Persistent Airway Inflammation but Accommodated

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and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last ... Nitrogen dioxide (NO2) is an air pollutant of concern since nu-.

Persistent Airway Inflammation but Accommodated Antioxidant and Lung Function Responses after Repeated Daily Exposure to Nitrogen Dioxide ANDERS BLOMBERG, MAMIDIPUDI T. KRISHNA, RAGNBERTH HELLEDAY, MARGARETA SÖDERBERG, MAJ-CARI LEDIN, FRANK J. KELLY, ANTHONY J. FREW, STEPHEN T. HOLGATE, and THOMAS SANDSTRÖM Department of Respiratory Medicine and Allergy, University Hospital, and National Institute for Occupational Health/Working Life, Medical Division, Umeå, Sweden; Air Pollution Group/Immunopharmacology Group, University Medicine, Southampton General Hospital, Southampton; and Cardiovascular Research, The Rayne Institute, St. Thomas’ Hospital, London, United Kingdom

Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant that may induce deterioration of respiratory health. In this study the effects of repeated daily exposure to NO2 on airway antioxidant status, inflammatory cell and mediator responses, and lung function were examined. Healthy nonsmoking subjects were exposed under controlled conditions to air (once) and to 2 ppm of NO2 for 4 h on four consecutive days. Lung function measurements were made before and immediately after the end of each exposure. Bronchoscopy with endobronchial biopsies, bronchial wash (BW), and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last exposure to NO2. Repeated NO2 exposure resulted in a decrease in neutrophil numbers in the bronchial epithelium. The BW revealed a twofold increase in content of neutrophils (p , 0.05) and a 1.5-fold increase in myeloperoxidase (MPO) (p , 0.01) indicative of both migration and activation of neutrophils in the airways. After the fourth NO2 exposure, antioxidant status of the airway fluid was unchanged. Significant decrements in FEV1 and FVC were found after the first exposure to NO2, but these attenuated with repeated exposures. Together, these data indicate that four sequential exposures to NO2 result in a persistent neutrophilic inflammation in the airways, whereas changes in pulmonary function and airway antioxidants are resolved. We conclude that NO2 is a proinflammatory air pollutant under conditions of repeated exposure. Blomberg A, Krishna MT, Helleday R, Söderberg M, Ledin M-C, Kelly FJ, Frew AJ, Holgate ST, Sandström T. Persistent airway inflammation but accommodated antioxidant and lung function responses after repeated daily expoAM J RESPIR CRIT CARE MED 1999;159:536–543. sure to nitrogen dioxide.

Nitrogen dioxide (NO2) is an air pollutant of concern since numerous epidemiologic studies have indicated an increase in respiratory illness in people exposed to NO2 in polluted ambient air (1, 2). There are also data suggesting an increased susceptibility to airway infection because of NO2 exposure (3–5). NO2 is one of the most common air pollutants in ambient air, in indoor air in some industrial workplaces, and in homes with gas stoves, where 24-h averages may reach 0.5 ppm and peak concentrations 1 to 2 ppm (1, 6). Exposure to NO2 concentrations of 2 ppm or more may be encountered in workplaces such as ferries, Ro-Ro ships, mines, and tunnels (7–9). In mine environments the mean annual NO2 concentration may be as high as 1.7 ppm, with 10% of the daily mean values above or (Received in original form November 17, 1997 and in revised form July 30, 1998) Supported by the Swedish Asthma and Allergy Association, the Swedish Work Environmental Fund, and the Swedish Heart and Lung Foundation. Correspondence and requests for reprints should be addressed to Dr. Anders Blomberg, M.D., Ph.D., Department of Respiratory Medicine and Allergy, University Hospital, S-901 85 Umeå, Sweden. Am J Respir Crit Care Med Vol 159. pp 536–543, 1999 Internet address: www.atsjournals.org

equal to 3 ppm and short-term exposure values of as much as 8 ppm (10, 11). Occasional studies have demonstrated bronchoconstriction after a single exposure to 1.5 to 5 ppm of NO2 in healthy subjects, whereas some have been unable to detect an effect despite an exposure concentration as high as 4 ppm (12). Folinsbee (13), in a recent meta-analysis, estimated bronchial hyperresponsiveness to increase after exposure to NO2 at concentrations above 1 ppm. To date, the effects of repeated exposure to NO2 on lung function have been addressed in only one study. Rubinstein and coworkers (14) in San Francisco investigated five healthy subjects exposed to 0.6 ppm on four occasions within 6 d, but they were unable to detect any lung function change after the exposure series. The paucity of data in this respect is most likely due to the laborsome study designs needed to elucidate effects of multiple-day exposures. It is well recognized that exposure to NO2 causes an inflammatory response in the airways. We recently reported that a single exposure to 2 ppm of NO2 for 4 h resulted in an increase in interleukin (IL)-8 (1.5 h) and neutrophils (6 h) in the proximal airways of normal healthy subjects, but no signs of inflammatory cell changes were detected in bronchial biopsies (15).

