Prostaglandins 44:101-110, 1992 RELEASE OF ARACHIDONIC ACID METABOLITES FROM ISOLATED HUMAN ALVEOLAR TYPE II CELLS
Ove.~eld 1, P.G. Jo~ens 1, W.A. De Sack~r I,
F.J. van M. Rampart
~ , L.
Bossaert
°
and
P.A.
Vermeire
~
Depts. of i) Respiratory Medicine, 2) Pharmacology, and 3) Intensive Care, University of Antwerp (UIA), B-2610 Antwerpen, Belgium ~n~ACT
Human alveolar type II cells are thought to play a role in the pathogenesis of lung injury. Patterns of mediator release of arachidonic acid metabolism by type II cells were therefore studied after challenge with calcium ionophore A23187, opsonized zymosan and hydrogen peroxide. A time- and concentration dependent release of cyclooxygenase products was observed, with release of PGE 2 > 6-keto-PGFla > TxB 2. Addition of glutathione or bicarbonate further increased the production of PGE 2. N-ethylmaleimide, a sulfhydryl (SH) reactant, induced a dose-dependent increase in the release of TxB 2 and 6-keto-PGFla, but not of PGE 2. This relates most likely to the SH-dependency and glutathione requirement of the PGE2 isomerase and SH-independence of thromboxane and prostacyclin isomerase. INTRODUCTION Alveolar type II cells are important cells for normal lung function because they synthesize and secrete surface active material, maintain the alveolar epithelium by serving as stem cells for both type II and type I cells, and transport fluid from the alveolar subphase into the interstitium (1). The secretory function of type II cells has been studied principally for the production of surfactant. The other secretory functions of type II cells are less well known, although some studies have been done with cells from animals showing production and release of arachidonic acid (AA) metabolites (2,3). Both alveolar type I and type II cells are known targets for many toxic agents (4,5) and are affected in adult respiratory distress syndrome (ARDS) (6). After lung injury, type I1 cells retain the important ability to replace type I cells and to epithelialize the alveolus (7). In ARDS, deficiency of surfactant plays a major role (3). Recent studies have also shown that AA metabolites could regulate surfactant secretion. Enhancement of surfactant secretion was observed with leukotrienes, whereas prostaglandins appeared to control its secretion (8,9). Although these type II pneumocytes only constitute 4% of the alveolar epithelial area, they represent 60% of alveolar epithelial cells by number (10). An improvement of our knowledge of the pathogenesis of ARDS may thus depend on a better understanding of the acute toxicology of these cells. This prompted us to initiate in vitro studies of purified preparations of human type II cells. Copyright © 1992 Butterworth-Heinemann
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Prostaglandins
MATERIAI~ AND M E I ~ O D S Materials Enriched Tyrode's buffer (TGMD) contained 137 mM NaC1, 2.7 mM KC1, 0.4 mM NaH2PO4.2H20, 1.2 mM MgC12.6H20, 1% gelatin (Merck, Darmstadt, FRG), 60 mg deoxyribonuclease type I (EC 3.1.21.1) (DNAse) (Boehringer, Mannheim, FRG), 5.6 mM glucose and 1 U/ml penicillin and 1 ~g/ml streptomycin (pH 7.4, 20°C) (Sigma Chemical Co., St. Louis, MO, USA). Before and after the cell isolation, tissue and cells were kept in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (Gibco Ltd., Paisley, UK). The enzyme solutions were prepared by dissolving 1 mg/ml collagenase (EC 3.4.24.3) (Boehringer) and 0.1 U/ml elastase type I (3.4.21.11) (Sigma) in TGMD. The isotonic shock solution contained 155 mM NH4C1, 10 mM KHCO3, 0.1 mM EDTA and 10 mg/1 phenol red (pH 7.4, 0°C). The osmolality was adjusted to 290 mOsm, as measured by freezing point depression. Percoll (Pharmacia, Uppsala, Sweden) densities of 1.040 and 1.090 g/ml were prepared by diluting a stock-solution of 1.100 g/ml with phosphate buffered saline (PBS). Osmolality of the Percoll densities was 290 mOsm/kg. Human collagen type IV, calcium ionophore A23187, zymosan and N-ethylmaleimide were purchased from Sigma. PGE 2, TxB2, 6-keto-PGFla were obtained from Upjohn, Kalamazoo, MI, USA. Their 3H-labelled forms were purchased from New England Nuclear, Boston, MA. All other chemicals were reagent grade and they were purchased from Merck. Cell isolation Isolation of alveolar type II cells was performed as described previously (11). Briefly, cells were isolated from a macroscopically disease-free part of human lung tissue obtained during resection of bronchial tumours. The tissue was minced into 1 to 2 mm pieces. The fragments were washed extensively with RPMI-1640/10% FCS to remove blood cells as much as possible. Subsequently the lung fragments were digested during four consecutive 30 minute exposures at 37°C to the enzyme solution. Dispersed cells were collected, filtered through sterile gauze, centrifuged and resuspended in TGMD. Erythrocytes were removed by an isotonic shock procedure (12). The cell suspension at 1.5 to 2 x 106 cells/ml was cultured for 90 min in 25 cm 2 plastic flasks (7 ml/flask) (Gibco/Nunc, Ghent, Belgium) during which time most of the alveolar macrophages adhered to the plastic. The nonadherent cells were removed and resuspended in 1 to 2 ml TGMD (max. 35 x 106 cells/ml). A discontinuous Percoll density gradient was prepared by layering a less dense suspension (1.040 g/ml) above a denser one (1.090 g/ml). The cell suspension was then layered onto the gradient and tubes were centrifuged at 400 xg for 25 min at room temperature. After centrifugation the cells that accumulated at the interface of both Percoll densities were removed, washed, resuspended in culture medium, RPMI-1640, 10% FCS, 1 U/ml penicillin and 1 gg/ml streptomycin, and incubated in collagen coated plates for 48 hrs at 37°C in a 5% CO2-95% air incubator. During this time type II cells adhered to the surface and contaminating nonadherent cells were decanted after 22 hrs.
Prostaglandins
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Morphological studies Cytocentrifuge smears of all cell fractions were prepared. Differential cell counts were performed by May-Griinwald Giemsa staining. Identification of alveolar type II cells was performed after fixation with glutaraldehyde and staining with tannic acid and a polychrome stain that showed both the lamellar bodies and the cell outlines (13).
