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Am J Physiol Lung Cell Mol Physiol 289: L111-L124, 2005. First published March 25, 2005; doi:10.1152/ajplung.00318.2004
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Activation of the aryl hydrocarbon receptor increases pulmonary neutrophilia and diminishes host resistance to influenza A virus

Sabine Teske,1 Andrea A. Bohn,2 Jean F. Regal,3 Joshua J. Neumiller,1 and B. Paige Lawrence1,2

1Department of Pharmaceutical Sciences, Pharmacology/Toxicology Graduate Program, College of Pharmacy, and 2Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington; and 3Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, Minnesota

Submitted 25 August 2004 ; accepted in final form 18 March 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Unlike their role in bacterial infection, less is known about the role of neutrophils during pulmonary viral infection. Exposure to pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) results in excess neutrophils in the lungs of mice infected with influenza A virus. TCDD is the most potent agonist for the aryl hydrocarbon receptor (AhR), and exposure to AhR ligands has been correlated with exacerbated inflammatory lung diseases. However, knowledge of the effects of AhR agonists on neutrophils is limited. Likewise, the factors regulating neutrophil responses during respiratory viral infections are not well characterized. To address these knowledge gaps, we determined the in vivo levels of KC, MIP-1{alpha}, MIP-2, LIX, IL-6, and C5a in infected mouse lungs. Our data show that these neutrophil chemoattractants are generally produced transiently in the lung within 12–24 h of infection. We also report that expression of CD11a, CD11b, CD49d, CD31, and CD38 is increased on pulmonary neutrophils in response to influenza virus. Using AhR-deficient mice, we demonstrate that excess neutrophilia in the lung is mediated by activation of the AhR and that this enhanced neutrophilia correlates directly with decreased survival in TCDD-exposed mice. Although AhR activation results in more neutrophils in the lungs, we show that this is not mediated by deregulation in levels of common neutrophil chemoattractants, expression of adhesion molecules on pulmonary neutrophils, or delayed death of neutrophils. Likewise, exposure to TCDD did not enhance pulmonary neutrophil function. This study provides an important first step in elucidating the mechanisms by which AhR agonists exacerbate pulmonary inflammatory responses.

pulmonary inflammation; chemokines; adhesion molecules; aryl hydrocarbon receptor-knockout mice; dioxin

DESPITE OF ADVANCES IN HEALTH CARE, morbidity and mortality from respiratory pathogens have not declined in the past 20 years, and the incidence of chronic inflammatory lung diseases has increased. According to the World Health Organization, respiratory diseases are among the top 10 leading causes of illness and death, resulting in approximately four million deaths annually (100b). Among lower respiratory tract infections, infection with influenza virus constitutes one of the major factors in this morbidity and mortality (100a). In addition to infectious agents, chronic inflammatory disorders of the lung further contribute to the burden caused by respiratory diseases. For example, chronic obstructive pulmonary disease (COPD) is a group of diseases of unknown etiology characterized by airflow obstruction and inflammation. COPD is the third leading cause of mortality in elderly adults, and in the United States alone, current estimates indicate that ~31.3 million people have been diagnosed with this disease (4, 55).

Although it is not clear why certain individuals are more susceptible to chronic inflammatory diseases of the lung, one etiologic factor is infectious disease. In particular, defects in the ability to fight respiratory infections correlate with enhanced incidence and severity of chronic inflammatory disorders of the lower respiratory tract (40, 79). In addition, increasing evidence exists that exposure to pollutants increases inflammatory responses in the lung (19, 24–26, 31). The mechanisms by which infectious agents or pollutants increase pathology in the lung are not entirely understood, but knowledge of the effects of pollutants on the pulmonary immune system is an important consideration in our efforts to reduce the incidence and severity of pulmonary inflammatory diseases.

One category of pollutants known to affect the lung are ligands for the aryl hydrocarbon receptor (AhR). The AhR is an orphan nuclear receptor that is widely expressed in mammalian tissue, including the lung and cells of the immune system (21, 42). Activation of AhR by exogenous compounds is of interest for several reasons. First, ligands for the AhR include an extensive list of exogenous compounds, many of which are very abundant and persistent airborne pollutants. In particular, agonists for the AhR include dioxin-like compounds and polyaromatic hydrocarbons (PAH) found in cigarette smoke and diesel exhaust. Second, the AhR is a member of the per-arnt-sim protein family of basic helix-loop-helix transcription regulators. This family of proteins controls a wide spectrum of complex biological processes that includes toxin metabolism, circadian rhythms, the response to hypoxia, cell cycle progression, and cell lineage commitment (18, 62, 72). Therefore, inappropriate AhR activation affects numerous biological processes. Finally, many AhR ligands have well-characterized immunomodulatory properties (9, 10, 22, 32, 35, 93).

Of all the AhR ligands identified to date, the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) has the highest affinity for the receptor and has therefore been used for many studies characterizing the toxic and biological effects of AhR activation. In addition to being a prototypic AhR ligand, TCDD is among the 33 chemicals listed as priority air toxics in the U.S. Environmental Protection Agency's Integrated Urban Air Toxics Strategy. The potent suppressive effects of TCDD on the cell-mediated and humoral immune responses to a large variety of pathogenic and nonpathogenic antigens have been particularly well characterized (33). Furthermore, studies using AhR ligands that vary in receptor affinity and using AhR-deficient mice indicate that impaired function of the adaptive immune system is mediated by and dependent on activation of the AhR (35, 37, 101).

In contrast to suppression of adaptive immune responses, our laboratory (96, 99) and others (15, 36, 53, 58) have reported that exposure to TCDD enhances inflammatory responses. Although the levels of inflammatory cells and molecules affected by exposure to TCDD vary among experimental systems, this exacerbated inflammatory response is consistently characterized by an increase in the number of neutrophils at the site of antigen challenge. With regard to the lung, exposure to TCDD results in twice as many neutrophils in airways of infected mice compared with vehicle control-treated, infected mice (53, 96, 99). This effect of TCDD on neutrophils is observed exclusively in the context of infection, suggesting that exposure to TCDD alters the immunoregulatory balance in the lung, such that upon infection, adaptive responses are suppressed and inflammatory responses are enhanced.

