|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Previous studies have shown that a single exposure of animals to ozone (O3) can induce protection or adaptation to the acute injurious effects of a subsequent O3 challenge. Although a number of mechanisms have been proposed to account for this response, none appear to be fully explanatory. We examined the role interleukin (IL)-6 may play in the induction of adaptation to O3-induced pulmonary injury. A statistically significant 29-fold increase in bronchoalveolar lavage fluid IL-6 levels was observed in rats exposed to 0.5 ppm O3 during nighttime hours when compared with daytime hours even though similar kinetics of inflammation were induced by each exposure. Animals receiving an initial nighttime O3 exposure showed a lesser degree of inflammation following a subsequent O3 exposure when compared with animals which received an initial daytime exposure. Rats pretreated with IL-6 both intratracheally and intraperitoneally and subsequently exposed to O3 showed a lesser degree of cellular inflammation when compared with respective controls. Pretreatment of rats with anti-IL-6-receptor antibodies (ra) prior to the nighttime O3 exposure completely abrogated the O3-induced cellular adaptive response without effecting the inflammatory response induced by the initial nighttime O3 exposure. In fact, administration of anti-IL-6ra augmented the neutrophil influx following the second O3 exposure. Anti-IL-6ra treatment did not alter the pulmonary edema adaptive response, suggesting that the O3-induced cellular and edema adaptive responses are regulated by different mechanisms. Our data indicate that mobilization of pulmonary antioxidants does not play a role in the IL-6-mediated early cellular adaptive response and suggest that IL-6 is an essential mediator of the O3-induced cellular adaptive response.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Pulmonary inflammation following an acute exposure to ozone (O3) has been demonstrated in humans and animals as an increase in pulmonary edema as well as an influx of neutrophils into the airspace (1). However, when the O3 exposure is repetitive the resultant inflammatory response is largely attenuated (5). This phenomenon of apparent recovery to normal values is frequently referred to as "adaptation."
A number of mechanisms have been proposed to explain the adaptive response, yet none appear to be completely satisfactory. Most notably, antioxidant induction has been suggested by many investigators to explain the adaptive response (8). However, these reported increases in antioxidant levels following O3 injury occur within 3-4 days following the initial exposure. In contrast, adaptation is observed as early as 18 h following an acute O3 exposure and appears to precede the mobilization of antioxidants (12, 13). Thus while antioxidant induction may participate in the persistence of the adaptive response (late adaptive response), this proposed mechanism fails to explain observations of O3-induced adaptation occurring within 18 h after a single acute exposure (early adaptive response). During the initial characterization of the O3-induced adaptive response, investigators realized that the higher the tolerated pretreatment concentration of O3, the stronger and longer the duration of the protection with respect to resistance to subsequent lethal challenges (14, 15). This correlation suggests that the intensity of the initial O3-induced injury/inflammation may modulate the adaptive response to subsequent O3-induced injury as well as O3-induced lethality.
Cytokines have been implicated as potential mediators
of protection against oxidative injury (16). Recent studies
have shown that interleukin (IL)-6 levels are increased in
the bronchoalveolar lavage (BAL) fluid of humans and
rats following acute O3 exposures (17, 18). IL-6 concentrations measured in the BAL fluid of rats was observed to
be dose-dependent (18). Furthermore, exposure to near-ambient concentrations of O3 (< 0.5 ppm) were shown to induce IL-6 mRNA expression in rats with no change in the
mRNA levels of other cytokines such as IL-1
, KC, macrophage inflammatory protein-2 (MIP-2), tumor necrosis
factor-
(TNF-), transforming growth factor- (TGF-),
and monocyte chemotactic protein-1 (MCP-1) (19).
IL-6 may be secreted into the lung lining fluid by O3-stimulated alveolar macrophages and/or epithelial cells (20, 21). Although initially thought to be a proinflammatory cytokine, recent data suggest that IL-6 also possesses anti-inflammatory properties. IL-6 has been shown to play a protective or suppressive role in hypersensitivity pneumonitis, O2-induced toxicity, bacterial and viral infection, turpentine-induced toxicity, and airway hyperresponsiveness to bronchoconstrictor agents (22).
Here we investigated the role of IL-6 in O3-induced adaptation using various models. One model utilized differences in IL-6 levels immediately following initial daytime or nighttime O3 exposures to implicate IL-6 in the O3-induced adaptive response. Overall data from the various models support the hypothesis that induction of IL-6 during an initial O3 exposure plays an integral role in the development of the early (< 24 h) adaptive cellular response to this air pollutant. In addition, we suggest possible IL-6-mediated mechanisms for bridging the early adaptive response with the late adaptive response.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Animals
Male Wistar rats (Charles River Breeding Laboratories, Inc., Raleigh, NC), 60-90 days old, were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care-approved animal care facility at a temperature of 72-75°F and humidity of 50-60% with fixed day (6:00 A.M.-6:00 P.M.) and night (6:00 P.M.-6:00 A.M.) periods. Rats (300-380 g) received food (Purina Rodent Lab Chow; Ralston Purina, St. Louis, MO) and water ad libitum. Animals were allowed to acclimatize for 1 wk prior to any treatment. The study group sizes range from five to 15 animals and are noted specifically in the figure legends and tables.
Ozone Exposure
Exposures to 0.5 ppm O3 were performed in 0.3 m3 Rochester-type exposure chambers (Bertke and Young, Cincinnati, OH) with O3 generated from 100% O2 using a silent-arc-discharge O3 generator (OREC, Phoenix, AZ) and mixed with preconditioned clean air to provide a flow of 1 chamber volume/min. The concentration of O3 was monitored and maintained continuously via computer feedback using chemiluminescent O3 analyzers (Bendix, Lewisburg, WV), calibrated biweekly using a Dasibi transfer standard, which is referenced quarterly to a primary ultraviolet O3 standard (National Technical Information Service). During exposure the monitored O3 concentration did not vary by more than 1% of the target concentration.