Blomberg, Krishna, Helleday, et al.: Airway Inflammation after Repeated Exposure to NO2

A single, acute exposure to NO2 has also been shown to transiently modify the protective antioxidant defense network in the respiratory tract lining fluid with consumption of ascorbic acid (AH2) and uric acid (UA) and increased reduced glutathione (GSH) levels (16). Previous studies evaluating the inflammatory effects of repeated exposure to NO2 have revealed different effects compared with a single acute exposure. Repeated exposure to 1.5 or 4 ppm of NO2 for 20 min every second day on six occasions resulted in decreased CD161561 and CD191 cells in bronchoalveolar lavage (BAL), 24 h after the final exposure. In contrast, neutrophils, mast cells, and total lymphocytes were unaffected (17, 18). The soluble mediators hyaluronan (HA) and myeloperoxidase (MPO) increased, whereas methyl-histamine content decreased. The levels of fibronectin, tryptase, albumin, total protein, leukotriene B4 (LTB4), PGE2, and PGF2a were all unaffected (19). Repeated exposure to a low (0.6 ppm) NO2 concentration induced a slightly greater proportion of natural killer (NK)-cells in BAL as early as 2 h after the last of four NO2 exposures (14). The aim of the present study was to evaluate the effects of repeated NO2 exposure on four consecutive days on cells, inflammatory markers, and antioxidants in the bronchial and bronchoalveolar regions of the lung. In addition, bronchial biopsies were taken to assess whether repeated exposure to NO2 would induce an inflammatory response in the bronchial mucosa. Furthermore, lung function was followed to determine whether bronchoconstriction occurred and whether this was modified on subsequent exposure days.

METHODS Subjects Twelve healthy, nonsmoking volunteers (eight male and 4 female; mean age, 26 yr; range, 21 to 32 yr) without any history of asthma or other respiratory disease were recruited. All had negative skin prick tests and normal lung function (FEV1 and FVC . 80% of predicted). No subject had a history of airway infection for at least 6 wk prior to the first exposure or during the study. No nonsteroid anti-inflammatory drugs (NSAIDs) or aspirin or additional intake of vitamin C (ascorbic acid) and vitamin E (a-tocopherol) were allowed.

Study Design The subjects were exposed once to filtered air and on four consecutive days to air containing 2 ppm of NO2 in an environmental chamber according to a previously described standard protocol (20, 21). During the exposures light exercise (75 W) on a bicycle ergometer was alternated with rest in 15-min intervals. Each exposure lasted for 4 h. The air exposure and the four exposures to NO2 were performed in random order, separated by at least 3 wk. Flexible fiberoptic bronchoscopy with mucosal biopsies, bronchial wash (BW), and BAL was performed 1.5 h after the air exposure and the fourth and last NO2 exposure, and it has been described elsewhere (15). In this study, the effects on the airways of four daily exposures to 2 ppm of NO2 were compared with results achieved after a single air exposure. A doubleblinded randomized study with four air exposures as well as the four conducted NO2 exposures would have been optimal, but it was not possible because of practical reasons and resources. Ideally, the NO2induced effects on airway reactivity during the repeated exposure series should also have been addressed in this study. This was not possible, however, since the bronchial provocation tests may elicit airway inflammation themselves and, hence, influence the inflammatory markers (22, 23). Informed consent was obtained from the subjects, and the study was approved by the Ethics Committee of Umeå University.

Nitrogen Dioxide Exposure The exposures were performed at the Medical Division of the National Institute for Working Life at Umeå according to the principles previously described (17, 18, 20, 21, 24) and the NO2 exposure technique has been presented elsewhere (16). In short, the exposures were

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performed in an exposure chamber built of aluminum. Windows and an intercommunication system enable the operator to maintain visual contact and communication with the subject inside the chamber. The concentration of NO2 within the chamber was continuously monitored and registered. During this exposure series the chamber air temperature was kept at 20.0 6 0.58 C, and the relative humidity was 50.1 6 1.0%. During the NO2 exposures the NO2 concentration was 2.0 6 0.16 ppm.

