[61]
LTC4
SYNTHESIS
BY PMNL
AND VASCULAR
CELLS
559
THF. Add 30/zl of 2 N LiOH followed by 15/zl water. Stir this mixture at 4 ° for 24 hr. Saponification can be verified by reversed-phase HPLC using a Hamilton PRP-1 column eluted with acetonitrile/0.01 M borate, pH 10 (Fig. 1).is The concentration of LTA4 can be determined by UV spectroscopy at 280 nm and an extinction coefficient of 52,000. ~s M. Wynalda, D. Morton, R. Kelly, and F. A. Fitzpatrick, Anal. Chem. 54, 1079 (1982).
[ 6 1 ] L e u k o t r i e n e C4 B i o s y n t h e s i s d u r i n g P o l y m o r p h o n u c l e a r Leukocyte-Vascular Cell Interactions B y STEVEN J. FEINMARK
Polymorphonuclear leukocytes (PMNL) must adhere to and cross the endothelial cell (EC) lining in order to emigrate from the circulation to sites of developing inflammation. The sustained contact between EC and PMNL that ensues enhances the potential for novel biochemical interactions between these cells. One such interaction is the augmented production ofleukotriene (LT)C4 as a consequence o f P M N L - E C coincubation in vitro. ~ A similar interaction occurs between aortic smooth muscle (SMC) and PMNL. 2 Several approaches have been taken to demonstrate the transcellular nature of L T C 4 production during these incubations. Each cell type was evaluated individually to determine which metabolites could be produced either from endogenous arachidonate or from exogenous biosynthetic intermediates. Then, cultured cells and PMNL were coincubated and the products quantified. Finally, the glutathione (GSH) pools of each cell type were labeled individually and incorporation of this label into leukotriene was used to determine the site of the final synthetic step, the conversion of LTA4 to LTC4. Cell Preparations
Human Peripheral Blood Leukocytes Blood is collected from normal drug-free volunteers who had granted informed consent. Typically, blood is drawn through a 19-gauge butterfly S. J. Feinmark and P. J. Cannon, J. Biol. Chem. 261, 16466 (1986). 2 S. J. Feinmark and P. J. Cannon, Biochim. Biophys. Acta 922, 125 (1987).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
560
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
into 60-ml syringes and immediately transferred to EDTA-containing vacutainers (Becton Dickinson, Rutherford, NJ). The anticoagulated blood is pooled into 50-ml polycarbonate tubes and subjected to centrifugation at 200 g for 15 min at room temperature. The platelet-rich plasma (upper layer) is removed and the residual blood (approx. one-half of the original volume) is transferred to a Nalgene cylinder and combined with an equal volume of dextran [prepared by dissolving 6 g of dextran T-500 (Pharmacia, Piscataway, NJ) plus 2.7 g of NaCI in 300 ml of water]. After several inversions to ensure adequate mixing, the blood is left at room temperature to permit the aggregated erythrocytes to sediment. When the blood/ dextran solution separates into two roughly equal phases (approx. 45-60 min), the PMNL-rich upper phase is collected and concentrated by centrifugation at 200 g for 15 min at room temperature. The cell pellets are gently resuspended in 5-10 ml of lysis buffer [prepared by dissolving 2.06 g of Tris(hydroxymethyl)aminoethane and 7.47 g of ammonium chloride/liter of water and finally adjusting the pH to 7.4) and kept at 37° for 7 min. The tubes are gently agitated and a sodium metrizoate/Ficoll solution (density 1.077 g/ml; Lymphoprep, Nycomed) is gently layered under the cell suspension to form a discontinuous gradient (5 ml is slowly dispensed through an 18-gauge needle placed at the bottom of the centrifuge tube). These tubes are spun at 400 g for 40 min at room temperature. The cell pellets are resuspended in a physiological salt solution (for example, Dulbecco's phosphate-buffered saline, pH 7.4; PBS; GIBCO) with bovine serum albumin (BSA; essentially fatty acid-free, Sigma, St. Louis, MO), if desired. The resulting suspension is counted in a hemocytometer and diluted as necessary. This preparation contains primarily neutrophils with some eosinophils (mononuclear cells are generally less than 1%). 3,4 Porcine Aortic Endothelial Cells
Porcine aortas are collected within minutes of slaughter at a local abatoir under a license from the United States Department of Agriculture (USDA). The vessels are packed in sterile bags on ice and transported to the laboratory within 2-3 hr. Subsequent steps are carried out in a sterile hood using standard sterile technique. The vessels are washed free of blood with Dulbecco's modified Eagle medium (DMEM; GIBCO), opened longitudinally, and laid on a piece of sterile foil. The exposed intima is gently scraped with a scalpel taking care to avoid collateral vessels, any residual blood clots, and the cut ends of the tissue. Cells are collected by gently swirling the scalpel blade in DMEM (10 ml in a sterile tube) followed by centrifugation (300 g, 15 min). The cell pellet is resuspended in DMEM 3 A. B6yum, Scand. J. lmmunol. 5 (Suppl. 5), 9 (1976). 4 H.-E. Claesson and S. J. Feinmark, Biochim. Biophys. Acta 804, 52 (1984).
