Chemoattractant and Leukotriene B4 Production from Rat Alveolar Macrophages Exposed to Nitrogen Dioxide Timothy W. Robison, Dennis P. Duncan, and Henry Jay Forman Cell Biology Group, Department of Pediatrics, Department of Pathology, and Institute for Toxicology, University of Southern California, Childrens Hospital of Los Angeles, Los Angeles, California
The present study examined the hypothesis that exposure of alveolar macrophages to nitrogen dioxide (N02) resulted in enhanced production of a lipophilic chemotactic agent for neutrophils, possibly leukotriene B. (LTB.). Neutrophil migration was significantly increased in response to the reconstituted ethyl acetate extract of the medium surrounding macrophages exposed for 1 h to 5 or 20 ppm N0 2 • Compared with air-treated macrophages, production of LTB. was found to be significantly increased by exposure to 5 ppm N0 2 , but unchanged by exposure to 20 ppm N0 2 • Treatment of macrophages with the calcium ionophore A23187 at a concentration of 2 t-tM for 15 min following a l-h exposure to 5 ppm N02 led to a significant increase in the production of LTB. compared with A23187-treated air controls; however, LTB. production in response to the calcium ionophore was unchanged following exposure to 20 ppm N0 2 • Thus, while increased neutrophil migration in response to products from macrophages exposed to 5 ppm N02 correlated with the increased production of LTB., increased migration in response to products from macrophages exposed to 20 ppm N02 suggested the presence of another chemotactic lipid. Lipid peroxidation processes induced by N02 at 5 ppm may lead to the formation of hydroperoxides that enhance the formation of LTB.; yet at 20 ppm, significantly higher concentrations of hydroperoxides may be responsible for impaired LTB. formation. Phorbol ester-stimulated macrophage superoxide production was significantly inhibited in a dose-dependent manner following exposure to N0 2 concentrations of 1, 5, or 20 ppm.
Nitrogen dioxide (N0 2) is a toxic atmospheric pollutant formed as a result of combustion processes, most notably those that result in cigarette smoke and automobile exhaust (1- 3). The oxidation of membrane unsaturated fatty acids to fatty acid peroxides is suspected to be the initiating toxic event in injury caused by N02 (3, 4). Lipid peroxidation processes initiated by N02 can lead to membrane damage and cell death (3). The sites of lung injury by N02 is influenced by the high reactivity of this rather insoluble gas (1). The proximal alveolar region, adjacent to the terminal bronchioles, is a major site of N0 2 toxicity (1). Pulmonary alveolar macrophages are phagocytic cells Key Words: alveolar macrophages, nitrogen dioxide, leukotriene B., chemotaxis, superoxide production, eicosanoids (Received in original form August 23, 1989 and in revised form November 8, 1989) Address correspondence to: Henry Jay Forman, Cell Biology Group, Department of Pediatrics, University of Southern California, Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. Abbreviations: formylmethionylleucylphenylalanine, FMLP; hydroxyeicosatetraenoic acid, HETE; high-performance liquid chromatography, HPLC; leukotriene B., LTB.; nitric oxide, NO; nitrogen dioxide, N02 ; pulmonary alveolar macrophage, PAM; polymorphonuclear neutrophil, PMN; radioimmunoassay, RJA. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 21-26, 1990
that are found in high concentration in the proximal alveolar region, principally residing on the surface of the alveolar lining (1, 5). Macrophages have been found to be a major target of oxidant injury (6, 7) such as that found with N02 toxicity. Morphologic studies have shown that macrophages accumulate and proliferate following N0 2 exposure. With the capacity of these cells to release an array of biologically active molecules, including chemotactic factors, alveolar macrophages are considered to be an important regulator of inflammatory reactions in the lung (8, 9). The most potent chemotactic factor synthesized by alveolar macrophages has been identified as leukotriene B. (LTB.) (10). LTB. is synthesized from arachidonic acid through a pathway beginning with the action of the enzyme 5-lipoxygenase (10). LTB. is a potent chemotactic factor for polymorphonuclear neutrophils (PMN) in vitro (8-10). In addition, LTB. causes PMN to adhere to vascular endothelium and to migrate into extravascular tissues in vivo (8-10). The capacity of macrophages to synthesize LTB. and consequently to recruit PMN to a site of inflammation may be an important defense mechanism in the lung. Its synthesis can be enhanced by lipid hydroperoxides, which are frequently found in many types of inflammatory reactions (11, 12). These peroxides can liberate arachidonic acid as well as interact directly with 5-lipoxygenase to stimulate the
22
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
formation of LTB. (11-13). Hydroperoxides generated from the action of N02 on the plasma membrane of alveolar macrophages may significantly influence the ability of these cells to synthesize LTB., leading to significant changes in the development of inflammatory reactions. N02 exposure is known to incite an inflammatory response in the lower respiratory tract and morphologic descriptions have included an infiltration of PMN (14-16). The present study assessed whether an observed increase in neutrophil chemotaxis in response to products from alveolar macrophages exposed to N02 was due to an enhanced biosynthesis of LTB. or another lipophilic chemotactic substance. Neutrophil chemotaxis was measured in response to products elicited from macrophages exposed to N02 alone or treated with A23187 following exposure to N02 • Synthesis of LTB. by macrophages was measured either following exposure to N02 alone or after subsequent treatment with the calcium ionophore A23187, which has been shown to be a potent agonist for the formation of this lipoxygenase product. The functionality of macrophages, following N0 2 exposure, was assessed by measurement of superoxide production, while the structural integrity was determined by measurement of cellular lactate dehydrogenase (LDH) content.
