Departments of 1 Environmental Health, 2 Molecular and Cellular Physiology, and 3 Medicine, University of Cincinnati, Cincinnati 45267; and 4 Human and Environmental Safety Division and Corporate Research Division, Procter and Gamble Company, Cincinnati, Ohio 45239
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pulmonary inflammation has been observed in humans
and in many animal species after ozone exposure. Inflammatory cell
accumulation involves local synthesis of chemokines, including
neutrophil chemoattractants such as macrophage inflammatory protein-2
(MIP-2), and monocyte chemoattractants, such as monocyte
chemoattractant protein-1 (MCP-1). To better understand the mechanism
of ozone-induced inflammation, we exposed mice and rats to ozone for 3 h and measured MIP-2 and MCP-1 gene expression. In C57BL/6
mice, steady-state mRNA levels for MCP-1 in the lung increased at 0.6 parts/million (ppm) ozone and were maximal at 2.0 ppm ozone. After
exposure to 2 ppm ozone, MIP-2 mRNA levels peaked at 4 h postexposure,
whereas MCP-1 mRNA levels peaked at 24 h postexposure.
Neutrophils and monocytes recovered in bronchoalveolar lavage fluid
peaked at 24 and 72 h, respectively. The accumulation of monocytes was
thus delayed relative to that of neutrophils, consistent with the
sequential expression of the corresponding chemokines. The role of
MCP-1 in monocyte accumulation was evaluated in greater detail in rats. Ozone caused an increase in monocyte chemotactic activity in
bronchoalveolar fluid that was inhibited by an antibody directed
against MCP-1. Ozone-induced MCP-1 mRNA levels were higher in lavage
cells than in whole lung tissue, indicating that lavage cells are an
important source of MCP-1. In these cells, nuclear factor-B, a
nuclear transcription factor implicated in MCP-1 gene regulation, was also activated 20-24 h after ozone exposure. These findings
indicate that monocyte accumulation subsequent to acute lung injury can be mediated through MCP-1 and that nuclear factor-
B may play a role
in ozone-induced MCP-1 gene expression.
air pollution; asthma; inflammatory mediators; ozone
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
OZONE, A COMMON URBAN AIR POLLUTANT, can alter pulmonary structure and function. Short-term exposure leads to immediate epithelial cell injury through protein oxidation (25) and ozonolysis of unsaturated fatty acids on or in the plasma membrane (19, 29, 31). This is followed by an early inflammatory response (6-24 h) characterized by neutrophil accumulation (33). Once activated, these cells produce additional damage through the release of reactive oxygen species and proteolytic enzymes. This period is marked by acute bronchospasm, cough, mucus secretion, and airway hyperreactivity (21). A third phase (48-72 h) follows, characterized by monocyte accumulation, particularly at the bronchoalveolar junction (7). During this period and after prolonged exposure, the proximal alveolar region develops interstitial monocyte infiltration, edema, hypertrophy, and finally fibrosis. Thus chronic monocytic accumulation may lead to persistent tissue damage and remodeling.
Chemokines belong to a family of cytokines responsible for attracting
and activating leukocytes (11, 23). Chemokines can be divided into two
groups based on the sequence encompassing the first two conserved
cysteines, which are either C-X-C or C-C, where X is any amino acid.
Human C-X-C chemokines are potent neutrophil chemoattractants and
include interleukin (IL)-8, gro- [homologous to rat
cytokine-induced neutrophil chemoattractant (CINC) and mouse KC],
and gro-
and gro-
[homologous to murine macrophage inflammatory protein (MIP)-2]. These cytokines can be synthesized by a variety of pulmonary cells; e.g., MIP-2 can be expressed in
epithelial cells and macrophages (11). Ozone exposure increases mRNA
for mouse MIP-2 and rat CINC in whole lung homogenates, which may
contribute to ozone-induced neutrophil infiltration (12, 15). Human C-C
chemokines are monocyte chemoattractants and include monocyte
chemoattractant protein-1 (MCP-1; homologous to mouse MCP-1/JE), MIP-1,
RANTES (regulated on activation normal T cell expressed and secreted),
and I309. MCP-1, a potent monocyte chemotactic protein, is expressed in
many pulmonary cells including airway and alveolar epithelial cells,
macrophages, and endothelial cells (4, 30). Increases in MCP-1 release
have been found in patients with acute respiratory distress syndrome
(14) and chronic inflammatory diseases including pulmonary sarcoidosis and idiopathic pulmonary fibrosis (6). Whether ozone can induce accumulation of MCP-1 transcripts and protein in the lung is unknown.
In addition, the mechanism by which ozone increases chemokine gene
expression is not clear. Most chemokines are regulated by
transcriptional initiation that is under the control of transcription factors. Two common transcription factor binding sites, nuclear factor-B (NF-
B) and activating protein-1, have been
found in the 5' untranslated region of the human MCP-1 gene (27,
35). Of these two transcription factors, NF-
B is thought to be more important because deletion of its binding site diminishes the transcription stimulated with tumor necrosis factor-
(TNF-
), IL-1, or 12-O-tetradecanoylphorbol
13-acetate (TPA) (35). In many cells, NF-
B is redox sensitive and
can be regulated by oxidants including
H2O2
(32). Ozone as a potent oxidant may increase intracellular oxidative
stress through hydroperoxide formation (19, 31). Therefore, ozone may
activate the MCP-1 gene through activation of NF-
B.