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULAR CELLS
561
containing fetal bovine serum (FBS, 10%), penicillin (400 U/ml), streptomycin (400 tzg/ml), L-glutamine (2 mM), chlortetracycline (50 tzg/ml), and Fungizone (2.5 /zg/ml), and washed twice more with this medium before being plated into 25-cm 2 tissue culture flasks and grown overnight. The following day, the primary cultures are washed vigorously to remove any nonadherent cells or debris and refed with the same medium. On the second day, the cultures are rewashed and refed with normal growth medium consisting of DMEM plus FBS (10%), penicillin (I00 U/ml), streptomycin (I00/zg/ml), and e-glutamine (2 mM). Cultures are routinely fed every 3-4 days and subcultured as needed. Confluent cultures are washed with calcium, magnesium-free PBS (GIBCO) to remove the growth medium, and subdivided with I-2 ml of trypsin-EDTA solution (containing 0.5 mg/ml trypsin and 0.2 mg/ml Na4EDTA in calcium, magnesium-free Hanks' balanced salt solution; GIBCO). The cells round up and can be detached within minutes by gentle tapping of the tissue culture flask. The cell suspensions are then diluted in growth medium and subdivided as needed. From time to time, cultures are grown in the absence of antibiotics and shown to be free of mycoplasma (MycoTect, BRL, Gaithersburg, MD). Cultures are verified to be endothelium by their typical cobblestone morphology and by the organization of actin-based cytoskeleton (by rhodamine-phalloidin) and factor VIII staining, and the absence of detectable smooth muscle actin. 5 Human umbilical vein endothelial cells (HUVEC) are prepared from term umbilical cords by collagenase treatment essentially as reported by others. 6"7 Cells are maintained in HEPES-buffered M-199 supplemented with human serum (15%), penicillin (50 U/ml), streptomycin (50/zg/ml), L-glutamine (2 mM), endothelial cell growth factor (ECGF) (15/zg/ml), and heparin (90 tzg/ml) 8 and subcultured as necessary. Vascular smooth muscle cells are cultured by explant from porcine aortas after removal of the endothelium by intimal scraping. The aortic media is isolated by blunt dissection and chunks of vascular smooth muscle (approximately 1 mm 3) are placed in a culture dish. The tissue is fed with DMEM plus FBS (10%), penicillin (100 U/ml), streptomycin (I00 tzg/ml), and e-glutamine (2 mM ) and covered with a sterile glass coverslip. The dishes are left undisturbed until cells are observed to be growing out of the explants. The tissue pieces and coverslip are then removed and
A. 1. Gotlieb, W. Spector, M. K. K. Wong, and C. Lacey, Arteriosclerosis 4, 91 (1984). 6 E. A. Jaffe, R. L. Nachman. C. G. Becker, and C. R. Minick, J. Clin. Invest. 52, 2745 (1973). 7 G. A. Zimmerman, R. E. Whatley, T. M. Mclntyre, D. M. Benson, and S. M. Prescott, this volume [57]. s S. C. Thornton, S. N. Mueller, and E. M. Levine, Science 222, 623 (1983).
562
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
the primary cultures refed and grown to confluence. These cells are subcultured as described above. The cultures are determined to be smooth muscle by morphological criteria and the presence of smooth muscle actin.
Incubation and Purification Procedures Exogenous LTA4. Synthetic LTA4 methyl ester is obtained from Upjohn Co. (Kalamazoo, MI, through the courtesy of Dr. F. Sun) or from BIOMOL Research Laboratories (Plymouth Meeting, PA). The ester is hydrolyzed by dissolution in methanol: 50% NaOH (9/1, v/v; usually at a concentration of 5 mM) and kept at - 2 0 ° overnight. In many cases, the LTA4 is tested to confirm that the hydrolysis is complete and that the labile epoxide is intact. An aliquot of the hydrolyzate is diluted in acidic methanol which rapidly converts intact LTA4 into two isomers of 5(S )-hydroxy12-methoxyeicosatetraenoic acid. 9 Samples are then dried, redissolved in mobile phase, and analyzed by HPLC on a Nucleosil C~8 column (4.6 x 250 mm, Alltech, Deerfield, IL) eluted with methanol : water : acetic acid (75:25:0.01, v/v/v) at 1 ml/min. The products are detected by UV absorbance at 270 nm. The methanolysis products of LTA4 are readily separated from the isomeric 5(S),12-dihydroxyeicosatetraenoic acids which result from prior decomposition of LTA4. In addition, the free acids resolve from their corresponding methyl esters thus confirming that the ester hydrolysis is complete. LTA4 is added directly to cultured cells bathed in PBS buffered with HEPES (15 raM, pH 7.4). Media from these experiments are directly injected on to the HPLC which is fitted with a Nucleosil C~8 column and eluted with methanol : water : acetic acid (67 : 33 : 0.08, v/v/v) buffered to pH 5.8 with ammonium hydroxide and flowing at 1 ml/min. Columns are routinely treated with EDTA. 1oThe eluent is monitored at 280 nm or with a photodiode array spectrophotometer. Fractions (1-ml) are collected for subsequent assay. PMNL/Vascular Cell Coincubations. Cultured cells are used at confluence (typically in 25-cm 2 flasks). PMNL are purified as described above and resuspended in PBS plus BSA (0.5-1%). PMNL, 1-2 × 107, in I ml are added to a washed culture of vascular cells or incubated alone. A23187 (final concentration, 5/xM) is added to the incubation buffer in a concentrated ethanolic solution (final solvent concentrations never exceeded 0.2%). Simultaneous addition of arachidonic acid is achieved by the dissolution of a measured amount of fatty acid in the A23187 solution, followed 9 p. Borgeat and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 76, 3213 (1979). io S. A. Metz, M. E. Hall, T. W. Harper, and R. C. Murphy, J. Chromatogr. 233, 193 (1982).
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULAR CELLS
563
by brief sonication, fMLP (formylmethionylleucylphenylalanine) stimulation is carded out by the initial addition of arachidonic acid (10/zM) in ethanol followed 1 min later by fMLP (1 /zM) in dimethyl sulfoxide (DMSO). In some experiments, EC monolayers are treated with aspirin (ASA; 0.2 mM) added in ethanol for 30 min. The cultures are then washed twice more and the PMNL suspension added. Incubations are continued for 30 min at 37 ° in a humidified CO2 incubator (5% in air). After the completion of the incubation, the media are mixed with ice-cold methanol (1-2 volumes) containing tracer amounts of [3H]LTC4 as internal standard and kept at - 2 0 ° for several hours; any protein precipitate is removed by centrifugation. Aliquots of the samples are evaporated to dryness, reconstituted in mobile phase, and applied to the reversed-phase HPLC as above.