Exposure to Nitrogen Dioxide N02 possesses a low solubility in aqueous solution, necessitating that cells only be covered by a thin layer of medium to allow direct interaction with gas (17). Each well was filled with 1 ml of KRPH buffer, which contained 125 mM NaCl, 5 mM KCl, 1.0 mM MgSO., 10 mM sodium phosphate, 5 mM glucose, 1.3 mM CaCI2 , and 10 mM HEPES, pH 7.4, at 37° C. Cells adhered to the membrane inside the Transwell are kept moist by fluid from below the membrane, while essentially not covered with fluid on top. N02 (0.1, 1.0, 5.0, or 20 ppm) or air were passed into a sealed Teflon" beaker containing water warmed to 37° C. The gas was bubbled into the water, allowing it to be warmed and humidified before it passed into sealed polycarbonate chambers containing the macrophages in Transwell plates. The chambers were placed in a water bath warmed to 37° C. In addition, each chamber contained a small dish of water warmed to 37° C. This exposure system makes it possible to keep cells warmed to 37° C in a humidified environment. Exposure time was 1 h. Following exposure to N02 , the media in each Transwell was collected for measurement of LTB., acidified to pH 4.5, and then kept frozen at -80° C overnight until extraction.
Materials and Methods
Treatment with A23187 Exposed cells were immediately treated with A23187 following exposure to N02 • A23187 was dissolved in dimethylsulfoxide (DMSO) and then added to medium. The final concentration of DMSO was 0.1%, which was without any effect on cellular arachidonic acid metabolism. The cells were covered with 2 rnl KRPH buffer containing 2 J.l.M A23187. An additional 1 ml of buffer containing A23187 was placed below the membrane of the Transwell. Cells were incubated with A23187 for 15 min at 37° C. Following treatment with the calcium ionophore, the media was processed as described above.
A [3H]leukotriene B. radioimmunoassay kit was obtained from Amersham Corporation (Arlington Heights, IL). Fatty acid-free bovine serum albumin, A23187, N-formylmethionylleucylphenylalanine (FMLP), and RPMI-1640 (Catalog No. R8005) were obtained from Sigma Chemical Co. (St. Louis, MO). Agarose was obtained from ICN Chemical Co. (Irvine, CA). Fetal bovine serum and Medium 199 X 2 containing Earle's modified salts were obtained from GIBCO (Grand Island, NY). Nitrogen dioxide in air at concentrations of 0.1, 1.0, 5.0, and 20 ppm were obtained from Scott Specialty Gases (San Bernadino, CA). Gas concentrations were certified to an analytical accuracy of ~ 5.0%. Compressed air (medical grade) was obtained from Liquid Carbonics (Los Angeles, CA). All other reagents were of analytical grade and obtained from standard commercial sources. All solvents used were of HPLC grade. Preparation and Culture of Alveolar Macrophages Alveolar macrophages were prepared from specific pathogen-, viral-free, 250- to 350-g Sprague-Dawley rats (Crl: CD(SD)BR; Charles River, Wilmington, MA). Animals were killed by intrapertioneal injection of pentobarbital, 100 mg/kg. Alveolar macrophages were obtained by pulmonary lavage as previously described (7). Cells were resuspended at a concentration of 1 million/rnl in sterile filtered RPMI1640 with added 0.1% bovine serum albumin, 100 U penicillin G/rnl, and 100 J.l.g streptomycin/ml, pH 7.2 (RPMI-1640 buffer). Cells (3 million) were added to tissue culture-treated 24.5-mm-diameter Transwells (Costar, Cambridge, MA). Cells were allowed to adhere to the membrane inside the Transwell for 1 hat 37° C. After 1 h, Transwells were rinsed with 0.01 M sodium phosphate-buffered saline (PBS), pH 7.4, to remove nonadherent cells. Adhered macrophages were incubated in RPMI-1640 buffer for 4 h at 37° C.
