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Ingested Aggregates of Ultrafine Carbon Particles and Interferon-γ Impair Rat Alveolar Macrophage Function
Environmental Research Section A 81, 309}315 (1999) Article ID enrs.1999.3992, available online at http://www.idealibrary.com on
Ingested Aggregates of Ultrafine Carbon Particles and Interferon-c Impair Rat Alveolar Macrophage Function Margot Lundborg,1 Anne Johansson, Lena La> stbom, and Per Camner Division of Inhalation Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Received December 28, 1998
after infections, and there might be an increased risk for additional infections. Moreover, during an episode of high ambient particle concentration the inhaled particles will not be efAciently phagocytized and may thereby damage the lung tissue.
Alveolar macrophages (AM), obtained by lavage from the rat lung, were allowed to ingest aggregated ultraAne carbon particles, about 1 lg/106 AM, which is a realistic result of long-term exposure to ambient air. The effects of the ingested carbon on the phagocytosis of test particles and oxidative metabolism of the AM were studied. In addition, the effects of short-term (40 min or 2 h) and long-term (28 or 44 h) incubation with interferon gamma (IFN-c) on AM loaded and unloaded with carbon were investigated. Phagocytic activity was studied using Buorescein-labeled 3.2-lm silica particles. The attachment and ingestion processes were evaluated separately. The ingested carbon markedly impaired the phagocytosis of silica particles; the accumulated attachment (sum of attached and ingested particles per AM) decreased from 5.0 to 4.2 particles/AM and the ingested fraction (number of ingested particles per AM divided with accumulated attachment) from 0.42 to 0.27. The short-term incubation with IFN-c tended to increase the accumulated attachment (from 5.0 to 5.7 particles/AM) and decreased the ingested fraction (from 0.42 to 0.34) in unloaded AM. Long-term incubation with IFN-c markedly impaired both the accumulated attachment (to 3.8 particles/AM) and the ingested fraction (to 0.24) in unloaded AM and the carbon load further decreased the accumulated attachment to 2.8 particles/AM, and the ingested fraction to 0.21. The oxidative metabolism was not effected by the ingested carbon or the short-term incubation with IFN-c, but the long-term incubation with IFN-c increased it with a factor of almost 3. Our results suggest that ingested environmental particles in AM may markedly impair their phagocytic capacity, especially during long-term exposure to IFN-c as
( 1999 Academic Press
Key Words: particulate matter; ultraAne particles; alveolar macrophages; phagocytosis; oxidative metabolism; interferon-c; infections; carbon.
INTRODUCTION
The interest in health effects caused by ambient air particles has increased in recent times. One reason is that a large number of epidemiological studies show relationships between rather moderate levels of particles in the ambient air and mortality as well as lung morbidity (EPA, 1996; Pope et al., 1995; Brunekreef et al., 1995). Another reason is that long-term exposure to particles of rather innocuous material such as carbon particles and titanium oxide particles has caused pathological changes, including tumors, in the rat lung (EPA, 1996; Heinrich et al., 1995; Nikula et al., 1995). The mechanisms behind the effects documented in the epidemiological and experimental studies are not known. It is reasonable to believe that alveolar macrophages (AM) play a role in these mechanisms. By taking up particles deposited in the lungs, the AM protect other, more sensitive cells. Since the mechanical clearance from the alveolar region in humans is extremely slow, the half-time being about 5 years (Philipson et al., 1996), an accumulation of ingested particles within the AM must take place. If these ingested particles would impair the phagocytic capacity of the AM, other cells might be damaged by particles inhaled during episodes with high particle concentration in the ambient air. There might also be an increased risk for infections. Moreover, if the
1 To whom correspondence should be addressed at Division of Inhalation Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden. Fax: #46 8 30 33 90.
309 0013-9351/99 $30.00 Copyright ( 1999 by Academic Press All rights of reproduction in any form reserved.
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particles ingested by the AM trigger an oxidative burst with release of reactive oxygen metabolites, there would be a risk of tissue damage in the lung. The purpose of the present study was to clarify if commonly occurring amounts of ingested carbon particles in AM impair their ability to phagocytize other particles and if the oxidative metabolism of the AM would be affected. In an attempt to increase the differences between AM loaded and not loaded with carbon particles, we also incubated AM with interferon-c (IFN-c). IFN-c is considered to have a fundamental role in activating white blood cells, including macrophages. The change is antigen-induced via T-lymphocyte activation and is enhanced by infections by microorganisms or exposure to other antigens (Baron et al., 1991; Curfs et al., 1997).
FIG. 1. Relationship between the concentration of suspended carbon particles and optical density (OD).
MATERIAL AND METHODS
Animals and Lung Lavage Alveolar macrophages were obtained by lavage from 12 healthy rats (Sprague-Dawley, Charles River, Uppsala, Sweden) weighing 250}300 g. The rats were sacri7ced by an overdose of sodium pentobarbital. The lungs were excised and lavaged with Hank’s balanced salt solution (without Ca2` and Mg2` pH 7.4, 37@C) using brief massage. Around 40 ml lavage 8uid, containing 10}15]106 cells, was obtained. In the light microscope, more than 90% of the cells were estimated to be AM. The cells were washed once by a 10-min, 300g centrifugation at room temperature; the resulting cell pellet was resuspended in Hepes buffered medium 199, pH 7.4 (GIBCO, Paisly, Scotland), with 10% fetal calf serum, penicillin 100 units/ml, and streptomycin 100 lg/ml (complete medium). The number of cells was counted in a BuK rker hemocytometer.
Experimental Design For each of the studies of phagocytosis and oxidative metabolism, three sets of experiments were performed in parallel with AM from the same rats at the same time. In the 7rst sets of experiments (Exp. 1), AM loaded and not loaded with carbon were compared concerning the two functions. The second sets of experiments (Exp. 2) were performed as the 7rst sets with the exception that the AM were incubated with IFN-c during the tests of phagocytosis and oxidative metabolism (short-term incubation). The third sets of experiments (Exp. 3) were performed as the second sets with the exception that IFN-c was present not only during the test but also during the loading of AM with carbon particles (long-term incubation). Loading of AM with Carbon Particles
Preparation of Carbon Particle Suspension Aggregates of ultra7ne carbon powder (GuK nther Wayner, Hannover, Germany) were used. Before use, the powder was suspended in medium 199 (with penicillin 100 units/ml and streptomycin 100 lg/ml) and sonicated for 3 h with a short break every 20 min for vortexing. A stable suspension of carbon particles was obtained in this way. There was a linear relationship between carbon particle concentration and optical density measured spectrophotometrically (Shimadzu, UV-160A), facilitating estimation and control of the carbon particles in suspension, see Fig. 1. The 7nal carbon suspension contained 20 lg carbon/ml in the complete medium.
