Alterations in Eicosanoid Production by Rat Alveolar Type II Cells Isolated after Silica-induced Lung Injury Ralph J. Panos, Norbert F. Voelkel, Gary R. Cott, Robert J. Mason, and Jay Y. Westcott University of Colorado Health Sciences Center, National Jewish Center for Immunology and Respiratory Medicine, Cardiovascular Pulmonary Research Laboratory, Denver, Colorado
Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-ketoPGF 1" or thromboxane B2 (TxB2) . Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE 2 and 6-keto-PGF 1" production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE 2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE 2 between the normotrophic and the hypertrophic type II cells. After 1 day in culture, the cell size differences in PGE 2 and TxB2 production between the normotrophic and hypertrophic type II cells isolated from silica-treated animals were no longer present. We conclude that the metabolism of AA by isolated type II cells is altered by in vivo silica-induced lung injury and that hypertrophic and normotrophic type II cells isolated from silica-treated animals have distinct eicosanoid production profiles that distinguish these populations of type II cells from each other and from type II cells isolated from untreated animals. Increased basal production of PGE 2 and PGI 2 by freshly isolated hypertrophic type II cells suggests that these cells may playa reparative role in the resolution oflung injury.
Arachidonic acid (AA) metabolites are important paracrine factors that provide local physiologic control in the lung (1-3). Prostaglandins modulate blood vessel and airway contraction and dilation (4-6), regulate fibroblast growth and extracellular matrix production (7-12), and influence inflammatory cell activation and migration within the lung (3). Numerous pulmonary cell types including endothelial cells,
macrophages, and epithelial cells have been shown to release and metabolize AA (2, 3, 13-15). Recently, alveolar type II cells in primary culture have been shown to produce eicosanoids from both endogenous and exogenous AA through both the cyclooxygenase and the lipoxygenase pathways (16-20). The principal arachidonic acid metabolites produced by isolated type II cells are prostaglandin (PG) E2 , 6-keto-PGF thromboxane B2 (TxB2 ) , and leukotriene (LT) C4 (18-20). Alveolar type II cells proliferate and differentiate into type I cells, restoring the integrity of the alveolar epithelium after acute lung injury. Both the number and the size of alveolar type II cells increase in many types of pulmonary disease and lung injury models. After intratracheal silica instillation in the nit, there is a time- and dose-dependent increase in the percentage of hypertrophic type II cells (21, 22). We have demonstrated that these hypertrophic type II cells are enriched with proliferating type II cells that maintain their j" ,
(Received in original form July 15, 1991 and in revised form October 7, 1991)
Address correspondence to: Ralph J. Panos, M.D., Department of Medicine, Northwestern University School of Medicine, Room 456, Wesley Pavilion, 250 East Superior Street, Chicago, IL 60611-2950. Abbreviations: arachidonic acid, AA; Dulbecco's modified Eagle's medium, DMEM; fetal bovine serum, FBS; leukotriene, LT; prostaglandin, PG; specific pathogen-free, SPF; thin layer chromatography, TLC; thromboxane B2 , TxB2 • Am. J. Respir. CeU Mol. BioI. Vol. 6. pp. 430-438, 1992
Panos, Voelkel, Cott et al.: Type II Cell Eicosanoid Production after Lung Injury
commitment to DNA synthesis in primary culture (23, 24). Klein and Adamson (25) have shown that silica alters eicosanoid production by alveolar type II cells in primary culture in a concentration-dependent manner and can stimulate a 6-fold increase in PGE2 secretion. Medium conditioned by silica-treated type II cells inhibits fibroblast proliferation and incorporation of proline into collagen. This inhibition is abrogated by indomethacin, suggesting that it is mediated by prostanoids. Thus, the alteration in eicosanoid production by alveolar type II cells after silica-induced injury may be important in the regulation of fibroblast proliferation and extracellular matrix production. Although alveolar type II cells have been demonstrated to produce AA metabolites and exposure of alveolar type II cells to silica in vitro increases the secretion of PGE 2 , the production of prostaglandins by alveolar type II cells isolated after acute lung injury has not been evaluated. Therefore, we investigated the production of eicosanoids by alveolar type II cells isolated after silica-induced lung injury in the rat. To determine if alveolar type II cell size influences eicosanoid production, we separated type II cells isolated after silicainduced lung injury into normotrophic and hypertrophic fractions by centrifugal elutriation and determined their basal and stimulated secretion of eicosanoids on the day of isolation and after 1 day in culture.
