Department of Environmental Health Sciences, The Johns Hopkins University School of Public Health, Baltimore, Maryland 21205
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ABSTRACT |
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We
tested the hypotheses that 1) inducible nitric oxide
synthase (iNOS) mediates ozone (O3)-induced lung
hyperpermeability and 2) mRNA levels of the gene for iNOS
(Nos2) are modulated by Toll-like receptor 4 (Tlr4) during O3 exposure. Pretreatment of O3-susceptible C57BL/6J mice with a specific inhibitor of
total NOS (NG-monomethyl-L-arginine)
significantly decreased the mean lavageable protein concentration (a
marker of lung permeability) induced by O3 (0.3 parts/million for 72 h) compared with vehicle control mice.
Furthermore, lavageable protein in C57BL/B6 mice with targeted disruption of Nos2 [Nos2(/
)] was 50% less
than the protein in wild-type [Nos2(+/+)] mice after
O3. To determine whether Tlr4 modulates
Nos2 mRNA levels, we studied C3H/HeJ (HeJ) and C3H/HeOuJ mice that differ only at a missense mutation in Tlr4 that
confers resistance to O3-induced lung hyperpermeability in
the HeJ strain. Nos2 and Tlr4 mRNA levels were
significantly reduced and correlated in resistant HeJ mice after
O3 relative to those in susceptible C3H/HeOuJ mice.
Together, the results are consistent with an important role for iNOS in
O3-induced lung hyperpermeability and suggest that
Nos2 mRNA levels are mediated through Tlr4.
innate immunity; epithelium; inflammation; polymorphism; knockout mouse
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INTRODUCTION |
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IN HEALTHY HUMAN SUBJECTS, ozone (O3) causes decrements in pulmonary function and induces airway inflammation and hyperpermeability. Although O3 continues to be a major public health concern (1, 9, 17, 31, 48), the mechanisms of injury and associated susceptibility factors are not completely understood. Kleeberger et al. (23) previously demonstrated that susceptibility to lung hyperpermeability induced by subacute O3 exposure is a quantitative trait in inbred mice. Genetic linkage analyses with O3-susceptible C57BL/6J (B6) and O3-resistant C3H/HeJ (HeJ) mice identified significant and suggestive quantitative trait loci for susceptibility to O3-induced hyperpermeability on chromosomes 4 and 11, respectively. Within the chromosome 4 quantitative trait locus, Toll-like receptor 4 (Tlr4) was identified as a candidate gene for differential O3 susceptibility. Tlr4 has been identified as the gene that determines susceptibility to endotoxin challenge in mice (6, 40, 42). Strong supportive evidence for this gene candidate in O3 susceptibility was provided by two observations. First, O3-induced lung permeability was significantly different between HeJ and C3H/HeOuJ (OuJ) mice, which differ only at a missense mutation in the third exon of the Tlr4 gene (40). Second, Tlr4 mRNA levels in the lungs of HeJ mice were decreased relative to those in OuJ mice after exposure to O3, which suggested that downregulation of Tlr4 gene expression may contribute to O3 resistance in HeJ mice.
The mechanism(s) through which Tlr4 mediates differential O3-induced hyperpermeability is not understood. Nitric oxide (NO) has received considerable attention over the last decade for its homeostatic and "protective" roles as well as for its pathological and "injurious" roles in many tissue and organ systems. NO is produced from L-arginine with citrulline (a by-product) by nitric oxide synthase (NOS) activity in many tissue types. Three isoforms of NOS have been identified and the genes for each have been cloned. Endothelial NOS and neuronal NOS are constitutive forms that were initially characterized in endothelial and neuronal cells, respectively (10). Endothelial NOS has an important role in the maintenance of vascular tone, whereas neuronal NOS is a neurotransmitter that activates soluble guanylate cyclase and increases intracellular cGMP (see Ref. 10 for review). An inducible form of NOS, iNOS, was initially characterized in murine macrophages and has subsequently been identified in many cell types. In phagocytic cells of mice and other species, induction of iNOS results in prolonged production of NO and is a primary mechanism in the phagocytic killing of viruses, bacteria, and tumor cells (5, 10, 34). For example, iNOS-derived NO has antimicrobial properties during infection with Leishmania major (3) and Klebsiella pneumoniae (49). Inhaled NO has also been shown to enhance ventilation and perfusion matching in subgroups of patients with acute respiratory distress syndrome (28, 43).
