- Email: [email protected]
Reactions of macrophages exposed to particles <10 μm
Environmental Research 91 (2003) 35–44
Reactions of macrophages exposed to particles o10 mm
$
Christian Monn,* Roland Naef, and Theo Koller Institute for Hygiene and Applied Physiology, Environmental Hygiene, ETH-Zurich, Clausiusstrasse 25, 8092 Zu¨rich, Switzerland Received 10 October 2001; received in revised form 18 July 2002; accepted 20 August 2002
Abstract This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNFa) in a cell line of rat lung macrophages exposed to aqueous extracts from ambient air particles o10 mm (PM10) collected on Teflon filters. The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland. Cytotoxic effects, determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons. Taking together the data from all sites and seasons, a dose–response function was observed between the particle mass on the filter and toxicity (r2 ¼ 0:633; linear regression). The release of the pro-inflammatory cytokine TNFa as well as of oxidative radicals was most pronounced in particles collected in spring–summer and autumn. While at Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zu¨rich and Lugano. It is interpreted that at high PM10 levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects. Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein. This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophage activity. All together, the experiments indicate that in the PM10 fraction, water-soluble macrophagetoxic and macrophage-stimulating compounds are present. The data offer an explanation for at least some of the known harmful effects of PM10, and confirm endotoxin as a possible reactant. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Particles; PM10; Macrophages; Endotoxin; Cytokines
1. Introduction In the past decade, epidemiological studies have shown associations between suspended particulate matter and morbidity and mortality (Dockery et al., 1993; Schwartz, 1996; Ackermann-Liebrich et al., 1997). The strongest effects were observed for inhalable size fractions smaller than 10 mm (PM10) and 2.5 mm (PM2.5). The question of how particulate matter contributes to these effects, is largely unanswered. Hypotheses concerning inflammation, cardiovascular effects, and pulmonary microcirculation are discussed by Hester and Harrison (1998). A way to study physiological effects of particles is the use of in vitro experiments with epithelial cells or with lung macrophages (Becker et al., 1996; $ This study was supported by institutional funds of the Federal Institute of Technology. All experiments were performed in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. *Corresponding author. Fax: +49-1-632-13-18. E-mail address: [email protected] (C. Monn).
Allermann and Poulsen, 2000). In our experiments we have chosen lung macrophages, the important target cells of fine particles (o2.5 mm) in the alveoli. Macrophages act as phagocytes and have the ability to release proinflammatory cytokines and oxidative radicals (for review, see Riott, 1997). Moreover, macrophages are antigen-presenting cells for T-helper cells and modulate the polarization between Th1 and Th2 cells, important in allergy (Steerenberg et al., 1999). In vitro experiments with human and rat alveolar macrophages exposed to urban particulate matter showed the production of oxygen species and release of various cytokines, experiments which emphasized that macrophages are target cells of particulate pollution (Becker et al., 1996; Pritchard et al., 1996; Carter et al., 1997). Stimulated macrophages produced tumor growth factor alpha (TNFa), a cytokine for the recruitment and activation of inflammatory cells (e.g., neutrophils) (Driscoll et al., 1997). Oxidative responses of macrophages can be attributed to the presence of transition metals (Cr, V, Fe) as demonstrated in experiments with
0013-9351/03/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 3 - 9 3 5 1 ( 0 2 ) 0 0 0 2 1 - X
36
C. Monn et al. / Environmental Research 91 (2003) 35–44
residual oil fly ash (ROFA) (Pritchard et al., 1996). In contrast, experiments with inert particles showed upregulation of scavenger receptors and phagocytosis without inflammation (Goldsmith et al., 1997). In toxicological experiments with rodents, particle acidity and transition metals caused inflammation and interfered with host-defense functions of lung macrophages (Chen et al., 1992; Dreher et al., 1996; Kodavanti et al., 1997). In another set of in vitro experiments, alveolar macrophages exposed to urban and rural particulates were stimulated to release the proinflammatory cytokines IL-6 and IL-8 (Becker et al., 1996; Monn and Becker, 1999). The cytokine-inducing moiety was a water-soluble compound of the particles and most effects could largely be inhibited by removing endotoxin, a cell-wall component of gram-negative bacteria. These experiments indicated that endotoxin may be present in ambient air particles and play a modulating role in the release of cytokines. In occupational studies, endotoxins are well-known agents causing inflammation in the respiratory tract, but data on their role in ambient air are sparse (Rylander and Haglind, 1984). All these in vitro experiments demonstrated the importance of transition metals and endotoxins. While the emission sources of transition metals are mainly of anthropogenic origin (combustion processes), the sources of endotoxin remain unclear (Pritchard et al., 1996). We speculate that airborne endotoxin originates from resuspended dust and from fuels. In the study of Miguel et al. (1996), the contribution of deposited biological material to ambient PM10 was quite large. We performed in vitro experiments with lung macrophages in order to address effects such as direct toxicity and the release of radicals and cytokines. Rat macrophages were exposed to filter extracts from ambient PM10 particles. The particles were collected at four different sites (urban, rural, alpine) during all seasons. The goal of the study was to compare site-specific and seasonal effects in macrophages reflecting differences in the pollution composition and suggesting explanations for at least some of the in vivo observations by epidemiology.
The other two sites are located in the north of the Alps about 200 km from each other. Particles were collected during four weeks (4 1 week) in winter, spring, summer, and autumn 1997. Gravimetrical analyses (Mettler: AT 261, Delta Range) were conducted after conditioning of the filters for 24 h at 211C and 52% relative humidity. 2.2. Extraction of collected air pollutants Filters were stored at room temperature (RT). Extraction of water-soluble material was performed in sterile snap cap tubes (Falcon) using 1.5 mL of sterile phosphate-buffered saline (PBS, Fakola). Filters were submerged in PBS and placed for 20 min in an ice-cold ultrasound bath (ABS) followed by 30 min on a horizontal shaker (Baxter) at 4500 rpm. Filters were removed after a quick spin at 1500 rpm, and extracts were stored at –201C until further use. A blank filter was used as a negative control and treated identically, while a solution of 10 ng/mL of lipopolysaccharide (LPS, from Salmonella typhimurium, Sigma) was used as a positive control and a well-known inducer of oxidative radicals and cytokines by macrophages (Becker et al., 1996). 2.3. Cell culture The NR8383 rat alveolar macrophage cell line was obtained from the American Type Culture Collection (CRL-2192, American Type Culture Collection (ATCC), Virginia) as a homogenous and easily expandable source of alveolar monocyte/macrophage-like cells. They were cultured in F12K medium supplemented with 15% of endotoxin-free fetal bovine serum (FBS, heat inactivated for 30 min at 561C), 50 mg/mL gentamicin, and 4 mM l-glutamine (all from Life Technologies) at 371C, 7.5% CO2, and 95% relative humidity. Cells were allowed to expand to 8 105/mL and subsequently split to 2 105/mL in order to maintain optimal density for exponential growth according to ATCC. Viability was checked to be within the recommended range prior to each passage using Trypan Blue (Sigma). 2.4. Exposure of phagocytes
2. Materials and methods 2.1. PM10 sampling PM10 was measured with Harvard low-volume sharp cut impactors (Marple et al., 1987). At the urban sites Zu¨rich (410 m.s.l.) and Lugano (280 m.s.l.), the rural site Payerne (460 m.s.l.), and the alpine site Montana (1350 m.s.l.), weekly integrated samples were collected on Teflon filters (Gelman Sci., R2PJ041, 47 mm diameter, 2 mm pore size). Lugano and Montana are located in the south of the Alps but in different valleys.
Rat NR8383 cells were plated in a 96-well dish at a final density of 4 105/mL with 10% of total volume (50 mL particle extract in 450 mL cell media) of particle extracts and controls in triplicate for each of the MTT, cytokine, and NO 2 assays (see below) and incubated for 40 h. 2.5. Metabolic activity test (MTT) The MTT test (Roche Diagnostics) was used to determine the cytotoxicity of the particle extracts and
C. Monn et al. / Environmental Research 91 (2003) 35–44
controls for NR8383 cells, according to the manufacturer’s instructions. The MTT assay uses a mitochondrial dehydrogenase-dependent conversion of a yellow tetrazolium salt to purple formazan crystals by life cells. For the metabolic activity test, the 96-well dish (see above) was incubated for 40 h prior to addition of 10 mL of the MTT reagent and another 24 h afterward under the same conditions as above. Incubation times for the MTT reaction were set to 4 h. The conversion rate was measured photometrically. A standard curve was established based on a maximum of 40,000 cells (=100% metabolic activity or 0% toxicity) and eight diluted concentrations. Toxicity in percent is expressed as 100%-metabolic activity (%). A comparison with the Trypan blue assay (Sigma, Bioscience T8154) showed good correlation between the two assays.
37
endotoxin-neutralizing-protein (ENP)-coated resin (Associates of Cape Cod). After incubation at room temperature for 4 h and spinning down at 1200 g, supernatants were stored at 201C. 2.9. Statistical analyses Statistical analyses were performed with the Systat Version 8.0 program. Differences between the sites for mass, toxicity, NO 2 , and TNFa were tested by analysis of variance. The correlation matrix was calculated with the Spearman correlation. Linear multiple regression models were calculated in order to control for the toxicity in the mass–TNFa and mass–NO 2 relationships.