Materials and Methods Animals and Silica Instillation Specific pathogen-free (SPF) male Sprague-Dawley rats, weighing 170 to 250 g, were obtained from Bantin and Kingman (Fremont, CA). The rats were housed in a horizontal laminar airflow hood and were transferred to conventional animal quarters after silica instillation. They were given free access to standard laboratory chow and water. Silica instillation was performed as described previously (23). Briefly, crystalline silica (Berkeley Min-U-Sil, less than 5 j.tm particle size; Pennsylvania Glass Sand Corporation, Pittsburgh, PA) was suspended in sterile 0.15 M NaCI and sonicated before use. Rats received 1.25 ml/kg of an anesthetic solution (ketamine hydrochloride; Bristol Laboratories, Syracuse, NY), xylazine (Miles Laboratories, Shawnee, KS), and 0.15 M NaCI (2:1:1, vol/vol/vol) by intraperitoneal injection and were intubated orally with an 18-gauge Teflon catheter. The catheter position was ascertained by lung insufflation. The rats then received silica (100 mg/kg) in 0.5 ml of sterile 0.15 M NaCI followed by 5 ml of air. The animals were ventilated with a Harvard small rodent ventilator until they resumed spontaneous respirations. Isolation and Elutriation of Alveolar Type II Cells Alveolar type II cells were isolated by elastase dissociation and partially purified on discontinuous metrizamide density gradients as described previously (23, 26). Type II cells were isolated from untreated SPF rats and from silica-treated rats 14 days after instillation. The purity of alveolar type II cells was determined by modified Papanicolaou staining and cell viability was assessed by erythrocin B exclusion (27). The alveolar type II cells were further purified by centrifugal elutriation (18, 23, 28). Briefly, the cells were suspended in 3.75 mM Hepes, 1 mg/ml bovine serum albumin
431
(Sigma Chemical Co., St. Louis, MO), 1 mg/ml deoxyribonuclease I (Sigma), 100 U/ml penicillin, and 100 j.tg/ml streptomycin in Hanks' balanced salt solution (GIBCO, Grand Island, NY). The cells were pumped into the rotor separation chamber at a flow rate of 9 ml/min with a rotor speed of 2,000 rpm (23,28). Cells were eluted by incremental increases in flow rate, and the cells in each fraction were centrifuged at 230 x g for 8 min at 4 0 C and pooled. Protein was determined by the folin phenol method (29). To determine cell number and plating efficiency, cells were harvested with 0.05% trypsin in 0.053 mM EDTA (GIBCO), the trypsin was inactivated with media containing 10% fetal bovine serum (FBS), and the cells counted in a hemocytometer. Alveolar Type II Cell Culture Freshly isolated elutriated type II cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine, 10 j.tg/ml gentamicin, 2.5 j.tg/ml amphotericin B, 100 U/ml penicillin, and 100 j.tg/ml streptomycin, and plated onto 48-well plates (Costar, Cambridge, MA) at 3 x 105 cells/well. The cells were maintained at 370 C in a humidified incubator containing 90% air/10% CO2 for 30 min before stimulation. For the day 1 experiments, the alveolar type II cells were cultured in media supplemented with 10% FBS for 20 to 22 h. The media were removed, and the cultures were washed with unsupplemented media to remove serum and nonadherent cells. DMEM (0.5 ml) was added to each well, and basal and stimulated eicosanoid production was determined. Eicosanoid synthesis was stimulated by the addition of either 2 j.tM A23187 (Sigma) or 33 j.tM AA (Calbiochem). A23187 was dissolved in dimethyl sulfoxide. After stimulation for 1 h in a humidified incubator containing 90 % air/ 10% COz, the medium was removed and centrifuged to remove nonadherent cells. The supernatants Werefrozen in an ethanol and dry ice bath and stored at -70 0 C before determination of eicosanoid content. All specimens were assayed within 24 h of collection. Each condition was tested in three to eight independent experiments, and, in each experiment, each condition (basal, A23187, and arachidonate) was tested in triplicate. Eicosanoid Assays Eicosanoids were assayed in unpurified cellular supernatants by enzyme immunoassay as described previously (18, 30). The antibodies used for prostaglandins were specific for the measured prostanoids with less than 1% cross-reactivity with other prostanoids likely to be present. The 6-ketoPGF 1" antiserum was a generous gift from Dr. K. Allen (Colorado State University, Fort Collins, CO), and the PGE2 and TxB2 antisera were gifts from Dr. F. Fitzpatrick (University of Colorado Health Sciences Center, Denver, CO). The leukotriene antibody was purchased from Advanced Magnetics (Cambridge, MA) and has 100% crossreactivity for LTD4 , 56% for LTC4 , and 51% for LTE4 • Eicosanoid Metabolism To determine if the measured differences in PGE 2 were due to differences in PG~ catabolism, the metabolism of PGE2 by both normotrophic and hypertrophic type II cells isolated
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
