; P < 0.05) incubated with
PM10 caused a similar temporary increase in circulating
PMNs (Fig. 3A). Circulating band cell counts also increased after instillation of the supernatants in the human AM plus PM10 group at 6 and 9 h and in
the rabbit AM plus PM10 group at 6 h (Fig.
3B). There was no significant difference in the PMN and band
cell counts between the human AM plus PM10 and rabbit AM
plus PM10 groups.

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Fig. 3.
Circulating polymorphonuclear leukocyte (PMN;
A) and band cell (B) counts after instillation of
supernatants from cultured rabbit (n = 4) and human
(n = 6) AMs with PM10 and from human AMs
cultured without PM10 (n = 5).
5-Bromo-2'-deoxyuridine (BrdU) was used to label the cells. Values are
means ± SE. Note the increase in PMNs and band cells induced by
supernatants from human and rabbit AMs 6 h after instillation.
Band cells were also significantly increased 9 h after
instillation of supernatants from human AMs stimulated with
PM10. * P < 0.05 compared with human
AMs cultured without PM10.
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Figure 4 shows the percentage
(A) and number (B) of PMNBrdU in the
circulation over time. Supernatants from both human and rabbit AMs
stimulated with PM10 shifted these curves to the left (Fig.
4). Figure 5 shows the G3 cells that
represent the transit of PMNs through the postmitotic pool, and Fig.
6 shows the G1 cells that represent the
transit of PMNs through the mitotic plus postmitotic pools. Both G3
(Fig. 5) and G1 (Fig. 6) cells show that the supernatants from rabbit
and human AMs stimulated with PM10 causes a left shift of
the curve compared with that from control human AMs.

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Fig. 4.
Percentage (A) and number (B) of
all BrdU-labeled PMNs in the circulation after instillation of
supernatant from AMs cultured with and without PM10. Values
are means ± SE. Note the early peaks at 36 h and rapid
decline in circulating BrdU-labeled PMNs after instillation of
supernatants from the human (n = 6) AM plus
PM10 and rabbit (n = 5) AM plus
PM10 groups compared with those from the human
(n = 4) AM group. * P < 0.05 compared with human AMs cultured without PM10.
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Fig. 5.
Percentage (A) and number (B) of G3
cells (see text for description) in the circulation after instillation
of supernatants from AMs cultured with and without PM10.
Values are means ± SE. Note the early peaks at 36 h in the
human (n = 6) AM plus PM10 and rabbit
(n = 5) AM plus PM10 groups compared with
those in the human (n = 4) AM group.
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Fig. 6.
Percentage (A) and number (B) of G1
cells (see text for description) in the circulation after instillation
of supernatants from AMs cultured with and without PM10.
Values are means ± SE. Note the lower values at 144 h in the
human (n = 6) AM plus PM10 and rabbit
(n = 5) AM plus PM10 groups compared with
those in the human (n = 4) AM group.
* P < 0.05 compared with the human AMs cultured
without PM10.
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Table 2 shows the calculated
transit time of all the different populations of PMNBrdU
(all PMNBrdU and G3, G2, and G1 cells). The data show that
compared with the effect of the instillation of supernatants from human
AMs incubated without particles, supernatants from human
(P < 0.01) and rabbit (P < 0.05) AMs
incubated with PM10 shortened the transit time of PMNs
through the bone marrow. The transit time of PMNs through the
postmitotic (G3; P < 0.01) and mitotic plus
postmitotic (G1; P < 0.05) pools were shorter in human
AMs incubated with than without PM10. There was no
difference in PMN transit times between the instillation of
supernatants from human and rabbit AMs stimulated with PM10
(Table 2) and supernatants incubated only in RPMI 1640 medium
(29).
Urban air particles contain small amounts of endotoxin (4, 34,
35), and the PM10 (0.1 mg/ml) dose used in this
study contained trace amounts of endotoxin (0.096 endotoxin unit/ml). Instillation of RPMI 1640 medium containing the same amount of endotoxin present in 0.1 mg/ml of PM10 into rabbit lungs
(n = 4) produced transit times of PMNs through the bone
marrow that were similar to those of human AMs alone (all PMNs,
105.5 ± 4.1 vs. 104.9 ± 2.4 h; G3 cells, 65.5 ± 3.3 vs. 65.7 ± 2.7 h; G2 cells, 90.0 ± 3.5 vs.
92.3 ± 2.3 h; G1 cells, 136.6 ± 5.4 vs. 130.7 ± 3.4 h).
 |
DISCUSSION |
The results of this study show that when human AMs phagocytose
atmospheric PM10, they secrete mediators such as TNF-
in
a dose-dependent manner. They also show that when supernatants from human AMs incubated with PM10 are instilled into the lungs
of rabbits, the transit time of PMNs through both the mitotic and postmitotic bone marrow pools are shortened. Together these results show that mediators released from AMs after phagocytosis of atmospheric particles are important in eliciting the systemic response associated with particulate air pollution.
The biological mechanisms responsible for the adverse health effects of
breathing particulate air pollution are not clear. Seaton et al.
(25) proposed the hypothesis that the inhalation of fine
particles provokes a low-grade inflammatory response in the lung that
causes an exacerbation of lung disease and a change in blood
coagulability that results in increased pulmonary and cardiovascular
deaths. Because AMs are important in processing airborne particles, we
postulate that they are critical in secreting the mediators that
initiate a systemic response that includes the release of leukocytes
from the bone marrow. The present study shows that human AMs exposed to
PM10 collected in an urban environment are stimulated to
secrete mediators such as TNF-
(Fig. 1) and that the mediators
secreted by these human AMs and instilled into the lung of the rabbit
stimulate the bone marrow (Table 2). This suggests that AMs are an
important source of inflammatory mediators responsible for the systemic
response after PM10 exposure.
