[58]
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535
Acknowledgments This work was supported by the Nora Eccles Treadwell Foundation and by Grants from the National Institutes of Health (HL 34127 and HL 35828) and the American Heart Association (Grant-in-Aid 871147). Drs. Prescott and Zimmerman are Established Investigators of the American Heart Association. Dr. Whatley was supported by a National Research Service Award (HL 07529). We thank Dan Fennell for photographs of EC monolayers and Julie Wald for preparing the manuscript.
[58] U s e o f C u l t u r e d C e l l s to S t u d y A r a c h i d o n i c Acid Metabolism By ROBERT R. GORMAN, MICHAEL J. B1ENKOWSKI, an d CHRISTOPHER W. BENJAMIN
Introduction As is often the case in modern biology, there is no one cell system that is ideal for the study of a particular metabolic pathway and arachidonic acid metabolism is no exception. For example, the human platelet is an excellent cell in which to study thromboxane A2 (TxAa) synthesis, and the endothelial cell is useful for studies of prostacyclin (PGI2) synthesis, but, neither cell type makes both PGI2 and TxA2. However, there are enough cultured cells available to study the synthesis of a majority of the various eicosanoids. In this chapter we describe the culture of the human lymphoma cell line U937 and the human lung fibroblast WI-38 as useful cell lines in which to study thromboxane A2 synthesis, human umbilical vein endothelial cells to investigate PGI2 biosynthesis, and the murine fibroblast line NIH-3T3 to study PGE2 synthesis and the influence of cellular transfection and transformation on arachidonate metabolism. These same cell culture systems and techniques can also be used to study the regulation of the various enzymes of the arachidonate cascade as well as the phospholipase(s) that regulate arachidonate concentration in cells. Thromboxane A2 Synthesis by U937 Cells U937 cells were originally isolated from the pleural fluid of a patient with diffuse histiocytic lymphoma.1 These cells exist as immature myelomonocytic cells that can be terminally differentiated by treatment with C. Sundstrom and K. Nilsson, Int. J. Cancer 17, 565 (1976).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
536
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[58]
phorbol 12-myristate 13-acetate (PMA) into cells with the phenotypical characteristics of monocytes/macrophages.2 These differentiated cells can produce TxA2 as the major product in response to a variety of agonists, 3 and are an excellent system in which to study the influence of glucocorticoids on eicosanoid biosynthesis. Reagents. U937 cells (American Type Culture Collection, Rockville, MD); RPMI-1640, Fungi Bact, and fetal calf serum (FCS) (Irvine Scientific, Santa Ana, CA); phorbol 12-myristate 13-acetate (PMA), Ca 2÷ ionophore A23187, lipopolysaccharide (E. coli serotype 055 : B5), zymosan A, and dexamethasone (Sigma Chemical, St. Louis, MO); arachidonic acid, NuCheck Preps (Elysian, MN); EIA Kit (Cayman Chemical Co., Ann Arbor, MI). Cell Culture Procedure. U937 cells are maintained in suspension culture in RPMI-1640 supplemented with l x FungiBact, 10% heatinactivated FCS. For differentiation, cells are plated at 2.0 × 105 cells/ml in medium containing 100 nM PMA and allowed to attach for 48 hr. The cells are then fed with PMA-free medium and cultured overnight prior to use.
Measurement o f Synthesis 1. Differentiated U937 cell monolayers are washed twice with serumfree RPMI-1640 followed by addition of fresh medium containing 1% FCS and either E. coli LPS (10/.~g/ml), heat-activated zymosan A (500/xg/ml), A23187 (10/zg/ml), or arachidonic acid (10/xM) both initially dissolved in dimethyl sulfoxide. 2. After 4 hr (LPS or zymosan) or 15 min (A23187 or arachidonic acid), the medium is removed and TxB2 levels measured using a TxB2-specific enzyme immunoassay (EIA) (see [3], this volume). 3. Steroid treatments (i.e., dexamethasone) are performed by adding the appropriate steroid to RPMI-1640 medium containing 1% FCS and pretreating the cells for varying lengths of time. The medium is removed, replaced with steroid-free medium containing the appropriate agonist, and TxAz synthesis quantified as described above. Table I shows a typical result following exposure of differentiated U937 cells to LPS, zymosan A, arachidonic acid, or A23187:10/zg/ml LPS stimulates 2.7 ng TxA2/106 cells, 500/zg/ml zymosan A stimulates 1.5 ng TxA2/106 cells in 4 hr, 1/~g/ml A23187 stimulates 1.0 ng TxA2/106 cells, and 10/zM arachidonate stimulates 0.8 ng TxA2/106 cells in 15 min. 2 p. Harris and R. Peter, J. Leukocyte Biol. 37, 407 (1985). 3 M. J. Bienkowski, M. A. Petro, and L. J. Robinson, J. Biol. Chem. 264, 6536 (1989).
[58]
537
CULTURED CELLS AND ARACHIDONATE
TABLE I THROMBOXANE SYNTHESIS IN U937 CELLSa Agonist
ng TxA2/I
None, basal LPS, 10/xg/ml Zymosan A, 500 p,g/ml Arachidonic acid 10 t~M A23187, 1/~g/ml
0.54 2.7 1.5 0.8 1.0
× 10 6
cells
-+ 0.04 ± 0.15 ± 0.09 +- 0.06 ± 0.10
a U937 cells are differentiated and cultured as described. TxAz synthesis is initiated by washing the monolayer with serum-free RPMI-1640 and replacing the culture medium with fresh medium containing 10 ~g/ml LPS, 500 ~g/ml zymosan A, 10 t~M arachidonic acid, 1/.Lg/ml A23187. After 4 hr (LPS or zymosan) or 15 rain (A23187, arachidonate) at 37°, the culture medium is removed and the TxB2 content determined by EIA.
