Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of in vivo coal dust exposure on arachidonic acid metabolism in the rat alveolar macrophage Douglas C. Kuhn , Charles F. Stanley , Nadia El‐Ayouby & Laurence M. Demers To cite this article: Douglas C. Kuhn , Charles F. Stanley , Nadia El‐Ayouby & Laurence M. Demers (1990) Effect of in vivo coal dust exposure on arachidonic acid metabolism in the rat alveolar macrophage, Journal of Toxicology and Environmental Health, 29:2, 157-168, DOI: 10.1080/15287399009531380 To link to this article: http://dx.doi.org/10.1080/15287399009531380
Published online: 15 Oct 2009.
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Article views: 2
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Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 13:00
EFFECT OF IN VIVO COAL DUST EXPOSURE O N ARACHIDONIC ACID METABOLISM IN THE RAT ALVEOLAR MACROPHAGE Douglas C. Kuhn
Downloaded by [University of Florida] at 13:01 06 November 2015
Department of Pathology, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, Pennsylvania Charles F. Stanley, Nadia El-Ayouby Department of Mechanical and Aerospace Engineering, West Virginia University, Morgantown, West Virginia Laurence M. Demers Department of Pathology, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, Pennsylvania
Oxygenated metabolites of arachidonic acid (AA) are produced by the alveolar macrophage (AM) and have been shown to mediate inflammatory reactions. We therefore assessed the production of eicosanoids by AM harvested from the lungs of rats exposed to a bituminous coal dust for 2 wk in an inhalation chamber in order to determine if AA metabolism was altered in a manner that may promote an inflammatory response in the lung. Exposure to coal dust resulted in a 66% increase in the number of AM harvested, an increase in thromboxane A2 (TxA2) and leukotriene B4 (LTB4) production to 222% and 181% of control values, respectively, and a decrease in prostaglandin E2 (PGE2) production to 62% of control values. In AM harvested from rats allowed to breath clean air for 2 wk following coal dust exposure, PCE2 production returned to control levels but TxA2 and LTB4 production remained elevated. The TxA2 synthesis inhibitor UK 38,485 reduced TxA2 production in dust-exposed AM both immediately and 2 wk following exposure. Thus, exposure of rats to coal dust significantly alters the metabolism of AA in AM, with potentially important aspects of AA metabolism remaining altered even after a 2-wk recovery period. Based on the established role of eicosanoids in inflammatory and fibrotic processes, these results suggest that the alteration of AM eicosanoid production as a result of the inhalation of coal mine dust may be an important factor in the pathophysiology of coal workers' pneumoconiosis.
The authors are grateful to Joan Greenwood and Barbara Scheetz for their excellent technical assistance and to Brenda Pavone for the preparation of the manuscript. This research was supported by the U.S. Department of The Interior's Mineral Institute Program administered through the Generic Mineral Technology Center for Respirable Dust under grant 1135142. Request for reprints should be sent to Douglas C. Kuhn, Ph.D., Department of Pathology, The M.S. Hershey Medical Center, Hershey, PA 17033.