Lung Function Assessments The dynamic spirometry variables (FVC and FEV1) were measured before and immediately after each exposure using a whole-body plethysmograph with SensorMedics 2100 system (SensorMedics Autobox 2800; SensorMedics, Yorba Linda, CA). At least three satisfactorily performed well-cooperated measurements of each variable were performed as judged by an experienced lung function technician and according to the guidelines of the American Thoracic Society (25).

Bronchoscopies Fiberoptic bronchoscopy was performed as previously described (15, 16, 26). In short, lidocaine was used for topical anesthesia and mild sedation was achieved with propofol given intravenously. The bronchoscope was inserted through the mouth with the subject in supine position. At each of the two bronchoscopies three endobronchial mucosal biopsies were taken using fenestrated forceps (FB-21C; Olympus, Tokyo, Japan) either from the anterior aspect of the main carina and the subcarinae of the third and fourth generation airways of the right side or from the posterior aspect of the main carina and the corresponding subcarinae on the left side, with lavages undertaken on the contralateral side, in a predetermined randomized way. The alternative locations were used during the second bronchoscopy to avoid biopsy artefacts at former biopsy sites. A bronchial wash with 2 3 20 ml and a BAL with 3 3 60 ml sterile phosphate-buffered saline (PBS) at pH 7.3 and 378 C were performed and the fluid was gently withdrawn into a siliconized container placed in iced water. The chilled lavage fluid was filtered through a nylon filter (pore diameter, 100 mm; Syntab Product AB, Malmö, Sweden) and centrifuged at 400 3 g for 15 min. The supernatants were separated from the cell pellets, immediately analyzed for albumin and protein, and stored at 2708 C until subsequent cytokine and mediator analyses. The cell preparations, including flow cytometry, were performed as previously outlined (15). The following antibodies were used for flow cytometry; CD3, CD4, and CD8 receptors on T-cells, CD19 for B-cells, CD25 as a marker for IL-2 receptor, CD16 together with CD56 for natural killer cells, CD45RO for memory T-cells, CD69 as an activation marker for mononuclear cells, and HLA-DR as an activation marker for macrophages (Becton-Dickinson AB, Stockholm, Sweden). Albumin and total protein were measured with assays from Boehringer Mannheim (Mannheim, Germany) in an autoanalyzer at the Department of Clinical Chemistry, University Hospital of Northern Sweden, Umeå, Sweden. IL-8 was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc., Minneapolis, MN). MPO, HA, and methyl-histamine (M-his) were analyzed by radioimmunoassay (RIA) (Kabi Pharmacia Diagnostics, Uppsala, Sweden). The recovery from the first 20 ml BW and the first 10 ml of the instilled second 20 ml were analyzed separately, and the recovery from the 3 3 60 ml was pooled. Cell differential counts, total protein, albumin, MPO, and HA concentrations were determined in the first BW and in the BAL. IL-8 was measured in the first BW, whereas flow cytometry was performed only on cells recovered from the BAL fluid since the cell number recovered by the first BW was too small for this analysis. Antioxidant concentrations were determined in both the second BW and the BAL.

Determination of Antioxidants The methods used for the determination of these moieties in lavage fluid have been described in detail previously (16). Reduced and oxidized glutathione were determined using the enzyme recycling method of Teitze, adapted for use on a plate reader (27). UA and AH2 were determined using reverse phase HPLC with electrochemical detection (16).

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TABLE 1 NAMES AND SOURCES OF ANTIBODIES USED FOR IMMUNOHISTOCHEMISTRY Name CD3 CD4 CD8 AA1 NE ICAM-1 (RR1)

Marker

Cells

Source

CD3 CD4 CD8 Tryptase Elastase ICAM-1

T-cells T-cells T-cells Mast cells Neutrophils Microvasculature

DAKO, High Wycombe, UK Becton Dickinson, Oxford, UK DAKO, High Wycombe, UK Dr. A. Walls, Univ. of Southampton, UK DAKO, High Wycombe, UK Dr. Robert Rothlein, Boehringer Ingelheim, Ridgefield, CT