Measurement of Cellular LDH Activity and Protein Transwell membranes with attached cells were cut out of the holder and placed into 0.1%Triton X-IOO in PBS for measurement of LDH activityusing a LDH assay kit (Sigma) or protein using a Bio-Rad Protein Assay Kit (Richmond, CA). Lipid Extraction Acidified medium was extracted with 3 vol ethyl acetate (4 rnl each for samples treated with air or N02 alone or 6 ml each for samples treated with A23187). The ethyl acetate was dried under a stream of nitrogen (99.999 %). The residue was reconstituted in radioimmunoassay (RIA) buffer for measurement of LTB. as described below. Measurement of Leukotriene B. by Radioimmunoassay LTB. levels in medium following a 1-h exposure to either air or N02 were measured using a RIA kit (Amersham Corp.). Ethyl acetate-extracted samples were reconstituted in 1 ml 0.05 M Tris buffer (pH 8.6) containing 0.1% gelatin. A range-finding assay was performed to determine approximate levels of LTB. present in samples. An aliquot of 50 J.l.l was used for measurement of LTB. from samples exposed to either N02 or air alone. For samples treated with A23187
Robison, Duncan, and Forman: Production of Chemoattractant by Macrophages Exposed to N0 2
following exposure to either air or N0 2 , an aliquot of 100 p.l of a 1:50 dilution was used. Relative cross-reactivity for LTB4 antiserum was 100% for LTB4 and less than 0.5 % for other eicosanoids examined. The sensitivity limit of the RIA was 3.0 pg per tube. Macrophage Production of Chemoattractants The measurement of PMN chemotaxis was performed using the under agarose method (18). A 0.6% agarose gel was poured onto a 60 x 15 mm petri dish, and six sets of triplicate wells 2.4 mm in diameter were made (19). A suspension of 250,000 PMN was placed in the center well, and stimulant or buffer was placed in the respective adjacent wells. FMLP at a concentration of 100 nM was used as a positive control. After 2 h of incubation at 37° C, the plates were fixed with methanol and formalin and the agarose was removed. The PMN remained fixed to the petri dish. The ethyl acetate extract of media from 12 million macrophages (pooled contents of 4 wells of 3 million cells each) exposed to air or varying concentrations of N02 were reconstituted in KRPH buffer containing 0.1% ethanol and used. In addition, the ethyl acetate extract of media from 6 million macrophages (2 wells of 3 million cells each) treated with 2 p.M A23187 following exposure to air or N02 were also used. Chemotaxis was measured using a video analysis system (19). Superoxide Production Following exposure to N02 , alveolar macrophages, adhered to Transwell membranes, were placed into 4 ml KRPH buffer containing 20 p.Mferricytochrome c (7). The respiratory burst was initiated by the addition of 50 ng/ml PMA. Cells were incubated for 30 min at 37° C. The rate of superoxide production was found to be linear over 30 min. The reaction was terminated by removal of the cells from the solution. The rate of superoxide production, which was not superoxide dismutase (SOD)-inhibitable, was determined by the addition of 45 U SOD prior to the addition of PMA (7). Samples were scanned using a Beckman DU-7 spectrophotometer to determine the absorbance at 540 and 550 nm. The absorbance at 540 nm was subtracted from that at 550 nm, and the stimulated superoxide production was then calculated by subtracting the rate of cytochrome c reduction, which was not SOD-inhibitable, from this value. Protein was measured, and values were expressed as nanomoles of superoxide produced per 30 min per milligram of protein. Statistical Analysis The data are expresed as the mean ± SEM. The Student's t test (unpaired) was performed with the Crunch Interactive Statistical Package (San Francisco, CA) to examine differences in eicosanoid levels between N02 and air-treated samples.
