IL-8 is one of the major chemokines produced by monkey airway
epithelium after ozone-induced injury
Mary Mann-Jong
Chang1,2,
Reen
Wu1,2,3,
Charles G.
Plopper1,2, and
Dallas M.
Hyde1,2
1 Center for Comparative
Respiratory Biology and Medicine,
2 Department of Anatomy,
Physiology and Cell Biology, School of Veterinary Medicine, and
3 Pulmonary and Critical Care
Medicine, School of Medicine, University of California, Davis,
California 95616
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ABSTRACT |
A rhesus monkey interleukin (IL)-8 cDNA clone
with >94% homology to the human IL-8 gene was isolated by
differential hybridization from a cDNA library of distal airways after
ozone inhalation. In situ hybridization and immunohistochemistry showed
increased IL-8 mRNA and protein levels in epithelial cells at 1 h but
not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine
in acute ozone-induced airway inflammation in primates.
chemotaxis; neutrophil; differential hybridization; in situ
hybridization; immunohistochemistry; interleukin-8
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INTRODUCTION |
OZONE IS A KEY air pollutant contaminating the
atmosphere of most industrialized cities and is known to cause
short-term pulmonary function impairment and chronic lung disease in
humans (18). The respiratory system is the primary target of ozone
toxicity (30). Acute exposure of humans or animals to ambient levels of
ozone results in reversible changes in pulmonary function and pulmonary
inflammation characterized by increased neutrophil influx (16).
Although data have shown that ozone inhalation induces the production
of different chemoattractants such as leukotreine B4, macrophage inflammatory
protein-2, and interleukin (IL)-8 in various cases (13,
21), there is no direct in vivo evidence linking neutrophil influx and
those chemoattractants. Therefore, the mechanism of neutrophil influx
resulting from ozone inhalation is still not entirely clear.
To investigate the relationship between granulocyte migration and
epithelial injury, we exposed rhesus monkeys to 0.96 part/million (ppm)
ozone for 8 h and observed that epithelial necrosis and repair were
associated with the presence of granulocytes in the epithelium and
interstitium of the tracheobronchial airways during the week-long
postexposure period (16). To understand the early cellular and
molecular events that are important in the recruitment of neutrophils
into the tracheobronchial airways, we hypothesized that one of the key
target zones for ozone-induced injury, the respiratory bronchioles,
upregulates gene expression and production of chemokines. To
investigate the nature of the chemokine response to ozone, we used the
microdissecting technique (23) to isolate different airway regions to
establish a cDNA library for screening differentially expressed genes.
We used the same ozone-exposed monkey lungs in which we have documented
injured epithelial cells and increased neutrophil influx into
respiratory bronchioles (16). This approach has allowed the isolation
and characterization of several monkey ozone-responsive (MOR) genes
(19). One of these differentially hybridized MOR cDNA clones, MOR6, had
a DNA sequence with 94% homology to the human IL-8 gene. We used this
clone in the present paper along with a human IL-8 antibody to
investigate the presence of IL-8 mRNA and protein in respiratory
bronchioles of monkeys after exposure to ozone. To evaluate the
relative importance of IL-8 as a chemokine for neutrophil migration in
human and monkey primary tracheobronchial epithelium after ozone
exposure, we exposed primary human and monkey and human transformed
(BEAS-2B) tracheobronchial epithelial cells to ozone and established
that IL-8 is a key chemokine in neutrophil migration in response to
ozone-induced epithelial cell injury.
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MATERIALS AND METHODS |
In vivo ozone exposure of monkeys.
Rhesus monkeys were exposed individually in exposure chambers of 4.2-m
capacity that were ventilated at a rate of 30 changes per hour with
chemical-biological and radiological-filtered air (FA) at 24 ± 2°C and 40-50% relative humidity. Ozone concentrations were
measured using an ultraviolet ozone monitor and were reported with
respect to the ultraviolet photometric standard. For additional details
of the exposure and monitoring, please refer to the previous paper
(16).
Isolation and characterization of monkey IL-8 cDNA
clone. The airway microdissection technique described
by Plopper et al. (23) was adapted in this study to isolate the distal
airway region of monkey lungs after ozone or FA exposure.