In the study of phagocytosis of test particles, 1 ml of carbon particle suspension was added to a test tube with 1]106 precipitated AM and to another without AM. In the study of oxidative metabolism, 2 ml of carbon particle suspension was added to a test tube with 2]106 precipitated AM and to another without AM. The samples were placed in a shaking water bath at 37@C for 6 h (study of phagocytosis) or for 20 h (study of oxidative metabolism) in order to obtain AM with attached or ingested carbon particles. The tubes were then centrifuged at 300g for 10 min. The supernatants from the tubes with and without AM were measured in the spectrophotometer and the difference in carbon particle
ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES
concentration was taken as a measure of attached and ingested particles by AM. Centrifugation of the carbon particle suspension from tubes without AM had only a negligible effect on the concentration of suspended particles. Fresh complete medium was added to the precipitated cells and in order to get rid of remaining free carbon particles, an additional washing was made. Subsequently, fresh, complete medium was added to the AM and the tubes were placed in a shaking water bath for 20 h at 37@C to ensure that all carbon particles attached to the AM would be ingested. Control AM were treated in exactly the same way from lavage to the end of the phagocytosis and oxidative metabolism experiments, only leaving out the carbon particles. Short-Term and Long-Term Incubation with IFN-c In the studies of short-term effects of recombinant rat IFN-c (Sigma) on phagocytosis of test particles and oxidative metabolism, AM were treated as in the 7rst sets of experiments with the exception that IFN-c was added immediately before the tests of phagocytosis and oxidative metabolism. In the studies of long-term effects of IFN-c on phagocytosis of test particles and oxidative metabolism, AM were treated as in the 7rst set of experiments with the exception that IFN-c was present during the loading of the AM with the carbon particles and the entire experimental procedure. The concentration of IFN-c was 12.5 U/ml in all experiments. This concentration was originally chosen from a dose-response curve of IFN-c concentration on NO production of AM (Gross et al., 1999). 12.5 U IFN-c/ml induced a marked increase in the NO production, which was 10}15% of the maximum increase, obtained at 250 U/ml. Preliminary studies on long-term incubation with IFN-c (24}48 h) on the oxidative metabolism (NBT test) of AM indicated that a concentration higher than 12.5 U/ml resulted in about the same reduction in the oxidative metabolism. Assay of Phagocytosis Phagocytic ability of AM with and without ingested carbon particles was studied in samples from six rats. Spherical particles of amorphous silica (Spherisorb S 3, NH 2, Phase Separations Ltd, Queens Ferry, Clwyd, UK) were labeled with 8uorescein isothiocyanate (FITC) (Nyberg et al., 1996). The size of the silica particles was measured in a light microscope (Viscopan projection microscope, Reichert, Austria) and was found to be 3.2$0.4 lm
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(mean$SD). A modi7cation (Wiernik et al., 1983) of the method described by Hed (1977) was used for the assessment of phagocytic function. After the 20 h of incubation (see above), which was made in order to ensure that attached carbon particles had been ingested by the AM, the tubes with the cell suspensions were centrifuged at 300g for 10 min. One milliliter of complete medium was added to the cell precipitate in each test tube; from this suspension (106 AM/ml), samples of 250 ll were added to glass slides with round-bottomed wells (Nova Kemi Ltd, Sweden). The cells were allowed to attach to the glass during incubation for 30 min at 373C with 5% CO2 in air and 80% relative humidity. Complete medium (250 ll) containing silica particles at a concentration of 10]106 particles/ml was added to the cells, i.e., 10 particles per AM. After incubation for 40 min, the slides were placed in ice-cold Ringer acetate (to interrupt the phagocytosis) and unattached particles were rinsed off. The cells were then stained with trypan blue (0.4 mg/ml) for 30 s. The number of particles ingested by and attached to an AM was then directly counted in a Zeiss microscope, which permits examination in both visible light and UV light (8uorescence). When the microscope is turned on the visible light mode, the attached particles are clearly visible by the trypan blue staining, while the ingested ones are not. When the microscope is switched over to the UV light mode, only the ingested particles are seen as the 8uorescence from the attached particles is quenched by trypan blue. In each sample 100 consecutive macrophages were scored. The parameters accumulated attachment and ingested fraction were introduced in order to differentiate between the attachment and the ingestion processes. The accumulated attachment is the sum of the numbers of attached and ingested particles per AM. As all ingested particles must have been attached to the AM, the accumulated attachment is the integrated number of particles attached to the AM during the time of the test. Therefore, this parameter is a measure of the attachment process. The ingested fraction is a number of ingested particles per AM divided by the accumulated attachment, i.e., the ingested particles are related to the integrated number of attached particles. This parameter, thus, represents a measure of the ingestion process and is rather independent of the accumulation process. Assay of Oxidative Metabolism The oxidative metabolism (‘‘spontaneous’’ metabolism with no stimulation by agents like zymozan)
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times and enlarged about 3 times, and the diameters of individual carbon particles as well as of aggregates of particles were measured. Latex particles, 1.099 lm in diameter (Serva Entwichlungslabor, Heidelberg, Germany), were used as standards for calibration purposes.
of AM was measured by using the ability of the produced superoxide to reduce yellow nitroblue tetrazolium (NBT) to blue formazan, according to the method described by Jarstrand et al. (1978). After the 20 h in a shaking 373C water bath, the cell suspensions were centrifuged at 300g for 10 min. One milliliter of complete medium and 1 ml of NBT solution were added to the cell precipitate in each tube. The tubes were incubated for 2 h in a shaking 373C water bath. The reaction was stopped by adding 1 ml of 0.5% HCl to each tube. The tubes were centrifuged at 2500g for 30 min. One milliliter of dimethyl sulfoxide was added to each tube to dissolve the produced formazan and the tubes were sonicated for 30 min. The absorption of the solution from each tube was measured in a spectrophotometer (Shimadzu, UV-160A) at wavelength 572 nm.
Statistical Analysis The paired t test was used with a level of signi7cance of 0.05. A two-tailed test was made to evaluate the effects of IFN-c and a one-tailed test to evaluate the effects of carbon. RESULTS
EM Data The carbon particle in the suspension occurred as aggregates with a mean diameter of 0.165$ 0.077 lm ($SD) (0.050}0.347 lm). The diameter of the primary carbon particles was 0.044$0.010 lm.