L TC4 Quantification
LTC4 is measured by radioimmunoassay after HPLC purification. Aliquots of the HPLC fractions are tested for the presence of [3H]LTC4 internal standard to unequivocally identify the LTC4-containing fractions and to correct for purification losses. The remainder of each HPLC fraction is evaporated to dryness and reconstituted in PBS. Each fraction is assayed in duplicate for the presence of immunoreactive LTC4. Details of the radioimmunoassay (DuPont-NEN, Wilmington, DE) have been described previously. 11 The anti-LTC4 antiserum has a cross-reactivity of 55.3% with LTD4 and 8.6% with LTE4.12 The intraassay variability is 8.5%, the interassay variability is 19-22%. Measurement of Cellular Integrity PMNL or cultured vascular cells are labeled with ~lln-oxine by a modification of published methods. 1'13'14 Monolayer cultures (grown in 24-well plates) and PMNL are washed twice with Hanks' balanced salt solution (Hanks' BSS; GIBCO). PMNL are resuspended in Hanks' (2 × 107/ml) and the cultures covered with the same medium (0.5 ml). lllInoxine (2/zCi) is added to each well or the suspended PMNL and the cells are left at room temperature for 15 rain. H D. Piomelli, S. J. Feinmark, and P. J. Cannon, J. Pharmacol. Exp. Ther. 241, 763 (1987). ~z E. C. Hayes, D. L. Lombardo, Y. Girard, A. L. Maycock, J. Rokach, A. S. Rosenthal, R. N. Young, R. W. Egan, and H. J. Zweerink, J. lmmunol. 131, 429 (1983). t3 B. Zakhireh, M. L. Thakur, H. L. Malech, M. S. Cohen, A. Gottschaik, and R. K. Root, J. Nucl. Med. 20, 741 (1979). ~4 T. Collins, A. M. Krensky, C. Clayberger, W. Fiers, M. A. Gimbrone, Jr., S. J. Burakoff, and J. S. Pober, J. lmmunol. 133, 1878 (1984).
564
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
After washing the monolayers with Tyrode's buffer containing 4% BSA (Tyrode's-BSA), the cells are covered with the same buffer and incubated at 37° for 90 min. Each well is rewashed with Tyrode's-BSA and then incubated as described with unlabeled PMNL. At the end of the incubation, the buffer is recovered, centrifuged (200 g, room temperature) to remove any cells, and aliquots are assayed for the presence of l~In by gamma counting. The cells remaining in the wells are lysed by the addition of 2% Nonidet P-40 (NP-40). Aliquots of the detergent lysate are assayed for l llln and the percentage of total I i qn released is calculated. PMNL are washed twice in Hanks' BSS after the labeling period and then resuspended either in phosphate-buffered saline (PBS) or PBS-BSA as appropriate and incubated as described above. At the end of the incubation, the buffer is recovered and the PMNL are concentrated by centrifugation at 200 g, at room temperature. The pellet is lysed with 2% NP-40 and aliquots of the supernatant and of the lysed pellet are assayed for the presence of ~~tIn.
Incorporation o f Label into Cellular Glutathione Pools PMNL (2 × 107/ml) or monolayer cultures are washed twice with PBS plus 1% BSA and then incubated for 1 hr at 37° with [35S]cysteine (5/zCi; >600 Ci/mmol) in PBS-BSA. Unincorporated cysteine is removed by two washes with PBS-BSA. Prelabeled cells are incubated as described above and the incubation media purified by HPLC. The fractions are analyzed for the presence of 35S-labeled material by liquid scintillation counting.
e~
f
13
'
i5
RETENTION TIME (min)
FIG. 1. Monolayercultures of humanumbilicalveinendothelialcells are incubatedwith LTA4 (10 ~tM) in HEPES-bufferedPBS plus 1% BSA. The culture mediumis recovered, dilutedwithmethanol(2 volumes),and directlyinjectedon the HPLCas describedin the text.
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULARCELLS
565
TABLE I
LTC4PRODUCTION BY MIXED P M N L
AND VASCULAR CELLS a
LTC4 production (pmol/107 PMNL-flask) Stimulus A23187 (5/xM) A23187 (5/~M) +20:4 (150/~M) fMLP (1/zM) +20:4 (10/xM)
PMNL 3.8 (n 10.5 (n 0.75 (n
= ± = ± =
1.8 8) 2.1 11) 0.39 5)
PMNL+EC 15.9 (n 24.3 (n 0.23 (n
--- 6.1 = 3) ± 6.7 = 3) - 0.08 = 5)
PMNL+SMC
PMNL+EC (ASA)
11.0 ± 3.5(n = 5) 39.8 ± 8.1 (n = 5) 1.7 -+ 0.4
(n = 5)
PMNL and vascular cells are coincubated as described in the text with the stimuli noted above. LTC4 is purified by HPLC and quantified by radioimmunoassay. Portions of these data have been reported elsewhere in a different form. 1.2,19
Results
LTA4 Metabolism HUVEC are incubated with exogenous synthetic LTA4 and the p r e s e n c e o f m e t a b o l i t e s in t h e m e d i u m m e a s u r e d b y H P L C . S e v e r a l p e a k s o f U V - a b s o r b i n g m a t e r i a l a r e d e t e c t e d at 280 n m ( F i g . I). T h e t w o l a r g e s t p e a k s a r e a l s o f o u n d in c o n t r o l i n c u b a t i o n s a n d c o r r e s p o n d to t h e n o n e n z y m a t i c d e c o m p o s i t i o n p r o d u c t s o f L T A 4 . I n t h e cell i n c u b a t i o n s , a t h i r d p e a k a p p e a r s t h a t e l u t e s at t h e r e t e n t i o n t i m e o f s y n t h e t i c L T C 4 . T h e p r o d u c t i o n o f L T B 4 is n o t o b s e r v e d in this e x p e r i m e n t . T h e a b s e n c e o f L T B 4 c a n n o t b e e x p l a i n e d b y its m e t a b o l i c r e m o v a l s i n c e n e i t h e r E C l'15 n o r S M C 2 m e t a b o l i z e this lipid. C l a e s s o n a n d H a e g g s t r 6 m , ~5 h o w e v e r , have reported that HUVEC produce LTB4 under these conditions. Earlier s t u d i e s o f H U V E C , t6 p o r c i n e E C , l o r p o r c i n e S M C 2 d i d n o t d e t e c t t h e c o n v e r s i o n o f L T A 4 to L T B 4 .