Viability Viability was monitored by determining the percentage of dispersed lung cells that excluded 0.02% trypan blue dye from their nuclei.
Challenge of type H pneumocytes To study their mediator release after challenge with different secretagogues, type II cells (5 x 105; counted after trypsinisation) were challenged in 250 ~1 Dulbecco's Salt Solution with the optimal concentrations of calcium-ionophore A23187 (10 ~M), opsonized zymosan (OZ) (1.25 mg), or 10-5 to 10-2 M hydrogen peroxide for increasing time periods (0-60 min) at 37°C. Incubations were also p e r f o r m e d in presence of 10-6 to 10-3 M N-ethylmaleimide (NEM), 5 mM glutathione (GSH) or 100 mM bicarbonate (HCO3-). Mediator release was stopped by the addition of 250 /~1 ice-cold buffer supplemented with 2 mM EDTA followed by immediate centrifugation (250 x g) for 3 minutes at 4°C. The supernatant was removed and assayed for metabolites of the AA cascade. Metabolites were determined by means of radioimmunoassay (RIA) and their formation was expressed as ng/106 type II cells. The amount of released mediators was corrected for the spontaneous release in absence of any stimulus. The RIAs were developed and performed in the Dept. of Pharmacology (14,15). The antisera were raised in rabbits. Cross reactivities on the 50% level of the binding curve were: for 6-keto-PGFla-antiserum: P G F l a 1%, 15-HETE (hydroxyeicosatetraenoic acid) 0.01%, 15-HPETE (hydroperoxyeicosatetraenoic acid) 0.01%, PGE2, 15-keto-PGE 2, TxB 2 and AA < 0.01%; for the TxB2-antiserum: P G D 2 8.9%, PGF2a 1%, P G E 2 0.9%, 6-keto-PGFla 0.1%, 15-keto-13,14-dihydro-PGF2a , AA, 15-HETE and 15-HPETE < 0.01%; for the PGE2-antiserum: PGE 1 70.5%, 15-keto13,14-dihydro-PGE 2 12.4%, PGA 2 7.8%, PGB 2 3.2%, PGF2a 1.1%, 6-keto-PGFla 0.4%, TxB 2 0.03%, P G D 2, AA < 0.02%. Samples of 0.1 and 0.2 ml were analysed directly. Minimal detection limits varied between 20 and 30 pg (100 and 150 pg/ml). The intra-assay variation coefficient for R I A of 6 - k e t o - P G F l a and TxB 2 were 15 + 1% (n = 103) and 13 + 1% (n = 111) respectively. Prostaglandin content of the samples was calculated using a computer programme developed by Munson, Rodbard and Jaff6 (NIH, Bethesda, MD, USA).
Statistical analysis Results are expressed as means with standard error of the mean (SEM), with n denoting the number of experiments performed. The two-tailed Student's t-test was used to determine the statistical difference between the mean values of two sets of data. A P-value lower than 0.05 was considered statistically significant. P < 0.05 and P < 0.01 were marked with one or two asterisks respectively.
Prostaglandins
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RESULTS
Cell isolation Enzymatic digestion of human lung tissue yielded 13.4 + 1.9 x 106 (n = 12) dispersed cells per gram wet lung tissue. The obtained cell suspension contained 32.0 _+ 7.9% type II cells. After differential adherence of macrophages the type II cell purity was increased to 59.8 + 9.3% (P < 0.05). The recovery of type II cells was 88.0 + 10.4%. F u r t h e r increase of their purity to 77.0 _+ 9.0% (P < 0.05) was achieved with centrifugation across the discontinuous Percoll gradient. Percoll separation also effectively removed cellular debris (above the first Percoll suspension) and remaining red blood cells (bottom of the tube). After gradient centrifugation the recovery of type II cells was 81.7 +_ 6.3%. T h e i r total recovery was 55.2 +_ 2.0%. The total time required for isolation so far, was approximately 5.5 hours (including adherence for 90 min). Incubation of the isolated cells for 22 hrs at 37°C resulted in type II cells of 92.0 + 2.5% purity with a seeding efficiency of 67.9 + 10.1%. The contaminating cells were identified as neutrophils and lymphocytes. Viability of the nucleated lung cells exceeded 92% at each isolation step. Viability observed after incubating type II ceils with concentrations of H 2 0 2 as high as 10 mM was 90%. In our method differential adherence to remove alveolar macrophages preceded gradient centrifugation to prevent choking up of the gradient by macrophages. Stimulation of type H pneumocytes After short culture of human alveolar type II cells, a release of TxB 2, PGE 2 and 6-keto-PGFla was observed after challenge during 60 minutes with optimal concentrations of opsonized zymosan (Table 1) or calcium ionophore A23187 (Table 2). Table 1: Release of AA metabolites by bnman alveolar type II cells challenged with 1.25 nag opsonized zymosan for 60 minutes.
Control +GSH +HCO 3 +Dazoxiben (10 -7 M) +Dazoxiben (10-3 M) Mean + S.E.M.; n = 4 ; *) P < 0.05
TxB 2
PGE 2
6-keto-PGFla
ng/106 cells
ng/106 cells
ng/106cells
0.58 0.87 0.80 0.48 0.37
6.8 + 1.8 9.3 + 1.4" 9.2 + 2.8* 6.8 _+ 0.3 13.6 + 0.1"
2.3 3.0 3.0 2.4 2.8
+ 0.22 + 0.10' _+ 0.23 + 0.21 ~ 0.17"
+ 0.7 + 0.5 +_ 1.1 + 0.2 + 0.4
Prostaglandins
105
Table 2: Release of AA metabofites by human alveolar type H cells challenged with 10 ~M calcium ionophore A23187 for 60 minutes.
Control +GSH +HCO 3
TxB 2
PGE 2
6-keto-PGFla
ng/106 cells
ng/106 cells
ng/106 cells
0.18 + 0.08 0.18 + 0.10 0.14 + 0.44
2.1 + 0.2 3.8 + 0.5* 3.9 _+ 0.4*
1.0 + 0.1 0.9 + 0.1 1.1 + 0.1
Mean + S.E.M.; n = 4 ; *) P < 0.05 The basal values of release of AA metabolites by non-challenged cells were not detectable. Addition of GSH or HCO 3- increased the formation of TxB 2 (only after opsonized zymosan) and PGE 2. The thromboxane synthetase inhibitor dazoxiben showed a dose-dependent decrease in TxB 2 production and a similar increase in P G E 2. Release of 6-keto-PGFla remained unaffected by all stimuli. Figure 1.