To better understand the processes underlying ozone-induced
inflammation, we investigated the concentration-response and temporal relationships between expression of MIP-2 and MCP-1 mRNA after exposure
to ozone and the recruitment of inflammatory cells to the lung. To
further determine the role of MCP-1 in ozone-induced monocyte
infiltration, we examined the chemotactic activity in bronchoalveolar
lavage (BAL) fluid after exposure to ozone and the inhibitory effect of
MCP-1-neutralizing antibody on the activity. To understand the cell
source of ozone-induced MCP-1, we also compared MCP-1 gene expression
in BAL cells and whole lung tissue. In addition, to determine whether
NF-B might be involved in the ozone-induced MCP-1 gene expression,
we measured the time course of NF-
B binding activity in rat BAL
cells after ozone exposure.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental design. Previously, Driscoll et al. (12) found that ozone induced a rapid increase in MIP-2 mRNA and neutrophil infiltration in the lungs of C57BL/6 mice. This strain was selected because Kleeberger et al. (17) reported that C57BL/6 mice are sensitive to ozone-induced neutrophil infiltration. To determine whether MCP-1 mRNA preceded monocyte infiltration in these mice, we exposed C57BL/6 mice to ozone and extended the time course (obtaining samples up to 72 h after cessation of exposure). The results of neutrophil and monocyte infiltration were compared with samples obtained from the same groups of mice, followed by a determination of the concentration-response relationship between ozone and the MCP-1 message level.
To determine whether MCP-1 mRNA increases are associated with NF-B
activation, we exposed C57BL/6 mice to ozone and measured NF-
B levels in the lung. In preliminary tests, NF-
B activity increased in the lung 24 h after exposure (data not shown). However, the autoradiographs of the gel mobility shift assay could not be
quantified due to high backgrounds of nonspecific protein from lung
disaggregates. To circumvent this problem, NF-
B activity was
measured in the nuclei of cells recovered from lung lavage. This
required a species shift from mouse to rat to provide enough nuclear
protein for the assay. This was accomplished in three steps. First, to
confirm that the ozone response of Wistar rats was similar to that of
C57BL/6 mice, monocyte infiltration and MCP-1 mRNA levels in
the lung were measured in rats. Second, we measured the monocyte
chemotactic activity of the cell-free lavage fluid. Specificity was
assessed by the addition of either an immunoglobulin (Ig) G antibody
directed to MCP-1 or a nonspecific IgG antibody (antibody control).
Third, the NF-
B activity in rat lavage cells was assayed by an
electrophoretic gel mobility shift assay.
Animals and ozone exposure. Six- to
eight-week-old C57BL /6 mice and fourteen- to
sixteen-week-old Wistar rats (Harlan Laboratory, Indianapolis, IN) were
housed in a reverse light cycle (5:00 AM to 5:00 PM dark, 5:00 PM to
5:00 AM light) for 1 wk before each experiment. Ozone was generated by
passing oxygen through an ultraviolet ozone generator (OREC Ozonator
model O3V1, Ozone Research & Equipment, Phoenix, AR), mixed with
filtered air, and introduced into a stainless steel chamber. The ozone
concentration was continuously monitored by an ultraviolet photometric
ozone analyzer (model 1008-PC, Dasibi Environmental, Glendale, CA) and
controlled within ±5% of the reported value. To determine the time
course of response, mice were exposed to 2 parts/million (ppm) ozone
for 3 h and were killed 0-72 h after exposure for
either whole lung RNA or BAL cell count. Unexposed mice were used as
control animals. To determine the concentration-response
relationship, mice were killed 24 h after exposure to 0 (filtered
air)-2.0 ppm ozone for 3 h. For comparison of ozone-induced MCP-1
in BAL cells and whole lung tissue, measurement of ozone-induced MCP-1
chemotactic activity, or measurement of NF-B activity in BAL cells,
the rats were killed 0-24 h after exposure to 2 ppm ozone for 3 h.
BAL was performed, and whole lung tissue was obtained. To determine the
time course of monocyte infiltration in rats, animals were killed
0-72 h after exposure. Filtered air-exposed rats were used as
control animals.
BAL and cell count. After exposure, the mice were killed by cervical dislocation and exsanguination. The lungs were lavaged three times with 1 ml of Hanks' balanced salt solution (137 mM NaCl, 5.4 mM KCl, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 4.2 mM NaHCO3, and 5.6 mM glucose). BAL fluid was pooled and cooled to 4°C immediately. Cell number was determined with a hemocytometer. Differential cell counts were performed on Diff-Quick-stained (Baxter Diagnostics, McGaw Park, IL) cytospin (Cytospin3, Shandon Scientific) slides of cells from 200 µl of BAL fluid. Three to four hundred cells per slide were counted.
Rats were anesthetized with 50 mg/kg of pentobarbital sodium and killed by exsanguination. The lungs were lavaged [6 × 5 ml of phosphate-buffered saline (PBS; 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4)]. The BAL fluid was pooled, and cell counts were conducted as mentioned above.
Mouse lung RNA extraction. Total RNA
was isolated according to the method described by Chomczynski and
Sacchi (8). Immediately after the mice were killed, the lungs were
removed, placed into liquid nitrogen, and stored at 70°C
until RNA extraction. The frozen lung was placed into 3 ml of
solution D [4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7), 0.5% Antifoam A, and 100 mM
-mercaptoethanol] and homogenized. Sequentially, 0.1 volume of
2 M sodium acetate (pH 4.0), 1 volume of water-saturated phenol, and
0.2 volume of a chloroform-isoamyl alcohol mixture (49:1) were added,
with thorough vortexing after each addition. The final suspension was
vortexed vigorously for 10 s and set in ice for 25 min. After
centrifugation at 14,000 g for 20 min
at 4°C, the aqueous phase was mixed with an equal volume of
isopropanol and placed at
20°C for 1 h to precipitate RNA.
