Published ahead of print on June 30, 2004, doi:10.1164/rccm.200403-423OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200403-423OC Metal-rich Ambient Particles (Particulate Matter2.5) Cause Airway Inflammation in Healthy SubjectsFraunhofer Institute of Toxicology and Experimental Medicine, Hannover; Institut für Umweltmedizinische Forschung, Duesseldorf; and GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany Correspondence and requests for reprints should be addressed to Norbert Krug, M.D., Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, Hannover 30625, Germany. E-mail: krug{at}item.fraunhofer.de
Epidemiologic studies have shown an increased prevalence of allergic asthma in children living in a German smelter area (Hettstedt) compared with a cohort who live in a nonindustrialized area (Zerbst). However, it is not known whether ambient particles (particulate matter2.5 [PM2.5]) from these areas induce distinct lung inflammation, which might be an explanation for this difference. Therefore, 100 µg of PM2.5 suspensions, collected simultaneously in the two areas, were instilled through a bronchoscope into contralateral lung segments of 12 healthy volunteers. PM2.5 from both Hettstedt and Zerbst increased the number of leukocytes in the bronchoalveolar lavage performed 24 hours later. PM2.5 from Hettstedt, but not Zerbst, induced a significant influx of monocytes (Hettstedt: 7.0% vs. Zerbst: 4.3%) without influencing the expression of surface activation markers on monocytes and alveolar macrophages. Oxidant radical generation of bronchoalveolar lavage cells and cytokine concentration (interleukin-6 and tumor necrosis factor-  ) in bronchoalveolar lavage fluid was significantly increased after instillation of Hettstedt PM2.5. We conclude that environmentally relevant concentrations of PM2.5 from the smelter area induced distinct airway inflammation in healthy subjects with a selective influx of monocytes and increased generation of oxidant radicals. The higher concentration of transition metals in PM2.5 from Hettstedt might be responsible for this increased inflammation.
Key Words: air pollution, bronchoscopy • monocytes • oxidants Cross-sectional epidemiologic studies in eastern Germany have clearly shown that regional differences in ambient particulate matter (PM) concentration are associated with differences in the prevalence of respiratory and allergic diseases. School children living in the smelter area of Hettstedt, which was strongly impacted by PM during the 1980s and in the early 1990s, had a significantly higher prevalence of allergies and bronchitis compared with children living in the rural control area of Zerbst (1). After the German reunification, levels of total PM were reduced and PM concentrations converged between different areas throughout the 1990s. The differences in the prevalence rates of nonallergic respiratory symptoms between the two cities decreased and lung function impairment improved along with improved air quality (2–4). However, the difference in the prevalence of allergies remained (4), which indicates that the composition of Hettstedt PM may contribute to the higher prevalence of allergies in that area independently of particle mass. This question has been investigated by Gavett and coworkers, who applied Hettstedt and Zerbst particles in a mouse model of ovalbumin-induced allergic airway disease (5). They demonstrated that an intratracheal instillation of an equal mass of PM2.5 collected in 1999 in Hettstedt, but not that collected in Zerbst, caused a significant increase in airway responsiveness and lung inflammation. Levels of transition metals (including zinc, copper, and cadmium) and oxidant generation (6) were several fold increased in PM2.5 from Hettstedt compared with Zerbst. Although epidemiology and animal toxicology demonstrate convincing consistency, it is unclear whether humans subjected to equal masses of PM2.5 from these areas react with distinct inflammatory reactions of the lung. Therefore, in a first approach, the aim of this study was to instill PM2.5 suspensions collected simultaneously from Hettstedt and Zerbst into two different lung segments of healthy nonallergic subjects. To reflect environmentally relevant conditions, low masses of 100 µg PM2.5 from both cities were used. We tested the hypothesis that PM2.5 from both cities would enhance bronchial inflammation and oxidative stress, and that the effect of PM from Hettstedt would be more pronounced than that from Zerbst. Some of the results of this study have been previously reported in the form of an abstract (7).
