Reduced Release of Leukotrienes B4 and of Rats with Silicosis
C4
from Alveolar Macrophages
Carsten Mohr, Gerald S. Davis, Christine Graebner, Susanne Amann, 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
Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-a (TNF-a) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-a production, which was higher in AM from silicaexposed than from control rats. To study the interrelation between leukotriene and TNF-a release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosaninduced TNF-a release from AM in a dose-dependent manner, and TNF-a production could be restored almost completely by addition of LTB4 • These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-a production during silicosis.
The inhalation of silica dust leads to silicosis, a lung disease characterized by interstitial inflammation and fibrosis. Alveolar macrophages (AM) respond to silica inhalation by increased secretion of a variety of mediators (1, 2), which may be a key event in the development of silicosis. Many messengers such as tumor necrosis factor-a (TNF-a) (3-5), interleukin-I (IL-I) (3), and prostanoids (6) probably contribute to the complex network of signals that ultimately results in leukocyte accumulation and fibroblast proliferation. Alterations of leukotriene metabolism in silicosis are poorly defined. AM represent a particularly rich source of leukotrienes (7, 8), and in vitro silica particles have been shown to stimulate AM to an increase in leukotriene B4 (LTB4) secretion (9, 10). The release of LTB4 may be one of the mechanisms that induce interstitial lung inflammation by increasing leukocyte adherence and by chemotactically attracting neutrophils, monocytes, and macrophages (11, (Received in originalform March 2,1992 and in revisedform May 19,1992) Address correspondence to: Diethard Gemsa, M. D., Institute of Immunology, Philipps University, Robert-Koch-Str, 17, D-3550, Marburg, Germany. Abbreviations: alveolar rnacrophage(s), AM; interleukin, IL; leukotriene, LT; tumor necrosis factor-a, TNF-a. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 542-547, 1992
12). LTB4 may also be involved in the regulation of cytokine release from lung macrophages; suppression of the 5-lipoxygenase pathwaydownregulates (13, 14), and addition of LTB4 upregulates, cytokine secretion from mononuclear phagocytes (15, 16). With respect to lung pathology, it has been shown that administration of 5-lipoxygenase inhibitors resulted in a reduction of bleomycin-induced pulmonary fibrosis (17) and in a suppression of Schistosoma mansoniinduced lung granuloma formation (18). We have previously described a rat model of chronic silicosis (5). In response to quartz aerosol inhalation, rats developed interstitial lung fibrosis and multiple foci of inflammatory leukocyte accumulations 4 to 9 months after silica exposure. AM from silica-exposed rats were characterized by an enhanced TNF-a (5) and prostanoid (6) release in comparison with control AM. In this report, we demonstrate that inhalation of silica particles resulted in a downregulation of leukotriene synthesis from AM 5 to 7 mo after exposure.
Materials and Methods Animals Male Fischer 344 rats from a cesarean-derived, barriermaintained colony were obtained from the National Cancer Institute (Bethesda, MD). Rats weighed approximately 175 g
Mohr, Davis, Graebner et al.: Reduced Leukotriene Release from AM in Silicosis
upon arrival, were housed in wire cages with filtered air, and were given sterilized food and water ad libitum. The rats gained weight normally, appeared healthy, and were regularly screened for viral and mycoplasmal infections during up to 7 mo of observation. Housing conditions and mineral exposure met National Institutes of Health and German guidelines. Silica Particle Exposure Aerosol exposure to a-quartz (Min-U'-Sil; US Silica Products, Pittsburgh, PA)has been described previously in detail (5, 19, 20). Briefly, two groups of rats were simultaneously exposed to either the mineral aerosol or the carrier air (sham control) in horizontal flow chambers for 5 h/day for a period of 8 days. The aerosol contained particles of respirable size (0.9 Jtm median diameter ± 2.4 Jtm SD) at an average air concentration of 44.47 ± 0.81 mg/m' (mean ± SD). After silica or sham exposure in Burlington, Vermont, both groups of rats were shipped by plane to Germany after 2 to 3 mo for further experimentation. Animals were killed 5 to 7 mo after exposure for bronchoalveolar lavage. In each experiment, rats exposed to a-quartz were compared with