Poly(A)+ mRNAs were isolated from
these tissues and used to establish cDNA libraries using the Lambda
GEM-2 system (Promega, Madison, WI). The sizes of the cDNA
libraries were 5 and 2 × 105
phages/library for FA and ozone-exposed tissues, respectively. With the
use of differential hybridization (19), MOR genes were isolated. Among
these MOR clones, MOR6 was sequenced using an automatic DNA sequencer
(ABI sequencer), and we found a 94% homology to the human IL-8 gene in
the 491-bp inserts of MOR6 (Fig. 1).

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Fig. 1.
Monkey ozone-responsive (MOR) 6 clone sequence. Gene sequence of monkey
MOR6 clone (bottom) compared with
human interleukin (IL)-8 gene (top).
Common sequences appear in boxes. Note the high degree of homology
between the monkey and human genes (GenBank search).
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In situ hybridization. By subcloning
the MOR6 into a pGEM4Z plasmid (Promega), sense and anti-sense IL-8
cRNAs were generated from MOR6 and labeled with
35S using the Maxiscript in vitro
transcript kit (Ambion, Austin, TX). Paraffin sections from lung tissue
of ozone- and FA-treated monkeys were used for hybridization, with
conditions and washing following the procedure described by Angerer et
al. (2). Autoradiography was carried out over a 7-day interval, and the
developed slides were counterstained with methylene green.
Immunohistochemical staining with anti-human IL-8
antibody. Tissue sections were treated with
methanol-acetone and then stained immunologically with a mouse
anti-human IL-8 antibody (R&D Systems, Minneapolis, MI) using the
Vectastain ABC Kit (Vector Laboratory, Burlingame, CA), an
avidin-biotin peroxidase method to detect the reaction product.
Controls used no primary antibody.
Cell culture. Tracheobronchial
epithelial (TBE) cells were isolated from monkey or human as primary
culture; cell isolation and medium conditions were carried out as
previously described (31). Human tissues were obtained from organ donor
patients or from autopsy through the University of California, Davis,
Medical Center. Rhesus monkey tracheobronchial airways were obtained
from the California Regional Primate Research Center, University of California, Davis. Two human bronchial epithelial cell lines were used
in this study. BEAS-2B S-6 (S; serum-sensitive) clonal cell line, an
immortalized cell line of normal human bronchial epithelium derived by
transfection of primary cells with SV40 early-region genes, was
obtained by courtesy of Dr. J. Lechner (Lovelace Inhalation Toxicology
Research Institute, Albuquerque, NM). The HBE1 cell line, a
papillomavirus immortalized human bronchial epithelial cell line, was
generously provided by Dr. J. Yankaskas (University of North Carolina,
Chapel Hill, NC). Cells were plated onto the membrane inserts (pore
size 0.4 µm) of Costar six-well clustered Transwell chambers (Corning
Costar, Cambridge, MA) at 2 × 105 cells/well and maintained in
serum-free media with 1 ml on top of the cells (inside the insert,
apical phase) and 2 ml medium beneath the cells (in the dish,
basolateral phase). In the case of primary cultures, collagen was
coated on top of the membrane inserts before the cells were plated,
whereas in the case of BEAS-2B S cells and HBE1 cells, no collagen was
added to the membrane inserts. After 7-10 days, when cells had
reached confluency and maintained confluency for a couple of days, they
were used for FA or ozone exposure. Immediately before exposure, the
serum-free media were removed from both sides of the membrane inserts
and washed two to three times with Hanks' balanced salt solution
(HBSS), and then 1 ml of HBSS was added to the basolateral compartment so the cell could have direct contact with FA or ozone.
In vitro ozone exposure. The in vitro
system for exposing airway epithelial cells to ozone has been
previously described (28). To allow gases to interact with the cells,
no medium was added to the apical compartment during exposure, and only
1 ml of HBSS was added to the basolateral phase.
Conditioned media. After ozone
exposure, HBSS was removed, samples were further washed two times with
HBSS, 1 ml of serum-free media was added to both the apical and
basolateral compartments, cells were incubated for 2 or 4 h in a
37°C incubator, and then the media were collected as conditioned
media. If the assays were not performed immediately, the supernatant
was saved at
70°C until the assay was run, and the
supernatant was never thawed more than one time. For the time-course
study, at 0 or 16 h after ozone or FA exposure, cells were washed two
times with HBSS and replaced with fresh media; conditioned media were
collected after 4 h of incubation (4 and 20 h after ozone exposure).