2.6. Measurement of oxidative response
3. Results
We utilized the specific potential of rodent macrophage cells to form reactive nitrogen species as an oxidative response to inflammatory stimuli by measuring nitrite (NO 2 ) levels as the stable end product in the supernatant of NR8383 cultures using the Griess reaction (Lewats et al., 1993). NR8383 cells were incubated with particle extracts and controls for 40 h and spun for 10 min at 120 g, and 50 mL of supernatant was transferred into a new 96-well dish to which 50 mL of a 1% sulfanilamide (Sigma), solution followed by 50 mL of a 0.1% solution of N(1-Naphtyl)-ethylendiamine dihydrochloride (Sigma), each in 2.5% orthophosphoric acid (Fluka), were added. Color formation was measured photometrically at 550 nm and the resulting NO 2 concentration was calculated relative to a standard 1:2 dilution curve of a sodium nitrite (Sigma) solution in culture medium ranging from 100 to 0.78 mM.
Particles smaller than 10 mm (PM10) were collected with low-volume samplers at four different sites (two urban, one rural, one alpine) during all seasons for 4 weeks in 1997. Collecting time was 1 week and the filter medium was Teflon. After gravimetric analyses for the determination of PM10 air pollution levels, the filters were extracted with phosphate buffer (PBS) for 20 min in an ultrasonic bath. Rat lung macrophages were exposed to these extracts in order to assess cytotoxic effects, oxidative response (NO 2 ), and release of the proinflammatory cytokine TNFa. The experiments were repeated three times. Within each series, aliquots of the same batch of rat macrophages were used and, additionally, in all experiments negative (unexposed blank filters) and positive controls (10 ng/mL LPS) were run in parallel to the particle extracts. The levels of the collected particle mass are presented in Fig. 1. The highest mass concentrations were found at Lugano and Zu¨rich (both urban), followed by Montana (alpine) and the rural site Payerne, in agreement with previous observations (Monn et al., 1995). Based on analysis of variance, the differences were significant between Lugano and Montana (po0.05) and between Lugano and Payerne (po0.05). The levels and the seasonal variations at the four sites were quite different, reflecting differences in climate, population densities, human activities, and life styles (Martin et al., 1997). A sampled particle mass of 800 mg is equivalent to an airborne PM10 level of 20 mg/m3. Average PM10 levels were 27 mg/m3 at Lugano, 17 mg/m3 at Zu¨rich, 9 mg/m3 at Montana, and 8 mg/m3 at Payerne. Rat macrophages exposed to the aqueous extracts from the particles were tested by three different assays in order to evaluate the cytotoxic effect (MTT-assay) and the production of NO 2 and TNFa. Fig. 2a shows the result of the MTT-assays. In the negative controls (blank filters) toxicity was around 5%; in the positive
2.7. Cytokine measurements Induction of the proinflammatory cytokine TNFa by NR8383 cells due to particle extracts and controls were measured with a rat TNFa ELISA (BioConcept) according to the manufacturer’s instructions. Cells were incubated for 40 h and spun at 120 g for 10 min, and supernatants were stored at –801C until further use. Supernatants were diluted 1.5-fold with the exception of the positive controls (10 ng/mL LPS), which were diluted five-fold in order to remain in the assay’s recommended concentration range. 2.8. Inhibition of endotoxin Particle extracts were treated with inhibitors for endotoxin as previously described (Monn and Becker, 1999): 500 mL of each extract were added to END-X B15
C. Monn et al. / Environmental Research 91 (2003) 35–44
38
2000 1800
40 µg/m3
particle mass (µg)
1600 1400 1200 1000
20 µg/m3
800 600 400 200
Lugano
Zürich
Montana
autumn
spring
summer
winter
autumn
summer
winter
spring
autumn
summer
winter
spring
autumn
summer
winter
spring
0
Payerne
Fig. 1. Airborne particles o10 mm (PM10) were collected on Teflon filters using low-volume sharp-cut impactors at four sites during winter, spring, summer, and autumn period in 1997. Four measurements were performed during each season with a cumulative sampling period of 7 days. The columns represent averages of the collected mass of each site and season and the bars represent the standard deviation. On average, largest mass concentrations were observed at the urban sites Lugano and Zu¨rich, followed by Montana and Payerne. Significant differences in an analysis of variance were found between Lugano and Montana (po0.05) and between Lugano and Payerne (po0.05). Note that a particle mass of 800 mg is equivalent to an airborne PM10 concentration of 20 mg/m3.
LPS control toxicity reached 80%. (A 100% toxicity means that all cells exposed to the particle extracts lost their mitochondrial dehydrogenase activity.) The toxicity of the environmental particle extracts ranged between 10% and 65%. At Zu¨rich and Payerne, extracts from winter had the strongest cytotoxic activity. At Montana (alpine), the spring and summer samples were more toxic than the winter particles. At Lugano, summer and winter extracts induced the strongest toxicity. Taking together the samples from all seasons, Lugano differed significantly from Montana (po0.01) and Payerne (po0.001). Zu¨rich and Payerne differed significantly (po0.001). The other differences between sites were not significant. All exposures to particle extracts differed significantly from the negative control (blank filter) (po0.001). In the second experiment, cells exposed to particle extracts were tested with respect to the NO 2 production. Fig. 2b shows the result for all sites and seasons. In
contrast to the cytotoxic effects, the strongest production of NO 2 appeared mostly in the nonwinter seasons. At Montana, Payerne, and Zu¨rich, the strongest peaks were found in the summer. Taking together the values of all seasons, Lugano differed significantly (analysis of variance) from Zu¨rich (po0.01). In the third experiment (Fig. 2c), the production of TNFa in cells exposed to particle extracts was measured. Several of the extracts did not show a significant stimulation of the TNFa production with respect to the negative control. The seasonal variation tends to have the strongest inductions outside winter. Highest peaks were observed at Payerne (summer), Zu¨rich (summer) and Montana (spring). Taking together the values of all seasons, there were no significant differences (analysis of variance) between the sites. The observations found in Figs. 2a–c were statistically analyzed. Table 1 presents the Spearman rank correlations between the variables ‘‘mass,’’ ‘‘toxicity,’’ ‘‘NO’’ 2 ,
Fig. 2. (a–c) Particles collected on filters were submitted to an extraction in physiologically buffered saline. Each particle extract was added to the same number of rat macrophages (NR8383) for 40 h. Measurements of the toxicity (MTT-assay) (a), of NO 2 (b), and TNFa (c) were performed in three individual experiments. The columns represent the average levels of the three experiments (bars: 7 one standard deviation). The controls represent extracts from unexposed blank filters (neg) and 10 ng/mL LPS (pos) which were run in parallel in the same experiments. (a) Toxicity is expressed as 100% minus the metabolic activity in % (100% represents 40,000 live cells). On the average, strongest toxicity was observed with the urban extracts, Lugano and Zu¨rich. Statistically significant differences (analysis of variance, data from all seasons) were observed between Lugano and Montana (po0.01), Lugano and Payerne (po0.001) and Zu¨rich and Payerne (po0.001). All other differences between the sites were not significant. All values differed significantly from the negative control (po0.001). (b) Statistically significant differences of the NO 2 concentrations (analysis of variance, data from all seasons) were observed between Lugano and Zu¨rich (po0.01). All other differences were not significant. Values which differed significantly from the negative controls (po0.001) are marked by asterisks. (c) For the TNFa values, no significant differences (analysis of variance, data from all seasons) between the sites (p40.05) were observed. Values which differed significantly from the negative controls (po0.001) are marked by asterisks.
(c)
control
Lugano
Zürich
Montana
* autumn
summer
spring
winter
*
autumn
* *
spring
Montana
summer
* autumn
Montana
winter
*
autumn
912 summer
*
spring
35
summer
Zürich winter
*
spring
Zürich
winter
16
autumn
500 autumn
18
summer
spring
*
spring
* winter
autumn
Lugano
summer
Lugano
winter
450 summer
*
autumn
winter spring
14
summer
neg
control
spring
control
winter
(b) pos
NO2- (µM) (a)
pos
neg
TNFα (pg/ml)
autumn
summer
spring
winter
autumn
summer
spring
winter
autumn
summer
spring
winter
autumn
summer
spring
winter
pos
neg
toxicity (%)
C. Monn et al. / Environmental Research 91 (2003) 35–44 39
100 90
80
70
60
50
40
30
20
10
0
Payerne
* *
*
12
* *
10
8
6
4
2
0
Payerne
*
*
400
350
300
250
200
150
100
50
0
Payerne
C. Monn et al. / Environmental Research 91 (2003) 35–44
40
and ‘‘TNFa’’, based on the data from all sites and seasons. The mass levels measured on the filters correlated best and positive with the toxic effects (r ¼ 0:797). On the other hand, NO 2 and TNFa values were not well correlated with the mass but they correlated well with each other (r ¼ 0:834). The effect of the mass levels on the production of NO 2 and TNFa was also calculated in a multiple regression model with the controlling variable ‘‘toxicity.’’ However, the influence of mass on NO 2 and TNFa remained insignificant in this multiple regression model. The scatterplot in Fig. 3 shows the influence of mass on the toxic effect of the aqueous extracts. In a linear regression model, the relationship is positive and highly significant, with an R2 of 0.633 over all data. For all sites, the toxicity of the extracts increased with increasing mass levels. Note that for Payerne, almost no variation in mass data occurred, so that no relationship between mass and toxicity can be assessed. Since the NO 2 and TNFa values correlated strongly (see Table 1), a ‘‘stimulation’’ factor was calculated,
taking together the NO 2 and TNFa values expressed as percentage of the respective LPS control. Figs. 4a and b show the scatterplots between mass and the stimulation factor for Montana on the one hand and Lugano and Zu¨rich on the other hand. In contrast to the relationship between mass and toxicity, considerable differences were observed between the mass and the stimulation factor at the different sites. At the low end of the mass levels (Montana, Fig. 4a) the relationship was positive. At the two urban sites Zu¨rich and Lugano, which are located at the upper end of the mass levels, the relationship became negative (Fig. 4b). At Payerne, where the variability of the mass levels was small (see Fig. 1), no correlation can be assessed. According to the findings from Becker et al. (1996) and Monn and Becker (1999), endotoxins may partly be responsible to cause some of the effects described above. We tested the influence of endotoxin in parallel experiments by removing endotoxins by an ENP. Figs. 5a–c show the effects of the untreated (set at 100%) and ENP-treated (relative to the untreated
Table 1 Spearman correlation matrix between the particle mass, toxicity, and NO 2 and TNFa concentrations Toxicity
1.000 0.797 0.137 0.179
1.000 0.075 0.082
1.000 0.834
TNFa
1.000
Montana
50 stimulation
Mass Toxicity NO 2 TNFa
Mass
NO 2
55
The analysis is based on data from all sites and seasons. The matrix shows strong and significant correlations between the collected mass levels and the toxicity and between the NO 2 and TNFa concentrations.
