Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Long‐term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages Katsumi Mochitate , Kunihiko Ishida , Takumi Ohsumi & Takashi Miura To cite this article: Katsumi Mochitate , Kunihiko Ishida , Takumi Ohsumi & Takashi Miura (1992) Long‐term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages, Journal of Toxicology and Environmental Health, 35:4, 247-260, DOI: 10.1080/15287399209531615 To link to this article: http://dx.doi.org/10.1080/15287399209531615
Published online: 20 Oct 2009.
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LONG-TERM EFFECTS OF OZONE AND NITROGEN DIOXIDE O N THE METABOLISM AND POPULATION OF ALVEOLAR MACROPHAGES
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Katsumi Mochitate, Kunihiko Ishida, Takumi Ohsumi Environmental Health Sciences Division, National Institute for Environmental Studies, Ibaraki, Japan Takashi Miura Regional Environmental Division, National Institute for Environmental Studies, Ibaraki, Japan
To investigate how alveolar macrophages adapt themselves to oxidative pollutants in the long term, rats were exposed to a strong oxidant, ozone (O3), or a weak oxidant, nitrogen dioxide (NO2), for a maximum duration of 12 wk. After exposures, alveolar macrophages were collected by pulmonary lavage. Throughout 11 wk of exposure to 0.2 ppm O3r the specific activities of glucose-6phosphate dehydrogenase (G6PDH) and glutathione peroxidase of the peroxidative metabolic pathway and pyruvate kinase and hexokinase of the glycolytic pathway were 40-70% elevated over the controls in alveolar macrophages. The population of alveolar macrophages was consistently 60% higher than the controls. The small-sized macrophages, immature macrophages, preferentially increased. To the contrary, the thymidine incorporation per cell was always 20-30% lower than in the controls, although the total incorporation remained unchanged. No infiltration of polymorphonuclear leukocytes occurred. By 12 wk of exposures to 1.2 and 4.0 ppm NO2, the population of alveolar macrophages increased 30% over the control. Among the enzymes examined, however, only the C6PDH activity increased 10% for 4.0 ppm NO2. No increase in the enzyme activities occurred for 1.2 ppm NO2. Based on these results, alveolar macrophages adapt themselves to the long-term exposure of O3 or NO2 by recruiting immature macrophages through an apparent influx of monocytes. During the exposure to O3, the peroxidative metabolic and glycolytic pathways are enhanced persistently in alveolar macrophages, whereas both pathways were not enhanced by the exposures to NO2
The authors thank Yuko Katahoka for her technical assistance. Requests for reprints should be sent to Katsumi Mochitate, Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan.
247 Journal of Toxicology and Environmental Health, 35:247-260, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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INTRODUCTION Ozone (O3) and nitrogen dioxide (NOJ are major components of oxidative pollutants and are found in urban and industrial atmospheres (Eschenroeder, 1977). Both gases impair the pulmonary epithelial cells in a similar fashion. Type I alveolar epithelial cells and ciliated cells at terminal bronchiole are the most sensitive to these gases and are easily damaged, leading to necrosis and desquamation (Parkinson and Stephens, 1973; Schwartz et al., 1976; Stephens et al., 1972, 1974a, 1974b). In contrast, type 2 alveolar epithelial cells and Clara cells appear to be tolerant. When impaired, type 1 epithelial cells and ciliated cells are soon replaced by new ones derived from their progenitors, such as type 2 alveolar cells and Clara cells (Evans et al., 1973, 1975, 1976a, 1976b, 1986), respectively. Through these processes, the sensitive cells seem to become tolerant to O3 and NO2 (Evans et al., 1985). Ozone and NO2 also impair alveolar macrophages (Gardner, 1984; Schlesinger, 1989). Their functions such as bactericidal activity, phagocytosis (Coffin et al., 1968; Devlin et al., 1991; Driscoll et al., 1987; Goldstein et al, 1978; Parker et al., 1989) and lysosomal hydrolysis (Goldstein et al., 1978; Hurst et al., 1970; Kimura and Goldstein, 1981) are damaged by these gases. However, it is not clear how alveolar macrophages repair damages and adapt to O3 and NO2. We have investigated the metabolic and populational responses of alveolar macrophages to 0.2 ppm O3 or 4.0 ppm NO2 for a maximum duration of 2 wk (Mochitate and Miura, 1989; Mochitate et al., 1986) to clarify their processes for adaptation. When exposed to 0.2 ppm O3, alveolar macrophages appear to repair damages through the persistent enhancement of the peroxidative metabolic and glycolytic pathways. When exposed to 4.0 ppm NO2, however, both metabolic pathways temporarily increase and then reduce to the control levels. The population of immature macrophages is elevated consistently during both exposures, following the temporary enhancement of DNA synthesis. Therefore, to clarify the adaptation processes it is necessary to examine whether alveolar macrophages persistently enhance the peroxidative metabolic and glycolytic pathways or fluctuate between the elevated and control levels during long-term exposure to O3 or NO2. It is also of interest to investigate whether alveolar macrophages repeat the temporary enhancement of DNA synthesis and maintain the elevated level of immature macrophages. In this report, we show that the peroxidative metabolic and glycolytic pathways in alveolar macrophages were enhanced persistently during long-term exposure to 0.2 ppm O3, whereas no enhancement occurred with exposures to 1.2 and 4.0 ppm NO2. Immature macrophages, however, increased to a similar extent without enhancement of DNA synthesis by these exposures.
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MATERIALS AND METHODS
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Materials
The following reagents were purchased from Boehringer Mannheim Yamanouchi Co. (Tokyo, Japan): phosphoenolpyruvate, glucose-6phosphate, pyruvate, fructose-1,6-bisphosphate, reduced glutathione, ATP, ADP, NADH, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase. We also obtained NADP+, NADPH, and glutathione reductase from Oriental Yeast Co. (Tokyo, Japan); HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) from Dojindo Laboratories (Kumamoto, Japan); Eagle's minimum essential medium from Nissui Seiyaku Co. (Tokyo, Japan); and fetal bovine serum from Flow Laboratories Inc. (McLean, Va.). [Methyl-14C]thymidine (61 mCi/ mmol) and L-[3,4,5-3H(N)]leucine (152 Ci/mmol) were the products of Amersham (Buckinghamshire, England) and New England Nuclear (Boston, Mass.), respectively. Animals
Male Jcl: Wistar rats (specific-pathogen-free) at 10 wk of age were obtained from CLEA-Japan Co. (Tokyo, Japan). Until exposures, rats were fed a sterilized rodent diet (CE-2, CLEA-Japan Co.) and water ad libitum and kept specific-pathogen-free (SPF). Exposure Conditions
In the 1-wk exposure to O3, each group of rats (16 wk old, 6 rats/ group) was continuously exposed to 0.10 ± 0.01 or 0.20 ± 0.01 ppm O3 in exposure chambers of the same model, which were controlled to 25°C and 55-60% relative humidity throughout the exposures. In longterm exposures, each group (21 wk old, six rats/group) was exposed to 0.20 ppm O3 for 6-11 wk or 0.4, 1.2, and 4.0 ppm NO2 for 12 wk in the same way. As the controls, an equal number of rats was allowed to breathe only filtered clean air in another identical chamber at the same time. After exposures, rats were supplied to the following experiments. No significant differences of body weights and wet weights of lung tissues were observed between the exposed and control groups. The SPF status was maintained throughout the exposure periods, and respiratory diseases did not occur in any rats used. Preparation of Alveolar Macrophages
Alveolar macrophages were prepared according to Myrvik et al. (1961), with slight modifications (Mochitate and Miura, 1989; Mochitate et al., 1986). After exposures, rats were anesthetized with sodium pentobarbital injected intraperitoneally and were exsanguinated through the carotid artery. The lungs were then perfused thoroughly with cold 0.95% NaCI solution injected from the right ventricle. After perfusion,
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the lungs were removed from the thorax and lavaged by intratracheal instillation with a total of 35 ml (first 15 ml, second and third 10 ml each) of isotonic HEPES buffer, which consisted of 136 mM NaCI, 5.3 mW KCI, 2.5 mM sodium phosphate buffer, 5.5 mM glucose, and 10 mM HEPES, adjusted to pH 7.4. Alveolar free cells were recovered from the lavage fluid by centrifugation at 220 x g for 10 min and washed with HEPES buffer. The viability was determined by the trypan blue exclusion test. The viabilities of alveolar macrophages were always larger than 89% in both exposed and control groups and were not affected by the exposures. The population of lavaged-out cells was measured with a Coulter counter (type Industrial D, Coulter Electronics Ltd., Luton Beds, England), which was calibrated with latex microspheres of 10 /jm in diameter (6602796, Coulter Electronics Ltd.) and adjusted to count particles larger than 9 /*m in diameter. The adjusted counter yielded the same result as counting macrophages with a hematocytometer. Each population of alveolar macrophages differing in size was determined by successively changing the adjustment in the counter to count cells larger than 9, 11, 13, 15, 17, 19, and 21 /xm in diameter and calculating the differences in population between nearest diameters. The composition of alveolar free cells collected by pulmonary lavage was determined by Giemsa staining of smear preparations. More than 96% of lavaged-out cells were always macrophages. Therefore, the lavaged cells are hereafter referred to as alveolar macrophages. Homogenization of Alveolar Macrophages
Half of each alveolar macrophage preparation was suspended to 1.00 ml in cold 0.25 M sucrose solution containing 10 mM Tris-HCl (pH 7.4) and 0.5 mM EDTA and homogenized in a tapered Potter-Elvehjem Teflon homogenizer (358133, Wheaton Scientific, Millville, N.J.) on an ice bath. The homogenates were centrifuged at 105,000 x g for 60 min at 4°C. The supernatants were recovered for the measurements of protein concentration and enzyme activities. Assays of Enzyme Activities
The enzyme activities of glucose-6-phosphate dehydrogenase and pyruvate kinase were assayed at 30°C as described by Lohr and Waller (1974) and Mochitate and Miura (1989), respectively. The glutathione peroxidase activity was determined at 37°C according to Chiu et al. (1976), with slight modifications (Mochitate and Miura, 1989). The assay mixture consisted of 100 mM Tris-HCl (pH 7.6), 2.7 mM reduced glutathione, 0.25 mM cumene hydroperoxide, 0.2 mM NADPH, 0.1 mM EDTA, and 2 units/ml of glutathione reductase. The activities of lactate dehydrogenase and hexokinase were measured at 30°C as described by Bergmeyer et al. (1974).
