Department of Environmental Health Sciences, The Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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This study was designed to
investigate the mechanisms through which tumor necrosis factor
(Tnf) modulates ozone (O3)-induced pulmonary
injury in susceptible C57BL/6J (B6) mice. B6 [wild-type (wt)] mice and B6 mice with targeted disruption (knockout)
of the genes for the p55 TNF receptor [TNFR1(/
)], the
p75 TNF receptor [TNFR2(
/
)], or both receptors
[TNFR1/TNFR2(
/
)] were exposed to 0.3 parts/million
O3 for 48 h (subacute), and lung responses were
determined by bronchoalveolar lavage. All TNFR(
/
) mice had
significantly less O3-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared
with air-exposed control mice, O3 elicited upregulation of
lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated
TNFR1 and TNFR2 mRNAs in TNFR2(
/
) and
TNFR1(
/
) mice, respectively. Airway hyperreactivity induced by acute O3 exposure (2 parts/million for 3 h)
was diminished in knockout mice compared with that in wt
mice, although lung inflammation and permeability remained elevated.
Results suggested a critical role for TNFR signaling in subacute
O3-induced pulmonary epithelial injury and inflammation and
in acute O3-induced airway hyperreactivity.
susceptibility; tumor necrosis factor receptor knockout; pulmonary injury; gene targeting
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INTRODUCTION |
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OZONE (O3) is a highly toxic principal oxidant found in urban environments and workplaces throughout the world. Although airway toxicity to O3 has been studied extensively in laboratory animals and humans (4), the precise mechanisms underlying pathogenesis of the pulmonary airways after exposure to O3 are not clear.
Recent studies have raised concern about the potentially susceptible subpopulations that are at increased risk to the effects of environmental pollutants including O3. Smoking status, preexisting pulmonary disease, age, gender, and race may affect the variability of airway responses to O3 (4, 28, 37, 39). Interindividual variation in the pulmonary function responses to O3 has also been observed in healthy nonsmokers (28, 41, 43). Similarly, a wide range of neutrophilic inflammation has been reported from bronchial biopsies or bronchoalveolar lavage (BAL) from healthy humans after acute O3 exposure (2, 9, 12). These observations have led investigators to suggest that genetic background is a host risk factor in humans that contributes to the differential susceptibility to the adverse health effects of this air pollutant.
Significant differences in O3 sensitivity have also been
reported in animal models (8, 11, 33). Kleeberger and
colleagues (16, 18) previously demonstrated that
C57BL/6J (B6) inbred mice were more sensitive to pulmonary injury with
subacute [0.3 parts/million (ppm) for 72 h] and acute (2 ppm for
3 h) O3 exposures than C3H/HeJ (C3) mice. To
investigate the genetic factors that determine differential
susceptibility, a genomewide linkage analysis was done with a
B6C3F2 cohort phenotyped for inflammatory responses to 0.3 ppm O3 (19). In that study, significant and
suggestive quantitative trait loci (QTLs) were identified on
chromosomes 17 and 11, respectively. The chromosome 17 QTL included a
potential candidate gene [tumor necrosis factor (Tnf)]
that encodes TNF-. TNF-
is known to be a key proinflammatory
cytokine released after O3 exposure from lung cells
including alveolar macrophages and epithelial cells (1).
It has also been proposed as a central mediator in airway
hyperresponsiveness and inflammation in rodent airways after
O3 exposure (22). Pretreatment of susceptible B6 mice with anti-TNF-
antibody significantly attenuated
O3-induced pulmonary injury and provided strong evidence
for Tnf as an O3 susceptibility gene
(19). However, the role of TNF-
in acute lung injury
and inflammation has not been thoroughly studied.