Processing and Quantification of Bronchial Mucosal Biopsies The bronchial biopsies were placed in ice-cooled acetone containing protease inhibitors and processed in glycolmethacrylate (GMA) resin as previously outlined (28). The blocks were stored in airtight containers at 2208 C until used for immunostaining. Sections were cut and stained with monoclonal antibodies as previously described (15). In short, sections were cut at 2 mm thickness and treated to block endogenous peroxide, and nonspecific antibody binding was blocked with undiluted culture supernatant. The primary monoclonal antibodies (mAbs) (Table 1) were added and incubated at room temperature overnight. After rinsing, biotinylated rabbit antimouse IgG Fab (Dako, High Wycombe, UK) was applied followed by the streptavidin-biotinhorseradish-peroxide complex. Finally, the stained areas were developed with aminoethyl carbazole (AEC) in acetate buffer and hydrogen peroxide to yield a red color. The sections were then counterstained with Mayer’s hematoxylin. The expression of the endothelial adhesion molecules in the submucosal blood vessels was quantified by expressing the number of vessels staining with specific antiadhesion mAb as a proportion of the total vessel complement revealed by staining with the panendothelial mAb EN4, as previously described (29). Immunostained inflammatory cells were counted separately in the epithelium and in the submucosa, the total number of positive cells being expressed as cells/mm of the epithelium and cells/mm2 of the submucosa, respectively. Immunostaining was identified under the light microscope, and the length of the basement membrane and the area of the submucosa were measured using computer-assisted image analysis Colour Vision Software (Improvision, Birmingham, UK). Smooth muscle, glands, large blood vessels, and torn or folded tissue within the sections were not included in the areas analyzed.

Blood Samples The peripheral blood samples were drawn prior to the bronchoscopies. Analyses of blood cells and differential counts were performed with an autoanalyzer at the Department of Clinical Chemistry, University Hospital of Northern Sweden, Umeå, according to clinical routine.

vestigated. Lung function data were analyzed with Shapiro-Wilk test for normality together with paired t test and analysis of variance. Statistical analyses were carried out with SPSS version 6.1.2 for Windows (SPSS, Inc., Chicago, IL). A p value , 0.05 was considered significant.

RESULTS Immunohistochemistry

When analyzing the bronchial biopsy sections with neutrophil elastase and mast cell tryptase, cytoplasmic staining was observed, whereas ring staining was seen for CD31, CD41, and CD81. The number of neutrophils in the epithelium showed a significant decrease after NO2 compared with after air exposure (1.19, 0.40 to 2.97 versus 0.00, 0.00 to 0.70) (median, interquartile range) (p , 0.05). No other changes in the number of inflammatory cells were detected in the epithelium or submucosa. Expression of intercellular adhesion molecule-1 (ICAM-1) was also unchanged after the NO2 exposures (Table 2). Cell Parameters in BW and BAL

Lavage recoveries after air exposure were: BW I, 6.5 ml (5.0 to 8.6); BW II, 10.0 ml (10.0 to 10.0); BAL, 132 ml (120 to 135), respectively. The amounts recovered after NO2 exposure did not differ significantly. The total number of neutrophils increased in BW after repeated NO2 exposure compared with after air exposure (0.80, 0.30 to 1.40 versus 1.54, 0.49 to 2.79) (p , 0.05). In BAL, no significant changes in total cell numbers and differential counts were found after repeated NO2 exposure (Table 3). Flow cytometry, however, revealed a significant increase in the percentage of CD251 lymphocytes (0.85, 0.55 to 1.68 versus 1.30, 0.62 to 2.45; p , 0.05) and HLA-DR1 macrophages (3.45, 2.05 to 5.32 versus 4.35, 1.88

TABLE 2

Statistics In all analyses, each subject acted as his or her own control for comparisons after air and the last NO2 exposure. As the exposure sequence was randomized, no period effect was expected. The sample size calculation was based on our previous experience in similar studies (15, 17–19). The following parameters were defined a priori as primary end points: neutrophil counts in biopsies and BW, concentration of MPO and HA in BW, and the percentage of NK-cells in BAL. Other end points were included in order not to overlook other possible effects by the NO2 exposures. These parameters were selected depending on their biologic plausibility to be affected by NO2. As the study design was very laborsome and resource demanding, it was not possible to design a series of studies each investigating a limited number of primary end points. The magnitude of the NO2 effects on secondary parameters in BAL and biopsies was not corrected for multiple tests used, as this has been the practice in this Journal (15, 30). Instead, any changes in these parameters were interpreted cautiously. Wilcoxon’s nonparametric signed-rank test for paired observations was used as a conservative test for changes in BAL and biopsy data. Such data are not normally distributed within the size of the cohort in-

CELL COUNTS IN EPITHELIUM (cells/mm) AND SUBMUCOSA (cells/mm2 ) OF BRONCHIAL BIOPSIES* mAb CD3 (epithelium) CD3 (submucosa) CD4 (epithelium) CD4 (submucosa) CD8 (epithelium) CD8 (submucosa) NE (epithelium) NE (submucosa) AA1 (epithelium) AA1 (submucosa) ICAM-1, %‡