Electron Microscopy Carbon particles from a stable suspension in distilled water were studied with transmission electron microscopy. Drops of the suspension were allowed to dry on to copper grids (200 mesh) covered by collodion 7lm. The grids were examined in a Jeol 100S electron microscope (Jeol Ltd, Tokyo, Japan). Photos were taken at a primary magni7cation of 10,000
Phagocytosis Data The data on the accumulated attachment and the ingested fraction for the three experiments are shown in Table 1. In the short-term incubation of AM with IFN-c (AM not loaded with carbon, Exp. 2),
TABLE 1 Accumulated Attachment (Acc. att.) and Ingested Fraction (Ingest fract.) of Test Particles of Amorphous Silica in the Carbon, Carbon and Short-Term IFN-c and Carbon and Long-Term IFN-c Experimentsa Carbon experiment (Exp. 1) AM Rat no. 1 2 3 4 5 6 Mean SD a
Carbon and short-term IFN-c experiment Carbon and long-term IFN-c experiment (Exp. 2) (Exp. 3)
AM#carbon
AM
AM#carbon
AM#carbon
Acc. att. part/AM 4.1 6.7 6.9 4.1 3.1 5.2
Ingest. fract. 0.50 0.44 0.43 0.49 0.35 0.33
Acc. att. part/AM 3.5 5.9 6.4 4.1 1.7 3.8
Ingest. fract. 0.29 0.29 0.25 0.27 0.23 0.18
Acc. att. part/AM 5.6 7.8 8.6 4.4 3.1 4.8
Ingest. fract. 0.45 0.35 0.27 0.41 0.29 0.25
Acc. att. part/AM 3.8 6.3 5.7 4.3 1.8 3.9
Ingest. fract. 0.34 0.33 0.26 0.23 0.27 0.18
Acc. att. part/AM 2.7 5.4 5.2 3.8 2.3 3.5
Ingest fract. 0.33 0.22 0.27 0.26 0.17 0.20
Acc. att. part/AM 2.3 4.7 3.5 1.9 1.3 3.2
Ingest. fract. 0.30 0.13 0.26 0.26 0.15 0.16
5.0 1.5
0.42 0.09
4.2b 1.7
0.27b 0.07
5.7 2.1
0.34g 0.08
4.3c 1.6
0.27d 0.06
3.8g 1.3
0.24g 0.06
2.8e 1.2
0.21f 0.07
All experiments were carried out at the same time with cells from the same rats. P(0.01 one-tailed test, compared to AM in Exp. 1. c P(0.001 one-tailed test, compared to AM and short-term IFN-c in Exp. 2. d P(0.05 one-tailed test, compared to AM and short-term IFN-c in Exp. 2. e P(0.01 one-tailed test, compared to AM and long-term IFN-c in Exp. 3. f P(0.05 one-tailed test, compared to AM and long-term IFN-c in Exp. 3. g P(0.01 two-tailed test, compared to AM in Exp. 1. b
AM
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ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES
there was a tendency to increased accumulated attachment (P"0.1, two-tailed test) and a signi7cantly decreased ingested fraction compared to AM alone. Long-term incubation with IFN-c (Exp. 3) caused a signi7cant decrease in both the accumulated attachment and the ingested fraction in carbon-free AM. Within all three experiments, both the accumulated attachments and ingested fractions were signi7cantly lower in the samples with carbonloaded AM than in the samples with carbon-free AM. In all three experiments the average mass was about 1 lg/106 AM (Table 2).
TABLE 3 Amount of Formazan Reduced from NBT by Superoxide Anions from AM in the Carbon (Exp. 1), Carbon and ShortTerm IFN-c (Exp. 2), and Carbon and Long-Term IFN-c (Exp. 3) Experiments Optical density (arbitrary units)
Carbon experiment (Exp. 1)
Carbon and short-term IFN-c experiment (Exp. 2)
Carbon and long-term IFN-c experiment (Exp. 3)
Rat No.
AM
AM# carbon
AM
AM# carbon
AM
AM# carbon
7 8 9 10 11 12
0.55 0.40 0.32 0.38 0.77 0.50
0.48 0.44 0.42 0.40 0.78 0.48
0.59 0.41 0.35 0.43 0.62 0.61
0.47 0.45 0.37 0.43 0.63 0.65
0.17 0.11 0.17 0.13 0.17 0.09
0.14 0.41 0.20 0.14 0.15 0.06
Mean SD
0.49 0.16
0.50 0.14
0.50 0.12
0.50 0.11
0.14a 0.04
0.18 0.12
Oxidative Metabolism Data The amount of formazan reduced from NBT by superoxide anions in the three experiments is shown in Table 3. Short-term incubation with IFN-c had no effect on the amount of produced formazan, but long-term incubation markedly reduced the production in AM unloaded with carbon by a factor of 3. In none of the three experiments was there any difference in formazan production between carbon-loaded and carbon-free AM. The ingested mass was signi7cantly lower in the carbon and long-term IFN-c experiment (Exp. 3) than in the two other experiments (Table 2). DISCUSSION
In an in vitro experiment, it is important to establish that the dose used is relevant for the in vivo TABLE 2 Mass (in lg) of Carbon Particles Taken Up by 106 AM, in the Carbon, Carbon and Short-Term IFN-c, and Carbon and Long-Term IFN-c Experiments in the Studies of Phagocytosis and Oxidative Metabolisma
Carbon experiment (lg/106 AM) (Exp. 1) Study of phagocytic activity Study of oxidative metabolism a
1.3$0.5 (n"6) 1.2$0.5b (n"5) 1.6$0.7 (n"6)
Carbon and short-term IFN-c experiment (Exp. 2)
Carbon and long-term IFN-c experiment (Exp. 3)
0.9$0.2b
1.1$0.3 (n"6) 1.1$0.3b (n"5) 0.9$0.5c (n"6)
1.5$0.6 (n"6)
Data are given as mean $SD. Value from one rat missing due to technical error. c Values for the 7ve rats used in all experiments. d P(0.05 two-tailed test compared to Exp. 1 and Exp. 2. b
a
P(0.01 two-tailed test compared to AM only in Exp. 1.
situation, which in the present context is inhalation of ambient air. The particle load to AM in this study can be compared with the load in an in vivo study with known exposure conditions. OberdoK rster et al. (1994) exposed rats to aggregates of ultra7ne TiO2 particles at a concentration of 23.5 mg/m3 during 30 h/week for 12 weeks. After the exposure, they found around 100 lg/106 AM. Assuming that an exposure of 24 h/day for 7 days/week results in 5.4 times higher load than an exposure for 30 h/week, the present concentration of 1 lg/106 AM should correspond to a concentration of 40 lg/m3. This is a crude estimate, we have not taken into account that the half-time increases for high lung burdens, which should give a higher estimate, or that the lung burden has not reached equilibrium after 12 weeks exposure, which should result in a lower estimate. However, these calculations indicate that the AM load may occur after long-term ambient air exposure. IFN-c have a fundamental role in the activation of leukocytes (Curfs et al., 1997). It has been shown that this cytokine increases the oxidative metabolism in neutrophils, monocytes, and macrophages obtained from the blood (Nathan et al., 1983; Cassatella et al., 1985, 1988; Kowanko and Ferrante, 1987). The tendency to increase in the accumulated attachment in AM by the short-term incubation with IFN-c (40 min) agrees with these previous results. However, short-term incubation with IFN-c
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LUNDBORG ET AL.