Release of arachidonic acid metabolites (ng/106 cells) by human alveolar type II cells after challenge with 10"5-10-2 M H202 for 60 minutes.
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[----} TxB2 PGE2 6-keto-PGF~o
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e"
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6.0 ID
0
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5
4
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-log Molar H202
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Prostaglandins
106
Time-dependent production of arachidonie acid metabolites (pg/10 cells) by type 11 ceils after challenge with 10-5 M H202.
Figure 2.
0.8
• 6-ket°-PGFla • PGE2 A TXBz
09 ll)
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O
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09
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20
30
40
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50
60
Time (rain} Figure 3.
09
The influence of different concentrations of N-ethylmaleimide on mediator production (ng/106 ceUs) by human alveolar type II cells after challenge with 1.25 mg opsonized zymosan for 60 minutes.
4.8
TxB2 r777] PGE2 6-keto-PGF~a
o 4.0 O •-- 3.2 t-
-I
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6
5
-log Molar NEM
4
3
Prostaglandins
107
Upon incubation of alveolar type II cells with H 2 0 2 a dose-dependent release of prostacyclin and P G E 2 was found (Figure 1). Their maximum release was observed at 0.1 mM H202. Comparable results were obtained for TxB 2 with a maximum release at 1 mM H202. However, the absolute amount of P G E 2 release per 106 cells was about 10-fold higher than for TxB2 and prostacyclin. This release of AA metabolites was not only dose-dependent, but also time-dependent (Figure 2). Release of PGE 2 and TxB 2 started after 10 minutes incubation with 10-4 M H202, while prostacyclin release was only detectable after 30 minutes. Addition of NEM enhanced the release of 6-keto-PGFla and TxB 2 compared to challenge with opsonized zymosan alone, the effect being maximal at 10 -5 M NEM. In contrast, an increasing concentration of NEM decreased the amount of PGE 2 release (Figure 3). Challenge with the calcium ionophore gave similar results. DISCUSSION In the present study we have purified type lI pneumocytes from human lung tissue. It is believed that these cells are likely to represent normal adult type II cells, as identifiable on the basis of morphological criteria. The tannic acid stain also strongly suggests that 90% of the cells in culture are type II cells. And in addition, their secretory pattern tells strongly in favour of type II cells, because the production of AA metabolites upon stimulation with opsonized zymosan or calcium ionophore consisted mainly of PGE 2 and 6-keto-PGFla and a small amount of TxB 2. This is in accordance with other studies in which rat type II cells released PGE 2 and/or prostacyclin as the major eicosanoids synthesized and TxB 2 was released in a smaller amount (2,16-18). Although macrophages can be difficult to differentiate from type II cells, they are usually easily recognized by the absence of stained inclusions and the presence of peripheral cytoplasmic extensions, which are well preserved by the glutaraldehyde fixation (13). Since alveolar macrophages produce much more thromboxane than prostaglandins (18), this criterium could be used for discrimination of macrophages and type II cells. Therefore a pilot study was performed in an effort to examine the secretory pattern of isolated and pure alveolar macrophages from lung tissue or lavage fluid. The results of this study were in the same range as described by Cott et al. (18). Both morphological criteria, and even more the respective secretory patterns of type II cells and alveolar macrophages from human lung provide strong evidence for the purity of our isolated type II cells. In a few studies it has been shown that the use of enzymes does not seem to alter cell viability and that receptors of the cell were not impaired (19,20). However, it was demonstrated that/3-adrenergic receptors lost all their high-affinity binding sites, but after a recuperation period high-affinity sites and muscarinic cholinergic receptors were gradually restored in time (21,22). Considerable evidence suggests that oxygen metabolites could cause prostaglandin generation by interacting at several sites in the pathway of AA metabolism (23-25). Taking into account that the neutrophil is one of the actual sources of oxygen metabolites in acute lung injury (26), it is difficult to estimate the local concentration of oxygen metabolites at the neutrophil-target cell interface. Although it was reported that type II cells can be injured in a dose-dependent fashion (27), we
Prostaglandins
108
found a viability of 90%, even at concentrations of 10-3 M H 2 0 2. It remains possible that concentrations of reactive oxygen species generated at the neutrophil-type II cell interface are sufficiently higher. However, we can not exclude the decrease in production of AA metabolites observed at higher concentrations of H 2 0 2 results from cytotoxicity due to the peroxide, since earlier studies demonstrated a type II cell lysis of 50% at 0.56 mM (28). Chemical reactivity with thiol or sulfhydryl (SH) groups is a property shared by heavy metals, oxidants and alkylating agents, all of which possess proinflammatory and toxic activities. Therefore, it is of interest that SH reactants have been shown to stimulate AA release and cyclooxygenasejaroduct formation in a variety of cell types (29-31). NEM at a concentration of 10-SM stimulated the synthesis of TxB 2 and prostacyclin, but not PGE 2. Indeed, production of PGE 2 decreased when release of the other metabolites was upregulated. In rat alveolar macrophages it was already demonstrated that this effect most likely relates to the SH-dependence and glutathione requirement of endoperoxide E isomerase and the SH-independence of thromboxane synthase (31). In conclusion, human alveolar type II cells have been isolated and purified using differential adherence and Percoll gradient centrifugation. They were shown to release products of the cyclooxygenase pathway of arachidonate metabolism upon induction with opsonized zymosan, ionophore A23187 and H 2 0 2. This production could be modulated by addition of GSH, HCO 3- or NEM. So, type II cells have the capability of cyclooxygenase activity. The SH-dependency and glutathione requirement of the PGE 2 isomerase was also demonstrated. Since the release of cyclooxygenase products could have significant physiological consequences, their exact role and the role of alveolar type II cells in ARDS needs further investigation in the future. ACKNOWI~DGEMEI~S The authors would like to thank Prof. E. Schoofs of the University Hospital, Dr D. Deleersnijder of the St. Augustinus Hospital and Dr J. Tombeur of the Middelheim Hospital of Antwerp for kindly providing human lung specimens. The technical assistance of Mrs R. Vandenbossche is gratefully acknowledged. REFERENCES 1.