NO may also have detrimental effects by directly inducing tissue damage (30, 52), leading to compromised epithelial integrity and increased epithelial permeability (13, 50, 51). In particular, a role for NO in the endotoxin-induced change in acute lung injury (29, 32), vascular tone (12, 47), and alveolar epithelial permeability (32) has been demonstrated. Furthermore, Pendino and colleagues and Punjabi et al. have shown that 3 h of exposure to 2 parts/million (ppm) O3 causes NO production in macrophages (37-39) and type II cells (41) of rats, whereas Haddad et al. (14) demonstrated iNOS induction in rats after 6 h of exposure to 3 ppm O3.
Because Tlr4 mediates lung responses to endotoxin and
O3 and iNOS contributes to endotoxin-induced lung
hyperpermeability, we hypothesized that iNOS similarly mediates a lung
permeability change induced by subacute O3 exposure. There
were two objectives of this study. The first was to determine the role
of iNOS in the pathogenesis of O3-induced lung
hyperpermeability in mice. To initially address this objective, we
evaluated the effect of NG-monomethyl-L-arginine (L-NMMA),
an inhibitor of total NOS, on O3-induced hyperpermeability
in B6 mice. We also evaluated the specific role of iNOS in B6 mice with
a site-directed mutation (knockout) of the gene for iNOS
[Nos2(/
)]. Nos2(
/
) mice were exposed to
O3, and the hyperpermeability response was compared with
that in wild-type [Nos2(+/+)] control mice. The second
objective of this study was to test the hypothesis that
O3-induced Nos2 mRNA levels are regulated by
Tlr4. Nos2 mRNA transcripts in lung homogenates
from O3-exposed HeJ and OuJ mice were amplified by reverse
transcriptase-polymerase chain reaction, quantitated, and compared
between strains. Results of these experiments are consistent with a
strong role for iNOS in O3-induced lung hyperpermeability and suggest that Tlr4 is an important determinant of
Nos2 expression.
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METHODS |
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General.
Male (6- to 8-wk-old, 20- to 25-g) inbred strains of mice {HeJ, OuJ,
Nos2(+/+) [C57BL/6J-Nos2(+/+)], and
Nos2(/
) [C57BL/6J-Nos2(
/
)]} were
purchased from Jackson Laboratories (Bar Harbor, ME). All animals were
housed in a virus- and antigen-free room. Water and mouse chow were
provided ad libitum. Cages were placed in laminar flow hoods with
high-efficiency particulate-filtered air. Sentinel animals were
examined periodically (titers and necropsy) to ensure that the animals
had remained free of infection. All experimental protocols conducted in
the mice were carried out in accordance with the standards established
by the US Animal Welfare Acts and set forth in National Institutes of
Health guidelines and the Policy and Procedures Manual
(Johns Hopkins University School of Hygiene and Public Health,
Baltimore, MD, Animal Care and Use Committee).
O3 generation and 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- and 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). Simultaneous exposures to filtered air were done in age- and strain-matched mice to serve as O3 controls.
Lung lavage and cell preparation. Bronchoalveolar lavage (BAL) was performed immediately after O3 exposure. The mice were killed by cervical dislocation, and the lungs were lavaged in situ four times with Hanks' balanced salt solution (35 ml/kg; pH 7.2-7.4). In the gene expression experiments, a suture was tied around the left main stem bronchus of each mouse, and the lung was removed distal to the suture and immediately frozen in liquid nitrogen. The right lung was then lavaged (17 ml/kg) as described above. Recovered BAL fluid was immediately cooled to 4°C and centrifuged. The cell pellets were resuspended in 1 ml of Hanks' balanced salt solution, and the cells were counted with a hemacytometer. Aliquots (10 µl) were cytocentrifuged, and the cells were stained with Diff-Quik for differential cell analysis. The supernatants were assayed for total protein (a marker of lung permeability) by the Bradford (4) assay.
Nos2 and Tlr4 mRNA levels.