Figure I shows a similar experiment when cells are pretreated with dexamethasone (10-5-10 -ll M) for 2 hr prior to stimulation with LPS (10 ~g/ml) or zymosan A (500 t~g/ml) for 4 hr, or arachidonic acid (10 ~M) or A23187 (1 ~g/ml) for 15 min. Dexamethasone has no effect on A23187 or 3.5 3.0...A
-J
2.5
~
2.0
X
l & 1.5-. o ,,r-
g...
,~
1.0 0.5
0.0
I
-11
I0
-1
I
I
I
I
I
-9
-8
-7
-6
-5
LOGDEXAMETHASONE[M] FIG. I. Dexamethasone inhibition of TxAz synthesis initiated by various agonists. Cells are pretreated with various concentrations of dexamethasone for 2 hr. They are then stimulated with either 10 ~g/ml LPS ( O - - © ) or 500 tzg/ml zymosan (&--&) for 4 hr or with 10 tzM arachidonic acid ([~--D) or 1 Ixg/ml A23187 ( A - - A ) for 15 min in steroid-free medium, and the TxB~ content of the medium determined by EIA. Results are expressed as mean +- SE.
538
CELLMODELSOF LIPID MEDIATORPRODUCTION
[58]
arachidonate stimulation of TxA2, however, it dose-dependently inhibits both LPS- and zymosan A-stimulated TxA2 release. Thromboxane A2 Synthesis by WI-38 Cells WI-38 cells are unique because they are the only fibroblast cell line that has been shown to produce thromboxane A2.4 The following outlines step by step procedures for measuring thromboxane synthesis in these cells. Reagents. Human lung fibroblast WI-38 (Flow Laboratories, Rockville, MD); 75 mm tissue culture flasks (Falcon Plastics, Oxnard, CA); 35-mm tissue culture wells (Costar Plastics, Cambridge, MA); Eagle's minimum essential medium (Earle's base), FCS (inactivated 56°, 30 rain), and EDTA-trypsin (Irvine Scientific, Santa Ana, CA); precoated silica gel GF thin-layer plates (Analtech, Inc., Newark, DE); 9,1 l-azoprosta-5,13dienoic acid (azo analog I) and prostaglandin standards (Upjohn Co., Kalamazoo, MI); arachidonic acid (NuCheck Preps., Elysian, MN). [1-14C]PGH2 is synthesized according to Gorman et al. 5 Cell Culture Procedure. Cells are split 1 : 5 using EDTA-trypsin, into culture bottles (75 cm 2) and grown in Eagle's minimum essential medium (Earle's base) supplemented with 10% FCS. This medium with serum will be referred to as MEM-10. Confluent cells are fed with 25 ml of MEM-10 every 5 days, and passaged every 13-14 days. For individual biochemical experiments, the cells are seeded into 35-mm Costar wells, and grown under a humidified atmosphere of 95% air-5% CO2 at 37° until confluent (3-4 days). Final density is approximately 1 × l06 cells/35-mm well. Experimental Procedures
1. Confluent WI-38 cells in 35-ram wells are washed twice with icecold MEM and incubated for 15 min at 37 ° with fresh MEM. 2. After 15 rain, the cells are exposed to 0.1-1.0 ~ M [1-14C]PGH2 (dissolved in MEM immediately before use) or 0.l-10 /xM [1-14C]arachidonic acid (initially dissolved in ethanol) and incubated for 5-30 min at 37°. 3. The reactions are stopped by acidification with 100/zl of 1 N HCI, followed by three extractions with ethyl acetate. All tubes then receive 1/xg of unlabeled TxB2, PGFz~, PGE2, and PGD2 to assist recoveries. 4. The ethyl acetate extracts are pooled, taken to dryness, and resuspended in 100/zl of benzene. 4 N. K. Hopkins, F. F. Sun, and R. R. Gorman, Biochem. Biophys. Res. Commun. 85, 827 (1978). 5 R. R. Gorman, F. F. Sun, O. V. Miller, and R. A. Johnson, Prostaglandins 13, 1043(1977).
[58]
CULTURED CELLS AND ARACHIDONATE
539
5. The resuspended samples are then spotted onto a silica gel GF plate, and chromatographed in a solvent system of 1% acetic acid, 99% ethyl acetate. 6. The appropriate regions are then scraped from the thin-layer plate and quantitated by liquid scintillation spectroscopy. Table II shows a typical experiment in which 0.1/~M [1-14C]PGH2 is added to WI-38 cells in culture, and TxB2 production quantitated by thin-layer chromatography. The data show that WI-38 cells produce nearly equivalent amounts of TxB2 and PGE2 from PGH2, with lesser amounts of PGF2~ and PGD2 being formed. There is also a considerable amount of HHT formed from nonenzymatic breakdown of the endoperoxide in aqueous medium. If radioactive PGH2 is not readily available, either [1-14C]- or [3H]arachidonic acid can be substituted for [1-14C]PGH2. Alternatively, unlabeled PGH2 or arachidonic acid can be used, and thromboxane quantitated by radioimmunoassay or EIA. Human Umbilical Vein Endothelial Cells Human umbilical vein endothelial cells are an ideal system in which to study PGI2 (prostacyclin) biosynthesis. The PGI2 synthase can be studied directly by adding exogenous cold or [1-14C]PGH2, and the cyclooxygenase and PGI2 synthase can be studied simultaneously by adding exogenous unlabeled or radiolabeled arachidonic acid, or the entire activation sequence, including phospholipase activity, can be studied by stimulating the release of endogenous arachidonic acid by exposing the cells to thrombin. The procedures for using exogenous PGH2 or arachidonate are essentially identical to those outlined for WI-38 cells, except that the thin-layer chromatography system is the organic phase of ethyl acetate-acetic acidisooctane-water (110 : 20 : 50 : 100). The following outlines the step-bystep procedures for using thrombin to initiate PGI2 synthesis in endothelial cells. TABLE I1 ll-14C]PGH2 METABOLISMIN Wl-38 CELLSa cpm/radioactive zone Additions
PGF2a
PGE2
TxB2
PGD2
HHT
[1-~4C]PGH2, 0.6/xM
852 -+ 76
4126 -+ 312
4419 -+ 511
977 - 35
7154 +_ 762
WI-38 cells are incubated with 0.60 ~M PGH2 for 15 rain at 22°. Zones of radioactivity that corresponded to standard prostaglandins are scrapped and quantitated by liquid scintillation counting. Data reported as mean -+ SEM of triplicate determinations.