Day versus Night Ozone-induced Lung Inflammation and Adaptation
To determine the time course of O3-induced inflammation following either a nighttime or daytime exposure, rats were exposed to O3 (0.5 ppm) or air either during the daytime (12:00-4:00 P.M.) or nighttime (7:00-11:00 P.M.) hours. BAL fluid IL-6, as well as cellular and biochemical biomarkers of lung inflammation were determined at various times after exposure. To determine the effect of a daytime or nighttime exposure on O3-induced adaptation, rats were similarly exposed to O3 (0.5 ppm) or air during the daytime or nighttime hours as described previously. At 16 h after exposure, animals received a second O3 exposure (0.5 ppm, 4 h). BAL fluid samples were collected and analyzed for cellular and biochemical biomarkers of lung inflammation 25 h following the second exposure.
IL-6 and Anti-IL-6 Receptor Antibody Pretreatment
To determine the role of IL-6 in O3-induced adaptation, 90-d-old rats were anesthetized with halothane (Aldrich Chemical Co, Milwaukee, WI) and administered IL-6 (recombinant mouse IL-6, approximate specific activity of 106 units/µg; Genzyme, Cambridge, MA), 0.3 ml (30 ng or 30,000 units) intratracheally (i.t.) and 0.5 ml (30 ng or 30,000 units) intraperitoneally (i.p.) in carrier buffer (1% vol/vol rat plasma in pyrogen-free saline) or carrier buffer alone during the daytime (12:00 P.M.). At 16 h after IL-6 administration, rats were exposed to O3 (0.5 ppm) for 4 h. At 25 h after O3 exposure, BAL fluid samples were collected and analyzed for cellular and biochemical biomarkers of lung inflammation. To determine whether IL-6- mediated protection involved a systemic or local mechanism, IL-6 or carrier buffer was administered either i.t. or i.p. and animals were treated as described above.
Monoclonal rat antimouse IL-6 receptor antibody derived from clone 15A7 was employed to determine the role of IL-6 in O3-induced pulmonary adaptation. The production and characterization of this antibody has been described elsewhere (28, 29). This monoclonal antibody recognizes both mouse and rat IL-6 receptors and inhibits IL-6 in vitro functional activity (clone 15A7; Genzyme). Rats were administered monoclonal rat antimouse IL-6 receptor antibodies (clone 15A7; Genzyme) or rat IgG2b kappa control antibody (Pharmingen, San Diego, CA) i.p. (350 µg in 0.5 ml of carrier buffer) 12 h prior to an initial nighttime O3 exposure (0.5 ppm, 7:00-11:00 P.M.). A second dose of antibodies was administered i.t. (150 µg in 0.3 ml of carrier buffer) 1 h prior to the initial nighttime O3 exposure (0.5 ppm, 7:00-11:00 P.M.). The initial O3 exposure was followed 16 h later by an additional exposure to the same concentration of O3. BAL fluid was collected and analyzed for cellular and biochemical endpoints 16 h following the initial O3 exposure and 25 h after the second O3 exposure.
To assess whether antioxidants played a role in IL-6-mediated adaptation to O3, IL-6 (i.t/i.p.) or carrier buffer (i.t./i.p.) was administered as described previously. In addition, we assessed whether the interaction of O3 and IL-6 induce levels of antioxidants which are different from control, and animals were exposed during nighttime hours as described previously. Sixteen hours following IL-6 treatment or an acute O3 exposure, BAL fluid samples were collected and analyzed for water-soluble ascorbic acid (AA), uric acid (UA), and glutathione (GSH), as well as protein concentration. In addition, lung tissues were extracted and samples were assayed for glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GSHrd), glutathione peroxidase (GSHpx), glutathione transferase (GSHtr), superoxide dismutase (SOD) activity, and total tissue protein (described below).
BAL Analysis
Rats were anesthetized with pentobarbital (50 mg/kg) and exsanguinated by severing the abdominal aorta. The lungs were lavaged 3 times with the same aliquot (35 ml/kg body wt/rat) of isotonic saline or phosphate-buffered saline (PBS) (30). Recovered BAL fluid volumes ranged between 75 and 85% of instilled saline or PBS. There was no statistically significant difference in recovered BAL fluid volumes between control and experimental groups. Cell counts were determined using a Coulter counter (Coulter Electronic, Hialeah, FL). Cell differential analysis was performed on Diff-Quik (American Scientific Products, McGaw Park, IL)-stained cytospins of BAL fluid samples. A total of 300 cells were counted per slide. Protein concentrations were determined using Pierce Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL) (31) using a bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO)-derived standard curve.
BAL Fluid and Tissue Antioxidant Analysis
BAL samples were centrifuged at 700 × g for 20 min. The
BAL supernate, 1 ml, was added to 35 µl of 60% perchloric acid and stored at 
70°C for later analysis of AA,
GSH, and UA as well as soluble total protein. The left
lung was homogenized using a Polytron tissue homogenizer (Brinkman Instrument Co., Westbury, NY) in 50 mM Tris buffer with 1.15% KCl (5 ml/liter g wt). The homogenate was centrifuged at 20,000 × g for 20 min. The
supernatant (0.5 ml) was stored at 70°C and later analyzed for total SOD and protein. The remainder of the supernate (2 ml) was centrifuged at 100,000 × g for 1 h and
stored at 70°C for later analysis of G6PDH, GSHrd,
GSHpx, and GSHtr activity and tissue protein.
The right apical lung lobe was extracted with 3 ml of
cold 3% perchloric acid and centrifuged at 4°C at 20,000 × g
for 20 min. The supernate was recovered and stored at 70°C
for later analyses for AA, UA, and GSH. The pellet was
dissolved in 1 ml of 0.25 N NaOH at 45°C overnight and
subsequently frozen at 70°C for later protein analysis.