Enhanced inflammatory responses characterized by excess numbers of neutrophils constitute an important component of the pathology associated with inflammatory lung diseases. Neutrophils produce cytotoxic molecules, such as reactive oxygen species (ROS) and degradative enzymes. Although controlled production of these cytotoxic mediators aids the host and helps to control infection, excess levels are detrimental (3, 56, 87). Thus enhanced numbers or function of neutrophils leads to increased production of cytotoxic mediators, which ultimately contribute to the pathology observed in inflammatory lung diseases (3, 13, 31, 38, 68, 73).

We have shown that infected mice treated with TCDD exhibit a dose-dependent decrease in host resistance to infection with a nonlethal dose of virus (96, 99). Viral titer assays have shown that these mice do not die from excess viral burden. In fact, TCDD-treated mice clear the virus from their lungs with kinetics similar to vehicle-treated mice (45, 60). Moreover, although CD8+ T cells are fewer in number, influenza virus is cleared by a CD8+ T cell-dependent mechanism (60). Given that exposure to TCDD does not impair viral clearance mechanisms, another mechanism likely accounts for the impaired host resistance. We believe that, after infection with influenza virus, increased numbers of pulmonary neutrophils are detrimental to the host.

Knowledge of the effects of AhR agonists on neutrophils or on factors that control neutrophil migration is highly limited. Likewise, there is relatively little known regarding the in vivo levels of neutrophil chemoattractants in the mouse lung during infection with influenza A virus. To address these gaps in knowledge, we conducted experiments to characterize the levels of common neutrophil chemoattractants in the lung over the course of infection with influenza virus. In the presented studies, we also determined whether there is a positive correlation between enhanced neutrophil numbers in the lung and decreased survival following infection observed in TCDD-treated mice and whether the exacerbated pulmonary neutrophilia occurs through an AhR-dependent mechanism. We then sought to examine whether exposure to TCDD enhances the level of neutrophil chemoattractants in the lung, alters neutrophil function or alters the expression of adhesion molecules on pulmonary neutrophils. In addition to examining the relationship between AhR activation and deregulation of multiple components of the pulmonary innate immune response, our findings provide novel information regarding the in vivo kinetics and levels of common neutrophil chemoattractants in the lungs of mice infected with influenza A virus.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Animals. Female C57BL/6 mice (6–8 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) or NCI-Frederick (Frederick, MD). Breeding pairs of C57BL/6-AhR-deficient (AhR–/–) mice (deficient for exon 2 of AhR) (78) were purchased from The Jackson Laboratory. The female AhR-deficient mice used in this study were obtained by breeding AhR-heterozygous (AhR–/+) females with AhR–/+ males. We extracted DNA from ear punches (7) and used PCR to determine AhR status of offspring using the following primers: CAG TGG GAA TAA GGC AAG AGT GA and AGG GAG ATG AAG TAT GTG TAT GTA (Qiagen, Alameda, CA). Samples from an AhR–/– mouse yield a 260-bp product, whereas DNA from an AhR+/+ mouse yields a 300-bp product. AhR–/+ mice have both the 260- and 300-bp products. The PCR conditions were as follows: 95°C for 5 min, 30 cycles at 95°C for 30 s, 30 cycles at 60°C for 30 s, 30 cycles at 72°C for 30 s, 72°C for 5 min. Gel electrophoresis of 20 µl of PCR products was performed on a 2.5% NuSieve 3:1 agarose gel (Cambrex Bio Science Rockland, Rockland, ME).

All mice were housed under pathogen-free conditions in microisolator units (three to five mice per cage) and provided with water and food ad libitum. All animal treatment was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee.

Animal treatment. TCDD (≥99% pure; Cambridge Isotope Laboratories, Woburn, MA) was dissolved in anisole and diluted in peanut oil to 10 µg/ml. Mice were gavaged with a single dose of TCDD (10 µg/kg body wt). Vehicle-control mice received a single dose of peanut oil-anisole vehicle. One day following exposure to vehicle or TCDD, mice were infected intranasally (i.n.) with murine-adapted human influenza A virus, strain HKx31 (H3N2). Mice received 120 hemagglutinating units (HAU) influenza virus in a final volume of 25 µl of endotoxin-tested PBS under general anesthesia (Avertin, 2,2,2-tribromoethanol; Aldrich, Milwaukee, WI). Infection with 120 HAU does not usually cause mortality in vehicle-treated control mice (45, 99). Mock-infected mice were treated with vehicle or TCDD and given 25 µl of endotoxin-tested PBS (i.n.). Data from these mice are defined in all figures as the zero time point. Mice were killed on multiple days after infection by either anesthetic overdose or CO2 asphyxiation. Depending on the experiment, immune cells were collected from the airways only or from both airways and interstitial spaces.

Bronchoalveolar lavage cells and fluid. Lungs were lavaged as previously described (99). In brief, three sequential washes with serum-free RPMI 1640 medium containing 1% BSA and 10 mM HEPES were performed. The first wash was collected separately [bronchoalveolar lavage (BAL) fluid]. BAL cells were separated from BAL fluid by centrifugation. BAL fluid was stored at –80°C until further analysis. BAL cells from all washes were pooled and enumerated using a Coulter counter (Beckman Coulter, Miami, FL).

Total lung-derived immune cells. To obtain total lung-derived immune cells, lungs were digested with collagenase (48). Lungs were incubated for 25 min at 37°C, 5% CO2 with RPMI 1640 containing 2.5% FBS (Hyclone, Logan, UT), 1 mg/ml collagenase type 2 (Worthington Biochemical, Lakewood, NJ), and 30 µg/ml deoxyribonuclease I (Sigma-Aldrich, St. Louis, MO). After digestion with collagenase, the lung cell suspension was layered over Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada) and centrifuged (900 g) for 20 min at room temperature to separate immune cells from parenchymal cells, dead cells, and erythrocytes. Lung-derived immune cells were counted with a Coulter counter.

