Enhanced Release of Prostaglandin E 2 from Macrophages of Rats with Silicosis Carsten Mohr, Gerald S. Davis, Christine Graebner, David R. Hemenway, and Diethard Gemsa Institute of Immunology, Philipps University, Marburg, Germany, and the Departments of Medicine (Pulmonary Unit) and Civil and Mechanical Engineering, University of Vermont, Burlington, Vermont
The pathogenesis of silicosis results, in part, from interactions between silica particles and alveolar macrophages (AM) with release of cytokines and other mediators. Different arachidonic acid metabolites have been shown to promote or to suppress inflammation and fibrosis. We designed experiments to study the production of cyclooxygenase metabolites and tumor necrosis factor-a (TNF-a) from macrophages during active silicosis. Macrophages were harvested from rats 5 to 7 mo after an 8-day silica aerosol exposure. Upon in vitro culture of AM, the spontaneous release of prostaglandin E2 (PGE 2) , thromboxane B2 (TXB2) , and prostaglandin D2 (PGD 2) of silica-exposed animals was higher than that of sham-exposed animals. Moreover, AM from silicotic rats displayed an increased sensitivity to low concentrations of lipopolysaccharide (LPS, 10 ng/ml) and released copious amounts of PGE 2 and TXB2 • When compared with similarly enhanced release of TNF-a from AM of silica-exposed rats, PGE2 production occurred later and started to increase when TNF-a production declined. Addition of the cyclooxygenase blocker indomethacin augmented TNF-a production, whereas the addition of PGE2 counteracted TNF-a release. Also peritoneal macrophages, which did not have direct contact with silica particles, released enhanced levels of PGE 2 in response to low LPS doses. We conclude that AM and other macrophages from silica-exposed rats are preactivated and display an enhanced prostanoid production that could serve anti-inflammatory or immunomodulating roles in silicosis.
A prolonged inhalation of silica particles may lead to chronic inflammatory and fibrotic lung disease. Silica particles deposited in the alveolar space are mainly phagocytosed by alveolar macrophages (AM), and this crucial event appears to form the pathogenic basis leading ultimately to silicosis (1). This disease has been thought to be the sequela of various mediators and cytokines that are persistently released from macrophages interacting with silica particles (2). Among mediators, various eicosanoids (3, 4) and, more recently, cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-l have been implicated in causing a chronic inflammation, granuloma formation, and lung fibrosis (5-8). Until now, it has remained uncertain by which mechanism silica particles may stimulate macrophages to an enhanced release of inflammatory and fibrosis promoting mediators. Two principally different ideas have been forwarded: (1) silica particles damage macrophages, which in (Received in original form June 4, 1991 and injinalform September 5, 1991) Address correspondence to: Oiethard Gemsa, M.D., Institute of Immunology, Philipps University, Robert-Koch-Strasse 17, 0-3550 Marburg, Germany. Abbreviations: alveolar macrophages, AM; lipopolysaccharide, LPS; prostaglandin, PG; peritoneal macrophages, PM; tumor necrosis factor-a, TNF-a; thromboxane, TX. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 390-396, 1992
turn respond with release of mediators (3, 9, 10), and (2) silica particles stimulate or activate macrophages without apparent cell toxicity, which may lead to a persistent mediator production (1, 2, 7). Evidence for the first assumption has predominantly been found under in vitro conditions, whereas the second idea has mainly been supported by in vivo investigations. Recently, we described a rat model of chronic silicosis that was characterized by a typical local pulmonary response (8). After 4 to 12 mo after aerosol exposure to silica, rats developed an interstitial fibrosis and an inflammatory accumulation of mononuclear leukocytes and neutrophils in association with dust deposits. In addition, a systemic preactivation of macrophages was found with respect to lipopolysaccharide (LPS)-inducible TNF-a release. Among macrophage products, prostanoids such as prostaglandin (PG) E2 may play an important role as mediators that control various leukocyte and fibroblast functions (11-13). Because production ofTNF-a has been shown to be downregulated by macrophage-derived PGE 2 , a mutual interrelation of both mediators has been postulated (14-16). Inthis report, we show that both AM and peritoneal macrophages (PM) from silicotic rats display an enhanced spontaneous and LPS-induced release of PGE 2 , which supports the notion that a systemic preactivation of macrophages may prevail in silicosis.