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The protein concentration was determined by the method of Lowry et al. (1951).
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Incorporation of [14C]Thymidine and [3H]Leucine into Alveolar Macrophages
The remaining portion of each alveolar macrophage preparation was used for the experiment of thymidine and leucine incorporations as described before (Mochitate and Miura, 1989; Mochitate et al., 1986). Alveolar macrophages (2.0 x 106 cells) were suspended in 4.0 ml of Eagle's minimum essential medium supplemented with 10% fetal calf serum, 50 nCi/ml of [14C]thymidine, and 1 /iCi/ml of [3H]leucine and incubated in a humidified 5% CO2 atmosphere at 37°C for 3.5 h. After incubation, the alveolar macrophages were recovered with a rubber policeman. The free radioactive thymidine and leucine were removed by washing the recovered macrophages three times with Ca2+, Mg 2+ free Dulbecco's phosphate-buffered saline supplemented with 1% fetal calf serum. The radiolabeled macrophages were then mixed with scintillator (Aquasol-2, New England Nuclear) and the incorporated radioactivities were counted with a scintillation counter (model 3255, Packard Instrument Co., Downers Grove, III.). Statistical Analysis
Analyses of significant differences between exposure and control groups were performed by means of Student's t test or Welch's t test after the analysis of variance. RESULTS Short-Term Effects of Ozone on the Enzyme Activities and Population of Alveolar Macrophages
To examine whether the metabolism involved in clearance of peroxidates and ATP-production in alveolar macrophages responds to an ambient level of ozone, rats were exposed to 0.1 and 0.2 ppm O3 for 1 wk. The specific activities of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase (GPx) in the peroxidative metabolic pathway (PMP) and of pyruvate kinase (PK), lactate dehydrogenase (LDH), and hexokinase (HK) in the glycolytic pathway (GP) increased 20-70% over the controls by 0.2 ppm O3 (Table 1). By 0.1 ppm O3, the key enzyme activities in both pathways were also 30-50% enhanced. The population of alveolar macrophages increased in a dosedependent manner (Table 1 and Fig. 16). The smaller-sized macrophages increased preferentially (Fig. 1a). With exposure to 0.2 ppm O3, for example, the macrophages of 9-11, 11-13, 13-15, and 15-17 jim in diameter increased 120, 90, 60, and 20% over the controls, respectively.
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TABLE 1. Changes in the Specific Activities of Glucose-6-Phosphate Dehydrogenase (G6PDH), Glutathione Peroxidase (GPx), Pyruvate Kinase (PK), Lactate Dehydrogenase (LDH), and Hexokinase (HK) and the Population of Alveolar Macrophages at 1 Week of Exposure to Ozone Parameter
Control
Protein 6 G6PDHC GPxc PKC LDHC HKC Population^
0.86 169 2130 2040 940 81 11.2
± ± ± ± + ± ±
0.06 17 230 260 80 8 0.8
0.1 ppm
E/Ca
0.2 ppm
E/Ca
1.04 248 3050 2850 1050 108 14.8
1.21 1.47 1.44 1.39 1.12 1.34 1.33
1.15 287 3260 3330 1100 112 17.5
1.34 1.69 1.53 1.63 1.17 1.39 1.56
± ± ± ± ± ± ±
0.05'* 14' 340' 170' 110 8' 1.4'*
± ± ± ± ± ± ±
0.04'* 24' 120' 450' 110e 6' 1.4'*
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposed group to control. 6 l n mg supernatant protein/macrophage preparation (mean ± SD, n — 6). c ln ftmol/min/g supernatant protein (mean ± SD, n — 6). ^Population x 106 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposed and control groups. 'Significant at p < .001 between exposed and control groups. Significant at p < .05 between 0.1 and 0.2 ppm O 3 groups.
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FIGURE 1. (a) Changes in the size distribution of alveolar macrophages by in vivo exposures of rats to 0.1 ppm and 0.2 ppm O 3 for 1 wk. Each population of alveolar macrophages in different size was determined by successively changing the adjustment in Coulter counter to count cells larger than 9, 11, 13, 15, 17, 19, and 21 ^m in diameter and calculating the differences in population between nearest diameters. Abscissa indicates the cell size: 7-9, 9-11,11-13,13-15,15-17,17-19, and 19-21 jim in diameter. Open, dark-shaded, and light-shaded columns show the population in the control and 0.1 and 0.2 ppm O 3 groups, respectively. Values are expressed as mean ± SD (n 6). Asterisk indicates significant at p < .05 between exposed and control groups; double asterisk, significant at p < .01; and triple asterisk, significant at p < .001. (6) Changes in the whole population of alveolar macrophages by 1 wk of exposures to 0.1 and 0.2 ppm O 3 . The alveolar free cells larger than 9 /»m in diameter were counted as macrophages.
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TABLE 2. Changes in the Specific Activities of Clucose-6-Phosphate Dehydrogenase (G6PDH), Glutathione Peroxidase (GPx), Pyruvate Kinase (PK), Lactate Dehydrogenase (LDH), and Hexokinase (HK) and the Population of Alveolar Macrophages over 11 Weeks of Exposure to 0.2 ppm Ozone
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Exposure time Parameter
Group
6wk
Protein 6
Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed
0.92 1.38 214 327 2280 3650 2520 3260 930 1210 86 115 9.9 16.7
G6PDHC GPxc PKC LDHC HKC Population 0 '
E/Ca ± 0.07 ± 0.16s ± 15 ± 43' ± 300 ± 250 s ± 300 ± 110' ±160 ± 60' ± 8 ± 18e ± 0.9 ± 3.0'
1.51 1.52 1.60 1.30 1.31 1.33 1.69
11 wk 0.90 1.42 197 303 2060 3490 2310 3070 860 1260 85 107 12.2 19.2
± 0.05 ± 0.12s ± 15 ± 18 s ± 140 ± 90 s ± 190 ± 120 s ± 110 ± 70 s ± 5 ± 5s ±1.4 ± 1.3s
E/Ca
1.58 1.54 1.69 1.33 1.47 1.25 1.57
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposed group to control. b l n mg supernatant protein/macrophage preparation (mean ± SD, n — 6). c ln /*mol/min/g supernatant protein (mean ± SD, n — 6). ^Population x 10 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposed and control groups. 'Significant at p < .01. ^Significant at p < .001.