We used TNF-
as a marker of AM stimulation by PM10, and
the increased secretion of TNF-
is consistent with a previous report (4) with different urban air pollutants. AMs are also
capable of producing numerous other mediators such as hematopoietic
growth factors (granulocyte colony-stimulating factor and
granulocyte-macrophage colony-stimulating factor), IL-1, IL-6, and IL-8
(14, 18), all capable of stimulating the bone marrow.
These mediators could spill over into the circulation and stimulate the
bone marrow directly or indirectly via their effect on other effector
cells in the lung or other organs. Other effector cells in the lung such as airway epithelial cells also produce inflammatory mediators when exposed to PM10 (21), but their role in
this systemic inflammatory response is not well established. IL-1 and
IL-6 play a critical role in the systemic inflammatory response by
initiating the production of acute-phase proteins such as C-reactive
protein, fibrinogen, complement, and antiproteases by the liver
(10). Therefore, secondary mediator secretion by effector
cells in the lung or distant organs such as the liver could also
contribute to the observed bone marrow response.
The urban PM10 used in this study is a complex aggregate of
elemental and organic carbons, metals, sulfates, nitrates, and organic
contaminants (35). The metal components in these particles are thought to contribute to their ability to cause oxidant stress and
cytokine production in AMs (11). Becker et al.
(4) have suggested that endotoxin contributes to cytokine
production by AMs because the cytokine response to PM10 was
partly inhibitable by polymyxin B (4). Trace amounts of
endotoxin were detected on the EHC-93 particles used in this study, but
instillation of a similar amount of endotoxin into the lungs of
rabbits did not alter the PMN transit time through the bone
marrow. This suggests that the endotoxin contamination cannot explain
the bone marrow stimulation induced by EHC-93 particles. Other soluble
components of EHC-93 particles have been shown to induce an acute
inflammatory response in the lungs of mice (2), although
the dose used was 500 times higher than the calculated dose we used in
our study (Table 1). Therefore, the small dose of particles we used in our experiments makes it unlikely that the soluble elements of the
EHC-93 in the supernatants contribute to the bone marrow stimulation we
observed. Furthermore, the dose of PM10 we used is not
cytotoxic to AMs when structural integrity end points are measured
(16), indicating that proteases and esterases released
from damaged AMs are responsible for bone marrow stimulation. Further
studies are needed to determine which component(s) of PM10
is responsible for cytokine production by AMs and which mediator or
mediators cause the bone marrow release of PMNs.
A major question that we addressed in this study is whether the rabbit
model we used to study bone marrow stimulation is suitable to study the
effect of mediators produced by human AMs. The results show that the
supernatants from AMs incubated with PM10 caused a
shortening of these transit times and that this effect was similar when
either human or rabbit AMs were incubated with the same amount of
particles (Table 2). This suggests a large degree of interspecies homology between the cytokines of humans and rabbits (22,
32), allowing the rabbit to be used to study the effects of
mediator release from human AMs. This model provides an advantage
because the thymidine analog BrdU used to measure the transit time of PMNs through the mitotic and postmitotic bone marrow pools (17, 27-31) cannot be used in humans. It also allows us
to study the functional characteristics and intravascular behavior of
the newly released cells. The AMs used in this study were obtained from smokers who are urban dwellers, both factors that could potentially influence the response of these cells to particles. Although AMs from
young healthy nonsmoking volunteers would minimize variables such as
the effect of chronic smoking on the AM response, the subjects in our
study represented the target population vulnerable to the adverse
health effects of exposure to particle air pollution (6, 7, 19,
20).
This study shows a shortening of the transit time of PMNs through the
bone marrow and an increased number of band cells in the circulation
induced by PM10, indicating that the number of circulating
immature PMNs has increased. Lichtman and Weed (15) showed
that immature PMNs harvested from the bone marrow are larger and less
deformable than their circulating counterparts. Our laboratory has
shown that immature PMNs newly released from the bone marrow by acute
pneumonia (23) and cigarette smoke exposure
(31) preferentially sequester in the pulmonary
microvessels. These immature PMNs were also slow to migrate out of the
capillaries into an inflammatory site (23), and it is
reasonable to postulate that activation of these sequestered PMNs in
lung microvessels could cause endothelial damage. We speculate that the
increased burden of immature PMNs in the lung could contribute to the
observed decrease in lung function associated with chronic exposure to particulate air pollutants. Furthermore, cytokines such as IL-6 stimulate the bone marrow to produce platelets and the liver to produce
coagulation factors (10, 25), and these prothrombotic factors could contribute to the cardiovascular events associated with
exposure to particulate air pollution.
In summary, our results show that mediators released from AMs after
phagocytosis of ambient particulate matter collected in a major North
American city induces a systemic inflammatory response that includes
stimulation of the bone marrow. This bone marrow stimulation results in
the release of immature PMNs into the circulation, and we speculate
that both these leukocytes and the circulating inflammatory mediators
released from AMs play an important role in the pathogenesis of the
pulmonary and cardiovascular diseases associated with particulate air pollution.
 |
ACKNOWLEDGEMENTS |
We thank Jennifer Hards and Mark Elliott for technical support and
Health Canada (Ottawa, ON) for making the EHC-93 available.
 |
FOOTNOTES |
The work was supported by Medical Research Council of Canada Grant 4219 and the British Columbia Lung Association.
Address for reprint requests and other correspondence: S. F. van Eeden, Pulmonary Research Laboratory, Univ. of British Columbia, St. Paul's Hospital, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6
(E-mail: svaneeden{at}mrl.ubc.ca).
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.
Received 30 November 1999; accepted in final form 9 June 2000.
 |
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