45 40 35 30 25 20 0
500
(a)
1000 mass
1500
2000
55
70 stimulation
60 50 toxicity (%)
urban:Lugano, Zürich
50
40
45 40 35 30
30
25
20
20 0
10
(b)
0 0
500
1000 mass
1500
2000
Fig. 3. Toxicity (%) is plotted versus the collected mass levels (mg) for all sites and seasons. Linear regression over all data: y ¼ 0:025x þ 20:6; R2 ¼ 0:6338: Overall, with increasing mass the toxicity increased. Payerne is an exception with mass levels of low variance. (Legend: triangle, Montana; circle, Payerne; square, Zu¨rich; small square: Lugano).
500
1000 mass
1500
2000
Fig. 4. (a–b) A ‘‘stimulation’’ factor was calculated, taking together the NO 2 and TNFa values (expressed as percentage of the respective LPS control) from the alpine site Montana and the urban sites Zu¨rich and Lugano. This factor is plotted against the collected mass levels (mg). (a) At Montana (lower mass range), a positive relationship was observed. Regression model: y ¼ 0:0216x þ 28:2; R2 ¼ 0:831: (b) At the urban sites Lugano and Zu¨rich (upper mass range), the relationship was negative. Regression model: y ¼ 0:0066x þ 44:5; R2 ¼ 0:582: (Note that the use of the single variables ‘‘TNFa’’ or ‘‘NO 2 ’’ leads to similar figures.)
C. Monn et al. / Environmental Research 91 (2003) 35–44
41
toxicity
120 100 percent
80 60 40 20 0 (a)
neg
pos
Lugano
Zürich
Montana Payerne
NO2-
120
percent
100 80 60 40 20 0 (b)
neg
pos
Lugano
Zürich
Montana
Payerne
Zürich
Montana
Payerne
TNF alpha
120 100 percent
80 60 40 20 0 (c)
neg
pos
Lugano
Fig. 5. (a–c) Rat macrophages (NR8383) were exposed to particle extracts treated with an ENP, to untreated particle extracts and to controls (blank, LPS) in parallel experiments. An assessment of the toxicity (a), of the NO 2 levels (b), and of the TNFa levels (c) was performed. In all experiments, a negative (neg) (unexposed blank filter) and a positive control (pos) (10 ng/mL LPS) were run in parallel with the particle extracts. The first columns (grey) show the ENP-untreated values set at 100%, the second columns present the values of the ENP-treated extracts (in percent) (dark columns). (a) Highly significant differences (pairwise analysis: untreated versus treated) in toxicity were observed after treatment with ENP for LPS and for all sites (po0.001). (b) Highly significant differences (pairwise analysis: untreated versus treated) of the NO 2 release were observed after treatment with ENP for LPS and for all sites (po0.001). (c) Significant differences (pairwise analysis: untreated versus treated) of TNFa release were observed after treatment with ENP for LPS (po0.001) and for Zu¨rich (po0.05) and Payerne (po0.05). Note that the TNFa levels compared to the negative controls (see Fig. 2) were generally low in these experiments. Only data where the values were higher than the blank were included in this analysis.
samples in %) samples and the negative (blank filter) and positive (LPS) controls. The effect of ENP on the reduction of toxicity was highly significant at all sites and for the positive control (po0.001) (Fig. 5a). Similar effects were observed for the NO 2 production with
significant reductions at all sites and for the positive control (po0.001) (Fig. 5b). For TNFa, weak reductions (po0.05) were observed at Zu¨rich and Payerne and not at Lugano and Montana. In this analysis, only data with significant stimulations (compared to the
42
C. Monn et al. / Environmental Research 91 (2003) 35–44
negative controls) were used as in about 45% of the cases, the particle extracts did not induce TNFa levels above the blank background levels (see Fig. 2). In the LPS control, the reduction was highly significant after treatment with ENP (po0.001) (Fig. 5c).
4. Discussion Macrophages are target cells for particles in the alveolar tract, where they take part in particle scavenging and modulate inflammation (for review see Riott, 1997). An in vitro system with a cell line from rat macrophages was used to analyze the effects of aqueous extracts from ambient air PM10 particles. Compared to freshly prepared phagocytes or monocytes, the use of a cell line provides standardized conditions in different sets of experiments. Such in vitro studies make it possible to visualize a single reaction mechanism contributing to a complex in vivo situation and to show differences in the reactions induced by different pollution samples. Cytotoxic effects were assessed with a mitochondrialdehydrogenase-dependent test (MTT-assay). A comparison with the trypan blue test, where dead and live cells are counted microscopically, showed good agreement between the two assays (r ¼ 0:85; data not shown). Therefore, we assume that the MTT-assay expressed as toxicity mainly reflects the proportion of dead cells, although the presence of living cells with reduced mitochondrial dehydrogenase activity cannot be excluded. The advantage of the MTT-assay lies in the precise and easy photometric measurement. Our experiments show mainly effects of water-soluble compounds in the PM10 sample since we extracted the filters with a buffer solution only. The chemical composition of our aqueous extract was not determined for technical reasons. It is known that allergens, endotoxins, ions, soluble metal salts, and some polar organic compounds can be present in aqueous particle extracts (Vrtala et al., 1993, 1988; Ghio et al., 1996). After our extraction procedure, the filter pieces were still quite blackened and we assume that only a small proportion of solid particles was washed off into the extract. Moreover, we assume that the apolar organic and carbon black fraction remained mostly in the filters. Unfortunately, it is not possible to quantify the mass extracted as the difference between an extracted and unextracted filter is too small and as the buffer salt may even increase the total mass after extraction. Our experiments showed that cells exposed to particle extracts from urban, rural, or alpine sites had reduced viability compared to cells exposed to extracts from blank control filters. The strongest effects were observed with extracts collected during winter, except for the alpine site Montana and for Lugano, where the summer extracts also had strong toxic effects.
Taking together data from all sites and seasons, a good correlation between the particle mass on the filters and the toxicity of the extracts (R2=0.633, linear regression) was observed, suggesting that toxic water-soluble compounds were trapped on the filter and washed off in our extraction procedure. The correlation between mass and toxicity is remarkable as the chemical composition between sites and seasons might vary considerably. However, this finding correlates with observations in epidemiological studies where health effects were independent of the chemical composition of particles and the associations were attributable to the mass concentration of PM10 (or PM2.5) in ambient air (Schwartz, 1996). The observed toxic effects may reflect health hazards caused by hampering scavenging and defence mechanisms. There is support for this explanation from an in vitro study by Becker and Soukup (1999) where macrophages exposed to ambient air particle extracts had reduced capability of virus uptake and reduced production of monocyte chemoattractants (e.g., MCP-1) which are important in anti-bacterial defense. Our experiments on the release of oxidative radicals and cytokines showed that the strongest effects occurred with PM10 filter extracts collected in spring and summer, especially from nonurban sites. Taking together data from all sites and seasons, no correlation between the production of oxidative radicals or TNFa and the PM10 mass concentration was observed. On the other hand, the correlation between NO 2 and TNFa values was good, indicating similar signalling. Because of this significant correlation, we calculated a macrophage stimulation factor comprising both the NO 2 and the TNFa values. By plotting this stimulation factor versus the particle mass of the corresponding filters, we noticed a profound difference between the urban and the alpine sites. In Montana with rather low particle mass collected, the correlation was positive. In Zu¨rich and Lugano with high particle mass, the correlation was negative. For Payerne, where the variation of the particle mass was small (Fig. 1), no obvious correlation was found. Since mass and toxicity correlate well, we interpret that at low PM10 levels as in Montana (Fig. 4a), the macrophages are increasingly stimulated by increasing particle mass. On the other hand, at high PM10 levels, as in Lugano and Zu¨rich (Fig. 4b), macrophage stimulation is inhibited with increasing anthropogenic air pollution by increasing toxic effects. Alternatively, we can interpret this observation by the contribution of biological material to the particle mass, being stronger at the alpine site Montana compared to the urban sites Lugano and Zu¨rich (Hu¨glin et al., 1999; Spieksma, 1995). The production of high radical levels exhibits a self-toxic effect on cells, i.e., in the presence of transition metals (Pritchard et al., 1996). The poor statistical correlation between the bulk of our NO 2 values and the toxicity is not in contradiction to this
C. Monn et al. / Environmental Research 91 (2003) 35–44
mechanism, as with an increasing number of dead cells in the Lugano and Zu¨rich extracts, the production of NO 2 is inhibited (part of the data of Fig. 4b). However, transition metals may not be important in our case, since values measured in a parallel study ranged between 50 and 370 ng/m3 for Fe and between o1 and 2.4 ng/m3 for Cr (unpublished data). These values are more than 10 times lower than concentration levels detected in urban PM10 of US cities (US Environmental Protection Agency, 1996). We tested whether the presence of endotoxin plays a role in the observed effects by inactivating endotoxins with an ENP. After inactivation, the cytotoxic effects were significantly reduced, as well as, NO 2 being released. Cytokine release was slightly reduced in the samples of Zu¨rich and Payerne. These observations suggest that endotoxin is at least one factor in the toxic effects and the oxidative radical production of our particle extracts. Endotoxins exhibit a toxic potential by activating enzymes in the caspase family (Mariani et al., 1997). Part of the endotoxin, the Lipid A portion, is also responsible for inducing inflammatory cytokines (Someya et al., 1996). Endotoxin levels in the particle extracts were also determined with the Limulus assay. However, the data were hardly above background, as the levels of endotoxin were very low (data not shown). In the studies from Monn and Becker (1999), and Becker et al. (1996) the mere presence of endotoxin was sufficient to produce cytokines and there was no relationship with the particle mass. In contrast to the studies from Becker et al. (1996), Monn and Becker (1999), and Pritchard et al. (1996), this study is based on air samples from different seasons collected in the lower air pollution range. The stimulatory effects were smaller than those found in the other studies due to lower endotoxin and transition metal levels. However, despite the low content, of these compounds, our particle extracts exhibited toxic effects with a clear dose–response relationship with the collected mass on the filters. In conclusion, our experiments offer explanations for at least some of the harmful effects of PM10.They indicate that PM10 contains water-soluble macrophagetoxic and macrophage-stimulating compounds, some of them of biological origin (e.g., endotoxin, allergens) (Miguel et al., 1996; Knox et al., 1997). The experiments help to explain observations made by epidemiology and show differences in the pollution pattern between urban, rural, and alpine sites and between different seasons.