Lung homogenates. Lavaged lungs were homogenized in 1.5 ml ice-cold sucrose buffer containing Tris and protease inhibitors [0.25 M sucrose, 10 mM Tris, pH 7.4, with 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin]. PMSF, aprotinin, and leupeptin were purchased from Sigma-Aldrich. After the homogenization, debris was removed by centrifugation (8,000 g) for 10 min at 4°C. The protein concentration of the lung homogenates was determined with the Pierce BCA assay (Pierce Biotechnology, Rockford, IL).

In vivo depletion of neutrophils. To deplete neutrophils (Gr-1+ cells) in vivo, we used a rat monoclonal antibody Gr-1 (RB6–8C5; Ly-6G), which targets the neutrophil surface antigen Gr-1 (15, 90). This antibody was generously provided by Drs. Robert Coffman (Dynavax Technologies, Berkeley, CA) and Nancy Kerkvliet (Oregon State University, Corvallis, OR). Preliminary studies were conducted to define an effective dosing regimen of anti-Gr-1. Mice were treated with vehicle or TCDD 1 day before intraperitoneal administration of 300 µg anti-Gr-1 or rat IgG control (Jackson ImmunoResearch Laboratories, West Grove, PA). Two hours after antibody administration, mice were infected with 120 HAU influenza virus i.n. Mice received a second 300-µg dose of anti-Gr-1 or rat IgG control 4 days after infection. Depletion efficacy was determined by flow cytometry and differential cell counts and was >80%.

Lung histology. Vehicle- or TCDD-treated mice given the anti-Gr-1 antibody or rat IgG control were killed 7 days after infection with influenza virus. Lungs were tracheally perfused with 10% buffered formalin, excised, and placed into the fixative. After fixation, the lungs were paraffin embedded, and 5-mm tissue slices were cut, mounted, and stained with hematoxylin and eosin at the Washington Animal Disease Diagnostic Laboratory and Center for Reproductive Biology (Washington State University, Pullman, WA).

Immunophenotypic analyses. Cells were stained with the following combinations of monoclonal antibodies purchased from BD Pharmingen (San Diego, CA) or Caltag Laboratories (Burlingame, CA): biotinylated anti-CD11a (lymphocyte function-associated antigen-1), FITC-labeled anti-CD11b (Mac-1), FITC-labeled anti-CD31 (platelet-endothelial cell adhesion molecule-1), biotinylated anti-CD38, biotinylated anti-CD49d (very late antigen-4), FITC-labeled anti-Gr-1, and phycoerythrin (PE)-labeled anti-Gr-1. SpectralRed (SPRD)-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, AL) was used as the secondary reagent for the biotinylated antibodies. Appropriately labeled, isotype-matched antibodies were used as negative controls for nonspecific fluorescence. Data from 20,000–50,000 cells were collected by list-mode acquisition, using a FACSort flow cytometer (Becton Dickenson, San Jose, CA). Data analyses were performed using WinList software (Verity Software, Topsham, ME).

ROS detection. Detection of ROS levels in neutrophils was assessed as described previously (44). In brief, BAL cells were incubated with either 6-carboxy-2',7'-dichlorofluorescein diacetate (DCFH-DA) or dihydroethidine (HE; both from Molecular Probes, Eugene, OR) for 15 min at 37°C in the dark. Excess dye was removed by sequential washes with PAF (endotoxin-tested PBS containing 0.02% sodium azide and 4% FBS). The BAL cells were stained with PE-labeled anti-Gr-1 for the DCF staining set or FITC-labeled anti-Gr-1 for the HE staining set and analyzed using a FACSort flow cytometer.

Myeloperoxidase activity. Myeloperoxidase (MPO) activity was assessed according to LeVine et al. (49). Briefly, we used a neutrophil-specific gradient isolation medium, Mono-Poly resolving medium (ICN Biomedicals, Aurora, OH), to obtain neutrophils from BAL cells. For the vehicle and TCDD treatment group, the neutrophil-specific fraction contained 50% neutrophils. Neutrophils were suspended in a final volume of 50 µl of hexadecyltrimethylammonium bromide and incubated for 1 h at 37°C to allow for release of MPO. After the incubation, 100 µl of freshly prepared assay buffer containing 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide were added to each well. Absorbance readings (optical density) were taken immediately on a microplate reader for a period of 4 min in intervals of 12 s.

Cytokine and chemokine analyses. Cytokines and chemokines were analyzed using matched antibody pairs in sandwich ELISAs. ELISA reagents for macrophage inflammatory protein (MIP)-1{alpha}, MIP-2, and keratinocyte chemoattractant (KC) were supplied by R&D Systems (Minneapolis, MN). IL-6 ELISA reagents were purchased from Pierce Biotechnology. ELISAs were performed according to the manufacturer's recommended protocols. The limits of detection were 125 pg/ml for MIP-1{alpha}, 31 pg/ml for MIP-2 and KC, and 250 pg/ml for IL-6. The lipopolysaccharide-induced CXC chemokine (LIX)-specific ELISA was performed in the laboratory of Dr. Daniel Remick (Univ. of Michigan, Ann Arbor, MI). The limit of detection for the LIX ELISA was 20 pg/ml.

