Bio~irni~ et Biophvsica Acta 927 (1987) 43-54 43 Elsevier

BBA 11848

Studies on the mechanism of interleukin 1 stimulation of platelet activating

factor synthesis in human endothelial cells in culture

F. B u s s o l i n o a, F. B r e v i a r i o c, M. A g l i e t t a b, F. S a n a v i o b, A. B o s i a a

a n d E. D e j a n a c

, Cattedra di Chimica e Propedeutica Biochimica, Universith di Torino, h Clinica Medica A. Dipartimento di Scienze Biomediche e Oncologia, Sezione Clinica, Universitfi di Torino, Turin

and " lstituto di Richerche Farrnacologiche Mario Negri. Milan (ltalv)

(Received 1 May 1986) (Revised manuscript received 2 October 1986)

Key words: Interleukin 1 ; Platelet activating factor; PAF, lyso; (Human endothelial cell)

lnterleukin 1 promotes the conversion of the biologically inactive lyso-platelet activating factor (lyso-PAF) to the bioactive platelet activating factor (PAF) by an acetylation reaction in cultured human endothelial cells. After 2 h stimulation with interleukin 1, l-O-alkyl-2-1ysoglycero-3-phosphocholine (GPC): acetyl CoA acetyltransferase is activated, reaching a plateau after 6 h and then declining to the basal value within 24 h. This time course is comparable to that of PAF production. These cells are able to incorporate [3H]acetate and [3Hllyso-PAF into PAF. Synthetized [3H]PAF is then catabolized in [3Hlalkylacyl phosphoglycerides. l-O-alkyl-2-acetylglyceroh CDP-choline cholinephosphotransferase and l-O-alkyl-2-acetyI-GPC: acetyl- hydrolase activities are both present in endothelial cells, but are not activated under our conditions of stimuli. These findings indicate that interleukin 1 induces the PAF synthesis by a deacylation/reacetylation mechanism into human endothelial cells.

Introduction

Platelet activating factor (PAF) is a potent mediator of inflammation and cell-to-cell interac- tion, and has antihypertensive and vasopermeab- ilizing properties. PAF is produced and released by several cell types and tissues, including neu- trophils, basophils, eosinophils, macrophages,

Abbreviations: PAF, platelet activating factor; GPC, glycero- 3-phosphocholine; PC, phosphatidylcholine; PE, phosphatidyl- ethanolamine; AGEPE, "l-O-alkyl-2-acetyl-sn-glycero-3-phos- phoethanolamine.

Correspondence: Dr. F. Bussolino, Cattedra di Chimica e Propedeutica Biochimica, V. Santena 5 bis, 10126 Torino, Italy.

monocytes, platelets, kidney, liver and heart (for reviews see Refs. 1-3).

Recently, immunological and non-immunologi- cal stimuli, such as antibodies anti-angiotensin converting enzyme, antibodies anti-factor VIII, thrombin, angiotensin II, vasopressin, ATP, histamine, bradykinin and ionophore A23187, have been found to induce PAF production by human endothelial cells [4-7]. We have recently reported that interleukin 1, a monokine produced by stimulated monocytes and macrophages, known to induce differentiation and proliferation of T lymphocytes (for a review see Ref. 8), induces PAF generation and release by human endothelial cells in culture [9]. In contrast to the above stimuli which promote rapid synthesis of PAF reaching a plateau after 10-15 min [4-7], interleukin 1 in-

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

44

duces slow PAF production by human endothelial cells after a lag phase of 1-2 h, reaching the maximum after 4-6 h and declining after 18 h [9].

Two specific enzymatic reactions have been documented in the PAF biosynthesis. 1-O-alkyl- 2-1ysoglycero-3-phosphocholine: acetyl CoA acetyltransferase catalyzes the acetylation of inac- tive lyso-PAF into bioactive PAF (for reviews see Refs. 3 and 10), whereas 1-O-alkyl-2-acetyl-sn- glycerol: CDP-choline choline phosphotransferase transfers the phosphobase from CDP-choline to 1-O-alkyl-2-acetyl-sn-glycerol [11]. The acetyl- transferase seems to be the active enzyme in PAF synthesis in neutrophils [12], macrophages [13,14], eosinophils [15] and renal glomeruli [16] and its activity increases after the cells are challenged with stimuli inducing PAF production. By con- trast, neutrophil cholinephosphotransferase activ- ity is not modified during the phagocytosis of zymosan and does not correlate with PAF release [12].

PAF is then rapidly catabolized and biologi- cally inactivated through the action of an acetyltransferase distinct from conventional phos- pholipase A2 (for a review see Ref. 3). The lyso- PAF formed is reacylated with a long acyl residue, primarily arachidonic acid derived from phos- phatidylcholine [3,17-21].

In view of the characteristics of PAF produc- tion from human endothelial cells stimulated by interleukin 1, in the present study we investigated the PAF activation/inactivation cycle. Our inves- tigation shows that interleukin 1 activates acetyltransferase but not cholinephosphotrans- ferase in a time-dependent manner similar to that of PAF production and that a deacylation/ reacetylation occurs.