impaired the ingested fraction and did not result in increased oxidative metabolism. This might be explained by the relatively low concentration of IFN-c used in the present experiment. Long-term incubation with IFN-c (28 or 44 h) markedly impaired the phagocytosis of test particles, both the attachment and the ingestion processes, and resulted in decreased oxidative metabolism in AM. The results concerning the incubation of AM with IFN-c, especially the long-term incubation, are unexpected from the literature data. One explanation for this discrepancy might be that studies on IFN-c and leukocytes have focused on responses to microorganisms and antigens and not to inorganic particles. The results of the studies of phagocytosis of the test particles agree well with the amounts of carbon particles, taken up by the AM. In the phagocytosis study of long-term IFN-c-incubated, carbon-loaded AM (Exp. 3), the carbon particles were only taken up during 6 h and the carbon particle mass/106 AM did not differ signi7cantly from the masses in the two experiments where IFN-c was not present during the loading with carbon. In the study of oxidative metabolism, the carbon particles were taken up during a much longer time, 20 h, and the ingested carbon particle mass 106 AM was signi7cantly lower in the carbon and long-term IFN-c experiment (Exp. 3) than in the two other experiments where IFN-c was not present during the loading with carbon. Raised levels of oxygen metabolites can cause cell and tissue damage. The results of our study do not support the hypothesis that small amounts of ingested carbon particles enhance the production of such metabolites. We have earlier with the same method as in the present study investigated oxidative metabolism of macrophages in vitro 24 h after phagocytosis of yeasts (Candida albicans and Saccharomyces cerevisae), conidia of Aspergillus species, and particles of amorphous silica (Nyberg et al., 1996; Nessa et al., 1997). A few ingested fungi/AM induced a twoto threefold increase in the oxidative metabolism and a few silica particles/AM induced a small but signi7cant increase. The difference between our earlier results and our present results might be due to the different particle material and also that the ingested mass of silica particles and fungi (a few particles/AM) was 10}100 times larger than in the present study. The loading of AM with carbon particles markedly impaired both the attachment and the ingestion processes of the test particles, and there was a combined effect of long-term IFN-c incubation and presence of ingested carbon particles. From these data, it seems reasonable to assume that accumulated ingested environmental particles in AM markedly impair the
cell’s phagocytic capacity, especially during longterm exposures to IFN-c, as after infections. Consequently, there might be an increased risk for additional infections. Moreover, inhaled particles during an episode with high particle concentration in the ambient air may not be ef7ciently phagocytized by AM and, thus, might damage the lung tissue. ACKNOWLEDGMENTS We are grateful for the skillful assistance by Ms Ulla Bergsten. The carbon powder from GuK nther Wayner, Hannover, Germany, was kindly provided from Professor BjoK rn Afzelius. The study was supported by grants from the Swedish Heart-Lung Foundation and research funds of Karolinska Institute.
REFERENCES Baron, S., Tyring, S. T., Fleischmann, R. W., Coppenhaver, D. H., Niesel, D. W., Klimpel, G. R., Stanton, G. J., and Hughes, T. K. (1991). The interferons. Mechanisms of action and clinical applications. J. Am. Med. Assoc. 266, 1375}1383. Brunekreef, B., Dockery, D. W., and Krzyzanowski, M. (1995). Epidemiologic studies on short-term effects of low levels of major ambient air pollution components. Environ. Health Perspect. 103 (Suppl. 2), 3}13. Cassatella, M. A., Bianca, V. D., Berton, G., and Rossi, F. (1985). Activation by gamma interferon of human macrophage capability to produce toxic oxygen molecules is accompanied by decreased Km of the superoxide-generating NADPH oxidase. Biochem. Biophys. Res. Commun. 132, 908}914. Cassatella, M. A., Cappelli, R., Bianca, D., Grzeskowiak, M., Dusi, S., and Berton, G. (1988). Interferon-gamma activates human neutrophil oxygen metabolism and exocytosis. Immunology 63, 499}506. Curfs, J. H. A. J., Meis, J. F. G. M., and Hoogkamp-Korstanje, J. A. A. (1997). A primer on cytokines: Sources, receptors, effects, and inducers. Clin. Microbiol. Rev. 10, 742}780. Diamond, R. D., Llyman, C. A., and Wysong, D. R. (1991). Disparate effects of interferon-c and tumor necrosis factor-d on early neutrophil respiratory burst and fungicidal responses to Candida albicans hyphae in vitro. J. Clin. Invest. 87, 711}720. EPA (1996). Air quality criteria for particulate matter. US EPA/600/P-95/00 1bF, Research Triangle Park, NC. Gross, N. T., Nessa, K., Camner, P., and Jarstrand, C. (1999). Production of nitrite by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus. Med. Mycol. 37, 151}157. Hed, J. (1977). The extinction of 8uorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis. FEMS Microbiol. Lett. 1, 357}361. Heinrich, U., Fahst, R., Rittinghausen, S., Creutzenberg, O., Bellman, B., Koch, W., and Leven, K. (1995). Chronic inhalation exposure of Wistar rats and two different strains of mice to diesel engine exhaust, carbon black, and titanium dioxide. Inhal. Toxicol. 7, 533}556. Jarstrand, C., Lundborg, M., Wiernik, A., and Camner, P. (1978). Alveolar macrophage function in nickel dust exposed rabbits. Toxicology 11, 353}359.
ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES Kowanko, I. C., and Ferrante, A. (1987). Stimulation of neutrophil respiratory burst and lysosomal enzyme release by human interferon-gamma. Immunology 62, 149}151. Nathan, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y. (1983). Identi7cation of interferon-c as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158, 670}689. Nessa, K., Jarstrand, C., Johansson, A., and Camner, P. (1997). In vitro interaction of alveolar macrophages and Aspergillus fumigatus. Environ. Res. 74, 54}60. Nikula, K. J., Snipes, M. B., Barr, E. G., Grif7th, W. C., Henderson, R. F., and Mauderly, J. L. (1995). Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. Fundam. Appl. Toxicol. 25, 80}94.
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Nyberg, K., Nessa, K., Johansson, A., Jarstrand, C., and Camner, P. (1996). Alveolar macrophage response to yeasts and to inert particles in humans. J. Med. Vet. Mycol. 34, 11}17. OberdoK rster, G., Ferin, J., and Lehnert, B. E. (1994). Correlation between particle size, in vivo particle persistence, and lung injury. Environ. Health Perspect. 102, 173}179. Philipson, K., Falk, R., Gustafsson, J., and Camner, P. (1996). Long-term lung clearance of 195Au labelled Te8on particles in humans. Exp. Lung Res. 22, 65}83. Pope III, C. A., Bates, D. V., and Raizenne, M. E. (1995). Health effects of particulate air pollution: Time for reassessment. Environ. Health Perspect. 103, 472}480. Wiernik, A., Johansson, A., Jarstrand, C., and Camner, P. (1983). Rabbit lung after inhalation of soluble nickel. I. Effects on alveolar macrophages. Environ. Res. 30, 129}141.