The cellular effects of TNF- are mediated by two structurally
related but functionally distinct receptors: TNF receptor type 1 (TNFR1; 55 kDa) and TNF receptor type 2 (TNFR2; 75 kDa). TNFR1 and
TNFR2 are members of the nerve growth factor/TNFR superfamily of
proteins that are characterized by their conserved extracellular cysteine-rich repeat (5). Both receptors are coexpressed
on the surface of most cells and may be proteolytically released as
soluble molecules to inhibit TNF-
action in the inflammatory response (42, 44). TNF-
binds to the two receptors with
similar affinity (24) and induces the transcription of
genes that regulate acute inflammation, including early-response
cytokines (e.g., interleukin-1
), chemokines (e.g., macrophage
inflammatory protein-2), and adhesion molecules (e.g., intercellular
adhesion molecule-1) (21, 34, 44).
The present study was designed to investigate the roles of TNFR1 and
TNFR2 signaling pathways in the development of O3-induced lung injury in mice. Wild-type (wt) B6 mice and
gene-knockout mice deficient in either TNFR1
[TNFR1(/
)], TNFR2 [TNFR2(
/
)], or both
TNFR1 and TNFR2 [TNFR1/TNFR2(
/
)] were exposed to 0.3 ppm O3 for 48 h (subacute) or 2 ppm O3 for
3 h (acute), and lung inflammatory and injury responses were
compared. Deficiency of TNFR1 and TNFR2 provided significant protection
from O3-induced inflammation and epithelial injury (at 0.3 ppm) and airway hyperreactivity (at 2 ppm) in murine lungs. These
studies demonstrated the importance of TNFR-mediated responses in the
detrimental pulmonary effects of O3.
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METHODS |
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General
Male (6-8 wk old) inbred B6 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Breeding pairs of TNFR1(O3 Exposure
Mice were placed individually in stainless steel wire cages with free access to food and water during the exposure. The cages were set inside one of two separate 700-liter laminar flow inhalation chambers (Baker, Sanford, ME) that were equipped with a charcoal-filtered air and a high-efficiency particulate-filtered air supply. Chamber air was renewed at the rate of ~20 changes/h, with 50-65% relative humidity and a temperature of 20-25°C. O3 was generated by directing air (2 l/min) through an ultraviolet light O3 generator (Orec, Phoenix, AZ) that was upstream from one of the exposure chambers. The O3-air mixture was metered into the inlet airstream with computer-operated stainless steel mass flow controllers. O3 concentrations were monitored regularly at different levels within the chamber with an O3 ultraviolet light photometer (Dasibi model 1003 AH, Dasibi Environmental, Glendale, CA). The Dasibi model 1003 AH was calibrated regularly against a standard source (Dasibi model 1008-PC, Dasibi Environmental). Mice from each genotype were exposed to 0.3 ppm O3 for 3, 24, or 48 h to assess the subacute effect of O3. Other mice from each genotype were exposed to 2 ppm O3 for 3 h and then put in room air for 6 or 24 h for recovery to assess the acute effect of O3. Mice assigned to corresponding control groups were exposed to filtered air in the inhalation chambers for the same duration.Necropsy and BAL
Mice were anesthetized with pentobarbital sodium (104 mg/kg), and the lungs were lavaged in situ four times with Hanks' balanced salt solution (35 ml/kg, pH 7.2-7.4). Recovered BAL fluid was immediately cooled to 4°C and centrifuged. The supernatant from the first lavage return was assayed for total protein (a marker of lung permeability) with the Bradford assay. The four cell pellets were resuspended and pooled in 1 ml of Hanks' balanced salt solution, and the cells were counted with a hemacytometer. An aliquot (10 µl) of BAL cell suspension was cytocentrifuged and stained with Wright-Giemsa stain (Diff-Quik, Baxter Scientific Products, McGaw Park, IL) for differential cell analysis. Differential counts for epithelial cells (a marker of epithelial damage), neutrophils, lymphocytes, and macrophages (markers of inflammation) were done by identifying 300 cells according to standard cytological technique (36).Measurement of Airway Pressure-Time Index