Quantitative Aspects o f P M N L - Vascular Cell Interactions A23187 Stimulation. A 2 3 1 8 7 - s t i m u l a t e d P M N L p r e p a r a t i o n s g e n e r a t e LTC4 and the levels are enhanced by the addition of exogenous arachid o n i c a c i d ( T a b l e I). T h e p r e c i s e c e l l u l a r s o u r c e o f this l e u k o c y t e p r o d u c t is n o t c l e a r s i n c e p o r t i o n s o f t h e L T C 4 p r o d u c t i o n m a y be d u e t o e o s i n o -
~5 H.-E. Claesson and J. HaeggstrOm, Eur. J. Biochem. (1988). 16 B. O. Ibe and W. B. Campbell, Biochim. Biophys. Acta 960, 309 0988).
566
CELL MODELS OF LIPID MEDIATOR PRODUCTION
2000n_
/
l
/
1500-
II
[61]
°-° D PMNL
A--A
LABELEDPMNL
c)
_
I.0,< 0 ~:
1000
• 500
Df
0
5
15
25
(rain) FiG. 2. Either PMNL or EC are prelabeled with [35S]cysteine and then washed. PMNL and EC are coincubated, stimulated with A23187 (5 ~ M ) and arachidonic acid (150 ~M), and the incubation buffer recovered. The LTC4 is purified by HPLC as described in the text and fractions analyzed for the presence of 35S-labeled products. Data have been normalized to equal LTC4 mass production from a paired incubation. RETENTION TIME
phils 17 or to transcellular metabolism of neutrophil-derived LTA4 by contaminating platelets.~8 Nevertheless, A23187 stimulation of PMNL in the presence of vascular cells invariably leads to increased LTC4 production compared to matched control incubations of PMNL alone (Table I). Exogenous arachidonate also enhances the leukotriene production during these experiments. fMLP Stimulation/EC Feedback Regulation. The bacterial tripeptide fMLP is a very weak inducer of PMNL LTC4 synthesis, generating less than 20% of the product released after A23187 treatment (Table I). Unlike A23187, fMLP stimulation of mixed PMNL plus EC fails to produce a quantitative increase in LTC4 production but rather, PMNL LTC4 synthesis is significantly inhibited (Table I).19 EC-dependent inhibition of fMLPinduced LTC4 synthesis is abolished by treatment of the EC cultures with the cyclooxygenase inhibitor, aspirin (0.2 mM). fMLP stimulation of PMNL in the presence of aspirin-treated EC generates more LTC4 than either PMNL alone or PMNL plus untreated EC. Thus, transcellular ~7 p. F. Weller, C. W. Lee, D. W. Foster, E. J. Corey, K. F. Austen, and R. A. Lewis, Proc. Natl. Acad. Sci. U.S.A. 80, 7626 (1983). t8 j. A. Maciouf and R. C. Murphy, J. Biol. Chem. 263, 174 (1986). 19 S. J. Feinmark, J. Edasery, and P. J. Cannon, Adv. Prostaglandin Thromboxane Leukotriene Res. 19, 255 (1989).
[62]
I N T E R C E L L U L A R T R A N S F E R OF L T A 4
567
metabolism of L T A 4 to L T C 4 c a n be demonstrated even after relatively weak stimulation. [35S]LTC4 Production during PMNL/Vascular Cell Coincubation. To demonstrate that the conversion of PMNL-derived LTA4 to L T C 4 o cc u r r e d within the vascular cell cultures, [35S]cysteine is used to label the cellular GSH pools. Prelabeled PMNL incubated alone or with unlabeled vascular cells releases low levels of [35S]LTC4 after A23187 stimulation. However, [35S]cysteine-prelabeled EC 1 or SMC z incubated with A23187stimulated unlabeled PMNL produces significantly m o r e [358]LTC4 than in the reverse-labeling experiment (Fig. 2). Conclusions Using the methods described in this chapter, it has been possible to demonstrate that activated PMNL release LTA4 which can be converted to LTC4 by adjacent cultured vascular cells. The level of LTC4 produced depends on the activating stimulus. Weak PMNL activators, such as fMLP, may permit the observation of a feedback regulation loop in which PMNL leukotriene synthesis is inhibited by a vascular cell product which is probably a prostaglandin. Acknowledgments Some of the experiments described in this chapter were supported by AI-26702, HL-38312, and HL-21006. Portions of this work were done during the tenure of an Established lnvestigatorship award from the American Heart Association and Boehringer lngelheim, Inc. The author would like to thank Dr. J. Brett for the morphological examination of the cultured cells and Dr. R. R. Sciacca for statistical analyses and critical reading of this manuscript.
[62] R e l e a s e a n d M e t a b o l i s m o f L e u k o t r i e n e A4 in Neutrophil-Mast Cell Interactions
By CLEMENS A. DAmNDEN and URs WmTHMUELLER Introduction The cellular sources, biosynthesis, structure, and different biological activities of the 5-1ipoxygenase (5-LOX) metabolites leukotriene (LT) B4, L T C 4 , LTD4, and L T E 4 a r e well established. However, there is an interesting particularity of the 5-LOX pathway in that at each metabolic step the precursor molecules are formed in amounts largely exceeding the METHODS IN ENZYMOLOGY, VOL. 187
Copyright (~3 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
LTC4
SYNTHESIS
BY PMNL
AND VASCULAR
CELLS
559
THF. Add 30/zl of 2 N LiOH followed by 15/zl water. Stir this mixture at 4 ° for 24 hr. Saponification can be verified by reversed-phase HPLC using a Hamilton PRP-1 column eluted with acetonitrile/0.01 M borate, pH 10 (Fig. 1).is The concentration of LTA4 can be determined by UV spectroscopy at 280 nm and an extinction coefficient of 52,000. ~s M. Wynalda, D. Morton, R. Kelly, and F. A. Fitzpatrick, Anal. Chem. 54, 1079 (1982).