shamair exposed control rats. Bronchoalveolar Lavage After anesthesia, the trachea was cannulated and the heart and lungs were removed en bloc. Alveolar cells were harvested by bronchoalveolar lavage with phosphate-buffered saline as previously described (5). The cell suspensions were centrifuged at 400 X g and resuspended in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 Jtg/ml), Hepes (20 mM), and nonessential amino acids. Cytocentrifuge preparations were stained by the May-Grunwald-Giemsa technique for differential counts of cell types. Macrophage Cultures AM were adjusted to 0.5 X 1()6/ml in RPMI 1640 + 10% heat-inactivated fetal calf serum and seeded into 24-well tissue culture plates (Primaria; Becton-Dickinson, Heidelberg, Germany). Macrophages were allowed to adhere for 1 hat 37°C in a 5 % CO z/95 % air atmosphere, and the nonadherent cells were removed by two washings with culture medium. Adherent cells regularly amounted to 80 ± 3 % of the originally seeded cells whether derived from silica- or sham-exposed animals. The remaining cells consisted of > 85 % macrophages, as determined by morphology, carbon particle phagocytosis, and staining for nonspecific esterase (21). In standard experiments, macrophages were incubated in the presence or absence of opsonized zymosan particles (l X 107/ml) at a cell:particle ratio of 1:20 in RPMI medium without additional serum. Zymosan particles (Serva, Heidelberg, Germany) were opsonized by incubation with noninactivated fetal calf serum for 30 min at 37°C. The amount of 1 X 107 zymosan particles contained < 0.1 ng endotoxin contamination, as determined by the limulus amebocyte test (Whittaker Bioproducts Inc., Walkersville, MD). At the end of the incubation period, the macrophage supernatants were rendered cell free by centrifugation and were stored at -70°C until tested for leukotrienecontent or TNF-a bioactivity.
543
Assays for Leukotriene Release LTB, and LTC, in macrophage supernatants were determined by a radioimmunoassay that had previously been developed in our laboratory (22). Briefly, 100 Jtl of culture supernatants or synthetic [3H]LTB, or [3H]LTC, (specific activity, 60 Ci/mmol; NEN, Dreieich, Germany) and 100 Jtl of a rabbit anti-Ll'B, or LTC. antiserum were incubated together with Tris/bovine serum albumin buffer in 1.5-mlpolypropylene test tubes. A goat anti-rabbit IgG serum (100 Jtl) was added, and incubation was continued overnight. The tubes were centrifuged at 12,000 X g for 30 min at 4°C. The precipitate was dissolved in 500 Jtl of 0.1 N NaOH, diluted with 4 ml scintillation cocktail (Rotiszint; Roth, Karlsruhe, Germany), and radioactivity was measured in a liquid scintillation counter. A calibration curve was established with synthetic LTB, or LTC, in a range from 0.0625 to 16 ng/ml. The cross-reactivities of the LTB, antibodies were < 1% for LTC" 5-HETE, and other eicosanoids. The cross-reactivities of the LTC, antiserum were < 1% for LTB" 5-HETE, and other eicosanoids. Assay for TNF-o: Release TNF-o: bioactivity in macrophage culture supernatants was measured as cytotoxicity against L929 cells as recently described in detail (5). Briefly, L929 cells (5 X 10'/0.2 ml) were grown to confluency, and thereafter incubated for 18 h with actinomycin D (l Jtg/ml)and various dilutions of macrophage culture supernatants or of murine recombinant TNF-o: (kindly provided by Dr. G. R. Adolf, Ernst-BoehringerInstitute, Vienna, Austria). The viability of L929 cells was determined by staining with crystal violet (0.25% in 20% methanol) for 10 min. Thereafter, the dye was extracted and photometrically measured at 550 nm. TNF-o: activity is expressed in U/ml, in which a unit is defined as the TNF-o: amount required to lyse 50% of L929 target cells. Murine recombinant TNF-a was used to calibrate the assay system. Specificity of TNF-o: determinations in culture supernatants was corroborated by parallel use of TNF-a-insensitive L929 cells (23, 24) and neutralization of TNF-a bioactivity by a highly specific rabbit anti-murine TNF-a antiserum (kindly supplied by Dr. D. Mannel, German Cancer Research Park, Heidelberg, Germany). Agents (VZ 65, LTB,) added to macrophages cultures did not interfere with TNF-o:cytotoxicity on L929 cells (data not shown). Reagents The lipoxygenase inhibitor VZ 65 (l-(3,4-dihydroxyphenyl)ll-hydroxyundeca-l,9 diin) was kindly donated by Grunenthal GmbH (Stolberg, Germany). LTB, was purchased from Calbiochem GmbH (Frankfurt, Germany). Statistical Evaluation Statistically significant differences were calculated by the Student's t test.