Mohr, Davis, Graebner et al.: Enhanced PGE z Release from Macrophages in Silicosis
Materials and Methods Animals Male Fischer 344 rats from a cesarean-derived, barriermaintained colony were obtained from the National Cancer Institute (Bethesda, MD). Rats with an average weight of 175 g were housed in wire cages in a filtered air atmosphere and received sterilized food and water ad libitum. The rats remained healthy without apparent signs of infections and gained weight during up to 12 mo of observation. Housing conditions and mineral exposure met National Institutes of Health and German guidelines. Silica Particle Exposure Aerosols of a-quartz (Min-U-Sil 5; U.S. Silica Products, Pittsburgh, PA)were generated by a Wright dust feed apparatus. The characteristics of the exposure system have been described previously in detail (17, 18). Briefly,rats were placed into horizontal flow chambers and were exposed for 5 h/day for 8 days to an aerosol containing particles of respirable size (0.9 p,mmedian diameter, 2.4 p,mSD) at an average air concentration of 44.47 ± 0.81 mg/m' (mean ± SD). Matched groups of rats were exposed simultaneously in adjacent chambers to either the mineral aerosol or to the carrier air (sham control). Experimental Design A total of 24 silica- and 24 sham-exposed rats were used for this study. Cells harvested from one silica- and one shamexposed rat 5 to 7 mo after aerosol exposure were compared in each experiment. Four replicate cultures from each animal were established for each condition tested, and the prostanoid content or cytokine activity of each supernatant was assayed separately. The data are shown as the mean ± SD of the four replicates. This experimental design was repeated 3 to 5 times for each of the measurements made. The results of the repeated experiments were qualitatively similar, but absolute values for the amounts of prostanoids. or TNF-a varied among experiments. The results of a representative experiment for each measurement are shown in Figures 1 through 4 and Table 2. Bronchoalveolar and Peritoneal Lavage After anesthesia, the trachea was cannulated and heart and lungs were removed en bloc. Alveolar and peritoneal cells were harvested by bronchoalveolar or peritoneal lavage with phosphate-buffered saline as previously described (8). After harvest, cells were washed by centrifugation at 400 x g and resuspended in RPMI 1640 medium supplemented with i.-glutamine (2 mM), penicillin (100 U/ml) , streptomycin (100 p,g/ml), Hepes (20 mM), and nonessential amino acids. Differential counts were performed by staining cytocentrifuge smears with May-Grunwald-Giemsa dye. Purification and Culture of Macrophages AM or PM were adjusted to 0.5 x 106/ml in RPMl1640 medium plus 10% heat-inactivated (56 0 C, 30 min) fetal calf serum to promote adherence and were placed into 24-well tissue culture plates (Primaria; Becton-Dickinson, Heidelberg, Germany). Four replicate cultures from each animal were established for each of the conditions tested. After
391
1 h of incubation at 37 0 C in a 5 % CO z/95 % air atmosphere, the nonadherent cells were removed by two washings with culture medium. The remaining monolayer consisted of > 85 % macrophages as determined by carbon particle phagocytosis and staining for nonspecific esterase (19). The incubation was continued in RPMI 1640 medium without the addition of serum in the presence or absence of LPS from Escherichia coli 0127:B8 (Difco, Detroit, MI). At the end of the incubation, the culture supernatants were removed and centrifuged. The cell-free culture supernatants were stored at -70 0 C until tested for prostanoid content or TNF-a activity. Assays for Prostanoid Release PGEz in macrophage culture supernatants was determined by an enzyme-linked immunosorbent assay, which was developed in our laboratory using a streptavidin-biotin-peroxidase detecting system. Monoclonal anti-Pflli, antibodies were kindly supplied by Dr. M. Reinke (Erlangen, Germany). Sensitivity of the PGEz enzyme-linked immunosorbent assay was 24 pg/ml and crossreactivity of the antiserum with PGE was 2 % and < 0.1% with all other prostanoids. The content of prostacyclin (PGl z) was determined by its metabolite 6-keto-PGF 1a and of thromboxane (TX) A z by its metabolite TXB z• For both metabolites, radioimmunoassays that had been developed in our laboratory were used (20). Sensitivity of both assays was 20 pg/mI and 25 pg/ml, respectively, and crossreactivity with other prostanoids was < 0.2 %. PGD z was determined by a commercially available radioimmunoassay from Amersham-Buchler (Braunschweig, Germany). Sensitivity of the assay was 30 pg/ml and crossreactivity with TXB z was 0.3 % and < 0.1% with other prostanoids. j
Assay for TNF-a Release Release of TNF-a from macrophages was determined by bioassay as cytotoxicity of culture supernatants against TNFa-sensitive L929 cells, as recently described in detail (8, 16, 21, 22). TNF-a activity is expressed in units/mI, in which a unit is defined as the activity required to lyse 50% ofL929 target cells. Murine recombinant TNF-a was used to standardize the assay system. Specificity of TNF-a determinations in macrophage culture supernatants was corroborated by: (1) parallel use ofTNF-a-insensitive L929 cells (23) and (2) neutralization of TNF-a activity by highly specific rabbit anti-murine TNF-a antibodies (kindly provided by Dr. D. Manuel, German Cancer Research Center, Heidelberg, Germany). Statistical Analysis Statistical analysis was performed by the paired Student's t test; P values < 0.05 were considered significant.