The airway pressure-time index (APTI) procedure for assessment of airway reactivity in mice has been previously described in detail (23). Briefly, each animal was initially anesthetized with an inspired concentration of 1.5% halothane. After the trachea was cannulated, each mouse was given ketamine (50 mg/kg iv in sterile saline), paralyzed with decamethonium (25 mg/kg iv in sterile saline; Sigma, St. Louis, MO), and ventilated at 120 breaths/min with a constant tidal volume (0.2 ml). These drugs do not have apparent strain-specific effects on airway caliber that may influence interpretation of airway responses to agonists. Airway pressure was measured at a distal port of the tracheal cannula and recorded on a strip chart. Acetylcholine (ACh; 50 µg/kg in sterile saline; Sigma) was injected into the inferior vena cava, and airway reactivity was estimated as the integrated change in airway pressure from the initial change until the return to baseline airway pressure (APTI; in cmH2O · s). Airway reactivity was measured 24 h after a 3-h exposure because Zhang et al. (46) had previously demonstrated the greatest difference in ACh responsiveness between air- and O3-exposed B6 mice occurred at this time. We did not measure airway responsiveness in mice exposed to 0.3 ppm O3 because we found that this exposure does not significantly increase responsiveness to ACh in C57BL/6J mice (Kleeberger, unpublished observations).Analysis of TNFR1 (p55) and TNFR2 (p75) mRNA Expression
Total RNA was isolated from lung homogenates according to the method of Chomczynski and Sacchi (7) as indicated in the TRIzol reagent (Life Technologies, Gaithersburg, MD) specifications. Five hundred nanograms of pooled RNA from mice of each group were reverse transcribed into cDNA in a volume of 50 µl containing 1× PCR buffer (50 mM KCl and 10 mM Tris, pH 8.3), 5 mM MgCl2, 1 mM each deoxynucleotide triphosphate, 125 ng of oligo(dT)15, and 50 U of Moloney murine leukemia virus (MMLV) RT (Life Technologies) at 45°C for 15 min and 95°C for 5 min with a Gene Amp PCR system 9700 (PerkinElmer Applied Biosystems, Foster City, CA). PCR amplification was performed with an aliquot of cDNA (10 µl) with a final concentration of 1× PCR buffer, 4 (TNFR1) or 2 (TNFR2 andLung Tissue Preparation for Histopathology
Left lung lobes excised from mice exposed to either 0.3 ppm O3 or filtered air for 48 h were fixed with zinc formalin, embedded in paraffin, and sectioned 5 µm thick. Tissue sections were histochemically stained with hematoxylin and eosin for morphological comparison of pulmonary injury between genotypes. The terminal bronchioles and alveoli were the primary focus of study because a subacute exposure to 0.3 ppm O3 causes histologically evident inflammation and epithelial lesions in these regions of the mouse lung (see Ref. 19).Statistics
All data are expressed as group means ± SE. The data were natural log (ln) transformed, if necessary, to normalize the distribution and make the variances approximately equal. Two-way ANOVA was used to evaluate the effects of the subacute O3 exposure on pulmonary toxicity in wt and TNFR-deficient mice. The factors in the analysis were exposure (air or O3) and genotype [wt, TNFR1( ![]() |
RESULTS |
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Role of TNFR in O3-Induced Pulmonary Inflammation and Injury
To determine the role of TNFR-mediated responses in O3-induced lung inflammation, epithelial injury, and hyperpermeability, wt and TNFR-knockout mice were exposed to 0.3 ppm O3 for 48 h (subacute) or 2.0 ppm O3 for 3 h (acute). The number of neutrophils, macrophages, lymphocytes (markers of inflammation), and epithelial cells (a marker of epithelial injury) and the concentration of total protein (a marker of the permeability change) in BAL returns were determined as indexes of response.Subacute O3 exposure.
In mice exposed to air, there were no significant differences in the
number of inflammatory and epithelial cells or in the amount of protein
between genotypes [i.e., wt, TNFR1(/
),
TNFR2 (
/
), and TNFR1/TNFR2(
/
); Figs.
1 and 2].
Compared with air exposure, O3 caused significant increases
in the number of neutrophils, macrophages, lymphocytes, and epithelial
cells in wt mice (24-, 2.5-, 6-, and 4-fold, respectively).