157 Journal of Toxicology and Environmental Health, 29:157-168, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
158
D. C. KUHN ET AL.
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INTRODUCTION Pulmonary alveolar macrophages (AM) are activated by foreign particles and microorganisms that enter the small airways (Kass et al., 1966; Herscowitz, 1985). This defensive reaction involves phagocytosis, lysosomal enzyme release, and the generation of reactive oxygen species by the AM (Snella, 1986; Rich et al., 1987). A number of agents, including phorbol esters, zymosan, adenosine diphosphate, organic hydroperoxides, and calcium ionophore, have been shown to activate the AM in vitro (Badger, 1986; Marshall and Lands, 1986; Snella, 1986). The AM response to these activators appears to be mediated in part by the oxygenated metabolites of arachidonic acid (AA) (Bertram et al., 1988; Demers et al., 1987; McLeish et al., 1987). The AM may metabolize AA through cyclooxygenase- and lipoxygenase-catalyzed pathways to produce a variety of eicosanoids including prostaglandin (PG) E2, thromboxane (Tx) A2, leukotriene (LT) B4, LTQ, LTD4, and several hydroxyeicosatetraenoic acid derivatives (HETEs) (Willis, 1970; Camp et al., 1983; Poubelle et al., 1986). The production of these eicosanoids may enhance the defensive ability of the challenged lung since they stimulate chemotactic cell recruitment to the site of invasion, enhance extravasation of circulating immune cells, and promote local vasodilatation. However, continued stimulation of the AM population, with the consequent production of proinflammatory eicosanoids, may ultimately enhance the disease process by contributing to chronic bronchoconstriction, fibrosis, and the persistent release of toxic oxygen species. Coal workers' pneumoconiosis (CWP) is an example of a disease process resulting from chronic exposure of the lung to foreign particles. We have therefore analyzed AA metabolism in AM harvested from rats exposed to respirable coal dust under closely controlled conditions. The results of these studies suggest that a relatively short exposure to coal dust results in the increased production of eicosanoids with vasoconstrictor and chemotactic activity and that some, but not all, aspects of AA metabolism return to normal when rats are allowed to breath normal air for 2 wk following dust exposure. In addition, the specific TxA2 synthesis inhibitor UK 38,485 reduced TxA2 production to normal levels when incubated in vitro with AM from dust-exposed animals. Coal dust exposure therefore altered AA metabolism in the primary defensive cell in the lung in a manner that may, on the one hand, enhance the primary defensive reaction by recruiting additional defensive cells to the site of invasion while, on the other hand, contributing to disease in longterm exposure by increasing the production of eicosanoids with mitogenic activity.
AA METABOLISM IN COAL DUST-EXPOSED AM
159
METHOD
Downloaded by [University of Florida] at 13:01 06 November 2015
Radiolabeled AA ([5,6,8,9,11,12,14,15-3H(N)]arachidonic acid) was purchased from Dupont/New England Nuclear Research Products. Powdered medium 199 (M199) (without phenol red) was purchased from Sigma Chemical Co.; UK 38,485 [3-(1H-imidazol-1-yl-methyl)-2-methyl-1Hindole-1-propanoic acid] was the generous gift of Pfizer Central Research, Crotón, Conn. All solvents were HPLC grade and purchased from Fisher Scientific. The bituminous coal dust samples of respirable quality were generously provided by the Department of Mineral Engineering, The Pennsylvania State University. Coal Dust Exposure
Coal mine dust from the Pittsburgh seam was initially sized using a Donaldson Acucut Classifier model A-12, which produced dust with a mean particle diameter of 4-5 (im (80% of the particles were between 2 and 7 /¿m in diameter). Respirable dust was introduced into the animal chamber by a fluidized-bed aerosol generator (Thermal Systems, Inc.). Aerosolized dust was mixed with filtered air and flowed through an aerosol neutralizer before entering the chamber. The air flow rate was 425 l/min (15 air changes per hour). Each chamber had a volume of 64 ft3 and was capable of housing 24 animals in individual cages. The dust concentration was uniform throughout the chamber (25 mg/m3); however, animal cages were rotated every other day in order to ensure uniform exposure. Temperature and humidity were monitored and