from silica-treated animals was examined. PH]PGE2 (0.04 j.tCi, 150 Ci/mM; New England Nuclear, Boston, MA) was added to 0.5 ml of DMEM alone or to the supernatant of hypertrophic (cells eluting at 34 ml/min) or normotrophic (cells eluting at 18 ml/min) alveolar type II cells for 1 h. The media and supernatants were removed, acidified, and extracted with ethyl acetate twice. Aliquots of the ethyl acetate extract were spotted on a Whatman 4D silica thin layer chromatography (TLC) plate and developed in a solvent of ethyl acetate/methanol/acetic acid (100:10:1, vol/vol/vol). Areas on the TLC plate were scraped off, and radioactivity eluting with standard PG~ was determined by scintillation spectrophotometry. To determine if leukotrienes were metabolized by type II cells, PH]LTC4 (0.06 j.tCi, 40 Ci/mM; New England Nuclear) was added to normotrophic cells isolated from untreated animals or to medium alone and incubated for 1 h. The medium was removed and injected into an octadecylsilyl cartridge. Eicosanoids were eluted with methanol: water (80: 20, vol/vol). Recovery of added label was 80 to 100 %. After evaporation of the methanol extract, the samples were injected onto an Altex HPLC column using a gradient solvent system as previously described (31). In this system, LTC4 elutes between 29 and 31 min and LTD4/LTE4 elutes between 37 and 39 min. Radioactivity eluting in each 1 ml/min fraction was determined by scintillation spectrophotometry.
lated from the untreated animals eluting at flow rates of 18 and 22 ml/min, respectively. Plating effi- ciency after 1 day in culture was 31 % for the type II cells isolated from untreated animals and 32 % for the cells isolated from the silicatreated rats. There were no differences in the plating efficiencies of the elutriated fractions of type II cells from either the untreated or the silica-treated animals. Eicosanoid production was normalized to cell number in all experiments and expressed as pg/3 x 105 cells. Basal Eicosanoid Production by Freshly Isolated Type II Cells To determine the eicosanoid profile of the freshly isolated alveolar type II cells, we measured basal release of the cyclooxygenase products, TxB2 , 6-keto-PGF 1a , and PGE 2 and the lipoxygenase product, LTC4 (Figure 1). In type II cells isolated from untreated animals, the basal production of PGE 2 was greater than 6-keto-PGF 1a and TxB2 • LTC4 was not dePGE2 1200 Ul
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Statistical Analysis Data are expressed as mean ± SEM. For statistical analysis, the data were grouped into normotrophic cells (cells eluting at 18 and 22 ml/min) and hypertrophic cells (cells eluting at 28 and 34 ml/min). The natural logarithms of the data were analyzed using a repeated measures ANOVA. Significance was set at P < 0.05 and was corrected for multiple comparisons using Sidak's adjustment.
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Results Normotrophic and Hypertrophic Alveolar Type II Cells To determine the relationship between alveolar type II cell size and eicosanoid production and to maximize the purity of the freshly isolated cells, type II cells were purified and separated by centrifugal elutriation into different-sized fractions. We have shown previously that greater than 90 % of alveolar type II cells isolated from untreated SPF rats elute at flow rates of 18 and 22 ml/min (23, 32, 33). Therefore, these cells were considered normotrophic, and the type II cells eluting at flow rates of 28 and 34 ml/min were considered hypertrophic (23, 28). The separation of the type II cells by cell size was confirmed by light microscopy and flow cytometric analysis of forward light scatter (24). The hypertrophic type II cells contained more protein per cell than the normotrophic cells, 120.8 ± 12.7, 148.0 ± 7.3, 162.7 ± 11.5, and 182.3 ± 12.5 j.tg/1 x 106 cells in the cells eluting at flow rates of 18, 22, 28, and 34 ml/min, respectively. The distribution of the isolated type II cells was the same as that reported previously (23, 28). After elutriation, the alveolar type II cell purity was 88.0 ± 1.8, 94.3 ± 2.1, 94.5 ± 2.1, and 94.5 ± 1.3 % in the cells isolated from the silica-treated animals eluting at flow rates of 18, 22, 28, and 34 ml/min, respectively, and 93.0 ± 1.5 and 89.4 ± 1.8% in the cells iso-
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Silica Normotrophic Hypertrophic
Figure 1. Basal prostanoid production is increased in hypertrophic alveolar type II cells. Alveolar type II cells were isolated from untreated and silica-treated animals by elastase dissociation and purification on discontinuous metrizamide gradients and separated by centrifugal elutriation into groups of increasing cell size. (In-. creasing flow rate corresponds to increasing cell size. Cells eluting at 18 and 22 mllmin are normotrophic and cells eluting at 28 and 34 ml/min are hypertrophic.) The cells were plated onto 48-well plates at 3 X 105 cells/well. After incubation in Dulbecco's modified Eagle's medium (DMEM) for 1 h, prostanoids were determined by enzyme immunoassay.