Materials and Methods

Materials. The chemicals used and their sources were as follows: 1-O-hexadecyl-2-acetyl-GPC (synthetic PAF), 1-O-hexadecyl-2-1yso-GPC (syn- thetic lyso-PAF), 1-O-hexadecyl-sn-glycerol and 1-palmitoyl-rac-glycerol from Bachem Feinkemi- kalien (Bubendorf, Switzerland); acetyl-CoA, bovine serum albumin, fraction V, phosphati- dylcholine (PC) from bovine brain, phosphatidy- lethanolamine (PE) from bovine liver, lysoPC from

bovine liver, phospholipase A2 from pig pancreas, phospholipase C from Bacillus cereus, lipase A1 from Rhizophus arrhizus, dithiothreitol, EGTA, butylated hydroxytoluene and deoxycholate from Sigma Chemical Co. (St. Louis, MO, U.S.A.); ionophore A23187 from Calbiochem (La Jolla, CA, U.S.A.); (RS)-2-methyl-3-(octadecyl-carbam- oxyloxy)propyl-2-(3-thiazolio)ethylphosphate (CV-3988) from Takeda Chemical Industries (Osaka, Japan); silicic acid from Mallinkrodt (St. Louis, MO, U.S.A.); human recombinant inter- leukin 2 from Biogen Res. Corp. (Cambridge, MA, U.S.A.); human recombinant a- and y-interferon from Hoffman La Roche (La Roche Inc., NY, U.S.A.); natural human interferon /3 from Serono (Rome, Italy); ultrapure interleukin 1 lipopolysac- charide-free as assessed by the negative Limulus assay (Sigma) from Genzyme Inc. (Boston, MA, U.S.A.); 1-O-[3H]alkyl-2-acetyl-GPC ([3H]PAF, 120 Ci/mmol), 1-O-[3H]octadecyl-2-1yso-GPC ([3H]lyso-PAF, 90 Ci/mmol), [3H]acetic acid, sodium salt (2 Ci/mmol), [3H]acetyl-CoA (1 Ci / mmol; the spec. act. was adjusted by addition of unlabelled acetyl-CoA), [1-14C]palmitoyl-2-1yso- GPC (55 mCi/mmol), CDP-methyl-[14C]choline, ammonium salt (59 mCi/mmol), [3H]acetic any- dride (500 mCi/mmol) and OCS from Amersham Int. (Bucks. U.K.); sodiumdihydrobis-(2-methoxy- ethoxy)aluminate, pyridine, thin-layer chromatog- raphy (TLC) plates (60F254) and acetic anhydride from Merck (Darmstadt, F.R.G.); phytohemagg- lutinin from Wellcome Res. Labs. (Beckenham, U.K.). All culture reagents were purchased from Flow Laboratories (McLean, VA, U.S.A.); the plastic flasks and Petri dishes came from Falcon Labware (Division of Becton-Dickinson Co., Oxnard, CA, U.S.A.); 1-O-alkyl-2-acetyl-sn- glycero-3-phosphoethanolamine (AGEPE) was a gift from Prof. D.J. Hanahan (Department of Biochemistry, The University of Texas Health Sci- ence Center, San Antonio, TX, U.S.A.).

All solvents used for extraction and characteri- zation of the lipids were graded and contained 50 mg/1 butylated hydroxytoluene. High-pressure liquid chromatography (HPLC) grade solvents (Merck) were filtered before use through Millex-SR (0.5 /~pore diameter, Millipore Co., Bedford, MA, U.S.A.).

Before use, all lipids were submitted to TLC

using ch lo ro fo rm/m e t hano l / wa t e r (65 : : 35 : 6, v / v ) as solvent system.

1-O-Hexadecyl-2-[3H]acetyl-GPC and 1-[14C]

palmitoyl-2-acetyl-GPC was prepared by incubat- ing respectively 5 mg of lyso-PAF with 50 mCi [3H]acetic anydride and 200 /~Ci of [1-14C]pal - mitoyl-2-1yso-GPC with 2 ml acetic anhydride in the presence of catalytic amounts of pyridine, overnight at room temperature [22].

Labelled and unlabelled 1-O-alkyl-2-acetyl-sn- glycerol were prepared according to Waku et al. [23]. Briefly, to 0.5 mCi (13.2 ~g) of [3H]PAF in 1 ml each diethyl ether and 0.1 M Tris-HC1 buffer (pH 7.4) containing 1 mM CaC12, 0.15 mg of phospholipase C was added and the medium was incubated at 22°C for 18 h with constant stirring. The ether layer was dried under N 2 and the lipids were separated by TLC on silica gel G plates prepared in 4% boric acid using chloroform/ methanol (98 : 2, v / v ) as solvent. The plates stained b y I 2 vapours, showed one single spot. The lipid concentration was calculated measuring the glycerol according to Nagele et al. [24]. In order to prevent isomerization of the molecule, 1-O-alkyl- 2-acetyl-sn-glycerol was prepared the day before use and dissolved in acetone (500/~Ci/0.5 ml).

1-O-Hexadecyl-2,3-diacetylglycerol and 1- palmitoyl-2,3-diacetylglycerol were prepared by acetylation of 1-O-hexadecyl-sn-glycerol (0.1 mg) and 1-palmitoyl-rac-glycerol (0.1 mg), respectively, in the presence of 2 ml acetic anhydride and 2 ml pyridine as described above. TLC analysis per- formed as described for the preparation of 1-O-al- kyl-2-acetyl-sn-glycerol showed two spots: the first comigrating with 1-O-hexadecyl-sn-glycerol or 1- palmitoyl-rac-glycerol, respect ive ly (range; 33-47% of the total lipid used for the process of acetylation); the second with the diradylglycerol. The produced diradylglycerols were used im- mediately.

Ionophore A23187 was solubilized in dimethyl- sulfoxide as a 5 mM stock solution, and diluted in Iscove's medium containing 0.1% bovine serum albumin. The final concentration of dimethyl- sulfoxide in assay never exceeded 0.2% which did not interfere with the experiments. CV-3988 was solubilized immediately before use in 0.15 M NaC1 by heating at 60°C, and was then buffered to pH 7.4 with 1 N NaOH.

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Crude interleukin 1 was obtained from culture supernatants of human monocytes stimulated with lipopolysaccharide as described [25,26].

Crude and ultrapure interleukin 1 activities were evaluated in a co-stimulatory assay with C 3 H / H e J thymocytes as responding cells and phytohemag- glutinin as stimulus [27]. A partially purified inter- leukin 1 preparation was assigned 1000 uni ts /ml and used as a standard in each assay [28].