2. 3.
Mason, R.J., C.C. Leslie, and D.R. Voelker. Alveolar type II cells in primary culture. In: The Cells of the Alveolar Unit. (G. Favez, A. Junod, and P. Leuenberger, eds.) Huber, Bern, 1983, p.24. Chauncey, J.B., M. Peters-Golden, and R.H. Simon. Arachidonic acid metabolism by rat alveolar epithelial cells. Lab. Invest. 58: 133, 1988. Maghni, K., C. Robidoux, J. Laporte, A. Hall~e, and P. Sirois. Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes. Prostaglandins 40: 217, 1990.
Prostaglandins 4.
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Freeman, G., L.T. Juhas, N.J. Furiosi, R. Mussenden, R.J. Stephens, and M.J. Evan. Pathology of pulmonary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. Health 2___99:203, 1974. Crapo, J.D., B.E. Barry, H.A. Foscue, and J. Shelburne. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen.Am. Rev. Respir. Dis. 122: 123, 1980. Hasleton, P.S. Adult respiratory distress syndrome-A review. Histopathology 7: 307, 1983. Adamson, I.Y.R., and D.H. Bowden. The type II cell as a progenitor of alveolar regeneration: A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30: 35, 1974. Gilifillan, A.M., and S.A. Rooney. Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes. Biochim. Biophys. Acta 87_66:22, 1986. Oyrazun, M.J., and J.A. Clements. Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. Am. Rev. Respir. Dis. 117: 879, 1978. Crapo, J.D., B.E. Barry, P. Gehr, M. Bachofen, and E.R. Weibel. Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 125: 740, 1982. Van Overveld, F.J., W.A. De Backer, and P.A. Vermeire. A modified method for isolating viable alveolar type II cells from human lung tissue. J. Immunol. Methods 132: 145, 1990. Roos, D., and J.A. Loos. Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohaemagglutinin. Biochim. Biophys. Acta 222: 565, 1970. Mason, R.J., S.R. Walker, B.A. Shields, J.E. Henson, and M.C. Williams. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain.Am. Rev. Respir. Dis. 13_!1:786, 1985. Laekeman, G.M., I.B. Vergote, G.M. Keersmaekers, J. Heiremans, C.F. Haensch, G. De Roy, F.L. Uyttenbroeck, and A.G. Herman. Prostacyclin and thromboxane in benign and malignant breast tumours. Br. J. Cancer 54:431, 1986. Dupont, A.G., E. Gerlo, P. Van der Niepen, G. Laekeman, and A. Piepsz. Renal and pharmacodynamic effects of torasemide and furasemide in normal man. Arzneimittel-Forschung 38: 172, 1988. Taylor, L., P. Polgar, J.A. McAteer, and W.H.J. Douglas. Prostaglandin production by type II alveolar epithelial cells. Biochim. Biophys. Acta 572: 502, 1979. Xu, G.L., K. Sivarajah, R. Wu, P. Nettesheim, and T. Eling. Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells. Exp. Lung Res. 10: 101, 1986. Cott, G.R., J.J. Westcott, and N.F. Voelkel. Prostaglandin and leukotriene production by alveolar type II cells.Am. J. Physiol. 258: L179, 1990. Paterson, N.A.M., J.F. Burka, and J.D. Craig. Release of slow-reacting substance of anaphylaxis from dispersed pig lung cells: effect of cyclooxygenase and lipoxygenase inhibitors. J. Allergy CIin. lmmunol. 67: 426, 1981.
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MacGlashan Jr, D.W., R.P. Schleimer, S.P. Peters, E.S. Schulman, G.K. Adams III, H.H. Newball, and L.M. Lichtenstein. Generation of leukotrienes by purified human lung mast cells. J. Clin. Invest. 70: 747, 1982. Raaijmakers, J.A.M., H.J. Harmsen, E. de Boer, C. Beneker, and A.J. van Rozen. Phospholipase A2 products induce pulmonary/~-adrenoceptor deviations. Eur. J. Pharrnacol. 127: 163, 1986. Van Overveld, F.J. Some aspects of mast cell subtypes; a comparison between normal individuals and patients with chronic obstructive lung disease. Thesis, Utrecht, 1988, p.35. Tate, R.M., H.G. Morris, W.R. Schroeder, and J.E. Repine. Oxygen metabolites stimulate thromboxane production and vasoconstriction in isolated salineperfused rabbit lungs. J. Clin. Invest. 74: 608, 1984. Panganamala, R.V., H.M. Sharma, H. Sprecher, J.C. Geer, and D.G. Cornwell. A suggested role for hydrogen peroxide in the biosynthesis of prostaglandins. Prostaglandins 8: 3, 1974. Lands, W.E.M., J. Sauter, and G.W. Stone. Oxygen requirement for prostaglandin biosynthesis. Prostaglandins Med. 1: 117, 1978. Johnson, K.J., and P.A. Ward. Role of oxygen metabolites in immune complex injury of lung. J. Immunol. 126: 2365, 1981. Crim, C., and R.H. Simon. Effects of oxygen metabolites on rat alveolar type II cell viability and surfactant metabolism. Lab. Invest. 58: 428, 1988. Simon,R.H., P.D. Dehart, and R.F. Todd III. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J. Clin. Invest. 78: 1375, 1986. Shier, W.T., and D.J. DuBourdieu. Stimulation of phospholipid hydrolysis and cell death by mercuric chloride: evidence for mercuric ion acting as a calciummimetic agent. Biochem. Biophys. Res. Commun. 1113:758, 1983. Grundfest, C.C., J. Chang, and D. Newcombe. Acrolein: a potent modulator of lung macrophage arachidonic acid metabolism. Biochim. Biophys. Acta 713: 149, 1982. Peters-Golden, M., and C. Shelly. Modulation of alveolar macrophage-derived 5-1ipoxygenase products by the sulfhydryl reactant N-ethylmaleimide. J. Biol. Chem. 262: 10594, 1987.
Editor:
R.
Krell
Received:
11-19-92
Accepted:
5-4-92
Ove.~eld 1, P.G. Jo~ens 1, W.A. De Sack~r I,
F.J. van M. Rampart
~ , L.
Bossaert
°
and
P.A.