[ 6 1 ] L e u k o t r i e n e C4 B i o s y n t h e s i s d u r i n g P o l y m o r p h o n u c l e a r Leukocyte-Vascular Cell Interactions B y STEVEN J. FEINMARK
Polymorphonuclear leukocytes (PMNL) must adhere to and cross the endothelial cell (EC) lining in order to emigrate from the circulation to sites of developing inflammation. The sustained contact between EC and PMNL that ensues enhances the potential for novel biochemical interactions between these cells. One such interaction is the augmented production ofleukotriene (LT)C4 as a consequence o f P M N L - E C coincubation in vitro. ~ A similar interaction occurs between aortic smooth muscle (SMC) and PMNL. 2 Several approaches have been taken to demonstrate the transcellular nature of L T C 4 production during these incubations. Each cell type was evaluated individually to determine which metabolites could be produced either from endogenous arachidonate or from exogenous biosynthetic intermediates. Then, cultured cells and PMNL were coincubated and the products quantified. Finally, the glutathione (GSH) pools of each cell type were labeled individually and incorporation of this label into leukotriene was used to determine the site of the final synthetic step, the conversion of LTA4 to LTC4. Cell Preparations
Human Peripheral Blood Leukocytes Blood is collected from normal drug-free volunteers who had granted informed consent. Typically, blood is drawn through a 19-gauge butterfly S. J. Feinmark and P. J. Cannon, J. Biol. Chem. 261, 16466 (1986). 2 S. J. Feinmark and P. J. Cannon, Biochim. Biophys. Acta 922, 125 (1987).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
560
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
into 60-ml syringes and immediately transferred to EDTA-containing vacutainers (Becton Dickinson, Rutherford, NJ). The anticoagulated blood is pooled into 50-ml polycarbonate tubes and subjected to centrifugation at 200 g for 15 min at room temperature. The platelet-rich plasma (upper layer) is removed and the residual blood (approx. one-half of the original volume) is transferred to a Nalgene cylinder and combined with an equal volume of dextran [prepared by dissolving 6 g of dextran T-500 (Pharmacia, Piscataway, NJ) plus 2.7 g of NaCI in 300 ml of water]. After several inversions to ensure adequate mixing, the blood is left at room temperature to permit the aggregated erythrocytes to sediment. When the blood/ dextran solution separates into two roughly equal phases (approx. 45-60 min), the PMNL-rich upper phase is collected and concentrated by centrifugation at 200 g for 15 min at room temperature. The cell pellets are gently resuspended in 5-10 ml of lysis buffer [prepared by dissolving 2.06 g of Tris(hydroxymethyl)aminoethane and 7.47 g of ammonium chloride/liter of water and finally adjusting the pH to 7.4) and kept at 37° for 7 min. The tubes are gently agitated and a sodium metrizoate/Ficoll solution (density 1.077 g/ml; Lymphoprep, Nycomed) is gently layered under the cell suspension to form a discontinuous gradient (5 ml is slowly dispensed through an 18-gauge needle placed at the bottom of the centrifuge tube). These tubes are spun at 400 g for 40 min at room temperature. The cell pellets are resuspended in a physiological salt solution (for example, Dulbecco's phosphate-buffered saline, pH 7.4; PBS; GIBCO) with bovine serum albumin (BSA; essentially fatty acid-free, Sigma, St. Louis, MO), if desired. The resulting suspension is counted in a hemocytometer and diluted as necessary. This preparation contains primarily neutrophils with some eosinophils (mononuclear cells are generally less than 1%). 3,4 Porcine Aortic Endothelial Cells
Porcine aortas are collected within minutes of slaughter at a local abatoir under a license from the United States Department of Agriculture (USDA). The vessels are packed in sterile bags on ice and transported to the laboratory within 2-3 hr. Subsequent steps are carried out in a sterile hood using standard sterile technique. The vessels are washed free of blood with Dulbecco's modified Eagle medium (DMEM; GIBCO), opened longitudinally, and laid on a piece of sterile foil. The exposed intima is gently scraped with a scalpel taking care to avoid collateral vessels, any residual blood clots, and the cut ends of the tissue. Cells are collected by gently swirling the scalpel blade in DMEM (10 ml in a sterile tube) followed by centrifugation (300 g, 15 min). The cell pellet is resuspended in DMEM 3 A. B6yum, Scand. J. lmmunol. 5 (Suppl. 5), 9 (1976). 4 H.-E. Claesson and S. J. Feinmark, Biochim. Biophys. Acta 804, 52 (1984).