Preparation of human endothelial cells. Human endothelial cells were isolated from normal um- bilical cord veins [29], grown in Iscove's medium supplemented with 15% fetal calf serum and char- acterized as previously described [4]. Primary cul- tures were plated in 25 cm 2 flasks for the experi- ments with labelled precursors and in 35 mm diameter wells for the acetyltransferase, choline- phosphotransferase and acetylhydrolase assays and for PAF production. After 4 -7 days, the cultures were at confluence and the cell number was 5 • 105 + 6.3 - 104 in the flasks and 1.7 • 105 + 5.6 • 10 4 in the wells (mean + S.D. of 12 determinations of separated cultures).

PAF production. Human endothelial cells were washed three times with Iscove's medium contain- ing 0.25% bovine serum albumin to remove fetal calf serum, then stimulated with 10 U / m l crude or ultrapure interleukin 1, with 1 ~tM A23187, 10-20 U / m l interleukin 2, or 500 U / m l inter- feron a, fl and "/ in 2 ml of the same medium at 37°C in an incubator (5% CO2). In some experi- ments the cells were preincubated for 5 min at 37°C with 100 #M acetyl-CoA. After different intervals, the medium was removed and endo- thelial cells were harvested by use of a rubber policeman in the presence of 1 ml of methanol. PAF was extracted from medium and cells accord- ing to a modified procedure of Bligh and Dyer [30], in which formic acid was added to lower the aqueous phase to pH 3 .0+0 .1 [17]. PAF was purified as previously described by two sequential chromatographic steps on a silicic AR column followed by TLC (solvent system, ch loroform/ methanol /water , 65 : 35 : 6, v /v ) [31].

For the studies with the labelled precursor of PAF, cells were incubated with 12.5 btCi [3H]- acetate, or 2.5 /LCi [3H]lyso-PAF or 2.5 /~Ci 1-O- [3H]alkyl-2-acetyl-sn-glycerol in 3 ml of Iscove's medium containing 0.25% bovine serum albumin

46

and was then stimulated with crude or ultrapure interleukin 1 (10 U/ml ) for different times. The lipids were extracted from cells as described above.

After 2 h incubation, incorporation of [3H]- acetate and [3H]lyso-PAF in the total extracted lipids reached a plateau (0.44 + 0.08% and 13.3 ± 0.63% of total added radioactivity, respectively; mean + S.D. of four experiments). 4 h of incuba- tion were needed for 1-O-[3H]alkyl-2-acetyl-sn - glycerol to reach a plateau (14.4 + 0.42% of total added radioactivity; mean + S.D. of four experi- ments). After these incubation times, no labelled lipid comigrated with synthetic PAF when [3H]acetate and [3H]lyso-PAF were used as pre- cursors; in contrast 0.004 ± 0.0003% (mean ± S.D. of four experiments) of total 1-O-[3H]alkyl-2 - acetyl-sn-glycerol labelling human endothelial cells comigrated with synthetic PAF after 4 h of in- cubation.

On the basis of these data, human endothelial cells were stimulated with interleukin 1 when the incorporation of different radiolabelled precursors was maximal (2 h for [3H]acetate and [3H]lyso- PAF and 4 h for 1-O-[3H]alkyl-2-acetyl-sn - glycerol).

The viability of endothelial cells was de- termined by Trypan blue exclusion and ranged between 88-95% after 20 h of incubation under the different conditions described.

P A F assay. PAF was detected by aggregation of washed rabbit platelets [32] and quantified over a calibration curve of synthetic PAF constructed for each test [4]. The amount of PAF was expressed in ng/1 .5 • 105 cells.

P A F characterization. TLC-purified PAF from each sample was characterized by its retention time on a HPLC apparatus (Mill±pore Chromato- graphic Division, Waters, Milford, MA, U.S.A.) equipped with a Microporasil column (Waters). Elution was carried out with dichloromethane/ methanol /water (60: 50: 5, v /v ) [33] at a flow rate of 1.5 ml /min . Synthetic PAF, PC and lyso- PC were used as standard lipids. The lipid coe- luted with synthetic PAF (retention time 14-17 min) in a sole peak containing the biologically active material. PAF was characterized by the following criteria: sensitivity to phospholipase A2 [34], phospholipase C [23,34] and base-catalyzed methanolysis [31] and insensitivity to lipase A1

[34] and acidic condition [31]. The acetylation of PAF treated by phospholipase A2 or base-cata- lyzed methanolysis partially restored the biologi- cal activity (83.6 + 8.8% of the activity; mean ± S.D. of seven experiments). Furthermore, CV-3988, a specific antagonist of PAF [35], completely in- hibited the biological activity of HPLC-purified samples.

Characterization of the labelled products. 1-O-AI- kyl-2-[3H]acetyl-GPC and 1-O-[3H]octadecyl-2 - acetyl-GPC synthesized respectively from [3H]- acetate and [3H]lyso-PAF, were purified on TLC plates using chloroform/methanol /ace t ic ac id / water ( 5 0 : 2 5 : 8 : 4 , v /v) as a solvent system [32]. 1-O-[ 3 H]Alkyl-2-acetyl-GPC synthesized by 1-O- [3 H]alkyl-2-acetyl-sn-glycerol was purified on TLC plates using chloroform/methanol /ace t ic ac id / water (50 : 25 : 8 : 4, v /v ) or ch lo ro fo rm/ methanol /ammonium hydroxide 28% (70 : 30 : 5, v /v ) as solvent systems [36]. Synthetic PAF, lyso- PAF, 1-O-alkyl-2-acetyl-sn-glycerol, AGEPE, PE and PC were used as standard lipids. The plate was scraped in 0.5 cm and radioactivity was counted after addition of OCS scintillation liquid.

Acetolysis of the labelled products was per- formed according to Hanahan et al. [37] and the treated samples were submitted to TLC using hexane/e thyl ether/acet ic acid (60:40: 1, v /v) as solvent and 1-O-hexadecyl-2,3-diacetylglycerol (Rf 0.51) and 1-palmitoyl-2,3-diacetylglycerol (Rf 0.67) as standards. Acetolysis of the lipid corn- ±grating with synthetic PAF yielded, as expected, only a peak of radioactivity comigrating with 1- O-hexadecyl-2,3-diacetylglycerol.