Panos, Voelkel, Cott et al.: Type II Cell Eicosanoid Production after Lung Injury
TABLE 1
duced significantly more PGE 2 and 6-keto-PGF l a than did the normotrophic type II cells isolated from silica-treated animals (cells eluting at 18 and 22 ml/min). There was not a significant difference in TxB2 production between the normotrophic and the hypertrophic type II cells isolated from silica-treated animals. Even when normalized to cellular protein content, eicosanoid production still increased with increasing cell size (Table 1).
Prostanoid production normalized to cellular protein content* Elutriation Fraction
PGE 2
6-Keto-PGF\
TxB 2
(ml/min)
(pglJlg protein)
(pglJlg protein)
(pglJlg protein)
18 22 28 34
6.75 7.15 10.21 11.49
± ± ± ±
2.87 2.70 5.93 6.56
1.61 2.56 2.91 3.41
± ± ± ±
0.44 0.80 0.64 0.96
2.38 2.15 2.45 3.43
± ± ± ±
433
0.80 0.77 0.53 1.75
A23187-stimulated Eicosanoid Production To evaluate changes in the production of eicosanoids under stimulated conditions, we incubated the freshly isolated alveolar type II cells with the calcium ionophore, A23187. This agonist stimulates eicosanoid production by alveolar type II cells in primary culture and the concentration used (2 J.tM) has been shown to induce maximal stimulation of eicosanoid production without inducing cellular injury (18-20). Calcium ionophore increased prostanoid production in all the fractions of type II cells (Figure 2). PGE 2 production approximately doubled after addition of A23187 in the normotrophic groups of cells from both untreated and silicatreated rats, whereas there was a less than 50 % increase in the hypertrophic type II cells isolated from silica-treated animals. Calcium ionophore stimulated a less than 2-fold increase in 6-keto-PGF l a production. This increase was significant in the normotrophic groups of type II cells from both untreated and silica-treated animals. After ionophore stimulation, the levels of PGE 2 and 6-keto-PGF 1a production by the hypertrophic type II cells were no longer significantly different from the normotrophic type II cells isolated from silica-treated animals. In contrast to PGE2 and 6-keto-PGF 1a production, A23187 greatly enhanced TxB2 and LTC4 production by normotrophic type II cells from both untreated and silicatreated animals (Figure 2). The pattern of A23187 stimulation of TxB2 and LTC4 production was similar in the nor-
* Cells were isolated as described in Figure I. After incubation in DMEM for 1 h, prostanoids were determined by enzyme immunoassay. Protein was determined by the folin phenol method (29) and prostanoid production was normalized to cellular protein content.
tected (detection limit, 100 pg/ml). There were no significant differences in prostanoid production between the normotrophic type II cells isolated from the untreated animals and the normotrophic cells isolated from the silica-treated rats. Under basal conditions, the type II cells isolated from the silica-treated animals produced more PGE 2 than 6-ketoPGF Jc or TxB2 (Figure 1). Leukotriene production was not detected in the basal state in any of the normotrophic and hypertrophic type II cells isolated after silica-induced lung injury. Production of PGE 2 and 6-keto-PGF 1a increased with increasing cell size. The smallest type II cells isolated from silica-treated animals (cells eluting at 18 ml/min) produced 245 ± 104 pg/3 x 105 cells of PGE 2 , 86 ± 29 pg/3 x 105 cells of TxB2 , and 59 ± 16 pg/3 X 105 cells of 6-keto-PGF 1a , whereas the largest type II cells isolated from silica-treated animals (cells eluting at 34 ml/min) produced 628 ± 309 pg/3 x 105 cells of PGE 2 , 188 ± 96 pg/ 3 x 105 cells of TxB2 , and 187 ± 53 pg/3 x 105 cells of 6-keto-PGF 1a • The hypertrophic type II cells isolated from silica-treated animals (cells eluting at 28 and 34 ml/min) pro-
6 KETO PGF 1 ALPHA
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300
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Although alveolar type II cells in primary culture have been shown to produce eicosanoids and exposure of type II cells to silica in vitro alters eicosanoid production, the production of eicosanoids by alveolar type II cells isolated after acute lung injury in vivo has not been evaluated. Therefore, we investigated the production of arachidonic acid (AA) metabolites by alveolar type II cells isolated after silica-induced lung injury. Alveolar type II cells were isolated from rats 14 days after intratracheal silica instillation and from untreated animals. Type II cells were separated into normotrophic and hypertrophic populations by centrifugal elutriation, and secreted eicosanoids were determined under basal and stimulated conditions by enzyme immunoassay on the day of isolation and after 1 day in culture. Under basal conditions, freshly isolated type II cells from silica-treated animals produced more prostaglandin (PG) E2 than 6-ketoPGF 1" or thromboxane B2 (TxB2) . Production of all three prostanoids increased with