Results General Observations We established a rat model of chronic silicosis that was previously described in detail (5). Aerosol exposure to silica particles resulted in an influx of inflammatory mononuclear
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
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C4
from Alveolar Macrophages
Carsten Mohr, Gerald S. Davis, Christine Graebner, Susanne Amann, 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
Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-a (TNF-a) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-a production, which was higher in AM from silicaexposed than from control rats. To study the interrelation between leukotriene and TNF-a release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosaninduced TNF-a release from AM in a dose-dependent manner, and TNF-a production could be restored almost completely by addition of LTB4 • These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-a production during silicosis.
The inhalation of silica dust leads to silicosis, a lung disease characterized by interstitial inflammation and fibrosis. Alveolar macrophages (AM) respond to silica inhalation by increased secretion of a variety of mediators (1, 2), which may be a key event in the development of silicosis. Many messengers such as tumor necrosis factor-a (TNF-a) (3-5), interleukin-I (IL-I) (3), and prostanoids (6) probably contribute to the complex network of signals that ultimately results in leukocyte accumulation and fibroblast proliferation. Alterations of leukotriene metabolism in silicosis are poorly defined. AM represent a particularly rich source of leukotrienes (7, 8), and in vitro silica particles have been shown to stimulate AM to an increase in leukotriene B4 (LTB4) secretion (9, 10). The release of LTB4 may be one of the mechanisms that induce interstitial lung inflammation by increasing leukocyte adherence and by chemotactically attracting neutrophils, monocytes, and macrophages (11, (Received in originalform March 2,1992 and in revisedform May 19,1992) Address correspondence to: Diethard Gemsa, M. D., Institute of Immunology, Philipps University, Robert-Koch-Str, 17, D-3550, Marburg, Germany. Abbreviations: alveolar rnacrophage(s), AM; interleukin, IL; leukotriene, LT; tumor necrosis factor-a, TNF-a. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 542-547, 1992
12). LTB4 may also be involved in the regulation of cytokine release from lung macrophages; suppression of the 5-lipoxygenase pathwaydownregulates (13, 14), and addition of LTB4 upregulates, cytokine secretion from mononuclear phagocytes (15, 16). With respect to lung pathology, it has been shown that administration of 5-lipoxygenase inhibitors resulted in a reduction of bleomycin-induced pulmonary fibrosis (17) and in a suppression of Schistosoma mansoniinduced lung granuloma formation (18). We have previously described a rat model of chronic silicosis (5). In response to quartz aerosol inhalation, rats developed interstitial lung fibrosis and multiple foci of inflammatory leukocyte accumulations 4 to 9 months after silica exposure. AM from silica-exposed rats were characterized by an enhanced TNF-a (5) and prostanoid (6) release in comparison with control AM. In this report, we demonstrate that inhalation of silica particles resulted in a downregulation of leukotriene synthesis from AM 5 to 7 mo after exposure.
Materials and Methods Animals Male Fischer 344 rats from a cesarean-derived, barriermaintained colony were obtained from the National Cancer Institute (Bethesda, MD). Rats weighed approximately 175 g
Mohr, Davis, Graebner et al.: Reduced Leukotriene Release from AM in Silicosis
upon arrival, were housed in wire cages with filtered air, and were given sterilized food and water ad libitum. The rats gained weight normally, appeared healthy, and were regularly screened for viral and mycoplasmal infections during up to 7 mo of observation. Housing conditions and mineral exposure met National Institutes of Health and German guidelines. Silica Particle Exposure Aerosol exposure to a-quartz (Min-U'-Sil; US Silica Products, Pittsburgh, PA)has been described previously in detail (5, 19, 20). Briefly, two groups of rats were simultaneously exposed to either the mineral aerosol or the carrier air (sham control) in horizontal flow chambers for 5 h/day for a period of 8 days. The aerosol contained particles of respirable size (0.9 Jtm median diameter ± 2.4 Jtm SD) at an average air concentration of 44.47 ± 0.81 mg/m' (mean ± SD). After silica or sham exposure in Burlington, Vermont, both groups of rats were shipped by plane to Germany after 2 to 3 mo for further experimentation. Animals were killed 5 to 7 mo after exposure for bronchoalveolar lavage. In each experiment, rats exposed to a-quartz were compared with shamair exposed control rats. Bronchoalveolar