The total reduction of [3H]PAF and that of the labelled phospholipids comigrating with PC to 1-[3H]alkylglycerol, was achieved by treating the samples with sodiumdihydrobis (2-methoxy- ethoxy)aluminate as described by Snyder et al. [38]. The samples were chromatographed on TLC plates using ethyl e ther /water (100 : 0.5, v /v) as a solvent system and 1-O-hexadecyl-sn-glycerol and 1-palmitoyl-rac-glycerol as standard lipids.

Sodiumdihydrobis(2-methoxyethoxy)aluminate reduction of the lipids comigrating with synthetic PAF and PC in the experiments with [3H]lyso-PAF and 1-O-[3H]alkyl-2-acetyl-sn-glycerol as pre- cursors, yielded a product with the same R e as 1-O-hexadecylglycerol (Rf 0.36), indicating that

the label was still in the ether chain and no oxidative cleavage had occurred at sn-1.

TLC-purified [3H]PAF shared physicochemical characteristics and lipases sensitivity with syn- thetic PAF according to the above mentioned criteria.

A cetyltransferase, cholinephosphotransferase and acetylhydrolase assay. To test acetyltransferase ac- tivity, human endothelial cells stimulated for dif- ferent periods of time at 37°C in an incubator (CO 2 5%) with crude or ultrapure interleukin 1 (10 U/ml) , A23187 (1 /~M), interleukin 2 (20 U /ml ) or interferon a, fl and -/ (500 U /ml ) were processed as previously described [9] according to Wykle et al. [40]. Protein was determined by the method of Lowry et al. [39]. The reaction was performed at 37 ° C for 10 rain. Enzymatic activity was linear as a function of the concentration of lysate proteins (up to 60 ktg) and the incubation time (up to 20 min). The reaction was stopped with 3 ml chloroform/methanol (1:2, v /v) and the lipid extracted was submitted to TLC (solvent system, chloroform/methanol /water , 65 : 35 : 6, v/v) . The layer was scraped in 0.5 cm increments and radioactivity was counted in OCS scintillation liquid. The radioactivity corresponding to the R f of synthetic PAF (0.21) was used to measure the enzymatic activity. The results were corrected for the radioactivity losses in lipid extraction and TLC purification using 1-[aaC]palmitoyl-2-acetyl - GPC as internal standard in both cases. The absolute recovery of 0.05 /tCi 1-[t4C]palmitoyl-2 - acetyl-GPC after lipid extraction and TLC purifi- cation was 86.3 _+ 7.6% (mean _+ S.D. of four ex- periments experiments).

To test cholinephosphotransferase activity, the cells were stimulated in Iscove's or Dulbecco's calcium-free phosphate-buffered saline in the same way as for acetyltransferase, with the only dif- ference that endothelial cells were harvested and sonicated in 0.25 M sucrose in 10 mM Tris-HC1 (pH 7.4). The reaction was performed for 20 min at 37°C in 1 ml of 0.1 M Tris-HC1 (pH 8.0) containing 0.5 mM EGTA, 10 mM MgCI 2, 5 mM dithiothreitol, 0.5/~Ci CDP-methyl-[a4c]choline in 10/~1 ethanol, 5/~M 1-O-alkyl-2-acetyl-sn-glycerol and 50 /tg protein [11]. Enzymatic activity was linear as a function of the concentration of lysate protein (up to 80/~g) and the incubation time (up

47

to 40 min). Lipid extraction, TLC and the mea- surement of the enzymatic activity were carried out as above.

To test acetylhydrolase, the cells were stimu- lated with 10 U / m l ultrapure interleukin 1 as described for the acetyltransferase assay. The standard incubation contained: 1 /~M 1-O- hexadecyl-2-[3H]acetyl-GPC (0.2 ~tCi), 30 /~g lysate protein in 0.5 ml 150 mM NaC1 and 10 mM N a2 H P O 4 /N aH 2 P O 4 (pH 8.0) [41]. The incuba- tion was performed for 10 min at 37°C. The enzymatic activity was linear as a function of the protein concentration (up to 70 ~g) and the time of incubation (up to 20 rain). Boiled lysate pre- parations were used as controls.

Results

Interleukin 1-stimulated PAF level: effect on acetyltransferase cholinephosphotransferase and acetylhydrolase activities

In a previous study [9], we reported that inter- leukin 1 stimulated PAF production in human endothelial cells in a concentration-dependent way. The minimal active concentration of interleukin 1 was 1 U / m l and the stimulatory activity reached a plateau at 10 U/ml . Therefore, in this study, 10 U / m l was used routinely.

The time-dependent effects of crude interleukin 1 and A23187 on PAF production and on the acetyltransferase and cholinephosphotransferase activities of human endothelial cells are shown in Fig. 1 and Table I, respectively. Crude interleukin 1 increased PAF production and acetyltransferase activity after a lag phase of 2 h reaching the maximum after 6 h of incubation (Fig. 1). At this time, interleukin 1 (10 U /m l ) induced the produc- tion of 6 .4+ 1.8 ng P A F /1 .5 .1 0 5 cells in the presence of 100 /tM acetyl CoA and 2.7 + 0.2 ng PAF/1 .5 • 105 cells in its absence (mean + S.D. of four experiments) and increased acetyltransferase activity to 9.6 + 1.4 n m o l /m in per mg protein (mean + S.D. of five experiments). The control data were: 0.40 + 0.2 ng PAF/1 .5 • 105 cells both with and without acetyl CoA (P < 0.002 for the experiments with acetyl CoA and P < 0.01 without) and 2.9 + 1.3 n m o l / m i n per mg protein ( P < 0.001). After 12 h, PAF product ion diminished, and was close to the basal value within