In the three genotypes of TNFR-knockout mice exposed to
O3, there was a marked decrease in macrophages (30%),
neutrophils (80-90%), and epithelial cells (40-50%)
compared with those in wt mice exposed to O3. No
significant effects of O3 were detected on lymphocyte
infiltration in TNFR1(
/
), TNFR2(
/
), and
TNFR1/TNFR2(
/
) mice. There were no significant differences in the number of inflammatory and epithelial cells among
TNFR1(
/
), TNFR2(
/
), and
TNFR1/TNFR2(
/
) mice exposed to O3.
|
|
Acute O3 exposure.
Neutrophil infiltration and hyperpermeability were assessed 6 and
24 h, respectively, after acute O3 exposure because
Kleeberger et al. (16) previously determined that the
responses peak at these times. As opposed to the subacute
O3 exposure model, we have not found that the number of BAL
fluid macrophages, lymphocytes, or epithelial cells are significantly
elevated in B6 mice (i.e., wt) after acute O3
exposure (16). Compared with air-exposed wt
control mice, however, there was an ~20-fold greater BAL fluid neutrophilic inflammation in wt mice exposed to
O3 (Fig. 3A). In
contrast to subacute exposure, acute O3 exposure induced a significant elevation in neutrophil number in both
TNFR1(/
) and TNFR2(
/
) mice, and the
O3-induced neutrophilic inflammation in
TNFR1(
/
) and TNFR2(
/
) mice was not
significantly different from that in wt mice.
|
Role of TNFR in Acute O3-Induced Airway Hyperreactivity
The role of TNFR in the acute O3-induced pulmonary functional response was assessed by measuring airway reactivity as determined by APTI. Mean baseline airway pressures measured 3 min before ACh challenge were not significantly different between wt and TNFR-knockout mice after air or O3 exposure (data not shown). O3 significantly increased (twofold) the APTI response to ACh in wt mice (Fig. 4). However, the ACh response in O3-exposed TNFR1(
|
Histopathology of Lungs From wt and TNFR2(/
) Mice After
Subacute O3 Exposure
|
TNFR mRNA Expression
Subacute O3 exposure.
To ensure the absence of TNFR1 (p55) and TNFR2 (p75) mRNA expression in
TNFR1(/
) and TNFR2(
/
) mice, respectively,
and to determine whether transcriptional activation of the
TNFR gene is associated with activation of TNF-
signaling
in O3-sensitive mice, TNFR1 and TNFR2 mRNA expression was
determined by RT-PCR in murine lung tissues after 3, 24, and 48 h
of 0.3 ppm O3 exposure. The normal mouse lung
constitutively expressed both TNFR1 and TNFR2 mRNA (Fig.
6A). As expected, no TNFR1
mRNA was detected in the TNFR1(
/
) mice exposed to either
air or O3. Similarly, TNFR2 mRNA was not detected in
TNFR2(
/
) mice exposed to either air or O3.
The basal expression of TNFR1 mRNA in TNFR2(
/
) mice (25%) and TNFR2 mRNA in TNFR1(
/
) mice (fourfold) was
higher than that in wt mice (Fig. 6B). However,
O3 enhanced the steady-state expression of TNFR1 and TNFR2
mRNAs only in wt mice as early as 3 h of exposure (56%
and threefold, respectively), and mRNA levels remained elevated at
48 h of exposure in these mice. After a 24-h O3
exposure, the expression of TNFR1 mRNA in TNFR2(
/
) mice
and TNFR2 mRNA in TNFR1(
/
) mice was decreased to levels
lower than those in wt mice. At 48 h of exposure, the
level of TNFR1 mRNA in TNFR2(
/
) mice was slightly
elevated but did not exceed that of wt mice.
|
Acute O3 exposure.
Lung TNFR mRNA expression levels were determined by RT-PCR 6 and
24 h after a 3-h exposure to 2 ppm O3. As determined
in the subacute O3 exposure experiment, the basal
expression of TNFR1 mRNA in TNFR2(/
) mice and TNFR2 mRNA
in TNFR1(
/
) mice was higher than those in wt
mice (Fig. 7). The differences in
TNFR2 expression between wt and TNFR1(
/
) mice
after acute and subacute air exposure may be attributed to exposure
(chamber) effects or, more likely, may simply reflect
between-experiment variation in gene expression and quantitation.