Ingested Aggregates of Ultrafine Carbon Particles and Interferon-c Impair Rat Alveolar Macrophage Function Margot Lundborg,1 Anne Johansson, Lena La> stbom, and Per Camner Division of Inhalation Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Received December 28, 1998
after infections, and there might be an increased risk for additional infections. Moreover, during an episode of high ambient particle concentration the inhaled particles will not be efAciently phagocytized and may thereby damage the lung tissue.
Alveolar macrophages (AM), obtained by lavage from the rat lung, were allowed to ingest aggregated ultraAne carbon particles, about 1 lg/106 AM, which is a realistic result of long-term exposure to ambient air. The effects of the ingested carbon on the phagocytosis of test particles and oxidative metabolism of the AM were studied. In addition, the effects of short-term (40 min or 2 h) and long-term (28 or 44 h) incubation with interferon gamma (IFN-c) on AM loaded and unloaded with carbon were investigated. Phagocytic activity was studied using Buorescein-labeled 3.2-lm silica particles. The attachment and ingestion processes were evaluated separately. The ingested carbon markedly impaired the phagocytosis of silica particles; the accumulated attachment (sum of attached and ingested particles per AM) decreased from 5.0 to 4.2 particles/AM and the ingested fraction (number of ingested particles per AM divided with accumulated attachment) from 0.42 to 0.27. The short-term incubation with IFN-c tended to increase the accumulated attachment (from 5.0 to 5.7 particles/AM) and decreased the ingested fraction (from 0.42 to 0.34) in unloaded AM. Long-term incubation with IFN-c markedly impaired both the accumulated attachment (to 3.8 particles/AM) and the ingested fraction (to 0.24) in unloaded AM and the carbon load further decreased the accumulated attachment to 2.8 particles/AM, and the ingested fraction to 0.21. The oxidative metabolism was not effected by the ingested carbon or the short-term incubation with IFN-c, but the long-term incubation with IFN-c increased it with a factor of almost 3. Our results suggest that ingested environmental particles in AM may markedly impair their phagocytic capacity, especially during long-term exposure to IFN-c as
( 1999 Academic Press
Key Words: particulate matter; ultraAne particles; alveolar macrophages; phagocytosis; oxidative metabolism; interferon-c; infections; carbon.
INTRODUCTION
The interest in health effects caused by ambient air particles has increased in recent times. One reason is that a large number of epidemiological studies show relationships between rather moderate levels of particles in the ambient air and mortality as well as lung morbidity (EPA, 1996; Pope et al., 1995; Brunekreef et al., 1995). Another reason is that long-term exposure to particles of rather innocuous material such as carbon particles and titanium oxide particles has caused pathological changes, including tumors, in the rat lung (EPA, 1996; Heinrich et al., 1995; Nikula et al., 1995). The mechanisms behind the effects documented in the epidemiological and experimental studies are not known. It is reasonable to believe that alveolar macrophages (AM) play a role in these mechanisms. By taking up particles deposited in the lungs, the AM protect other, more sensitive cells. Since the mechanical clearance from the alveolar region in humans is extremely slow, the half-time being about 5 years (Philipson et al., 1996), an accumulation of ingested particles within the AM must take place. If these ingested particles would impair the phagocytic capacity of the AM, other cells might be damaged by particles inhaled during episodes with high particle concentration in the ambient air. There might also be an increased risk for infections. Moreover, if the
1 To whom correspondence should be addressed at Division of Inhalation Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden. Fax: #46 8 30 33 90.
309 0013-9351/99 $30.00 Copyright ( 1999 by Academic Press All rights of reproduction in any form reserved.
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particles ingested by the AM trigger an oxidative burst with release of reactive oxygen metabolites, there would be a risk of tissue damage in the lung. The purpose of the present study was to clarify if commonly occurring amounts of ingested carbon particles in AM impair their ability to phagocytize other particles and if the oxidative metabolism of the AM would be affected. In an attempt to increase the differences between AM loaded and not loaded with carbon particles, we also incubated AM with interferon-c (IFN-c). IFN-c is considered to have a fundamental role in activating white blood cells, including macrophages. The change is antigen-induced via T-lymphocyte activation and is enhanced by infections by microorganisms or exposure to other antigens (Baron et al., 1991; Curfs et al., 1997).
FIG. 1. Relationship between the concentration of suspended carbon particles and optical density (OD).
MATERIAL AND METHODS
Animals and Lung Lavage Alveolar macrophages were obtained by lavage from 12 healthy rats (Sprague-Dawley, Charles River, Uppsala, Sweden) weighing 250}300 g. The rats were sacri7ced by an overdose of sodium pentobarbital. The lungs were excised and lavaged with Hank’s balanced salt solution (without Ca2` and Mg2` pH 7.4, 37@C) using brief massage. Around 40 ml lavage 8uid, containing 10}15]106 cells, was obtained. In the light microscope, more than 90% of the cells were estimated to be AM. The cells were washed once by a 10-min, 300g centrifugation at room temperature; the resulting cell pellet was resuspended in Hepes buffered medium 199, pH 7.4 (GIBCO, Paisly, Scotland), with 10% fetal calf serum, penicillin 100 units/ml, and streptomycin 100 lg/ml (complete medium). The number of cells was counted in a BuK rker hemocytometer.
Experimental Design For each of the studies of phagocytosis and oxidative metabolism, three sets of experiments were performed in parallel with AM from the same rats at the same time. In the 7rst sets of experiments (Exp. 1), AM loaded and not loaded with carbon were compared concerning the two functions. The second sets of experiments (Exp. 2) were performed as the 7rst sets with the exception that the AM were incubated with IFN-c during the tests of phagocytosis and oxidative metabolism (short-term incubation). The third sets of experiments (Exp. 3) were performed as the second sets with the exception that IFN-c was present not only during the test but also during the loading of AM with carbon particles (long-term incubation). Loading of AM with Carbon Particles
Preparation of Carbon Particle Suspension Aggregates of ultra7ne carbon powder (GuK nther Wayner, Hannover, Germany) were used. Before use, the powder was suspended in medium 199 (with penicillin 100 units/ml and streptomycin 100 lg/ml) and sonicated for 3 h with a short break every 20 min for vortexing. A stable suspension of carbon particles was obtained in this way. There was a linear relationship between carbon particle concentration and optical density measured spectrophotometrically (Shimadzu, UV-160A), facilitating estimation and control of the carbon particles in suspension, see Fig. 1. The 7nal carbon suspension contained 20 lg carbon/ml in the complete medium.