increasing cell size. The calcium ionophore A23187 stimulated a less than 2-fold increase in PGE 2 and 6-keto-PGF 1" production in all groups of cells. In contrast, this calcium ionophore greatly enhanced TxB2 and leukotriene C4 (LTC4) production by normotrophic type II cells from both untreated and silica-treated animals. Incubation with exogenous AA suggested that the increased capability of the hypertrophic cells to synthesize PGE 2 and TxB2 was due primarily to an increase in arachidonate availability. The hypertrophic type II cells also appear to have increased prostacyclin synthase activity. There were no differences in the catabolism of PGE 2 between the normotrophic and the hypertrophic type II cells. After 1 day in culture, the cell size differences in PGE 2 and TxB2 production between the normotrophic and hypertrophic type II cells isolated from silica-treated animals were no longer present. We conclude that the metabolism of AA by isolated type II cells is altered by in vivo silica-induced lung injury and that hypertrophic and normotrophic type II cells isolated from silica-treated animals have distinct eicosanoid production profiles that distinguish these populations of type II cells from each other and from type II cells isolated from untreated animals. Increased basal production of PGE 2 and PGI 2 by freshly isolated hypertrophic type II cells suggests that these cells may playa reparative role in the resolution oflung injury.
Arachidonic acid (AA) metabolites are important paracrine factors that provide local physiologic control in the lung (1-3). Prostaglandins modulate blood vessel and airway contraction and dilation (4-6), regulate fibroblast growth and extracellular matrix production (7-12), and influence inflammatory cell activation and migration within the lung (3). Numerous pulmonary cell types including endothelial cells,
macrophages, and epithelial cells have been shown to release and metabolize AA (2, 3, 13-15). Recently, alveolar type II cells in primary culture have been shown to produce eicosanoids from both endogenous and exogenous AA through both the cyclooxygenase and the lipoxygenase pathways (16-20). The principal arachidonic acid metabolites produced by isolated type II cells are prostaglandin (PG) E2 , 6-keto-PGF thromboxane B2 (TxB2 ) , and leukotriene (LT) C4 (18-20). Alveolar type II cells proliferate and differentiate into type I cells, restoring the integrity of the alveolar epithelium after acute lung injury. Both the number and the size of alveolar type II cells increase in many types of pulmonary disease and lung injury models. After intratracheal silica instillation in the nit, there is a time- and dose-dependent increase in the percentage of hypertrophic type II cells (21, 22). We have demonstrated that these hypertrophic type II cells are enriched with proliferating type II cells that maintain their j" ,
(Received in original form July 15, 1991 and in revised form October 7, 1991)
Address correspondence to: Ralph J. Panos, M.D., Department of Medicine, Northwestern University School of Medicine, Room 456, Wesley Pavilion, 250 East Superior Street, Chicago, IL 60611-2950. Abbreviations: arachidonic acid, AA; Dulbecco's modified Eagle's medium, DMEM; fetal bovine serum, FBS; leukotriene, LT; prostaglandin, PG; specific pathogen-free, SPF; thin layer chromatography, TLC; thromboxane B2 , TxB2 • Am. J. Respir. CeU Mol. BioI. Vol. 6. pp. 430-438, 1992
Panos, Voelkel, Cott et al.: Type II Cell Eicosanoid Production after Lung Injury
commitment to DNA synthesis in primary culture (23, 24). Klein and Adamson (25) have shown that silica alters eicosanoid production by alveolar type II cells in primary culture in a concentration-dependent manner and can stimulate a 6-fold increase in PGE2 secretion. Medium conditioned by silica-treated type II cells inhibits fibroblast proliferation and incorporation of proline into collagen. This inhibition is abrogated by indomethacin, suggesting that it is mediated by prostanoids. Thus, the alteration in eicosanoid production by alveolar type II cells after silica-induced injury may be important in the regulation of fibroblast proliferation and extracellular matrix production. Although alveolar type II cells have been demonstrated to produce AA metabolites and exposure of alveolar type II cells to silica in vitro increases the secretion of PGE 2 , the production of prostaglandins by alveolar type II cells isolated after acute lung injury has not been evaluated. Therefore, we investigated the production of eicosanoids by alveolar type II cells isolated after silica-induced lung injury in the rat. To determine if alveolar type II cell size influences eicosanoid production, we separated type II cells isolated after silicainduced lung injury into normotrophic and hypertrophic fractions by centrifugal elutriation and determined their basal and stimulated secretion of eicosanoids on the day of isolation and after 1 day in culture.