Total RNA was isolated from lung tissues of HeJ and OuJ mice by
homogenization in TRIzol reagent (Life Technologies) following the
manufacturer's recommended protocol. cDNA was prepared by reverse
transcribing 5 µg of total RNA primed with oligo(dT) with the
SuperScript preamplification system (Life Technologies). Amplification was done under the following conditions: 1.5 mM MgCl2,
70°C annealing temperature, extension time of 1 (Nos2) or
2.5 (Tlr4) min, and 30 cycles. Primers for Nos2
and Tlr4 were synthesized according to Kleinert et al.
(24) and Poltorak et al. (40), respectively. -Actin was simultaneously amplified as an internal (reference) control. Fragments were analyzed on 1.3% agarose gels. The amplified cDNA fragments were scanned and quantitated with a Bio-Rad Gel Doc 2000 system (Bio-Rad Laboratories, Hercules CA).
Statistics. Statistical analyses of the O3-induced change in lung permeability were done by ANOVA (SuperANOVA statistical package, Abacus Concepts, Berkeley, CA). The Student-Newman-Keuls a posteriori test was used for comparisons of means (45). Statistical analyses of the relative changes in mRNA levels were done by nonparametric Kruskall-Wallis two-factor ANOVA (45). The factors were strain (HeJ and OuJ) and time (0 and 90 min and 3, 6, 24, 48, and 72 h). Significance for all comparisons of means was accepted at P < 0.05.
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RESULTS |
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Role of NOS in O3-induced
increase in lung permeability.
The role of NOS in the O3-induced increase in lung
permeability was assessed by treating B6 mice with L-NMMA
(2.1 mg/mouse ip; Sigma) immediately before exposure to O3
or filtered air was begun. A second, boosting dose (1 mg/mouse ip) was
administered after 24 h of exposure. Compared with that in
vehicle-treated mice, L-NMMA did not significantly affect
total BAL fluid protein in mice exposed to air for 72 h (Fig.
1). However, the mean BAL fluid protein
concentration in L-NMMA-treated mice exposed to O3 for 72 h (399 ± 85 µg/ml) was significantly
attenuated by 35% relative to that in O3-exposed vehicle
control mice (615 ± 52 µg/ml; P < 0.05; Fig.
1).
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Regulation of O3-induced increase in
Nos2 levels by Tlr4.
Kleeberger et al. (23) demonstrated previously that
basal levels of Tlr4 mRNA in the lungs of HeJ and OuJ mice
(which differ only at the Tlr4 locus) were not significantly
different from each other. Exposure to O3 for 72 h
caused Tlr4 mRNA levels to decrease beyond detection in the
lungs of O3-resistant HeJ mice but increase in
O3-susceptible OuJ mice (23). To determine
whether differential susceptibility to O3-induced
hyperpermeability in HeJ and OuJ mice is regulated through iNOS, we
compared expression kinetics of Nos2 and Tlr4 in
both strains. Mice were exposed to filtered air or O3 for
1.5, 3, 6, 24, 48, and 72 h. The mean total BAL fluid protein
concentration was not significantly different between the strains at
any time after air exposure. No differences in BAL fluid protein
between HeJ and OuJ mice were found after 1.5, 3, or 6 h of
O3 exposure. However, as previously demonstrated (23), BAL fluid protein concentration was significantly
elevated in OuJ mice compared with that in HeJ mice after 24, 48 and
72 h of exposure to O3 (Table
3).
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DISCUSSION |
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In the present study, we demonstrated that NOS and, in particular, iNOS have an important role in O3-induced lung hyperpermeability in B6 mice. This conclusion is supported by two experiments. First, treatment of B6 mice with a NOS inhibitor (L-NMMA) significantly decreased the change in mean BAL fluid protein induced by O3 relative to that in vehicle-treated mice. Second, the change in mean BAL fluid protein induced by O3 in Nos2 knockout mice (Nos2-deficient mice) was significantly reduced compared with that in wild-type B6 mice after 48 and 72 h of exposure. It is important to note that neither experiment accounted for all the O3-induced permeability change. In Nos2 knockout mice, ~50% of the hyperpermeability was inhibited relative to that in wild-type mice, whereas L-NMMA inhibited 35% of the O3 effect relative to vehicle control mice. Additional contributors to O3-induced hyperpermeability may include lipid mediators and cytokines. For example, platelet-activating factor has been shown to have a potent effect on airway permeability in the mouse, and a role for this mediator has been demonstrated in O3-induced acute lung injury (33).