In the study of phagocytosis of test particles, 1 ml of carbon particle suspension was added to a test tube with 1]106 precipitated AM and to another without AM. In the study of oxidative metabolism, 2 ml of carbon particle suspension was added to a test tube with 2]106 precipitated AM and to another without AM. The samples were placed in a shaking water bath at 37@C for 6 h (study of phagocytosis) or for 20 h (study of oxidative metabolism) in order to obtain AM with attached or ingested carbon particles. The tubes were then centrifuged at 300g for 10 min. The supernatants from the tubes with and without AM were measured in the spectrophotometer and the difference in carbon particle
ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES
concentration was taken as a measure of attached and ingested particles by AM. Centrifugation of the carbon particle suspension from tubes without AM had only a negligible effect on the concentration of suspended particles. Fresh complete medium was added to the precipitated cells and in order to get rid of remaining free carbon particles, an additional washing was made. Subsequently, fresh, complete medium was added to the AM and the tubes were placed in a shaking water bath for 20 h at 37@C to ensure that all carbon particles attached to the AM would be ingested. Control AM were treated in exactly the same way from lavage to the end of the phagocytosis and oxidative metabolism experiments, only leaving out the carbon particles. Short-Term and Long-Term Incubation with IFN-c In the studies of short-term effects of recombinant rat IFN-c (Sigma) on phagocytosis of test particles and oxidative metabolism, AM were treated as in the 7rst sets of experiments with the exception that IFN-c was added immediately before the tests of phagocytosis and oxidative metabolism. In the studies of long-term effects of IFN-c on phagocytosis of test particles and oxidative metabolism, AM were treated as in the 7rst set of experiments with the exception that IFN-c was present during the loading of the AM with the carbon particles and the entire experimental procedure. The concentration of IFN-c was 12.5 U/ml in all experiments. This concentration was originally chosen from a dose-response curve of IFN-c concentration on NO production of AM (Gross et al., 1999). 12.5 U IFN-c/ml induced a marked increase in the NO production, which was 10}15% of the maximum increase, obtained at 250 U/ml. Preliminary studies on long-term incubation with IFN-c (24}48 h) on the oxidative metabolism (NBT test) of AM indicated that a concentration higher than 12.5 U/ml resulted in about the same reduction in the oxidative metabolism. Assay of Phagocytosis Phagocytic ability of AM with and without ingested carbon particles was studied in samples from six rats. Spherical particles of amorphous silica (Spherisorb S 3, NH 2, Phase Separations Ltd, Queens Ferry, Clwyd, UK) were labeled with 8uorescein isothiocyanate (FITC) (Nyberg et al., 1996). The size of the silica particles was measured in a light microscope (Viscopan projection microscope, Reichert, Austria) and was found to be 3.2$0.4 lm
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(mean$SD). A modi7cation (Wiernik et al., 1983) of the method described by Hed (1977) was used for the assessment of phagocytic function. After the 20 h of incubation (see above), which was made in order to ensure that attached carbon particles had been ingested by the AM, the tubes with the cell suspensions were centrifuged at 300g for 10 min. One milliliter of complete medium was added to the cell precipitate in each test tube; from this suspension (106 AM/ml), samples of 250 ll were added to glass slides with round-bottomed wells (Nova Kemi Ltd, Sweden). The cells were allowed to attach to the glass during incubation for 30 min at 373C with 5% CO2 in air and 80% relative humidity. Complete medium (250 ll) containing silica particles at a concentration of 10]106 particles/ml was added to the cells, i.e., 10 particles per AM. After incubation for 40 min, the slides were placed in ice-cold Ringer acetate (to interrupt the phagocytosis) and unattached particles were rinsed off. The cells were then stained with trypan blue (0.4 mg/ml) for 30 s. The number of particles ingested by and attached to an AM was then directly counted in a Zeiss microscope, which permits examination in both visible light and UV light (8uorescence). When the microscope is turned on the visible light mode, the attached particles are clearly visible by the trypan blue staining, while the ingested ones are not. When the microscope is switched over to the UV light mode, only the ingested particles are seen as the 8uorescence from the attached particles is quenched by trypan blue. In each sample 100 consecutive macrophages were scored. The parameters accumulated attachment and ingested fraction were introduced in order to differentiate between the attachment and the ingestion processes. The accumulated attachment is the sum of the numbers of attached and ingested particles per AM. As all ingested particles must have been attached to the AM, the accumulated attachment is the integrated number of particles attached to the AM during the time of the test. Therefore, this parameter is a measure of the attachment process. The ingested fraction is a number of ingested particles per AM divided by the accumulated attachment, i.e., the ingested particles are related to the integrated number of attached particles. This parameter, thus, represents a measure of the ingestion process and is rather independent of the accumulation process. Assay of Oxidative Metabolism The oxidative metabolism (‘‘spontaneous’’ metabolism with no stimulation by agents like zymozan)
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times and enlarged about 3 times, and the diameters of individual carbon particles as well as of aggregates of particles were measured. Latex particles, 1.099 lm in diameter (Serva Entwichlungslabor, Heidelberg, Germany), were used as standards for calibration purposes.
of AM was measured by using the ability of the produced superoxide to reduce yellow nitroblue tetrazolium (NBT) to blue formazan, according to the method described by Jarstrand et al. (1978). After the 20 h in a shaking 373C water bath, the cell suspensions were centrifuged at 300g for 10 min. One milliliter of complete medium and 1 ml of NBT solution were added to the cell precipitate in each tube. The tubes were incubated for 2 h in a shaking 373C water bath. The reaction was stopped by adding 1 ml of 0.5% HCl to each tube. The tubes were centrifuged at 2500g for 30 min. One milliliter of dimethyl sulfoxide was added to each tube to dissolve the produced formazan and the tubes were sonicated for 30 min. The absorption of the solution from each tube was measured in a spectrophotometer (Shimadzu, UV-160A) at wavelength 572 nm.
Statistical Analysis The paired t test was used with a level of signi7cance of 0.05. A two-tailed test was made to evaluate the effects of IFN-c and a one-tailed test to evaluate the effects of carbon. RESULTS
EM Data The carbon particle in the suspension occurred as aggregates with a mean diameter of 0.165$ 0.077 lm ($SD) (0.050}0.347 lm). The diameter of the primary carbon particles was 0.044$0.010 lm.