48

0 2 4 6 8 10 12 14 16 18 20 22 24

h o u r s

Fig. 1. PAF production (It), acetyltransferase (,i) and cholinephosphotransferase (O) activities in human endothelial cells stimulated by interleukin 1. Human endothelial cells (1.5-105) were stimulated at 37°C for different times with interleukin 1 (10 U/ml) in 2 ml of Iscove's medium containing 0.25% bovine serum albumin and 100 /~M acetyl CoA in the PAF extraction experiments. The reactions were stopped by removing the medium, and scraping the cells in the presence of 2 ml methanol, for the purpose of PAF extraction, or 1 ml of 0.25 M sucrose containing 1 mM dithiothreitol, for acetyltransferase activity and 1 ml of 0.25 M sucrose in Tris- HC1 10 mM (pH 7.4) for cholinephosphotransferase activity. Results are expressed as means of duplicate determinations of one single experiment out of four with similar results. Varia- tion between experiments was _+ 20%. Percentages of control values were based on the basal value of untreated cells in this experiment: extractable PAF from endothelial cells, 0.75 ng/ 1.5-105 cells; acetyltransferase activity, 3.68 nmol/min per mg protein; cholinephosphotransferase activity, 1.70 nmol/min per mg protein.

24 h (Fig. 1). Acetyltransferase persisted at the maximum from 6 to 12 h and declined to the

control at 18 h. We have already shown that only 20-25% of

the PAF extracted from the cells is released in the medium with a time-course similar to that of acetyltransferase activity [9].

A23187 caused a 5-6-fo ld s t imulat ion of the acetyltransferase activity, which correlated with PAF product ion (Table I). After 60 min of incubat ion, both acetyltransferase activity and the PAF extracted from the cells declined.

Inter leukin 1 and A23187 did not affect cholinephosphotransferase activity. To exclude an

TABLE I

THE EFFECT OF A23187 (1 #M) ON PAF PRODUCTION, ACETYLTRANSFERASE AND CHOLINEPHOSPHO- TRANSFERASE ACTIVITY IN HUMAN ENDOTHELIAL CELLS

Human endothelial cells were stimulated for different times in 2 ml of Iscove's medium containing 0.25% bovine serum al- bumin with 1 ~M A23187. Data represent results of a typical experiment performed in duplicate (three times).

Min PAF (nmol/min per mg protein)

(ng/1.5-105 EC) a c e t y l cholinephos- transferase photransferase

0 0.80_+0.32 3.12+0.56 1.20_+0.44 5 1.23 +0.71 4.23+1.08 1.14_+0.23

10 5.11+1.62 6.13+0.92 1.31+0.45 20 12.35_+2.83 21.72+3.72 1.03+0.19 30 11.34_+3.18 18 .10+2 .98 1.23+0.41 60 5.30_+1.14 4.76_+1.96 1.21+0.37

inh ib i tory role of extracellular calcium on

cholinephosphotransferase, several experiments were made on endothelial cells st imulated by in-

terleukin 1 in Dulbecco 's calcium-free phosphate- buffered saline. Inter leukin 1 did not activate this

enzyme (data not shown).

Lipopolysaccharide seemed to play no role either in the induct ion of PAF product ion or

acetyltransferase activity by interleukin 1. In fact,

l ipopolysaccharide-free ul t rapure interleukin 1 (0.1 b tg /ml l ipopolysaccharide in a solution conta in ing 100 U / m l inter leukin 1 as detected in the Limulus assay) induced PAF generat ion in the cells in the presence or absence of acetyl CoA (6.3 + 1.3 and 2.7 + 0.7 ng P A F / 1 . 5 - 105 cells, respectively;

mean + S.D. of three experiments) and increased

acetyltransferase activity (9.1 _+ 2.0 n m o l / m i n per mg protein; mean + S.D. of three experiments)

after 6 h of st imulation. In order to establish the specificity of inter-

leukin 1-induced PAF product ion and acetyl- transferase st imulation, we tested the effect of lymphokines known to affect other endothelial cell properties. Inter leukin 2 (10 U / m l ) and inter- feron a, fl and ~, (500 U / m l ) did not promote the PAF product ion and did not affect acetyltrans- ferase or cholinephosphotransferase after 6 h of s t imulat ion (data not shown).

The decrease of the PAF levels was not corre-

lated to a rise in the acetylhydrolase activity, which remained constant up to 20 h in interleukin 1-treated cells (4.81 _+ 0.32 nmol /min per mg pro- tein at 6 h; 4.76 + 0.51 nmol /min per mg protein at 20 h; mean _+ S.D. of three experiments) as well as in the controls (4.56 _+ 0.27 nmol /min per mg protein at the start of the incubation; 4.18 _+ 0.29 nmol /min per mg protein at 6 h; 4.49_+ 0.68 nmol /min per mg protein at 20 h; mean + S.D. of three experiments).

[ 3H]Acetate studies Initial experiments were made to establish

whether [3H]acetate could be incorporated into the PAF molecule by interleukin 1-stimulated hu- man endothelial cells. As shown in Fig. 2 (panel A), TLC analysis of the total lipid extract from interleukin 1-stimulated human endothelial cells revealed a peak of radioactivity which migrated with synthetic PAF by using chloroform/metha-

49

nol/acet ic acid/water (50:25 : 8:4, v/v) (Rf 0.32). Endothelial cells incubated under the same condition without interleukin 1 did not synthesize [3 H]PAF. The time course of [3 H]acetate incorpo- ration into PAF (Fig. 2, panel B) shows the ap- pearance of [3H]PAF by 2 h. This synthesis re- ached the maximum level by 6 h, and fell to 34% (25-39%) of the maximum by 20 h. Also after 20 h, unstimulated endothelial cells produced no [3H]PAF (data not shown).

Similar results were obtained with ultrapure interleukin 1 (10 U/ml) after 6 h of incubation (data not shown).