After Air

After NO2

p Value†

3.38 (0.57–9.85) 42.4 (3.06–68.9) 0.93 (0.00–2.18) 17.1 (3.22–51.0) 1.54 (0.00–3.50) 18.4 (1.53–31.5) 1.19 (0.40–2.97) 70.3 (47.7–99.1) 0.00 (0.00–0.05) 28.5 (10.1–41.8) 83 (56–100)

4.62 (0.98–8.08) 35.3 (11.7–79.4) 0.32 (0.12–2.97) 17.3 (6.43–55.4) 2.31 (1.35–6.66) 15.7 (6.73–27.6) 0.00 (0.00–0.70) 69.0 (40.3–81.8) 0.00 (0.00–1.57) 31.1 (17.6–45.4) 61 (38–84)

NS NS NS NS NS NS , 0.05 NS NS NS NS

Definition of abbreviations: AA1 5 mast cell tryptase; ICAM-1 5 intercellular adhesion molecule-1; NE 5 neutrophil elastase; NS 5 nonsignificant. * Data given as medians and interquartile ranges. † Wilcoxon’s paired rank sum. ‡ ICAM-1 is quantified as a percentage of total EN4 staining vessels.

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Blomberg, Krishna, Helleday, et al.: Airway Inflammation after Repeated Exposure to NO2 TABLE 3 DIFFERENTIAL CELL COUNTS IN BW AND BAL*

BW After air After NO2 p Value† BAL After air After NO2 p Value†

Neutrophils

Macrophages

Lymphocytes

Mast Cells

All Cells (3 104 3 ml)

(3 104 3 ml)

(%)

(3 104 3 ml)

(%)

(3 104 3 ml)

(%)

(3 104 3 ml)

(%)

18.0 13.3–29.6 25.5 16.3–33.8 NS

0.80 0.30–1.40 1.54 0.49–2.79 , 0.05

3.0 1.2–8.8 7.0 3.0–13.5 NS

20.4 12.1–29.0 23.4 14.1–31.1 NS

93.5 82.2–96.0 89.5 82.0–91.8 NS

0.72 0.22–1.13 0.95 0.29–1.90 NS

2.5 2.0–6.5 4.0 2.5–5.8 NS

0.007 0.003–0.014 0.009 0.003–0.020 NS

0.02 0.02–0.05 0.05 0.02–0.08 NS

9.65 7.65–13.8 12.1 9.1–14.8 NS

0.10 0.07–0.24 0.12 0.10–0.18 NS

1.0 1.0–1.0 1.0 1.0–1.8 NS

9.15 6.59–12.5 10.8 8.28–12.7 NS

89.5 86.0–94.8 90.0 85.5–91.0 NS

0.81 0.40–1.80 1.10 0.70–1.50 NS

9.0 4.2–12.0 8.5 7.0–12.0 NS

0.004 0.001–0.016 0.008 0.002–0.015 NS

0.05 0.01–0.16 0.05 0.02–0.11 NS

Definition of abbreviations: BAL 5 bronchoalveolar lavage; BW 5 bronchial wash; NS 5 nonsignificant. * Data are given as medians and interquartile ranges. † Wilcoxon’s paired rank sum test.

to 7.15; p , 0.05) after the NO2 exposures. No changes were found in the percentages of CD31, CD41, CD81, CD191, CD161561, CD45RO1, or CD691 cells. Soluble Mediators and Cytokines

An increase in MPO (p , 0.01) and a decrease in albumin (p , 0.05) together with a trend towards a decrease in total protein (p 5 0.07) were found in BW after repeated NO2 exposure. No changes were found in the concentrations of IL-8, HA, and methyl-histamine. In BAL fluid, no significant changes were detected in any of the measured soluble components (Table 4). Antioxidants

No difference in the antioxidant status of either the BW or the BAL fluids were found after the four exposures to NO2 compared with air (Table 5). Blood Parameters

Total white cell number and differential counts were all unchanged after the last NO2 exposure compared with after air (data not shown).