Results Cellular Viability For air-treated alveolar macrophages adhered in Transwells, cellular viability was found to be greater than 90 %, as measured by cellular LDH content, for a 6-h exposure period. The viability of these air-treated cells was similar to that for
23
cells covered with medium and kept at 37° C for an identical period of time. For a l-h exposure period to either 0.1 or 1 ppm N02 , cellular viability was unaffected as compared to air-treated cells. For a l-h exposure period to 5 ppm N0 2 , cellular viability was found to be 90% of air-treated cells, and for 20 ppm N0 2 , it was found to be 85%. LTB4 Production in Response to N02 Alone LTB4 levels measured by RIA following a l-h exposure to either air or 0.1, 1, 5, or 20 ppm N02 are shown in Figure 1. LTB4 levels were unchanged between a l-h exposure to either air or 0.1, 1 or 20 ppm N02 ; however, levels were significantly elevated following a l-h exposure to 5 ppm N02 • Control levels of LTB4 released by macrophages exposed to air for 1 h were 1.03 ± 0.08 ng/mg protein. Response to A23187 following a One-hour Exposure to N02 Figure 2 displays changes in eicosanoid levels produced by alveolar macrophages treated with 2 p.M A23187 for 15 min following a l-h exposure to various concentrations of N0 2 • Values are expressed as a percentage of air-treated macrophages. No changes in A23187-induced formation of LTB4 were found between air and 0.1 or 1.0 ppm N02 • A23187 treatment following exposure of 5 ppm N02 significantly enhanced the formation of LTB4 • In contrast, A23187 treatment following exposure to 20 ppm N02 did not produce a significant change in LTB4 levels. Control LTB4 levels produced by macrophages treated for 15 min with 2 p.MA23187 following a l-h exposure to air were 35.2 ± 9.4 ng/mg protein. Changes in LTB4 production by macrophages treated with A23187 following exposure to N02 were independently confirmed through the use of (3H]arachidonic acid incorporated into macrophage phospholipid and separation of eicosanoids by HPLC (data not shown). Production of Chemotactic Agents Figure 3 displays changes in neutrophil chemotaxis (leading front-random migration [LF-RM]) in response to products elicited from alveolar macrophages exposed for 1 h to air or ~
200 p (0.05
0
1:;
c 0
u
~
150
The present study examined the hypothesis that exposure of alveolar macrophages to nitrogen dioxide (N02) resulted in enhanced production of a lipophilic chemotactic agent for neutrophils, possibly leukotriene B. (LTB.). Neutrophil migration was significantly increased in response to the reconstituted ethyl acetate extract of the medium surrounding macrophages exposed for 1 h to 5 or 20 ppm N0 2 • Compared with air-treated macrophages, production of LTB. was found to be significantly increased by exposure to 5 ppm N0 2 , but unchanged by exposure to 20 ppm N0 2 • Treatment of macrophages with the calcium ionophore A23187 at a concentration of 2 t-tM for 15 min following a l-h exposure to 5 ppm N02 led to a significant increase in the production of LTB. compared with A23187-treated air controls; however, LTB. production in response to the calcium ionophore was unchanged following exposure to 20 ppm N0 2 • Thus, while increased neutrophil migration in response to products from macrophages exposed to 5 ppm N02 correlated with the increased production of LTB., increased migration in response to products from macrophages exposed to 20 ppm N02 suggested the presence of another chemotactic lipid. Lipid peroxidation processes induced by N02 at 5 ppm may lead to the formation of hydroperoxides that enhance the formation of LTB.; yet at 20 ppm, significantly higher concentrations of hydroperoxides may be responsible for impaired LTB. formation. Phorbol ester-stimulated macrophage superoxide production was significantly inhibited in a dose-dependent manner following exposure to N0 2 concentrations of 1, 5, or 20 ppm.