Complement split product C5a analysis. BAL fluid was collected by lavaging mice once with 1 ml of endotoxin-tested PBS. Levels of C5a were measured in BAL fluid by immunoblotting (54), which was modified from previous studies for guinea pig C3a (74). C5a in BAL fluid was separated from intact C5 by SDS-PAGE under denaturing conditions using a 20% acrylamide gel (41). Proteins were electrophoretically transferred to a 0.2-µm nitrocellulose membrane (BA-S 83; Schleicher & Schuell, Keene, NH). The primary antibody used for immunodetection was the IgG fraction of a rabbit polyclonal antibody to the 15 carboxy-terminal amino acids of murine C5a. The nitrocellulose blot was incubated in 3% BSA overnight at 4°C and probed with anti-C5a-peptide antibody at 1:2,500 dilution for 2 h at 25°C followed by goat anti-rabbit IgG coupled to horseradish peroxidase at 1:10,000 dilution (Pierce Biotechnology). ECL Plus reagents were used to develop the blot (Amersham Biosciences UK, Buckinghamshire, UK). Images of light emission were recorded on X-ray film, digitized, and quantified by densitometric analysis using Scion Image for Windows (public domain NIH Image program, developed at the U.S. National Institutes of Health). A standard pool of yeast-activated complement (YAC) was prepared by activating pooled mouse serum with yeast cell walls. A dilution series of YAC was used to construct a standard curve and regression equation for each immunoblot. Relative amounts of C5a in each sample are expressed as YAC equivalents, based on the signal intensity of YAC dilution.

Neutrophil apoptosis and necrosis. To identify neutrophils, we stained cells with biotinylated anti-Gr-1 followed by allophycocyanin-conjugated streptavidin (BD Pharmingen). Cells were then stained with FITC-labeled annexin V and 7-amino-actinomycin to assess apoptosis and necrosis (BD Pharmingen) (59). For the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining method, the in situ cell death detection kit was purchased from Roche Diagnostics (Indianapolis, IN). To identify neutrophils using the TUNEL staining method, cells were stained with biotinylated anti-Gr-1 followed by SPRD-conjugated streptavidin (BD Pharmingen; Southern Biotechnology Associates), and Gr-1+ TUNEL+ cells were enumerated by flow cytometry.

Statistical analysis. All statistical analyses were performed using StatView statistical software (SAS, Cary, NC). Significant treatment effects were determined by one-way ANOVA, followed by a Fisher's protected least significant difference post hoc test, to compare the mean values from each treatment group at a specific point in time. To examine infection-associated changes, the mean values in a treatment group were compared over time within that group. Values of P ≤ 0.05 serve as the basis for designation of statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In vivo depletion of neutrophils improves the survival of TCDD-treated mice infected with influenza A virus. In previous studies, we have found that neutrophil numbers are increased in the airways of TCDD-treated mice infected with influenza virus (96, 99) and that this excess neutrophilia peaks ~7 days after infection (99). The same phenomenon was observed when we examined the number of neutrophils in total lung-derived immune cells (i.e., airways and interstitial spaces). That is, exposure to TCDD increases the number of neutrophils in the interstitial spaces as well as the airways, and this increase peaks 7 days after infection (Fig. 1A).



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  		Fig. 1. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to excess pulmonary neutrophilia, decreased host resistance to influenza virus, and severe bronchopneumonia. A: mice were treated with vehicle (open bars) or 10 µg/kg of TCDD (solid bars) 1 day before infection with 120 hemagglutinating units (HAU) influenza virus i.n. Mice were killed on the indicated days relative to infection, and lungs were digested with collagenase to isolate immune cells. Using flow cytometry, the number of Gr-1+ cells in the lungs of vehicle- or TCDD-treated mice was determined (6 mice per treatment group/day). Mock-infected mice treated with vehicle or TCDD were used as controls (4 mice per treatment group), and average values from these groups are shown at the zero time point. *Significant difference from the vehicle group (P ≤ 0.05). B: mice (20 per group) were treated with vehicle or TCDD 1 day before infection with influenza virus. The next day, 300 µg of anti-Gr-1 or rat IgG control were administered (i.p.) 2 h before infection with 120 HAU influenza virus (strain HKx31). A second 300-µg dose of anti-Gr-1 or rat IgG control was given 4 days after infection. Neutrophil depletion efficacy was >80%. Survival was monitored for all 4 treatment groups until 14 days after infection. C: mice (5 per group) were treated as described in A and killed 7 days after infection for histological examination of the lungs. Photomicrographs of representative fields were taken with an Olympus MicroFire digital camera. Hematoxylin and eosin stain; x100 magnification.

 
Concomitant with the elevated neutrophilia in the lungs of TCDD-treated mice, we have repeatedly observed a decrease in survival following a nonlethal infection with influenza virus compared with vehicle-exposed mice (45, 96, 99). This observation prompted us to investigate whether a relationship exists between excess number of neutrophils and impaired survival after infection with influenza virus. To accomplish this, we depleted neutrophils in mice treated with vehicle or TCDD using the monoclonal antibody anti-Gr-1 and monitored survival for 14 days after infection. Interestingly, in vivo depletion of neutrophils did not detrimentally affect survival in vehicle-treated mice (Fig. 1B). Consistent with our previous observations (45, 96, 99), survival was severely compromised in mice treated with TCDD (Fig. 1B). In contrast, depletion of neutrophils improved survival from 40% in the TCDD-treated, rat IgG group to 70% in the TCDD-treated, neutrophil-depleted group (Fig. 1B). These data strongly suggest that the excess number of neutrophils in the lungs of mice exposed to TCDD impairs survival following infection with influenza virus.

In addition to monitoring host resistance, we compared lung tissue from mice treated with vehicle or TCDD to investigate whether neutrophil depletion reduces lung pathology (Fig. 1C). We examined lungs on day 7, the peak day of enhanced neutrophilia in TCDD-treated mice. Bronchointerstitial pneumonia was present in all treatment groups. Administration of anti-Gr-1 ablated the severity of the cellular infiltrate observed in TCDD-treated, infected mice relative to those given the rat IgG control (Fig. 1C).