Lung Function Measurements

The FEV1 and FVC values before and after air exposure and the four exposures to NO2 are presented in Table 6. Differences between the preexposure and postexposure lung function values after each NO2 exposure were compared with the difference after air exposure (Figure 1). The data were shown to be drawn from a parametric population and thus allowing the use of a paired t test. There were no changes in preexposure FEV1 or FVC across the five exposure days. Significant decrement in FEV1 (20.09 6 0.13 L versus 0.04 6 0.16 L; mean and SD) was seen over the first NO2 exposure versus air (p , 0.05). Similar results were seen for FVC (20.16 6 0.12 L versus 20.06 6 0.11 L) (p , 0.05). After subsequent exposures to NO2 the differences in FEV1 and FVC did not differ significantly from the changes after air exposure. However, the changes between preexposure and postexposure FVC at the first NO2 exposure were significantly greater than the changes seen during the second and third exposure to NO2 (p , 0.01 and p , 0.005, respectively). The difference in FEV1 over the first NO2 exposure was also significantly greater than the changes at the last exposure to NO2 (p , 0.05). Analysis of variance revealed that the group mean of lung function changes

TABLE 4 SOLUBLE MEDIATORS IN BW AND BAL*

BW After air After NO2 p Value† BAL After air After NO2 p Value†

Albumin (mg/ml)

Protein (mg/ml)

IL-8 (pg/ml)

Methyl-histamine (pg/ml)

Myeloperoxidase (ng/ml)

Hyaluronic Acid (ng/ml)

47.0 16.2–71.2 22.0 13.2–51.8 , 0.05

74.0 26.5–129 28.0 20.0–94.0 NS (p 5 0.075)

56.0 38.0–86.0 65.0 50.0–156 NS

88 72–116 81 65–103 NS

5.85 3.65–8.62 8.35 5.21–17.9 , 0.01

16.0 13.4–18.6 16.3 13.4–19.4 NS

29.0 23.5–37.8 40.5 20.7–47.8 NS

38.0 20.0–49.8 51.0 29.8–59.2 NS

NA

56 49–70 53 49–61 NS

2.20 1.94–2.89 2.20 1.82–2.88 NS

13.5 12.2–16.6 14.0 13.5–15.4 NS

Definition of abbreviations: NA 5 not analyzed. For other definitions, see Table 3. * Data are given as medians and interquartile range. † Wilcoxon’s paired rank sum test.

NA

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BW After air After NO2 p Value† BAL After air After NO2 p Value†

GSSG (mmol/L)

Ascorbic Acid (mmol/L)

Uric Acid (mmol/L)

0.42 0.07–1.04 0.61 0.11–1.39 NS

0.16 0.01–1.20 0.40 0.01–1.20 NS

0.52 0.01–1.36 0.46 0.23–1.64 NS

0.79 0.33–0.78 0.54 0.39–0.58 NS

0.44 0.01–0.64 0.54 0.08–1.67 NS

0.16 0.01–0.97 0.02 0.01–0.69 NS

0.65 0.01–1.64 0.55 0.03–1.48 NS

0.59 0.33–0.67 0.50 0.33–0.72 NS

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In agreement with findings from the single exposure of normal subjects to 2 ppm of NO2, four consecutive daily exposures to NO2 did not result in inflammatory cell recruitment into the bronchial mucosa in the major airways (15). Likewise, as after single exposure, the expression of ICAM-1 was unchanged after repeated exposure to 2 ppm of NO2, indicating that a major transit of cells at the sampled bronchial mucosal sites was unlikely to have occurred. Surprisingly, neutrophils present in the epithelium after air exposure had largely disappeared after the repeated exposures to NO2, suggesting that these cells had migrated from the epithelium into the airway lumen. Repeated NO2 exposure may have initiated stimuli that directed neutrophils to migrate from the bronchial epithelium, the first cell surface to encounter the inhaled NO2. A small volume BW is believed to better identify inflammation in the airways than a large volume BAL, which may more reflect conditions in the alveoli (31). In a previous study we found an early increase in the C-X-C chemokine IL-8 in BW after a single exposure to NO2 and we suggested that IL-8 could be an important chemoattractant for recruiting neutrophils after exposure to NO2 (15). In the present study, IL-8 was not elevated in BW after the fourth NO2 exposure. This was probably due to attenuation by repeated exposure. The neutrophilia in the BW was accompanied by an increase in MPO, indicating that the neutrophils were activated by NO2. The current data, as well as recent lavage findings (32), suggest that repeated exposure to a relatively high concentration of NO2 on four consecutive days results in airway inflammatory changes that are mainly located more peripherally in the airways than it is possible to biopsy. The major site for the NO2-induced inflammatory changes has been suggested to be the terminal bronchioles (33), which here may be reflected in the finding of increased neutrophil numbers in the BW. Interestingly, however, repeated exposure to NO2 also seems to effect the more proximal bronchi where migration of neutrophils into the airway lumen appears to be initiated. In contrast to the findings after a single exposure to NO2, where consumption of AH2 and UA together with an increase in GSH levels were demonstrated in airway lavage (16), no changes in the concentration of these antioxidants were seen after repeated NO2 exposure. Hence, it is likely that the exposure burden used in this study induced counter regulatory mechanisms that attenuated the immediate effects seen after a