Nitrogen dioxide (N0 2) is a toxic atmospheric pollutant formed as a result of combustion processes, most notably those that result in cigarette smoke and automobile exhaust (1- 3). The oxidation of membrane unsaturated fatty acids to fatty acid peroxides is suspected to be the initiating toxic event in injury caused by N02 (3, 4). Lipid peroxidation processes initiated by N02 can lead to membrane damage and cell death (3). The sites of lung injury by N02 is influenced by the high reactivity of this rather insoluble gas (1). The proximal alveolar region, adjacent to the terminal bronchioles, is a major site of N0 2 toxicity (1). Pulmonary alveolar macrophages are phagocytic cells Key Words: alveolar macrophages, nitrogen dioxide, leukotriene B., chemotaxis, superoxide production, eicosanoids (Received in original form August 23, 1989 and in revised form November 8, 1989) Address correspondence to: Henry Jay Forman, Cell Biology Group, Department of Pediatrics, University of Southern California, Childrens Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027. Abbreviations: formylmethionylleucylphenylalanine, FMLP; hydroxyeicosatetraenoic acid, HETE; high-performance liquid chromatography, HPLC; leukotriene B., LTB.; nitric oxide, NO; nitrogen dioxide, N02 ; pulmonary alveolar macrophage, PAM; polymorphonuclear neutrophil, PMN; radioimmunoassay, RJA. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp. 21-26, 1990
that are found in high concentration in the proximal alveolar region, principally residing on the surface of the alveolar lining (1, 5). Macrophages have been found to be a major target of oxidant injury (6, 7) such as that found with N02 toxicity. Morphologic studies have shown that macrophages accumulate and proliferate following N0 2 exposure. With the capacity of these cells to release an array of biologically active molecules, including chemotactic factors, alveolar macrophages are considered to be an important regulator of inflammatory reactions in the lung (8, 9). The most potent chemotactic factor synthesized by alveolar macrophages has been identified as leukotriene B. (LTB.) (10). LTB. is synthesized from arachidonic acid through a pathway beginning with the action of the enzyme 5-lipoxygenase (10). LTB. is a potent chemotactic factor for polymorphonuclear neutrophils (PMN) in vitro (8-10). In addition, LTB. causes PMN to adhere to vascular endothelium and to migrate into extravascular tissues in vivo (8-10). The capacity of macrophages to synthesize LTB. and consequently to recruit PMN to a site of inflammation may be an important defense mechanism in the lung. Its synthesis can be enhanced by lipid hydroperoxides, which are frequently found in many types of inflammatory reactions (11, 12). These peroxides can liberate arachidonic acid as well as interact directly with 5-lipoxygenase to stimulate the
22
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
formation of LTB. (11-13). Hydroperoxides generated from the action of N02 on the plasma membrane of alveolar macrophages may significantly influence the ability of these cells to synthesize LTB., leading to significant changes in the development of inflammatory reactions. N02 exposure is known to incite an inflammatory response in the lower respiratory tract and morphologic descriptions have included an infiltration of PMN (14-16). The present study assessed whether an observed increase in neutrophil chemotaxis in response to products from alveolar macrophages exposed to N02 was due to an enhanced biosynthesis of LTB. or another lipophilic chemotactic substance. Neutrophil chemotaxis was measured in response to products elicited from macrophages exposed to N02 alone or treated with A23187 following exposure to N02 • Synthesis of LTB. by macrophages was measured either following exposure to N02 alone or after subsequent treatment with the calcium ionophore A23187, which has been shown to be a potent agonist for the formation of this lipoxygenase product. The functionality of macrophages, following N0 2 exposure, was assessed by measurement of superoxide production, while the structural integrity was determined by measurement of cellular lactate dehydrogenase (LDH) content.
Exposure to Nitrogen Dioxide N02 possesses a low solubility in aqueous solution, necessitating that cells only be covered by a thin layer of medium to allow direct interaction with gas (17). Each well was filled with 1 ml of KRPH buffer, which contained 125 mM NaCl, 5 mM KCl, 1.0 mM MgSO., 10 mM sodium phosphate, 5 mM glucose, 1.3 mM CaCI2 , and 10 mM HEPES, pH 7.4, at 37° C. Cells adhered to the membrane inside the Transwell are kept moist by fluid from below the membrane, while essentially not covered with fluid on top. N02 (0.1, 1.0, 5.0, or 20 ppm) or air were passed into a sealed Teflon" beaker containing water warmed to 37° C. The gas was bubbled into the water, allowing it to be warmed and humidified before it passed into sealed polycarbonate chambers containing the macrophages in Transwell plates. The chambers were placed in a water bath warmed to 37° C. In addition, each chamber contained a small dish of water warmed to 37° C. This exposure system makes it possible to keep cells warmed to 37° C in a humidified environment. Exposure time was 1 h. Following exposure to N02 , the media in each Transwell was collected for measurement of LTB., acidified to pH 4.5, and then kept frozen at -80° C overnight until extraction.