References Ackermann-Liebrich, U., Leuenberger, Ph., Schwartz, J., Schindler, Ch., Monn, Ch., Bolognini, G., et al., and SAPALDIA Team, 1997. Lung function and long term exposure to air pollutants in Switzerland. Am. J. Respir. Crit. Care Med. 155, 122–129.
43
Allermann, L., Poulsen, O.M., 2000. Inflammatory potential of dust from waste handling facilities measured as IL-8 secretion rom lung epithelial cells in vitro. Ann. Occup. Hyg. 44 (4), 259–269. Becker, S., Soukup, J.M., Gilmour, M.I., Devlin, R.B., 1996. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol. Appl. Pharmacol. 141, 637–648. Becker, S., Soukup, J.M., 1999. Exposure to urban air particulates alters the macrophage-mediated inflammatory response to respiratory viral infection. J. Toxicol. Environ. Health 57 (7), 445–457. Carter, J.D., Ghio, A., Samet, J.M., Devlin, R.B., 1997. Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol. Appl. Pharmacol. 146, 180–188. Chen, L.C, Fine, J.M., Qu, Q.S., Amdur, M.O., Gordon, T., 1992. Effects of fine and ultra-fine sulfuric acid aerosols in guinea pigs: alterations in alveolar macrophage function and intracellular pH. Toxicol. Appl. Pharmacol. 113 (1), 109–117. Dockery, D.W., Pope III, C.A., Xu, X., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris Jr., B.G., Speizer, F.E., 1993. An association between air pollution and mortality in six US cities. N. Engl. J. Med. 329, 1753–1759. Dreher, K., Jaskot, R., Kodavanti, U., Lehmann, J., Winsett, D., Costa, D., 1996. Soluble transition metals mediate the acute pulmonary injury and airway hyperreactivity induced by residual oil fly ash articles. Chest 109 (3 Suppl), 33S–34S. Driscoll, K.E., Carter, J.M., Hassenbein, D.G., Howard, B., 1997. Cytokines and particle-induced inflammatory cell recruitment. Environ. Health Perspect. 105 (Suppl 5), 1159–1164. Ghio, A.J., Stonehuerner, J., Pritchard, R.J., Piantadosi, C.A., Quigley, D.R., Dreher, K.L., Costa, D.L., 1996. Human-like substances in air pollution particulates correlates with concentrations of transition metals and oxidant response. Inhalation Toxicol. 8, 479–494. Goldsmith, C.A., Frevert, Ch., Imrich, A., Sioutas, C., Kobzik, L., 1997. Alveolar macrophage interaction with air pollution particulates environ. Health Perspect. 105 (Suppl 5), 1191–1195. Hester, R.E., Harrison, R.M. (Eds.), 1998. Air Pollution and Health. Issues in Environmental Science and Technology, Vol. 10. Roy. Soc. Chem. Herts., UK. Hu¨glin, Ch., Gehrig, R., Devos, W., Moor, Ch., Baltensperger, U., Monn, Ch., 1999. Chemical composition and source apportionment of PM10 and PM25 in Switzerland. VDI Rep. 1443, 307–312. Knox, R.B., Suphioglu, C., Taylor, P., Desai, R., Watson, H.C., Peng, J.L., Bursill, L.A., 1997. Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution. Clin. Exp. Allergy 27, 246–251. Kodavanti, U.P., Jaskot, R.H., Su, W.Y., Costa, D.L., Ghio, A.J., Dreher, K.L., 1997. Genetic variability in combustion particleinduced chronic lung injury. Am. J. Physiol. 272 (3 Part 1), L521–L532. Lewats, J., Mumford, J., Everson, R.B., Hulka, B., Wilcosky, T., Kozumbo, W., Thompson, C., George, M., Dobias, L., Sram, R., 1993. Environ. Health Perspect. 99, 89–97. Mariani, S.M., Matiba, B., Armandola, E.A., Krammer, P.H., 1997. Interleukin 1 beta converting enzyme related protease/caspase are involved in TRAIL-induced apoptosis of myeloma and leukemia cells. J. Cell Bio. 137, 221–229. Marple, V.A., Rubow, K.L., Turner, W., Spengler, D., 1987. Low flow-rate sharp-cut impactors for indoor air sampling: design and calibration. J Air Pollut. Control. Assoc. 37 (11), 1303–1307. Martin, B., Ackermann, U., Leuenberger, Ph., Ku¨nzli, N., Zemp, E., Keller, R., Zellweger, J.-P., Wu¨thrich, B., Monn, Ch., et al., SAPALDIA Team, 1997. SAPALDIA: methods and participation in the cross sectional part of the Swiss Study on Air Pollution and Lung Function in Adults. Soz. Pra¨ventivmed. 42, 67–84.
44
C. Monn et al. / Environmental Research 91 (2003) 35–44
Miguel, A.G., Cass, G.R., Weiss, J., Glovsky, M.M., 1996. Latex allergens in tire dust and airborne particles. Environ. Health Perspect. 104, 1180–1185. Monn, Ch., Bra¨ndli, O., Scha¨ppi, G., Ackermann, U., Leuenberger, Ph., SAPALDIA Team, 1995. Particulate matter and total suspended particulates in urban, rural and alpine air in Switzerland. Atmos. Environ. 29 (19), 2565–2573. Monn, Ch., Becker, S., 1999. Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (PM 2.5) and coarse particles (PM10-2.5) in outdoor and indoor air. Toxicol. Appl. Pharmacol. 155, 245–252. Pritchard, R.J., Ghio, A.J.J., Lehmann, J.R., Winsett, D.W., Tepper, J.S., Park, P., Gilmour, M.I., Dreher, K.L., Costa, D.L., 1996. Oxidant generation and lung injury after particulate air pollutants exposure increase with the concentrations of associated metals. Inhalation Toxicol. 8, 457–477. Riott, I.M., 1997. Essential Immunology, 9th Edition. Blackwell, Oxford. Rylander, R., Haglind, P., 1984. Airborne endotoxins and humidifier disease. Clin. Allergy 14 (1), 109–112.
Schwartz, J., 1996. Air pollution and hospital admissions for respiratory disease. Epidemiology 7, 20–28. Someya, K., Tsutomi, Y., Soga, T., Akahane, K., 1996. A lipid A analog inhibits LPS-induced cytokine expression and improves survival in endotoxemic mice. Immunopharmacol. Immunotoxicol. 18 (4), 477–495. Spieksma, F.Th.M., 1995. Aerobiology of inhalatory allergen carriers. Allergol. Immunopathol. 23, 20–23. Steerenberg, P.A., Van Amsterdam, J.G.C., Vernderbriel, R.J., Vos, J.G., Van Bree, L., Van Loveren, H., 1999. Environmental and lifestyle factors may act in concert to increase prevalence of respiratory allergy including asthma. Clin. Exp. Allergy 29, 1304–1308. US Environmental Protection Agency, 1996. Air Quality Criteria for Particulate Matter, Vol. I, Report: EPA/600/P-95/001aF, Research Triangle Park, NC. Vrtala, S., Grote, M., Duchene, M., van-Ree, R., Kraft, D., Scheiner, O., Valenta, R., 1993. Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity. Int. Arch. Allergy Immunol. 102 (2), 160–169.