Cell counting. Cells were trypsinized
and stained with trypan blue, and then the viable, nonstained cells
were counted using a microscope and a hemocytometer.
Quantitation of IL-8 by ELISA. The
level of IL-8 in conditioned media was quantified by an
ELISA kit obtained from Biosource International (Camarillo, CA).
In vitro neutrophil chemotaxis assay.
Neutrophils were prepared from monkey or human blood according to the
method of Haslett et al. (14) and then were labeled with
51Cr. We used a 24-well Transwell
dish with inserts of 3-µm pore size membrane to evaluate chemotaxis
according to the method of Casale and Abbas (5). To the outside of the
filter insert, 450 µl of conditioned media (cell culture supernatant)
were added to fill the well, whereas to the inside of the filter
insert, 100 µl (1 × 106
cells) of freshly isolated and
51Cr-labeled neutrophils were
added. The incubation was at 37°C for 3 h. The number of
neutrophils that migrated to the bottom of the dish was measured by the
51Cr counts of the cell lysate.
The chemotactic activity (CA) was calculated as follows
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where
background is defined as neutrophil migration in the well with fresh
medium and total counts indicates the total count of the 200-µl
neutrophils loaded onto the insert.
Northern blot hybridization. Total RNA
was isolated by the single-step acid guanidium
thiocyanate-phenol-chloroform extraction method described previously
(8). Northern blot hybridization was done as previously described (15).
32P-labeled MOR6 and
PG11-480 were used as probes, where MOR6 is the clone described
above and PG11-480 is a clone using PGEM4Z (Promega) as the
vector, and ligated with an insert of 469 bp of the human IL-8 sequence
produced by the PCR method according to the published human IL-8 gene
sequence.
Anti-IL-8 neutralizing antibody
treatment. The conditioned media (collected 4 h after
ozone exposure) were preincubated with increasing concentrations of
1) a mouse monoclonal anti-human IL-8 IgG (R&D Systems) or 2) a mouse
anti-human mucin IgG (control antibody developed in our laboratory) for
1 h at 37°C. After this preincubation period, the pretreated (PCM)
and untreated (UCM) conditioned media were evaluated in a chemotaxis
assay. The inhibition of migration (IM) due to the pretreatment of
antibody was calculated as follows
Statistics. All quantitative
data were evaluated by group by ANOVA, and individual group comparisons
were evaluated using Fisher's least significant difference test
(SYSTAT 7.0, Chicago, IL). Differences were considered to be
significant at P
0.05. Some data
were evaluated using regression analysis.
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RESULTS |
Identification of a monkey IL-8 cDNA clone as one of
the MOR genes. Only the cDNA library derived from
ozone-exposed monkeys was used in this study to isolate the
ozone-inducible cDNA clones. From a screening of ~5 × 105 phages, 16 MOR cDNA clones
were isolated after several rounds of screening. Among these clones,
MOR4 and MOR6 were identified as ozone-induced genes. DNA sequence
information identified MOR4 as a monkey surfactant protein C cDNA
clone, whereas the MOR6 clone had a DNA sequence with 94% homology to
the human IL-8 gene (Fig. 1). In this study, we focused on the
expression of the IL-8 gene after ozone exposure.
In situ hybridization and immunohistochemical
identification of monkey IL-8. To further support the
differential hybridization result,
35S-cRNA probes in both sense and
antisense directions were generated from the MOR6 cDNA clone and used
for in situ hybridization with paraffin lung sections derived from
monkeys after the FA or ozone exposure. Similarly, a monoclonal
antibody specific to the human IL-8 protein (R&D Systems), which has
been shown to be monospecific to the monkey chemokine in a Western blot
(unpublished data), was used for immunohistochemical evaluation on
these monkey paraffin sections. Dark-field photomicrographs of in situ
hybridization evaluation showed an elevated IL-8 mRNA steady-state
level in monkey airway epithelium after acute exposure to ozone (Fig.
2B). In
contrast, hybridization with sections derived from FA-exposed monkeys
showed background staining (Fig. 2A)
similar to the control hybridization with sense probe (Fig.
2C). Similar to in situ
hybridization, immunohistochemical staining also showed enhanced IL-8
protein levels in monkey airway epithelium of sections after ozone
exposure (Fig. 3). This increase in IL-8
protein level was found immediately after exposure (Fig.
3B) but declined 24 h after exposure
to ozone (1 ppm for 8 h) (Fig. 3C).