[ 3H]Lyso-PAF studies In experiments to establish whether exogenous

[3H]lyso-PAF could be a substrate for PAF synthesis, the stimulation of [3H]lyso-PAF-pre- labelled endothelial cells with crude or ultrapure interleukin 1 (10 U/ml) led to synthesis of labelled

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Fig. 2. Analysis of products synthesized by human endothelial cells in the presence of [3H]acetate after 6 h of incubation with or without interleukin 1 (panel A) and (panel B) the time course of incorporation of [3 H]acetate into [ 3 H]PAF. Human endothelial cells were labelled for 2 h at 37°C with 12.5 t~Ci [3H]acetate in 3 ml Iscove's medium containing 0.25% bovine serum albumin and then stimulated with crude interleukin 1 (10 U / m l ) or the same amount of Iscove's medium (5 t~l/ml). The medium was removed and the reaction was blocked by adding 2 ml of methanol. The lipids extracted according to the modified technique of Bligh and Dyer [27] (see Materials and Methods) were separated by TLC using chloroform/methanol /ace t ic ac id /water ( 50 :25 :8 :4 , v /v ) as solvent system. The chromatogram was divided in 30 bands (0.5 cm width) and radioactivity was counted. The profiles are typical of four experiments and represent 25% of the total cellular extract. Panel A depicts schematically a representative of TLC analysis of phospholipids extracted from stimulated and unstimulated endothelial cells after 6 h of incubation. 0.044% (range 0.037-0.046%) of the exogenous substrate was incorporated into the lipids corresponding to the R f of synthetic PAF. The radioactivity recovered from the plate was 91% (range 88-93%). At the solvent front, the cpm were 31283 (range 28217-34119 cpm). Control, - - - - - - ; interleukin 1, - - . Panel B depicts the time course of [3H]acetate incorporation into phospholipids comigrating with synthetic PAF in the absence ( - - - - - - ) or presence ( ) of interleukin 1. Each time point represents the total radioactivity migrating with synthetic PAF. 0.0032% (range 0.0021-0.0035%), 0.028 (range 0.021-0.030%), 0.04% (0.036-0.045%), 0.031% (range 0.027-0.034%) and 0.007% (range 0.003-0.008%) of the exogenous substrate was incorporated at 2, 4, 6, 12 and 20 h of interleukin 1 stimulation. Values are the mean of four experiments.

50

1OOOO- @

®

E 5000- C}..

E (3- u

©

E O.

O

O 1 0 0 0 0 -

5 0 0 0 -

0,,

0

1 0 0 0 0 -

5000-

Lyso PAF PAF F~ t~

0.34 %

0.31 - 0.40 )

0.24 °0

( 0.21 - 0 .29 )

0 .22 %

( 0.20 - 0.23 )

~ 0.14 %

.12 - 0 .16

I

0 5

PC r - - 1

0.30 %

( 0 . 2 8 - 0.33 )

5 F

1 7

10 c m 15

0.40 %

0.38 - 0.41 )

5 F

1 I

0 c m 15

0 .24 %

0.22 - 0.26 )

)

S F

1 1

0 15 ¢ m

Fig. 3. Analysis of products synthesized by human endothelial cells in the presence of [3H]lyso-PAF (panels A, B and C). Human endothelial cells were labelled for 2 h at 37°C with 2.5 ~Ci [3H]lyso-PAF in 3 ml of Iscove's medium containing 0.25% bovine serum albumin and were then stimulated with crude interleukin 1 (10 U/ml) or the same amount of Iscove's medium (5 /zl/ml). Lipid extraction and TLC analysis was performed as described in Fig. 3. Each profile, obtained from one experiment out of four with similar results, represents the 12.5% of the total cellular extract. Panels A, B and C depict schematically a representative TLC analysis of the extracted phospholipids after labelling time (2 h) (A), after 6 h incuba- tion without (B) or with (C) crude interleukin 1. The numbers represent the percentage of total exogenous labelled substrate

P A F at sn-1. As shown by TLC analysis (solvent system, c h l o r o f o r m / m e t h a n o l / a c e t i c a c i d / w a t e r , 5 0 : 3 5 : 8 : 4 , v / v ) (Fig. 3, panel A), after 2 h of incubat ion with [3H]lyso P A F without inter leukin 1, we recovered two peaks of radioact ivi ty , the first comigra t ing with synthet ic l y s o - P A F (Rf 0.20) and the second comigra t ing with PC (R r 0.72). Af te r 6 h of incubat ion in the same condi t ions (Fig. 3, panel B), the peak cor responding to syn- thetic l y so -PAF was reduced by 29.5%, whereas that cor responding to PC was increased by 33%, without the appea rance of [3H]PAF. Af ter 6 h, inter leukin 1 induced the synthesis of [3H]PAF with a marked reduct ion (40%) in the peak of rad ioac t iv i ty cor responding to PC compared to that of uns t imula ted cells (Fig. 3, panel C).

This effect has been bet ter demons t ra t ed by the t ime course of [3H]PAF product ion . Fig. 4 depicts an increase of [3H]PAF at 6 h coupled with a decrease the radioac t iv i ty migrat ing with PC, whereas [3 H] Iyso-PAF decl ined in a l inear manner in s t imula ted and uns t imula ted endothel ia l cells. These da ta indicate that in uns t imula ted cells, [3H]Iyso-PAF was not conver ted to [3H]PAF within 6 h but to phospho l ip ids comigra t ing with PC. In contrast , in inter leukin 1-s t imulated cells, [3H]PAF was formed concomi tan t ly with a reduc- t ion of l ipids comigra t ing with PC.

In inter leukin 1-s t imulated cells, the radioact iv- i ty comigra t ing with synthet ic P A F began to de- cline after 12 h and re turned to the basal value within 20 h, indica t ing its ca tabol ism. At the same time, the PC peak increased up to 20 h. The rate of rad ioac t iv i ty loss in [3H] lyso-PAF was not ap- prec iab ly al tered by exposure of endothel ia l cells to inter leukin 1.