540
CELL MODELS OF LIVID MEDIATOR PRODUCTION
[58]
Reagents. Human umbilical cords; T-75 plastic tissue culture flasks (Costar Plastics, Cambridge, MA); bovine thrombin, collagenase, and trypsin (Sigma Chemical, St. Louis, MO); medium 199, Earle's base, HEPES buffer, and L-glutamine (Microbiological Associates, Walkersville, MD); sodium penicillin G (Upjohn Co., Kalamazoo, MI); streptomycin sulfate (Eli Lilly, Indianapolis, IN). Human serum is prepared from fresh whole blood. Cord buffer consists of 137 mM NaC1, 4 mM KCI, 10 mM HEPES, pH 7.5, and 11 mM glucose. Cell Culture Procedure. Endothelial cells are derived from human umbilical cord veins as described by Jaffe et al. 6 Cords are stored in sterile containers with 20 ml of cord buffer. A disposable syringe is attached to a blunt 18-gauge needle and the vein flushed with 50 ml of cord buffer to remove any blood. The vein is then perfused with 10 ml of 0.1% collagenase in cord buffer, and incubated at 37° in a bath of cord buffer for 10 min. The collagenase-cell mixture is flushed from the vein with 30 ml of cord buffer into a plastic centrifuge tube that contains 10 ml of medium 199 and centrifuged at 200 g for 5 min at 22°. The cell pellet is resuspended in 10 ml of fresh medium 199 + 20% serum (v/v) and added to a T-75 flask. Cells are exposed to an atmosphere of 95% air-5% CO2 and are fed twice a week until subculturing. Confluency is usually reached in 4-5 days. For subculturing, the medium is removed and the cells rinsed once with cord buffer. A 5-min incubation with a 1 : 1 mixture of 0.02% EDTA, 0.2% collagenase followed by centrifugation is used to harvest the cells. Cells are grown in either T-150 culture flasks or distributed into 35-mm wells. Suspended cells are counted using a Coulter Model ZB-I cell counter. Experimental Procedures 1. Endothelial cells are grown to confluency in 35-mm dishes, and washed twice with ice-cold medium 199-HEPES (50 mM, pH 7.5) and allowed to equilibrate with an additional 1.0 ml of medium 199-HEPES for 10 min at 37°. Cells grown in this manner are characterized by (a) presence of Weibel-Palade bodies as observed by electron microscopyT; (b) the •ability of the medium from cultured cells to support ristocetin-induced agglutination of washed human plateletsS; and (c) immunofluorescence studies of Factor VIII antigens .9
6 E. A. Jaffe, R. L. Nachman, C. G. Becker, and C. R. Minick, J. Clin. Invest. 52, 2745 (1973). 7 E. R. Weibel and G. E. Palade, J. CellBiol. 23, 101 (1964). 8 E. A. Jaffe, L. W. Hoyer, and R. L. Nachman, Proc. Natl. Acad. Sci. U.S.A. 71, 1906 (1974). 9 E. A. Jaffe, L. W. Hoyer, and R. L. Nachman, J. Clin. Invest. 52, 2757 (1973).
[58]
CULTURED CELLS AND ARACHIDONATE
541
2. After the equilibration period, the cells are challenged with 0.1-3.0 U/ml thrombin, and allowed to incubate for the appropriate time interval at 37°. 3. The media is then immediately withdrawn and quickly frozen using liquid nitrogen. 4. Aliquots of the media are subsequently assayed for 6-keto-PGF~, by radioimmunoassay or enzyme-linked immunoassay (EIA) (see [3], this volume). Table III shows a typical profile of PGI2 (measured as 6-keto-PGFl,) synthesis in thrombin-stimulated endothelial cells. Synthesis of PGI2 increases 10-fold with 2 U/ml thrombin and this synthesis is completely blocked by cyclooxygenase inhibitors such as aspirin or indomethacin. In addition, the addition of exogenous PGH2 or arachidonic acid can markedly stimulate PGI2 production. NIH-3T3 Murine Fibroblasts NIH-3T3 cells are very useful because they produce prostaglandin E2 in response to growth factors, and can be easily transfected and transformed by a number of oncogenes. These characteristics make it possible to study the influence of various transfected gene products or drugs on growth factor-regulated eicosanoid metabolism. Reagents. NIH-3T3 cells (American Type Culture Collection (Rockville, MD); Dulbecco's modified Eagle's medium (DMEM), penicillinstreptomycin, glutamine, sodium pyruvate, EDTA, fetal calf serum (Irvine Scientific, Santa Aria, CA); sterile disposable plasticware (Costar Plastics, Cambridge, MA); platelet-derived growth factor (purified according to
T A B L E III THROMBIN-STIMULATED PGI2 BIOSYNTHESIS a Treatment N o n e , basal Thrombin, 2 U T h r o m b i n + 1 m M aspirin T h r o m b i n + 10/~M i n d o m e t h a c i n
6-keto-PGFl,d 106 cells (ng) 1.7 18.8 1.9 1.6
-+ 0.2 +- 1.0 -+ 0.3 - 0.3
o Confluent m o n o l a y e r s of endothelial cells in 35-mm wells are w a s h e d once with 2.0 ml of w a r m M E M containing 25 m M H E P E S . T h e cells are allowed to equilibrate with 1.0 ml of M E M - H E P E S with or without the c y c l o o x y g e n a s e inhibitors for 10 min at 37 ° a n d t h e n stimulated with 2.0 U / m l thrombin. Data are reported as m e a n +- S E M of triplicate samples.