With exposure to 0.1 ppm O3, the small macrophages similarly increased. Long-Term Effects of Ozone on Alveolar Macrophages
For 11 wk of exposure to 0.2 ppm O3, all the enzyme activities examined and the population of alveolar macrophages were elevated to the same levels as for the 1-wk exposure (Table 2). The small-sized macrophages preferentially increased in the same way as for the 1-wk exposure (Fig. 2). Therefore, the elevated levels both of the enzyme activities and of the population of small-sized macrophages were retained throughout the exposure period. To examine populational changes in other kinds of alveolar free cells, differential cell counts were performed on Giemsa-stained smears (Table 3). Alveolar macrophages always accounted for more than 96% of the whole cells in both exposure and control groups. Although lympho-
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254 8 7 |
6
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= 5
ow-9
,
It 13 15 17 19 21 "9 II 13 15 17 19 21 Cell Size (/im)
FIGURE 2. Changes in the size distribution of alveolar macrophages during 11 wk of exposure to 0.2 ppm O3: (a, b) exposure periods of 6 and 11 wk, respectively.
cytes were inclined to increase, polymorphonuclear leukocytes did not increase during the whole period of exposure. Next, to examine if protein and DNA synthesis in alveolar macrophages corresponded to the increase in population, alveolar macrophages were incubated with [3H]leucine and [14C]thymidine for 3.5 h (Table 4). Although the enzyme activities of the PMP and GP examined were all enhanced by the exposure to 0.2 ppm O3, the protein synthesis per cell showed no increase. Contrary to the populational increase in small-sized macrophages, the DNA synthesis per cell was 20-30% lower than in the controls during the exposure, whereas the total incorporaTABLE 3. Effects of 0.2 ppm Ozone on the Composition of Alveolar Free Cells Recovered by Alveolar Lavage Composition (%f Exposure time
Group
AM3>>
1 wk
Control Exposed Control Exposed Control Exposed
97.3 96.1 98.0 97.3 98.3 96.6
6wk 11 wk
± ± ± + ± ±
Lymc 1.4 2.0 1.0 1.3 1.0 0.6'
1.7 3.2 1.0 1.7 1.5 2.7
± ± ± ± ± ±
1.0 1.5 0.8 1.3 0.9 0.5e
PMN d
Degenerated
0.5 ± 0.5 0.3 ± 0.3 0.0 0.0 0.0 0.1 ± 0.1
0.5 0.5 1.0 1.0 0.2 0.6
Note. The composition of alveolar free cells was determined by Giemsa staining. Values were expressed as mean ± SD (n - 6). ^Alveolar macrophage. lymphocyte. ''Polymorphonuclear leukocyte. Significant at p < .05 between exposed and control groups. 'Significant at p < .01. a
± ± ± ± ± ±
0.5 0.6 1.0 0.9 0.2 0.5
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TABLE 4. Changes in Incorporation of [ 3 H]Leucine and [ 14 C]Thymidine into Alveolar Macrophages over 11 Weeks of Exposure to 0.2 ppm Ozone Incorporation (dpm/2 X 105 cells) Exposure time
Group
[ 3 H]Leucine a
1 wk
Control Exposed Control Exposed Control Exposed
37,500 36,800 45,300 48,300 35,500 41,100
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6wk 11 wk
± ± ± ± ± ±
4400 3500 6900 4200 7900 4000
E/Cb
0.98 1.07 1.16
[ 14 C]Thymidine a 4760 3360 5640 3950 4180 3520
± ± ± ± ± +
810 580c 600 810c 720 570
E/Cfa
0.71 0.70 0.84
Note. Alveolar macrophages (2 x 106 cells) were suspended in 4.0 ml of Eagle's minimum essential medium supplemented with 10% fetal bovine serum, [ 3 H]leucine (1 /»Ci/ml), and [ 14 C]thymidine (50 nCi/ml) and incubated at 37°C for 3.5 h. a Values were expressed as mean ± SD (n - 6). b Ratio of exposed group to control. Significant at p < .05 between exposed and control groups.
tion of thymidine remained unchanged. Therefore, the increase in the population of alveolar macrophages may be due to an influx of small macrophages. Long-Term Effects of Nitrogen Dioxide on the Enzyme Activities and Population of Alveolar Macrophages
To compare long-term effects of ozone with those of nitrogen dioxide, rats were exposed to 0.4,1.2, and 4.0 ppm NO2 for 12 wk. G6PDH and PK were chosen as indicator enzymes for their high susceptibilities to O3 and NO2. The specific activity of G6PDH increased only for exposure to 4.0 ppm NO2, while the PK activity did not change (Table 5). The population of alveolar macrophages, however, increased 30% over the control for exposures to 1.2 and 4.0 ppm NO2. Therefore, alveolar macrophages increased to the same degree as for 1-wk exposure to 0.1 ppm O3, without enhancement of the peroxidative metabolic and glycolytic pathways. DISCUSSION There were differences in the responses of alveolar macrophages to the long-term exposures to a strong oxidant, O3, and a weak oxidant, NO2 (Latimer, 1952). Throughout 11 wk of exposure to 0.2 ppm O3, all the enzyme activities examined in the peroxidative metabolic pathway (PMP) and glycolytic pathway (CP) were retained at the elevated levels of the 1-wk exposure. By 12 wk of exposure to 0.4-4.0 ppm NO2, however, only G6PDH activity responded at 4.0 ppm, although both G6PDH and PK activities were 20-30% enhanced over the controls at 1 wk of
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TABLE 5. Changes in the Specific Activities of Glucose-6-phosphate Dehydrogenase (C6PDH), Pyruvate Kinase (PK), and the Population of Alveolar Macrophages by 12 Weeks of Exposures to 0.4, 1.2, and 4.0 ppm NO 2 Parameter
NO 2
Level
Protein 6
Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm
0.91 0.95 1.02 0.97 210 210 208 232 2200 2380 2240 2490 12.0 14.2 15.2 15.6
C6PDHC
PKC
Population*7
E/Ca + ± ± ± ± ± + ± ± ± ± ± ± ± ± ±
0.09 0.04 0.09 0.11 15 25 10 11 e 240 190 204 370 2.3 3.0 2.1 e 2.1 e
1.04 1.12 1.06 1.00 0.99 1.10 1.08 1.02 1.13 1.18 1.27 1.30
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposure group to control. 6 l n mg supernatant protein/macrophage preparation (mean ± SD, n - 6). c ln pmol/min/g supernatant protein (mean ± SD, n - 6). Population x 106 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposure and control groups.
exposure to 4.0 ppm NO2 (Mochitate et al., 1986). In contrast to the exposure to O3, the enzyme activities in both pathways did not stay at the elevated levels in the case of long-term exposures to NO2. The difference in the responses of the enzyme activities in PMP and GP may reflect the difference in adaptation processes of alveolar macrophages to O3 versus NO2. As indicator enzymes of the peroxidative metabolism and energy-generation, we chose G6PDH and GPx of PMP and PK, HK, and LDH of GP for their high susceptibilities to O3 and NO2 (Mochitate et al., 1985, 1986; Mochitate and Miura, 1989). GPx reduces peroxides, consuming reduced glutathione and NADPH. Glutathione is reduced with NADPH by glutathione reductase, and NADPH is mainly supplied through G6PDH. PK and HK are the key enzymes of GP (Minakami and Yoshikawa, 1966), through which the energy is supplied mainly in alveolar macrophages (Karnovsky et al., 1970). Because the specific activities of these enzymes increased during long-term exposure to O3, the capacities of peroxidative metabolism and energy generation appear to have been elevated in alveolar macrophages throughout the exposure period. The enhancement of both metabolic pathways
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may be necessary for alveolar macrophages to repair damages and adapt themselves to O3. In the case of long-term exposures to NO2, however, both metabolic pathways appear not to be enhanced. The difference in the responses of both metabolic pathways to O3 versus NO2 may come from the difference in the oxidation-reduction potentials of O3 and NO2 (Latimer, 1952). As the protein synthesis per cell did not alter, the enhancement of both pathways was a specific response to O3. The increase in the population of small-sized macrophages during the long-term exposure to 0.2 ppm O3 was not necessarily dependent on enhancement of DNA synthesis. At 1 wk of exposure to 0.2 ppm O3 or 4.0 ppm NO2, the DNA synthesis per cell was temporarily enhanced and then small-sized macrophages increased (Mochitate et al., 1986; Mochitate and Miura, 1989). During the subsequent period of exposure to ozone, however, the DNA synthesis was persistently 20-30% lower than in the controls. The level of alveolar macrophages in rats and mice has been considered to depend on proliferation of resident mononuclear phagocytes as well as on monocyte influx from circulation (Shellito et al., 1987; van Furth and van oud Alblas, 1983). As the total incorporation of thymidine was kept unchanged, an enhancement of monocyte influx may account for the increase of small macrophages in alveoli. As the population of polymorphonuclear leukocytes did not increase at all, they do not appear to be associated with the increase in alveolar macrophages. The populational increase in alveolar macrophages was independent of enhancement of the enzyme activities in PMP and GP. The population of alveolar macrophages similarly increased 30% for 1-wk exposures to 0.1 ppm O3 and 4.0 ppm NO2. However, the extent of enhancement in the G6PDH and PK activities was 40-50% at 0.1 ppm O3, but 20-30% maximum on d 4 for 4.0 ppm NO2 (Mochitate et al., 1986). In the case of long-term exposure to 4.0 ppm NO2, the population of alveolar macrophages was retained at the elevated level of the 1-wk exposure throughout the exposure period, in spite of the subsequent reduction in the G6PDH and PK activities. Therefore, the enhancement of the enzyme activities in PMP and GP may be regulated by another mechanism different from that of the populational increase in alveolar macrophages. It is quite interesting to consider the relationship between the damages of bronchioloalveolar epithelial cells and the induction of small macrophages by O3 and NO2. One of the main functions of alveolar macrophages has been considered to be to eliminate the inflammatory agents and the necrotic tissue through phagocytosis (Rabinovitch, 1970). Exposures to O3 and NO2 are shown to impair type I alveolar epithelial cells around the terminal bronchioloalveolar duct region and proximal alveoli, followed by necrosis and desquamation of these cells.