1- 0-[ 3H]A lkyl-2 -acetyl-sn-glycerol studies These exper iments were carr ied out to de-

termine whether P A F could be synthesized from 1-O-alkyl-2-acetyl-sn-glycerol, a subst ra te of 1-O- alkyl-2-acetyl-sn-glycerol : CDP-chol ine choline- phosphot rans fe rase [11], in s t imula ted or un-

added to endothelial cells which is incorporated into the lipids corresponding to the R t of lyso-PAF, PAF and PC in this experiment. The range of four experiments is shown in brack- ets. Radioactivity recovered from the plates ranged from 88 to 95%.

a~oo

1ooc.

6 ~ o .

4 ~

2 0 ~

1

I I - - - . . . . . . . . . I1"- . . . . . . . . . . . . . - I I /

- ~ / / . A f -

II, "~.¢/ z" / "

l k , / / "

/ - / -

/ " d , , \ . . . . . ,,--""

0 2 ,4- 6 8 1 0 1 2 1 4 1 6 1 8 2 0

h o u r s

Fig. 4. Time course of [3H]lyso PAF incorporation into lipids comigrating with synthetic PAF ( ), lyso PAF (-- --) or PC ( . . . . . ) in interleukin 1 (10 U/ml) stimulated (A) or unstimulated (11) cells. The figure is representative of four experiments.

stimulated endothelial cells. When human endo- thelial cells were preincubated for 4 h with 2.5 btCi 1-O-[ 3 H]alkyl-2-acetyl-sn-glycerol, TLC analysis of the labelled lipids showed seven radioactivity peaks (solvent system, ch loroform/methanol /ammoni- um hydroxyde 28%, 70 :35 :8 , v /v) with the fol- lowing Rf values: 0.15, 0.27, 0.35, 0.45, 0.55, 0.71 and 0.95. The lipids with Rf values of 0.15, 0.27, 0.35, 0.55 and 0.95 comigrated with synthetic PAF, PC, AGEPE, PE and 1-O-alkyl-2-acetyl-sn- glycerol. A similar pattern of migration was seen with acidic solvent (chloroform/methanol /acet ic acid/water , 50 : 25 : 8 : 4, v/v) . After preincuba- tion (4 h), the fraction of labelled lipid comigrat- ing with synthetic PAF was 0.004% (range 0.0031-0.0043%) of the total radioactivity added. 4 h after stimulation with crude interleukin 1, and in the control, the fraction of labelled lipids com- igrating with synthetic PAF increased by 332% and by 320%, respectively. The peak reached a maximum level after 6 h (0.02% of total radioac- tivity incorporated in the lipid) and persisted up to 20 h in the control and in interleukin 1 stimu- lated cells. After 6 h, in the controls and in endothelial cells stimulated with interleukin 1, only small amounts of the exogenous 1-O-[3H]alkyl-2 -

51

acetyl-sn-glycerol comigrated at TLC analysis with standard PC (range, 0.084-0.130%), AGEPE (range, 0.017-0.021%) and PE (range, 0.043- 0.050%). Under similar conditions, higher amounts of exogenous radioactivity comigrated with l-O- alkyl-2-acetyl-sn-glycerol (range, 1.28-1.32%).

Discussion

Recently we have shown that human endo- thelial cells stimulated by crude, ultrapure or re- combinant interleukin 1 produce PAF, which re- mains largely associated with the cells [9]. In order to study the pathway by which PAF is synthesized in cultured human endothelial cells stimulated by crude or ultrapure interleukin 1, we investigated acetyltransferase, cholinephosphotransferase and acetylhydrolase activities and the incorporation of radiolabelled substrate into the PAF molecule.

The time course of PAF production and the activation of acetyltransferase in interleukin 1- stimulated human endothelial cells were closely correlated. Interleukin 1 induced an increase of enzymatic activity after 2 h, remaining activated for 6 h. By contrast, A23187 caused rapid activa- tion as observed in other cell types [12-15],42]. It is difficult to speculate about the mechanism of action of the two agonists. A23187 might activate acetyltransferase per se, as suggested by Albert and Snyder [14], or through the entry of extracell- ular calcium [43,44]. The lag time necessary for enzyme activation by interleukin 1 excludes direct action of the monokine on the enzyme and sug- gests the activation of an intermediate biochemical step between interleukin 1 stimulation and PAF production. We have previously described inter- leukin 1 increasing the Vm~ of the acetyltransferase in endothelial cells, while the K m w a s not affected [9]. These data are consistent both with an activa- tion or a de novo synthesis of the enzyme. Fur- thermore, it has been reported that acetyltrans- ferase activity might be enhanced by a phosphor- ylation step [45] and that protein kinase A [46] increased the Vm~ x of the enzyme without affecting the Km. Interleukin 1 could promote a phosphory- lation process as demonstrated in the K562 cell line [47] and thus activate the acetyltransferase.

In contrast with acetyltransferase, interleukin 1 did not activate cholinephosphotransferase, even

52

in the absence of extracellular calcium, which may inhibit this enzyme [11]. A23187 showed similar characteristics. These results are in agreement with those of Alonso et al. [12] showing that opsonized zymosan induced PAF production in neutrophils by activating acetyltransferase but not choline- phosphotransferase.