542
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[58]
Raines and ROSS 1°) or recombinant c-sis BB-PDGF (Amgen Biologicals, Thousand Oaks, CA); plasma-derived serum (PDS) from freshly drawn human blood (prepared as described by Habenicht et al. ~!); PGE2 EIA Kit (Cayman Chemical, Ann Arbor, MI). Cell Culture Procedure. NIH-3T3 fibroblasts are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS (EC-10 media). The cells are incubated at 37° in 5% CO2/95% air, and 90% relative humidity. Semiconfluent cultures are passed twice weekly at a 1/10 dilution, by washing the monolayer with EDTA, followed by trypsinization. The suspended cells are centrifuged at 300 g for 5 min at 22°, resuspended in EC-10, and plated in 10-cm 2 tissue culture dishes. Experimental Procedures. NIH-3T3 cells can be stimulated to produce prostaglandin E2 by the addition of 5-20 ng/ml of either purified human platelet-derived growth factor (PDGF) or the recombinant B-chain homodimer of PDGF, c-sis PDGF. 12 The addition of nonexternal receptor agonists, such as phorbol myristate acetate and/or the calcium ionophore, A23187, will also elicit a response from these cells. 13 1. Cells are grown in EC-10 media in 35-mm culture dishes, or 35-mm "6-pack" culture dishes, to about 75% confluency. The media is replaced with DMEM containing either 1.25% PDS, or 0.2% FCS to remove endogenous growth factor and thus maximize the response 18 hr prior to an experiment. 2. To begin the experiment, the media is replaced with DMEM with no serum, and 5-20 ng/ml of PDGF (or c-sis PDGF) or vehicle (0. I M acetic acid) is added. 3. Media are collected at various times and frozen at - 7 0 ° until analysis of PGE2 by radioimmunoassay or EIA (see [3], this volume). 4. Exogenous arachidonate metabolism may be studied by adding 0.33-33/xM arachidonate to cultures treated as above, without addition of PDGF. Again, media is collected and frozen at - 7 0 °, and PGE2 levels determined by RIA or EIA. Figure 2 shows a typical experiment in which purified PDGF is added to four stably transfected NIH-3T3 cell lines and PGE2 measured by EIA over time. The four cell lines are: (1) control, cotransfected with calf thymus DNA; (2) v-src, transformed by pSRA-2, a permuted clone of Rous
~o E. W. Raines, and R. Ross, this series, Vol. 109, p. 733. H A. J. R. Habenicht, J. A. Glomset, and R. Ross, J. Biol. Chem. ,~5, 5134 (1980). 12 C. W. Benjamin, W. G. Tarpley, and R. R. Gorman, Proc. Natl. Acad. Sci. U.S.A. 84, 546 (1987). ~3 C. W. Benjamin, W. G. Tarpley, and R. R. Gorman, Biochem. Biophys. Res. Commun. 145, 1254 (1987).
[58]
543
CULTURED CELLS AND ARACHIDONATE
6000. 5500. 5000.
t"~ ~ - ~
4500 -
1
40003500. D,.
D.
t
//
3000~
/ /
///
2500. .o
"~"
/ "'~ // /I~'"~"~. / / ~/ /
2000-
/
1500-[
,oool
I.....
i,{/I iii1 ~/
_~--i
//
oobT - ° 7J/
0 ,~'~,----~
0
30
-
60
,
~,--_----_--~---¢
90
120 150 180 210 240
Time (minutes) FIG. 2. PDGF-stimulated PGE2 release from control, EJ-ras-, v-src-, and c-rastransfected cells. Cells (0.6 x l06 cells per 35-mm well) are grown for 24 hr in 1.25% PDS and then stimulated by PDGF at 20 ng/ml. PGE2 levels are quantitated by EIA at the indicated times. Data are presented as mean --- SEM of triplicate determinations. O - - O , Control + PDGF; -- O, v-src transfected + PDGF; [ ] - - R , c-ras transfected + PDGF; A - - A , EJ-ras transfected + PDGF.
sarcoma virus DNA; (3) EJ-ras, transformed by the EJ human bladder carcinoma oncogene; and (4) c-ras, a cell line that overexpresses the normal human c-Harvey-ras allele, but is not transformed. PDGF markedly stimulates PGE2 release in control cells, with maximal stimulation at 2 hr. v-src-Transformed cells show a similar pattern of PDGFstimulated PGE2 synthesis, but EJ-ras-transformed cells show no PDGFstimulated E2 synthesis. Finally, cells overexpressing c-ras display slightly reduced PDGF-stimulated PGE2 levels. This type of approach led to the discovery that the ras gene product inhibits phospholipase C activity in NIH-3T3 cells. 14 14 C. W. Benjamin, J. A. Connor, W. G. Tarpley, and R. R. Gorman, Proc. Natl. A c a d . Sci. U . S . A . 85, 4345 (1988).