Collection of Fine Particles (PM2.5) and Filter Preparation The sample period was between January 8, 2002 and June 18, 2002. We used a Graseby Anderson Dichotomous Sampler Series 240 (Atlanta, GA) with Anderson Teflon membrane filters (37 mm diameter, pore size of 2 µm) for particle collection. Further details have been previously described (8). PM was removed by a procedure including repetitive manual agitation (5 minutes) followed by sonication in a sonication water bath for 10 minutes in sterile, pyrogen-free water (8). The resulting suspension was diluted to a concentration of 200 µg/ml based on filter loading. LPS was determined using the quantitative kinetic Limulus Amebocyte Lysate method (Kinetic-QCL, BioWhittaker, Walkerville, MD), as previously described (8). Hydroxyl radical generation of the particle suspensions was measured using electron paramagnetic resonance, as described previously (6). Inductively coupled plasma mass spectrometry (ICP-MS, ELEMENT2; Finnigan MAT, Bremen, Germany) was used to determine the concentrations of transition metals in the aqueous suspensions of PM. For the instillation experiments, PM2.5 suspensions from each area with low endotoxin concentration were selected (Figure 1). Samples from Weeks 1, 6, and 8–11 were pooled, adjusted to 50 µg/ml PM using sterile saline, aliquoted in 2 ml, and stored at –80°C until the day of bronchoscopy. Before instillation, samples were adjusted to 10 ml with sterile saline solution to a resulting PM2.5 concentration of 100 µg/10 ml.
Study Subjects The study population consisted of 12 healthy subjects (8 women and 4 men, mean age 27 ± 2.5 years). Each subject had a normal physical examination, normal lung function test results, and no clinically relevant laboratory test findings. None of the subjects had elevated serum IgE (> 100 IU/ml), bronchial hyperresponsivesness (PC20 [provocative concentration] > 8 mg/ml methacholine), a positive skin-prick test (20 major inhalative allergens), or infections of the respiratory tract within the last month. Current smokers, persons living or working with smokers, and former smokers, who smoked during the last 5 years or more than 5 pack-years, were excluded from the study. The exposure to tobacco smoke was controlled by the measurement of cotinine excretion in the urine at both bronchoscopy days (inclusion criteria: cotinine concentration < 20 ng/ml). The study protocol was approved by the ethics committee of the Hannover Medical School. Before study inclusion, a written informed consent was obtained from each subject.
Bronchoscopy and Bronchoalveolar Lavage
Processing of BAL Cells For immunofluorescence labeling, 1.5 x 105 BAL cells were used for each test. Biotinylated monoclonal antibodies against CD14, CD16 (Beckmann Coulter, Krefeld, Germany), CD64, HLA-DR (Becton Dickinson, Heidelberg, Germany), CD71 (Caltag, Hamburg, Germany), CD54, CD11b, 27E10, CD163 (Dianova, Hamburg, Germany), or isotype controls (Caltag) were added according to the manufacturer's recommendations and incubated for 30 minutes at 4°C. After washing with phosphate-buffered saline (PBS), cells were incubated with phycoerythrine-cyanine 5.1-conjugated Streptavidin (Beckman Coulter) for 30 minutes. The samples were lysed, fixed (Lysing solution; Becton Dickinson), washed, resuspended in PBS, and kept on ice until flow cytometric analysis.
Flow Cytometric Phenotyping of BAL Cells
Oxygen Radical Generation of BAL Cells
Biochemical Assays in BAL Fluid
Statistics
Analysis of PM2.5 Suspensions Figure 1 shows the endotoxin concentrations and the hydroxyl radical generation in the PM2.5 suspensions for each of the 21 weekly collection periods in Hettstedt and Zerbst. For the instillation, we selected samples with low endotoxin concentrations and with no obvious differences between samples of both locations (Weeks 1, 6, 8–11). The total PM2.5 mass on filters from these selected weeks was 13.1 mg for Hettstedt and 16.1 mg for Zerbst. The metal concentrations in the selected samples are given in Table 1. The LPS concentration in the pooled PM2.5 suspensions from the selected weeks was close to the detection limit of 0.005 EU/ml in both samples (Hettstedt: 0.012 EU/ml; Zerbst: 0.010 EU/ml).
Differential Cell Count of BAL Fluid BAL fluid recovery was slightly less in the baseline BAL from the left lower lobe (68.5 ± 13.7 ml), but recovery was not different between the BAL samples from the challenged segments (saline: 72.3 ± 10.0 ml, Hettstedt: 79.8 ± 6.4 ml, Zerbst: 78.0 ± 7.9 ml). The PM2.5 from both Hettstedt and Zerbst led to a significant increase in the total BAL cells (Figure 2). There was no significant difference in the percentages of neutrophils, lymphocytes, and macrophages after instillation of either PM compared with saline, although a trend toward an increased percentage of neutrophils was seen after instillation of Hettstedt dust (Table 2).