The increase in neutrophil number in TCDD-treated mice is AhR dependent. Studies using AhR-deficient mice have shown that the AhR directly mediates suppression of adaptive immune responses caused by exposure to TCDD (37, 98). Based on this, we hypothesized that the excess neutrophilia and the underlying decrease in survival in TCDD-treated mice is likely mediated by the AhR. Because there are no studies to date investigating whether TCDD-mediated neutrophilia is AhR dependent, we treated AhR-deficient mice with vehicle or TCDD 1 day before infection and measured the number of Gr-1+ cells in the lungs 7 days after infection. In contrast to TCDD-treated wild-type mice, AhR-deficient mice exposed to TCDD did not have excess numbers of neutrophils in the lungs after challenge with influenza virus (Fig. 2). Interestingly, no mortality was observed in infected AhR-deficient mice treated with TCDD, whereas ~60% of infected wild-type mice treated with TCDD died by day 7 (data not shown). These findings indicate that activation of the AhR directly mediates the excessive numbers of neutrophils in the lungs of infected mice.



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  		Fig. 2. Activation of the aryl hydrocarbon receptor (AhR) mediates the enhanced neutrophilia in the lungs of TCDD-exposed mice. Mice were treated as described in Fig. 1. The average number of Gr-1+ cells in the lungs of infected AhR wild-type (AhR+/+) and AhR-deficient (AhR–/–) mice was determined on day 7 postinfection, the peak day of enhanced neutrophilia in TCDD-treated wild-type mice. Results are representative of 3 separate experiments. In each experiment, there were 2–4 mice in each group. Error bars represent SE. *Significant difference compared with vehicle (Veh) treatment group (P ≤ 0.05).

 
Exposure to TCDD does not alter neutrophil function. During infection with influenza virus, neutrophils produce ROS (27). Although some levels of these ROS are host beneficial, excess levels damage healthy host tissue and are detrimental (56, 87). Some reports show that exposure to TCDD leads to an increased production of ROS in mice (80, 81). Therefore, we examined whether exposure to TCDD enhances the production of ROS by pulmonary neutrophils by staining with DCF, an indicator of hydrogen peroxide production (5), and HE, an indicator of superoxide anion production (66). We observed a very small increase in the percentage and mean channel fluorescence (MCF), an indicator of staining intensity, of DCF+ neutrophils on days 5 and 9 postinfection in mice treated with TCDD (Fig. 3, A and B). Exposure to TCDD also caused a very slight increase in the percentage of HE+ neutrophils on days 5 and 7 postinfection (Fig. 3C). However, the MCF of the HE+ neutrophils was equivalent in both treatment groups throughout the course of infection (Fig. 3D), suggesting that on a per cell basis, superoxide anion levels are not different in neutrophils derived from mice treated with vehicle or TCDD.



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  		Fig. 3. Pulmonary neutrophils from vehicle- and TCDD-treated mice are functionally equivalent. Mice were treated as described in Fig. 1. On the indicated days after infection, mice were killed, and bronchoalveolar lavage (BAL) cells were collected (5–7 mice per treatment group/day). With flow cytometry, the percentage of neutrophils (Gr-1+ cells) that stained positive for DCF (dichlorofluorescein, A) and dihydroethidine (HE, C) was examined in infected mice treated with vehicle (open bars) or TCDD (solid bars). Mean channel fluorescence (MCF) of DCF+ neutrophils (B) and HE+ neutrophils (D) was also assessed. E: myeloperoxidase (MPO) activity, an indicator of neutrophil function, was measured 7 days after infection in a total of 50,000 neutrophil-enriched BAL cells. Bars represent the average for the vehicle (3 mice) and TCDD treatment group (4 mice). Error bars represent SE. *Significant difference compared with vehicle-treated mice (P ≤ 0.05).

 
Another method to examine neutrophil function is to measure MPO activity. MPO is an enzyme unique to the cytoplasmic granules of neutrophils and catalyzes the formation of hypochlorous acid from hydrogen peroxide and chloride anion (75). We assessed MPO activity in lung-derived neutrophils 7 days after infection (Fig. 3E) and found that cells from vehicle- or TCDD-treated mice exhibited equivalent MPO activity. Given that hydrogen peroxide and superoxide anion levels were unchanged or only very slightly elevated and MPO levels were equivalent, we conclude that exposure to TCDD enhances the number of neutrophils in the lungs of infected mice, but they appear functionally equivalent to neutrophils from vehicle-treated, infected mice.

Profile of neutrophil chemoattractants in the lungs of mice infected with influenza A virus. To delineate the mechanism underlying the excess neutrophilia, we examined the chemokines KC, MIP-1{alpha}, and MIP-2, which have well-characterized chemoattractant properties for neutrophils (20, 85, 94). In response to infection, we found that levels of KC, MIP-1{alpha}, and MIP-2 in lung lavage fluid rapidly and coordinately reached peak levels at 12 h and returned to baseline after 24 h (Fig. 4, A–F). In addition to lung lavage fluid, we assessed levels of KC, MIP-1{alpha}, and MIP-2 in lung homogenates at 12 h and 1, 3, 5, 7, and 9 days after infection (Fig. 4, G and H). KC was present in lung homogenates throughout the course of infection; however, there was no effect of exposure to TCDD on the amount of KC (Fig. 4G). Very low levels of MIP-1{alpha} were detected in homogenates throughout the course of infection (Fig. 4H), and levels of MIP-2 were below the limit of detection in lung homogenates (data not shown).



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  		Fig. 4. Kinetics of chemokine induction in response to infection with influenza virus. Mice were treated as described in Fig. 1. The levels of keratinocyte chemoattractant (KC; A, D, G), macrophage inflammatory protein (MIP-1{alpha}; B, E, H), and MIP-2 (C, F) were measured by ELISA in BAL fluid or lung homogenates derived from 3 separate time course studies: I) 1.5–24 h postinfection in BAL fluid (3–4 mice per treatment group/day); II) days 1 to 9 postinfection in BAL fluid (6–7 mice per treatment group/day); and III) 12 h–9 days after infection in lung homogenates (7–8 mice per treatment group/day). Mock-infected mice treated with vehicle or TCDD served as controls (3–4 mice per treatment group for experiments I and II; 6 mice per treatment group for experiment III). Error bars represent SE. *Significant difference compared with the vehicle treatment group (P ≤ 0.05); #P ≤ 0.09.