Within-experiment RT-PCR results (Figs. 6 and 7) were highly
reproducible. In wt mice, O3 caused a slight
elevation (~14%) in TNFR1 mRNA expression 6 and 24 h postexposure, although the increased TNFR1 mRNA level did not exceed
the basal expression level of TNFR1 mRNA in TNFR2(
/
) mice (Fig. 7B). O3 did not enhance TNFR2 mRNA in
wt mice. There was no marked change in TNFR1 mRNA in
TNFR2(
/
) mice after O3 exposure. However,
the basal level of TNFR2 mRNA in TNFR1(
/
) mice was
decreased by 24 h postexposure to a level lower than that in
wt mice. TNFR1 mRNA was not detected in
TNFR1(
/
) mice, and TNFR2 mRNA was not detected in
TNFR2(
/
) mice (Fig. 7A).
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DISCUSSION |
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Results of this study indicated that TNFR-mediated responses play
a key role in the O3-induced injury and hyperreactivity in
pulmonary airways of the mouse. Mice lacking TNFR1, TNFR2, or both were
markedly less sensitive than wt mice to lung inflammation and epithelial damage by subacute O3 exposure (0.3 ppm for
48 h). On acute exposure to 2 ppm O3 (3 h), airway
hyperreactivity to ACh in wt mice was diminished in mice
lacking either TNFR1 or TNFR2, independent of pulmonary inflammation
and epithelial injury. These observations strongly suggested that
molecular and cellular mechanisms triggered by TNFR are critical in the
development of pulmonary O3 toxicity in mice and supported
TNF- as a subacute O3 susceptibility gene in murine
lungs. The current findings also implied that independent mechanisms
control 1) lung inflammatory and permeability changes in
response to subacute O3 and 2) pulmonary inflammation induced by two levels of O3 (i.e.,
subacute and acute). In addition, O3-induced lung
hyperpermeability may not be dependent on TNF-
signaling regardless
of O3 levels.
TNF- is recognized as a key mediator in the pathogenesis of various
inflammatory disorders. Numerous studies (27, 29, 30) have
demonstrated that TNF-
plays a pivotal role in acute lung injury and
inflammation induced by environmental toxicants such as bacterial
endotoxin and silica. However, only a few studies (e.g., Refs.
19, 31) have examined the deleterious effects of TNF-
on airway responses to O3. The specific roles of
the two TNFRs in TNF-
-mediated biological activities triggered by inflammatory stimuli such as lipopolysaccharide have been investigated using receptor-specific antibodies or ligands and receptor knockout mice. TNFR1 is known as the primary signaling receptor through which
the majority of inflammatory and cytotoxic responses attributed to
TNF-
occur (3). In contrast, TNF-
-mediated T cell
apoptosis and proliferation or skin necrosis are directed
through TNFR2 (38). Additionally, TNFR2 has been
postulated to function as a TNF-
antagonist by neutralizing TNF-
and as a TNF-
agonist by recruiting and delivering TNF-
to
facilitate the interaction between TNF-
and TNFR1 at the cell
surface (ligand passing) (32, 39). In vivo studies with
silica exposure (29, 30) demonstrated the importance of
TNFR signaling in lung inflammation, injury, and fibrosis. However,
little is known about the behavior of TNFR1 and TNFR2 and TNF-
signaling events in the pathogenesis of airway O3 toxicity.
In the present study, a similar magnitude of attenuation in
inflammation and epithelial injury responses to subacute O3 exposure was observed in the absence of either TNFR1 or TNFR2. The same
trend was detected in the airway hyperreactivity response to acute
O3 exposure. The present study also revealed that the suppressive effects on pulmonary injury and inflammation in the double-receptor knockout mice [TNFR1/TNFR2(
/
)] exposed
to subacute O3 were not greater than those in the
single-receptor knockout mice.