Materials and Methods Animals and Silica Instillation Specific pathogen-free (SPF) male Sprague-Dawley rats, weighing 170 to 250 g, were obtained from Bantin and Kingman (Fremont, CA). The rats were housed in a horizontal laminar airflow hood and were transferred to conventional animal quarters after silica instillation. They were given free access to standard laboratory chow and water. Silica instillation was performed as described previously (23). Briefly, crystalline silica (Berkeley Min-U-Sil, less than 5 j.tm particle size; Pennsylvania Glass Sand Corporation, Pittsburgh, PA) was suspended in sterile 0.15 M NaCI and sonicated before use. Rats received 1.25 ml/kg of an anesthetic solution (ketamine hydrochloride; Bristol Laboratories, Syracuse, NY), xylazine (Miles Laboratories, Shawnee, KS), and 0.15 M NaCI (2:1:1, vol/vol/vol) by intraperitoneal injection and were intubated orally with an 18-gauge Teflon catheter. The catheter position was ascertained by lung insufflation. The rats then received silica (100 mg/kg) in 0.5 ml of sterile 0.15 M NaCI followed by 5 ml of air. The animals were ventilated with a Harvard small rodent ventilator until they resumed spontaneous respirations. Isolation and Elutriation of Alveolar Type II Cells Alveolar type II cells were isolated by elastase dissociation and partially purified on discontinuous metrizamide density gradients as described previously (23, 26). Type II cells were isolated from untreated SPF rats and from silica-treated rats 14 days after instillation. The purity of alveolar type II cells was determined by modified Papanicolaou staining and cell viability was assessed by erythrocin B exclusion (27). The alveolar type II cells were further purified by centrifugal elutriation (18, 23, 28). Briefly, the cells were suspended in 3.75 mM Hepes, 1 mg/ml bovine serum albumin
431
(Sigma Chemical Co., St. Louis, MO), 1 mg/ml deoxyribonuclease I (Sigma), 100 U/ml penicillin, and 100 j.tg/ml streptomycin in Hanks' balanced salt solution (GIBCO, Grand Island, NY). The cells were pumped into the rotor separation chamber at a flow rate of 9 ml/min with a rotor speed of 2,000 rpm (23,28). Cells were eluted by incremental increases in flow rate, and the cells in each fraction were centrifuged at 230 x g for 8 min at 4 0 C and pooled. Protein was determined by the folin phenol method (29). To determine cell number and plating efficiency, cells were harvested with 0.05% trypsin in 0.053 mM EDTA (GIBCO), the trypsin was inactivated with media containing 10% fetal bovine serum (FBS), and the cells counted in a hemocytometer. Alveolar Type II Cell Culture Freshly isolated elutriated type II cells were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine, 10 j.tg/ml gentamicin, 2.5 j.tg/ml amphotericin B, 100 U/ml penicillin, and 100 j.tg/ml streptomycin, and plated onto 48-well plates (Costar, Cambridge, MA) at 3 x 105 cells/well. The cells were maintained at 370 C in a humidified incubator containing 90% air/10% CO2 for 30 min before stimulation. For the day 1 experiments, the alveolar type II cells were cultured in media supplemented with 10% FBS for 20 to 22 h. The media were removed, and the cultures were washed with unsupplemented media to remove serum and nonadherent cells. DMEM (0.5 ml) was added to each well, and basal and stimulated eicosanoid production was determined. Eicosanoid synthesis was stimulated by the addition of either 2 j.tM A23187 (Sigma) or 33 j.tM AA (Calbiochem). A23187 was dissolved in dimethyl sulfoxide. After stimulation for 1 h in a humidified incubator containing 90 % air/ 10% COz, the medium was removed and centrifuged to remove nonadherent cells. The supernatants Werefrozen in an ethanol and dry ice bath and stored at -70 0 C before determination of eicosanoid content. All specimens were assayed within 24 h of collection. Each condition was tested in three to eight independent experiments, and, in each experiment, each condition (basal, A23187, and arachidonate) was tested in triplicate. Eicosanoid Assays Eicosanoids were assayed in unpurified cellular supernatants by enzyme immunoassay as described previously (18, 30). The antibodies used for prostaglandins were specific for the measured prostanoids with less than 1% cross-reactivity with other prostanoids likely to be present. The 6-ketoPGF 1" antiserum was a generous gift from Dr. K. Allen (Colorado State University, Fort Collins, CO), and the PGE2 and TxB2 antisera were gifts from Dr. F. Fitzpatrick (University of Colorado Health Sciences Center, Denver, CO). The leukotriene antibody was purchased from Advanced Magnetics (Cambridge, MA) and has 100% crossreactivity for LTD4 , 56% for LTC4 , and 51% for LTE4 • Eicosanoid Metabolism To determine if the measured differences in PGE 2 were due to differences in PG~ catabolism, the metabolism of PGE2 by both normotrophic and hypertrophic type II cells isolated
432
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992