Sedimentation at 14,000 g for 20 min
was again performed. The resulting RNA pellet was dissolved in 0.4 volume of solution D and precipitated
with an equal volume of isopropanol at
20°C for 1 h. After
centrifugation for 20 min at 4°C, the RNA pellet was washed with 2 volumes of 75% ethanol, air-dried, and dissolved in 0.2 volume of
H2O at 65°C for 5 min. The RNA
solution was stored at
70°C.
Rat lung and BAL cell RNA extraction.
To obtain sufficient numbers of BAL cells for study, rats were used.
Total cellular RNA was isolated by using RNeasy kits (QIAGEN,
Chatsworth, CA). After the lungs were lavaged 10 times with 5 ml of
Hanks' balanced salt solution, the lungs were removed, placed into
liquid nitrogen, and stored at 70°C until RNA extraction.
The frozen lung was placed into 25 ml of solution
D and homogenized. The total RNA in the homogenate was
extracted according to the manufacturer's protocol. The BAL fluid was
centrifuged at 400 g for 10 min, and the cell pellet obtained was lysed with 650 µl of lysis buffer RLT
(QIAGEN). The cell lysate was homogenized by centrifugation through
QIAshredder (QIAGEN), and the total RNA in the homogenate was extracted
according to the manufacturer's protocol.
Reverse transcription-polymerase chain reaction. Mouse MCP-1 mRNA was amplified by short sequence-specific reverse transcription (RT)-polymerase chain reaction (PCR) (26). Two hundred fifty nanograms of total lung RNA were reverse transcribed by incubation with 25 U of Superscript (GIBCO BRL, Grand Island, NY) in first-strand buffer (GIBCO BRL) containing 10 mM dithiothreitol (DTT), 1 µM short sequence-specific primer (GAG AGG GAA AAA TGG), 1 mM of each deoxynucleotide triphosphate, and 10 U of RNasin (Promega, Madison, WI) in a total volume of 10 µl for 10 min at 25°C (annealing) and 45 min at 42°C (extension) followed by 5 min at 94°C. To amplify the reverse-transcribed cDNA, 40 µl of the PCR mixture were added to give a final solution containing 1× Taq buffer (GIBCO BRL), 1.5 mM MgCl2, 0.2 µM of each sense (GCC CAG CAC CAG CAC CAG) and antisense (GGC ATC ACA GTC CGA GTC ACA C) primer, and 0.75 U of Taq polymerase. PCR was performed in a 96-well UNO-thermoblock (Biometra, Tampa, FL) for 20 s at 94°C, 30 s at 59.6°C, and 30 s plus 1 s per cycle at 72°C for 30 cycles.
Mouse MIP-2 and -actin mRNA were amplified by RT-PCR as previously
described (12). Two hundred fifty nanograms of total lung RNA were
reverse transcribed by using 100 U of murine Moloney leukemia virus
reverse transcriptase (GIBCO BRL) in the first-strand buffer containing
antisense primer (GGC ACA TCA GGT ACG ATC CAG for MIP-2 or CAG GAT GGC
GTG AGG GAG AGC for
-actin) for 10 min at 22°C, 60 min at
37°C, and 5 min at 94°C. The PCR solution and conditions were
the same as above except for the sense primers (ACC CTG CCA AGG GTT GAC
TTC for MIP-2 and AAG GTG TGA TGG TGG GAA TGG for
-actin) and
annealing conditions (1 min at 58°C for MIP-2 and 30 s at 58°C
for
-actin). A total of 30 cycles were used for MIP-2 and 18 cycles
for
-actin.
Rat MCP-1 and -actin were amplified by RT-PCR as described for mouse
MCP-1 and
-actin except for the primers, annealing condition, and
cycle number. For rat MCP-1, sense (CTG CTG CTA CTC ATT CAC TGG) and
antisense (TCT GTC ATA CTG GTC ACT TCT ACA) primers were used.
Annealing was for 30 s at 54°C, and PCR was run for a total of 26 cycles. For rat
-actin, mouse
-actin primers were used. The
annealing condition was the same as for the mouse, but the cycle number
was 17 instead of 18.
Quantitative measurement of PCR
product. The PCR products were quantitated by
densitometry measurements. PCR products were separated by
electrophoresis on a 2% agarose gel containing 0.5 µg/ml of ethidium
bromide and 1× 90 mM tris(hydroxymethyl)aminomethane phosphate-2
mM EDTA. PCR products were visualized on a transilluminator (model
FBTIV-816, Fisher Scientific) at a 312-nm wavelength and photographed
with Polaroid 667 film. The band images were obtained by scanning the
Polaroid with a ScanJet 3P (Hewlett-Packard). The total intensity
(average intensity × total pixels) of each band was measured with
Mocha software (Jandel Scientific Software, San Rafael, CA). In each
RT-PCR, serial dilutions of a reference RNA were included. The relative
level of mRNA for each sample was determined from the linear portion of
the reference curve. All MIP-2 and MCP-1 mRNA data were normalized to
-actin mRNA.
Chemotaxis assay. To determine whether
increases in MCP-1 mRNA are associated with chemotactic activity in rat
BAL fluid, direct monocyte migration was assayed. After ozone exposure,
the rats were killed, and the lungs were lavaged (4 × 5 ml of
PBS, 37°C). Samples were pooled, centrifuged (400 g for 15 min) to remove the cells, and
stored at 20°C.