Our experiments were not designed to understand the detailed mechanisms of the two receptors mediating pulmonary O3 toxicity. However, results suggested that both TNFR1 and TNFR2 can independently modulate specific pulmonary responses to O3 (i.e., epithelial injury, inflammation, and hyperreactivity) in mice. Although the lack of additive suppression in double-receptor knockout mice seems not to support the ligand-passing concept in which TNFR1-mediated responses are enhanced by TNFR2, we cannot rule out a possible overlap between signaling pathways under two receptors in the development of O3-induced pulmonary injury. Further studies will be necessary to elucidate the role of intracellular signaling events triggered by two receptors in the pathogenesis of O3-induced airway injury and hyperreactivity.
The pattern of TNFR gene expression in the lungs of wt (upregulation) and receptor knockout (downregulation) mice by subacute O3 exposure was correlated with the pulmonary injury responses in both genotypes of mice. The relationship between TNFR gene expression and pulmonary injury has been reported in a recent study (30) in which the inducible expression of TNFR2 mRNA was believed to contribute to the silica- and bleomycin-induced lung fibrosis. Results of the present study suggested that upregulation of the steady-state levels of TNFR mRNA precedes the TNFR-dependent pulmonary injury responses. There was an elevation in basal TNFR1 mRNA expression in mice lacking TNFR2, and the converse was also observed. It appears that the two receptors in mouse lungs act in a compensatory manner in the absence of the other. To our knowledge, this is the first report that supports this concept, and the mechanisms through which compensation occurs have not been characterized. It is also unclear why O3 downregulated TNFR1 or TNFR2 mRNA expression in the lungs of single-knockout mice. It is possible that the absence of signaling mediated by one TNFR may lead to dysregulation of the other receptor gene during lung injury processes. The gene encoding TNFR2 is known to be more readily inducible than the gene encoding TNFR1 (5), and we observed greater inducible levels of TNFR2 mRNA than of TNFR1 mRNA in wt mice exposed to subacute O3.
The present study also demonstrated that O3-induced permeability changes in murine lungs are not attributed to TNFR-mediated responses. The apparent dissociation between inflammatory and hyperpermeability responses to O3 has been suggested by a number of previous studies. For example, Reinhart et al. (35) induced neutrophilic inflammation in rats (with 1% rabbit serum) before acute exposure to 0.8 ppm O3 and found that enhanced neutrophilic infiltration did not augment the pulmonary hyperpermeability with inhaled O3. In another study, mice were treated with cyclophosphamide, colchicine, or indomethacin to inhibit or deplete neutrophils before acute exposure to 2 ppm O3, and all treatments had no effect on O3-induced lung hyperpermeability (17). Although the mechanisms of O3-induced changes in lung permeability are not understood, two separate studies (14, 26) with rodents pretreated by a platelet-activating factor (PAF) receptor antibody indicated that the PAF receptor mediates pulmonary permeability and inflammation induced by acute O3. More recently, genetic linkage analysis of the lung hyperpermeability response to subacute O3 exposure identified a significant QTL on chromosome 4 (20). A candidate susceptibility gene, Toll-like receptor-4 (Tlr4), was identified within the QTL and tested. In that study, an association of differential O3 sensitivity in resistant (C3) and susceptible (C3H/HeOuJ) mice with a polymorphism in the coding region of Tlr4 was demonstrated. Furthermore, an opposite pattern of Tlr4 mRNA expression was shown in C3H/HeOuJ (upregulation) and C3 (downregulation) mice after subacute O3 exposure, consistent with the hypothesis that decreased expression of Tlr4 conferred resistance to O3-induced hyperpermeability. Further studies of Tlr4 signal pathways may provide a better understanding of the molecular mechanisms of O3-induced pulmonary hyperpermeability.