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Alveolar macrophages tend to make clusters or aggregates on the type I alveolar cells around the openings of alveoli where the impairment is most severe (Evans et al., 1976; Plopper et al., 1973; Schwartz et al., 1976; Stephens et al., 1974a, 1974b). After experimental animals are returned to clean air, the damages to the type I alveolar cells soon disappear and the clusters of alveolar macrophages diminish (Plopper et al., 1978). Therefore, under the present conditions of exposure bronchioloalveolar epithelial cells were quite likely to be damaged to necrosis (Barry et al., 1985; Boorman et al., 1980; Castleman et al., 1977; Terada et al., 1981), which may have recruited immature macrophages. In summary, alveolar macrophages adapt themselves to the longterm exposures to O3 and NO2 by recruiting immature macrophages without enhancement of DNA synthesis. The recruitment of immature macrophages by an apparent influx of monocytes may be associated with the damages of bronchioloalveolar epithelial cells rather than with the infiltration of polymorphonuclear leukocytes. The enhancement of the peroxidative metabolic and glycolytic pathways is apparently induced depending on the oxidation-reduction potentials of the pollutants. REFERENCES Barry, B. E., Miller, F.J., and Crapo, J. D. 1985. Effects of inhalation of 0.12 and 0.25 parts per million ozone on the proximal alveolar region of juvenile and adult rats. Lab. Invest. 53:692-704. Bergmeyer, H. U., Gawehn, K., and Grassl, M. 1974. Enzymes as biochemical reagent. In Methods of Enzymatic Analysis, ed. H. U. Bergmeyer, vol. 1, p. 473, and vol. 2, p. 574. New York: Academic Press. Boorman, G. A., Schwartz, L. W., and Dungworth, D. L. 1980. Pulmonary effects of prolonged ozone insult in rats. Lab. Invest. 43:108-115. Castleman, W. L., Tyler, W. S., and Dungworth, D. L. 1977. Lesions in respiratory bronchioles and conducting airways of monkeys exposed to ambient levels of ozone. Exp. Mol. Pathol. 26:384400. Chiu, D. T. Y., Stults, F. H., and Tappel, A. L. 1976. Purification and properties of rat lung soluble glutathione peroxidase. Biochim. Biophys. Acta 445:558-566. Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. 1968. Influence of ozone on pulmonary cell population. Arch. Environ. Health 16:633-636. Devlin, R. B., McDonnell, W. F., Mann, R., Becker, S., House, D. E., Schreinemachers, D., and Koren, H. S. 1991. Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung. Am. J. Respir. Cell Mol. Biol. 4:72-81. Driscoll, K. E., Vollmuth, T. A., and Schlesinger, R. B. 1987. Acute and subchronic ozone inhalation in the rabbit: Response of alveolar macrophages. J.Toxicol. Environ. Health 21:27-43. Eschenroeder, A. Q. 1977. Atmospheric concentrations of photochemical oxidants. In Ozone and Other Photochemical Oxidants, ed. Committee on Medical and Biologic Effects of Environmental Pollutants, Division of Medical Sciences Assembly of Life Sciences National Research Council, pp. 126-194. Washington, DC: National Academy of Sciences. Evans, M. J., Cabral, L. J., Stephens, R. J., and Freeman, G. 1973. Renewal of alveolar epithelium in the rat following exposure to NO 2 . Am. J. Pathol. 70:175-198. Evans, M. J., Cabral, L. J. Stephens, R. J., and Freeman, G. 1975. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO 2 . Exp. Mol. Pathol. 22:142-150.
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Evans M.J., Dekker, N. P., Cabral, A. L., and Shami, S. G. 1985. Morphological basis of tolerance to ozone. Exp. Mol. Pathol. 42:366-376. Evans, M. J., Johnson, L. V., Stephens, R. J., and Freeman, C. 1976a. Cell renewal in the lungs of rats exposed to low levels of ozone. Exp. Mol. Pathol. 24:70-83. Evans, M. J., Johnson, L. V., Stephens, R. J., and Freeman, G. 1976b. Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO 2 or O 3 . Lab. Invest. 35:246-257. Evans, M. J., Shami, S. G., Cabral-Anderson, L. J., and Dekker, N. P. 1986. Role of nonciliated cells in renewal of the bronchial epithelium of rats exposed to NO 2 . Am. J. Pathol. 123:126-133. Garnder, D. E. 1984. Alterations in macrophage functions by environmental chemicals. Environ. Health Perspect. 55:343-358. Goldstein, E., Bartlema, H. C., van der Ploeg, M., van Duijn, P., van der Stap, J. G. M. M., and Lippert, W. 1978. Effect of ozone on lysosomal enzymes of alveolar macrophages engaged in phagocytosis and killing of inhaled Staphylococcus aureus. J. Infect. Dis. 138:299-311. Hurst, D. J., Gardner, D. E., and Coffin, D. L. 1970. Effect of ozone on acid hydrolases of the pulmonary alveolar macrophages. J. Reticuloendothel. Soc. 8:288-300. Karnovsky, M. L., Simmons, S., Glass, E. A., Shafer, A. W., and Hart, P. D'A. 1970. Metabolism of macrophages. In Mononuclear Phagocytes, ed. R. van Furth, pp. 103-120. The Hague, Netherlands: Martinus Nijhoff. Kimura, A., and Goldstein, E. 1981. Effect of ozone on concentrations of lysozyme in phagocytizing alveolar macrophages. J. Infect. Dis. 143:247-251. Latimer, W. M. 1952. Oxidation Potentials. New York: Prentice Hall. Löhr, G. W., and Waller, H. D. 1974. Glucose-6-phosphate dehydrogenase. In Methods of Enzymatic Analysis, ed. H. U. Bergmeyer, vol. 2, pp. 636-643. New York: Academic Press. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chew. 193:265-273. Minakami, S., and Yoshikawa, H. 1966. Studies on erythrocyte glycolysis. II. Free energy changes and rate limiting steps in erythrocyte glycolysis. J. Biochem. (Tokyo) 59:139-145. Mochitate, K., and Miura, T. 1989. Metabolic enhancement and increase of alveolar macrophages induced by ozone. Environ. Res. 49:79-92. Mochitate, K., Miura, T., and Kubota, K. 1985. An increase in the activities of glycolytic enzymes in rat lungs produced by nitrogen dioxide. J. Toxicol. Environ. Health 15:323-331. Mochitate, K., Takahashi, Y., Ohsumi, X, and Miura, T. 1986. Activation and increment of alveolar macrophages induced by nitrogen dioxide. J. Toxicol. Environ. Health 17:229-239. Myrvik, Q. N., Leake, E. S., and Fariss, B. 1961. Studies on pulmonary alveolar macrophages from the normal rabbit: A technique to produce them in a high state of purity. J. Immunol. 86:128132. Parker, R. F., Davis, J. K., Cassell, G. H., White, H., Dziedzic, D., Blalock, D. K., Thorp, R. B., Simecka, J. W. 1989. Short-term exposure to nitrogen dioxide enhances susceptibility to murine respiratory mycoplasmosis and decreases intrapulmonary killing of Mycoplasma pulmonis. Am. Rev. Respir. Dis. 140:502-512. Parkinson, D. R., and Stephens, R. J. 1973. Morphological surface changes in the terminal bronchiolar region of NO2-exposed rat lung. Environ. Res. 6:37-51. Plopper, C. G., Chow, C. K., Dungworth, D. L., Brummer, M., and Nemeth, T. J. 1978. Effect of low level of ozone on rat lungs. II. Morphological responses during recovery and re-exposure. Exp. Mol. Pathol. 29:400-411. Plopper, C. G., Dungworth, D. L., and Tyler, W. S. 1973. Pulmonary lesions in rats exposed to ozone. A correlated light and electron microscopic study. Am. J. Pathol. 71:375-394. Rabinovitch, M. 1970. Phagocytic recognition. In Mononuclear Phagocytes, ed. R. van Furth, pp. 299-315. Oxford: Blackwell Press. Schlesinger, R. B. 1989. Comparative toxicity of ambient air pollutants: Some aspects related to lung defense. Environ. Health Perspect. 81:123-128. Schwartz, L. W., Dungworth, D. L, Mustafa, M. G., Tarkington, B. K., and Tyler, W. S. 1976. Pulmonary responses of rats to ambient levels of ozone. Effects of 7-day intermittent or continuous exposure. Lab. Invest. 34:565-578.