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULAR CELLS
561
containing fetal bovine serum (FBS, 10%), penicillin (400 U/ml), streptomycin (400 tzg/ml), L-glutamine (2 mM), chlortetracycline (50 tzg/ml), and Fungizone (2.5 /zg/ml), and washed twice more with this medium before being plated into 25-cm 2 tissue culture flasks and grown overnight. The following day, the primary cultures are washed vigorously to remove any nonadherent cells or debris and refed with the same medium. On the second day, the cultures are rewashed and refed with normal growth medium consisting of DMEM plus FBS (10%), penicillin (I00 U/ml), streptomycin (I00/zg/ml), and e-glutamine (2 mM). Cultures are routinely fed every 3-4 days and subcultured as needed. Confluent cultures are washed with calcium, magnesium-free PBS (GIBCO) to remove the growth medium, and subdivided with I-2 ml of trypsin-EDTA solution (containing 0.5 mg/ml trypsin and 0.2 mg/ml Na4EDTA in calcium, magnesium-free Hanks' balanced salt solution; GIBCO). The cells round up and can be detached within minutes by gentle tapping of the tissue culture flask. The cell suspensions are then diluted in growth medium and subdivided as needed. From time to time, cultures are grown in the absence of antibiotics and shown to be free of mycoplasma (MycoTect, BRL, Gaithersburg, MD). Cultures are verified to be endothelium by their typical cobblestone morphology and by the organization of actin-based cytoskeleton (by rhodamine-phalloidin) and factor VIII staining, and the absence of detectable smooth muscle actin. 5 Human umbilical vein endothelial cells (HUVEC) are prepared from term umbilical cords by collagenase treatment essentially as reported by others. 6"7 Cells are maintained in HEPES-buffered M-199 supplemented with human serum (15%), penicillin (50 U/ml), streptomycin (50/zg/ml), L-glutamine (2 mM), endothelial cell growth factor (ECGF) (15/zg/ml), and heparin (90 tzg/ml) 8 and subcultured as necessary. Vascular smooth muscle cells are cultured by explant from porcine aortas after removal of the endothelium by intimal scraping. The aortic media is isolated by blunt dissection and chunks of vascular smooth muscle (approximately 1 mm 3) are placed in a culture dish. The tissue is fed with DMEM plus FBS (10%), penicillin (100 U/ml), streptomycin (I00 tzg/ml), and e-glutamine (2 mM ) and covered with a sterile glass coverslip. The dishes are left undisturbed until cells are observed to be growing out of the explants. The tissue pieces and coverslip are then removed and
A. 1. Gotlieb, W. Spector, M. K. K. Wong, and C. Lacey, Arteriosclerosis 4, 91 (1984). 6 E. A. Jaffe, R. L. Nachman. C. G. Becker, and C. R. Minick, J. Clin. Invest. 52, 2745 (1973). 7 G. A. Zimmerman, R. E. Whatley, T. M. Mclntyre, D. M. Benson, and S. M. Prescott, this volume [57]. s S. C. Thornton, S. N. Mueller, and E. M. Levine, Science 222, 623 (1983).
562
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
the primary cultures refed and grown to confluence. These cells are subcultured as described above. The cultures are determined to be smooth muscle by morphological criteria and the presence of smooth muscle actin.
Incubation and Purification Procedures Exogenous LTA4. Synthetic LTA4 methyl ester is obtained from Upjohn Co. (Kalamazoo, MI, through the courtesy of Dr. F. Sun) or from BIOMOL Research Laboratories (Plymouth Meeting, PA). The ester is hydrolyzed by dissolution in methanol: 50% NaOH (9/1, v/v; usually at a concentration of 5 mM) and kept at - 2 0 ° overnight. In many cases, the LTA4 is tested to confirm that the hydrolysis is complete and that the labile epoxide is intact. An aliquot of the hydrolyzate is diluted in acidic methanol which rapidly converts intact LTA4 into two isomers of 5(S )-hydroxy12-methoxyeicosatetraenoic acid. 9 Samples are then dried, redissolved in mobile phase, and analyzed by HPLC on a Nucleosil C~8 column (4.6 x 250 mm, Alltech, Deerfield, IL) eluted with methanol : water : acetic acid (75:25:0.01, v/v/v) at 1 ml/min. The products are detected by UV absorbance at 270 nm. The methanolysis products of LTA4 are readily separated from the isomeric 5(S),12-dihydroxyeicosatetraenoic acids which result from prior decomposition of LTA4. In addition, the free acids resolve from their corresponding methyl esters thus confirming that the ester hydrolysis is complete. LTA4 is added directly to cultured cells bathed in PBS buffered with HEPES (15 raM, pH 7.4). Media from these experiments are directly injected on to the HPLC which is fitted with a Nucleosil C~8 column and eluted with methanol : water : acetic acid (67 : 33 : 0.08, v/v/v) buffered to pH 5.8 with ammonium hydroxide and flowing at 1 ml/min. Columns are routinely treated with EDTA. 1oThe eluent is monitored at 280 nm or with a photodiode array spectrophotometer. Fractions (1-ml) are collected for subsequent assay. PMNL/Vascular Cell Coincubations. Cultured cells are used at confluence (typically in 25-cm 2 flasks). PMNL are purified as described above and resuspended in PBS plus BSA (0.5-1%). PMNL, 1-2 × 107, in I ml are added to a washed culture of vascular cells or incubated alone. A23187 (final concentration, 5/xM) is added to the incubation buffer in a concentrated ethanolic solution (final solvent concentrations never exceeded 0.2%). Simultaneous addition of arachidonic acid is achieved by the dissolution of a measured amount of fatty acid in the A23187 solution, followed 9 p. Borgeat and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 76, 3213 (1979). io S. A. Metz, M. E. Hall, T. W. Harper, and R. C. Murphy, J. Chromatogr. 233, 193 (1982).
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULAR CELLS
563
by brief sonication, fMLP (formylmethionylleucylphenylalanine) stimulation is carded out by the initial addition of arachidonic acid (10/zM) in ethanol followed 1 min later by fMLP (1 /zM) in dimethyl sulfoxide (DMSO). In some experiments, EC monolayers are treated with aspirin (ASA; 0.2 mM) added in ethanol for 30 min. The cultures are then washed twice more and the PMNL suspension added. Incubations are continued for 30 min at 37 ° in a humidified CO2 incubator (5% in air). After the completion of the incubation, the media are mixed with ice-cold methanol (1-2 volumes) containing tracer amounts of [3H]LTC4 as internal standard and kept at - 2 0 ° for several hours; any protein precipitate is removed by centrifugation. Aliquots of the samples are evaporated to dryness, reconstituted in mobile phase, and applied to the reversed-phase HPLC as above.