Materials and Methods
Treatment with A23187 Exposed cells were immediately treated with A23187 following exposure to N02 • A23187 was dissolved in dimethylsulfoxide (DMSO) and then added to medium. The final concentration of DMSO was 0.1%, which was without any effect on cellular arachidonic acid metabolism. The cells were covered with 2 rnl KRPH buffer containing 2 J.l.M A23187. An additional 1 ml of buffer containing A23187 was placed below the membrane of the Transwell. Cells were incubated with A23187 for 15 min at 37° C. Following treatment with the calcium ionophore, the media was processed as described above.
A [3H]leukotriene B. radioimmunoassay kit was obtained from Amersham Corporation (Arlington Heights, IL). Fatty acid-free bovine serum albumin, A23187, N-formylmethionylleucylphenylalanine (FMLP), and RPMI-1640 (Catalog No. R8005) were obtained from Sigma Chemical Co. (St. Louis, MO). Agarose was obtained from ICN Chemical Co. (Irvine, CA). Fetal bovine serum and Medium 199 X 2 containing Earle's modified salts were obtained from GIBCO (Grand Island, NY). Nitrogen dioxide in air at concentrations of 0.1, 1.0, 5.0, and 20 ppm were obtained from Scott Specialty Gases (San Bernadino, CA). Gas concentrations were certified to an analytical accuracy of ~ 5.0%. Compressed air (medical grade) was obtained from Liquid Carbonics (Los Angeles, CA). All other reagents were of analytical grade and obtained from standard commercial sources. All solvents used were of HPLC grade. Preparation and Culture of Alveolar Macrophages Alveolar macrophages were prepared from specific pathogen-, viral-free, 250- to 350-g Sprague-Dawley rats (Crl: CD(SD)BR; Charles River, Wilmington, MA). Animals were killed by intrapertioneal injection of pentobarbital, 100 mg/kg. Alveolar macrophages were obtained by pulmonary lavage as previously described (7). Cells were resuspended at a concentration of 1 million/rnl in sterile filtered RPMI1640 with added 0.1% bovine serum albumin, 100 U penicillin G/rnl, and 100 J.l.g streptomycin/ml, pH 7.2 (RPMI-1640 buffer). Cells (3 million) were added to tissue culture-treated 24.5-mm-diameter Transwells (Costar, Cambridge, MA). Cells were allowed to adhere to the membrane inside the Transwell for 1 hat 37° C. After 1 h, Transwells were rinsed with 0.01 M sodium phosphate-buffered saline (PBS), pH 7.4, to remove nonadherent cells. Adhered macrophages were incubated in RPMI-1640 buffer for 4 h at 37° C.
Measurement of Cellular LDH Activity and Protein Transwell membranes with attached cells were cut out of the holder and placed into 0.1%Triton X-IOO in PBS for measurement of LDH activityusing a LDH assay kit (Sigma) or protein using a Bio-Rad Protein Assay Kit (Richmond, CA). Lipid Extraction Acidified medium was extracted with 3 vol ethyl acetate (4 rnl each for samples treated with air or N02 alone or 6 ml each for samples treated with A23187). The ethyl acetate was dried under a stream of nitrogen (99.999 %). The residue was reconstituted in radioimmunoassay (RIA) buffer for measurement of LTB. as described below. Measurement of Leukotriene B. by Radioimmunoassay LTB. levels in medium following a 1-h exposure to either air or N02 were measured using a RIA kit (Amersham Corp.). Ethyl acetate-extracted samples were reconstituted in 1 ml 0.05 M Tris buffer (pH 8.6) containing 0.1% gelatin. A range-finding assay was performed to determine approximate levels of LTB. present in samples. An aliquot of 50 J.l.l was used for measurement of LTB. from samples exposed to either N02 or air alone. For samples treated with A23187
Robison, Duncan, and Forman: Production of Chemoattractant by Macrophages Exposed to N0 2