controlled by microcomputer and maintained mean levels of 73°F and 48%, respectively, over the entire exposure period. Female rats (F344 strain, 180 g, Charles River Labs) were divided into control and experimental groups. Both groups were acclimated to identical temperature- and humidity-controlled chambers for 3 d. Subsequently, the experimental group was subjected to whole-body exposure to respirable bituminous coal dust for 16-h periods each day for 2 wk. The dust concentration and daily exposure time were chosen to provide a substantial dust deposition in the lungs during the 2-wk exposure period. Macrophage Harvest Alveolar macrophages were collected by bronchoalveolar lavage on the day following the last period of exposure. Rats were anesthetized with sodium pentobarbital (10 mg ip) and then exsanguinated through the abdominal aorta. The chest cavity was opened and a section of ribs
Downloaded by [University of Florida] at 13:01 06 November 2015
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approximately 0.7 cm on either side of the sternum was removed to allow for maximal lung expansion. The trachea was exposed and a cannula was inserted and secured. The lungs were inflated with lavage fluid [phosphate-buffered saline containing 0.1% EDTA (w/v), 7-8 ml, 37°C] and washed five times. Each wash was instilled and withdrawn five times. Cells were then centrifuged (600 x g, 10 min), washed with 2 ml M199, recentrifuged, and finally pooled in 2 ml medium 199 containing 0.7% HEPES, 100 U/ml penicillin, and 100 /tg/ml streptomycin. Cells were enumerated in a hemacytometer and viability (>95%) was determined by the exclusion of trypan blue. The number of cells containing coal dust was assessed by counting at least 100 cells in a hemacytometer grid and dividing the number of cells containing coal dust by the total cell count. Cells from each group were diluted to 5 x 105 cells/ml in medium 199 and 1 ml of cell suspension was placed in wells of a culture plate. Following a 1-h preincubation period (37°C in a humidified atmosphere of 5% CO2 in air), medium containing nonadherent cells was removed and replaced with fresh medium. Incubation was then continued for 4 h following the addition of either (1) radiolabeled AA (100 Ci/mmol, 1 ¡iCUwell) plus unlabeled AA (1.3 /¿g/well), (2) unlabeled AA (1.3 fig/weW), or (3) AA in combination with UK 38,485 (14.2 /xg/well). An identical protocol was followed to assess AA metabolism by AM from control rats and dust-exposed rats who had been allowed to breath normal air for 2 wk following the last day of exposure. Analytical Methods
T (5000 cpm) was added to samples containing radiolabeled AA in order to quantitate extraction efficiency (85-90%). Incubation supernatants were extracted twice with 2 ml ethyl acetate/cyclohexane (1:1) following adjustment of the supernatant pH to 3.0 with 1 N HCI. Extracts were stored at -20°C until analysis. Extracts from incubations containing radiolabeled AA were subjected to HPLC separation of AA metabolites on a Waters HPLC system using a C-18, reverse-phase, radial compression column. The following gradient profile was used to affect baseline separation of the eicosanoids: 0-20 min, isocratic elution with 100% Solvent A (25% acetonitrile, 74% H2O, pH 3.0 with H3PO4); 20-80 min, linear gradient elution to 22% Solvent A, 78% Solvent B (95% acetonitrile, 5% H2O, pH 3.0 with H3PO4). The solvent flow rate was 3 ml/min. Radioactivity in 3-ml column fractions was determined by liquid scintillation spectrometry. The radioactivity profile was compared to that generated by authentic eicosanoid standards. Extracts from incubations containing unlabeled AA were subjected to radioimmunoassay (RIA) of TxB2 (the stable metabolite of TxA2), PGE2, and LTB4 as previously described (Demers, 1983). The RIA reagents were pur-
AA METABOLISM IN COAL DUST-EXPOSED AM
16T
chased from Dupont/New England Nuclear Products. The antibodies utilized in the RIAs were specific and cross-reacted less than 1% with other eicosanoids (with the exception of LTB4 antibody, which showed 3.6% cross-reactivity with 5,12-diHETE, and PGE2 antibody, which showed 52% cross-reactivity with PGE^. Statistical significance of differences in mean eicosaniod levels between groups was determined by Student's f-test for independent samples. Mean eicosanoid levels in experimental groups were then expressed as percent of mean control levels for each eicosanoid.