Reactions of macrophages exposed to particles o10 mm
$
Christian Monn,* Roland Naef, and Theo Koller Institute for Hygiene and Applied Physiology, Environmental Hygiene, ETH-Zurich, Clausiusstrasse 25, 8092 Zu¨rich, Switzerland Received 10 October 2001; received in revised form 18 July 2002; accepted 20 August 2002
Abstract This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNFa) in a cell line of rat lung macrophages exposed to aqueous extracts from ambient air particles o10 mm (PM10) collected on Teflon filters. The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland. Cytotoxic effects, determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons. Taking together the data from all sites and seasons, a dose–response function was observed between the particle mass on the filter and toxicity (r2 ¼ 0:633; linear regression). The release of the pro-inflammatory cytokine TNFa as well as of oxidative radicals was most pronounced in particles collected in spring–summer and autumn. While at Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zu¨rich and Lugano. It is interpreted that at high PM10 levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects. Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein. This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophage activity. All together, the experiments indicate that in the PM10 fraction, water-soluble macrophagetoxic and macrophage-stimulating compounds are present. The data offer an explanation for at least some of the known harmful effects of PM10, and confirm endotoxin as a possible reactant. r 2002 Elsevier Science (USA). All rights reserved. Keywords: Particles; PM10; Macrophages; Endotoxin; Cytokines
1. Introduction In the past decade, epidemiological studies have shown associations between suspended particulate matter and morbidity and mortality (Dockery et al., 1993; Schwartz, 1996; Ackermann-Liebrich et al., 1997). The strongest effects were observed for inhalable size fractions smaller than 10 mm (PM10) and 2.5 mm (PM2.5). The question of how particulate matter contributes to these effects, is largely unanswered. Hypotheses concerning inflammation, cardiovascular effects, and pulmonary microcirculation are discussed by Hester and Harrison (1998). A way to study physiological effects of particles is the use of in vitro experiments with epithelial cells or with lung macrophages (Becker et al., 1996; $ This study was supported by institutional funds of the Federal Institute of Technology. All experiments were performed in accordance with national and institutional guidelines for the protection of human subjects and animal welfare. *Corresponding author. Fax: +49-1-632-13-18. E-mail address: [email protected] (C. Monn).
Allermann and Poulsen, 2000). In our experiments we have chosen lung macrophages, the important target cells of fine particles (o2.5 mm) in the alveoli. Macrophages act as phagocytes and have the ability to release proinflammatory cytokines and oxidative radicals (for review, see Riott, 1997). Moreover, macrophages are antigen-presenting cells for T-helper cells and modulate the polarization between Th1 and Th2 cells, important in allergy (Steerenberg et al., 1999). In vitro experiments with human and rat alveolar macrophages exposed to urban particulate matter showed the production of oxygen species and release of various cytokines, experiments which emphasized that macrophages are target cells of particulate pollution (Becker et al., 1996; Pritchard et al., 1996; Carter et al., 1997). Stimulated macrophages produced tumor growth factor alpha (TNFa), a cytokine for the recruitment and activation of inflammatory cells (e.g., neutrophils) (Driscoll et al., 1997). Oxidative responses of macrophages can be attributed to the presence of transition metals (Cr, V, Fe) as demonstrated in experiments with
0013-9351/03/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 3 - 9 3 5 1 ( 0 2 ) 0 0 0 2 1 - X
36
C. Monn et al. / Environmental Research 91 (2003) 35–44
residual oil fly ash (ROFA) (Pritchard et al., 1996). In contrast, experiments with inert particles showed upregulation of scavenger receptors and phagocytosis without inflammation (Goldsmith et al., 1997). In toxicological experiments with rodents, particle acidity and transition metals caused inflammation and interfered with host-defense functions of lung macrophages (Chen et al., 1992; Dreher et al., 1996; Kodavanti et al., 1997). In another set of in vitro experiments, alveolar macrophages exposed to urban and rural particulates were stimulated to release the proinflammatory cytokines IL-6 and IL-8 (Becker et al., 1996; Monn and Becker, 1999). The cytokine-inducing moiety was a water-soluble compound of the particles and most effects could largely be inhibited by removing endotoxin, a cell-wall component of gram-negative bacteria. These experiments indicated that endotoxin may be present in ambient air particles and play a modulating role in the release of cytokines. In occupational studies, endotoxins are well-known agents causing inflammation in the respiratory tract, but data on their role in ambient air are sparse (Rylander and Haglind, 1984). All these in vitro experiments demonstrated the importance of transition metals and endotoxins. While the emission sources of transition metals are mainly of anthropogenic origin (combustion processes), the sources of endotoxin remain unclear (Pritchard et al., 1996). We speculate that airborne endotoxin originates from resuspended dust and from fuels. In the study of Miguel et al. (1996), the contribution of deposited biological material to ambient PM10 was quite large. We performed in vitro experiments with lung macrophages in order to address effects such as direct toxicity and the release of radicals and cytokines. Rat macrophages were exposed to filter extracts from ambient PM10 particles. The particles were collected at four different sites (urban, rural, alpine) during all seasons. The goal of the study was to compare site-specific and seasonal effects in macrophages reflecting differences in the pollution composition and suggesting explanations for at least some of the in vivo observations by epidemiology.
The other two sites are located in the north of the Alps about 200 km from each other. Particles were collected during four weeks (4 1 week) in winter, spring, summer, and autumn 1997. Gravimetrical analyses (Mettler: AT 261, Delta Range) were conducted after conditioning of the filters for 24 h at 211C and 52% relative humidity. 2.2. Extraction of collected air pollutants Filters were stored at room temperature (RT). Extraction of water-soluble material was performed in sterile snap cap tubes (Falcon) using 1.5 mL of sterile phosphate-buffered saline (PBS, Fakola). Filters were submerged in PBS and placed for 20 min in an ice-cold ultrasound bath (ABS) followed by 30 min on a horizontal shaker (Baxter) at 4500 rpm. Filters were removed after a quick spin at 1500 rpm, and extracts were stored at –201C until further use. A blank filter was used as a negative control and treated identically, while a solution of 10 ng/mL of lipopolysaccharide (LPS, from Salmonella typhimurium, Sigma) was used as a positive control and a well-known inducer of oxidative radicals and cytokines by macrophages (Becker et al., 1996). 2.3. Cell culture The NR8383 rat alveolar macrophage cell line was obtained from the American Type Culture Collection (CRL-2192, American Type Culture Collection (ATCC), Virginia) as a homogenous and easily expandable source of alveolar monocyte/macrophage-like cells. They were cultured in F12K medium supplemented with 15% of endotoxin-free fetal bovine serum (FBS, heat inactivated for 30 min at 561C), 50 mg/mL gentamicin, and 4 mM l-glutamine (all from Life Technologies) at 371C, 7.5% CO2, and 95% relative humidity. Cells were allowed to expand to 8 105/mL and subsequently split to 2 105/mL in order to maintain optimal density for exponential growth according to ATCC. Viability was checked to be within the recommended range prior to each passage using Trypan Blue (Sigma). 2.4. Exposure of phagocytes
2. Materials and methods 2.1. PM10 sampling PM10 was measured with Harvard low-volume sharp cut impactors (Marple et al., 1987). At the urban sites Zu¨rich (410 m.s.l.) and Lugano (280 m.s.l.), the rural site Payerne (460 m.s.l.), and the alpine site Montana (1350 m.s.l.), weekly integrated samples were collected on Teflon filters (Gelman Sci., R2PJ041, 47 mm diameter, 2 mm pore size). Lugano and Montana are located in the south of the Alps but in different valleys.
Rat NR8383 cells were plated in a 96-well dish at a final density of 4 105/mL with 10% of total volume (50 mL particle extract in 450 mL cell media) of particle extracts and controls in triplicate for each of the MTT, cytokine, and NO 2 assays (see below) and incubated for 40 h. 2.5. Metabolic activity test (MTT) The MTT test (Roche Diagnostics) was used to determine the cytotoxicity of the particle extracts and
C. Monn et al. / Environmental Research 91 (2003) 35–44
controls for NR8383 cells, according to the manufacturer’s instructions. The MTT assay uses a mitochondrial dehydrogenase-dependent conversion of a yellow tetrazolium salt to purple formazan crystals by life cells. For the metabolic activity test, the 96-well dish (see above) was incubated for 40 h prior to addition of 10 mL of the MTT reagent and another 24 h afterward under the same conditions as above. Incubation times for the MTT reaction were set to 4 h. The conversion rate was measured photometrically. A standard curve was established based on a maximum of 40,000 cells (=100% metabolic activity or 0% toxicity) and eight diluted concentrations. Toxicity in percent is expressed as 100%-metabolic activity (%). A comparison with the Trypan blue assay (Sigma, Bioscience T8154) showed good correlation between the two assays.
37
endotoxin-neutralizing-protein (ENP)-coated resin (Associates of Cape Cod). After incubation at room temperature for 4 h and spinning down at 1200 g, supernatants were stored at 201C. 2.9. Statistical analyses Statistical analyses were performed with the Systat Version 8.0 program. Differences between the sites for mass, toxicity, NO 2 , and TNFa were tested by analysis of variance. The correlation matrix was calculated with the Spearman correlation. Linear multiple regression models were calculated in order to control for the toxicity in the mass–TNFa and mass–NO 2 relationships.