CULTURED CELLS AND ARACHIDONATE
535
Acknowledgments This work was supported by the Nora Eccles Treadwell Foundation and by Grants from the National Institutes of Health (HL 34127 and HL 35828) and the American Heart Association (Grant-in-Aid 871147). Drs. Prescott and Zimmerman are Established Investigators of the American Heart Association. Dr. Whatley was supported by a National Research Service Award (HL 07529). We thank Dan Fennell for photographs of EC monolayers and Julie Wald for preparing the manuscript.
[58] U s e o f C u l t u r e d C e l l s to S t u d y A r a c h i d o n i c Acid Metabolism By ROBERT R. GORMAN, MICHAEL J. B1ENKOWSKI, an d CHRISTOPHER W. BENJAMIN
Introduction As is often the case in modern biology, there is no one cell system that is ideal for the study of a particular metabolic pathway and arachidonic acid metabolism is no exception. For example, the human platelet is an excellent cell in which to study thromboxane A2 (TxAa) synthesis, and the endothelial cell is useful for studies of prostacyclin (PGI2) synthesis, but, neither cell type makes both PGI2 and TxA2. However, there are enough cultured cells available to study the synthesis of a majority of the various eicosanoids. In this chapter we describe the culture of the human lymphoma cell line U937 and the human lung fibroblast WI-38 as useful cell lines in which to study thromboxane A2 synthesis, human umbilical vein endothelial cells to investigate PGI2 biosynthesis, and the murine fibroblast line NIH-3T3 to study PGE2 synthesis and the influence of cellular transfection and transformation on arachidonate metabolism. These same cell culture systems and techniques can also be used to study the regulation of the various enzymes of the arachidonate cascade as well as the phospholipase(s) that regulate arachidonate concentration in cells. Thromboxane A2 Synthesis by U937 Cells U937 cells were originally isolated from the pleural fluid of a patient with diffuse histiocytic lymphoma.1 These cells exist as immature myelomonocytic cells that can be terminally differentiated by treatment with C. Sundstrom and K. Nilsson, Int. J. Cancer 17, 565 (1976).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
536
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[58]
phorbol 12-myristate 13-acetate (PMA) into cells with the phenotypical characteristics of monocytes/macrophages.2 These differentiated cells can produce TxA2 as the major product in response to a variety of agonists, 3 and are an excellent system in which to study the influence of glucocorticoids on eicosanoid biosynthesis. Reagents. U937 cells (American Type Culture Collection, Rockville, MD); RPMI-1640, Fungi Bact, and fetal calf serum (FCS) (Irvine Scientific, Santa Ana, CA); phorbol 12-myristate 13-acetate (PMA), Ca 2÷ ionophore A23187, lipopolysaccharide (E. coli serotype 055 : B5), zymosan A, and dexamethasone (Sigma Chemical, St. Louis, MO); arachidonic acid, NuCheck Preps (Elysian, MN); EIA Kit (Cayman Chemical Co., Ann Arbor, MI). Cell Culture Procedure. U937 cells are maintained in suspension culture in RPMI-1640 supplemented with l x FungiBact, 10% heatinactivated FCS. For differentiation, cells are plated at 2.0 × 105 cells/ml in medium containing 100 nM PMA and allowed to attach for 48 hr. The cells are then fed with PMA-free medium and cultured overnight prior to use.
Measurement o f Synthesis 1. Differentiated U937 cell monolayers are washed twice with serumfree RPMI-1640 followed by addition of fresh medium containing 1% FCS and either E. coli LPS (10/.~g/ml), heat-activated zymosan A (500/xg/ml), A23187 (10/zg/ml), or arachidonic acid (10/xM) both initially dissolved in dimethyl sulfoxide. 2. After 4 hr (LPS or zymosan) or 15 min (A23187 or arachidonic acid), the medium is removed and TxB2 levels measured using a TxB2-specific enzyme immunoassay (EIA) (see [3], this volume). 3. Steroid treatments (i.e., dexamethasone) are performed by adding the appropriate steroid to RPMI-1640 medium containing 1% FCS and pretreating the cells for varying lengths of time. The medium is removed, replaced with steroid-free medium containing the appropriate agonist, and TxAz synthesis quantified as described above. Table I shows a typical result following exposure of differentiated U937 cells to LPS, zymosan A, arachidonic acid, or A23187:10/zg/ml LPS stimulates 2.7 ng TxA2/106 cells, 500/zg/ml zymosan A stimulates 1.5 ng TxA2/106 cells in 4 hr, 1/~g/ml A23187 stimulates 1.0 ng TxA2/106 cells, and 10/zM arachidonate stimulates 0.8 ng TxA2/106 cells in 15 min. 2 p. Harris and R. Peter, J. Leukocyte Biol. 37, 407 (1985). 3 M. J. Bienkowski, M. A. Petro, and L. J. Robinson, J. Biol. Chem. 264, 6536 (1989).
[58]
537
CULTURED CELLS AND ARACHIDONATE
TABLE I THROMBOXANE SYNTHESIS IN U937 CELLSa Agonist
ng TxA2/I
None, basal LPS, 10/xg/ml Zymosan A, 500 p,g/ml Arachidonic acid 10 t~M A23187, 1/~g/ml
0.54 2.7 1.5 0.8 1.0
× 10 6
cells
-+ 0.04 ± 0.15 ± 0.09 +- 0.06 ± 0.10
a U937 cells are differentiated and cultured as described. TxAz synthesis is initiated by washing the monolayer with serum-free RPMI-1640 and replacing the culture medium with fresh medium containing 10 ~g/ml LPS, 500 ~g/ml zymosan A, 10 t~M arachidonic acid, 1/.Lg/ml A23187. After 4 hr (LPS or zymosan) or 15 rain (A23187, arachidonate) at 37°, the culture medium is removed and the TxB2 content determined by EIA.