L TC4 Quantification
LTC4 is measured by radioimmunoassay after HPLC purification. Aliquots of the HPLC fractions are tested for the presence of [3H]LTC4 internal standard to unequivocally identify the LTC4-containing fractions and to correct for purification losses. The remainder of each HPLC fraction is evaporated to dryness and reconstituted in PBS. Each fraction is assayed in duplicate for the presence of immunoreactive LTC4. Details of the radioimmunoassay (DuPont-NEN, Wilmington, DE) have been described previously. 11 The anti-LTC4 antiserum has a cross-reactivity of 55.3% with LTD4 and 8.6% with LTE4.12 The intraassay variability is 8.5%, the interassay variability is 19-22%. Measurement of Cellular Integrity PMNL or cultured vascular cells are labeled with ~lln-oxine by a modification of published methods. 1'13'14 Monolayer cultures (grown in 24-well plates) and PMNL are washed twice with Hanks' balanced salt solution (Hanks' BSS; GIBCO). PMNL are resuspended in Hanks' (2 × 107/ml) and the cultures covered with the same medium (0.5 ml). lllInoxine (2/zCi) is added to each well or the suspended PMNL and the cells are left at room temperature for 15 rain. H D. Piomelli, S. J. Feinmark, and P. J. Cannon, J. Pharmacol. Exp. Ther. 241, 763 (1987). ~z E. C. Hayes, D. L. Lombardo, Y. Girard, A. L. Maycock, J. Rokach, A. S. Rosenthal, R. N. Young, R. W. Egan, and H. J. Zweerink, J. lmmunol. 131, 429 (1983). t3 B. Zakhireh, M. L. Thakur, H. L. Malech, M. S. Cohen, A. Gottschaik, and R. K. Root, J. Nucl. Med. 20, 741 (1979). ~4 T. Collins, A. M. Krensky, C. Clayberger, W. Fiers, M. A. Gimbrone, Jr., S. J. Burakoff, and J. S. Pober, J. lmmunol. 133, 1878 (1984).
564
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[61]
After washing the monolayers with Tyrode's buffer containing 4% BSA (Tyrode's-BSA), the cells are covered with the same buffer and incubated at 37° for 90 min. Each well is rewashed with Tyrode's-BSA and then incubated as described with unlabeled PMNL. At the end of the incubation, the buffer is recovered, centrifuged (200 g, room temperature) to remove any cells, and aliquots are assayed for the presence of l~In by gamma counting. The cells remaining in the wells are lysed by the addition of 2% Nonidet P-40 (NP-40). Aliquots of the detergent lysate are assayed for l llln and the percentage of total I i qn released is calculated. PMNL are washed twice in Hanks' BSS after the labeling period and then resuspended either in phosphate-buffered saline (PBS) or PBS-BSA as appropriate and incubated as described above. At the end of the incubation, the buffer is recovered and the PMNL are concentrated by centrifugation at 200 g, at room temperature. The pellet is lysed with 2% NP-40 and aliquots of the supernatant and of the lysed pellet are assayed for the presence of ~~tIn.
Incorporation o f Label into Cellular Glutathione Pools PMNL (2 × 107/ml) or monolayer cultures are washed twice with PBS plus 1% BSA and then incubated for 1 hr at 37° with [35S]cysteine (5/zCi; >600 Ci/mmol) in PBS-BSA. Unincorporated cysteine is removed by two washes with PBS-BSA. Prelabeled cells are incubated as described above and the incubation media purified by HPLC. The fractions are analyzed for the presence of 35S-labeled material by liquid scintillation counting.
e~
f
13
'
i5
RETENTION TIME (min)
FIG. 1. Monolayercultures of humanumbilicalveinendothelialcells are incubatedwith LTA4 (10 ~tM) in HEPES-bufferedPBS plus 1% BSA. The culture mediumis recovered, dilutedwithmethanol(2 volumes),and directlyinjectedon the HPLCas describedin the text.
[61]
LTC4 SYNTHESIS BY P M N L AND VASCULARCELLS
565
TABLE I
LTC4PRODUCTION BY MIXED P M N L
AND VASCULAR CELLS a
LTC4 production (pmol/107 PMNL-flask) Stimulus A23187 (5/xM) A23187 (5/~M) +20:4 (150/~M) fMLP (1/zM) +20:4 (10/xM)
PMNL 3.8 (n 10.5 (n 0.75 (n
= ± = ± =
1.8 8) 2.1 11) 0.39 5)
PMNL+EC 15.9 (n 24.3 (n 0.23 (n
--- 6.1 = 3) ± 6.7 = 3) - 0.08 = 5)
PMNL+SMC
PMNL+EC (ASA)
11.0 ± 3.5(n = 5) 39.8 ± 8.1 (n = 5) 1.7 -+ 0.4
(n = 5)
PMNL and vascular cells are coincubated as described in the text with the stimuli noted above. LTC4 is purified by HPLC and quantified by radioimmunoassay. Portions of these data have been reported elsewhere in a different form. 1.2,19
Results
LTA4 Metabolism HUVEC are incubated with exogenous synthetic LTA4 and the p r e s e n c e o f m e t a b o l i t e s in t h e m e d i u m m e a s u r e d b y H P L C . S e v e r a l p e a k s o f U V - a b s o r b i n g m a t e r i a l a r e d e t e c t e d at 280 n m ( F i g . I). T h e t w o l a r g e s t p e a k s a r e a l s o f o u n d in c o n t r o l i n c u b a t i o n s a n d c o r r e s p o n d to t h e n o n e n z y m a t i c d e c o m p o s i t i o n p r o d u c t s o f L T A 4 . I n t h e cell i n c u b a t i o n s , a t h i r d p e a k a p p e a r s t h a t e l u t e s at t h e r e t e n t i o n t i m e o f s y n t h e t i c L T C 4 . T h e p r o d u c t i o n o f L T B 4 is n o t o b s e r v e d in this e x p e r i m e n t . T h e a b s e n c e o f L T B 4 c a n n o t b e e x p l a i n e d b y its m e t a b o l i c r e m o v a l s i n c e n e i t h e r E C l'15 n o r S M C 2 m e t a b o l i z e this lipid. C l a e s s o n a n d H a e g g s t r 6 m , ~5 h o w e v e r , have reported that HUVEC produce LTB4 under these conditions. Earlier s t u d i e s o f H U V E C , t6 p o r c i n e E C , l o r p o r c i n e S M C 2 d i d n o t d e t e c t t h e c o n v e r s i o n o f L T A 4 to L T B 4 .