2.6. Measurement of oxidative response
3. Results
We utilized the specific potential of rodent macrophage cells to form reactive nitrogen species as an oxidative response to inflammatory stimuli by measuring nitrite (NO 2 ) levels as the stable end product in the supernatant of NR8383 cultures using the Griess reaction (Lewats et al., 1993). NR8383 cells were incubated with particle extracts and controls for 40 h and spun for 10 min at 120 g, and 50 mL of supernatant was transferred into a new 96-well dish to which 50 mL of a 1% sulfanilamide (Sigma), solution followed by 50 mL of a 0.1% solution of N(1-Naphtyl)-ethylendiamine dihydrochloride (Sigma), each in 2.5% orthophosphoric acid (Fluka), were added. Color formation was measured photometrically at 550 nm and the resulting NO 2 concentration was calculated relative to a standard 1:2 dilution curve of a sodium nitrite (Sigma) solution in culture medium ranging from 100 to 0.78 mM.
Particles smaller than 10 mm (PM10) were collected with low-volume samplers at four different sites (two urban, one rural, one alpine) during all seasons for 4 weeks in 1997. Collecting time was 1 week and the filter medium was Teflon. After gravimetric analyses for the determination of PM10 air pollution levels, the filters were extracted with phosphate buffer (PBS) for 20 min in an ultrasonic bath. Rat lung macrophages were exposed to these extracts in order to assess cytotoxic effects, oxidative response (NO 2 ), and release of the proinflammatory cytokine TNFa. The experiments were repeated three times. Within each series, aliquots of the same batch of rat macrophages were used and, additionally, in all experiments negative (unexposed blank filters) and positive controls (10 ng/mL LPS) were run in parallel to the particle extracts. The levels of the collected particle mass are presented in Fig. 1. The highest mass concentrations were found at Lugano and Zu¨rich (both urban), followed by Montana (alpine) and the rural site Payerne, in agreement with previous observations (Monn et al., 1995). Based on analysis of variance, the differences were significant between Lugano and Montana (po0.05) and between Lugano and Payerne (po0.05). The levels and the seasonal variations at the four sites were quite different, reflecting differences in climate, population densities, human activities, and life styles (Martin et al., 1997). A sampled particle mass of 800 mg is equivalent to an airborne PM10 level of 20 mg/m3. Average PM10 levels were 27 mg/m3 at Lugano, 17 mg/m3 at Zu¨rich, 9 mg/m3 at Montana, and 8 mg/m3 at Payerne. Rat macrophages exposed to the aqueous extracts from the particles were tested by three different assays in order to evaluate the cytotoxic effect (MTT-assay) and the production of NO 2 and TNFa. Fig. 2a shows the result of the MTT-assays. In the negative controls (blank filters) toxicity was around 5%; in the positive
2.7. Cytokine measurements Induction of the proinflammatory cytokine TNFa by NR8383 cells due to particle extracts and controls were measured with a rat TNFa ELISA (BioConcept) according to the manufacturer’s instructions. Cells were incubated for 40 h and spun at 120 g for 10 min, and supernatants were stored at –801C until further use. Supernatants were diluted 1.5-fold with the exception of the positive controls (10 ng/mL LPS), which were diluted five-fold in order to remain in the assay’s recommended concentration range. 2.8. Inhibition of endotoxin Particle extracts were treated with inhibitors for endotoxin as previously described (Monn and Becker, 1999): 500 mL of each extract were added to END-X B15
C. Monn et al. / Environmental Research 91 (2003) 35–44
38
2000 1800
40 µg/m3
particle mass (µg)
1600 1400 1200 1000
20 µg/m3
800 600 400 200
Lugano
Zürich
Montana
autumn
spring
summer
winter
autumn
summer
winter
spring
autumn
summer
winter
spring
autumn
summer
winter
spring
0
Payerne
Fig. 1. Airborne particles o10 mm (PM10) were collected on Teflon filters using low-volume sharp-cut impactors at four sites during winter, spring, summer, and autumn period in 1997. Four measurements were performed during each season with a cumulative sampling period of 7 days. The columns represent averages of the collected mass of each site and season and the bars represent the standard deviation. On average, largest mass concentrations were observed at the urban sites Lugano and Zu¨rich, followed by Montana and Payerne. Significant differences in an analysis of variance were found between Lugano and Montana (po0.05) and between Lugano and Payerne (po0.05). Note that a particle mass of 800 mg is equivalent to an airborne PM10 concentration of 20 mg/m3.
LPS control toxicity reached 80%. (A 100% toxicity means that all cells exposed to the particle extracts lost their mitochondrial dehydrogenase activity.) The toxicity of the environmental particle extracts ranged between 10% and 65%. At Zu¨rich and Payerne, extracts from winter had the strongest cytotoxic activity. At Montana (alpine), the spring and summer samples were more toxic than the winter particles. At Lugano, summer and winter extracts induced the strongest toxicity. Taking together the samples from all seasons, Lugano differed significantly from Montana (po0.01) and Payerne (po0.001). Zu¨rich and Payerne differed significantly (po0.001). The other differences between sites were not significant. All exposures to particle extracts differed significantly from the negative control (blank filter) (po0.001). In the second experiment, cells exposed to particle extracts were tested with respect to the NO 2 production. Fig. 2b shows the result for all sites and seasons. In
contrast to the cytotoxic effects, the strongest production of NO 2 appeared mostly in the nonwinter seasons. At Montana, Payerne, and Zu¨rich, the strongest peaks were found in the summer. Taking together the values of all seasons, Lugano differed significantly (analysis of variance) from Zu¨rich (po0.01). In the third experiment (Fig. 2c), the production of TNFa in cells exposed to particle extracts was measured. Several of the extracts did not show a significant stimulation of the TNFa production with respect to the negative control. The seasonal variation tends to have the strongest inductions outside winter. Highest peaks were observed at Payerne (summer), Zu¨rich (summer) and Montana (spring). Taking together the values of all seasons, there were no significant differences (analysis of variance) between the sites. The observations found in Figs. 2a–c were statistically analyzed. Table 1 presents the Spearman rank correlations between the variables ‘‘mass,’’ ‘‘toxicity,’’ ‘‘NO’’ 2 ,
Fig. 2. (a–c) Particles collected on filters were submitted to an extraction in physiologically buffered saline. Each particle extract was added to the same number of rat macrophages (NR8383) for 40 h. Measurements of the toxicity (MTT-assay) (a), of NO 2 (b), and TNFa (c) were performed in three individual experiments. The columns represent the average levels of the three experiments (bars: 7 one standard deviation). The controls represent extracts from unexposed blank filters (neg) and 10 ng/mL LPS (pos) which were run in parallel in the same experiments. (a) Toxicity is expressed as 100% minus the metabolic activity in % (100% represents 40,000 live cells). On the average, strongest toxicity was observed with the urban extracts, Lugano and Zu¨rich. Statistically significant differences (analysis of variance, data from all seasons) were observed between Lugano and Montana (po0.01), Lugano and Payerne (po0.001) and Zu¨rich and Payerne (po0.001). All other differences between the sites were not significant. All values differed significantly from the negative control (po0.001). (b) Statistically significant differences of the NO 2 concentrations (analysis of variance, data from all seasons) were observed between Lugano and Zu¨rich (po0.01). All other differences were not significant. Values which differed significantly from the negative controls (po0.001) are marked by asterisks. (c) For the TNFa values, no significant differences (analysis of variance, data from all seasons) between the sites (p40.05) were observed. Values which differed significantly from the negative controls (po0.001) are marked by asterisks.
(c)
control
Lugano
Zürich
Montana
* autumn
summer
spring
winter
*
autumn
* *
spring
Montana
summer
* autumn
Montana
winter
*
autumn
912 summer
*
spring
35
summer
Zürich winter
*
spring
Zürich
winter
16
autumn
500 autumn
18
summer
spring
*
spring
* winter
autumn
Lugano
summer
Lugano
winter
450 summer
*
autumn
winter spring
14
summer
neg
control
spring
control
winter
(b) pos
NO2- (µM) (a)
pos
neg
TNFα (pg/ml)
autumn
summer
spring
winter
autumn
summer
spring
winter
autumn
summer
spring
winter
autumn
summer
spring
winter
pos
neg
toxicity (%)
C. Monn et al. / Environmental Research 91 (2003) 35–44 39
100 90
80
70
60
50
40
30
20
10
0
Payerne
* *
*
12
* *
10
8
6
4
2
0
Payerne
*
*
400
350
300
250
200
150
100
50
0
Payerne
C. Monn et al. / Environmental Research 91 (2003) 35–44
40
and ‘‘TNFa’’, based on the data from all sites and seasons. The mass levels measured on the filters correlated best and positive with the toxic effects (r ¼ 0:797). On the other hand, NO 2 and TNFa values were not well correlated with the mass but they correlated well with each other (r ¼ 0:834). The effect of the mass levels on the production of NO 2 and TNFa was also calculated in a multiple regression model with the controlling variable ‘‘toxicity.’’ However, the influence of mass on NO 2 and TNFa remained insignificant in this multiple regression model. The scatterplot in Fig. 3 shows the influence of mass on the toxic effect of the aqueous extracts. In a linear regression model, the relationship is positive and highly significant, with an R2 of 0.633 over all data. For all sites, the toxicity of the extracts increased with increasing mass levels. Note that for Payerne, almost no variation in mass data occurred, so that no relationship between mass and toxicity can be assessed. Since the NO 2 and TNFa values correlated strongly (see Table 1), a ‘‘stimulation’’ factor was calculated,
taking together the NO 2 and TNFa values expressed as percentage of the respective LPS control. Figs. 4a and b show the scatterplots between mass and the stimulation factor for Montana on the one hand and Lugano and Zu¨rich on the other hand. In contrast to the relationship between mass and toxicity, considerable differences were observed between the mass and the stimulation factor at the different sites. At the low end of the mass levels (Montana, Fig. 4a) the relationship was positive. At the two urban sites Zu¨rich and Lugano, which are located at the upper end of the mass levels, the relationship became negative (Fig. 4b). At Payerne, where the variability of the mass levels was small (see Fig. 1), no correlation can be assessed. According to the findings from Becker et al. (1996) and Monn and Becker (1999), endotoxins may partly be responsible to cause some of the effects described above. We tested the influence of endotoxin in parallel experiments by removing endotoxins by an ENP. Figs. 5a–c show the effects of the untreated (set at 100%) and ENP-treated (relative to the untreated
Table 1 Spearman correlation matrix between the particle mass, toxicity, and NO 2 and TNFa concentrations Toxicity
1.000 0.797 0.137 0.179
1.000 0.075 0.082
1.000 0.834
TNFa
1.000
Montana
50 stimulation
Mass Toxicity NO 2 TNFa
Mass
NO 2
55
The analysis is based on data from all sites and seasons. The matrix shows strong and significant correlations between the collected mass levels and the toxicity and between the NO 2 and TNFa concentrations.