Antioxidant Assays
Reduced AA and UA were assayed by liquid chromatography with electrochemical detection (32). Glutathione was measured in perchloric acid supernatants of BAL and tissues as previously described (33). GSHrd, GSHtr, and GSHpx activity was assayed as adapted to the Cobas Fara II centrifugal spectrophotometer (COBAS; Hoffman-La Roche, Branchbury, NJ) (34). G6PDH and total SOD activity were determined both as adapted to the COBAS (35, 36). The latter assay does not distinguish among the various forms of SOD.
IL-6 Assay
IL-6 activity was determined using the IL-6-dependent hybridoma cell line 7TD1 (37).
Statistics
The data were analyzed using one, two, or three-way analysis of variance (ANOVA) models (SAS Institute, Cary, NC) examining the main effects of each model as well as the interactive effects of two- and three-way ANOVA models. The significance level was set at 0.05. A significant interaction resulted in pairwise comparisons performed as a subtest of the ANOVA model adjusting the significance level for multiple comparisons using a modified Bonferroni procedure.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Effect of Nighttime versus Daytime O3 Exposure on Lung Inflammation and IL-6 Expression
The kinetics of lung inflammation induced following either a daytime or nighttime O3 exposure were similar. However, differences in recovered BAL neutrophil levels and edema peak values were observed (Figure 1). Significant neutrophil influx into the airways occurred earlier following the nighttime exposure to O3, with peak neutrophil influx occurring 24 h regardless of day- or nighttime exposure (Figure 1A). More neutrophils were present in BAL fluid recovered from rats exposed to ozone at night (Figure 1A). The increases in BAL fluid protein levels, an indicator of pulmonary edema, occurred more quickly following a daytime O3 exposure with peak values occurring 5 h after exposure and 10 h following a nighttime exposure (Figure 1B). The increases in pulmonary edema were similar following a day- or nighttime O3 exposure (Figure 1B).
|
A temporal effect on O3-induced IL-6 expression was observed in these studies (Figure 2). Ozone induced a statistically significant 29-fold higher BAL fluid IL-6 level immediately following the nighttime exposure when compared with corresponding daytime exposure. Quantitative analysis of BAL fluid IL-6 levels demonstrated the O3 nighttime exposure produced an average of 7 ng IL-6/rat (range: 2-29 ng IL-6/rat) whereas O3 daytime exposures produced an average of 0.24 ng IL-6/rat (range: 0.04-0.47 ng IL-6/rat). The O3-induced increases in IL-6 were highest immediately following the O3 exposure and declined toward control levels over the ensuing 36 h (Figure 2).
|
Effect of Daytime versus Nighttime Pre-exposure on O3-induced Adaptation
When the daytime or nighttime O3 exposure (as described previously) was followed by a second O3 exposure, animals initially exposed during the nighttime showed adaptation to the resultant injury (Figure 3). Rats exposed initially to a daytime O3 exposure showed an increase in lung inflammation following a second exposure (Figure 3). BAL fluid neutrophil concentrations in rats 16 h following the initial daytime O3 exposure were slightly, but not significantly, increased above control levels as compared with a significant 5.3-fold increase in BAL fluid neutrophil concentrations in rats exposed during an initial nighttime O3 exposure (Figure 3B). However, 25 h following the second O3 exposure, rats receiving an initial daytime O3 pre-exposure exhibited a 50% increase in BAL fluid neutrophil levels when compared with the initial daytime exposure-induced response. In comparison, animals receiving an initial nighttime O3 pre-exposure had a 65% decrease in neutrophil levels following the second O3 exposure when compared with the initial nighttime exposure-induced response (Figure 3B). A qualitatively similar (although less dramatic) response was observed with the changes in BAL fluid protein levels, an index of pulmonary edema (Figure 3C). BAL fluid protein levels measured 16 h following the initial daytime exposure to O3 were increased 53% above control as compared with a 63% increase in rats exposed during an initial nighttime O3 exposure (Figure 3C). However, 25 h following the second O3 exposure, animals receiving an initial daytime O3 pre-exposure exhibited a 63% increase in BAL fluid protein when compared with the initial daytime exposure-induced response. In comparison, animals receiving an initial nighttime O3 pre-exposure exhibited an 18% attenuation in BAL fluid protein levels when compared with the initial nighttime exposure-induced response (Figure 3C).
|
Effect of IL-6 and Anti-IL-6 Receptor Antibody Pretreatment on Adaptation to O3-induced Lung Inflammation
Rats were administered IL-6 (30 ng i.t./i.p.) in order to determine whether this cytokine could induce an adaptive response to an initial acute O3 exposure. The dose and routes of IL-6 administration were based on the following observations: (1) this amount of IL-6 was observed in BAL fluid recovered from rats immediately following a nighttime O3 exposure; and (2) pulmonary and circulatory IL-6 levels have been reported to increase by similar amounts following an acute O3 exposure in humans (38). Rats pretreated with IL-6 prior to a subsequent O3 exposure adapted to the resultant injury when compared with control and carrier buffer-treated rats (Figure 4). Control and carrier buffer-treated rats showed similar increases in BAL fluid neutrophil levels following O3 exposure. However, rats pretreated with IL-6 and subsequently exposed to air displayed pulmonary inflammation, indicating that IL-6 was proinflammatory (Figure 4B). In contrast, however, BAL fluid neutrophil levels of rats treated with IL-6 and subsequently exposed to O3 were at control levels, indicating a total inhibition of O3-induced pulmonary neutrophil influx (Figure 4B). Control and carrier buffer-treated rats showed similar increases in BAL fluid protein following O3 exposure. BAL fluid protein levels in IL-6 pretreated and air-exposed rats were only slightly increased over respective controls, suggesting that IL-6 alone displays proinflammatory properties (Figure 4C). However, BAL fluid protein levels in animals that were administered IL-6 and exposed to O3 were attenuated 18% when compared with control and carrier buffer-treated rats exposed to O3 (Figure 4C). These data indicate that IL-6 may play only a minor role in the attenuation of O3-induced increases in pulmonary edema. Interestingly, when IL-6 was administered separately by either i.p. or i.t. routes no attenuation of O3-induced increase in pulmonary neutrophil influx or pulmonary edema was observed (data not shown).