Results General Observation Although details of this rat model of silicosis have been presented previously (8), it should be recapitulated that 4 to 9 mo after silica exposure, dust deposits were detected in the lungs and thoracic organs but not elsewhere. Histopathology of lungs from silica-exposed rats showed multiple small foci
392
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
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responsive, even when stimulated with 100 ng/ml LPS. It should be noted that microscopically not all AM from silicotic animals contained silica particles (8). However, for the sake of brevity, those macrophages have been denoted as silica-exposed AM in the text, tables, and figures. To examine whether cyclooxygenase activity was generally enhanced in AM from silicotic rats, the spontaneous and LPS-stimulated secretion of additional arachidonic acid metabolites was determined. Table 1 shows that not only spontaneous release of PGEz but also of TXB z was enhanced in silica-exposed AM. When prostanoid production was additionally stimulated by LPS (10 ng/ml) , the difference became more evident in that exclusively AM from silicotic rats released markedly higher levels of PGE z and TXB z than AM from control animals. Although LPS-stimulated PGD z and 6-keto-PGg", production was also higher in AM from silicotic than from control rats, the total amounts were much lower than for PGE z or TXB z• The enhanced prostanoid production from silica-exposed AM was not exclusively LPS dependent. Other stimuli such as ionophore A23187 and zymosan were also efficient triggers and induced a higher PGE z release in silica-exposed AM than in control cells (Table 2).
T
5
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9
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100
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Figure 1. Lipopolysaccharide (LPS)-induced prostaglandin (PG) E, release. Alveolar macrophages (AM) (0.5 X IQ6/rnl) from silica-exposed (solid circles) or sham-exposed (open circles) rats were incubated with various concentrations of LPS. After 16 h of incubation, culture supernatants were harvested and assayed for PGE z content. Values are representative of four separately performed experiments and are the mean ± SD of four identically prepared cultures.