It has previously been reported that rat MCP-1 is chemotactic for human monocytes (36); therefore, human monocytes were used in the chemotaxis assays. Human blood mononuclear cells were isolated from the peripheral blood of healthy volunteers by utilizing Ficoll-Hypaque (Pharmacia) density gradient centrifugation. The cells were washed with PBS and resuspended (2.5 × 106 cells/ml) in RPMI 1640 without L-glutamine-25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-1% bovine serum albumin (BSA) medium.
The chemotaxis assays were conducted in 96-well microchemotaxis chambers (Neuro Probe, Cabin John, MD). Human mononuclear cells were added into the top well of the chamber and allowed to migrate through the membrane (5-µm pore size, polyvinylpyrrolidone-free polycarbonate membrane; Neuro Probe) toward the bottom wells containing the BAL fluid. Preliminary tests indicated that the highest chemotactic activity in the BAL fluid occurred at a 1:4 dilution; therefore, 1:4 dilutions of the rat BAL samples were prepared with RPMI 1640-HEPES-BSA before the samples were added into the well. To determine the specific MCP-1 activity in the BAL fluid, the diluted BAL fluid was incubated with 100 µg/ml of anti-murine JE (MCP-1) neutralizing antibody (R&D Systems) or nonimmune goat IgG for 30 min at room temperature before it was added to the bottom well. After the mixture was added, the assembly was incubated (90 min at 37°C, pH 7.4). After incubation, the membrane was removed, nonmigrating cells were wiped off, and the membrane was fixed and stained with LeukoStat (Fisher Scientific).
To assay activity for each lavage sample, BAL fluid was placed in three wells. In each well, the number of migrating monocytes (on the bottom of the membrane) per high-power field (×1,000) was obtained by counting five fields/well. The results represent the mean for four rats/treatment (three wells/rat) and are expressed as a percentage of the positive control value (obtained with 10 nM N-formyl-methionyl-leucyl-phenylalanine).
Nuclear protein extraction from rat BAL
cells. Nuclear protein extractions were obtained from
rat BAL cells with a modified protocol as described by Dignam et al.
(10). Rat BAL cells were obtained as described in BAL
and cell count, and the cell pellet was
lysed by vortexing for 40 s in 1 ml of lysis buffer [10 mM HEPES,
1.5 mM MgCl2, 10 mM KCl, 10 µM
leupeptin, 0.1 nM pepstatin, 0.5 mM -mercaptoethanol, 1% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM DTT].
The lysis solution was then centrifuged at 15,000 g for 2.5 min. The resulting nuclear
pellet was washed once with 200 µl of low-salt buffer (20 mM HEPES,
25% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 10 µM leupeptin, 0.1 nM pepstatin, 0.5 mM
-mercaptoethanol, 10 mM KCl, 1 mM PMSF, and 0.5 mM DTT), resuspended
in 50 µl of high-salt buffer (low-salt buffer containing 0.42 M NaCl
instead of KCl), and set on ice for 40 min. After centrifugation at 14,500 g
for 50 min, the supernatant was collected and dialyzed against buffer
(20 mM HEPES, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, and
0.5 mM DTT) at 4°C overnight. The dialysate was centrifuged at
14,500 g for 34 min, and the supernatant was stored at
70°C.
Electrophoretic mobility shift assay for
NF-B. The NF-
B activity in nuclear
protein extract was characterized by an electrophoretic mobility shift
assay. The NF-
B consensus probe (TCA GAG GGG ACT TTC CGA GAG GTC GA)
was end labeled with 32P using
bacteriophage T4 polynucleotide kinase. Five micrograms of
nuclear protein were incubated with 50 pg of
32P-labeled probe in binding
buffer (20 mM HEPES, pH 7.8, 1 mM EDTA, 1 mM DTT, 100 mM KCl, 10%
glycerol, 2.5 µg of BSA, and 0.5 µg of
polydeoxyinosinic-deoxycytidylic acid) in a total volume of 12.5 µl
at room temperature for 20 min. To determine specific binding, samples
were preincubated with a 100-fold molar excess of either unlabeled
consensus probe or a mutant probe (TCA GAG GCG ACT TTC CGA GAG GTC GA)
at 4°C for 15 min before the labeled probe was added. The binding
products were then resolved on a 4% 29:1 polyacrylamide gel,
dried, and autoradiographed. The intensities of the bands were
quantitated by densitometry, and the difference between the NF-
B
binding band and the corresponding position in the consensus
competition reaction was used as a measurement of NF-
B binding
activity.
Data analysis. Data are presented as
means ± SE. The cell count and mRNA data
(exposed/control) are not normally distributed. Therefore, multiple means were compared with Kruskal-Wallis analysis of
variance on ranks and post hoc comparison with Dunnett's method. For
comparison of two group means (mRNA data), the Mann-Whitney rank sum
test was used. The chemotactic activity and NF-B band intensity data
are normally distributed. Therefore, parametric analysis of variance
was used, with post hoc comparison of means (Student-Newman-Keuls
method) and comparison between sample and control groups (Dunnett's
method). Values with P
0.05 were considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mouse lung inflammatory cell infiltration and cytokine
mRNA levels after ozone exposure. Similar to what has
been reported previously (2, 12), ozone exposure increased the number
of inflammatory cells recovered in BAL fluid. The number of neutrophils in mouse BAL fluid increased and peaked at 24 h post-ozone exposure (Fig.
1A).
At 48 and 72 h postexposure, BAL neutrophils decreased; however, each
value remained higher than that of the control mice. In contrast, the
number of macrophages recovered from BAL fluid (Fig.