|
Anti-IL-6 receptor antibody (ra) pretreatment did not affect the inflammatory response induced by the initial acute nighttime O3 exposure since BAL fluid protein (218 ± 13 µg/ml O3 versus 194 ± 12 µg/ml O3 + anti-IL-6ra) and neutrophil (1.60 ± 0.06 × 104 cells/ml O3 versus 2.2 ± 0.38 × 104 cells/ml O3 + anti-IL-6ra) were not altered at 16 h after exposure (Figure 5A). However, antagonism of the IL-6 receptor by antibody pretreatment did alter the inflammatory response following a second O3 exposure (Figure 5). Pretreatment of rats with IL-6ra was found to reverse the cellular response observed following the second O3 exposure by producing a 5-fold increase in BAL fluid neutrophil levels when compared with those levels observed in adapted rats (Figure 5B). Pretreatment of rats with an irrelevant control antibody did not affect the cellular adaptation induced by sequential O3 exposures (Figure 5B). Rat anti-IL-6ra pretreatment had no effect on O3-induced changes in pulmonary edema because BAL fluid protein concentrations were not significantly altered (Figure 5C).
|
Effect of IL-6 Treatment and Acute O3 Exposure on BAL and Tissue Antioxidant Levels
The BAL and lung antioxidant substances and enzymes measured 16 h after IL-6 pretreatment or following an acute nighttime O3 exposure did not show significant changes between treatment/exposure groups and control groups (Table 1).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
We have provided evidence that IL-6 does not participate in
the acute inflammatory response induced by an initial O3 exposure. Our results demonstrate, however, that IL-6 induced
by an initial O3 exposure plays a significant role in mediating
the O3-induced cellular adaptive response to a second O3 exposure. Furthermore, our results suggest that pulmonary
edema adapts via an IL-6 independent mechanism or involves a different type of IL-6 receptor not recognized by the
monoclonal antibody used in this study. These results are consistent with similar studies which have suggested that cytokines such as tumor necrosis factor and IL-1 are markedly
protective against O2 toxicity (23, 39). However, these cytokines as well as IL-1, KC, MIP-2, TGF-, and MCP-1 do
not appear to be induced by exposure to ambient concentrations of O3 (19). Yet, at ambient O3 concentrations IL-6 levels are markedly increased in the BAL fluid of both humans
and rats (17, 18). Recently, IL-8 and the rat homologue cytokine-induced neutrophil chemoattractant (CINC) have
been measured in BAL fluid following exposure to O3, suggesting their role in cellular recruitment (17, 40).
Although nighttime and daytime O3-induced changes in biochemical and cellular endpoints of inflammation show similar response kinetics, quantitatively the nighttime exposures induced the greater increase in pulmonary neutrophil influx which correlated with higher IL-6 expression. Enhanced O3-induced inflammation in rats following nighttime as compared with daytime exposure has also been reported by Van Bree and coworkers (19). These investigators suggested that increased physical activity during the nighttime exposures may result in higher ventilation, resulting in increased inhalational uptake of O3. However, it appears that certain factors associated with nighttime O3 exposure-induced inflammation may impart protection to subsequent exposures to this air pollutant. This suggestion is prompted by the inordinately high IL-6 concentrations in BAL fluid samples following nighttime O3 exposures relative to those observed following the daytime O3 exposure. At present, the only in vivo O3 concentration-response relationship for IL-6 is unpublished data from this laboratory which showed a proportionate increase in IL-6 with O3 concentrations from 0.5 to 1.5 ppm. The marked disparity in IL-6 levels following daytime versus nighttime O3 exposure provided a model to investigate the role of endogenous IL-6 on O3-induced adaptation. Our data suggest that the increase in IL-6 following a nighttime exposure does not contribute to the acute inflammation but participates in attenuating further O3-induced cellular inflammatory response, whereas the small increase in IL-6 following a daytime O3 exposure is not sufficient to impart a protective response with respect to subsequent O3 challenge. DiCosmo and coworkers suggested that IL-6 may serve as an alarm alerting the body to injury as well as preparing the body to deal with the injury (27). Indeed, studies using turpentine-treated transgenic mice support this contention (25, 26). Studies conducted by Fattori and coworkers (26) used body weight as well as acute phase protein (APP) levels as indicators of injury response following the injection of turpentine. Normal mice initially lost weight following the administration of turpentine but began to regain weight within days following treatment. In contrast, transgenic mice incapable of producing IL-6 died within days following turpentine treatment. Furthermore, increases in APPs observed in the plasma of normal mice were absent in IL-6 knockout mice following turpentine administration (26). Likewise, transgenic IL-6 overexpressing mice have been shown to be markedly resistant to 100% O2 when compared to wild-type mice (25). Thus our contention is that the dramatic, though transient, increase in IL-6 immediately following a nighttime O3 exposure serves to initiate mechanisms designed to protect the lung from a second injury.
IL-6 may mediate the O3-induced cellular adaptive response by upregulation of APPs or, alternatively, may involve the induction of apoptosis of cells (in this case neutrophils) present at the site of inflammation (41). The APPs produced by the mammalian liver within hours of systemic injury or following IL-6 administration appear to participate in limiting the tissue damage at injury sites distant from the liver (42). For example C-reactive protein (CRP) has been shown to localize to sites of inflammation and inhibit neutrophil chemotaxis to a variety of chemoattractants in vitro and in vivo (45). In addition, CRP has been shown to be elevated in human plasma as well as BAL fluid following an acute O3 exposure (38).