Figure I shows a similar experiment when cells are pretreated with dexamethasone (10-5-10 -ll M) for 2 hr prior to stimulation with LPS (10 ~g/ml) or zymosan A (500 t~g/ml) for 4 hr, or arachidonic acid (10 ~M) or A23187 (1 ~g/ml) for 15 min. Dexamethasone has no effect on A23187 or 3.5 3.0...A
-J
2.5
~
2.0
X
l & 1.5-. o ,,r-
g...
,~
1.0 0.5
0.0
I
-11
I0
-1
I
I
I
I
I
-9
-8
-7
-6
-5
LOGDEXAMETHASONE[M] FIG. I. Dexamethasone inhibition of TxAz synthesis initiated by various agonists. Cells are pretreated with various concentrations of dexamethasone for 2 hr. They are then stimulated with either 10 ~g/ml LPS ( O - - © ) or 500 tzg/ml zymosan (&--&) for 4 hr or with 10 tzM arachidonic acid ([~--D) or 1 Ixg/ml A23187 ( A - - A ) for 15 min in steroid-free medium, and the TxB~ content of the medium determined by EIA. Results are expressed as mean +- SE.
538
CELLMODELSOF LIPID MEDIATORPRODUCTION
[58]
arachidonate stimulation of TxA2, however, it dose-dependently inhibits both LPS- and zymosan A-stimulated TxA2 release. Thromboxane A2 Synthesis by WI-38 Cells WI-38 cells are unique because they are the only fibroblast cell line that has been shown to produce thromboxane A2.4 The following outlines step by step procedures for measuring thromboxane synthesis in these cells. Reagents. Human lung fibroblast WI-38 (Flow Laboratories, Rockville, MD); 75 mm tissue culture flasks (Falcon Plastics, Oxnard, CA); 35-mm tissue culture wells (Costar Plastics, Cambridge, MA); Eagle's minimum essential medium (Earle's base), FCS (inactivated 56°, 30 rain), and EDTA-trypsin (Irvine Scientific, Santa Ana, CA); precoated silica gel GF thin-layer plates (Analtech, Inc., Newark, DE); 9,1 l-azoprosta-5,13dienoic acid (azo analog I) and prostaglandin standards (Upjohn Co., Kalamazoo, MI); arachidonic acid (NuCheck Preps., Elysian, MN). [1-14C]PGH2 is synthesized according to Gorman et al. 5 Cell Culture Procedure. Cells are split 1 : 5 using EDTA-trypsin, into culture bottles (75 cm 2) and grown in Eagle's minimum essential medium (Earle's base) supplemented with 10% FCS. This medium with serum will be referred to as MEM-10. Confluent cells are fed with 25 ml of MEM-10 every 5 days, and passaged every 13-14 days. For individual biochemical experiments, the cells are seeded into 35-mm Costar wells, and grown under a humidified atmosphere of 95% air-5% CO2 at 37° until confluent (3-4 days). Final density is approximately 1 × l06 cells/35-mm well. Experimental Procedures
1. Confluent WI-38 cells in 35-ram wells are washed twice with icecold MEM and incubated for 15 min at 37 ° with fresh MEM. 2. After 15 rain, the cells are exposed to 0.1-1.0 ~ M [1-14C]PGH2 (dissolved in MEM immediately before use) or 0.l-10 /xM [1-14C]arachidonic acid (initially dissolved in ethanol) and incubated for 5-30 min at 37°. 3. The reactions are stopped by acidification with 100/zl of 1 N HCI, followed by three extractions with ethyl acetate. All tubes then receive 1/xg of unlabeled TxB2, PGFz~, PGE2, and PGD2 to assist recoveries. 4. The ethyl acetate extracts are pooled, taken to dryness, and resuspended in 100/zl of benzene. 4 N. K. Hopkins, F. F. Sun, and R. R. Gorman, Biochem. Biophys. Res. Commun. 85, 827 (1978). 5 R. R. Gorman, F. F. Sun, O. V. Miller, and R. A. Johnson, Prostaglandins 13, 1043(1977).
[58]
CULTURED CELLS AND ARACHIDONATE
539
5. The resuspended samples are then spotted onto a silica gel GF plate, and chromatographed in a solvent system of 1% acetic acid, 99% ethyl acetate. 6. The appropriate regions are then scraped from the thin-layer plate and quantitated by liquid scintillation spectroscopy. Table II shows a typical experiment in which 0.1/~M [1-14C]PGH2 is added to WI-38 cells in culture, and TxB2 production quantitated by thin-layer chromatography. The data show that WI-38 cells produce nearly equivalent amounts of TxB2 and PGE2 from PGH2, with lesser amounts of PGF2~ and PGD2 being formed. There is also a considerable amount of HHT formed from nonenzymatic breakdown of the endoperoxide in aqueous medium. If radioactive PGH2 is not readily available, either [1-14C]- or [3H]arachidonic acid can be substituted for [1-14C]PGH2. Alternatively, unlabeled PGH2 or arachidonic acid can be used, and thromboxane quantitated by radioimmunoassay or EIA. Human Umbilical Vein Endothelial Cells Human umbilical vein endothelial cells are an ideal system in which to study PGI2 (prostacyclin) biosynthesis. The PGI2 synthase can be studied directly by adding exogenous cold or [1-14C]PGH2, and the cyclooxygenase and PGI2 synthase can be studied simultaneously by adding exogenous unlabeled or radiolabeled arachidonic acid, or the entire activation sequence, including phospholipase activity, can be studied by stimulating the release of endogenous arachidonic acid by exposing the cells to thrombin. The procedures for using exogenous PGH2 or arachidonate are essentially identical to those outlined for WI-38 cells, except that the thin-layer chromatography system is the organic phase of ethyl acetate-acetic acidisooctane-water (110 : 20 : 50 : 100). The following outlines the step-bystep procedures for using thrombin to initiate PGI2 synthesis in endothelial cells. TABLE I1 ll-14C]PGH2 METABOLISMIN Wl-38 CELLSa cpm/radioactive zone Additions
PGF2a
PGE2
TxB2
PGD2
HHT
[1-~4C]PGH2, 0.6/xM
852 -+ 76
4126 -+ 312
4419 -+ 511
977 - 35
7154 +_ 762
WI-38 cells are incubated with 0.60 ~M PGH2 for 15 rain at 22°. Zones of radioactivity that corresponded to standard prostaglandins are scrapped and quantitated by liquid scintillation counting. Data reported as mean -+ SEM of triplicate determinations.