Correlation between PGE z and TNF-a Release We previously described that inhaled silica particles could prime AM for an enhanced TNF-a production in response to LPS (8). Because PGE z had been found to suppress TNF-a synthesis (14-16), it was of interest in our experimental system to examine the production of both macrophage mediators on a kinetic basis. Figure 2 demonstrates that AM from silicotic rats rapidly released TNF-a in response to LPS, with a maximal peak around 12 h after LPS stimulation. In contrast, the onset of LPS-induced PGE z release was much slower, beginning to increase at maximal TNF-a production and reaching maximal levels around 20 h after incubation, i.e., when TNF-a levels had declined. Thus, it appeared that progressive PGE z accumulation closely correlated with TNF-a disappearance from the culture medium. Sham-exposed AM neither showed a comparable TNF-a nor PGE z production (data not shown). To study further the role of prostanoids in the regulation of TNF-a production, we incubated LPS-stimulated macrophages with the cyclooxygenase blocker indomethacin (Fig-
with accumulations of mononuclear leukocytes and an increased connective tissue matrix material. The bronchoalveolar lavage fluid revealed an increased cell number in silicotic animals, consisting mainly of macrophages, lymphocytes, and neutrophils. In peritoneal cell populations, no difference was found between silica- and sham-exposed animals. The goal of our present investigation was to study PGE z and TNF-a release at a representative time during evolving silicosis. Data are presented from rats 5 to 7 mo after the silica aerosol exposure. Similar results were found at selected times from 4 to 12 mo after exposure. Enhanced Prostanoid Release from AM of Silicotic Rats When AM from silica- or sham-exposed rats were incubated for 16 h with LPS, a marked difference in PGE z release was observed (Figure 1). AM from silicotic rats displayed an extremely enhanced LPS sensitivity and released PGE z at LPS concentrations as low as 1 to lOng/mi. In striking contrast, AM from control rats remained comparatively un-
TABLE 1
Release of prostanoids from AM of sham-exposed and silica-exposed rats *t
Prostanoid
PGEz PGDz TXB z
6-keto-PGF 1",
Release from Control AM
Release from Silica-exposed AM
(nglml)
(nglml)
Spontaneous
0.15 0.06 1.13 0.10
± ± ± ±
0.07 0.02 0.39 0.02
LPS-induced
0.36 0.15 1.35 0.14
± ± ± ±
0.16 0.03 0.45 0.02
Spontaneous
P Valuet
± ± ± ±
< 0.05 < 0.10 < 0.05
0.86 0.19 3.48 0.13
0.26 0.09 0.98 0.03
NS
LPS-induced
18.50 1.23 11.80 0.35
± ± ± ±
5.60 0.42 4.80 0.10
P Value§
< 0.05 < 0.05 < 0.05NS
Definition of abbreviations: AM = alveolar macrophages; LPS = lipopolysaccharide; PG = prostaglandin; TXB 2 = thromboxane B2 ; NS = not significant. * AM were incubated in the absence or presence of LPS (10 ng/rnl), and, after 16 h of incubation, prostanoids were determined in the cell-free culture supernatants. t All data are the mean ± SD of either four silica-exposed or four sham-exposed rats. Comparison of spontaneous release between AM from control rats versus AM from silica-exposed rats. § Comparison of LPS-induced release between AM from control rats versus AM from silica-exposed rats.
*
Mohr, Davis, Graebner et al.: Enhanced PGE2 Release from Macrophages in Silicosis
393
100
TABLE 2
Release of PGE2 from AM of sham-exposed and silica-exposed rats in response to various stimuli *f
---..
E
The pathogenesis of silicosis results, in part, from interactions between silica particles and alveolar macrophages (AM) with release of cytokines and other mediators. Different arachidonic acid metabolites have been shown to promote or to suppress inflammation and fibrosis. We designed experiments to study the production of cyclooxygenase metabolites and tumor necrosis factor-a (TNF-a) from macrophages during active silicosis. Macrophages were harvested from rats 5 to 7 mo after an 8-day silica aerosol exposure. Upon in vitro culture of AM, the spontaneous release of prostaglandin E2 (PGE 2) , thromboxane B2 (TXB2) , and prostaglandin D2 (PGD 2) of silica-exposed animals was higher than that of sham-exposed animals. Moreover, AM from silicotic rats displayed an increased sensitivity to low concentrations of lipopolysaccharide (LPS, 10 ng/ml) and released copious amounts of PGE 2 and TXB2 • When compared with similarly enhanced release of TNF-a from AM of silica-exposed rats, PGE2 production occurred later and started to increase when TNF-a production declined. Addition of the cyclooxygenase blocker indomethacin augmented TNF-a production, whereas the addition of PGE2 counteracted TNF-a release. Also peritoneal macrophages, which did not have direct contact with silica particles, released enhanced levels of PGE 2 in response to low LPS doses. We conclude that AM and other macrophages from silica-exposed rats are preactivated and display an enhanced prostanoid production that could serve anti-inflammatory or immunomodulating roles in silicosis.