Reports that IL-6 is released from damaged tissue into the bloodstream and appears to modulate hepatic plasma protein synthesis, as well as data which suggested that O3 exposure increases pulmonary IL-6, prompted our study design to include approaches involving administration of IL-6 i.t. and i.p. separately and together to ascertain whether either route could impart protection to O3-induced injury (38). We found that neither route alone was sufficient to induce adaptation; however, it is unclear whether these results relate to the total IL-6 dose rather than the route of administration. The need to pretreat animals with IL-6 by simultaneous i.t./i.p. routes to elicit adaptation to O3-induced lung injury would be consistent with previously reported results which suggested that BAL and plasma IL-6 levels increase to similar degrees following an acute O3 exposure (38). Indeed, increased circulatory levels of IL-6 following an acute O3 exposure may be responsible for the extrapulmonary effects of this air pollutant (48). Although APPs synthesized in the liver can migrate to sites of inflammation, recent data suggest that type II pulmonary cells are also capable of producing certain APPs that may also regulate local injury (49). Alternatively, the ineffective attenuation of the O3-induced increases in neutrophils when IL-6 was administered either i.t. or i.p. alone may suggest that a local as well as a systemic response is necessary for the adaptive response to occur. In order to assess whether traditional antioxidants play a role in the IL-6 mediated adaptive response to O3, BAL fluid and tissue antioxidants were measured 16 h after IL-6 treatment or O3 exposure (0.5 ppm, 7:00-11:00 P.M.). The BAL fluid and lung antioxidant metabolites and enzymes measured 16 h after IL-6 treatment or nighttime acute O3 exposure were not significantly altered. Although we measured total SOD and did not differentiate between copper/zinc SOD, extracellular SOD, and MnSOD, we observed no difference between treatment/exposure and control total SOD levels in lung tissue. Furthermore, others have reported no effect of IL-6 on MnSOD mRNA levels (23). Studies of antioxidant induction following O3 exposure indicate that antioxidant mRNAs are elevated in rats by Day 3 of a 5-d exposure (8- 11, 50). Similarly, Nambu and Yokoyama reported a gap between the time course of the enhancement of the antioxidant system and the development of O3-induced adaptation (51). These results indicate that pulmonary antioxidants do not play a role in the early adaptive response. Our studies suggest that IL-6 mediates a more rapidly inducible early protective response to O3-induced lung injury.
Confirmatory studies designed to establish IL-6 as a primary mediator of O3-induced early adaptation used anti-IL-6 receptor antibodies (anti-IL-6ra). This antibody blocks binding of rat IL-6 to its receptor, enabling it to neutralize IL-6 bioactivity in vitro as well as in vivo (28, 29). The interpretation of results obtained from studies using blocking antibodies specific for the cytokine rather than the cytokine receptor is not as straightforward, primarily because monoclonal antibodies against IL-6 appear to complex the cytokine and to prolong the functional activity in vitro (Dreher and colleagues, unpublished data) and in vivo (52). Our results show that the administration of anti-IL-6ra prior to an acute O3 exposure significantly abrogated the early cellular adaptive response to O3. However, this treatment had no effect on the inflammatory response following the initial O3-induced lung inflammation, suggesting that although IL-6 is induced early, it does not affect the initial O3-induced inflammation. Anti-IL-6ra pretreatment did not effect O3-induced pulmonary edema, which was not surprising because our earlier studies with administered IL-6 suggest that IL-6 had no significant effect on this response.
Administration of IL-6 without a subsequent O3 exposure induces an inflammatory response, as reported by Ulich and coworkers (55), who found that i.t. injection of IL-6 alone caused the accumulation of neutrophils while i.t. injection of saline did not (55). IL-6 may affect other cyto-kines, such as CINC. It has been suggested that CINC may be the primary chemoattractant responsible for neutrophil migration following an acute O3 exposure in rats (40). The slightly higher BAL protein concentration of animals administered IL-6 may result from IL-6-induced increases in endothelial permeability, which have been reported to occur in vitro (56). However, our data suggest that the pulmonary edema response and its adaptive mechanisms are not mediated by IL-6.
In conclusion, our findings provide evidence to support the role of IL-6 as a mediator of the early pulmonary cellular adaptive response to environmental levels of O3. Although the exact mechanism(s) by which IL-6 mediates this adaptive response is unclear, it appears that the mobilization of antioxidants is not involved. Other possible mechanisms include mobilization of APPs, apoptosis of inflammatory cells, and altered cellular adhesion molecule expression. Further study is needed to determine the mechanism(s) of IL-6-mediated adaptation to O3 exposure as well as its potential participation in lung adaptation to other environmental air pollutants.
| |
Footnotes |
|---|
Address correspondence to: Kevin L. Dreher, Pulmonary Toxicology Branch, NHEERL, MD 82, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. E-mail: Dreher.Kevin{at}epamail.epa.gov
(Received in original form February 14, 1997 and in revised form August 18, 1997).
Disclaimer: These studies were supported by the funds provided by the U.S. Environmental Protection Agency through a cooperative agreement with the Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, NC 27599. The research described in this article has been reviewed and approved for publication by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency. The contents do not necessarily reflect the views or the policies of the agency, nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.Acknowledgments: Results from this study have been presented at the 1994 and 1997 International Conference of the American Thoracic Society and published in abstract form (Am. J. Respir. Crit. Care Med. 1994;149:A156; Am. J. Respir. Crit. Care Med. 1997;155:A747). The authors thank Donald L. Doerfler (Research Support Division, U.S. Environmental Protection Agency) for statistical consultation, Joleen M. Soukup (Human Studies Division, U.S. EPA) for the quantification of IL-6, and Kay M. Crissman (Experimental Toxicology Division, U.S. EPA) for assisting in the quantification of antioxidants.