Vermeire
~
Depts. of i) Respiratory Medicine, 2) Pharmacology, and 3) Intensive Care, University of Antwerp (UIA), B-2610 Antwerpen, Belgium ~n~ACT
Human alveolar type II cells are thought to play a role in the pathogenesis of lung injury. Patterns of mediator release of arachidonic acid metabolism by type II cells were therefore studied after challenge with calcium ionophore A23187, opsonized zymosan and hydrogen peroxide. A time- and concentration dependent release of cyclooxygenase products was observed, with release of PGE 2 > 6-keto-PGFla > TxB 2. Addition of glutathione or bicarbonate further increased the production of PGE 2. N-ethylmaleimide, a sulfhydryl (SH) reactant, induced a dose-dependent increase in the release of TxB 2 and 6-keto-PGFla, but not of PGE 2. This relates most likely to the SH-dependency and glutathione requirement of the PGE2 isomerase and SH-independence of thromboxane and prostacyclin isomerase. INTRODUCTION Alveolar type II cells are important cells for normal lung function because they synthesize and secrete surface active material, maintain the alveolar epithelium by serving as stem cells for both type II and type I cells, and transport fluid from the alveolar subphase into the interstitium (1). The secretory function of type II cells has been studied principally for the production of surfactant. The other secretory functions of type II cells are less well known, although some studies have been done with cells from animals showing production and release of arachidonic acid (AA) metabolites (2,3). Both alveolar type I and type II cells are known targets for many toxic agents (4,5) and are affected in adult respiratory distress syndrome (ARDS) (6). After lung injury, type I1 cells retain the important ability to replace type I cells and to epithelialize the alveolus (7). In ARDS, deficiency of surfactant plays a major role (3). Recent studies have also shown that AA metabolites could regulate surfactant secretion. Enhancement of surfactant secretion was observed with leukotrienes, whereas prostaglandins appeared to control its secretion (8,9). Although these type II pneumocytes only constitute 4% of the alveolar epithelial area, they represent 60% of alveolar epithelial cells by number (10). An improvement of our knowledge of the pathogenesis of ARDS may thus depend on a better understanding of the acute toxicology of these cells. This prompted us to initiate in vitro studies of purified preparations of human type II cells. Copyright © 1992 Butterworth-Heinemann
102
Prostaglandins
MATERIAI~ AND M E I ~ O D S Materials Enriched Tyrode's buffer (TGMD) contained 137 mM NaC1, 2.7 mM KC1, 0.4 mM NaH2PO4.2H20, 1.2 mM MgC12.6H20, 1% gelatin (Merck, Darmstadt, FRG), 60 mg deoxyribonuclease type I (EC 3.1.21.1) (DNAse) (Boehringer, Mannheim, FRG), 5.6 mM glucose and 1 U/ml penicillin and 1 ~g/ml streptomycin (pH 7.4, 20°C) (Sigma Chemical Co., St. Louis, MO, USA). Before and after the cell isolation, tissue and cells were kept in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (Gibco Ltd., Paisley, UK). The enzyme solutions were prepared by dissolving 1 mg/ml collagenase (EC 3.4.24.3) (Boehringer) and 0.1 U/ml elastase type I (3.4.21.11) (Sigma) in TGMD. The isotonic shock solution contained 155 mM NH4C1, 10 mM KHCO3, 0.1 mM EDTA and 10 mg/1 phenol red (pH 7.4, 0°C). The osmolality was adjusted to 290 mOsm, as measured by freezing point depression. Percoll (Pharmacia, Uppsala, Sweden) densities of 1.040 and 1.090 g/ml were prepared by diluting a stock-solution of 1.100 g/ml with phosphate buffered saline (PBS). Osmolality of the Percoll densities was 290 mOsm/kg. Human collagen type IV, calcium ionophore A23187, zymosan and N-ethylmaleimide were purchased from Sigma. PGE 2, TxB2, 6-keto-PGFla were obtained from Upjohn, Kalamazoo, MI, USA. Their 3H-labelled forms were purchased from New England Nuclear, Boston, MA. All other chemicals were reagent grade and they were purchased from Merck. Cell isolation Isolation of alveolar type II cells was performed as described previously (11). Briefly, cells were isolated from a macroscopically disease-free part of human lung tissue obtained during resection of bronchial tumours. The tissue was minced into 1 to 2 mm pieces. The fragments were washed extensively with RPMI-1640/10% FCS to remove blood cells as much as possible. Subsequently the lung fragments were digested during four consecutive 30 minute exposures at 37°C to the enzyme solution. Dispersed cells were collected, filtered through sterile gauze, centrifuged and resuspended in TGMD. Erythrocytes were removed by an isotonic shock procedure (12). The cell suspension at 1.5 to 2 x 106 cells/ml was cultured for 90 min in 25 cm 2 plastic flasks (7 ml/flask) (Gibco/Nunc, Ghent, Belgium) during which time most of the alveolar macrophages adhered to the plastic. The nonadherent cells were removed and resuspended in 1 to 2 ml TGMD (max. 35 x 106 cells/ml). A discontinuous Percoll density gradient was prepared by layering a less dense suspension (1.040 g/ml) above a denser one (1.090 g/ml). The cell suspension was then layered onto the gradient and tubes were centrifuged at 400 xg for 25 min at room temperature. After centrifugation the cells that accumulated at the interface of both Percoll densities were removed, washed, resuspended in culture medium, RPMI-1640, 10% FCS, 1 U/ml penicillin and 1 gg/ml streptomycin, and incubated in collagen coated plates for 48 hrs at 37°C in a 5% CO2-95% air incubator. During this time type II cells adhered to the surface and contaminating nonadherent cells were decanted after 22 hrs.
Prostaglandins
103
Morphological studies Cytocentrifuge smears of all cell fractions were prepared. Differential cell counts were performed by May-Griinwald Giemsa staining. Identification of alveolar type II cells was performed after fixation with glutaraldehyde and staining with tannic acid and a polychrome stain that showed both the lamellar bodies and the cell outlines (13).
Viability Viability was monitored by determining the percentage of dispersed lung cells that excluded 0.02% trypan blue dye from their nuclei.