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Shellito, J., Esparza, C , and Armstrong, C. 1987. Maintenance of the normal rat alveolar macrophage cell population. The roles of monocyte influx and alveolar macrophage proliferation in situ. Am. Rev. Respir. Dis. 135:78-82. Stephens, R. J., Freeman, G., and Evans, M. J. 1972. Early response of lungs to levels of nitrogen dioxide. Light and electron microscopy. Arch. Environ. Health 24:160-179. Stephens, R. J., Sloan, M. F., Evans, M. J., and Freeman, G. 1974a. Early response of lung to low levels of ozone. Am. J. Pathol. 74:31-58. Stephens, R. J., Sloan, M. F., Evans, M. J., and Freeman, C. 1974b. Alveolar type 1 cell response to exposure to 0.5 ppm O 3 for short period. Exp. Mol. Pathol. 20:11-23. Terada, N., Suzuki, T., Ohsawa, M., Fukuda, M., Mizoguchi, I., and Kawai, K. 1981. Pathological changes in the lungs of rats exposed to nitrogen dioxide. Annu. Rep. Tokyo Metr. Res. Lab. 321:250-256. van Furth, R., and van oud Alblas, A. B. 1983. Origin of pulmonary macrophages in mice under normal conditions and during an inflammatory reaction by heat-killed BCC. In The Cells of the Alveolar Unit, eds. G. Favez, A. Junod, and P. Leuenberger, pp. 170-179. Bern, Switzerland: Hans Huber Press. Received April 11, 1990 Accepted December 2, 1997
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Long‐term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages Katsumi Mochitate , Kunihiko Ishida , Takumi Ohsumi & Takashi Miura To cite this article: Katsumi Mochitate , Kunihiko Ishida , Takumi Ohsumi & Takashi Miura (1992) Long‐term effects of ozone and nitrogen dioxide on the metabolism and population of alveolar macrophages, Journal of Toxicology and Environmental Health, 35:4, 247-260, DOI: 10.1080/15287399209531615 To link to this article: http://dx.doi.org/10.1080/15287399209531615
Published online: 20 Oct 2009.
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LONG-TERM EFFECTS OF OZONE AND NITROGEN DIOXIDE O N THE METABOLISM AND POPULATION OF ALVEOLAR MACROPHAGES
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Katsumi Mochitate, Kunihiko Ishida, Takumi Ohsumi Environmental Health Sciences Division, National Institute for Environmental Studies, Ibaraki, Japan Takashi Miura Regional Environmental Division, National Institute for Environmental Studies, Ibaraki, Japan
To investigate how alveolar macrophages adapt themselves to oxidative pollutants in the long term, rats were exposed to a strong oxidant, ozone (O3), or a weak oxidant, nitrogen dioxide (NO2), for a maximum duration of 12 wk. After exposures, alveolar macrophages were collected by pulmonary lavage. Throughout 11 wk of exposure to 0.2 ppm O3r the specific activities of glucose-6phosphate dehydrogenase (G6PDH) and glutathione peroxidase of the peroxidative metabolic pathway and pyruvate kinase and hexokinase of the glycolytic pathway were 40-70% elevated over the controls in alveolar macrophages. The population of alveolar macrophages was consistently 60% higher than the controls. The small-sized macrophages, immature macrophages, preferentially increased. To the contrary, the thymidine incorporation per cell was always 20-30% lower than in the controls, although the total incorporation remained unchanged. No infiltration of polymorphonuclear leukocytes occurred. By 12 wk of exposures to 1.2 and 4.0 ppm NO2, the population of alveolar macrophages increased 30% over the control. Among the enzymes examined, however, only the C6PDH activity increased 10% for 4.0 ppm NO2. No increase in the enzyme activities occurred for 1.2 ppm NO2. Based on these results, alveolar macrophages adapt themselves to the long-term exposure of O3 or NO2 by recruiting immature macrophages through an apparent influx of monocytes. During the exposure to O3, the peroxidative metabolic and glycolytic pathways are enhanced persistently in alveolar macrophages, whereas both pathways were not enhanced by the exposures to NO2
The authors thank Yuko Katahoka for her technical assistance. Requests for reprints should be sent to Katsumi Mochitate, Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305, Japan.
247 Journal of Toxicology and Environmental Health, 35:247-260, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
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INTRODUCTION Ozone (O3) and nitrogen dioxide (NOJ are major components of oxidative pollutants and are found in urban and industrial atmospheres (Eschenroeder, 1977). Both gases impair the pulmonary epithelial cells in a similar fashion. Type I alveolar epithelial cells and ciliated cells at terminal bronchiole are the most sensitive to these gases and are easily damaged, leading to necrosis and desquamation (Parkinson and Stephens, 1973; Schwartz et al., 1976; Stephens et al., 1972, 1974a, 1974b). In contrast, type 2 alveolar epithelial cells and Clara cells appear to be tolerant. When impaired, type 1 epithelial cells and ciliated cells are soon replaced by new ones derived from their progenitors, such as type 2 alveolar cells and Clara cells (Evans et al., 1973, 1975, 1976a, 1976b, 1986), respectively. Through these processes, the sensitive cells seem to become tolerant to O3 and NO2 (Evans et al., 1985). Ozone and NO2 also impair alveolar macrophages (Gardner, 1984; Schlesinger, 1989). Their functions such as bactericidal activity, phagocytosis (Coffin et al., 1968; Devlin et al., 1991; Driscoll et al., 1987; Goldstein et al, 1978; Parker et al., 1989) and lysosomal hydrolysis (Goldstein et al., 1978; Hurst et al., 1970; Kimura and Goldstein, 1981) are damaged by these gases. However, it is not clear how alveolar macrophages repair damages and adapt to O3 and NO2. We have investigated the metabolic and populational responses of alveolar macrophages to 0.2 ppm O3 or 4.0 ppm NO2 for a maximum duration of 2 wk (Mochitate and Miura, 1989; Mochitate et al., 1986) to clarify their processes for adaptation. When exposed to 0.2 ppm O3, alveolar macrophages appear to repair damages through the persistent enhancement of the peroxidative metabolic and glycolytic pathways. When exposed to 4.0 ppm NO2, however, both metabolic pathways temporarily increase and then reduce to the control levels. The population of immature macrophages is elevated consistently during both exposures, following the temporary enhancement of DNA synthesis. Therefore, to clarify the adaptation processes it is necessary to examine whether alveolar macrophages persistently enhance the peroxidative metabolic and glycolytic pathways or fluctuate between the elevated and control levels during long-term exposure to O3 or NO2. It is also of interest to investigate whether alveolar macrophages repeat the temporary enhancement of DNA synthesis and maintain the elevated level of immature macrophages. In this report, we show that the peroxidative metabolic and glycolytic pathways in alveolar macrophages were enhanced persistently during long-term exposure to 0.2 ppm O3, whereas no enhancement occurred with exposures to 1.2 and 4.0 ppm NO2. Immature macrophages, however, increased to a similar extent without enhancement of DNA synthesis by these exposures.