Abbreviations AA, ascorbic acid; anti-IL-6ra, anti-IL-6 receptor antibodies; APP, acute phase protein; BAL, bronchoalveolar lavage; CINC, cytokine-induced neutrophil chemoattractant; G6PDH, glucose-6-phosphate; GSH, glutathione; GSHpx, glutathione peroxidase; GSHrd, glutathione reductase; GSHtr, glutathione transferase; IL, interleukin; i.p., intraperitoneal(ly); i.t., intratracheal(ly); ra, receptor antibodies; SOD, superoxide dismutase; UA, uric acid.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1. Kehrl, H. R., L. M. Vincent, R. J. Kowalsky, D. H. Horstman, J. J. O'Neil, W. H. McCartney, and P. A. Bromberg. 1987. Ozone exposure increases respiratory epithelial permeability in humans. Am. Rev. Respir. Dis. 135: 1124-1128 [Medline].
2. Bhalla, D. K., R. C. Mannix, S. M. Lavan, R. F. Phalen, M. T. Kleinman, and T. T. Crocker. 1987. Tracheal and bronchoalveolar permeability changes in rats inhaling oxidant atmospheres during rest or exercise. J. Toxicol. Environ. Health 22: 417-437 [Medline].
3.
Seltzer, J.,
B. G. Bigby,
M. Stulborg,
M. J. Holtzman,
J. A. Nadel,
I. F. Ueki,
G. D. Leikauf,
E. J. Goetzl, and
H. A. Boushey.
1986.
Ozone-induced
change in bronchial reactivity to methacholine and airway inflammation in
humans.
J. Appl. Physiol
60:
1321-1326
4. Koren, H. S., R. B. Devlin, D. E. Graham, R. Mann, M. P. McGee, D. H. Horstman, W. J. Kozumbo, S. Becker, D. E. House, W. F. McDonnell, and P. A. Bromberg. 1989. Ozone-induced inflammation in the lower airways of human subjects. Am. Rev. Respir. Dis. 139: 407-415 [Medline].
5. Van Bree, L., P. J. A. Rombout, I. M. C. M. Rietjens, J. A. M. A. Dormans, and M. Marra. 1989. Pathobiochemical effects in rat lung related to episodic ozone exposure. In Atmospheric Ozone Research and its Policy Implications. T. Schneider, editor. Elsevier Science Publishers, Amsterdam. 723-732.
6. Devlin, R. B., W. F. McDonnell, R. Perez, S. Becker, D. E. House, and H. S. Koren. 1991. Prolonged exposure of humans to ambient levels of ozone causes cellular and biochemical changes in the lung. Am. Rev. Respir. Dis 4: 72-81 .
7. Wiester, M. J., J. S. Tepper, D. L. Doerfler, and D. L. Costa. 1994. Ozone adaptation in rats after chronic exposure to a simulated urban profile of ozone. Fundam. Appl. Toxicol 24: 42-51 .
8. Tepper, J. S., D. L. Costa, J. R. Lehmann, M. F. Weber, and G. E. Hatch. 1989. Unattenuated structural and biochemical alterations in the rat lung during functional adaptation to ozone. Am. Rev. Respir. Dis 140: 493-501 [Medline].
9.
Rahman, I. U.,
L. B. Clerch, and
D. Massaro.
1991.
Rat lung antioxidant
enzyme induction by ozone.
Am. J. Physiol. (Lung Cell Mol. Physiol. 4)
260:
L412-L418
10. Van Bree, L., M. Marra, and P. J. A. Rombout. 1992. Differences in pulmonary biochemical and inflammatory responses of rats and guinea pigs resulting from daytime or nighttime, single and repeated exposure to ozone. Toxicol. Appl. Pharmacol 116: 209-216 [Medline].
11.
Wiester, M. J.,
W. P. Watkinson,
D. L. Costa,
K. M. Crissman,
J. H. Richards,
D. W. Winsett, and
J. W. Highfill.
1996.
Ozone toxicity in the rat: III.
Effect of changes in ambient temperature on pulmonary parameters.
J.
Appl. Physiol
81:
1691-1700
12.
Alpert, S. M., and
T. R. Lewis.
1971.
Ozone tolerance studies utilizing unilateral lung exposure.
J. Appl. Physiol.
31:
243-246
13. Nambu, Z., and E. Yokoyama. 1983. Antioxidant system and ozone tolerance. Environ. Res 32: 111-117 [Medline].
14. Stokinger, H. E., W. D. Wagner, and P. G. Wright. 1956. Studies on ozone toxicity: I. Potentiating effects of exercise and tolerance development. Arch. Ind. Health 14: 158-162 .
15. Fairchild, E. J. II.. 1967. Tolerance mechanisms. Arch. Environ. Health 14: 111-126 [Medline].
16. Tsan, M. F., T. A. White, S. Y. Lee, and C. Y. Lee. 1991. Interleukin-1 protects rats against oxygen toxicity. J. Appl. Physiol. (Lung Cell Mol. Physiol. 2) 71: 688-697 .
17. Devlin, R. B., W. F. McDonnell, S. Becker, M. C. Madden, M. P. McGee, R. Perez, G. Hatch, D. E. House, and H. S. Koren. 1996. Time-dependent changes of inflammatory mediators in the lungs of humans exposed to 0.4 ppm ozone for two hours: a comparison of mediators found in bronchoalveolar lavage fluid 1 and 18 hours after exposure. Toxicol. Appl. Pharmacol 138: 176-185 [Medline].
18. Costa, D. L., J. S. Tepper, R. Devlin, M. Madden, G. E. Hatch, H. Koren, J. Boere, and P. Rombout. 1992. Differential sensitivity to acute ozone: a study of lung dysfunction and inflammation in three rat strains. The Toxicologist 12: 173 . (Abstr.) .
19. Van Bree, L., J. Wijnands, J. Dorman, R. Vandebriel, and H. Van Loveren. 1996. Differences in cytokine gene expression in a rat model of lung inflammation following acute and repeated exposure to ozone. Am. J. Respir. Crit. Care Med 153: A699 . (Abstr.) .