TABLE 5 ANTIOXIDANT CONCENTRATIONS IN BW AND BAL* GSH (mmol/L)

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Definition of abbreviations: GSSG 5 oxidized glutathione; GSH 5 reduced glutathione. For other definitions, see Table 3. * Data are given as medians and interquartile ranges. † Wilcoxon’s paired rank sum test.

after the first NO2 exposure significantly differed from the mean changes of all exposures (FVC, p , 0.005 and FEV1, p , 0.01, respectively).

DISCUSSION This study has demonstrated that four consecutive daily exposures to 2 ppm of NO2 induce a neutrophilic airway inflammation in the bronchial wash. No evidence was, however, obtained of a parallel increase in inflammatory cell numbers in mucosal biopsy specimens from the proximal airways. Instead there were signs of migration of neutrophils from the epithelium into the airway lumen. The acute decrement in lung function that occurred over the first exposure to NO2 was found to be attenuated during the subsequent exposures. Similarly, the initial loss of antioxidants in BAL that followed acute exposure to NO2 (16) is resolved after multiple daily exposures. Taken together, these data indicate that although the acute effects on lung function and antioxidants were attenuated with repeated exposure there were still signs of an ongoing neutrophilic inflammation in the airways.

TABLE 6 FEV1 AND FVC VALUES (LITERS) BEFORE AND AFTER AIR EXPOSURE AND THE FOUR CONSECUTIVE DAYS OF EXPOSURE TO 2.0 ppm NO2 Air Exposure Subject No. 1 2 3 4 5 6 7 8 9 10 11 12 Mean SD

FEV1

FVC

1st Exposure to NO2

2nd Exposure to NO2

3rd Exposure to NO2

4th Exposure to NO2

FEV1

FEV1

FEV1

FEV1

FVC

FVC

FVC

FVC

Before

After

Before

After

Before

After

Before

After

Before

After

Before

After

Before

After

Before

After

Before

After

Before

After

4.43 3.16 3.45 5.51 5.46 3.35 5.48 4.70 5.12 4.48 4.90 4.17 4.52 0.84

4.32 3.15 3.48 5.56 5.59 3.32 5.41 4.80 4.90 4.47 5.22 4.48 4.56 0.86

5.04 3.65 3.65 6.44 7.20 4.81 6.36 6.36 5.56 6.70 6.06 6.25 5.67 1.16

4.99 3.66 3.66 6.34 7.00 4.69 6.22 6.42 5.43 6.52 6.23 6.18 5.61 1.12

4.30 3.21 3.76 5.42 5.29 3.27 5.42 4.81 5.07 4.29 5.13 4.65 4.55 0.79

4.24 3.12 3.58 5.42 5.30 3.13 5.33 4.88 4.83 4.22 5.22 4.30 4.46 0.84

4.87 3.74 3.98 6.46 7.04 4.67 6.21 6.55 5.60 6.55 6.16 6.17 5.67 1.09

4.80 3.64 3.77 6.36 6.95 4.54 5.86 6.44 5.21 6.24 6.09 6.14 5.50 1.09

4.29 3.08 3.51 5.50 5.37 3.17 5.34 4.88 5.19 4.28 5.24 4.56 4.53 0.88

4.27 3.36 3.51 5.49 5.27 3.16 5.44 4.92 4.92 4.16 5.23 4.50 4.52 0.83

4.88 3.66 3.67 6.31 7.01 4.59 6.21 6.55 5.74 6.53 6.41 6.33 5.66 1.16

4.76 3.83 3.68 6.31 6.93 4.49 6.07 6.55 5.53 6.40 6.36 6.29 5.60 1.12

4.25 3.21 3.47 5.49 5.21 3.21 5.45 4.91 5.37 4.40 5.08 4.63 4.56 0.86

4.35 3.21 3.38 5.53 5.49 3.25 5.42 4.87 5.24 4.28 5.05 4.73 4.57 0.88

4.82 3.76 3.65 6.35 7.11 4.65 6.30 6.46 5.69 6.62 6.25 6.37 5.67 1.16

4.89 3.73 3.61 6.38 6.99 4.55 6.28 6.48 5.65 6.61 6.21 6.38 5.65 1.16

4.39 3.21 3.49 5.56 5.49 3.23 5.53 4.82 5.40 4.41 5.20 4.71 4.62 0.89

4.37 3.22 3.52 5.64 5.50 3.21 5.53 4.90 5.38 4.38 5.11 4.83 4.63 0.90

4.99 3.80 3.67 6.51 7.14 4.69 6.37 6.56 5.69 6.73 6.47 6.54 5.76 1.19

4.95 3.73 3.75 6.49 7.05 4.52 6.35 6.56 5.74 6.58 6.30 6.45 5.71 1.17

Blomberg, Krishna, Helleday, et al.: Airway Inflammation after Repeated Exposure to NO2