Quantitative Aspects o f P M N L - Vascular Cell Interactions A23187 Stimulation. A 2 3 1 8 7 - s t i m u l a t e d P M N L p r e p a r a t i o n s g e n e r a t e LTC4 and the levels are enhanced by the addition of exogenous arachid o n i c a c i d ( T a b l e I). T h e p r e c i s e c e l l u l a r s o u r c e o f this l e u k o c y t e p r o d u c t is n o t c l e a r s i n c e p o r t i o n s o f t h e L T C 4 p r o d u c t i o n m a y be d u e t o e o s i n o -
~5 H.-E. Claesson and J. HaeggstrOm, Eur. J. Biochem. (1988). 16 B. O. Ibe and W. B. Campbell, Biochim. Biophys. Acta 960, 309 0988).
566
CELL MODELS OF LIPID MEDIATOR PRODUCTION
2000n_
/
l
/
1500-
II
[61]
°-° D PMNL
A--A
LABELEDPMNL
c)
_
I.0,< 0 ~:
1000
• 500
Df
0
5
15
25
(rain) FiG. 2. Either PMNL or EC are prelabeled with [35S]cysteine and then washed. PMNL and EC are coincubated, stimulated with A23187 (5 ~ M ) and arachidonic acid (150 ~M), and the incubation buffer recovered. The LTC4 is purified by HPLC as described in the text and fractions analyzed for the presence of 35S-labeled products. Data have been normalized to equal LTC4 mass production from a paired incubation. RETENTION TIME
phils 17 or to transcellular metabolism of neutrophil-derived LTA4 by contaminating platelets.~8 Nevertheless, A23187 stimulation of PMNL in the presence of vascular cells invariably leads to increased LTC4 production compared to matched control incubations of PMNL alone (Table I). Exogenous arachidonate also enhances the leukotriene production during these experiments. fMLP Stimulation/EC Feedback Regulation. The bacterial tripeptide fMLP is a very weak inducer of PMNL LTC4 synthesis, generating less than 20% of the product released after A23187 treatment (Table I). Unlike A23187, fMLP stimulation of mixed PMNL plus EC fails to produce a quantitative increase in LTC4 production but rather, PMNL LTC4 synthesis is significantly inhibited (Table I).19 EC-dependent inhibition of fMLPinduced LTC4 synthesis is abolished by treatment of the EC cultures with the cyclooxygenase inhibitor, aspirin (0.2 mM). fMLP stimulation of PMNL in the presence of aspirin-treated EC generates more LTC4 than either PMNL alone or PMNL plus untreated EC. Thus, transcellular ~7 p. F. Weller, C. W. Lee, D. W. Foster, E. J. Corey, K. F. Austen, and R. A. Lewis, Proc. Natl. Acad. Sci. U.S.A. 80, 7626 (1983). t8 j. A. Maciouf and R. C. Murphy, J. Biol. Chem. 263, 174 (1986). 19 S. J. Feinmark, J. Edasery, and P. J. Cannon, Adv. Prostaglandin Thromboxane Leukotriene Res. 19, 255 (1989).
[62]
I N T E R C E L L U L A R T R A N S F E R OF L T A 4
567
metabolism of L T A 4 to L T C 4 c a n be demonstrated even after relatively weak stimulation. [35S]LTC4 Production during PMNL/Vascular Cell Coincubation. To demonstrate that the conversion of PMNL-derived LTA4 to L T C 4 o cc u r r e d within the vascular cell cultures, [35S]cysteine is used to label the cellular GSH pools. Prelabeled PMNL incubated alone or with unlabeled vascular cells releases low levels of [35S]LTC4 after A23187 stimulation. However, [35S]cysteine-prelabeled EC 1 or SMC z incubated with A23187stimulated unlabeled PMNL produces significantly m o r e [358]LTC4 than in the reverse-labeling experiment (Fig. 2). Conclusions Using the methods described in this chapter, it has been possible to demonstrate that activated PMNL release LTA4 which can be converted to LTC4 by adjacent cultured vascular cells. The level of LTC4 produced depends on the activating stimulus. Weak PMNL activators, such as fMLP, may permit the observation of a feedback regulation loop in which PMNL leukotriene synthesis is inhibited by a vascular cell product which is probably a prostaglandin. Acknowledgments Some of the experiments described in this chapter were supported by AI-26702, HL-38312, and HL-21006. Portions of this work were done during the tenure of an Established lnvestigatorship award from the American Heart Association and Boehringer lngelheim, Inc. The author would like to thank Dr. J. Brett for the morphological examination of the cultured cells and Dr. R. R. Sciacca for statistical analyses and critical reading of this manuscript.
[62] R e l e a s e a n d M e t a b o l i s m o f L e u k o t r i e n e A4 in Neutrophil-Mast Cell Interactions
By CLEMENS A. DAmNDEN and URs WmTHMUELLER Introduction The cellular sources, biosynthesis, structure, and different biological activities of the 5-1ipoxygenase (5-LOX) metabolites leukotriene (LT) B4, L T C 4 , LTD4, and L T E 4 a r e well established. However, there is an interesting particularity of the 5-LOX pathway in that at each metabolic step the precursor molecules are formed in amounts largely exceeding the METHODS IN ENZYMOLOGY, VOL. 187
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