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MATERIALS AND METHODS
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Materials
The following reagents were purchased from Boehringer Mannheim Yamanouchi Co. (Tokyo, Japan): phosphoenolpyruvate, glucose-6phosphate, pyruvate, fructose-1,6-bisphosphate, reduced glutathione, ATP, ADP, NADH, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase. We also obtained NADP+, NADPH, and glutathione reductase from Oriental Yeast Co. (Tokyo, Japan); HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid) from Dojindo Laboratories (Kumamoto, Japan); Eagle's minimum essential medium from Nissui Seiyaku Co. (Tokyo, Japan); and fetal bovine serum from Flow Laboratories Inc. (McLean, Va.). [Methyl-14C]thymidine (61 mCi/ mmol) and L-[3,4,5-3H(N)]leucine (152 Ci/mmol) were the products of Amersham (Buckinghamshire, England) and New England Nuclear (Boston, Mass.), respectively. Animals
Male Jcl: Wistar rats (specific-pathogen-free) at 10 wk of age were obtained from CLEA-Japan Co. (Tokyo, Japan). Until exposures, rats were fed a sterilized rodent diet (CE-2, CLEA-Japan Co.) and water ad libitum and kept specific-pathogen-free (SPF). Exposure Conditions
In the 1-wk exposure to O3, each group of rats (16 wk old, 6 rats/ group) was continuously exposed to 0.10 ± 0.01 or 0.20 ± 0.01 ppm O3 in exposure chambers of the same model, which were controlled to 25°C and 55-60% relative humidity throughout the exposures. In longterm exposures, each group (21 wk old, six rats/group) was exposed to 0.20 ppm O3 for 6-11 wk or 0.4, 1.2, and 4.0 ppm NO2 for 12 wk in the same way. As the controls, an equal number of rats was allowed to breathe only filtered clean air in another identical chamber at the same time. After exposures, rats were supplied to the following experiments. No significant differences of body weights and wet weights of lung tissues were observed between the exposed and control groups. The SPF status was maintained throughout the exposure periods, and respiratory diseases did not occur in any rats used. Preparation of Alveolar Macrophages
Alveolar macrophages were prepared according to Myrvik et al. (1961), with slight modifications (Mochitate and Miura, 1989; Mochitate et al., 1986). After exposures, rats were anesthetized with sodium pentobarbital injected intraperitoneally and were exsanguinated through the carotid artery. The lungs were then perfused thoroughly with cold 0.95% NaCI solution injected from the right ventricle. After perfusion,
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the lungs were removed from the thorax and lavaged by intratracheal instillation with a total of 35 ml (first 15 ml, second and third 10 ml each) of isotonic HEPES buffer, which consisted of 136 mM NaCI, 5.3 mW KCI, 2.5 mM sodium phosphate buffer, 5.5 mM glucose, and 10 mM HEPES, adjusted to pH 7.4. Alveolar free cells were recovered from the lavage fluid by centrifugation at 220 x g for 10 min and washed with HEPES buffer. The viability was determined by the trypan blue exclusion test. The viabilities of alveolar macrophages were always larger than 89% in both exposed and control groups and were not affected by the exposures. The population of lavaged-out cells was measured with a Coulter counter (type Industrial D, Coulter Electronics Ltd., Luton Beds, England), which was calibrated with latex microspheres of 10 /jm in diameter (6602796, Coulter Electronics Ltd.) and adjusted to count particles larger than 9 /*m in diameter. The adjusted counter yielded the same result as counting macrophages with a hematocytometer. Each population of alveolar macrophages differing in size was determined by successively changing the adjustment in the counter to count cells larger than 9, 11, 13, 15, 17, 19, and 21 /xm in diameter and calculating the differences in population between nearest diameters. The composition of alveolar free cells collected by pulmonary lavage was determined by Giemsa staining of smear preparations. More than 96% of lavaged-out cells were always macrophages. Therefore, the lavaged cells are hereafter referred to as alveolar macrophages. Homogenization of Alveolar Macrophages
Half of each alveolar macrophage preparation was suspended to 1.00 ml in cold 0.25 M sucrose solution containing 10 mM Tris-HCl (pH 7.4) and 0.5 mM EDTA and homogenized in a tapered Potter-Elvehjem Teflon homogenizer (358133, Wheaton Scientific, Millville, N.J.) on an ice bath. The homogenates were centrifuged at 105,000 x g for 60 min at 4°C. The supernatants were recovered for the measurements of protein concentration and enzyme activities. Assays of Enzyme Activities
The enzyme activities of glucose-6-phosphate dehydrogenase and pyruvate kinase were assayed at 30°C as described by Lohr and Waller (1974) and Mochitate and Miura (1989), respectively. The glutathione peroxidase activity was determined at 37°C according to Chiu et al. (1976), with slight modifications (Mochitate and Miura, 1989). The assay mixture consisted of 100 mM Tris-HCl (pH 7.6), 2.7 mM reduced glutathione, 0.25 mM cumene hydroperoxide, 0.2 mM NADPH, 0.1 mM EDTA, and 2 units/ml of glutathione reductase. The activities of lactate dehydrogenase and hexokinase were measured at 30°C as described by Bergmeyer et al. (1974).
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The protein concentration was determined by the method of Lowry et al. (1951).
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Incorporation of [14C]Thymidine and [3H]Leucine into Alveolar Macrophages
The remaining portion of each alveolar macrophage preparation was used for the experiment of thymidine and leucine incorporations as described before (Mochitate and Miura, 1989; Mochitate et al., 1986). Alveolar macrophages (2.0 x 106 cells) were suspended in 4.0 ml of Eagle's minimum essential medium supplemented with 10% fetal calf serum, 50 nCi/ml of [14C]thymidine, and 1 /iCi/ml of [3H]leucine and incubated in a humidified 5% CO2 atmosphere at 37°C for 3.5 h. After incubation, the alveolar macrophages were recovered with a rubber policeman. The free radioactive thymidine and leucine were removed by washing the recovered macrophages three times with Ca2+, Mg 2+ free Dulbecco's phosphate-buffered saline supplemented with 1% fetal calf serum. The radiolabeled macrophages were then mixed with scintillator (Aquasol-2, New England Nuclear) and the incorporated radioactivities were counted with a scintillation counter (model 3255, Packard Instrument Co., Downers Grove, III.). Statistical Analysis
Analyses of significant differences between exposure and control groups were performed by means of Student's t test or Welch's t test after the analysis of variance. RESULTS Short-Term Effects of Ozone on the Enzyme Activities and Population of Alveolar Macrophages
To examine whether the metabolism involved in clearance of peroxidates and ATP-production in alveolar macrophages responds to an ambient level of ozone, rats were exposed to 0.1 and 0.2 ppm O3 for 1 wk. The specific activities of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione peroxidase (GPx) in the peroxidative metabolic pathway (PMP) and of pyruvate kinase (PK), lactate dehydrogenase (LDH), and hexokinase (HK) in the glycolytic pathway (GP) increased 20-70% over the controls by 0.2 ppm O3 (Table 1). By 0.1 ppm O3, the key enzyme activities in both pathways were also 30-50% enhanced. The population of alveolar macrophages increased in a dosedependent manner (Table 1 and Fig. 16). The smaller-sized macrophages increased preferentially (Fig. 1a). With exposure to 0.2 ppm O3, for example, the macrophages of 9-11, 11-13, 13-15, and 15-17 jim in diameter increased 120, 90, 60, and 20% over the controls, respectively.
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TABLE 1. Changes in the Specific Activities of Glucose-6-Phosphate Dehydrogenase (G6PDH), Glutathione Peroxidase (GPx), Pyruvate Kinase (PK), Lactate Dehydrogenase (LDH), and Hexokinase (HK) and the Population of Alveolar Macrophages at 1 Week of Exposure to Ozone Parameter
Control
Protein 6 G6PDHC GPxc PKC LDHC HKC Population^
0.86 169 2130 2040 940 81 11.2
± ± ± ± + ± ±
0.06 17 230 260 80 8 0.8
0.1 ppm
E/Ca
0.2 ppm
E/Ca
1.04 248 3050 2850 1050 108 14.8
1.21 1.47 1.44 1.39 1.12 1.34 1.33
1.15 287 3260 3330 1100 112 17.5
1.34 1.69 1.53 1.63 1.17 1.39 1.56
± ± ± ± ± ± ±
0.05'* 14' 340' 170' 110 8' 1.4'*
± ± ± ± ± ± ±
0.04'* 24' 120' 450' 110e 6' 1.4'*
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposed group to control. 6 l n mg supernatant protein/macrophage preparation (mean ± SD, n — 6). c ln ftmol/min/g supernatant protein (mean ± SD, n — 6). ^Population x 106 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposed and control groups. 'Significant at p < .001 between exposed and control groups. Significant at p < .05 between 0.1 and 0.2 ppm O 3 groups.