45 40 35 30 25 20 0
500
(a)
1000 mass
1500
2000
55
70 stimulation
60 50 toxicity (%)
urban:Lugano, Zürich
50
40
45 40 35 30
30
25
20
20 0
10
(b)
0 0
500
1000 mass
1500
2000
Fig. 3. Toxicity (%) is plotted versus the collected mass levels (mg) for all sites and seasons. Linear regression over all data: y ¼ 0:025x þ 20:6; R2 ¼ 0:6338: Overall, with increasing mass the toxicity increased. Payerne is an exception with mass levels of low variance. (Legend: triangle, Montana; circle, Payerne; square, Zu¨rich; small square: Lugano).
500
1000 mass
1500
2000
Fig. 4. (a–b) A ‘‘stimulation’’ factor was calculated, taking together the NO 2 and TNFa values (expressed as percentage of the respective LPS control) from the alpine site Montana and the urban sites Zu¨rich and Lugano. This factor is plotted against the collected mass levels (mg). (a) At Montana (lower mass range), a positive relationship was observed. Regression model: y ¼ 0:0216x þ 28:2; R2 ¼ 0:831: (b) At the urban sites Lugano and Zu¨rich (upper mass range), the relationship was negative. Regression model: y ¼ 0:0066x þ 44:5; R2 ¼ 0:582: (Note that the use of the single variables ‘‘TNFa’’ or ‘‘NO 2 ’’ leads to similar figures.)
C. Monn et al. / Environmental Research 91 (2003) 35–44
41
toxicity
120 100 percent
80 60 40 20 0 (a)
neg
pos
Lugano
Zürich
Montana Payerne
NO2-
120
percent
100 80 60 40 20 0 (b)
neg
pos
Lugano
Zürich
Montana
Payerne
Zürich
Montana
Payerne
TNF alpha
120 100 percent
80 60 40 20 0 (c)
neg
pos
Lugano
Fig. 5. (a–c) Rat macrophages (NR8383) were exposed to particle extracts treated with an ENP, to untreated particle extracts and to controls (blank, LPS) in parallel experiments. An assessment of the toxicity (a), of the NO 2 levels (b), and of the TNFa levels (c) was performed. In all experiments, a negative (neg) (unexposed blank filter) and a positive control (pos) (10 ng/mL LPS) were run in parallel with the particle extracts. The first columns (grey) show the ENP-untreated values set at 100%, the second columns present the values of the ENP-treated extracts (in percent) (dark columns). (a) Highly significant differences (pairwise analysis: untreated versus treated) in toxicity were observed after treatment with ENP for LPS and for all sites (po0.001). (b) Highly significant differences (pairwise analysis: untreated versus treated) of the NO 2 release were observed after treatment with ENP for LPS and for all sites (po0.001). (c) Significant differences (pairwise analysis: untreated versus treated) of TNFa release were observed after treatment with ENP for LPS (po0.001) and for Zu¨rich (po0.05) and Payerne (po0.05). Note that the TNFa levels compared to the negative controls (see Fig. 2) were generally low in these experiments. Only data where the values were higher than the blank were included in this analysis.
samples in %) samples and the negative (blank filter) and positive (LPS) controls. The effect of ENP on the reduction of toxicity was highly significant at all sites and for the positive control (po0.001) (Fig. 5a). Similar effects were observed for the NO 2 production with
significant reductions at all sites and for the positive control (po0.001) (Fig. 5b). For TNFa, weak reductions (po0.05) were observed at Zu¨rich and Payerne and not at Lugano and Montana. In this analysis, only data with significant stimulations (compared to the
42
C. Monn et al. / Environmental Research 91 (2003) 35–44
negative controls) were used as in about 45% of the cases, the particle extracts did not induce TNFa levels above the blank background levels (see Fig. 2). In the LPS control, the reduction was highly significant after treatment with ENP (po0.001) (Fig. 5c).
4. Discussion Macrophages are target cells for particles in the alveolar tract, where they take part in particle scavenging and modulate inflammation (for review see Riott, 1997). An in vitro system with a cell line from rat macrophages was used to analyze the effects of aqueous extracts from ambient air PM10 particles. Compared to freshly prepared phagocytes or monocytes, the use of a cell line provides standardized conditions in different sets of experiments. Such in vitro studies make it possible to visualize a single reaction mechanism contributing to a complex in vivo situation and to show differences in the reactions induced by different pollution samples. Cytotoxic effects were assessed with a mitochondrialdehydrogenase-dependent test (MTT-assay). A comparison with the trypan blue test, where dead and live cells are counted microscopically, showed good agreement between the two assays (r ¼ 0:85; data not shown). Therefore, we assume that the MTT-assay expressed as toxicity mainly reflects the proportion of dead cells, although the presence of living cells with reduced mitochondrial dehydrogenase activity cannot be excluded. The advantage of the MTT-assay lies in the precise and easy photometric measurement. Our experiments show mainly effects of water-soluble compounds in the PM10 sample since we extracted the filters with a buffer solution only. The chemical composition of our aqueous extract was not determined for technical reasons. It is known that allergens, endotoxins, ions, soluble metal salts, and some polar organic compounds can be present in aqueous particle extracts (Vrtala et al., 1993, 1988; Ghio et al., 1996). After our extraction procedure, the filter pieces were still quite blackened and we assume that only a small proportion of solid particles was washed off into the extract. Moreover, we assume that the apolar organic and carbon black fraction remained mostly in the filters. Unfortunately, it is not possible to quantify the mass extracted as the difference between an extracted and unextracted filter is too small and as the buffer salt may even increase the total mass after extraction. Our experiments showed that cells exposed to particle extracts from urban, rural, or alpine sites had reduced viability compared to cells exposed to extracts from blank control filters. The strongest effects were observed with extracts collected during winter, except for the alpine site Montana and for Lugano, where the summer extracts also had strong toxic effects.
Taking together data from all sites and seasons, a good correlation between the particle mass on the filters and the toxicity of the extracts (R2=0.633, linear regression) was observed, suggesting that toxic water-soluble compounds were trapped on the filter and washed off in our extraction procedure. The correlation between mass and toxicity is remarkable as the chemical composition between sites and seasons might vary considerably. However, this finding correlates with observations in epidemiological studies where health effects were independent of the chemical composition of particles and the associations were attributable to the mass concentration of PM10 (or PM2.5) in ambient air (Schwartz, 1996). The observed toxic effects may reflect health hazards caused by hampering scavenging and defence mechanisms. There is support for this explanation from an in vitro study by Becker and Soukup (1999) where macrophages exposed to ambient air particle extracts had reduced capability of virus uptake and reduced production of monocyte chemoattractants (e.g., MCP-1) which are important in anti-bacterial defense. Our experiments on the release of oxidative radicals and cytokines showed that the strongest effects occurred with PM10 filter extracts collected in spring and summer, especially from nonurban sites. Taking together data from all sites and seasons, no correlation between the production of oxidative radicals or TNFa and the PM10 mass concentration was observed. On the other hand, the correlation between NO 2 and TNFa values was good, indicating similar signalling. Because of this significant correlation, we calculated a macrophage stimulation factor comprising both the NO 2 and the TNFa values. By plotting this stimulation factor versus the particle mass of the corresponding filters, we noticed a profound difference between the urban and the alpine sites. In Montana with rather low particle mass collected, the correlation was positive. In Zu¨rich and Lugano with high particle mass, the correlation was negative. For Payerne, where the variation of the particle mass was small (Fig. 1), no obvious correlation was found. Since mass and toxicity correlate well, we interpret that at low PM10 levels as in Montana (Fig. 4a), the macrophages are increasingly stimulated by increasing particle mass. On the other hand, at high PM10 levels, as in Lugano and Zu¨rich (Fig. 4b), macrophage stimulation is inhibited with increasing anthropogenic air pollution by increasing toxic effects. Alternatively, we can interpret this observation by the contribution of biological material to the particle mass, being stronger at the alpine site Montana compared to the urban sites Lugano and Zu¨rich (Hu¨glin et al., 1999; Spieksma, 1995). The production of high radical levels exhibits a self-toxic effect on cells, i.e., in the presence of transition metals (Pritchard et al., 1996). The poor statistical correlation between the bulk of our NO 2 values and the toxicity is not in contradiction to this
C. Monn et al. / Environmental Research 91 (2003) 35–44
mechanism, as with an increasing number of dead cells in the Lugano and Zu¨rich extracts, the production of NO 2 is inhibited (part of the data of Fig. 4b). However, transition metals may not be important in our case, since values measured in a parallel study ranged between 50 and 370 ng/m3 for Fe and between o1 and 2.4 ng/m3 for Cr (unpublished data). These values are more than 10 times lower than concentration levels detected in urban PM10 of US cities (US Environmental Protection Agency, 1996). We tested whether the presence of endotoxin plays a role in the observed effects by inactivating endotoxins with an ENP. After inactivation, the cytotoxic effects were significantly reduced, as well as, NO 2 being released. Cytokine release was slightly reduced in the samples of Zu¨rich and Payerne. These observations suggest that endotoxin is at least one factor in the toxic effects and the oxidative radical production of our particle extracts. Endotoxins exhibit a toxic potential by activating enzymes in the caspase family (Mariani et al., 1997). Part of the endotoxin, the Lipid A portion, is also responsible for inducing inflammatory cytokines (Someya et al., 1996). Endotoxin levels in the particle extracts were also determined with the Limulus assay. However, the data were hardly above background, as the levels of endotoxin were very low (data not shown). In the studies from Monn and Becker (1999), and Becker et al. (1996) the mere presence of endotoxin was sufficient to produce cytokines and there was no relationship with the particle mass. In contrast to the studies from Becker et al. (1996), Monn and Becker (1999), and Pritchard et al. (1996), this study is based on air samples from different seasons collected in the lower air pollution range. The stimulatory effects were smaller than those found in the other studies due to lower endotoxin and transition metal levels. However, despite the low content, of these compounds, our particle extracts exhibited toxic effects with a clear dose–response relationship with the collected mass on the filters. In conclusion, our experiments offer explanations for at least some of the harmful effects of PM10.They indicate that PM10 contains water-soluble macrophagetoxic and macrophage-stimulating compounds, some of them of biological origin (e.g., endotoxin, allergens) (Miguel et al., 1996; Knox et al., 1997). The experiments help to explain observations made by epidemiology and show differences in the pollution pattern between urban, rural, and alpine sites and between different seasons.