Downloaded by [University of Florida] at 13:01 06 November 2015
RESULTS AM Harvest
The AM harvested from both control and dust-exposed animals were >95% viable. Cell numbers were consistently higher in dust-exposed animals both immediately after exposure [4.2 x 106 (±3.6 x 105 SEM) cells/control rat, 7.0 x 106 (±7.5 x 105) cells/exposed rat] and 2 wk following recovery [4.6 x 106 (±2.9 x 105) cells/control rat, 7.8 x 106 (±6.4 x 105) cells/exposed rat]. Assessment by light microscopy showed that 60-70% of AM harvested immediately after dust exposure contained dust particles while 30-40% of AM harvested after 2 wk of recovery contained particles. Eicosanoid Production in Control and Dust-Exposed AM
As seen in Fig. 1, the major products of AA metabolism in control AM were TxB2, PGE2, LTB4, monohydroxy derivatives (HETEs), and hydroxyheptadecatrienoic acid (HHT, a product of thromboxane synthetase activity). The HPLC analyses of extracts from incubations containing [3H]AA qualitatively supported data developed by RIA throughout these studies. Effect of Exposure to Coal Dust In Vivo on AM Metabolism of AA
In vivo exposure of rat AM to respirable coal dust significantly altered AA metabolism (Fig. 2). While dust exposure reduced PGE2 production to 62% of control values, it increased TxB2 and LTB4 production to 222% and 181% of control values, respectively. The mean control values for PGE2, TxB2, and LTB4 production as determined by RIA were 580,150, and 1050 pg/ml, respectively. Following 2 wk without exposure to coal dust, PGE2 production had returned to levels that were not significantly different from control (Fig. 3). However, TxB2 and LTB4 production remained elevated at 214% and 204% of control values, respectively.
162
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CL
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ü
i
i
r
i
10 20 30 40
i
i
r
50 60 70 80
FRACTION NUMBER FIGURE 1. HPLC radioactivity profile of eicosanoids produced from [3H]AA by control rat PAM. 1, 6KPGF1a; 2, TxB2; 3, PGE2; 4, LTB4; 5, HHT; 6, 15-HETE; 7,12-HETE; 8, unconverted AA.
Effect of the Thromboxane Synthetase Inhibitor UK 38,485
Coincubation of AA with UK 38,485 significantly reduced TxB2 production in dust-exposed AM (Fig. 4). The TxB2 was reduced from 222% to 123% of control values by the addition of UK 38,485 to AM harvested immediately after dust exposure. Following the recovery period, the addition of UK 38,485 also resulted in a marked reduction in TxB2 production in dust-exposed AM. The administration of UK 38,485 had no effect on PGE2 production but enhanced LTB4 production in both control and dust-exposed AM harvested immediately after exposure. Similarly, following 2 wk of recovery, LTB4 production in control and dust-exposed AM remained elevated in the presence of UK 38,485. DISCUSSION AND CONCLUSIONS Many studies have been conducted to assess AA metabolism in phagocytes and to determine the role of the eicosanoids in the function of these important defensive cell types. The results of these studies clearly suggest a role for the eicosanoids that may range from the regulation of phagocytosis (Hsueh et al., 1987) to the release of oxygen radicals by the macrophage (McLeish et al., 1987). It is also now apparent that the
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AA METABOLISM IN COAL DUST-EXPOSED AM
163
spectrum of eicosanoids produced in any given phagocytic cell may depend on its state of differentiation (Chandler and Fulmer, 1987), the stimulus used to activate the cell (Humes et al., 1982), and perhaps the anatomic source of the cell. Thus, AM from different subpopulations, as defined by density differences, showed marked differences in eicosanoid production to various stimuli, and these differences may be reflected in differentiated function among macrophage subpopulations (Chandler and Fulmer, 1987). In addition, while zymosan has been shown to stimulate the production of cyclooxygenase- and lipoxygenase-derived products of AA in phagocytes, phorbol ester stimulates predominantly the cyclooxygenase pathway (Humes et al., 1982). Finally, although pulmonary intravascular macrophages produce 12-HETE, alveolar macrophages apparently do not (Bertram et al., 1988). Nonetheless, as in other systems such as the vasculature, an important consideration in the pathophysiologic state of the lung may involve the relative balance of the different eicosanoids, which can mediate different aspects of organ function. In the studies presented here, we found that exposure to coal dust not only altered the balance of eicosanoid production but that the number of cells harvested from dust-exposed rats was greater than from control animals. In addition, when the relative distribution of radioactivity 300 i
oc
ÜT
>. °> in
CO
200 -
« ± 4-1
O
•5 S3
" o^
4
CD
2
100 -
ra
PGE2
TxB2 METABOLITE
LTB4
FIGURE 2. Eicosanoid production by PAM harvested from control and coal dust-exposed rats was determined by RIA. Data are presented as percent control values for each metabolite. Significant differences were determined by the paired Student's t-test (*p < .05).