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FIGURE 1. (a) Changes in the size distribution of alveolar macrophages by in vivo exposures of rats to 0.1 ppm and 0.2 ppm O 3 for 1 wk. Each population of alveolar macrophages in different size was determined by successively changing the adjustment in Coulter counter to count cells larger than 9, 11, 13, 15, 17, 19, and 21 ^m in diameter and calculating the differences in population between nearest diameters. Abscissa indicates the cell size: 7-9, 9-11,11-13,13-15,15-17,17-19, and 19-21 jim in diameter. Open, dark-shaded, and light-shaded columns show the population in the control and 0.1 and 0.2 ppm O 3 groups, respectively. Values are expressed as mean ± SD (n 6). Asterisk indicates significant at p < .05 between exposed and control groups; double asterisk, significant at p < .01; and triple asterisk, significant at p < .001. (6) Changes in the whole population of alveolar macrophages by 1 wk of exposures to 0.1 and 0.2 ppm O 3 . The alveolar free cells larger than 9 /»m in diameter were counted as macrophages.
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TABLE 2. Changes in the Specific Activities of Clucose-6-Phosphate Dehydrogenase (G6PDH), Glutathione Peroxidase (GPx), Pyruvate Kinase (PK), Lactate Dehydrogenase (LDH), and Hexokinase (HK) and the Population of Alveolar Macrophages over 11 Weeks of Exposure to 0.2 ppm Ozone
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Exposure time Parameter
Group
6wk
Protein 6
Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed Control Exposed
0.92 1.38 214 327 2280 3650 2520 3260 930 1210 86 115 9.9 16.7
G6PDHC GPxc PKC LDHC HKC Population 0 '
E/Ca ± 0.07 ± 0.16s ± 15 ± 43' ± 300 ± 250 s ± 300 ± 110' ±160 ± 60' ± 8 ± 18e ± 0.9 ± 3.0'
1.51 1.52 1.60 1.30 1.31 1.33 1.69
11 wk 0.90 1.42 197 303 2060 3490 2310 3070 860 1260 85 107 12.2 19.2
± 0.05 ± 0.12s ± 15 ± 18 s ± 140 ± 90 s ± 190 ± 120 s ± 110 ± 70 s ± 5 ± 5s ±1.4 ± 1.3s
E/Ca
1.58 1.54 1.69 1.33 1.47 1.25 1.57
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposed group to control. b l n mg supernatant protein/macrophage preparation (mean ± SD, n — 6). c ln /*mol/min/g supernatant protein (mean ± SD, n — 6). ^Population x 10 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposed and control groups. 'Significant at p < .01. ^Significant at p < .001.
With exposure to 0.1 ppm O3, the small macrophages similarly increased. Long-Term Effects of Ozone on Alveolar Macrophages
For 11 wk of exposure to 0.2 ppm O3, all the enzyme activities examined and the population of alveolar macrophages were elevated to the same levels as for the 1-wk exposure (Table 2). The small-sized macrophages preferentially increased in the same way as for the 1-wk exposure (Fig. 2). Therefore, the elevated levels both of the enzyme activities and of the population of small-sized macrophages were retained throughout the exposure period. To examine populational changes in other kinds of alveolar free cells, differential cell counts were performed on Giemsa-stained smears (Table 3). Alveolar macrophages always accounted for more than 96% of the whole cells in both exposure and control groups. Although lympho-
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254 8 7 |
6
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= 5
ow-9
,
It 13 15 17 19 21 "9 II 13 15 17 19 21 Cell Size (/im)
FIGURE 2. Changes in the size distribution of alveolar macrophages during 11 wk of exposure to 0.2 ppm O3: (a, b) exposure periods of 6 and 11 wk, respectively.
cytes were inclined to increase, polymorphonuclear leukocytes did not increase during the whole period of exposure. Next, to examine if protein and DNA synthesis in alveolar macrophages corresponded to the increase in population, alveolar macrophages were incubated with [3H]leucine and [14C]thymidine for 3.5 h (Table 4). Although the enzyme activities of the PMP and GP examined were all enhanced by the exposure to 0.2 ppm O3, the protein synthesis per cell showed no increase. Contrary to the populational increase in small-sized macrophages, the DNA synthesis per cell was 20-30% lower than in the controls during the exposure, whereas the total incorporaTABLE 3. Effects of 0.2 ppm Ozone on the Composition of Alveolar Free Cells Recovered by Alveolar Lavage Composition (%f Exposure time
Group
AM3>>
1 wk
Control Exposed Control Exposed Control Exposed
97.3 96.1 98.0 97.3 98.3 96.6
6wk 11 wk
± ± ± + ± ±
Lymc 1.4 2.0 1.0 1.3 1.0 0.6'
1.7 3.2 1.0 1.7 1.5 2.7
± ± ± ± ± ±
1.0 1.5 0.8 1.3 0.9 0.5e
PMN d
Degenerated
0.5 ± 0.5 0.3 ± 0.3 0.0 0.0 0.0 0.1 ± 0.1
0.5 0.5 1.0 1.0 0.2 0.6
Note. The composition of alveolar free cells was determined by Giemsa staining. Values were expressed as mean ± SD (n - 6). ^Alveolar macrophage. lymphocyte. ''Polymorphonuclear leukocyte. Significant at p < .05 between exposed and control groups. 'Significant at p < .01. a
± ± ± ± ± ±
0.5 0.6 1.0 0.9 0.2 0.5
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TABLE 4. Changes in Incorporation of [ 3 H]Leucine and [ 14 C]Thymidine into Alveolar Macrophages over 11 Weeks of Exposure to 0.2 ppm Ozone Incorporation (dpm/2 X 105 cells) Exposure time
Group
[ 3 H]Leucine a
1 wk
Control Exposed Control Exposed Control Exposed
37,500 36,800 45,300 48,300 35,500 41,100
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6wk 11 wk
± ± ± ± ± ±
4400 3500 6900 4200 7900 4000
E/Cb
0.98 1.07 1.16
[ 14 C]Thymidine a 4760 3360 5640 3950 4180 3520
± ± ± ± ± +
810 580c 600 810c 720 570
E/Cfa
0.71 0.70 0.84
Note. Alveolar macrophages (2 x 106 cells) were suspended in 4.0 ml of Eagle's minimum essential medium supplemented with 10% fetal bovine serum, [ 3 H]leucine (1 /»Ci/ml), and [ 14 C]thymidine (50 nCi/ml) and incubated at 37°C for 3.5 h. a Values were expressed as mean ± SD (n - 6). b Ratio of exposed group to control. Significant at p < .05 between exposed and control groups.
tion of thymidine remained unchanged. Therefore, the increase in the population of alveolar macrophages may be due to an influx of small macrophages. Long-Term Effects of Nitrogen Dioxide on the Enzyme Activities and Population of Alveolar Macrophages
To compare long-term effects of ozone with those of nitrogen dioxide, rats were exposed to 0.4,1.2, and 4.0 ppm NO2 for 12 wk. G6PDH and PK were chosen as indicator enzymes for their high susceptibilities to O3 and NO2. The specific activity of G6PDH increased only for exposure to 4.0 ppm NO2, while the PK activity did not change (Table 5). The population of alveolar macrophages, however, increased 30% over the control for exposures to 1.2 and 4.0 ppm NO2. Therefore, alveolar macrophages increased to the same degree as for 1-wk exposure to 0.1 ppm O3, without enhancement of the peroxidative metabolic and glycolytic pathways. DISCUSSION There were differences in the responses of alveolar macrophages to the long-term exposures to a strong oxidant, O3, and a weak oxidant, NO2 (Latimer, 1952). Throughout 11 wk of exposure to 0.2 ppm O3, all the enzyme activities examined in the peroxidative metabolic pathway (PMP) and glycolytic pathway (CP) were retained at the elevated levels of the 1-wk exposure. By 12 wk of exposure to 0.4-4.0 ppm NO2, however, only G6PDH activity responded at 4.0 ppm, although both G6PDH and PK activities were 20-30% enhanced over the controls at 1 wk of
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TABLE 5. Changes in the Specific Activities of Glucose-6-phosphate Dehydrogenase (C6PDH), Pyruvate Kinase (PK), and the Population of Alveolar Macrophages by 12 Weeks of Exposures to 0.4, 1.2, and 4.0 ppm NO 2 Parameter
NO 2
Level
Protein 6
Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm Control 0.4 ppm 1.2 ppm 4.0 ppm
0.91 0.95 1.02 0.97 210 210 208 232 2200 2380 2240 2490 12.0 14.2 15.2 15.6
C6PDHC
PKC
Population*7
E/Ca + ± ± ± ± ± + ± ± ± ± ± ± ± ± ±
0.09 0.04 0.09 0.11 15 25 10 11 e 240 190 204 370 2.3 3.0 2.1 e 2.1 e
1.04 1.12 1.06 1.00 0.99 1.10 1.08 1.02 1.13 1.18 1.27 1.30
Note. The preparation of macrophage supernatants and the determination of protein content and enzyme activities were performed as described in Materials and Methods. a Ratio of exposure group to control. 6 l n mg supernatant protein/macrophage preparation (mean ± SD, n - 6). c ln pmol/min/g supernatant protein (mean ± SD, n - 6). Population x 106 cells/rat lung (mean ± SD, n - 6). Significant at p < .05 between exposure and control groups.