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Figure 1. Changes in FEV1 (top panel) and FVC (bottom panel) during air exposure and during the four consecutive days of exposure to 2.0 ppm of NO2. Bars indicate means and 95% confidence interval. *p , 0.05, **p , 0.01, and ***p , 0.005.

single NO2 exposure. There mechanisms are presently unclear but may include replenishment of lost antioxidants in the epithelial lining fluid, and/or the upregulation of other defenses, which may limit oxidative stress in the airways. In the present study we used a different study design than that employed in our previous repeated exposure studies in which short, 20-min exposures were repeated every second day (17–19). The intervals between, and the duration of, the exposures are likely to be important determinants of the induction of counter regulatory defense mechanisms in the airways. These mechanisms may not be elicited when subjects are exposed for short periods at longer intervals (i.e., 20 min every other day). A difference between the responses after daily exposures compared to alternate-day exposures was therefore not unexpected. The effects on lymphocyte subsets seen in previous human and animal studies of repeated NO2

exposure (14, 17, 18, 34) were not detected in the present study with four daily exposures. The time point for bronchoscopy is probably another important consideration when evaluating the effects of repeated exposure to NO2. Rubinstein and colleagues (14) found a slight increase in the percentage of NK-cells in BAL 2 h after the last of four 2-h exposures to 0.60 ppm of NO2 within 6 d. Our BAL data showed no effects on NK-cells, but a small, yet significant, increase in CD251 lymphocytes was seen after the repeated NO2 exposures, indicating lymphocyte activation. When a relatively similar protocol with only three daily sequential exposures to NO2 was used a significant decrease in CD41 cells was observed in BAL fluid at a much later time point: 18 h after the last exposure (32). In previous studies, the lung matrix component HA was increased in proximal as well as in peripheral airways (19), but the concentration of HA was unaffected in the present study.

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Levels of methyl-histamine were also unchanged. Differences in proinflammatory and compensatory mechanisms between the two exposure designs as well as the early time point for bronchoscopy in the present study could account for these different findings. After the first NO2 exposure we were able to detect an acute pulmonary response with small, but significant, decrements in both FEV1 and FVC in healthy subjects. However, after repeated exposures to NO2 these lung function changes were no longer apparent. This attenuation of lung function response after repeated NO2 exposure is consistent with the response pattern seen after repeated daily exposure to ozone, where a progressive attenuation of spirometric responses has been reported (35). Whether repeated exposure to NO2 at different exposure intervals and concentrations than used in the present study would give rise to different lung function responses is unknown. With few exceptions, the significant changes detected in this study were with the primary end points, determined a priori, based on preceding studies and upon their biological plausibility. However, in those instances where multiple tests are applied, some caution should always be applied while interpreting the results. In summary, repeated exposure to NO2 results in a neutrophilic inflammation in the lower airways that is detectable in the BW. This inflammatory response is not as pronounced as that seen after a single exposure to NO2 but may still be of importance given the adverse effects by NO2 on respiratory health as suggested by previous epidemiologic studies. The epithelial lining fluid antioxidant status is not altered by repeated NO2 exposure, suggesting that the antioxidant responses seen after acute single exposure to NO2 are accommodated with repeated exposure. Likewise, the acute lung function decrements detected after the first exposure to NO2 are attenuated with repeated exposures. Taken together, these data indicate that sequential daily exposures to NO2 result in a number of events that subsequently reduce, but do not abolish, the impact of NO2 on the lung. It is also evident that there are substantial differences in the airway responses between daily versus alternate-day exposure to NO2. Consequently, one must be cautious about drawing definitive conclusions from a single model of repeated NO2 exposures as many factors, including the number of exposures, duration, and time intervals, are all likely to influence the overall response. Acknowledgment : The writers are grateful to Jamshid Pourazar, Ann-Britt Lundström, Lena Skedebrant, Helén Burström, Ulf Hammarström, Annika Hagenbjörk-Gustafsson, and Gete Hestvik for technical assistance.

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