Interestingly, ozone-induced IL-8 expression appears to be somewhat
cell-type and region specific. Immunostaining showed that ciliated
cells are a primary target of ozone-induced IL-8 gene expression.
Furthermore, IL-8 staining was patchy in the proximal airways, whereas
it was more diffuse in the distal airways (Fig.
4). Most of the alveolar macrophages in
distal airways stained positive for IL-8 in monkeys immediately after
ozone exposure, but they were few in number compared with the
positively stained epithelial cells. Few alveolar macrophages in more
distal alveolar regions stained positive for IL-8. This staining
pattern is consistent with elevated neutrophil influx in respiratory
bronchioles compared with more proximal bronchi or more distal alveoli
in these same monkeys immediately after ozone inhalation (16).

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Fig. 2.
Detection of IL-8 mRNA in monkey lung by in situ hybridization. Frozen
lung sections from filtered air (FA)- or ozone (OZ)-exposed
monkey were hybridized with
35S-labeled antisense cRNA of MOR6
clone; hybridization signals seen as white grains in the dark-field
micrograph are concentrated on airway epithelium
(B). No significant amount of signal
was seen when sense cRNA was used as probe
(C) or when antisense cRNA probe was
used to hybridize the FA-exposed monkey frozen tissue
(A). Dark-field photomicrograph.
Scale bars, 20 µm.
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Fig. 3.
Comparison of IL-8 protein production in monkey respiratory bronchioles
at different times after OZ or FA exposure as shown by
immunohistochemical staining. Almost no detectable staining was shown
on the tissue processed 1 h after FA exposure
(A). Significant amount of IL-8
protein is shown on the tissue processed 1 h after OZ exposure
(B), whereas, at 24 h after OZ
exposure, the IL-8 production is hardly detectable
(C). Bars, 20 µm.
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Fig. 4.
Comparison of proximal versus distal airway staining at 1 h after OZ
exposure. A: typical negative staining
for IL-8 in a proximal airway. B:
positive focal staining for IL-8 in a proximal airway.
C: typical positive staining for IL-8
in a respiratory bronchiole. D:
negative control staining for IL-8. Bars, 20 µm.
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In vitro studies of ozone-induced IL-8 gene expression
in airway epithelial cells. To carry out in vitro ozone
exposure, both established cell lines and primary cells were used. In
most cases, monkey primary TBE cells were used; occasionally, human TBE
cells were also used. For cell lines, we used BEAS-2B S cells and HBE1 cells. After cells reached confluency, these chambers were exposed to
different ozone concentrations from 0.1 to 1.0 ppm for 60 min or to a
constant ozone level (0.5 ppm) for various times. The control chambers
were exposed to FA for the same period of time. The time- course study
showed that ozone enhanced IL-8 secretion immediately after the
exposure and that the enhancement was transient (Fig.
5). Also, ozone at 0.5 ppm caused
substantial dose-dependent cell damage on the cultured BEAS-2B S (Fig.
6) and HBE1 (data not shown) cell lines.
Concurrent to cell injury, there was a substantial increase in IL-8 in
the media of the surviving cells over the 2 h after ozone exposure. By
contrast, ozone exposure did not cause substantial damage to the
primary culture of monkey TBE cells, even though the elevated IL-8
secretion persisted (Fig. 7). This result
was also observed in primary cultures of human TBE cells (data not
shown). To further elaborate the level of the change of IL-8 production
by ozone, Northern blot hybridization was carried out to determine
whether the change occurs at the mRNA level. As shown in Fig.
8, IL-8 message in both monkey and human
primary TBE cells was increased by ozone exposure. These results show
that ozone altered gene expression in the accumulation of IL-8 mRNA in
culture.

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Fig. 5.
Comparison of IL-8 protein produced by primary monkey tracheobronchial
epithelial cells at different times after FA or OZ exposure as measured
by ELISA. Conditioned media with 4-h incubation period were collected
at 4 or 20 h after cells were exposed to FA or various concentrations
of OZ. Conditioned media at 4 h (containing the IL-8 production from 0 to 4 h postexposure) showed increased secretion of IL-8 in response to
increased dosage of OZ. Conditioned media at 20 h (contained the IL-8
secretion between 16 and 20 h postexposure) showed no difference
between FA or OZ exposure. To avoid collecting any IL-8 beyond the
specific 4-h period, cells were washed 2 times with Hanks'
balanced salt solution (HBSS) and replaced with fresh media for 4-h
incubation and then the conditioned media were
collected. ppm, Part/million. Values are means ± SE
of n = 3 experiments per
group. * Significantly greater than
(P 0.05) 20-h value.