D. C. KUHN ET AL.
164
300
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effect of in vivo coal dust exposure on arachidonic acid metabolism in the rat alveolar macrophage Douglas C. Kuhn , Charles F. Stanley , Nadia El‐Ayouby & Laurence M. Demers To cite this article: Douglas C. Kuhn , Charles F. Stanley , Nadia El‐Ayouby & Laurence M. Demers (1990) Effect of in vivo coal dust exposure on arachidonic acid metabolism in the rat alveolar macrophage, Journal of Toxicology and Environmental Health, 29:2, 157-168, DOI: 10.1080/15287399009531380 To link to this article: http://dx.doi.org/10.1080/15287399009531380
Published online: 15 Oct 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 06 November 2015, At: 13:00
EFFECT OF IN VIVO COAL DUST EXPOSURE O N ARACHIDONIC ACID METABOLISM IN THE RAT ALVEOLAR MACROPHAGE Douglas C. Kuhn
Downloaded by [University of Florida] at 13:01 06 November 2015
Department of Pathology, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, Pennsylvania Charles F. Stanley, Nadia El-Ayouby Department of Mechanical and Aerospace Engineering, West Virginia University, Morgantown, West Virginia Laurence M. Demers Department of Pathology, The Pennsylvania State University, The M.S. Hershey Medical Center, Hershey, Pennsylvania
Oxygenated metabolites of arachidonic acid (AA) are produced by the alveolar macrophage (AM) and have been shown to mediate inflammatory reactions. We therefore assessed the production of eicosanoids by AM harvested from the lungs of rats exposed to a bituminous coal dust for 2 wk in an inhalation chamber in order to determine if AA metabolism was altered in a manner that may promote an inflammatory response in the lung. Exposure to coal dust resulted in a 66% increase in the number of AM harvested, an increase in thromboxane A2 (TxA2) and leukotriene B4 (LTB4) production to 222% and 181% of control values, respectively, and a decrease in prostaglandin E2 (PGE2) production to 62% of control values. In AM harvested from rats allowed to breath clean air for 2 wk following coal dust exposure, PCE2 production returned to control levels but TxA2 and LTB4 production remained elevated. The TxA2 synthesis inhibitor UK 38,485 reduced TxA2 production in dust-exposed AM both immediately and 2 wk following exposure. Thus, exposure of rats to coal dust significantly alters the metabolism of AA in AM, with potentially important aspects of AA metabolism remaining altered even after a 2-wk recovery period. Based on the established role of eicosanoids in inflammatory and fibrotic processes, these results suggest that the alteration of AM eicosanoid production as a result of the inhalation of coal mine dust may be an important factor in the pathophysiology of coal workers' pneumoconiosis.
The authors are grateful to Joan Greenwood and Barbara Scheetz for their excellent technical assistance and to Brenda Pavone for the preparation of the manuscript. This research was supported by the U.S. Department of The Interior's Mineral Institute Program administered through the Generic Mineral Technology Center for Respirable Dust under grant 1135142. Request for reprints should be sent to Douglas C. Kuhn, Ph.D., Department of Pathology, The M.S. Hershey Medical Center, Hershey, PA 17033.
157 Journal of Toxicology and Environmental Health, 29:157-168, 1990 Copyright © 1990 by Hemisphere Publishing Corporation