Lavage After anesthesia, the trachea was cannulated and the heart and lungs were removed en bloc. Alveolar cells were harvested by bronchoalveolar lavage with phosphate-buffered saline as previously described (5). The cell suspensions were centrifuged at 400 X g and resuspended in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 Jtg/ml), Hepes (20 mM), and nonessential amino acids. Cytocentrifuge preparations were stained by the May-Grunwald-Giemsa technique for differential counts of cell types. Macrophage Cultures AM were adjusted to 0.5 X 1()6/ml in RPMI 1640 + 10% heat-inactivated fetal calf serum and seeded into 24-well tissue culture plates (Primaria; Becton-Dickinson, Heidelberg, Germany). Macrophages were allowed to adhere for 1 hat 37°C in a 5 % CO z/95 % air atmosphere, and the nonadherent cells were removed by two washings with culture medium. Adherent cells regularly amounted to 80 ± 3 % of the originally seeded cells whether derived from silica- or sham-exposed animals. The remaining cells consisted of > 85 % macrophages, as determined by morphology, carbon particle phagocytosis, and staining for nonspecific esterase (21). In standard experiments, macrophages were incubated in the presence or absence of opsonized zymosan particles (l X 107/ml) at a cell:particle ratio of 1:20 in RPMI medium without additional serum. Zymosan particles (Serva, Heidelberg, Germany) were opsonized by incubation with noninactivated fetal calf serum for 30 min at 37°C. The amount of 1 X 107 zymosan particles contained < 0.1 ng endotoxin contamination, as determined by the limulus amebocyte test (Whittaker Bioproducts Inc., Walkersville, MD). At the end of the incubation period, the macrophage supernatants were rendered cell free by centrifugation and were stored at -70°C until tested for leukotrienecontent or TNF-a bioactivity.
543
Assays for Leukotriene Release LTB, and LTC, in macrophage supernatants were determined by a radioimmunoassay that had previously been developed in our laboratory (22). Briefly, 100 Jtl of culture supernatants or synthetic [3H]LTB, or [3H]LTC, (specific activity, 60 Ci/mmol; NEN, Dreieich, Germany) and 100 Jtl of a rabbit anti-Ll'B, or LTC. antiserum were incubated together with Tris/bovine serum albumin buffer in 1.5-mlpolypropylene test tubes. A goat anti-rabbit IgG serum (100 Jtl) was added, and incubation was continued overnight. The tubes were centrifuged at 12,000 X g for 30 min at 4°C. The precipitate was dissolved in 500 Jtl of 0.1 N NaOH, diluted with 4 ml scintillation cocktail (Rotiszint; Roth, Karlsruhe, Germany), and radioactivity was measured in a liquid scintillation counter. A calibration curve was established with synthetic LTB, or LTC, in a range from 0.0625 to 16 ng/ml. The cross-reactivities of the LTB, antibodies were < 1% for LTC" 5-HETE, and other eicosanoids. The cross-reactivities of the LTC, antiserum were < 1% for LTB" 5-HETE, and other eicosanoids. Assay for TNF-o: Release TNF-o: bioactivity in macrophage culture supernatants was measured as cytotoxicity against L929 cells as recently described in detail (5). Briefly, L929 cells (5 X 10'/0.2 ml) were grown to confluency, and thereafter incubated for 18 h with actinomycin D (l Jtg/ml)and various dilutions of macrophage culture supernatants or of murine recombinant TNF-o: (kindly provided by Dr. G. R. Adolf, Ernst-BoehringerInstitute, Vienna, Austria). The viability of L929 cells was determined by staining with crystal violet (0.25% in 20% methanol) for 10 min. Thereafter, the dye was extracted and photometrically measured at 550 nm. TNF-o: activity is expressed in U/ml, in which a unit is defined as the TNF-o: amount required to lyse 50% of L929 target cells. Murine recombinant TNF-a was used to calibrate the assay system. Specificity of TNF-o: determinations in culture supernatants was corroborated by parallel use of TNF-a-insensitive L929 cells (23, 24) and neutralization of TNF-a bioactivity by a highly specific rabbit anti-murine TNF-a antiserum (kindly supplied by Dr. D. Mannel, German Cancer Research Park, Heidelberg, Germany). Agents (VZ 65, LTB,) added to macrophages cultures did not interfere with TNF-o:cytotoxicity on L929 cells (data not shown). Reagents The lipoxygenase inhibitor VZ 65 (l-(3,4-dihydroxyphenyl)ll-hydroxyundeca-l,9 diin) was kindly donated by Grunenthal GmbH (Stolberg, Germany). LTB, was purchased from Calbiochem GmbH (Frankfurt, Germany). Statistical Evaluation Statistically significant differences were calculated by the Student's t test.
Results General Observations We established a rat model of chronic silicosis that was previously described in detail (5). Aerosol exposure to silica particles resulted in an influx of inflammatory mononuclear
544
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 7 1992
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