1B) decreased at 4 and 24 h
postexposure, recovered to the level similar to the control at 48 h
postexposure, and increased over control at 72 h. Thus increased lung
macrophage infiltration occurs at 72 h postexposure, whereas peak
neutrophil infiltration occurs at 24 h.
|
Ozone increased mouse lung MIP-2 and MCP-1 mRNA levels in a time-dependent manner. The level of MIP-2 mRNA increased rapidly after ozone exposure, becoming maximal at 4 h postexposure and decreasing thereafter (Fig. 1C). In contrast, the level of MCP-1 mRNA peaked 24 h after ozone exposure (5.2-fold of control value) and then decreased to 2.2-fold of control value at 72 h postexposure (Fig. 1D).
Concentration response for ozone-induced murine MCP-1 mRNA expression. Ozone increased mouse lung MCP-1 mRNA in a concentration-dependent manner. The level of MCP-1 mRNA increased 24 h after a 0.6 ppm ozone exposure and reached maximum at 2 ppm ozone (Fig. 2). The threshold ozone concentration to increase MCP-1 mRNA was between 0.3 and 0.6 ppm.
|
Ozone-induced monocyte infiltration and MCP-1 mRNA levels in rat lung. An increase in macrophages in rat BAL fluid was also observed after ozone exposure. Similar to the time course in the mouse study, the increased number of macrophages in the BAL fluid occurred at 72 h postexposure (Fig. 3A). The magnitude of the increases was similar between the two species (1.5-fold of the control value in rat lungs vs. 1.4-fold in mouse lungs; Figs. 1B and 3A).
|
Increased rat MCP-1 mRNA levels were observed in both lung and BAL cells 24 h after exposure to 2 ppm ozone for 3 h (Fig. 3B). The increase in MCP-1 transcripts in rat lung tissue was 4.3-fold and similar to that in mouse lung (3.2- to 5.2-fold; Figs. 1D and 2). The increase in MCP-1 mRNA in BAL cells was higher than that in whole lung tissue (30.9- vs. 4.3-fold of the control value; P < 0.05).
Monocyte chemotactic activity in BAL fluid after ozone exposure. Ozone increased MCP-1 chemotactic activity in rat lavage fluid. Twenty-four hours after ozone exposure, an increased monocyte chemotactic activity was observed in rat lavage fluid (2.8-fold increase vs. the control value; Fig. 4). This activity was inhibited (87%) by MCP-1-neutralizing antibody (Fig. 4). In contrast, the control antibody had no significant effect.
|
Ozone-induced NF-B activity in
rats. NF-
B activity was increased by ozone (Fig.
5). This response was specific (Fig.
5A) in that the band ascribed to
NF-
B (lane 2) could be diminished by a consensus competitor (lane 3)
but not by a mutant competitor (lane
4). NF-
B activity increased 20-24 h after
exposure (Fig. 4B), with a time
course similar to that of MCP-1 mRNA (24 h) (Fig. 1D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Inflammatory cell accumulation has been observed in many animal species after ozone exposure. Acute high concentrations (1.8 ppm for 3 h) of ozone exposure in rats led to early lung neutrophil accumulation that could be observed morphologically immediately after exposure and by lavage 1 day after exposure (2). A similar exposure also caused neutrophil infiltration into the mouse lung beginning 2 h postexposure and with a maximal response at 6-24 h (12, 17). In rats exposed to ozone, the BAL monocytes were increased at 3 days and subsided by 5 days after exposure (2). In our study, the number of neutrophils in BAL fluid from C57BL/6 mice peaked at 24 h post-ozone exposure and decreased at 48 and 72 h postexposure. This time course was consistent with previous reports, indicating that neutrophil infiltration is an early event. In contrast, the macrophages in mouse BAL fluid decreased at 4 and 24 h postexposure, returned to a normal level at 48 h, and increased at 72 h postexposure. A similar time course of macrophage changes in the rat lung was also observed. This early decrease and later increase in macrophage numbers were also observed by other investigators (2, 13, 28). Bassett et al. (2) reported that the lactate dehydrogenase in BAL fluid was increased after ozone exposure and speculated that ozone-induced cell damage and membrane lysis might account for the decrease in the number of macrophages recovered in BAL fluid. In addition, high levels of ozone may damage pulmonary morphology (7, 28), and alterations of the lung morphology may reduce the yield of free cells by lavage. Nevertheless, 72 h after ozone, the BAL macrophages were significantly increased, and again, compared with neutrophils, the ozone-induced monocyte infiltration is a late event.
Studies on MIP-2 and CINC have indicated that they may be responsible for ozone-induced neutrophil infiltration (12, 15). The MIP-2 time-course data obtained in this study agrees with a previous study by Driscoll et al. (12) and indicates that MIP-2 mRNA increased rapidly and peaked at 4 h after ozone exposure. This peak in MIP-2 is also similar to that of CINC that occurred 2 h post-ozone exposure (15). The increases in the MIP-2 message preceding the neutrophil infiltration are consistent with a role for MIP-2 in neutrophil infiltration after ozone exposure.