20. Arsalane, K., P. Gosset, D. Vanhee, C. Voisin, Q. Hamid, A. Tonnel, and B. Wallaert. 1995. Ozone stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro. Am. J. Respir. Cell Mol. Biol. 13: 60-68 [Abstract].
21.
Devlin, R.,
K. McKinnon,
T. Noah,
S. Becker, and
H. Koren.
1994.
Ozone-induced release of cytokines and fibronectin by alveolar macrophages and
airway epithelial cells.
Am. J. Physiol. (Lung Cell Mol. Physiol. 10)
266:
L612-L619
22. Denis, M.. 1992. Interleukin-6 in mouse hypersensitivity pneumonitis: changes in lung free cells following depletion of endogenous IL-6 or direct administration of IL-6. J. Leukoc. Biol 52: 197-201 [Abstract].
23.
Tsan, M.,
J. E. White,
P. J. Del Vecchio, and
J. B. Shaffer.
1992.
IL-6 enhances TNF-- and IL-1-induced increase of Mn superoxide dismutase
mRNA and O2 tolerance. Am. J. Physiol. (
Lung Cell Mol. Physiol. 7)
263:
L22-L26
.
24. Einarsson, O., B. F. DiCosmo, R. Flavell, and J. A. Elias. 1995. CC-10-IL-6 transgenic mice manifest enhanced tolerance to 100% oxygen. Am. Rev. Respir. Dis 151: A326 . (Abstr.) .
25. Kopf, M., H. Baumann, G. Freer, M. Freudenberg, M. Lamers, T. Kishimoto, R. Zinkernagel, H. Bluethmann, and G. Kohler. 1994. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368: 339-342 [Medline].
26.
Fattori, E.,
M. Cappelletti,
P. Costa,
C. Sellitto,
L. Cantoni,
M. Carelli,
R. Faggioni,
G. Fantuzzi,
P. Ghezzi, and
V. Poli.
1994.
Defective inflammatory response in interleukin 6-deficient mice.
J. Exp. Med
180:
1243-1250
27. DiCosmo, B. F., G. P. Geba, D. Picarella, J. A. Elias, J. A. Rankin, B. R. Stripp, J. A. Whitsett, and R. A. Flavel. 1994. Airway epithelial cell expression of interleukin-6 in transgenic mice: uncoupling of airway inflammation and bronchial hyperreactivity. J. Clin. Invest 94: 2028-2035 .
28. Coulie, P. G., A. Vink, and J. Van-Snick. 1990. A monoclonal antibody specific for the murine IL-6 receptor inhibits the growth of a mouse plasmacytoma in vivo. Curr. Top. Microbiol. Immunol 166: 43-46 [Medline].
29.
Vink, A.,
P. Coulie,
G. Warnier,
J. C. Renauld,
M. Stevens,
D. Donckers, and
J. Van-Snick.
1990.
Mouse plasmacytoma growth in vivo: enhancement by interleukin-6 (IL-6) and inhibition by antibodies directed against
IL-6 or its receptor.
J. Exp. Med
172:
997-1000
30. Hatch, G. E., R. Slade, A. G. Stead, and J. A. Graham. 1986. Species comparison of acute inhalation toxicity of ozone and phosgene. J. Toxicol. Environ. Health 19: 43-53 [Medline].
31. Bradford, M. M. A.. 1976. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem 72: 248-254 [Medline].
32. Kutnink, M. A., J. H. Skala, H. E. Sauberlich, and S. T. Omaye. 1985. Simultaneous determination of ascorbic acid, isoascorbic acid (erythorbic acid) and uric acid in human plasma by high-performance liquid chromatography with amperometric detection. J. Liq. Chromatogr 8: 31-46 .
33. Anderson, M. E.. 1985. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113: 548-555 [Medline].
34. Jaskot, R. H., E. G. Charlet, E. C. Grose, M. A. Grady, and J. A. H. Roycroft. 1983. An automated analysis of glutathione peroxidase, S-transferase, and reductase activity in animal tissue. J. Anal. Toxicol 7: 86-88 [Medline].
35. Mustafa, M. G., A. D. Hacker, J. J. Ospital, M. Z. Husain, and S. D. Lee. 1977. Biochemical effects of environmental oxidant pollutants in animal lungs. In Biochemical Effects of Environmental Pollutants. S. D. Lee, editor. Ann Arbor Science Publishing, Ann Arbor, MI. 75-96.
36. Minami, M., and H. Yoshikawa. 1979. A simplified assay method of superoxide dismutase activity for clinical use. Clin. Chim. Acta 92: 337-342 [Medline].
37.
Van Damme, J.,
G. Opdenakker,
R. J. Simpson,
M. R. Rubira,
S. Cayphas,
A. Vink,
A. Billiau, and
J. Van Snick.
1987.
Identification of the human
26-kD protein, interferon 2 (IFN-2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor.
J.
Exp. Med.
165:
914-919
38. Malek, F. Y., M. J. Utell, R. J. Looney, P. E. Morrow, P. C. Levy, Y. Tsai, R. Barth, and M. W. Frampton. 1995. Does exposure to ozone induce systemic acute phase response in humans? Am. Rev. Respir. Dis 151: A27 . (Abstr.) .
39. Tsan, M. F., T. A. White, S. Y. Lee, and C. Y. Lee. 1990. Tracheal insufflation of tumor necrosis factor protects rats against oxygen toxicity. J. Appl. Physiol. (Lung Cell Mol. Physiol. 3) 68: 1211-1219 .
40.
Koto, H.,
M. Salmon,
E. B. Haddad,
T. J. Huang,
J. Zargorski, and
K. F. Chung.
1997.
Role of cytokine-induced neutrophil chemoattractant
(CINC) in ozone-induced airway inflammation and hyperresponsiveness.