Electron Microscopy Carbon particles from a stable suspension in distilled water were studied with transmission electron microscopy. Drops of the suspension were allowed to dry on to copper grids (200 mesh) covered by collodion 7lm. The grids were examined in a Jeol 100S electron microscope (Jeol Ltd, Tokyo, Japan). Photos were taken at a primary magni7cation of 10,000
Phagocytosis Data The data on the accumulated attachment and the ingested fraction for the three experiments are shown in Table 1. In the short-term incubation of AM with IFN-c (AM not loaded with carbon, Exp. 2),
TABLE 1 Accumulated Attachment (Acc. att.) and Ingested Fraction (Ingest fract.) of Test Particles of Amorphous Silica in the Carbon, Carbon and Short-Term IFN-c and Carbon and Long-Term IFN-c Experimentsa Carbon experiment (Exp. 1) AM Rat no. 1 2 3 4 5 6 Mean SD a
Carbon and short-term IFN-c experiment Carbon and long-term IFN-c experiment (Exp. 2) (Exp. 3)
AM#carbon
AM
AM#carbon
AM#carbon
Acc. att. part/AM 4.1 6.7 6.9 4.1 3.1 5.2
Ingest. fract. 0.50 0.44 0.43 0.49 0.35 0.33
Acc. att. part/AM 3.5 5.9 6.4 4.1 1.7 3.8
Ingest. fract. 0.29 0.29 0.25 0.27 0.23 0.18
Acc. att. part/AM 5.6 7.8 8.6 4.4 3.1 4.8
Ingest. fract. 0.45 0.35 0.27 0.41 0.29 0.25
Acc. att. part/AM 3.8 6.3 5.7 4.3 1.8 3.9
Ingest. fract. 0.34 0.33 0.26 0.23 0.27 0.18
Acc. att. part/AM 2.7 5.4 5.2 3.8 2.3 3.5
Ingest fract. 0.33 0.22 0.27 0.26 0.17 0.20
Acc. att. part/AM 2.3 4.7 3.5 1.9 1.3 3.2
Ingest. fract. 0.30 0.13 0.26 0.26 0.15 0.16
5.0 1.5
0.42 0.09
4.2b 1.7
0.27b 0.07
5.7 2.1
0.34g 0.08
4.3c 1.6
0.27d 0.06
3.8g 1.3
0.24g 0.06
2.8e 1.2
0.21f 0.07
All experiments were carried out at the same time with cells from the same rats. P(0.01 one-tailed test, compared to AM in Exp. 1. c P(0.001 one-tailed test, compared to AM and short-term IFN-c in Exp. 2. d P(0.05 one-tailed test, compared to AM and short-term IFN-c in Exp. 2. e P(0.01 one-tailed test, compared to AM and long-term IFN-c in Exp. 3. f P(0.05 one-tailed test, compared to AM and long-term IFN-c in Exp. 3. g P(0.01 two-tailed test, compared to AM in Exp. 1. b
AM
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ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES
there was a tendency to increased accumulated attachment (P"0.1, two-tailed test) and a signi7cantly decreased ingested fraction compared to AM alone. Long-term incubation with IFN-c (Exp. 3) caused a signi7cant decrease in both the accumulated attachment and the ingested fraction in carbon-free AM. Within all three experiments, both the accumulated attachments and ingested fractions were signi7cantly lower in the samples with carbonloaded AM than in the samples with carbon-free AM. In all three experiments the average mass was about 1 lg/106 AM (Table 2).
TABLE 3 Amount of Formazan Reduced from NBT by Superoxide Anions from AM in the Carbon (Exp. 1), Carbon and ShortTerm IFN-c (Exp. 2), and Carbon and Long-Term IFN-c (Exp. 3) Experiments Optical density (arbitrary units)
Carbon experiment (Exp. 1)
Carbon and short-term IFN-c experiment (Exp. 2)
Carbon and long-term IFN-c experiment (Exp. 3)
Rat No.
AM
AM# carbon
AM
AM# carbon
AM
AM# carbon
7 8 9 10 11 12
0.55 0.40 0.32 0.38 0.77 0.50
0.48 0.44 0.42 0.40 0.78 0.48
0.59 0.41 0.35 0.43 0.62 0.61
0.47 0.45 0.37 0.43 0.63 0.65
0.17 0.11 0.17 0.13 0.17 0.09
0.14 0.41 0.20 0.14 0.15 0.06
Mean SD
0.49 0.16
0.50 0.14
0.50 0.12
0.50 0.11
0.14a 0.04
0.18 0.12
Oxidative Metabolism Data The amount of formazan reduced from NBT by superoxide anions in the three experiments is shown in Table 3. Short-term incubation with IFN-c had no effect on the amount of produced formazan, but long-term incubation markedly reduced the production in AM unloaded with carbon by a factor of 3. In none of the three experiments was there any difference in formazan production between carbon-loaded and carbon-free AM. The ingested mass was signi7cantly lower in the carbon and long-term IFN-c experiment (Exp. 3) than in the two other experiments (Table 2). DISCUSSION
In an in vitro experiment, it is important to establish that the dose used is relevant for the in vivo TABLE 2 Mass (in lg) of Carbon Particles Taken Up by 106 AM, in the Carbon, Carbon and Short-Term IFN-c, and Carbon and Long-Term IFN-c Experiments in the Studies of Phagocytosis and Oxidative Metabolisma
Carbon experiment (lg/106 AM) (Exp. 1) Study of phagocytic activity Study of oxidative metabolism a
1.3$0.5 (n"6) 1.2$0.5b (n"5) 1.6$0.7 (n"6)
Carbon and short-term IFN-c experiment (Exp. 2)
Carbon and long-term IFN-c experiment (Exp. 3)
0.9$0.2b
1.1$0.3 (n"6) 1.1$0.3b (n"5) 0.9$0.5c (n"6)
1.5$0.6 (n"6)
Data are given as mean $SD. Value from one rat missing due to technical error. c Values for the 7ve rats used in all experiments. d P(0.05 two-tailed test compared to Exp. 1 and Exp. 2. b
a
P(0.01 two-tailed test compared to AM only in Exp. 1.