158
D. C. KUHN ET AL.
Downloaded by [University of Florida] at 13:01 06 November 2015
INTRODUCTION Pulmonary alveolar macrophages (AM) are activated by foreign particles and microorganisms that enter the small airways (Kass et al., 1966; Herscowitz, 1985). This defensive reaction involves phagocytosis, lysosomal enzyme release, and the generation of reactive oxygen species by the AM (Snella, 1986; Rich et al., 1987). A number of agents, including phorbol esters, zymosan, adenosine diphosphate, organic hydroperoxides, and calcium ionophore, have been shown to activate the AM in vitro (Badger, 1986; Marshall and Lands, 1986; Snella, 1986). The AM response to these activators appears to be mediated in part by the oxygenated metabolites of arachidonic acid (AA) (Bertram et al., 1988; Demers et al., 1987; McLeish et al., 1987). The AM may metabolize AA through cyclooxygenase- and lipoxygenase-catalyzed pathways to produce a variety of eicosanoids including prostaglandin (PG) E2, thromboxane (Tx) A2, leukotriene (LT) B4, LTQ, LTD4, and several hydroxyeicosatetraenoic acid derivatives (HETEs) (Willis, 1970; Camp et al., 1983; Poubelle et al., 1986). The production of these eicosanoids may enhance the defensive ability of the challenged lung since they stimulate chemotactic cell recruitment to the site of invasion, enhance extravasation of circulating immune cells, and promote local vasodilatation. However, continued stimulation of the AM population, with the consequent production of proinflammatory eicosanoids, may ultimately enhance the disease process by contributing to chronic bronchoconstriction, fibrosis, and the persistent release of toxic oxygen species. Coal workers' pneumoconiosis (CWP) is an example of a disease process resulting from chronic exposure of the lung to foreign particles. We have therefore analyzed AA metabolism in AM harvested from rats exposed to respirable coal dust under closely controlled conditions. The results of these studies suggest that a relatively short exposure to coal dust results in the increased production of eicosanoids with vasoconstrictor and chemotactic activity and that some, but not all, aspects of AA metabolism return to normal when rats are allowed to breath normal air for 2 wk following dust exposure. In addition, the specific TxA2 synthesis inhibitor UK 38,485 reduced TxA2 production to normal levels when incubated in vitro with AM from dust-exposed animals. Coal dust exposure therefore altered AA metabolism in the primary defensive cell in the lung in a manner that may, on the one hand, enhance the primary defensive reaction by recruiting additional defensive cells to the site of invasion while, on the other hand, contributing to disease in longterm exposure by increasing the production of eicosanoids with mitogenic activity.
AA METABOLISM IN COAL DUST-EXPOSED AM
159
METHOD
Downloaded by [University of Florida] at 13:01 06 November 2015
Radiolabeled AA ([5,6,8,9,11,12,14,15-3H(N)]arachidonic acid) was purchased from Dupont/New England Nuclear Research Products. Powdered medium 199 (M199) (without phenol red) was purchased from Sigma Chemical Co.; UK 38,485 [3-(1H-imidazol-1-yl-methyl)-2-methyl-1Hindole-1-propanoic acid] was the generous gift of Pfizer Central Research, Crotón, Conn. All solvents were HPLC grade and purchased from Fisher Scientific. The bituminous coal dust samples of respirable quality were generously provided by the Department of Mineral Engineering, The Pennsylvania State University. Coal Dust Exposure
Coal mine dust from the Pittsburgh seam was initially sized using a Donaldson Acucut Classifier model A-12, which produced dust with a mean particle diameter of 4-5 (im (80% of the particles were between 2 and 7 /¿m in diameter). Respirable dust was introduced into the animal chamber by a fluidized-bed aerosol generator (Thermal Systems, Inc.). Aerosolized dust was mixed with filtered air and flowed through an aerosol neutralizer before entering the chamber. The air flow rate was 425 l/min (15 air changes per hour). Each chamber had a volume of 64 ft3 and was capable of housing 24 animals in individual cages. The dust concentration was uniform throughout the chamber (25 mg/m3); however, animal cages were rotated every other day in order to ensure uniform exposure. Temperature and humidity were monitored and controlled by microcomputer and maintained mean levels of 73°F and 48%, respectively, over the entire exposure period. Female rats (F344 strain, 180 g, Charles River Labs) were divided into control and experimental groups. Both groups were acclimated to identical temperature- and humidity-controlled chambers for 3 d. Subsequently, the experimental group was subjected to whole-body exposure to respirable bituminous coal dust for 16-h periods each day for 2 wk. The dust concentration and daily exposure time were chosen to provide a substantial dust deposition in the lungs during the 2-wk exposure period. Macrophage Harvest Alveolar macrophages were collected by bronchoalveolar lavage on the day following the last period of exposure. Rats were anesthetized with sodium pentobarbital (10 mg ip) and then exsanguinated through the abdominal aorta. The chest cavity was opened and a section of ribs