Challenge of type H pneumocytes To study their mediator release after challenge with different secretagogues, type II cells (5 x 105; counted after trypsinisation) were challenged in 250 ~1 Dulbecco's Salt Solution with the optimal concentrations of calcium-ionophore A23187 (10 ~M), opsonized zymosan (OZ) (1.25 mg), or 10-5 to 10-2 M hydrogen peroxide for increasing time periods (0-60 min) at 37°C. Incubations were also p e r f o r m e d in presence of 10-6 to 10-3 M N-ethylmaleimide (NEM), 5 mM glutathione (GSH) or 100 mM bicarbonate (HCO3-). Mediator release was stopped by the addition of 250 /~1 ice-cold buffer supplemented with 2 mM EDTA followed by immediate centrifugation (250 x g) for 3 minutes at 4°C. The supernatant was removed and assayed for metabolites of the AA cascade. Metabolites were determined by means of radioimmunoassay (RIA) and their formation was expressed as ng/106 type II cells. The amount of released mediators was corrected for the spontaneous release in absence of any stimulus. The RIAs were developed and performed in the Dept. of Pharmacology (14,15). The antisera were raised in rabbits. Cross reactivities on the 50% level of the binding curve were: for 6-keto-PGFla-antiserum: P G F l a 1%, 15-HETE (hydroxyeicosatetraenoic acid) 0.01%, 15-HPETE (hydroperoxyeicosatetraenoic acid) 0.01%, PGE2, 15-keto-PGE 2, TxB 2 and AA < 0.01%; for the TxB2-antiserum: P G D 2 8.9%, PGF2a 1%, P G E 2 0.9%, 6-keto-PGFla 0.1%, 15-keto-13,14-dihydro-PGF2a , AA, 15-HETE and 15-HPETE < 0.01%; for the PGE2-antiserum: PGE 1 70.5%, 15-keto13,14-dihydro-PGE 2 12.4%, PGA 2 7.8%, PGB 2 3.2%, PGF2a 1.1%, 6-keto-PGFla 0.4%, TxB 2 0.03%, P G D 2, AA < 0.02%. Samples of 0.1 and 0.2 ml were analysed directly. Minimal detection limits varied between 20 and 30 pg (100 and 150 pg/ml). The intra-assay variation coefficient for R I A of 6 - k e t o - P G F l a and TxB 2 were 15 + 1% (n = 103) and 13 + 1% (n = 111) respectively. Prostaglandin content of the samples was calculated using a computer programme developed by Munson, Rodbard and Jaff6 (NIH, Bethesda, MD, USA).
Statistical analysis Results are expressed as means with standard error of the mean (SEM), with n denoting the number of experiments performed. The two-tailed Student's t-test was used to determine the statistical difference between the mean values of two sets of data. A P-value lower than 0.05 was considered statistically significant. P < 0.05 and P < 0.01 were marked with one or two asterisks respectively.
Prostaglandins
104
RESULTS
Cell isolation Enzymatic digestion of human lung tissue yielded 13.4 + 1.9 x 106 (n = 12) dispersed cells per gram wet lung tissue. The obtained cell suspension contained 32.0 _+ 7.9% type II cells. After differential adherence of macrophages the type II cell purity was increased to 59.8 + 9.3% (P < 0.05). The recovery of type II cells was 88.0 + 10.4%. F u r t h e r increase of their purity to 77.0 _+ 9.0% (P < 0.05) was achieved with centrifugation across the discontinuous Percoll gradient. Percoll separation also effectively removed cellular debris (above the first Percoll suspension) and remaining red blood cells (bottom of the tube). After gradient centrifugation the recovery of type II cells was 81.7 +_ 6.3%. T h e i r total recovery was 55.2 +_ 2.0%. The total time required for isolation so far, was approximately 5.5 hours (including adherence for 90 min). Incubation of the isolated cells for 22 hrs at 37°C resulted in type II cells of 92.0 + 2.5% purity with a seeding efficiency of 67.9 + 10.1%. The contaminating cells were identified as neutrophils and lymphocytes. Viability of the nucleated lung cells exceeded 92% at each isolation step. Viability observed after incubating type II ceils with concentrations of H 2 0 2 as high as 10 mM was 90%. In our method differential adherence to remove alveolar macrophages preceded gradient centrifugation to prevent choking up of the gradient by macrophages. Stimulation of type H pneumocytes After short culture of human alveolar type II cells, a release of TxB 2, PGE 2 and 6-keto-PGFla was observed after challenge during 60 minutes with optimal concentrations of opsonized zymosan (Table 1) or calcium ionophore A23187 (Table 2). Table 1: Release of AA metabolites by bnman alveolar type II cells challenged with 1.25 nag opsonized zymosan for 60 minutes.
Control +GSH +HCO 3 +Dazoxiben (10 -7 M) +Dazoxiben (10-3 M) Mean + S.E.M.; n = 4 ; *) P < 0.05
TxB 2
PGE 2
6-keto-PGFla
ng/106 cells
ng/106 cells
ng/106cells
0.58 0.87 0.80 0.48 0.37
6.8 + 1.8 9.3 + 1.4" 9.2 + 2.8* 6.8 _+ 0.3 13.6 + 0.1"
2.3 3.0 3.0 2.4 2.8
+ 0.22 + 0.10' _+ 0.23 + 0.21 ~ 0.17"
+ 0.7 + 0.5 +_ 1.1 + 0.2 + 0.4
Prostaglandins
105
Table 2: Release of AA metabofites by human alveolar type H cells challenged with 10 ~M calcium ionophore A23187 for 60 minutes.
Control +GSH +HCO 3
TxB 2
PGE 2
6-keto-PGFla
ng/106 cells
ng/106 cells
ng/106 cells
0.18 + 0.08 0.18 + 0.10 0.14 + 0.44
2.1 + 0.2 3.8 + 0.5* 3.9 _+ 0.4*
1.0 + 0.1 0.9 + 0.1 1.1 + 0.1
Mean + S.E.M.; n = 4 ; *) P < 0.05 The basal values of release of AA metabolites by non-challenged cells were not detectable. Addition of GSH or HCO 3- increased the formation of TxB 2 (only after opsonized zymosan) and PGE 2. The thromboxane synthetase inhibitor dazoxiben showed a dose-dependent decrease in TxB 2 production and a similar increase in P G E 2. Release of 6-keto-PGFla remained unaffected by all stimuli. Figure 1.
Release of arachidonic acid metabolites (ng/106 cells) by human alveolar type II cells after challenge with 10"5-10-2 M H202 for 60 minutes.