540
CELL MODELS OF LIVID MEDIATOR PRODUCTION
[58]
Reagents. Human umbilical cords; T-75 plastic tissue culture flasks (Costar Plastics, Cambridge, MA); bovine thrombin, collagenase, and trypsin (Sigma Chemical, St. Louis, MO); medium 199, Earle's base, HEPES buffer, and L-glutamine (Microbiological Associates, Walkersville, MD); sodium penicillin G (Upjohn Co., Kalamazoo, MI); streptomycin sulfate (Eli Lilly, Indianapolis, IN). Human serum is prepared from fresh whole blood. Cord buffer consists of 137 mM NaC1, 4 mM KCI, 10 mM HEPES, pH 7.5, and 11 mM glucose. Cell Culture Procedure. Endothelial cells are derived from human umbilical cord veins as described by Jaffe et al. 6 Cords are stored in sterile containers with 20 ml of cord buffer. A disposable syringe is attached to a blunt 18-gauge needle and the vein flushed with 50 ml of cord buffer to remove any blood. The vein is then perfused with 10 ml of 0.1% collagenase in cord buffer, and incubated at 37° in a bath of cord buffer for 10 min. The collagenase-cell mixture is flushed from the vein with 30 ml of cord buffer into a plastic centrifuge tube that contains 10 ml of medium 199 and centrifuged at 200 g for 5 min at 22°. The cell pellet is resuspended in 10 ml of fresh medium 199 + 20% serum (v/v) and added to a T-75 flask. Cells are exposed to an atmosphere of 95% air-5% CO2 and are fed twice a week until subculturing. Confluency is usually reached in 4-5 days. For subculturing, the medium is removed and the cells rinsed once with cord buffer. A 5-min incubation with a 1 : 1 mixture of 0.02% EDTA, 0.2% collagenase followed by centrifugation is used to harvest the cells. Cells are grown in either T-150 culture flasks or distributed into 35-mm wells. Suspended cells are counted using a Coulter Model ZB-I cell counter. Experimental Procedures 1. Endothelial cells are grown to confluency in 35-mm dishes, and washed twice with ice-cold medium 199-HEPES (50 mM, pH 7.5) and allowed to equilibrate with an additional 1.0 ml of medium 199-HEPES for 10 min at 37°. Cells grown in this manner are characterized by (a) presence of Weibel-Palade bodies as observed by electron microscopyT; (b) the •ability of the medium from cultured cells to support ristocetin-induced agglutination of washed human plateletsS; and (c) immunofluorescence studies of Factor VIII antigens .9
6 E. A. Jaffe, R. L. Nachman, C. G. Becker, and C. R. Minick, J. Clin. Invest. 52, 2745 (1973). 7 E. R. Weibel and G. E. Palade, J. CellBiol. 23, 101 (1964). 8 E. A. Jaffe, L. W. Hoyer, and R. L. Nachman, Proc. Natl. Acad. Sci. U.S.A. 71, 1906 (1974). 9 E. A. Jaffe, L. W. Hoyer, and R. L. Nachman, J. Clin. Invest. 52, 2757 (1973).
[58]
CULTURED CELLS AND ARACHIDONATE
541
2. After the equilibration period, the cells are challenged with 0.1-3.0 U/ml thrombin, and allowed to incubate for the appropriate time interval at 37°. 3. The media is then immediately withdrawn and quickly frozen using liquid nitrogen. 4. Aliquots of the media are subsequently assayed for 6-keto-PGF~, by radioimmunoassay or enzyme-linked immunoassay (EIA) (see [3], this volume). Table III shows a typical profile of PGI2 (measured as 6-keto-PGFl,) synthesis in thrombin-stimulated endothelial cells. Synthesis of PGI2 increases 10-fold with 2 U/ml thrombin and this synthesis is completely blocked by cyclooxygenase inhibitors such as aspirin or indomethacin. In addition, the addition of exogenous PGH2 or arachidonic acid can markedly stimulate PGI2 production. NIH-3T3 Murine Fibroblasts NIH-3T3 cells are very useful because they produce prostaglandin E2 in response to growth factors, and can be easily transfected and transformed by a number of oncogenes. These characteristics make it possible to study the influence of various transfected gene products or drugs on growth factor-regulated eicosanoid metabolism. Reagents. NIH-3T3 cells (American Type Culture Collection (Rockville, MD); Dulbecco's modified Eagle's medium (DMEM), penicillinstreptomycin, glutamine, sodium pyruvate, EDTA, fetal calf serum (Irvine Scientific, Santa Aria, CA); sterile disposable plasticware (Costar Plastics, Cambridge, MA); platelet-derived growth factor (purified according to
T A B L E III THROMBIN-STIMULATED PGI2 BIOSYNTHESIS a Treatment N o n e , basal Thrombin, 2 U T h r o m b i n + 1 m M aspirin T h r o m b i n + 10/~M i n d o m e t h a c i n
6-keto-PGFl,d 106 cells (ng) 1.7 18.8 1.9 1.6
-+ 0.2 +- 1.0 -+ 0.3 - 0.3
o Confluent m o n o l a y e r s of endothelial cells in 35-mm wells are w a s h e d once with 2.0 ml of w a r m M E M containing 25 m M H E P E S . T h e cells are allowed to equilibrate with 1.0 ml of M E M - H E P E S with or without the c y c l o o x y g e n a s e inhibitors for 10 min at 37 ° a n d t h e n stimulated with 2.0 U / m l thrombin. Data are reported as m e a n +- S E M of triplicate samples.