Although ozone-induced monocyte infiltration has been reported, the
responsible chemoattractants have not been identified. MCP-1 is a
potent monocyte chemoattractant. In vitro, MCP-1 attracts monocytes but
not neutrophils, with an optimal concentration of 109 M (20). In
addition to monocyte chemotaxis, MCP-1 augments monocyte cytostatic
activity against several tumor cell lines, stimulates monocyte
respiratory burst, and induces lysosomal enzyme release. MCP-1 can be
secreted by alveolar macrophages (4) and airway and alveolar epithelial
cells (30). Our study demonstrates that ozone increases lung MCP-1 gene
expression. The peak increase occurred at 24 h postexposure, preceding
the increased monocyte accumulation observed 72 h after exposure. The
sequential occurrence of increased MCP-1 expression, followed by
increased monocyte infiltration, suggested that MCP-1 might be
responsible for ozone-induced monocyte infiltration. Although an
increased pulmonary monocyte infiltration after ozone exposure has been
reported in both C57BL/6 mice (18) and Wistar rats (2), a direct
comparison has not been reported. Our results show that in both rats
and mice ozone induces similar monocyte infiltration and MCP-1 mRNA
levels in the lungs, indicating that Wistar rats and C57BL/6 mice have
a comparable ozone response.
Inasmuch as BAL cells are directly exposed to ozone when in the lung,
we compared MCP-1 mRNA levels in rat BAL cells with those in whole lung
tissue. Our results indicate that BAL cells had a higher MCP-1 mRNA
induction relative to whole lung tissue, suggesting that cells
recovered by BAL play an important role in ozone-induced monocyte
infiltration. After ozone exposure, the BAL cell population was
composed mainly of macrophages and neutrophils. MCP-1 gene expression
can be induced in either cell (3-5). In addition, these cells also
can be a source of IL-1 and TNF- (22), cytokines known to stimulate
MCP-1 transcription (3, 30). The increased level of MCP-1 mRNA after
ozone exposure could reflect the accumulation of neutrophils;
alternatively, IL-1 or TNF-
from neutrophils or macrophages could
activate MCP-1 gene expression in macrophages.
Although increased MCP-1 mRNA levels were observed in both lung tissue
and BAL cells, it is still not clear whether MCP-1 protein increases
after ozone exposure. In preliminary tests, we attempted to measure BAL
fluid MCP-1 levels by direct enzyme-linked immunosorbent assay (data
not shown). However, all samples were below the limit of detection
(109 M). Nevertheless, in a functional assay, ozone
increased lavage fluid monocyte chemotactic activity that could be
attributed primarily to MCP-1 because it was inhibited 87% by an MCP-1
antibody. The limit of detection of this assay (10
10 M)
was lower than that of the enzyme-linked immunosorbent assay. Thus
ozone not only induces MCP-1 mRNA but also increases MCP-1 activity in
the lung. The concurrence of increased MCP-1 activity in BAL fluid and
increased MCP-1 mRNA in BAL cells supports the important role that BAL
cells play in ozone-induced monocyte infiltration and also suggests
that the increased MCP-1 is regulated at the message level.
The mechanism by which ozone induces MCP-1 gene expression is not
clear. In human cell lines, NF-B contributes to IL-1-, TNF-
-, and
TPA-induced MCP-1 gene expression (35). An NF-
B binding site is
present in the 5' flanking region of the mouse MCP-1 gene.
NF-
B is a redox-sensitive transcription factor and is believed to be
regulated through intracellular oxidative stress (32, 34). Ozone is a
potent oxidant and can increase intracellular oxidative stress (24,
31), which, in turn, may activate NF-
B (32). Ozone exposure also can
increase IL-1 and TNF-
release from rat alveolar macrophages (1),
and IL-1 and TNF-
can increase NF-
B activity (32). Therefore, it
is possible that ozone stimulates MCP-1 gene expression through
multiple mechanisms that increase NF-
B activity.
Because the BAL cells exhibited a 30-fold increase in the MCP-1
transcript, we asked whether ozone induces NF-B activity in these
cells. Previously, Haddad et al. (15) reported that ozone exposure (3 ppm × 6 h) led to a peak in NF-
B activity in rat whole lung at
2 h postexposure, which returned to the control level at 24 h. In
contrast, here we find NF-
B activation persisted for 20-24 h
postexposure in rat BAL cells. This time course was similar to that for
the increase in MCP-1 mRNA in the mouse lung, which increased maximally
at 24 h. This relationship suggests a possible role of NF-
B
activation in ozone-induced MCP-1 gene expression.
The reason for the delay in ozone-induced NF-B activation in BAL
cells is unclear. The time course of the BAL cell NF-
B activity
matched that of neutrophil accumulation; however, NF-
B activation
has yet to be reported in neutrophils. Thus the role of neutrophils in
ozone-induced NF-
B is speculative. In addition, reactive oxygen
species released from activated neutrophils are known to activate
NF-
B and could mediate NF-
B activity in macrophages. Regardless
of marked neutrophilic infiltration, the macrophages remain the most
common cell type in BAL cells. The ability of macrophages to release
IL-1 and TNF-
after ozone exposure and to have increased NF-
B
after IL-1 or TNF-
treatment implicates macrophages as the probable
source for ozone-induced NF-
B activity in BAL cells. Increased IL-1
and TNF-
secretions from alveolar macrophages have been noted
18-20 h after ozone exposure (1), which is consistent with the
time course of ozone-induced NF-
B activity in BAL cells observed in
our study. Therefore, the delay in the increase of NF-
B activation
may be a consequence of secondary IL-1 or TNF-
secretion.
The threshold concentration of ozone necessary to produce MCP-1 expression in rodents was examined in our study. Our concentration-response data on MCP-1 mRNA indicate a threshold between 0.3 and 0.6 ppm in mice. Inasmuch as rodents are less sensitive to ozone than humans by factor of four to five (16), 0.6 ppm may reflect a human response at 0.12-0.15 ppm, a concentration comparable to the National Ambient Air Quality Standard (NAAQS). In addition, ozone levels of 0.12 ppm are known to cause neutrophil inflammation in the human lung (9), and the lungs of persons living in areas of high ozone and oxidant exposure are often marked by monocytic bronchiolitis (21).