9.0
[----} TxB2 PGE2 6-keto-PGF~o
0
o
// //
//
7.5
//
e"
// // //
6.0 ID
0
1.5
5
4
3
-log Molar H202
2
Prostaglandins
106
Time-dependent production of arachidonie acid metabolites (pg/10 cells) by type 11 ceils after challenge with 10-5 M H202.
Figure 2.
0.8
• 6-ket°-PGFla • PGE2 A TXBz
09 ll)
T
O
O
0.6
17} t--
09
t,
0.4
0.2
,m
|
0
......
10
1
I
I
20
30
40
I ......
I
50
60
Time (rain} Figure 3.
09
The influence of different concentrations of N-ethylmaleimide on mediator production (ng/106 ceUs) by human alveolar type II cells after challenge with 1.25 mg opsonized zymosan for 60 minutes.
4.8
TxB2 r777] PGE2 6-keto-PGF~a
o 4.0 O •-- 3.2 t-
-I
"-" 2.4 09 09
0.8
:~ o.4
control
6
5
-log Molar NEM
4
3
Prostaglandins
107
Upon incubation of alveolar type II cells with H 2 0 2 a dose-dependent release of prostacyclin and P G E 2 was found (Figure 1). Their maximum release was observed at 0.1 mM H202. Comparable results were obtained for TxB 2 with a maximum release at 1 mM H202. However, the absolute amount of P G E 2 release per 106 cells was about 10-fold higher than for TxB2 and prostacyclin. This release of AA metabolites was not only dose-dependent, but also time-dependent (Figure 2). Release of PGE 2 and TxB 2 started after 10 minutes incubation with 10-4 M H202, while prostacyclin release was only detectable after 30 minutes. Addition of NEM enhanced the release of 6-keto-PGFla and TxB 2 compared to challenge with opsonized zymosan alone, the effect being maximal at 10 -5 M NEM. In contrast, an increasing concentration of NEM decreased the amount of PGE 2 release (Figure 3). Challenge with the calcium ionophore gave similar results. DISCUSSION In the present study we have purified type lI pneumocytes from human lung tissue. It is believed that these cells are likely to represent normal adult type II cells, as identifiable on the basis of morphological criteria. The tannic acid stain also strongly suggests that 90% of the cells in culture are type II cells. And in addition, their secretory pattern tells strongly in favour of type II cells, because the production of AA metabolites upon stimulation with opsonized zymosan or calcium ionophore consisted mainly of PGE 2 and 6-keto-PGFla and a small amount of TxB 2. This is in accordance with other studies in which rat type II cells released PGE 2 and/or prostacyclin as the major eicosanoids synthesized and TxB 2 was released in a smaller amount (2,16-18). Although macrophages can be difficult to differentiate from type II cells, they are usually easily recognized by the absence of stained inclusions and the presence of peripheral cytoplasmic extensions, which are well preserved by the glutaraldehyde fixation (13). Since alveolar macrophages produce much more thromboxane than prostaglandins (18), this criterium could be used for discrimination of macrophages and type II cells. Therefore a pilot study was performed in an effort to examine the secretory pattern of isolated and pure alveolar macrophages from lung tissue or lavage fluid. The results of this study were in the same range as described by Cott et al. (18). Both morphological criteria, and even more the respective secretory patterns of type II cells and alveolar macrophages from human lung provide strong evidence for the purity of our isolated type II cells. In a few studies it has been shown that the use of enzymes does not seem to alter cell viability and that receptors of the cell were not impaired (19,20). However, it was demonstrated that/3-adrenergic receptors lost all their high-affinity binding sites, but after a recuperation period high-affinity sites and muscarinic cholinergic receptors were gradually restored in time (21,22). Considerable evidence suggests that oxygen metabolites could cause prostaglandin generation by interacting at several sites in the pathway of AA metabolism (23-25). Taking into account that the neutrophil is one of the actual sources of oxygen metabolites in acute lung injury (26), it is difficult to estimate the local concentration of oxygen metabolites at the neutrophil-target cell interface. Although it was reported that type II cells can be injured in a dose-dependent fashion (27), we
Prostaglandins
108
found a viability of 90%, even at concentrations of 10-3 M H 2 0 2. It remains possible that concentrations of reactive oxygen species generated at the neutrophil-type II cell interface are sufficiently higher. However, we can not exclude the decrease in production of AA metabolites observed at higher concentrations of H 2 0 2 results from cytotoxicity due to the peroxide, since earlier studies demonstrated a type II cell lysis of 50% at 0.56 mM (28). Chemical reactivity with thiol or sulfhydryl (SH) groups is a property shared by heavy metals, oxidants and alkylating agents, all of which possess proinflammatory and toxic activities. Therefore, it is of interest that SH reactants have been shown to stimulate AA release and cyclooxygenasejaroduct formation in a variety of cell types (29-31). NEM at a concentration of 10-SM stimulated the synthesis of TxB 2 and prostacyclin, but not PGE 2. Indeed, production of PGE 2 decreased when release of the other metabolites was upregulated. In rat alveolar macrophages it was already demonstrated that this effect most likely relates to the SH-dependence and glutathione requirement of endoperoxide E isomerase and the SH-independence of thromboxane synthase (31). In conclusion, human alveolar type II cells have been isolated and purified using differential adherence and Percoll gradient centrifugation. They were shown to release products of the cyclooxygenase pathway of arachidonate metabolism upon induction with opsonized zymosan, ionophore A23187 and H 2 0 2. This production could be modulated by addition of GSH, HCO 3- or NEM. So, type II cells have the capability of cyclooxygenase activity. The SH-dependency and glutathione requirement of the PGE 2 isomerase was also demonstrated. Since the release of cyclooxygenase products could have significant physiological consequences, their exact role and the role of alveolar type II cells in ARDS needs further investigation in the future. ACKNOWI~DGEMEI~S The authors would like to thank Prof. E. Schoofs of the University Hospital, Dr D. Deleersnijder of the St. Augustinus Hospital and Dr J. Tombeur of the Middelheim Hospital of Antwerp for kindly providing human lung specimens. The technical assistance of Mrs R. Vandenbossche is gratefully acknowledged. REFERENCES 1.
2. 3.