A prolonged inhalation of silica particles may lead to chronic inflammatory and fibrotic lung disease. Silica particles deposited in the alveolar space are mainly phagocytosed by alveolar macrophages (AM), and this crucial event appears to form the pathogenic basis leading ultimately to silicosis (1). This disease has been thought to be the sequela of various mediators and cytokines that are persistently released from macrophages interacting with silica particles (2). Among mediators, various eicosanoids (3, 4) and, more recently, cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-l have been implicated in causing a chronic inflammation, granuloma formation, and lung fibrosis (5-8). Until now, it has remained uncertain by which mechanism silica particles may stimulate macrophages to an enhanced release of inflammatory and fibrosis promoting mediators. Two principally different ideas have been forwarded: (1) silica particles damage macrophages, which in (Received in original form June 4, 1991 and injinalform September 5, 1991) Address correspondence to: Oiethard Gemsa, M.D., Institute of Immunology, Philipps University, Robert-Koch-Strasse 17, 0-3550 Marburg, Germany. Abbreviations: alveolar macrophages, AM; lipopolysaccharide, LPS; prostaglandin, PG; peritoneal macrophages, PM; tumor necrosis factor-a, TNF-a; thromboxane, TX. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 390-396, 1992
turn respond with release of mediators (3, 9, 10), and (2) silica particles stimulate or activate macrophages without apparent cell toxicity, which may lead to a persistent mediator production (1, 2, 7). Evidence for the first assumption has predominantly been found under in vitro conditions, whereas the second idea has mainly been supported by in vivo investigations. Recently, we described a rat model of chronic silicosis that was characterized by a typical local pulmonary response (8). After 4 to 12 mo after aerosol exposure to silica, rats developed an interstitial fibrosis and an inflammatory accumulation of mononuclear leukocytes and neutrophils in association with dust deposits. In addition, a systemic preactivation of macrophages was found with respect to lipopolysaccharide (LPS)-inducible TNF-a release. Among macrophage products, prostanoids such as prostaglandin (PG) E2 may play an important role as mediators that control various leukocyte and fibroblast functions (11-13). Because production ofTNF-a has been shown to be downregulated by macrophage-derived PGE 2 , a mutual interrelation of both mediators has been postulated (14-16). Inthis report, we show that both AM and peritoneal macrophages (PM) from silicotic rats display an enhanced spontaneous and LPS-induced release of PGE 2 , which supports the notion that a systemic preactivation of macrophages may prevail in silicosis.
Mohr, Davis, Graebner et al.: Enhanced PGE z Release from Macrophages in Silicosis
Materials and Methods Animals Male Fischer 344 rats from a cesarean-derived, barriermaintained colony were obtained from the National Cancer Institute (Bethesda, MD). Rats with an average weight of 175 g were housed in wire cages in a filtered air atmosphere and received sterilized food and water ad libitum. The rats remained healthy without apparent signs of infections and gained weight during up to 12 mo of observation. Housing conditions and mineral exposure met National Institutes of Health and German guidelines. Silica Particle Exposure Aerosols of a-quartz (Min-U-Sil 5; U.S. Silica Products, Pittsburgh, PA)were generated by a Wright dust feed apparatus. The characteristics of the exposure system have been described previously in detail (17, 18). Briefly,rats were placed into horizontal flow chambers and were exposed for 5 h/day for 8 days to an aerosol containing particles of respirable size (0.9 p,mmedian diameter, 2.4 p,mSD) at an average air concentration of 44.47 ± 0.81 mg/m' (mean ± SD). Matched groups of rats were exposed simultaneously in adjacent chambers to either the mineral aerosol or to the carrier air (sham control). Experimental Design A total of 24 silica- and 24 sham-exposed rats were used for this study. Cells harvested from one silica- and one shamexposed rat 5 to 7 mo after aerosol exposure were compared in each experiment. Four replicate cultures from each animal were established for each condition tested, and the prostanoid content or cytokine activity of each supernatant was assayed separately. The data are shown as the mean ± SD of the four replicates. This experimental design was repeated 3 to 5 times for each of the measurements made. The results of the repeated experiments were qualitatively similar, but absolute values for the amounts of prostanoids. or TNF-a varied among experiments. The results of a representative experiment for each measurement are shown in Figures 1 through 4 and Table 2. Bronchoalveolar and Peritoneal Lavage After anesthesia, the trachea was cannulated and heart and lungs were removed en bloc. Alveolar and peritoneal cells were harvested by bronchoalveolar or peritoneal lavage with phosphate-buffered saline as previously described (8). After harvest, cells were washed by centrifugation at 400 x g and resuspended in RPMI 1640 medium supplemented with i.