following exposure to either air or N0 2 , an aliquot of 100 p.l of a 1:50 dilution was used. Relative cross-reactivity for LTB4 antiserum was 100% for LTB4 and less than 0.5 % for other eicosanoids examined. The sensitivity limit of the RIA was 3.0 pg per tube. Macrophage Production of Chemoattractants The measurement of PMN chemotaxis was performed using the under agarose method (18). A 0.6% agarose gel was poured onto a 60 x 15 mm petri dish, and six sets of triplicate wells 2.4 mm in diameter were made (19). A suspension of 250,000 PMN was placed in the center well, and stimulant or buffer was placed in the respective adjacent wells. FMLP at a concentration of 100 nM was used as a positive control. After 2 h of incubation at 37° C, the plates were fixed with methanol and formalin and the agarose was removed. The PMN remained fixed to the petri dish. The ethyl acetate extract of media from 12 million macrophages (pooled contents of 4 wells of 3 million cells each) exposed to air or varying concentrations of N02 were reconstituted in KRPH buffer containing 0.1% ethanol and used. In addition, the ethyl acetate extract of media from 6 million macrophages (2 wells of 3 million cells each) treated with 2 p.M A23187 following exposure to air or N02 were also used. Chemotaxis was measured using a video analysis system (19). Superoxide Production Following exposure to N02 , alveolar macrophages, adhered to Transwell membranes, were placed into 4 ml KRPH buffer containing 20 p.Mferricytochrome c (7). The respiratory burst was initiated by the addition of 50 ng/ml PMA. Cells were incubated for 30 min at 37° C. The rate of superoxide production was found to be linear over 30 min. The reaction was terminated by removal of the cells from the solution. The rate of superoxide production, which was not superoxide dismutase (SOD)-inhibitable, was determined by the addition of 45 U SOD prior to the addition of PMA (7). Samples were scanned using a Beckman DU-7 spectrophotometer to determine the absorbance at 540 and 550 nm. The absorbance at 540 nm was subtracted from that at 550 nm, and the stimulated superoxide production was then calculated by subtracting the rate of cytochrome c reduction, which was not SOD-inhibitable, from this value. Protein was measured, and values were expressed as nanomoles of superoxide produced per 30 min per milligram of protein. Statistical Analysis The data are expresed as the mean ± SEM. The Student's t test (unpaired) was performed with the Crunch Interactive Statistical Package (San Francisco, CA) to examine differences in eicosanoid levels between N02 and air-treated samples.
Results Cellular Viability For air-treated alveolar macrophages adhered in Transwells, cellular viability was found to be greater than 90 %, as measured by cellular LDH content, for a 6-h exposure period. The viability of these air-treated cells was similar to that for
23
cells covered with medium and kept at 37° C for an identical period of time. For a l-h exposure period to either 0.1 or 1 ppm N02 , cellular viability was unaffected as compared to air-treated cells. For a l-h exposure period to 5 ppm N0 2 , cellular viability was found to be 90% of air-treated cells, and for 20 ppm N0 2 , it was found to be 85%. LTB4 Production in Response to N02 Alone LTB4 levels measured by RIA following a l-h exposure to either air or 0.1, 1, 5, or 20 ppm N02 are shown in Figure 1. LTB4 levels were unchanged between a l-h exposure to either air or 0.1, 1 or 20 ppm N02 ; however, levels were significantly elevated following a l-h exposure to 5 ppm N02 • Control levels of LTB4 released by macrophages exposed to air for 1 h were 1.03 ± 0.08 ng/mg protein. Response to A23187 following a One-hour Exposure to N02 Figure 2 displays changes in eicosanoid levels produced by alveolar macrophages treated with 2 p.M A23187 for 15 min following a l-h exposure to various concentrations of N0 2 • Values are expressed as a percentage of air-treated macrophages. No changes in A23187-induced formation of LTB4 were found between air and 0.1 or 1.0 ppm N02 • A23187 treatment following exposure of 5 ppm N02 significantly enhanced the formation of LTB4 • In contrast, A23187 treatment following exposure to 20 ppm N02 did not produce a significant change in LTB4 levels. Control LTB4 levels produced by macrophages treated for 15 min with 2 p.MA23187 following a l-h exposure to air were 35.2 ± 9.4 ng/mg protein. Changes in LTB4 production by macrophages treated with A23187 following exposure to N02 were independently confirmed through the use of (3H]arachidonic acid incorporated into macrophage phospholipid and separation of eicosanoids by HPLC (data not shown). Production of Chemotactic Agents Figure 3 displays changes in neutrophil chemotaxis (leading front-random migration [LF-RM]) in response to products elicited from alveolar macrophages exposed for 1 h to air or ~
200 p (0.05
0
1:;
c 0
u
~
150