exposure to 4.0 ppm NO2 (Mochitate et al., 1986). In contrast to the exposure to O3, the enzyme activities in both pathways did not stay at the elevated levels in the case of long-term exposures to NO2. The difference in the responses of the enzyme activities in PMP and GP may reflect the difference in adaptation processes of alveolar macrophages to O3 versus NO2. As indicator enzymes of the peroxidative metabolism and energy-generation, we chose G6PDH and GPx of PMP and PK, HK, and LDH of GP for their high susceptibilities to O3 and NO2 (Mochitate et al., 1985, 1986; Mochitate and Miura, 1989). GPx reduces peroxides, consuming reduced glutathione and NADPH. Glutathione is reduced with NADPH by glutathione reductase, and NADPH is mainly supplied through G6PDH. PK and HK are the key enzymes of GP (Minakami and Yoshikawa, 1966), through which the energy is supplied mainly in alveolar macrophages (Karnovsky et al., 1970). Because the specific activities of these enzymes increased during long-term exposure to O3, the capacities of peroxidative metabolism and energy generation appear to have been elevated in alveolar macrophages throughout the exposure period. The enhancement of both metabolic pathways
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may be necessary for alveolar macrophages to repair damages and adapt themselves to O3. In the case of long-term exposures to NO2, however, both metabolic pathways appear not to be enhanced. The difference in the responses of both metabolic pathways to O3 versus NO2 may come from the difference in the oxidation-reduction potentials of O3 and NO2 (Latimer, 1952). As the protein synthesis per cell did not alter, the enhancement of both pathways was a specific response to O3. The increase in the population of small-sized macrophages during the long-term exposure to 0.2 ppm O3 was not necessarily dependent on enhancement of DNA synthesis. At 1 wk of exposure to 0.2 ppm O3 or 4.0 ppm NO2, the DNA synthesis per cell was temporarily enhanced and then small-sized macrophages increased (Mochitate et al., 1986; Mochitate and Miura, 1989). During the subsequent period of exposure to ozone, however, the DNA synthesis was persistently 20-30% lower than in the controls. The level of alveolar macrophages in rats and mice has been considered to depend on proliferation of resident mononuclear phagocytes as well as on monocyte influx from circulation (Shellito et al., 1987; van Furth and van oud Alblas, 1983). As the total incorporation of thymidine was kept unchanged, an enhancement of monocyte influx may account for the increase of small macrophages in alveoli. As the population of polymorphonuclear leukocytes did not increase at all, they do not appear to be associated with the increase in alveolar macrophages. The populational increase in alveolar macrophages was independent of enhancement of the enzyme activities in PMP and GP. The population of alveolar macrophages similarly increased 30% for 1-wk exposures to 0.1 ppm O3 and 4.0 ppm NO2. However, the extent of enhancement in the G6PDH and PK activities was 40-50% at 0.1 ppm O3, but 20-30% maximum on d 4 for 4.0 ppm NO2 (Mochitate et al., 1986). In the case of long-term exposure to 4.0 ppm NO2, the population of alveolar macrophages was retained at the elevated level of the 1-wk exposure throughout the exposure period, in spite of the subsequent reduction in the G6PDH and PK activities. Therefore, the enhancement of the enzyme activities in PMP and GP may be regulated by another mechanism different from that of the populational increase in alveolar macrophages. It is quite interesting to consider the relationship between the damages of bronchioloalveolar epithelial cells and the induction of small macrophages by O3 and NO2. One of the main functions of alveolar macrophages has been considered to be to eliminate the inflammatory agents and the necrotic tissue through phagocytosis (Rabinovitch, 1970). Exposures to O3 and NO2 are shown to impair type I alveolar epithelial cells around the terminal bronchioloalveolar duct region and proximal alveoli, followed by necrosis and desquamation of these cells.
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Alveolar macrophages tend to make clusters or aggregates on the type I alveolar cells around the openings of alveoli where the impairment is most severe (Evans et al., 1976; Plopper et al., 1973; Schwartz et al., 1976; Stephens et al., 1974a, 1974b). After experimental animals are returned to clean air, the damages to the type I alveolar cells soon disappear and the clusters of alveolar macrophages diminish (Plopper et al., 1978). Therefore, under the present conditions of exposure bronchioloalveolar epithelial cells were quite likely to be damaged to necrosis (Barry et al., 1985; Boorman et al., 1980; Castleman et al., 1977; Terada et al., 1981), which may have recruited immature macrophages. In summary, alveolar macrophages adapt themselves to the longterm exposures to O3 and NO2 by recruiting immature macrophages without enhancement of DNA synthesis. The recruitment of immature macrophages by an apparent influx of monocytes may be associated with the damages of bronchioloalveolar epithelial cells rather than with the infiltration of polymorphonuclear leukocytes. The enhancement of the peroxidative metabolic and glycolytic pathways is apparently induced depending on the oxidation-reduction potentials of the pollutants. REFERENCES Barry, B. E., Miller, F.J., and Crapo, J. D. 1985. Effects of inhalation of 0.12 and 0.25 parts per million ozone on the proximal alveolar region of juvenile and adult rats. Lab. Invest. 53:692-704. Bergmeyer, H. U., Gawehn, K., and Grassl, M. 1974. Enzymes as biochemical reagent. In Methods of Enzymatic Analysis, ed. H. U. Bergmeyer, vol. 1, p. 473, and vol. 2, p. 574. New York: Academic Press. Boorman, G. A., Schwartz, L. W., and Dungworth, D. L. 1980. Pulmonary effects of prolonged ozone insult in rats. Lab. Invest. 43:108-115. Castleman, W. L., Tyler, W. S., and Dungworth, D. L. 1977. Lesions in respiratory bronchioles and conducting airways of monkeys exposed to ambient levels of ozone. Exp. Mol. Pathol. 26:384400. Chiu, D. T. Y., Stults, F. H., and Tappel, A. L. 1976. Purification and properties of rat lung soluble glutathione peroxidase. Biochim. Biophys. Acta 445:558-566. Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. 1968. Influence of ozone on pulmonary cell population. Arch. Environ. Health 16:633-636. Devlin, R. B., McDonnell, W. F., Mann, R., Becker, S., House, D. E., Schreinemachers, D., and Koren, H. S. 1991. Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes in the lung. Am. J. Respir. Cell Mol. Biol. 4:72-81. Driscoll, K. E., Vollmuth, T. A., and Schlesinger, R. B. 1987. Acute and subchronic ozone inhalation in the rabbit: Response of alveolar macrophages. J.Toxicol. Environ. Health 21:27-43. Eschenroeder, A. Q. 1977. Atmospheric concentrations of photochemical oxidants. In Ozone and Other Photochemical Oxidants, ed. Committee on Medical and Biologic Effects of Environmental Pollutants, Division of Medical Sciences Assembly of Life Sciences National Research Council, pp. 126-194. Washington, DC: National Academy of Sciences. Evans, M. J., Cabral, L. J., Stephens, R. J., and Freeman, G. 1973. Renewal of alveolar epithelium in the rat following exposure to NO 2 . Am. J. Pathol. 70:175-198. Evans, M. J., Cabral, L. J. Stephens, R. J., and Freeman, G. 1975. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO 2 . Exp. Mol. Pathol. 22:142-150.
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