from silica-treated animals was examined. PH]PGE2 (0.04 j.tCi, 150 Ci/mM; New England Nuclear, Boston, MA) was added to 0.5 ml of DMEM alone or to the supernatant of hypertrophic (cells eluting at 34 ml/min) or normotrophic (cells eluting at 18 ml/min) alveolar type II cells for 1 h. The media and supernatants were removed, acidified, and extracted with ethyl acetate twice. Aliquots of the ethyl acetate extract were spotted on a Whatman 4D silica thin layer chromatography (TLC) plate and developed in a solvent of ethyl acetate/methanol/acetic acid (100:10:1, vol/vol/vol). Areas on the TLC plate were scraped off, and radioactivity eluting with standard PG~ was determined by scintillation spectrophotometry. To determine if leukotrienes were metabolized by type II cells, PH]LTC4 (0.06 j.tCi, 40 Ci/mM; New England Nuclear) was added to normotrophic cells isolated from untreated animals or to medium alone and incubated for 1 h. The medium was removed and injected into an octadecylsilyl cartridge. Eicosanoids were eluted with methanol: water (80: 20, vol/vol). Recovery of added label was 80 to 100 %. After evaporation of the methanol extract, the samples were injected onto an Altex HPLC column using a gradient solvent system as previously described (31). In this system, LTC4 elutes between 29 and 31 min and LTD4/LTE4 elutes between 37 and 39 min. Radioactivity eluting in each 1 ml/min fraction was determined by scintillation spectrophotometry.
lated from the untreated animals eluting at flow rates of 18 and 22 ml/min, respectively. Plating effi- ciency after 1 day in culture was 31 % for the type II cells isolated from untreated animals and 32 % for the cells isolated from the silicatreated rats. There were no differences in the plating efficiencies of the elutriated fractions of type II cells from either the untreated or the silica-treated animals. Eicosanoid production was normalized to cell number in all experiments and expressed as pg/3 x 105 cells. Basal Eicosanoid Production by Freshly Isolated Type II Cells To determine the eicosanoid profile of the freshly isolated alveolar type II cells, we measured basal release of the cyclooxygenase products, TxB2 , 6-keto-PGF 1a , and PGE 2 and the lipoxygenase product, LTC4 (Figure 1). In type II cells isolated from untreated animals, the basal production of PGE 2 was greater than 6-keto-PGF 1a and TxB2 • LTC4 was not dePGE2 1200 Ul
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Statistical Analysis Data are expressed as mean ± SEM. For statistical analysis, the data were grouped into normotrophic cells (cells eluting at 18 and 22 ml/min) and hypertrophic cells (cells eluting at 28 and 34 ml/min). The natural logarithms of the data were analyzed using a repeated measures ANOVA. Significance was set at P < 0.05 and was corrected for multiple comparisons using Sidak's adjustment.
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Results Normotrophic and Hypertrophic Alveolar Type II Cells To determine the relationship between alveolar type II cell size and eicosanoid production and to maximize the purity of the freshly isolated cells, type II cells were purified and separated by centrifugal elutriation into different-sized fractions. We have shown previously that greater than 90 % of alveolar type II cells isolated from untreated SPF rats elute at flow rates of 18 and 22 ml/min (23, 32, 33). Therefore, these cells were considered normotrophic, and the type II cells eluting at flow rates of 28 and 34 ml/min were considered hypertrophic (23, 28). The separation of the type II cells by cell size was confirmed by light microscopy and flow cytometric analysis of forward light scatter (24). The hypertrophic type II cells contained more protein per cell than the normotrophic cells, 120.8 ± 12.7, 148.0 ± 7.3, 162.7 ± 11.5, and 182.3 ± 12.5 j.tg/1 x 106 cells in the cells eluting at flow rates of 18, 22, 28, and 34 ml/min, respectively. The distribution of the isolated type II cells was the same as that reported previously (23, 28). After elutriation, the alveolar type II cell purity was 88.0 ± 1.8, 94.3 ± 2.1, 94.5 ± 2.1, and 94.5 ± 1.3 % in the cells isolated from the silica-treated animals eluting at flow rates of 18, 22, 28, and 34 ml/min, respectively, and 93.0 ± 1.5 and 89.4 ± 1.8% in the cells iso-
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Elutriation Flow Rate (ml/min) Untreated
Silica Normotrophic Hypertrophic
Figure 1. Basal prostanoid production is increased in hypertrophic alveolar type II cells. Alveolar type II cells were isolated from untreated and silica-treated animals by elastase dissociation and purification on discontinuous metrizamide gradients and separated by centrifugal elutriation into groups of increasing cell size. (In-. creasing flow rate corresponds to increasing cell size. Cells eluting at 18 and 22 mllmin are normotrophic and cells eluting at 28 and 34 ml/min are hypertrophic.) The cells were plated onto 48-well plates at 3 X 105 cells/well. After incubation in Dulbecco's modified Eagle's medium (DMEM) for 1 h, prostanoids were determined by enzyme immunoassay.