542
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[58]
Raines and ROSS 1°) or recombinant c-sis BB-PDGF (Amgen Biologicals, Thousand Oaks, CA); plasma-derived serum (PDS) from freshly drawn human blood (prepared as described by Habenicht et al. ~!); PGE2 EIA Kit (Cayman Chemical, Ann Arbor, MI). Cell Culture Procedure. NIH-3T3 fibroblasts are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS (EC-10 media). The cells are incubated at 37° in 5% CO2/95% air, and 90% relative humidity. Semiconfluent cultures are passed twice weekly at a 1/10 dilution, by washing the monolayer with EDTA, followed by trypsinization. The suspended cells are centrifuged at 300 g for 5 min at 22°, resuspended in EC-10, and plated in 10-cm 2 tissue culture dishes. Experimental Procedures. NIH-3T3 cells can be stimulated to produce prostaglandin E2 by the addition of 5-20 ng/ml of either purified human platelet-derived growth factor (PDGF) or the recombinant B-chain homodimer of PDGF, c-sis PDGF. 12 The addition of nonexternal receptor agonists, such as phorbol myristate acetate and/or the calcium ionophore, A23187, will also elicit a response from these cells. 13 1. Cells are grown in EC-10 media in 35-mm culture dishes, or 35-mm "6-pack" culture dishes, to about 75% confluency. The media is replaced with DMEM containing either 1.25% PDS, or 0.2% FCS to remove endogenous growth factor and thus maximize the response 18 hr prior to an experiment. 2. To begin the experiment, the media is replaced with DMEM with no serum, and 5-20 ng/ml of PDGF (or c-sis PDGF) or vehicle (0. I M acetic acid) is added. 3. Media are collected at various times and frozen at - 7 0 ° until analysis of PGE2 by radioimmunoassay or EIA (see [3], this volume). 4. Exogenous arachidonate metabolism may be studied by adding 0.33-33/xM arachidonate to cultures treated as above, without addition of PDGF. Again, media is collected and frozen at - 7 0 °, and PGE2 levels determined by RIA or EIA. Figure 2 shows a typical experiment in which purified PDGF is added to four stably transfected NIH-3T3 cell lines and PGE2 measured by EIA over time. The four cell lines are: (1) control, cotransfected with calf thymus DNA; (2) v-src, transformed by pSRA-2, a permuted clone of Rous
~o E. W. Raines, and R. Ross, this series, Vol. 109, p. 733. H A. J. R. Habenicht, J. A. Glomset, and R. Ross, J. Biol. Chem. ,~5, 5134 (1980). 12 C. W. Benjamin, W. G. Tarpley, and R. R. Gorman, Proc. Natl. Acad. Sci. U.S.A. 84, 546 (1987). ~3 C. W. Benjamin, W. G. Tarpley, and R. R. Gorman, Biochem. Biophys. Res. Commun. 145, 1254 (1987).
[58]
543
CULTURED CELLS AND ARACHIDONATE
6000. 5500. 5000.
t"~ ~ - ~
4500 -
1
40003500. D,.
D.
t
//
3000~
/ /
///
2500. .o
"~"
/ "'~ // /I~'"~"~. / / ~/ /
2000-
/
1500-[
,oool
I.....
i,{/I iii1 ~/
_~--i
//
oobT - ° 7J/
0 ,~'~,----~
0
30
-
60
,
~,--_----_--~---¢
90
120 150 180 210 240
Time (minutes) FIG. 2. PDGF-stimulated PGE2 release from control, EJ-ras-, v-src-, and c-rastransfected cells. Cells (0.6 x l06 cells per 35-mm well) are grown for 24 hr in 1.25% PDS and then stimulated by PDGF at 20 ng/ml. PGE2 levels are quantitated by EIA at the indicated times. Data are presented as mean --- SEM of triplicate determinations. O - - O , Control + PDGF; -- O, v-src transfected + PDGF; [ ] - - R , c-ras transfected + PDGF; A - - A , EJ-ras transfected + PDGF.
sarcoma virus DNA; (3) EJ-ras, transformed by the EJ human bladder carcinoma oncogene; and (4) c-ras, a cell line that overexpresses the normal human c-Harvey-ras allele, but is not transformed. PDGF markedly stimulates PGE2 release in control cells, with maximal stimulation at 2 hr. v-src-Transformed cells show a similar pattern of PDGFstimulated PGE2 synthesis, but EJ-ras-transformed cells show no PDGFstimulated E2 synthesis. Finally, cells overexpressing c-ras display slightly reduced PDGF-stimulated PGE2 levels. This type of approach led to the discovery that the ras gene product inhibits phospholipase C activity in NIH-3T3 cells. 14 14 C. W. Benjamin, J. A. Connor, W. G. Tarpley, and R. R. Gorman, Proc. Natl. A c a d . Sci. U . S . A . 85, 4345 (1988).