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Fig. 6.
Response of BEAS-2B cells to OZ exposure. BEAS-2B cells were exposed to
FA or 0.5 ppm OZ for 30, 60, or 90 min. After the exposure, one-half of
the cells were used for cell counting; the number of viable cells
decreased in response to increased OZ dosage. The other half of the
cells were washed 2 times with HBSS and replaced with fresh media.
After 2 h of incubation, conditioned media were collected for ELISA.
Data showed increased IL-8 production on a per-cell basis. Nos. on
y-axis, ng IL-8 and cell number (millions).
* Significantly decreased (P 0.05) compared with time 0 (FA).
+ Significantly increased
(P 0.05) compared with
time 0 (FA).
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Fig. 7.
Amount of IL-8 protein produced in conditioned media by primary monkey
tracheobronchial epithelial cells as measured by ELISA. Conditioned
media collected 4 h after exposure to FA or varying concentrations of
OZ (0.2, 0.5, and 1 ppm) were quantified by ELISA. Values are means ± SE of n = 3 experiments per
group. There was a significant (P = 0.0001) correlation between IL-8 protein and dose of OZ.
* Significantly greater than (P 0.05) all other concentrations.
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Fig. 8.
Analysis of IL-8 mRNA expression in FA- or OZ (1 ppm)-exposed monkey
and human primary tracheobronchial epithelial cells. Autoradiograph of
the Northern blot is as shown. After scanning by a densitometer, IL-8
mRNA value is standardized by 18S, and the value is indicated below the
autoradiograph.
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Effects of ozone on epithelial cell-induced neutrophil
chemotactic activity. Because epithelial cells are
probably responsible for the first phase of inflammatory cell migration
in vivo, we wondered whether in vitro ozone exposure also induces the
secretion of neutrophil chemotactic activity. As shown in Fig.
9, ozone exposure enhanced neutrophil
migration, and the activity appears to be dose dependent. To
investigate the potential contribution of IL-8 in neutrophil chemotaxis
in ozone-exposed culture, anti-IL-8 neutralizing antibody was used. As
shown in Fig. 10, most neutrophil chemotactic activity in the conditioned medium from FA- and
ozone-exposed cultures was inhibited by the IL-8 neutralizing antibody.
This result supports the notion that IL-8 secretion by airway
epithelium is a key chemokine in the initial phase of neutrophil
chemotaxis in ozone-induced epithelial injury.

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Fig. 9.
Chemotactic activity. Migration of monkey neutrophils through 3-µm
membranes to conditioned media of FA- or OZ-exposed monkey primary
tracheobronchial epithelial cells was measured by chemotaxis assay.
* Significantly greater than 0.2 ppm OZ
(P = 0.032) and markedly increased
compared with FA (P = 0.059).
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Fig. 10.
Neutralization of chemotactic activity by IL-8 neutralizing antibody.
Monkey neutrophil chemotaxis to conditioned media from monkey primary
tracheobronchial epithelial cells exposed to 0.5 ppm OZ for 1 h was
inhibited in a dose-dependent fashion (%inhibition) by varying
concentrations of IL-8 neutralizing antibody. There was a significant
(P = 0.0001) correlation between the
concentration of IL-8 neutralizing antibody and %inhibition of
chemotaxis. Mouse anti-human mucin IgG antibody was used as the
control. * Significantly greater inhibition than
(P 0.05) all other
concentrations.
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DISCUSSION |
From the same monkeys in which neutrophil influx was associated with
epithelial necrosis (16), we isolated an IL-8 cDNA clone from the cDNA
library distal airway through differential hybridization. This clone
was used for in situ hybridization, and an antibody to human IL-8 was
used for immunohistochemical staining of monkey airways.
Increased production of IL-8 in ozone-exposed monkey airways correlated
well with our previous data showing an influx of neutrophils (16). When
we used monkey/human primary cells or BEAS-2B S/HBE1 cells for in vitro
evaluation, IL-8 production was also induced by ozone in a time- and
dose-responsive manner. The transient nature of IL-8 induction was
shown by a decrease in IL-8 production at 16 h after ozone exposure.