Expression of Surface Markers on Alveolar Monocytes and Macrophages BAL monocytes were quantified by flow cytometry on the basis of size, granularity, and staining characteristics (Figure 3). They showed an "immature" phenotype with CD71low, HLA-DRlow, CD14high, and lower autofluorescence intensity. Monocytes were smaller compared with macrophages that had a "mature" phenotype with CD71high, HLA-DRhigh, CD14low, and high autofluorescence. PM2.5 from Hettstedt (p = 0.01), but not from Zerbst, induced a significant increase in the percentage of recruited monocytes into the alveolar space compared with saline (saline: 3.7 ± 2.7%, Hettstedt: 7.0 ± 3.4%, Zerbst: 4.3 ± 2.0%; Figure 4). The expression of cell-surface markers, such as complement receptor CD11b, endotoxin receptor CD14, Fc receptors CD16 and CD64, the marker of acute (CD54, 27E10) or late phase of inflammatory process (CD163), as well as HLA-DR, and transferrin receptor CD71 on the alveolar marcrophages and on the recruited monocytes, were not statistically different to the control challenge in segments instilled with the PM2.5 from Zerbst or Hettstedt (Table 3).
Oxidant Radical Generation of Stimulated BAL Cells After zymosan stimulation, BAL cells from the segment exposed to PM2.5 from Hettstedt showed an increase in oxidant radical generation compared with cells after saline challenge (p = 0.01, Figure 5). There were no differences without stimulation (data not shown). PM2.5 from Zerbst had neither an influence on the spontaneous nor on the stimulated oxidant radical generation of BAL cells compared with the control challenge.
Biochemical Assays in BAL Fluid PM2.5 from Hettstedt, but not Zerbst, augmented IL-6 production (p = 0.01) and the concentration of tumor necrosis factor- (p = 0.02) in the challenged segment compared with the saline challenged segment (Table 4). However, there was no effect on IL-8 and IL-1 concentration. Instillation of PM2.5 from both Hettstedt and Zerbst did not result in statistically significant differences of total protein, albumin, and LDH in the BAL fluid compared with the control challenge with saline solution (data not shown).
This study was performed to investigate the effect of an endobronchial instillation of PM2.5 suspension from the two German cities, Hettstedt (industrial smelter area) and Zerbst (rural control area), in healthy subjects. The cities are located 80 km apart and are similar in climate and size. The environment of the Hettstedt region is contaminated with heavy metals as a result of an over 800-year history of mining and smelting of nonferrous metals, including lead and copper (11). Whereas both PM suspensions increased the total BAL cell count, only the instillation of the Hettstedt PM2.5 induced a selective influx of CD14high monocytes, together with increased cytokine secretion (IL-6, tumor necrosis factor- ) and elevated oxidant radical generation in BAL cells. Because an equal mass of 100 µg was instilled from both cities, specific dust components, but not the particle dose, must be responsible for this effect. The concentration of LPS was just above the detection limit for both filter extracts. This excludes an LPS effect on the different inflammatory responses. However, transition metals (in particular zinc and copper) were considerably elevated in Hettstedt PM2.5. Increased metal concentration and consequent oxidative stress is known to promote activation of cell signaling and transcription factors, which lead to increased release of proinflammatory mediator release. In particular zinc has been shown to be one of the major components of environmental PM, which induces pulmonary cell reactivity and epithelial damage in animals (12). Indeed, we show here that PM suspensions from Hettstedt had considerably elevated oxidant activity as compared with Zerbst, which leads to an increased generation of secondary oxidant radicals in BAL cells 24 hours after instillation. This was, however, not reflected in a decrease of glutathione and total extracellular antioxidants (data not shown).