Using simple breeding experiments and genetic cosegregation analyses,
Kleeberger et al. (18) demonstrated that separate genetic
mechanisms control inflammatory responses induced in mice by acute and
subacute O3 exposures. That study implied that mechanisms that confer differential susceptibility to acute exposure are not
necessarily predictive of susceptibility to subacute exposure. Results
of the present study suggest that TNFR signaling mechanisms are
fundamental to the pathogenesis of inflammation and epithelial injury
induced by subacute but not by acute O3 exposures and thus support the previous observations. This dissociation of mechanisms underlying injury responses by different O3 exposures is
perhaps not surprising because the magnitude and the peak time of the inflammatory response vary with O3 concentration and
exposure duration (6). The lack of TNFR gene induction in
wt mice exposed to 2 ppm O3 (as opposed to
subacute exposure results) also suggests that mechanisms other than
TNF- signaling are elicited directly by O3 and/or by
subsequent transient neutrophilic inflammation and lung injury. Studies
with PAF receptor antagonists (14, 26) and mast
cell-deficient mice (25) suggested that mast cells
contributed to a significant part of the lung inflammation and
epithelial injury induced by 2 ppm O3, perhaps by releasing soluble mediators including PAF. However, further studies are necessary
to understand the precise molecular and cellular mechanisms of lung
responses to acute O3 exposure.
O3-induced airway hyperreactivity has been demonstrated in
a number of species (4), but the mechanisms are not
completely understood. It has been demonstrated that parasympathetic
nerves mediate O3-induced hyperreactivity to histamine,
ACh, and methacholine in guinea pigs and dogs (see Ref.
45). Furthermore, it has been hypothesized that
dysfunction of inhibitory M2 muscarinic receptors is
critical to the development of O3-induced hyperreactivity
in the guinea pig (45). Depletion of polymorphonuclear
neutrophils abolished the O3 effect on reactivity and
suggested that inflammatory cell infiltration was a necessary component
to hyperreactivity (10). In the mouse, however,
polymorphonuclear neutrophil depletion did not affect
O3-induced hyperreactivity to ACh (46). The
differences between studies may be related to species specificity of
M2 receptor involvement in the development of
hyperreactivity. In the present study, mice lacking TNFR signaling did
not develop airway hyperreactivity due to acute O3 and
suggested that TNF- has an important role in the hyperreactivity
induced by this exposure. The potential role of TNF-
in airway
responsiveness has been previously demonstrated. Exogenous
recombinant human TNF-
induced methacholine hyperresponsiveness and
inflammation in human subjects (40), and anti-TNF-
antibody significantly inhibited lipopolysaccharide-induced airway
hyperresponsiveness to ACh in rats (15). It has been
postulated that the effect of TNF-
may be mediated through inducible
nitric oxide synthase, which, in turn, regulates mediators such as
interleukin-8 (or the mouse homolog macrophage inflammatory protein-2)
(13), but further studies are necessary to clarify these mechanisms.
In summary, we have demonstrated that TNFR-mediated responses play a
critical role in pulmonary inflammation, epithelial injury, and airway
hyperreactivity induced by O3 in mice. The results strongly
support Tnf as a pulmonary susceptibility gene in
inflammatory and epithelial responses induced by subacute
O3 exposure in mice. The inflammatory mechanisms stimulated
by acute O3 exposure differed from those with subacute
O3 exposure, and TNF- signaling did not contribute to
the acute inflammation in the mouse lungs. Although the pulmonary
inflammation and epithelial injury induced by subacute O3
exposure are largely but not completely mediated through TNF-
signaling, we cannot exclude the role of other candidate genes in the
lung injury and inflammatory responses. Further studies are needed to
clarify the TNFR-mediated signal transduction mechanisms and downstream
effector genes through which TNF-
exerts lung injury and
inflammation in response to subacute O3 exposure.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sekhar Reddy and Anne Jedlicka for critical review of the manuscript.
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FOOTNOTES |
---|
This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-57142 and National Institute of Environmental Health Sciences (NIEHS) Grant ES-03819 (Inhalation Facility, NIEHS Center).
Address for reprint requests and other correspondence: S. R. Kleeberger, Division of Physiology, Rm. 7006, Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205 (E-mail: skleeber{at}jhsph.edu).
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 7 June 2000; accepted in final form 12 October 2000.
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