Panos, Voelkel, Cott et al.: Type II Cell Eicosanoid Production after Lung Injury
TABLE 1
duced significantly more PGE 2 and 6-keto-PGF l a than did the normotrophic type II cells isolated from silica-treated animals (cells eluting at 18 and 22 ml/min). There was not a significant difference in TxB2 production between the normotrophic and the hypertrophic type II cells isolated from silica-treated animals. Even when normalized to cellular protein content, eicosanoid production still increased with increasing cell size (Table 1).
Prostanoid production normalized to cellular protein content* Elutriation Fraction
PGE 2
6-Keto-PGF\
TxB 2
(ml/min)
(pglJlg protein)
(pglJlg protein)
(pglJlg protein)
18 22 28 34
6.75 7.15 10.21 11.49
± ± ± ±
2.87 2.70 5.93 6.56
1.61 2.56 2.91 3.41
± ± ± ±
0.44 0.80 0.64 0.96
2.38 2.15 2.45 3.43
± ± ± ±
433
0.80 0.77 0.53 1.75
A23187-stimulated Eicosanoid Production To evaluate changes in the production of eicosanoids under stimulated conditions, we incubated the freshly isolated alveolar type II cells with the calcium ionophore, A23187. This agonist stimulates eicosanoid production by alveolar type II cells in primary culture and the concentration used (2 J.tM) has been shown to induce maximal stimulation of eicosanoid production without inducing cellular injury (18-20). Calcium ionophore increased prostanoid production in all the fractions of type II cells (Figure 2). PGE 2 production approximately doubled after addition of A23187 in the normotrophic groups of cells from both untreated and silicatreated rats, whereas there was a less than 50 % increase in the hypertrophic type II cells isolated from silica-treated animals. Calcium ionophore stimulated a less than 2-fold increase in 6-keto-PGF l a production. This increase was significant in the normotrophic groups of type II cells from both untreated and silica-treated animals. After ionophore stimulation, the levels of PGE 2 and 6-keto-PGF 1a production by the hypertrophic type II cells were no longer significantly different from the normotrophic type II cells isolated from silica-treated animals. In contrast to PGE2 and 6-keto-PGF 1a production, A23187 greatly enhanced TxB2 and LTC4 production by normotrophic type II cells from both untreated and silicatreated animals (Figure 2). The pattern of A23187 stimulation of TxB2 and LTC4 production was similar in the nor-
* Cells were isolated as described in Figure I. After incubation in DMEM for 1 h, prostanoids were determined by enzyme immunoassay. Protein was determined by the folin phenol method (29) and prostanoid production was normalized to cellular protein content.
tected (detection limit, 100 pg/ml). There were no significant differences in prostanoid production between the normotrophic type II cells isolated from the untreated animals and the normotrophic cells isolated from the silica-treated rats. Under basal conditions, the type II cells isolated from the silica-treated animals produced more PGE 2 than 6-ketoPGF Jc or TxB2 (Figure 1). Leukotriene production was not detected in the basal state in any of the normotrophic and hypertrophic type II cells isolated after silica-induced lung injury. Production of PGE 2 and 6-keto-PGF 1a increased with increasing cell size. The smallest type II cells isolated from silica-treated animals (cells eluting at 18 ml/min) produced 245 ± 104 pg/3 x 105 cells of PGE 2 , 86 ± 29 pg/3 x 105 cells of TxB2 , and 59 ± 16 pg/3 X 105 cells of 6-keto-PGF 1a , whereas the largest type II cells isolated from silica-treated animals (cells eluting at 34 ml/min) produced 628 ± 309 pg/3 x 105 cells of PGE 2 , 188 ± 96 pg/ 3 x 105 cells of TxB2 , and 187 ± 53 pg/3 x 105 cells of 6-keto-PGF 1a • The hypertrophic type II cells isolated from silica-treated animals (cells eluting at 28 and 34 ml/min) pro-
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