Mason, R.J., C.C. Leslie, and D.R. Voelker. Alveolar type II cells in primary culture. In: The Cells of the Alveolar Unit. (G. Favez, A. Junod, and P. Leuenberger, eds.) Huber, Bern, 1983, p.24. Chauncey, J.B., M. Peters-Golden, and R.H. Simon. Arachidonic acid metabolism by rat alveolar epithelial cells. Lab. Invest. 58: 133, 1988. Maghni, K., C. Robidoux, J. Laporte, A. Hall~e, and P. Sirois. Release of prostaglandins and thromboxanes by guinea pig isolated type II pneumocytes. Prostaglandins 40: 217, 1990.
Prostaglandins 4.
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109
Freeman, G., L.T. Juhas, N.J. Furiosi, R. Mussenden, R.J. Stephens, and M.J. Evan. Pathology of pulmonary disease from exposure to interdependent ambient gases (nitrogen dioxide and ozone). Arch. Environ. Health 2___99:203, 1974. Crapo, J.D., B.E. Barry, H.A. Foscue, and J. Shelburne. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen.Am. Rev. Respir. Dis. 122: 123, 1980. Hasleton, P.S. Adult respiratory distress syndrome-A review. Histopathology 7: 307, 1983. Adamson, I.Y.R., and D.H. Bowden. The type II cell as a progenitor of alveolar regeneration: A cytodynamic study in mice after exposure to oxygen. Lab. Invest. 30: 35, 1974. Gilifillan, A.M., and S.A. Rooney. Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes. Biochim. Biophys. Acta 87_66:22, 1986. Oyrazun, M.J., and J.A. Clements. Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandins in the rabbit. Am. Rev. Respir. Dis. 117: 879, 1978. Crapo, J.D., B.E. Barry, P. Gehr, M. Bachofen, and E.R. Weibel. Cell number and cell characteristics of the normal human lung. Am. Rev. Respir. Dis. 125: 740, 1982. Van Overveld, F.J., W.A. De Backer, and P.A. Vermeire. A modified method for isolating viable alveolar type II cells from human lung tissue. J. Immunol. Methods 132: 145, 1990. Roos, D., and J.A. Loos. Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes. I. Stimulation by phytohaemagglutinin. Biochim. Biophys. Acta 222: 565, 1970. Mason, R.J., S.R. Walker, B.A. Shields, J.E. Henson, and M.C. Williams. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain.Am. Rev. Respir. Dis. 13_!1:786, 1985. Laekeman, G.M., I.B. Vergote, G.M. Keersmaekers, J. Heiremans, C.F. Haensch, G. De Roy, F.L. Uyttenbroeck, and A.G. Herman. Prostacyclin and thromboxane in benign and malignant breast tumours. Br. J. Cancer 54:431, 1986. Dupont, A.G., E. Gerlo, P. Van der Niepen, G. Laekeman, and A. Piepsz. Renal and pharmacodynamic effects of torasemide and furasemide in normal man. Arzneimittel-Forschung 38: 172, 1988. Taylor, L., P. Polgar, J.A. McAteer, and W.H.J. Douglas. Prostaglandin production by type II alveolar epithelial cells. Biochim. Biophys. Acta 572: 502, 1979. Xu, G.L., K. Sivarajah, R. Wu, P. Nettesheim, and T. Eling. Biosynthesis of prostaglandins by isolated and cultured airway epithelial cells. Exp. Lung Res. 10: 101, 1986. Cott, G.R., J.J. Westcott, and N.F. Voelkel. Prostaglandin and leukotriene production by alveolar type II cells.Am. J. Physiol. 258: L179, 1990. Paterson, N.A.M., J.F. Burka, and J.D. Craig. Release of slow-reacting substance of anaphylaxis from dispersed pig lung cells: effect of cyclooxygenase and lipoxygenase inhibitors. J. Allergy CIin. lmmunol. 67: 426, 1981.
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Prostaglandins
MacGlashan Jr, D.W., R.P. Schleimer, S.P. Peters, E.S. Schulman, G.K. Adams III, H.H. Newball, and L.M. Lichtenstein. Generation of leukotrienes by purified human lung mast cells. J. Clin. Invest. 70: 747, 1982. Raaijmakers, J.A.M., H.J. Harmsen, E. de Boer, C. Beneker, and A.J. van Rozen. Phospholipase A2 products induce pulmonary/~-adrenoceptor deviations. Eur. J. Pharrnacol. 127: 163, 1986. Van Overveld, F.J. Some aspects of mast cell subtypes; a comparison between normal individuals and patients with chronic obstructive lung disease. Thesis, Utrecht, 1988, p.35. Tate, R.M., H.G. Morris, W.R. Schroeder, and J.E. Repine. Oxygen metabolites stimulate thromboxane production and vasoconstriction in isolated salineperfused rabbit lungs. J. Clin. Invest. 74: 608, 1984. Panganamala, R.V., H.M. Sharma, H. Sprecher, J.C. Geer, and D.G. Cornwell. A suggested role for hydrogen peroxide in the biosynthesis of prostaglandins. Prostaglandins 8: 3, 1974. Lands, W.E.M., J. Sauter, and G.W. Stone. Oxygen requirement for prostaglandin biosynthesis. Prostaglandins Med. 1: 117, 1978. Johnson, K.J., and P.A. Ward. Role of oxygen metabolites in immune complex injury of lung. J. Immunol. 126: 2365, 1981. Crim, C., and R.H. Simon. Effects of oxygen metabolites on rat alveolar type II cell viability and surfactant metabolism. Lab. Invest. 58: 428, 1988. Simon,R.H., P.D. Dehart, and R.F. Todd III. Neutrophil-induced injury of rat pulmonary alveolar epithelial cells. J. Clin. Invest. 78: 1375, 1986. Shier, W.T., and D.J. DuBourdieu. Stimulation of phospholipid hydrolysis and cell death by mercuric chloride: evidence for mercuric ion acting as a calciummimetic agent. Biochem. Biophys. Res. Commun. 1113:758, 1983. Grundfest, C.C., J. Chang, and D. Newcombe. Acrolein: a potent modulator of lung macrophage arachidonic acid metabolism. Biochim. Biophys. Acta 713: 149, 1982. Peters-Golden, M., and C. Shelly. Modulation of alveolar macrophage-derived 5-1ipoxygenase products by the sulfhydryl reactant N-ethylmaleimide. J. Biol. Chem. 262: 10594, 1987.
Editor:
R.
Krell
Received:
11-19-92
Accepted:
5-4-92