 
In a separate study we measured the levels of LIX, another chemokine with neutrophil-recruiting properties (14). Although a less extensive time course was performed, our data show that infection with influenza virus enhances levels of LIX in lung lavage fluid (Fig. 5). However, exposure to TCDD did not further augment levels of LIX compared with the vehicle treatment group.



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  		Fig. 5. Treatment with TCDD does not enhance the levels of LPS-induced CXC chemokine (LIX) in BAL fluid of infected mice. Mice were treated as described in Fig. 1. On days 5, 8, and 9 after infection, BAL fluid was collected from vehicle ({circ}) or TCDD-treated ({bullet}) mice (7–8 mice per treatment group/day). The level of LIX was measured by ELISA. Mock-infected mice treated with vehicle or TCDD served as controls (2 mice per treatment group). Error bars represent SE.

 
Another soluble mediator with neutrophil-recruiting properties is the cytokine IL-6 (30). Infection with influenza virus significantly increased levels of IL-6 in lung lavage fluid in both treatment groups (Fig. 6). This increase occurred very rapidly; however, unlike the other chemokines, it did not decline rapidly. We also measured IL-6 in lung homogenates at 12 h and 1, 3, 5, 7, and 9 days after infection in mice treated with vehicle or 10 µg/kg TCDD. In contrast to IL-6 levels in lung lavage fluid, levels of IL-6 in lung homogenates were below the limit of detection (data not shown).



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  		Fig. 6. Levels of IL-6 are elevated in lung lavage fluid throughout infection with influenza; however, exposure to TCDD does not increase the levels of IL-6. Mice (6–8 mice per treatment group/day) were treated with vehicle ({circ}) or TCDD ({bullet}) and infected 1 day later. On the indicated days relative to infection, mice were killed, and the level of IL-6 was assessed in lung lavage fluid by ELISA. Mock-infected mice treated with vehicle or TCDD served as controls (5 mice per treatment group). Results are representative of 4 separate experiments. Error bars represent SE.

 
In addition to these chemokines and cytokines, we examined complement split product C5a, which is not only chemoattractant for neutrophils (16) but is also elevated in the upper airways of humans during infection with influenza virus (8). We measured levels of C5a in lung lavage fluid throughout the course of infection in mice treated with vehicle or TCDD (Fig. 7). Surprisingly, neither infection with influenza virus nor exposure to TCDD enhanced levels of C5a, suggesting that mice may differ from humans with regard to enhanced levels of C5a during the immune response to influenza virus. Alternatively, differences in C5a levels between the upper and lower respiratory tract could account for the different expression profiles in our study.



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  		Fig. 7. C5a is constitutively present in BAL fluid. Mice (6–8 per treatment group/day) were treated as described in Fig. 1. On the indicated days after infection, levels of C5a in BAL fluid from mice treated with vehicle ({circ}) or TCDD ({bullet}) were measured by immunoblot. Levels of C5a were quantified as yeast-activated complement (YAC) equivalents. Mock-infected mice treated with vehicle (5 mice) or TCDD (6 mice) served as controls. Error bars represent SE.

 
In summary, the majority of neutrophil-recruiting mediators examined are produced rapidly after infection with influenza virus. Whereas increased levels of IL-6 were sustained throughout the course of infection, levels of KC, MIP-1{alpha}, and MIP-2 were no longer detected in lung lavage fluid after ~1 day. Together, these results indicate that these molecules are produced transiently at the site of infection and that their production occurs very early (within 48 h) following infection. Given this time frame and the levels detected, it is unlikely that altered expression of these molecules drives the excessive neutrophilia in the lungs of mice exposed to TCDD. In fact, if anything, levels of KC, MIP-1{alpha}, and MIP-2 were reduced in BAL fluid from TCDD-treated mice.

Infection with influenza virus induces expression of CD31, CD38, and CD49d on pulmonary neutrophils; however, treatment with TCDD does not markedly alter expression of these adhesion molecules. Some adhesion molecules important for neutrophil migration are CD11a, CD11b, CD31, CD38, and CD49d (64, 82, 84, 88). The effects of AhR agonists on the expression of adhesion molecules on neutrophils have not been previously characterized. However, it is known that treatment with TCDD alters expression of adhesion molecules on dendritic cells (97), suggesting that activation of the AhR potentially affects expression of adhesion molecules on other immune cells. Based on this logic, we examined whether treatment with TCDD increases expression of adhesion molecules on pulmonary neutrophils following infection with influenza virus. Our data indicate that CD11a and CD11b are constitutively expressed on neutrophils in the lung, whereas CD49d, CD31, and CD38 are not expressed on pulmonary neutrophils in the absence of infection (Fig. 8). Furthermore, infection increased the expression of all of these molecules on neutrophils in the lung. However, activation of the AhR by TCDD did not markedly upregulate the expression of any of these adhesion molecules on pulmonary neutrophils. Therefore, altered levels in the expression of these adhesion molecules do not offer a plausible mechanistic explanation for the enhanced pulmonary neutrophilia observed in TCDD-treated mice.



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  		Fig. 8. Expression profile of adhesion molecules on pulmonary neutrophils in response to infection with influenza virus. Mice (6 per treatment group/day) were treated as described in Fig. 1. To clear the pulmonary cavity of vascular blood, we flushed lungs with 3 ml of endotoxin-tested PBS containing 10 U/ml heparin before digestion with collagenase. A: representative histograms depict the staining of pulmonary neutrophils (Gr-1+ cells) with antibodies against the indicated adhesion molecule on day 7. The dashed gray line depicts background staining with an isotypic control antibody, and the solid gray and solid black lines indicate the staining of Gr-1+ cells from vehicle- and TCDD-treated mice, respectively. B, C: MCF and percentage of each adhesion molecule on Gr-1+ cells from lungs of mice treated with vehicle ({circ}) or TCDD ({bullet}) was examined 5, 7, and 8 days after infection. Mock-infected mice treated with vehicle or TCDD were used as controls (4 mice per treatment group). Error bars represent SE. *Significant difference compared with vehicle-treated mice (P ≤ 0.05). #P ≤ 0.09. We obtained the same results in a separate study in which the pulmonary cavity was not flushed of vascular blood (data not shown).