Downloaded by [University of Florida] at 13:01 06 November 2015
160
D. C. KUHN ET AL.
approximately 0.7 cm on either side of the sternum was removed to allow for maximal lung expansion. The trachea was exposed and a cannula was inserted and secured. The lungs were inflated with lavage fluid [phosphate-buffered saline containing 0.1% EDTA (w/v), 7-8 ml, 37°C] and washed five times. Each wash was instilled and withdrawn five times. Cells were then centrifuged (600 x g, 10 min), washed with 2 ml M199, recentrifuged, and finally pooled in 2 ml medium 199 containing 0.7% HEPES, 100 U/ml penicillin, and 100 /tg/ml streptomycin. Cells were enumerated in a hemacytometer and viability (>95%) was determined by the exclusion of trypan blue. The number of cells containing coal dust was assessed by counting at least 100 cells in a hemacytometer grid and dividing the number of cells containing coal dust by the total cell count. Cells from each group were diluted to 5 x 105 cells/ml in medium 199 and 1 ml of cell suspension was placed in wells of a culture plate. Following a 1-h preincubation period (37°C in a humidified atmosphere of 5% CO2 in air), medium containing nonadherent cells was removed and replaced with fresh medium. Incubation was then continued for 4 h following the addition of either (1) radiolabeled AA (100 Ci/mmol, 1 ¡iCUwell) plus unlabeled AA (1.3 /¿g/well), (2) unlabeled AA (1.3 fig/weW), or (3) AA in combination with UK 38,485 (14.2 /xg/well). An identical protocol was followed to assess AA metabolism by AM from control rats and dust-exposed rats who had been allowed to breath normal air for 2 wk following the last day of exposure. Analytical Methods
T (5000 cpm) was added to samples containing radiolabeled AA in order to quantitate extraction efficiency (85-90%). Incubation supernatants were extracted twice with 2 ml ethyl acetate/cyclohexane (1:1) following adjustment of the supernatant pH to 3.0 with 1 N HCI. Extracts were stored at -20°C until analysis. Extracts from incubations containing radiolabeled AA were subjected to HPLC separation of AA metabolites on a Waters HPLC system using a C-18, reverse-phase, radial compression column. The following gradient profile was used to affect baseline separation of the eicosanoids: 0-20 min, isocratic elution with 100% Solvent A (25% acetonitrile, 74% H2O, pH 3.0 with H3PO4); 20-80 min, linear gradient elution to 22% Solvent A, 78% Solvent B (95% acetonitrile, 5% H2O, pH 3.0 with H3PO4). The solvent flow rate was 3 ml/min. Radioactivity in 3-ml column fractions was determined by liquid scintillation spectrometry. The radioactivity profile was compared to that generated by authentic eicosanoid standards. Extracts from incubations containing unlabeled AA were subjected to radioimmunoassay (RIA) of TxB2 (the stable metabolite of TxA2), PGE2, and LTB4 as previously described (Demers, 1983). The RIA reagents were pur-
AA METABOLISM IN COAL DUST-EXPOSED AM
16T
chased from Dupont/New England Nuclear Products. The antibodies utilized in the RIAs were specific and cross-reacted less than 1% with other eicosanoids (with the exception of LTB4 antibody, which showed 3.6% cross-reactivity with 5,12-diHETE, and PGE2 antibody, which showed 52% cross-reactivity with PGE^. Statistical significance of differences in mean eicosaniod levels between groups was determined by Student's f-test for independent samples. Mean eicosanoid levels in experimental groups were then expressed as percent of mean control levels for each eicosanoid.