Furthermore, the neutrophil chemotaxis assay of the cell culture
supernatant indicated a direct relationship between the amount of IL-8
and neutrophil migration. When an IL-8 neutralization antibody was used, >85% of the chemotactic activity was blocked. From these studies, we concluded that ozone exposure of airway epithelial cells
can induce the production of IL-8 in vivo and in vitro and that IL-8 is
a key chemokine in neutrophil influx after ozone-induced epithelial
injury.
Numerous in vivo experiments of ozone inhalation by humans have shown
increased IL-8 secretion into bronchoalveolar lavage fluid. Aris et al.
(3) monitored lavage fluid of healthy volunteers after ozone inhalation
and showed a correlation between airway injury and increased IL-8
production. Additionally, McBride et al. (20) exposed subjects with
asthma to an ambient concentration of ozone and found a strong
correlation between IL-8 and white blood cell counts in nasal fluid.
Together, their results suggest that IL-8 plays an important role in
airway inflammation, although the specific lung site of IL-8 production
cannot be deduced by bronchoalveolar lavage fluid analysis alone. Our
investigations provide direct evidence of IL-8 production by airway
epithelial cells in response to ozone inhalation in monkeys. First, the
IL-8 monkey gene was isolated through differential hybridization from microdissected distal airways. This implies that ozone-exposed tissue
is differentially regulated at the genetic level in reference to IL-8
gene expression. This concept is supported by the work of Jaspers et
al. (17), which shows ozone exposure-induced DNA-binding activity of
transcription factors at the prospective IL-8 gene regulation sites,
nuclear factor-
B and nuclear factor IL-6 binding sites. Also, the
use of distal airways facilitated the demonstration of this
differentially expressed IL-8 gene, since previous pathological investigations into the effect of ozone exposure in monkeys revealed that the bronchiolar region showed the most consistent epithelial injury and neutrophil influx (16). Second, in vivo in situ
hybridization and immunohistochemistry support this conclusion of
direct IL-8 production by airway epithelial cells. These data show that
induction of IL-8 gene expression after ozone exposure is greatest in
airway epithelium, especially the distal airways. Epithelial cells are the first line of defense and the most predominant cells in the airway
system, and in vitro studies have demonstrated that epithelial cells
can produce a variety of cytokines and adhesion molecules (10). Our
data suggest that epithelial cells not only serve as a line of defense
but also play an important role in airway inflammation. Our data show
that epithelial cells are a major source of IL-8 production in vivo,
and its expression in epithelial cells can be modulated by ozone
exposure.
In concert with our in vivo investigations, we also showed elevated
IL-8 secretion and mRNA levels in cultures of immortalized human
bronchial epithelial cell lines and primary monkey/human TBE cells by
ozone using an in vitro exposure system. Similar in vitro observations
have been demonstrated in other laboratories (9, 11). Devalia et al.
(9) used an explant cultured system for human bronchial epithelial
cells to show the enhancement of IL-8 of four to five times over
background over a period of 24 h. However, the ozone exposure system
was different, primarily because of the nature of explant culture;
also, their exposure was at 100 parts per billion for 6 h. Our system
is more comparable with that of Devlin et al. (11). However, our data
yield several new observations as follows:
1) IL-8 production is dose dependent with ozone concentration; 2)
although both cell lines and primary cell culture enhanced IL-8
production after ozone exposure, the toxicity of ozone on cell lines
and primary cells is quite different; and
3) the enhancement of IL-8 is a
transient phenomenon, with IL-8 production being increased immediately
after ozone exposure but decreasing at 20 h after ozone exposure.
Established cell lines, which were cultured on membranes without
collagen, showed a significant amount of cell death. In contrast,
primary cell culture of monkey or human cells cultured on
collagen-coated membrane showed little cell death after ozone exposure.
Immunohistochemical staining with anti-IL-8 antibody of ozone-exposed
monkey distal airway sections also suggested a transient production of
IL-8 in vivo. The nature of the transient production of IL-8 is
unclear.