In summary, our data indicate that ozone can induce MCP-1 gene
expression in mouse and rat lungs and the time course of the MCP-1
message level is consistent with the ozone-induced monocyte infiltration. MCP-1 is the major monocyte chemoattractant in lavage fluid after ozone exposure, and an important source for this
chemoattractant is BAL cells. The NF-B activity in BAL cells peaks
at the same time as MCP-1 gene expression after ozone, implicating a
possible role in ozone-induced MCP-1 gene expression.
Concentration-response data suggest that ozone-induced increases in
MCP-1 mRNA can occur at or above the current NAAQS level
among people exposed.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Dr. Alvaro Puga and Brian Howard for helpful advice and technical assistance.
![]() |
FOOTNOTES |
---|
This study was supported by National Institute of Environmental Health Sciences Grants R01-ES-06562, R01-ES-06677, and P30-ES-06096 and National Heart, Lung, and Blood Institute Grant R01-HL-58275.
Q. Zhao is a recipient of a University of Cincinnati Graduate Assistantship, and this work was conducted in partial fulfillment of the requirements for the PhD degree at the University of Cincinnati.
Address for reprint requests: G. D. Leikauf, Dept. of Environmental Health, Univ. of Cincinnati, PO Box 670056, Cincinnati, OH 45267-0056.
Received 15 October 1996; accepted in final form 11 September 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arsalane, K.,
P. Gosset,
D. Vanhee,
C. Voisin,
Q. Hamid,
A. Tonnel,
and
B. Wallaert.
Ozone stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro.
Am. J. Respir. Cell Mol. Biol.
13:
60-68,
1995[Abstract].
2.
Bassett, D. J. P.,
E. Bowwen-Kelly,
E. L. Brewster,
C. L. Elbon,
S. S. Reichebaugh,
T. Bunton,
and
J. S. Kerr.
A reversible model of acute lung injury based on ozone exposure.
Lung
166:
355-369,
1988[Medline].
3.
Brieland, J. K.,
C. M. Flory,
M. L. Jones,
G. R. Miller,
D. G. Remick,
J. S. Warren,
and
J. C. Fantone.
Regulation of monocyte chemoattractant protein-1 gene expression and secretion in rat pulmonary alveolar macrophages by lipopolysaccharide, tumor necrosis factor-, and interleukin-1
.
Am. J. Respir. Cell Mol. Biol.
12:
104-109,
1995[Abstract].
4.
Brieland, J. K.,
M. L. Jones,
S. J. Clarke,
J. B. Baker,
J. S. Warren,
and
J. C. Fantone.
Effect of acute inflammatory lung injury on the expression of monocyte chemoattractant protein-1 (MCP-1) in rat pulmonary alveolar macrophages.
Am. J. Respir. Cell Mol. Biol.
7:
134-139,
1992[Medline].
5.
Burn, T. C.,
M. S. Petrovick,
S. Hohaus,
B. J. Rollins,
and
D. G. Tenen.
Monocyte chemoattractant protein-1 gene is expressed in activated neutrophils and retinoic acid-induced human myeloid cell lines.
Blood
84:
2776-2783,
1994
6.
Car, B. D.,
F. Meloni,
M. Luisetti,
G. Semenzato,
G. Gialdroni-Grassi,
and
A. Walz.
Elevated IL-8 and MCP-1 in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis.
Am. J. Respir. Crit. Care Med.
149:
655-659,
1994[Abstract].
7.
Castleman, W. L.,
D. L. Dungworth,
L. W. Schwartz,
and
W. S. Tyler.
Acute respiratory bronchiolitis: an ultrastructural and autoradiographic study of epithelial cell injury and renewal in rhesus monkeys exposed to ozone.
Am. J. Pathol.
98:
811-840,
1980[Abstract].
8.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
9.
Devlin, R. B.,
W. F. McDonnell,
R. Mann,
S. Becher,
D. E. House,
D. Schreinemachers,
and
H. S. Koren.
Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular biochemical changes in the lung.
Am. J. Respir. Cell Mol. Biol.
4:
72-81,
1991[Medline].
10.
Dignam, J. D.,
R. M. Lebovitz,
and
R. G. Roeder.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:
1475-1489,
1983[Abstract].
11.
Driscoll, K. E.
Macrophage inflammatory proteins: biology and role in pulmonary inflammation.
Exp. Lung Res.
20:
473-490,
1994[Medline].
12.
Driscoll, K. E.,
L. Simpson,
J. Carter,
D. Hassenbein,
and
G. D. Leikauf.
Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2.
Toxicol. Appl. Pharmacol.
119:
306-309,
1993[Medline].
13.
Driscoll, K. E.,
T. A. Vollmuth,
and
R. B. Schlesinger.
Acute and subchronic ozone inhalation in the rabbit: response of alveolar macrophages.
J. Toxicol. Environ. Health
21:
27-43,
1987[Medline].
14.
Goodman, R. B.,
R. M. Strieter,
D. P. Martin,
K. P. Steinberg,
J. A. Milberg,
R. J. Maunder,
S. L. Kunkel,
A. Walz,
L. D. Hudson,
and
T. R. Martin.
Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome.
Am. J. Respir. Crit. Care Med.
154:
602-611,
1996[Abstract].
15.