Apart from a small but nonsignificant influx of neutrophils, we observed a significant influx of monocytes into the airways after Hettstedt PM instillation. Previously, Maus and coworkers showed in a mouse model that this population of monocytes, with high expression of CD14 and increased tumor necrosis factor- An earlier clinical study applied a similar approach using the endobronchial instillation of metal-rich, water-soluble PM10 extracts from Utah Valley (17). Ghio and Devlin demonstrated that exposure to PM10 extracts collected before closure and after reopening of a steel mill provoked greater airway inflammation relative to PM extracts acquired during plant shutdown in healthy subjects. However, this trial has several major differences compared with our study. First of all, we used particle suspensions and not water-soluble, autoclaved extracts. This allows the interaction of particle core and biological constituents with bronchial epithelium and macrophages. Second, both the particle fraction (PM2.5 in our study vs. PM10 in the Utah Valley study) and the dose in our study (100 µg vs. 500 µg) are closer to the normal exposure of a lung segment to environmental particles. In the Utah Valley study, 500 µg of lyophilized water-soluble extract was instilled with an extraction fraction of 15–26% of the sampled PM10 mass, which means that at least a fourfold higher corresponding PM10 mass has to be considered. The low dose of 100 µg PM2.5 chosen in our study is similar to the amount that would be deposited into a lung lobe during 24 hours of normal breathing, when PM2.5 exceeds 100 µg/m3. This is a realistic scenario during wintertime in the investigated areas. Therefore, a strength of our study is that environmentally relevant PM concentrations were used. We show that this low exposure to metal-rich PM2.5 induces mild acute airway inflammation, which probably causes or aggravates chronic airway diseases, in particular when chronically inhaled. Third, the design of our study was not the comparison of particles from one location in time, as it was done in Utah Valley, but two locations sampled at the same time. Additionally, in our study, the two PM instillations were done within each subject at the same time. This reduces the degree of variation and the number of volunteers (n = 12). In the previous study, a parallel group design was used with three different groups (n = 30). The importance of the Utah Valley study is that it was possible to correlate the pulmonary effects of experimental human exposure with effects found in animal exposure studies and health outcomes observed in epidemiologic studies when the same material was inhaled under normal exposure conditions (18, 19). A similarly unique constellation is now apparent in Germany. Epidemiologic studies have clearly linked living in Hettstedt to increased prevalence of airway diseases and allergies (1, 2, 4, 20). This prompted animal studies, which demonstrate increased airway inflammation in an asthma model after exposure to Hettstedt PM (5). Our human exposure study adds to the scenario, which shows increased airway inflammation after exposure to Hettstedt PM. An important conclusion from both studies is that transition metals might play an important role in the in vivo toxicity of ambient particles. However, the possibility remains that other components of the particles, other than metal content and catalyzed oxidants, contribute to the proinflammatory effects. Furthermore, environmental exposures do not involve nonphysiologic instillation, which limits the comparison of instillation studies with panel studies. Ongoing studies with a detailed analysis of particle composition and strategies using controlled inhalation of the complete particles should answer some of the remaining questions. Harder and colleagues used such an approach to study the effect of ambient PM2.5 on the lung (21). In a controlled inhalation study, they exposed healthy subjects for 2 hours to concentrated ambient air particles from Chapel Hill, North Carolina that ranged from 23–311 µg/m3. They found only small changes in the percentage of neutrophils in BAL fluid 18 hours after the exposure. No differences in other inflammatory cells, including monocytes or cytokines, were found. In accordance with our study, they did not find changes in surface expression of activation markers on macrophages. Gong and colleagues (22) have confirmed these studies in healthy subjects and subjects with mild asthma using concentrated ambient air particles from Los Angeles in a range of 99–224 µg/m3. They were unable to detect clear inflammatory changes in induced sputum 24 hours after exposure. However, no data on metal content of the concentrated ambient air particles are available from either study.
Our study did not address whether subjects with asthma are more susceptible to exposure to Hettstedt PM in comparison to healthy subjects or the more difficult question of whether and how exposure to Hettstedt PM can initiate or trigger development of allergic sensitization. Answers to these questions could explain the increased prevalence of allergy in Hettstedt found in epidemiologic studies. Chronically elevated levels of IL-6 and tumor necrosis factor-
The authors thank B. Reubke-Gothe (Fraunhofer ITEM) for technical assistance, W. Bischof (University of Jena, Germany) for the LPS measurements, and Flemming Cassee (RIVM, Bilthoven, The Netherlands) for the metal measurements.
Supported by grants from Deutsche Forschungsgemeinschaft (SFB 587, project B9), Fraunhofer Society, Institut fuer Umweltmedizinische Forschung, and GSF-National Research Center for Environment and Health. Conflict of Interest Statement: F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.J.A.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; A.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; M.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.P.F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; B.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; N.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this article. Received in original form March 29, 2004; accepted in final form June 28, 2004
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