Hyde et al. (16) previously demonstrated in monkey lung that
neutrophils were recruited to airway lumen, with a peak at 12 h after
an 8-h ozone exposure and then a gradual decline to the baseline within
72 h postexposure. These times correlated well with the transient
enhancement of IL-8 in vivo and in vitro. This observation suggests a
possible role for airway epithelial cell-secreted IL-8 in the initial
phase of neutrophilic recruitment in vivo. This notion is further
supported by the in vitro chemotaxis assay of cultured medium from
ozone-exposed cultures. We demonstrated that the neutrophilic
chemotactic activity in culture medium of airway epithelial cells was
elevated by ozone and that it could be blocked by a monoclonal
anti-IL-8 neutralizing antibody. This inhibitory effect by anti-IL-8
neutralizing antibody strongly suggests that IL-8 is a key cytokine
contributing to ozone-induced airway inflammation. Abdelaziz et al. (1)
used conditioned media of primary human bronchial epithelial cells and
showed that chemotactic activity for neutrophils could be blocked by
IL-8 neutralizing antibody up to 50%. However, in our hands, the
blocking activity can be as high as 85%. Further investigations with
neutralizing anti-IL-8 antibody treatment in vivo with animals exposed
to ozone are needed to assess how critical the chemokine IL-8 is in the initial phase of ozone-induced airway inflammation.
One interesting observation emerging from the immunohistochemical study
is the demonstration that the ciliated cell type is the major
contributor in vivo in ozone-induced IL-8 gene expression. We observed
very little IL-8 gene expression in airway epithelium of control
monkeys exposed to FA. However, immediately after acute ozone exposure,
we saw increased staining for IL-8 in bronchiolar epithelium,
especially in ciliated cells. The nature of this increase is not
entirely clear. It has been demonstrated that ciliated cell type is a
primary target of ozone toxicity (12, 22). Although there is no good
explanation of why ciliated cell type is the major target of ozone
toxicity, previous work has shown that the ozone effect is attributed
to its ability to cause oxidation (6) or peroxidation (7) either
directly or with the formation of free radicals (24). A sequence of the
events may include lipid peroxidation, loss of functional groups of
enzymes, and alteration of membrane permeability (4, 32). Because the membranes of ciliated cells project into the airway lumen via their
cilia and have a much greater surface area compared with other airway
epithelial cell types, they are more vulnerable to oxidant-induced
injury. Therefore, it is reasonable to assume an association between
elevated IL-8 gene expression and ciliated cell injury in vivo.
However, we found it difficult to use "cell injury" to explain
the current data. In immortalized human bronchial epithelial cell line,
ozone exposure caused substantial damage in cells, although it also
caused an increase in IL-8 secretion. In contrast, ozone caused little
damage in primary human TBE cells, yet the enhanced IL-8 gene
expression persisted. Thus cell injury by ozone is not critical for
IL-8 gene expression. Our in vitro data show a strong correlation
between cell viability and IL-8 production, whereas
immunohistochemistry also showed staining of squamating
epithelial cells (survivors) in monkey respiratory bronchioles.
Nevertheless, the process of IL-8 gene activation may be part of the
injury process. Recently, several reports have demonstrated that cell
deformation (26) and cell detachment (29) in vitro can elevate IL-8
gene expression. Others (25, 27) have demonstrated that changes in the
cellular redox mechanism can regulate IL-8 gene expression through the
activation of transcriptional factors essential for IL-8 gene
expression. These observations are consistent with the current view in
which ozone may disrupt cell processes, and, as a consequence, IL-8
gene expression is activated.
Finally, we would like to comment on our general approach in this
study. Using the molecular cloning technique, we have identified several MOR cDNA clones in which expression in monkey lung is both
cell-type and regional specific and which is also altered by ozone. The
success in identifying IL-8 as one of the MOR genes, despite the use of
a genetic fishing expedition, demonstrates the utility of the airway
microdissecting technique for isolating targeted tissues for various
molecular, biochemical, and biological investigations. This type of
approach is inevitable since the lung is a very complicated organ and
not all conducting airways respond to environmental air pollutants in a
similar manner.
 |
ACKNOWLEDGEMENTS |
The technical support of Yu Hua Zhao on in situ hybridization and
immunohistochemical staining is deeply appreciated, and we thank Brian
Tarkington for assistance on ozone exposure.
 |
FOOTNOTES |
This work is supported in part by National Institute of Environmental
Health Sciences Grants ES-00628, ES-06553, and ES-06230.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. M.-J. Chang, Center for Comparative
Respiratory Biology and Medicine, School of Medicine, Univ. of
California, Davis, CA 95616.
Received 6 February 1998; accepted in final form 5 May 1998.
 |
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