Haddad, E. B.,
M. Salmon,
H. Koto,
P. J. Barnes,
I. Adcock,
and
K. F. Chung.
Ozone induction of cytokine-induced neutrophil chemoattractant (CINC) and nuclear factor-kb in rat lung: inhibition by corticosteroids.
FEBS Lett.
379:
265-268,
1996[Medline].
16.
Hatch, G. E.,
R. Slade,
L. P. Harris,
W. F. McDonnell,
R. B. Devlin,
H. S. Koren,
D. L. Costa,
and
J. McKee.
Ozone dose and effect in humans and rats: a comparison using oxygen-18 labeling and bronchoalveolar lavage.
Am. J. Respir. Crit. Care Med.
150:
676-683,
1994[Abstract].
17.
Kleeberger, S. R.,
D. J. P. Bassett,
G. J. Jakab,
and
R. C. Levitt.
A genetic model for evaluation of susceptibility to ozone-induced inflammation.
Am. J. Physiol.
258 (Lung Cell. Mol. Physiol. 2):
L313-L320,
1990
18.
Kleeberger, S. R.,
R. C. Levitt,
and
L. Y. Zhang.
Susceptibility to ozone-induced inflammation. II. Separate loci control responses to acute and subacute exposure.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L21-L26,
1993
19.
Leikauf, G. D.,
Q. Zhao,
S. Zhou,
and
J. Santrock.
Ozonolysis products of membrane fatty acids activate eicosanoid metabolism in human airway epithelial cells.
Am. J. Respir. Cell Mol. Biol.
9:
594-602,
1993[Medline].
20.
Leonard, E. J.,
and
T. Yoshimura.
Human monocyte chemoattractant protein-1 (MCP-1).
Immunol. Today
1:
97-101,
1990.
21.
Lippmann, M.
Ozone in Environmental Toxidants: Human Exposures and Their Health Effects. New York: Van Nostrand Reinhold, 1992, p. 465-519.
22.
Lloyd, A. R.,
and
J. J. Oppenheim.
Poly's lament: the neglected role of the polymorphonuclear neutrophil in the afferent limb of the immune response.
Immunol. Today
13:
169-172,
1992[Medline].
23.
Miller, M. D.,
and
M. S. Krangel.
Biology and biochemistry of the chemokines: a family of chemotactic and inflammatory cytokines.
Crit. Rev. Immunol.
12:
17-46,
1992[Medline].
24.
Morgan, D. L.,
and
D. G. Wenzel.
Free radical species mediating the toxicity of ozone for cultured rat lung fibroblast.
Toxicology
36:
243-251,
1985[Medline].
25.
Mudd, J. B.,
R. Leavitt,
A. Ongun,
and
T. T. McManus.
Reaction of ozone with amino acids and proteins.
Atmos. Environ.
3:
669-682,
1969[Medline].
26.
Pfeffer, U.,
E. Fecarotta,
and
G. Vidali.
Efficient one-tube RT-PCR amplification of rare transcripts using short sequence-specific reverse transcription primers.
Biotechniques
18:
204-206,
1995[Medline].
27.
Picard, D.,
and
W. Schaffner.
A lymphocyte-specific enhancer in the mouse immunoglobulin gene.
Nature
307:
80-82,
1984[Medline].
28.
Pino, M. V.,
J. R. Levin,
M. Y. Stovall,
and
D. M. Hyde.
Pulmonary inflammation and epithelial injury in response to acute ozone exposure in the rat.
Toxicol. Appl. Pharmacol.
112:
64-72,
1992[Medline].
29.
Pryor, W. A.,
B. Das,
and
D. F. Church.
The ozonation of unsaturated fatty acids: aldehydes and hydrogen peroxide as products and possible mediators of ozone toxicity.
Chem. Res. Toxicol.
4:
341-348,
1991[Medline].
30.
Rollins, B. J.
JE/MCP-1: an early-response gene encodes a monocyte-specific cytokine.
Cancer Cells
3:
517-524,
1991[Medline].
31.
Santrock, J.,
R. A. Gorski,
and
J. F. O'Gara.
Products and mechanisms of the reaction of ozone with phospholipids in unilamellar phospholipid vesicles.
Chem. Res. Toxicol.
5:
134-141,
1992[Medline].
32.
Schreck, R.,
P. Rieber,
and
P. A. Baeuerle.
Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1.
EMBO J.
10:
2247-2258,
1991[Abstract].
33.
Seltzer, J.,
B. G. Bigby,
M. Stulbarg,
M. J. Holtzman,
J. A. Nadel,
I. F. Ueki,
G. D. Leikauf,
E. J. Goetzl,
and
H. A. Boushey.
Ozone-induced change in bronchial reactivity to methacholine and airway inflammation in human.
J. Appl. Physiol.
60:
1321-1326,
1986
34.
Staal, F. J. T.,
M. Reeder,
and
L. A. Herzenberg.
Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus.
Proc. Natl. Acad. Sci. USA
87:
9943-9947,
1990[Abstract].
35.
Ueda, A.,
K. Okud,
S. Ohno,
A. Shirai,
T. Igarashi,
K. Matsunaga,
J. Fukushima,
S. Kawamoto,
Y. Ishigatsubo,
and
T. Okubo.
NF-B and Sp-1 regulate transcription of the human monocyte chemoattractant protein-1 gene.
J. Immunol.
153:
2052-2063,
1994
36.
Yoshimura, T.,
M. Takeya,
and
K. Takahashi.
Molecular cloning of rat monocyte chemoattractant protein-1 (MCP-1) and its expression in rat spleen cells and tumor lines.
Biochem. Biophys. Res. Commun.
174:
504-509,
1991[Medline].