References Ackermann-Liebrich, U., Leuenberger, Ph., Schwartz, J., Schindler, Ch., Monn, Ch., Bolognini, G., et al., and SAPALDIA Team, 1997. Lung function and long term exposure to air pollutants in Switzerland. Am. J. Respir. Crit. Care Med. 155, 122–129.
43
Allermann, L., Poulsen, O.M., 2000. Inflammatory potential of dust from waste handling facilities measured as IL-8 secretion rom lung epithelial cells in vitro. Ann. Occup. Hyg. 44 (4), 259–269. Becker, S., Soukup, J.M., Gilmour, M.I., Devlin, R.B., 1996. Stimulation of human and rat alveolar macrophages by urban air particulates: effects on oxidant radical generation and cytokine production. Toxicol. Appl. Pharmacol. 141, 637–648. Becker, S., Soukup, J.M., 1999. Exposure to urban air particulates alters the macrophage-mediated inflammatory response to respiratory viral infection. J. Toxicol. Environ. Health 57 (7), 445–457. Carter, J.D., Ghio, A., Samet, J.M., Devlin, R.B., 1997. Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol. Appl. Pharmacol. 146, 180–188. Chen, L.C, Fine, J.M., Qu, Q.S., Amdur, M.O., Gordon, T., 1992. Effects of fine and ultra-fine sulfuric acid aerosols in guinea pigs: alterations in alveolar macrophage function and intracellular pH. Toxicol. Appl. Pharmacol. 113 (1), 109–117. Dockery, D.W., Pope III, C.A., Xu, X., Spengler, J.D., Ware, J.H., Fay, M.E., Ferris Jr., B.G., Speizer, F.E., 1993. An association between air pollution and mortality in six US cities. N. Engl. J. Med. 329, 1753–1759. Dreher, K., Jaskot, R., Kodavanti, U., Lehmann, J., Winsett, D., Costa, D., 1996. Soluble transition metals mediate the acute pulmonary injury and airway hyperreactivity induced by residual oil fly ash articles. Chest 109 (3 Suppl), 33S–34S. Driscoll, K.E., Carter, J.M., Hassenbein, D.G., Howard, B., 1997. Cytokines and particle-induced inflammatory cell recruitment. Environ. Health Perspect. 105 (Suppl 5), 1159–1164. Ghio, A.J., Stonehuerner, J., Pritchard, R.J., Piantadosi, C.A., Quigley, D.R., Dreher, K.L., Costa, D.L., 1996. Human-like substances in air pollution particulates correlates with concentrations of transition metals and oxidant response. Inhalation Toxicol. 8, 479–494. Goldsmith, C.A., Frevert, Ch., Imrich, A., Sioutas, C., Kobzik, L., 1997. Alveolar macrophage interaction with air pollution particulates environ. Health Perspect. 105 (Suppl 5), 1191–1195. Hester, R.E., Harrison, R.M. (Eds.), 1998. Air Pollution and Health. Issues in Environmental Science and Technology, Vol. 10. Roy. Soc. Chem. Herts., UK. Hu¨glin, Ch., Gehrig, R., Devos, W., Moor, Ch., Baltensperger, U., Monn, Ch., 1999. Chemical composition and source apportionment of PM10 and PM25 in Switzerland. VDI Rep. 1443, 307–312. Knox, R.B., Suphioglu, C., Taylor, P., Desai, R., Watson, H.C., Peng, J.L., Bursill, L.A., 1997. Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution. Clin. Exp. Allergy 27, 246–251. Kodavanti, U.P., Jaskot, R.H., Su, W.Y., Costa, D.L., Ghio, A.J., Dreher, K.L., 1997. Genetic variability in combustion particleinduced chronic lung injury. Am. J. Physiol. 272 (3 Part 1), L521–L532. Lewats, J., Mumford, J., Everson, R.B., Hulka, B., Wilcosky, T., Kozumbo, W., Thompson, C., George, M., Dobias, L., Sram, R., 1993. Environ. Health Perspect. 99, 89–97. Mariani, S.M., Matiba, B., Armandola, E.A., Krammer, P.H., 1997. Interleukin 1 beta converting enzyme related protease/caspase are involved in TRAIL-induced apoptosis of myeloma and leukemia cells. J. Cell Bio. 137, 221–229. Marple, V.A., Rubow, K.L., Turner, W., Spengler, D., 1987. Low flow-rate sharp-cut impactors for indoor air sampling: design and calibration. J Air Pollut. Control. Assoc. 37 (11), 1303–1307. Martin, B., Ackermann, U., Leuenberger, Ph., Ku¨nzli, N., Zemp, E., Keller, R., Zellweger, J.-P., Wu¨thrich, B., Monn, Ch., et al., SAPALDIA Team, 1997. SAPALDIA: methods and participation in the cross sectional part of the Swiss Study on Air Pollution and Lung Function in Adults. Soz. Pra¨ventivmed. 42, 67–84.
44
C. Monn et al. / Environmental Research 91 (2003) 35–44
Miguel, A.G., Cass, G.R., Weiss, J., Glovsky, M.M., 1996. Latex allergens in tire dust and airborne particles. Environ. Health Perspect. 104, 1180–1185. Monn, Ch., Bra¨ndli, O., Scha¨ppi, G., Ackermann, U., Leuenberger, Ph., SAPALDIA Team, 1995. Particulate matter and total suspended particulates in urban, rural and alpine air in Switzerland. Atmos. Environ. 29 (19), 2565–2573. Monn, Ch., Becker, S., 1999. Cytotoxicity and induction of proinflammatory cytokines from human monocytes exposed to fine (PM 2.5) and coarse particles (PM10-2.5) in outdoor and indoor air. Toxicol. Appl. Pharmacol. 155, 245–252. Pritchard, R.J., Ghio, A.J.J., Lehmann, J.R., Winsett, D.W., Tepper, J.S., Park, P., Gilmour, M.I., Dreher, K.L., Costa, D.L., 1996. Oxidant generation and lung injury after particulate air pollutants exposure increase with the concentrations of associated metals. Inhalation Toxicol. 8, 457–477. Riott, I.M., 1997. Essential Immunology, 9th Edition. Blackwell, Oxford. Rylander, R., Haglind, P., 1984. Airborne endotoxins and humidifier disease. Clin. Allergy 14 (1), 109–112.
Schwartz, J., 1996. Air pollution and hospital admissions for respiratory disease. Epidemiology 7, 20–28. Someya, K., Tsutomi, Y., Soga, T., Akahane, K., 1996. A lipid A analog inhibits LPS-induced cytokine expression and improves survival in endotoxemic mice. Immunopharmacol. Immunotoxicol. 18 (4), 477–495. Spieksma, F.Th.M., 1995. Aerobiology of inhalatory allergen carriers. Allergol. Immunopathol. 23, 20–23. Steerenberg, P.A., Van Amsterdam, J.G.C., Vernderbriel, R.J., Vos, J.G., Van Bree, L., Van Loveren, H., 1999. Environmental and lifestyle factors may act in concert to increase prevalence of respiratory allergy including asthma. Clin. Exp. Allergy 29, 1304–1308. US Environmental Protection Agency, 1996. Air Quality Criteria for Particulate Matter, Vol. I, Report: EPA/600/P-95/001aF, Research Triangle Park, NC. Vrtala, S., Grote, M., Duchene, M., van-Ree, R., Kraft, D., Scheiner, O., Valenta, R., 1993. Properties of tree and grass pollen allergens: reinvestigation of the linkage between solubility and allergenicity. Int. Arch. Allergy Immunol. 102 (2), 160–169.