It is interesting that the L-NMMA treatment, which putatively blocks all NOS activity, was a less effective inhibitor of O3-induced hyperpermeability than targeting of the iNOS gene (Nos2). This apparent discrepancy is likely due to differences in the specificity of the two treatments. Although the dosing regimen for L-NMMA in B6 mice approximated the effective doses published elsewhere (e.g., Ref. 32), the duration of those studies was relatively short (4-6 h). The present experiments were considerably longer (3 days), and although a boosting dose was administered during the exposure, it is conceivable that the effective lung tissue dose was not optimal during the entire experimental protocol. On the other hand, targeted disruption of Nos2 effectively ablates all gene function and, therefore, eliminates the contribution of iNOS to injury or inflammatory processes. In any case, results of both experiments were consistent and supportive of a role for NOS in this model.
The effects of L-NMMA and Nos2 knockout on lung permeability were apparently not the result of reduced infiltrating inflammatory cells. In both experiments, O3-induced increases in lavageable PMNs and macrophages were not affected by inhibition of NOS. This suggests that NO is not involved in the chemotactic pathway that results in PMN and macrophage infiltration into the lower airways after subacute O3 exposure. These observations are consistent with those of Li et al. (32), who found that L-NMMA similarly inhibited lung hyperpermeability induced by instilled endotoxin in rats and that endotoxin-induced infiltration of macrophages and PMNs was not affected by the NOS inhibitor. However, another study (18) found that the NOS inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine reduced airway inflammation in guinea pigs exposed to 3 ppm O3 for 2 h. The treatment effect was not observed until 5 h after exposure, and later postexposure time points were not reported. The discrepancy between this study and others (32) may be due to differences in species and exposure. Airway inflammation induced in mice by acute and subacute O3 exposures have different genetic mechanisms (22), and it is possible that, in contrast to the subacute model presented here, the inflammatory cell response to acute O3 exposure has a NO-dependent component.
Our experiments do not exclude the possibility that macrophages and PMNs are important contributors to the permeability changes induced by O3. Both cell types have the capacity to produce NO when stimulated (8, 34) and thus may be important sources of NO in the lung response to subacute O3 exposure. Furthermore, it is likely that both treatments (inhibition of NOS and targeted disruption of Nos2) inhibited NO production in macrophages and PMNs. Experiments designed to deplete PMNs (e.g., Ref. 21) or inhibit macrophage function (e.g., Ref. 39) are necessary to address the specific role of these cells in NO-mediated changes in lung permeability. Experiments designed to inhibit cell function must be interpreted carefully, however, because PMNs and macrophages have the capacity to release many de novo synthesized and stored products that could contribute to hyperpermeability [e.g., platelet-activating factor (33)].
Collectively, the present studies are consistent with a role for NO in
O3-induced hyperpermeability in the mouse. However, the
mechanisms through which iNOS is regulated (and NO production increased) during O3 exposure are not understood. We
hypothesize that the involvement of iNOS in O3-induced
hyperpermeability is mediated through Tlr4. The role of
Tlr4 as a critical component in the innate immune responses
has been well described (26, 27). The involvement of NO in
the innate immune effector pathways has also been defined (see Ref.
3). Furthermore, Ohashi et al. (36) have
shown in bone marrow-derived macrophages from HeJ and
endotoxin-responsive C3H/HeN mice that NO formation is dependent on a
functional Tlr4. Previous work from our laboratory (23) and the present study also indicate that
Tlr4 is an important regulator of lung hyperpermeability
induced by O3 exposure. Stimulation of Tlr4
initiates an intracellular signaling pathway that utilizes a series of
adapter proteins and serine/threonine kinases that link to the protein
kinase nuclear factor (NF)-B-inducing kinase (26, 27).