-glutamine (2 mM), penicillin (100 U/ml) , streptomycin (100 p,g/ml), Hepes (20 mM), and nonessential amino acids. Differential counts were performed by staining cytocentrifuge smears with May-Grunwald-Giemsa dye. Purification and Culture of Macrophages AM or PM were adjusted to 0.5 x 106/ml in RPMl1640 medium plus 10% heat-inactivated (56 0 C, 30 min) fetal calf serum to promote adherence and were placed into 24-well tissue culture plates (Primaria; Becton-Dickinson, Heidelberg, Germany). Four replicate cultures from each animal were established for each of the conditions tested. After
391
1 h of incubation at 37 0 C in a 5 % CO z/95 % air atmosphere, the nonadherent cells were removed by two washings with culture medium. The remaining monolayer consisted of > 85 % macrophages as determined by carbon particle phagocytosis and staining for nonspecific esterase (19). The incubation was continued in RPMI 1640 medium without the addition of serum in the presence or absence of LPS from Escherichia coli 0127:B8 (Difco, Detroit, MI). At the end of the incubation, the culture supernatants were removed and centrifuged. The cell-free culture supernatants were stored at -70 0 C until tested for prostanoid content or TNF-a activity. Assays for Prostanoid Release PGEz in macrophage culture supernatants was determined by an enzyme-linked immunosorbent assay, which was developed in our laboratory using a streptavidin-biotin-peroxidase detecting system. Monoclonal anti-Pflli, antibodies were kindly supplied by Dr. M. Reinke (Erlangen, Germany). Sensitivity of the PGEz enzyme-linked immunosorbent assay was 24 pg/ml and crossreactivity of the antiserum with PGE was 2 % and < 0.1% with all other prostanoids. The content of prostacyclin (PGl z) was determined by its metabolite 6-keto-PGF 1a and of thromboxane (TX) A z by its metabolite TXB z• For both metabolites, radioimmunoassays that had been developed in our laboratory were used (20). Sensitivity of both assays was 20 pg/mI and 25 pg/ml, respectively, and crossreactivity with other prostanoids was < 0.2 %. PGD z was determined by a commercially available radioimmunoassay from Amersham-Buchler (Braunschweig, Germany). Sensitivity of the assay was 30 pg/ml and crossreactivity with TXB z was 0.3 % and < 0.1% with other prostanoids. j
Assay for TNF-a Release Release of TNF-a from macrophages was determined by bioassay as cytotoxicity of culture supernatants against TNFa-sensitive L929 cells, as recently described in detail (8, 16, 21, 22). TNF-a activity is expressed in units/mI, in which a unit is defined as the activity required to lyse 50% ofL929 target cells. Murine recombinant TNF-a was used to standardize the assay system. Specificity of TNF-a determinations in macrophage culture supernatants was corroborated by: (1) parallel use ofTNF-a-insensitive L929 cells (23) and (2) neutralization of TNF-a activity by highly specific rabbit anti-murine TNF-a antibodies (kindly provided by Dr. D. Manuel, German Cancer Research Center, Heidelberg, Germany). Statistical Analysis Statistical analysis was performed by the paired Student's t test; P values < 0.05 were considered significant.
Results General Observation Although details of this rat model of silicosis have been presented previously (8), it should be recapitulated that 4 to 9 mo after silica exposure, dust deposits were detected in the lungs and thoracic organs but not elsewhere. Histopathology of lungs from silica-exposed rats showed multiple small foci
392
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
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responsive, even when stimulated with 100 ng/ml LPS. It should be noted that microscopically not all AM from silicotic animals contained silica particles (8). However, for the sake of brevity, those macrophages have been denoted as silica-exposed AM in the text, tables, and figures. To examine whether cyclooxygenase activity was generally enhanced in AM from silicotic rats, the spontaneous and LPS-stimulated secretion of additional arachidonic acid metabolites was determined. Table 1 shows that not only spontaneous release of PGEz but also of TXB z was enhanced in silica-exposed AM. When prostanoid production was additionally stimulated by LPS (10 ng/ml) , the difference became more evident in that exclusively AM from silicotic rats released markedly higher levels of PGE z and TXB z than AM from control animals. Although LPS-stimulated PGD z and 6-keto-PGg", production was also higher in AM from silicotic than from control rats, the total amounts were much lower than for PGE z or TXB z• The enhanced prostanoid production from silica-exposed AM was not exclusively LPS dependent. Other stimuli such as ionophore A23187 and zymosan were also efficient triggers and induced a higher PGE z release in silica-exposed AM than in control cells (Table 2).