 
Neutrophil apoptosis and necrosis are not delayed in the lungs of infected mice exposed to TCDD. Neutrophils have a very short half-life at the site of infection (6–10 h), and neutrophil apoptosis is critical for the resolution of inflammation (69). There are numerous reports documenting the effects of TCDD, and other AhR ligands, on apoptosis (12, 17, 47, 65, 70, 83, 86). However, these studies focus on lymphocyte apoptosis, and to our knowledge there are no reports in which the effects of TCDD on neutrophil apoptosis have been examined. If exposure to TCDD causes a delay or defect in the death of neutrophils, then we would expect to detect fewer apoptotic or necrotic neutrophils in the lungs of TCDD-treated, infected mice. We investigated whether the excess numbers of neutrophils in the lungs of mice treated with TCDD results from a delay in neutrophil apoptosis (Fig. 9). Our results indicate that exposure to TCDD does not delay neutrophil apoptosis. We believe that the slight increase in the percentage of apoptotic neutrophils in cells from mice treated with TCDD (day 8) is not biologically significant because we did not detect this increase in a separate study using the TUNEL staining method (data not shown). Hence, delayed neutrophil death does not underlie the excess neutrophilia in the lungs of TCDD-treated, infected mice.



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  		Fig. 9. Exposure to TCDD does not affect pulmonary neutrophil apoptosis and necrosis. Mice were treated as described in Fig. 1. On days 5, 7, and 8 postinfection, mice treated with vehicle (open bars) or TCDD (closed bars) were killed, and lungs were digested with collagenase (6 mice per treatment group/day). Mock-infected mice treated with vehicle or TCDD served as controls (4 mice per treatment group). Apoptosis (A) and necrosis (B) of pulmonary neutrophils was measured by staining with annexin V and 7-amino-actinomycin (7-AAD). Apoptotic neutrophils were defined as Gr-1 positive, annexin V positive, and 7-AAD negative. Necrotic neutrophils were characterized as Gr-1 positive, annexin V positive, and 7-AAD positive. Results are representative of 2 separate experiments. Error bars represent SE. *Significant difference compared with the vehicle treatment group (P ≤ 0.05).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The lung expresses high levels of the AhR (101), and many AhR ligands are airborne pollutants, suggesting that the lung is a likely target for the immunotoxic action of a large group of abundant and persistent toxicants. Previous data from our laboratory (99, 103) and others (53) have shown that exposure to the prototypic AhR agonist TCDD decreases host resistance to infection with influenza virus, and this decrease in survival correlates with enhanced neutrophilia in the lung. However, there are currently no studies that attribute impaired host resistance to a particular cell type. Knowing that excess neutrophils are associated with the pathology that accompanies many inflammatory diseases, we found it logical to postulate that this increase is detrimental to the host's ability to survive infection. Data presented here strongly support this idea, as survival of TCDD-treated mice was greatly improved following neutrophil-depletion. Furthermore, our findings extend our understanding of how activation of the AhR adversely affects immune function, as we show for the first time that, in addition to the suppressive effects of TCDD on lymphocyte responses, the increased number of neutrophils occurs via an AhR-dependent mechanism.

Findings from these studies also provide novel information regarding the kinetics and magnitude of many aspects of the pulmonary innate immune response to infection with influenza A virus. We measured levels of the neutrophil-recruiting chemoattractants KC, MIP-1{alpha}, MIP-2, LIX, IL-6, and C5a in the lung. All of these mediators were detected, and, except for C5a, there was an infection-associated increase in the levels. KC, MIP-1{alpha}, and MIP-2 reached peak levels 12–24 h after infection. IL-6 was also elevated very rapidly, although instead of declining within a few days, levels remained elevated. The rapid production of these soluble mediators in response to infection with influenza virus is consistent with previous data from our laboratory (60) and studies by others (29, 76, 95), demonstrating that inflammatory mediators in the lung are elevated very rapidly following viral infection. Moreover, this timing is consistent with the very rapid recruitment of neutrophils to the lung following infection with influenza virus, as neutrophils are detected within 24 h of infection (95, 99). Yet, since neutrophils continue to emigrate to the lung until the infection is resolved, additional factors are likely responsible for the continued recruitment of neutrophils to the lungs during infection with influenza.

Adhesion molecules, such as CD11a, CD11b, CD49d, CD31, and CD38, are regulatory factors that are clearly important for neutrophil recruitment and extravasation (64, 82, 84, 88). However, there are no reports describing the effects of influenza virus infection on the expression of CD11a, CD11b, CD49d, CD31, and CD38 on neutrophils in vivo. The roles of CD31 and CD49d in neutrophil migration have been examined in models of Escherichia coli- and Streptococcus pneumoniae-mediated bacterial pneumonia (88, 89). Hartshorn and White (28) demonstrated that CD11b and CD11c are upregulated on neutrophils during in vitro infection with influenza virus. We show that in vivo infection with influenza virus induces expression of CD31, CD38, and CD49d and upregulates expression of CD11a and CD11b on pulmonary neutrophils. Furthermore, we show that their expression remains elevated even after the levels of common neutrophil chemoattractants have returned to baseline. This suggests that other factors likely regulate expression of adhesion molecules on neutrophils and their continued emigration to the lung during the response to influenza virus.

On the basis of reports that exposure to TCDD alters production of other types of cytokines (34, 63, 71, 99), it was logical to test the hypothesis that treatment with TCDD enhances levels of soluble mediators that are chemoattractant for neutrophils, thereby resulting in more neutrophils at the site of infection. Our findings do not support this, suggesting the activation of the AhR elevates the number of neutrophils in the lung through a mechanism that does not involve deregulated chemokine production. This conclusion is supported by other studies. For example, two other common proinflammatory and neutrophil-attracting cytokines not included in the study reported here are IL-1 and TNF-{alpha}. In separate studies, we characterized the effects of TCDD on IL-1{alpha}/{beta} and TNF-{alpha} levels in lung lavage fluid during the course of infection with influenza A virus. Similar to KC, MIP-1{alpha}, and MIP-2, levels peaked very early during infection, and there was no difference in IL-1 or TNF-{alpha} levels in BAL fluid from vehicle- and TCDD-treated mice (60). Our findings are also consistent with those of Lang et al. (42), who reported that exposure to TCDD did not alter the production of IL-6 and IL-8 by human lung cells or peripheral blood cells.