Downloaded by [University of Florida] at 13:01 06 November 2015
RESULTS AM Harvest
The AM harvested from both control and dust-exposed animals were >95% viable. Cell numbers were consistently higher in dust-exposed animals both immediately after exposure [4.2 x 106 (±3.6 x 105 SEM) cells/control rat, 7.0 x 106 (±7.5 x 105) cells/exposed rat] and 2 wk following recovery [4.6 x 106 (±2.9 x 105) cells/control rat, 7.8 x 106 (±6.4 x 105) cells/exposed rat]. Assessment by light microscopy showed that 60-70% of AM harvested immediately after dust exposure contained dust particles while 30-40% of AM harvested after 2 wk of recovery contained particles. Eicosanoid Production in Control and Dust-Exposed AM
As seen in Fig. 1, the major products of AA metabolism in control AM were TxB2, PGE2, LTB4, monohydroxy derivatives (HETEs), and hydroxyheptadecatrienoic acid (HHT, a product of thromboxane synthetase activity). The HPLC analyses of extracts from incubations containing [3H]AA qualitatively supported data developed by RIA throughout these studies. Effect of Exposure to Coal Dust In Vivo on AM Metabolism of AA
In vivo exposure of rat AM to respirable coal dust significantly altered AA metabolism (Fig. 2). While dust exposure reduced PGE2 production to 62% of control values, it increased TxB2 and LTB4 production to 222% and 181% of control values, respectively. The mean control values for PGE2, TxB2, and LTB4 production as determined by RIA were 580,150, and 1050 pg/ml, respectively. Following 2 wk without exposure to coal dust, PGE2 production had returned to levels that were not significantly different from control (Fig. 3). However, TxB2 and LTB4 production remained elevated at 214% and 204% of control values, respectively.
162
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CL
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ü
i
i
r
i
10 20 30 40
i
i
r
50 60 70 80
FRACTION NUMBER FIGURE 1. HPLC radioactivity profile of eicosanoids produced from [3H]AA by control rat PAM. 1, 6KPGF1a; 2, TxB2; 3, PGE2; 4, LTB4; 5, HHT; 6, 15-HETE; 7,12-HETE; 8, unconverted AA.
Effect of the Thromboxane Synthetase Inhibitor UK 38,485
Coincubation of AA with UK 38,485 significantly reduced TxB2 production in dust-exposed AM (Fig. 4). The TxB2 was reduced from 222% to 123% of control values by the addition of UK 38,485 to AM harvested immediately after dust exposure. Following the recovery period, the addition of UK 38,485 also resulted in a marked reduction in TxB2 production in dust-exposed AM. The administration of UK 38,485 had no effect on PGE2 production but enhanced LTB4 production in both control and dust-exposed AM harvested immediately after exposure. Similarly, following 2 wk of recovery, LTB4 production in control and dust-exposed AM remained elevated in the presence of UK 38,485. DISCUSSION AND CONCLUSIONS Many studies have been conducted to assess AA metabolism in phagocytes and to determine the role of the eicosanoids in the function of these important defensive cell types. The results of these studies clearly suggest a role for the eicosanoids that may range from the regulation of phagocytosis (Hsueh et al., 1987) to the release of oxygen radicals by the macrophage (McLeish et al., 1987). It is also now apparent that the
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spectrum of eicosanoids produced in any given phagocytic cell may depend on its state of differentiation (Chandler and Fulmer, 1987), the stimulus used to activate the cell (Humes et al., 1982), and perhaps the anatomic source of the cell. Thus, AM from different subpopulations, as defined by density differences, showed marked differences in eicosanoid production to various stimuli, and these differences may be reflected in differentiated function among macrophage subpopulations (Chandler and Fulmer, 1987). In addition, while zymosan has been shown to stimulate the production of cyclooxygenase- and lipoxygenase-derived products of AA in phagocytes, phorbol ester stimulates predominantly the cyclooxygenase pathway (Humes et al., 1982). Finally, although pulmonary intravascular macrophages produce 12-HETE, alveolar macrophages apparently do not (Bertram et al., 1988). Nonetheless, as in other systems such as the vasculature, an important consideration in the pathophysiologic state of the lung may involve the relative balance of the different eicosanoids, which can mediate different aspects of organ function. In the studies presented here, we found that exposure to coal dust not only altered the balance of eicosanoid production but that the number of cells harvested from dust-exposed rats was greater than from control animals. In addition, when the relative distribution of radioactivity 300 i
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FIGURE 2. Eicosanoid production by PAM harvested from control and coal dust-exposed rats was determined by RIA. Data are presented as percent control values for each metabolite. Significant differences were determined by the paired Student's t-test (*p < .05).
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