NF-
B subsequently induces key effector genes, including those for
proinflammatory cytokines (15, 53). There is accumulating evidence that Nos2 regulation is also mediated, in part,
through activation of NF-
B (10, 19, 34). Because of the
commonality between mechanisms of endotoxin- and O3-induced
lung hyperpermeability, we investigated whether the involvement of iNOS
in O3-induced hyperpermeability is mediated through
Tlr4. Differences in Nos2 gene expression between
O3-exposed HeJ and OuJ mice were consistent with this
hypothesis. Nos2 expression was markedly decreased in HeJ
mice after 6 h of exposure to O3, whereas
Nos2 expression in OuJ mice was elevated during exposure. To
further examine the relationship between Tlr4 and
Nos2, the expression kinetics of both genes were compared in
HeJ and OuJ mice. Interestingly, the kinetics of Tlr4 and
Nos2 expression were correlated in both strains after
exposure to O3. Although Tlr4 mRNA levels were
expressed constitutively and similarly in both strains, levels were
downregulated in HeJ lung homogenates as early as 90 min after exposure
to O3 and were maintained throughout the 72-h exposure. The
pattern of O3-induced change in Tlr4 gene
expression therefore resembled that of Nos2 in HeJ mice. In
contrast to HeJ mice, Tlr4 mRNA levels in the OuJ strain
increased early and remained elevated for up to 72 h. The
correlative patterns of gene expression in the two strains therefore
support a role for Tlr4 in the regulation of Nos2
during O3 exposure in the mouse.
The mechanisms through which NO causes a change in epithelial permeability in this model have not been determined. NO generated by NOS will react with superoxide anions to produce the highly oxidative molecule peroxynitrite, which may further lead to the formation of nitrotyrosine. Peroxynitrite and nitrotyrosine are highly cytotoxic and have been associated with acute lung injury (16, 25) and microvascular hyperpermeability (46). Thus it is conceivable that NO-mediated hyperpermeability caused by O3 exposure may be induced by secondary production of cytotoxic molecular species. It is also possible that hyperpermeability induced by O3 could be due, in part, to an uneven distribution of blood flow. Acute exposures to high concentrations of O3 cause increased bronchial circulation in sheep, which may contribute to the changes in lung permeability observed in this model (11, 44). Because NO has potent vasodilatory properties, O3-induced release of this gas into the microenvironment may alter blood flow and increase vessel permeability.
Another possibility is that NO effects are mediated through the
regulation of downstream effector genes. NO has been demonstrated to
modulate the transcription factor NF-B, which, in turn, regulates cytokines that are critical to the development and maintenance of
pulmonary inflammation (2). The effect of NO on NF-
B is primarily inhibitory. For example, de la Torre et al. (7)
demonstrated that lipopolysaccharide-mediated NO synthesis is
associated with S-nitrosylation of NF-
B p50 that inhibits
NF-
B-dependent gene transcription in ANA-1 murine macrophages.
However, NO may also activate NF-
B in some cell types (see Ref.
35). Clearly, further work is necessary to understand the
mechanisms through which NO may be mediating the hyperpermeability
effect of O3, and work is ongoing in our laboratory to
address this question.
Activation of TLR4 receptors by O3 is not clearly understood. It may be speculated that lipid ozonation products (LOPs) and other products of oxidant interaction with molecules in the epithelial lining fluid and cell membranes may stimulate TLR4 receptors. Kafoury et al. (20) have suggested that a cascade of events initiated by LOPs leads to inflammation in the lung, but it remains to be demonstrated whether LOPs initiate these processes via TLR4 receptors.
In summary, the inhibitory effects of L-NMMA on
O3-induced BAL fluid protein and differences in BAL fluid
protein between Nos2(+/+) and Nos2(/
) mice
after O3 exposure are consistent with a role for iNOS in
the development of oxidant-induced lung hyperpermeability. Furthermore,
differential expression of Nos2 in HeJ and OuJ mice during
O3 exposure suggests that Tlr4 has an important
role in the modulation of Nos2.
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ACKNOWLEDGEMENTS |
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We thank Kiana Brunson for excellent technical assistance.
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FOOTNOTES |
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This study was supported by National Institute of Environmental Health Sciences Grants ES-03819 and ES-09606; National Heart, Lung, and Blood Institute Grants R01-HL-57142 and R29-HL-58122; and Environmental Protection Agency Grant EPA R-826724.
Address for reprint requests and other correspondence: S. R. Kleeberger, Division of Physiology, Johns Hopkins Univ., 615 N. Wolfe St., Rm. 7006, 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 26 May 2000; accepted in final form 28 September 2000.
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