The lack of effect of TCDD on the production of common neutrophil chemoattractants or neutrophil adhesion molecules led us to examine whether, instead of enhancing levels of regulatory factors that cause more neutrophils to migrate to the lung, AhR activation decreases the rate of neutrophil apoptosis. Our data do not support this idea either. It is certainly possible that TCDD alters the production of another neutrophil chemoattractant, such as leukotriene (LT)B4, which is found in lung lavage fluid of mice infected with influenza virus (29). AhR-mediated alterations in arachidonic acid metabolism have been examined previously (43, 46). However, these studies found no evidence for an increase in LTB4 levels in TCDD-treated mice. Alternatively, instead of increasing positive signals, exposure to TCDD could prevent or dampen production of essential downregulators of inflammation, such as IL-10 or transforming growth factor (TGF)-{beta}. In fact, using lung lavage fluid from vehicle- and TCDD-treated mice, we examined levels of IL-10 and TGF-{beta} at multiple times relative to infection with influenza virus (from day 0 to day 9). We did not detect TGF-{beta} in BAL fluid at any time; however, IL-10 levels increased rapidly after infection in both treatment groups and returned to baseline levels 3 days after infection (data not shown). Exposure to TCDD did not alter the infection-associated production of IL-10. These findings indicate that altered levels of LTB4, IL-10, or TGF-{beta} are not likely the mechanistic explanation for the recruitment of excess neutrophils in the lung.

Thus elevated numbers of neutrophils at the site of antigen challenge are likely the result of some other mechanism. One possibility is that inappropriate activation of the AhR exerts its direct action on molecular targets in the lung, rather than via altered cytokine/chemokine production or targets within the hematopoietic system. For example, AhR activation may enhance the expression of adhesion molecules on lung endothelial or epithelial cells, or it may alter the levels of surfactant proteins. Several reports have attributed proinflammatory roles to surfactant protein (SP)-A and SP-D, including recruitment of neutrophils to the lung (11, 77). However, other reports suggest anti-inflammatory roles, as SP-A- and SP-D-deficient mice had exacerbated inflammatory processes (49, 50). Thus the precise relationship between SP-A and SP-D and pulmonary inflammation is not clear. Nevertheless, given the likely role for these lung-derived molecules in neutrophil recruitment and inflammation, it is possible that AhR-mediated alterations in surfactant proteins underlie the enhanced number of neutrophils observed in the lungs of infected mice treated with TCDD.

Information reported here fills a gap in our knowledge of the consequences of exposure to AhR ligands on the inflammatory mediators involved in the response to a common respiratory pathogen. Very little is known about the specific effects of AhR agonists on neutrophils in general, and even fewer studies have examined neutrophils as mediators of the toxicity associated with exposure to AhR ligands. With regard to the lung, other laboratories have reported effects of a variety of AhR ligands on the lung or cultured lung epithelial cells (6, 23, 39, 52, 57, 91, 92, 100). However, most of these studies focused on changes in cell growth, the induction of metabolic enzymes, and the relationship between AhR activation and cancer. The relationship between these biochemical effects of AhR ligands and pulmonary inflammation has received less attention. In vitro and in vivo studies using PAH or complex mixtures that contain PAH (e.g., diesel exhaust particles) generally demonstrate proinflammatory effects (26, 51, 61). Whether these reported alterations are mediated directly by the AhR has not been clearly defined. Nevertheless, these studies support the idea that exogenous AhR agonists affect pulmonary host defense mechanisms, which in turn likely alters susceptibility to infectious and inflammatory diseases. Our findings corroborate this idea, demonstrating that TCDD, via an AhR-dependent mechanism, increases the number of neutrophils in the lungs during infection.

Thus our observation that exposure to TCDD exacerbates the inflammatory response in the lungs suggests a possible mechanistic link between exposure to AhR ligands and chronic inflammatory lung diseases. Excess neutrophils within airways play a clear role in the pathology of these diseases; however, it is not clear why certain individuals are more prone to develop chronic inflammatory diseases of the lung. Exposure to toxicants and decreased resistance to respiratory infection have been suggested to be important factors, yet the mechanistic relationship between these events, and the onset of inflammatory disease remains unclear. Interestingly, retrospective study of a population heavily exposed to TCDD demonstrated a relationship between the level of exposure to TCDD and increases in COPD (67). Based on our data, we suggest that inappropriate AhR activation (i.e., by exogenous compounds) increases the numbers of neutrophils recruited to the lung in response to viral infection, and this excess number of neutrophils is detrimental to the host, causing neutrophil-mediated pathology and diminishing survival.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes for Environmental Health Sciences Grant RO1 ES-10619 (B. P. Lawrence).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Robert Coffman for permission to use the anti-Gr-1 antibody. We also thank Dr. Nancy Kerkvliet for generously providing the anti-Gr-1 antibody and Gr-1 hybridoma cells and for helpful discussion of our data. Additionally, we thank Dr. Daniel Remick for performing the LIX ELISA and Jennifer Cundiff and L. Nicole Harrison for excellent technical assistance. Finally, we gratefully acknowledge Drs. Beth Vorderstrasse (Washington State University) and Kristen Mitchell (University of Texas Medical Branch, Galveston, TX) for critical review of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. P. Lawrence, Dept. of Pharmaceutical Sciences, Wegner Hall, Washington State Univ., Pullman, WA 99164-6534 (E-mail bpl{at}mail.wsu.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.


   REFERENCES
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 DISCUSSION
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