PAF production by activation of acetyltrans- ferase is confirmed by the data obtained with the labelled precursors. The time course of [3 H]acetate and [3H]lyso-PAF incorporation into the PAF molecule agrees with the time course of acetyltransferase activation. The results obtained with [3H]acetate demonstrated that the cells can incorporate the exogenous precursor into PAF, as has been suggested in other cell types [18,19,48- 50], but do not clarify whether [3H]acetate is directly incorporated in lyso-PAF or into other lipids and then into PAF. The experiments with [3H]lyso-PAF suggest a deacylation/reacetylation pathway. In fact, in control cells, after 2 h of preincubation the radioactive precursor labels both 1-O-alkyl-2-1yso-GPC and 1-O-alkyl-acyl-GPC (lipids that comigrated with PC and that have an ether linkage at sn-1 as assessed by the sod iumdihydrobis (2-methoxye thoxy)a lumina te reduction). In interleukin 1-stimulated cells (at 6 h) the increase of the radioactivity of lipids com- igrating with synthetic PAF correlated with the decrease of that of PC (Fig. 4). Thus, we suggest that [3H]lyso-PAF is acylated to 1-O-[3H]-alkyl - 2-acyl-GPC and then interleukin 1 induces the synthesis of PAF from these lipids [14,48]. No evidence was obtained concerning the mechanisms involved in the deacylation process of 1-O-alkyl- 2-acyl-GPC.

After 12 h, the radioactivity in the peak of PAF starts to decrease, indicating a catabolic rearrange- ment of the molecule. The presence of an acetyl- hydrolase in endothelial cells, even if it is not affected by interleukin 1, coupled to the decrease of the acetyltransferase activity explains the drop in PAF production. Pertinent to this hypothesis is the recent demonstration that neutrophils [17], macrophages [18], tumor cells [19] and platelets [20,21] incubated with PAF inactivate the com- pound by deacetylating it to lyso-PAF, which is mostly acylated primarily with a tetraenoic acyl species. Rat capillary endothelial cells isolated

from epididymal adipose tissue [51] and human endothelial cells from cord veins [52] also catabo- lize exogenous PAF in this manner after 1 h of incubation, but lyso-PAF instead of alkyl-acyl- GPC is the major metabolic product. In our sys- tem, we did not observe the appearance of [3H]lyso-GPC concomitantly with the decrease of [3H]PAF, but only the rise in radioactivity corre- sponding to PC.

These differences could be due to the fact that we studied the catabolism of endogenously pro- duced PAF and not PAF added in the medium and to the different time course. However, we cannot exclude production of [3H]lyso-PAF dur- ing the PAF catabolism with only a fleeting time course of appearance. It is still not possible to decide whether PAF is inactivated by direct trans- acylation, as suggested by Pieroni and Hanahan [53], to 1-O-alkyl-2-acyl-GPC or whether lyso-PAF is an essential intermediate in PAF catabolism [18-21,51,521.

To confirm the lack of cholinephospho- transferase activation by interleukin 1, we pre- labelled human endothelial cells with 1-O-[3H]al - kyl-2-acetyl-sn-glycerol. 1-O-alkyl-2-acetyl-sn- glycerol is an active neutral lipid-inducing hypo- tension [54] and can be metabolized into PAF by rabbit platelets [36,55]. Human endothelial cells resting or stimulated by interleukin 1, metabolize 1-O-[3H]alkyl-2-acetyl-sn-glycerol into seven labelled lipids, five of which comigrate with syn- thetic PAF, PC, AGEPE, PE and 1-O-alkyl-2- acetyl-sn-glycerol. The major product synthetized comigrates at TLC with 1-O-alkyl-2-acetyl-glycerol and is probably the deacetylated product, 1-O-al- kyl-sn-glycerol as demonstrated by Blank et al. [52]. Only small amounts of exogenous precursor are incorporated into PAF molecules after a long period of incubation (4-6 h), suggesting that this pathway of PAF synthesis is not important in stimulated endothelial cells. The presence of a small amount of PAF in endothelial cells prein- cubated with 1-O-alkyl-2-acetyl-sn-glycerol leads us to hypothesize that cholinephosphotransferase can maintain the basal level of PAF in unstimu- lated cells [55,56]. However, it is possible that these results partially originate from the incom- plete treatment of [3H]PAF by phospholipase C used to prepare 1-O-alkyl-2-acetyl-sn-glycerol.

O u r s tudies o n h u m a n endo the l i a l cells s t imu- l a t e d b y i n t e r l e u k i n 1 i n d i c a t e t ha t : (1)

ace ty l t rans fe rase b u t n o t c h o l i n e p h o s p h o t r a n s -

ferase and ace ty lhydro lase is ac t ivated by this m o n o k i n e ; (2) the cells c an ut i l ize b o t h [3 H]aceta te

a n d [3H] lyso-PAF, b u t n o t 1-O-[3H]alkyl-2-acetyl -

sn-glycerol to yield P A F ; (3) 1-O-alkyl-2 a c y l - G P C is the e n d o g e n o u s p recursor for P A F synthes is

a n d could be the subs t ra te for a phospho l ipase A2

as suggested in macrophages , neu t roph i l s a n d pla te le ts (for a review see Ref. 3); (4) syn thes ized label led P A F is ca tabo l i zed to 1-O-alkyl -2-acyl -

G P C . This appears to be the first ev idence of ac t iva-

t ion of a me t abo l i c p a t h w a y m a k i n g avai lab le a

p o t e n t au t aco id such as P A F in s t imu la t ed en d o -

thel ia l cells b y the p r o d u c t of l y m p h o m o n o n u c l e a r cells.

Acknowledgments

W e wish to t h a n k prof. G. De G a e t a n o a n d Dr. A. M a n t o v a n i for f ru i t fu l d iscuss ion. Prof. D.J.

H a n a h a n for the gift of A G E P E . This work was

pa r t i a l ly suppo r t ed b y the C .N .R . R o m e (PF 'Bas i molecolar i del le Ma la t t i e Ered i ta r ie ' e P F ' O n c o -

logia ') , by Min i s t e ro del la P u b b l i c a I s t ruz ione , by R eg ione P i e m o n t e a n d b y Assoc iaz ione I t a l i ana pe r la Ricerca sul Cancro . F. Br. is the rec ip ien t fe l lowship f rom Avv. F. Ma i ron i , a n d F. Bu. is a

fe l low of Reg ions P iemonte . J. Baggett an d F. a n d V. de Cagl ia he lped p repa re the manusc r ip t .

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