Am. J. Respir. Crit. Care Med
156:
234-239
41.
Afford, S. C.,
J. Pongracz,
R. A. Stockley,
J. Crocker, and
D. Burnett.
1992.
The induction by human interleukin-6 of apoptosis in the promonocytic
cell line u937 and human neutrophils.
J. Biol. Chem.
267:
21612-21616
42. Geiger, T., T. Andus, J. Klapporth, T. Hirano, T. Kishimoto, and P. C. Heinrich. 1988. Induction of rat acute-phase proteins by interleukin 6 in vivo. Eur. J. Immunol 18: 717-721 [Medline].
43. Marinkovic, S., G. P. Jahreis, G. G. Wong, and H. Baumann. 1989. IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo. J. Immunol 142: 808-812 [Abstract].
44. Kushner, I.. 1982. The phenomenon of the acute phase response. Ann. NY Acad. Sci. 389: 39-48 [Medline].
45. Kew, R. R., T. M. Hyers, and R. O. Webster. 1990. Human C-reactive protein inhibits neutrophils chemotaxis in vitro: possible implications for the adult respiratory distress syndrome. J. Lab. Clin. Med 115: 339-345 [Medline].
46. Heuertz, R. M., C. A. Piquette, and R. O.Webster. 1993. Rabbits with elevated serum C-reactive protein exhibit diminished neutrophil infiltration and vascular permeability in C5a-induced alveolitis. Am. J. Pathol. 142: 319-328.
47.
Heuertz, R. M.,
D. Xia,
D. Samols, and
R. O. Webster.
1994.
Inhibition of
C5a des arg-induced neutrophil alveolitis in transgenic mice expressing
C-reactive protein.
Am. J. Physiol. (Lung Cell Mol. Physiol. 10)
266:
L649-L654
48. Laskin, D. L., K. J. Pendino, C. J. Punjabi, M. Rodriguez del Valle, and J. D. Laskin. 1994. Pulmonary and hepatic effects of inhaled ozone in rats. Environ. Health Perspect. 102: 61-64 .
49. Yang, F., W. E. Friedrichs, A. L. Navarijo-Ashbaugh, L. A. Degraffenried, B. H. Bowman, and J. J. Coalson. 1995. Cell type-specific and inflammatory-induced expression of haptoglobin gene in lung. Lab. Invest 73: 433-440 [Medline].
50. Rahman, I., L. B. Clerch, and D. Massaro. 1991. Rat lung antioxidant enzyme induction by ozone. Am. J. Physiol. 260: L412-L418 .
51. Nambu, Z., and E. Yokoyama. 1982. Experimental suppression of tolerance to ozone and cross-tolerance to (NO2-O3) in rats by actinomycin D and colchicine. Environ. Res 29: 62-69 [Medline].
52. Oldenburg, H. S. A., M. A. Rogy, D. D. Lazarus, K. J. Van Zee, B. P. Keeler, R. A. Chizzonite, S. F. Lowrys, and L. L. Moldawer. 1993. Cachexia and the acute-phase protein response in inflammation are regulated by interleukin-6. Eur. J. Immunol 23: 1889-1893 [Medline].
53. Strassman, G., M. Fong, S. Windsor, and R. Neta. 1993. The role of interleukin-6 in lipopolysaccharide-induced weight loss, hypoglycemia and fibrinogen production in vivo. Cytokine 5: 285-292 [Medline].
54. May, L. T., M. I. Ndubuisi, K. Patel, and D. Garcia. 1995. Interleukin-6 chaperones in blood. Ann. NY Acad. Sci 762: 120-128 [Abstract].
55. Ulich, T. R., S. Yin, K. Guo, S. E. Yi, D. Remick, and J. D. Castillo. 1991. Intratracheal injection of endoxin and cytokines: II. Interleukin-6 and transforming growth factor beta inhibit acute inflammation. Am. J. Pathol 138: 1097-1101 [Abstract].
56. Maruo, N., I. Morita, M. Shirao, and S. Murota. 1992. IL-6 increases endo-thelial permeability in vitro. Endocrinology 131: 710-714 [Abstract].
This article has been cited by other articles:
 |
|
 |
|
  H.-Y. Cho, D. L. Morgan, A. K. Bauer, and S. R. Kleeberger Signal Transduction Pathways of Tumor Necrosis Factor-mediated Lung Injury Induced by Ozone in Mice Am. J. Respir. Crit. Care Med., April 15, 2007; 175(8): 829 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-Y. Chen, A. C. Bonham, C. G. Plopper, and J. P. Joad Plasticity in Respiratory Motor Control: Selected Contribution: Neuroplasticity in nucleus tractus solitarius neurons after episodic ozone exposure in infant primates J Appl Physiol, February 1, 2003; 94(2): 819 - 827. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-X. Wu, B. E. Satterfield, J. S. Fedan, and R. D. Dey Interleukin-1beta -induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L909 - L917. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. Bhalla, P. G. Reinhart, C. Bai, and S. K. Gupta Amelioration of Ozone-Induced Lung Injury by Anti-Tumor Necrosis Factor-{alpha} Toxicol. Sci., October 1, 2002; 69(2): 400 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Wesselkamper, L. C. Chen, S. R. Kleeberger, and T. Gordon Genetic variability in the development of pulmonary tolerance to inhaled pollutants in inbred mice Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1200 - L1209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Wesselkamper, L. C. Chen, and T. Gordon Development of Pulmonary Tolerance in Mice Exposed to Zinc Oxide Fumes Toxicol. Sci., March 1, 2001; 60(1): 144 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. CHO, J. A. HOTCHKISS, C. B. BENNETT, and J. R. HARKEMA Neutrophil-Dependent and Neutrophil-Independent Alterations in the Nasal Epithelium of Ozone-Exposed Rats Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): 629 - 636. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Crit. Care Med. |