situation, which in the present context is inhalation of ambient air. The particle load to AM in this study can be compared with the load in an in vivo study with known exposure conditions. OberdoK rster et al. (1994) exposed rats to aggregates of ultra7ne TiO2 particles at a concentration of 23.5 mg/m3 during 30 h/week for 12 weeks. After the exposure, they found around 100 lg/106 AM. Assuming that an exposure of 24 h/day for 7 days/week results in 5.4 times higher load than an exposure for 30 h/week, the present concentration of 1 lg/106 AM should correspond to a concentration of 40 lg/m3. This is a crude estimate, we have not taken into account that the half-time increases for high lung burdens, which should give a higher estimate, or that the lung burden has not reached equilibrium after 12 weeks exposure, which should result in a lower estimate. However, these calculations indicate that the AM load may occur after long-term ambient air exposure. IFN-c have a fundamental role in the activation of leukocytes (Curfs et al., 1997). It has been shown that this cytokine increases the oxidative metabolism in neutrophils, monocytes, and macrophages obtained from the blood (Nathan et al., 1983; Cassatella et al., 1985, 1988; Kowanko and Ferrante, 1987). The tendency to increase in the accumulated attachment in AM by the short-term incubation with IFN-c (40 min) agrees with these previous results. However, short-term incubation with IFN-c
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impaired the ingested fraction and did not result in increased oxidative metabolism. This might be explained by the relatively low concentration of IFN-c used in the present experiment. Long-term incubation with IFN-c (28 or 44 h) markedly impaired the phagocytosis of test particles, both the attachment and the ingestion processes, and resulted in decreased oxidative metabolism in AM. The results concerning the incubation of AM with IFN-c, especially the long-term incubation, are unexpected from the literature data. One explanation for this discrepancy might be that studies on IFN-c and leukocytes have focused on responses to microorganisms and antigens and not to inorganic particles. The results of the studies of phagocytosis of the test particles agree well with the amounts of carbon particles, taken up by the AM. In the phagocytosis study of long-term IFN-c-incubated, carbon-loaded AM (Exp. 3), the carbon particles were only taken up during 6 h and the carbon particle mass/106 AM did not differ signi7cantly from the masses in the two experiments where IFN-c was not present during the loading with carbon. In the study of oxidative metabolism, the carbon particles were taken up during a much longer time, 20 h, and the ingested carbon particle mass 106 AM was signi7cantly lower in the carbon and long-term IFN-c experiment (Exp. 3) than in the two other experiments where IFN-c was not present during the loading with carbon. Raised levels of oxygen metabolites can cause cell and tissue damage. The results of our study do not support the hypothesis that small amounts of ingested carbon particles enhance the production of such metabolites. We have earlier with the same method as in the present study investigated oxidative metabolism of macrophages in vitro 24 h after phagocytosis of yeasts (Candida albicans and Saccharomyces cerevisae), conidia of Aspergillus species, and particles of amorphous silica (Nyberg et al., 1996; Nessa et al., 1997). A few ingested fungi/AM induced a twoto threefold increase in the oxidative metabolism and a few silica particles/AM induced a small but signi7cant increase. The difference between our earlier results and our present results might be due to the different particle material and also that the ingested mass of silica particles and fungi (a few particles/AM) was 10}100 times larger than in the present study. The loading of AM with carbon particles markedly impaired both the attachment and the ingestion processes of the test particles, and there was a combined effect of long-term IFN-c incubation and presence of ingested carbon particles. From these data, it seems reasonable to assume that accumulated ingested environmental particles in AM markedly impair the
cell’s phagocytic capacity, especially during longterm exposures to IFN-c, as after infections. Consequently, there might be an increased risk for additional infections. Moreover, inhaled particles during an episode with high particle concentration in the ambient air may not be ef7ciently phagocytized by AM and, thus, might damage the lung tissue. ACKNOWLEDGMENTS We are grateful for the skillful assistance by Ms Ulla Bergsten. The carbon powder from GuK nther Wayner, Hannover, Germany, was kindly provided from Professor BjoK rn Afzelius. The study was supported by grants from the Swedish Heart-Lung Foundation and research funds of Karolinska Institute.
REFERENCES Baron, S., Tyring, S. T., Fleischmann, R. W., Coppenhaver, D. H., Niesel, D. W., Klimpel, G. R., Stanton, G. J., and Hughes, T. K. (1991). The interferons. Mechanisms of action and clinical applications. J. Am. Med. Assoc. 266, 1375}1383. Brunekreef, B., Dockery, D. W., and Krzyzanowski, M. (1995). Epidemiologic studies on short-term effects of low levels of major ambient air pollution components. Environ. Health Perspect. 103 (Suppl. 2), 3}13. Cassatella, M. A., Bianca, V. D., Berton, G., and Rossi, F. (1985). Activation by gamma interferon of human macrophage capability to produce toxic oxygen molecules is accompanied by decreased Km of the superoxide-generating NADPH oxidase. Biochem. Biophys. Res. Commun. 132, 908}914. Cassatella, M. A., Cappelli, R., Bianca, D., Grzeskowiak, M., Dusi, S., and Berton, G. (1988). Interferon-gamma activates human neutrophil oxygen metabolism and exocytosis. Immunology 63, 499}506. Curfs, J. H. A. J., Meis, J. F. G. M., and Hoogkamp-Korstanje, J. A. A. (1997). A primer on cytokines: Sources, receptors, effects, and inducers. Clin. Microbiol. Rev. 10, 742}780. Diamond, R. D., Llyman, C. A., and Wysong, D. R. (1991). Disparate effects of interferon-c and tumor necrosis factor-d on early neutrophil respiratory burst and fungicidal responses to Candida albicans hyphae in vitro. J. Clin. Invest. 87, 711}720. EPA (1996). Air quality criteria for particulate matter. US EPA/600/P-95/00 1bF, Research Triangle Park, NC. Gross, N. T., Nessa, K., Camner, P., and Jarstrand, C. (1999). Production of nitrite by rat alveolar macrophages stimulated by Cryptococcus neoformans or Aspergillus fumigatus. Med. Mycol. 37, 151}157. Hed, J. (1977). The extinction of 8uorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis. FEMS Microbiol. Lett. 1, 357}361. Heinrich, U., Fahst, R., Rittinghausen, S., Creutzenberg, O., Bellman, B., Koch, W., and Leven, K. (1995). Chronic inhalation exposure of Wistar rats and two different strains of mice to diesel engine exhaust, carbon black, and titanium dioxide. Inhal. Toxicol. 7, 533}556. Jarstrand, C., Lundborg, M., Wiernik, A., and Camner, P. (1978). Alveolar macrophage function in nickel dust exposed rabbits. Toxicology 11, 353}359.
ULTRAFINE CARBON PARTICLES AND ALVEOLAR MACROPHAGES Kowanko, I. C., and Ferrante, A. (1987). Stimulation of neutrophil respiratory burst and lysosomal enzyme release by human interferon-gamma. Immunology 62, 149}151. Nathan, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y. (1983). Identi7cation of interferon-c as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158, 670}689. Nessa, K., Jarstrand, C., Johansson, A., and Camner, P. (1997). In vitro interaction of alveolar macrophages and Aspergillus fumigatus. Environ. Res. 74, 54}60. Nikula, K. J., Snipes, M. B., Barr, E. G., Grif7th, W. C., Henderson, R. F., and Mauderly, J. L. (1995). Comparative pulmonary toxicities and carcinogenicities of chronically inhaled diesel exhaust and carbon black in F344 rats. Fundam. Appl. Toxicol. 25, 80}94.
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Nyberg, K., Nessa, K., Johansson, A., Jarstrand, C., and Camner, P. (1996). Alveolar macrophage response to yeasts and to inert particles in humans. J. Med. Vet. Mycol. 34, 11}17. OberdoK rster, G., Ferin, J., and Lehnert, B. E. (1994). Correlation between particle size, in vivo particle persistence, and lung injury. Environ. Health Perspect. 102, 173}179. Philipson, K., Falk, R., Gustafsson, J., and Camner, P. (1996). Long-term lung clearance of 195Au labelled Te8on particles in humans. Exp. Lung Res. 22, 65}83. Pope III, C. A., Bates, D. V., and Raizenne, M. E. (1995). Health effects of particulate air pollution: Time for reassessment. Environ. Health Perspect. 103, 472}480. Wiernik, A., Johansson, A., Jarstrand, C., and Camner, P. (1983). Rabbit lung after inhalation of soluble nickel. I. Effects on alveolar macrophages. Environ. Res. 30, 129}141.