T
5
V
0::
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0
1
0
Control
9
0
100
10
LP$ (ng/ml)
Figure 1. Lipopolysaccharide (LPS)-induced prostaglandin (PG) E, release. Alveolar macrophages (AM) (0.5 X IQ6/rnl) from silica-exposed (solid circles) or sham-exposed (open circles) rats were incubated with various concentrations of LPS. After 16 h of incubation, culture supernatants were harvested and assayed for PGE z content. Values are representative of four separately performed experiments and are the mean ± SD of four identically prepared cultures.
Correlation between PGE z and TNF-a Release We previously described that inhaled silica particles could prime AM for an enhanced TNF-a production in response to LPS (8). Because PGE z had been found to suppress TNF-a synthesis (14-16), it was of interest in our experimental system to examine the production of both macrophage mediators on a kinetic basis. Figure 2 demonstrates that AM from silicotic rats rapidly released TNF-a in response to LPS, with a maximal peak around 12 h after LPS stimulation. In contrast, the onset of LPS-induced PGE z release was much slower, beginning to increase at maximal TNF-a production and reaching maximal levels around 20 h after incubation, i.e., when TNF-a levels had declined. Thus, it appeared that progressive PGE z accumulation closely correlated with TNF-a disappearance from the culture medium. Sham-exposed AM neither showed a comparable TNF-a nor PGE z production (data not shown). To study further the role of prostanoids in the regulation of TNF-a production, we incubated LPS-stimulated macrophages with the cyclooxygenase blocker indomethacin (Fig-
with accumulations of mononuclear leukocytes and an increased connective tissue matrix material. The bronchoalveolar lavage fluid revealed an increased cell number in silicotic animals, consisting mainly of macrophages, lymphocytes, and neutrophils. In peritoneal cell populations, no difference was found between silica- and sham-exposed animals. The goal of our present investigation was to study PGE z and TNF-a release at a representative time during evolving silicosis. Data are presented from rats 5 to 7 mo after the silica aerosol exposure. Similar results were found at selected times from 4 to 12 mo after exposure. Enhanced Prostanoid Release from AM of Silicotic Rats When AM from silica- or sham-exposed rats were incubated for 16 h with LPS, a marked difference in PGE z release was observed (Figure 1). AM from silicotic rats displayed an extremely enhanced LPS sensitivity and released PGE z at LPS concentrations as low as 1 to lOng/mi. In striking contrast, AM from control rats remained comparatively un-
TABLE 1
Release of prostanoids from AM of sham-exposed and silica-exposed rats *t
Prostanoid
PGEz PGDz TXB z
6-keto-PGF 1",
Release from Control AM
Release from Silica-exposed AM
(nglml)
(nglml)
Spontaneous
0.15 0.06 1.13 0.10
± ± ± ±
0.07 0.02 0.39 0.02
LPS-induced
0.36 0.15 1.35 0.14
± ± ± ±
0.16 0.03 0.45 0.02
Spontaneous
P Valuet
± ± ± ±
< 0.05 < 0.10 < 0.05
0.86 0.19 3.48 0.13
0.26 0.09 0.98 0.03
NS
LPS-induced
18.50 1.23 11.80 0.35
± ± ± ±
5.60 0.42 4.80 0.10
P Value§
< 0.05 < 0.05 < 0.05NS
Definition of abbreviations: AM = alveolar macrophages; LPS = lipopolysaccharide; PG = prostaglandin; TXB 2 = thromboxane B2 ; NS = not significant. * AM were incubated in the absence or presence of LPS (10 ng/rnl), and, after 16 h of incubation, prostanoids were determined in the cell-free culture supernatants. t All data are the mean ± SD of either four silica-exposed or four sham-exposed rats. Comparison of spontaneous release between AM from control rats versus AM from silica-exposed rats. § Comparison of LPS-induced release between AM from control rats versus AM from silica-exposed rats.
*
Mohr, Davis, Graebner et al.: Enhanced PGE2 Release from Macrophages in Silicosis
393
100
TABLE 2
Release of PGE2 from AM of sham-exposed and silica-exposed rats in response to various stimuli *f
---..
E
