Modulation of platelet-activating-factor production by incorporation of naturally occurring 1-o-alkylglycerols in phospholipids of human leukemic monocyte-like thp-1 cells

Eur. J. Biochem. 250, 242Ϫ248 (1997) Modulation of platelet-activating-factor production
by incorporation of naturally occurring 1-O-alkylglycerols in phospholipids
of human leukemic monocyte-like THP-1 cells
Aziz HICHAMI 1, Vale´rie DUROUDIER 1, Ve´ronique LEBLAIS1, Laurent VERNHET 1, Franc¸ois LE GOFFIC2, Ewa NINIO 3 and Alain LEGRAND 1 1 Laboratoire de Pharmacologie Mole´culaire, Faculte´ de Pharmacie, Universite´ de Rennes I, Rennes, France2 Ecole Nationale Supe´rieure de Chimie de Paris, CNRS EP 051, Paris, France INSERM U321, Hoˆpital de la Pitie´, France (Received 9 September 1997) Ϫ EJB 97 1293/1 1-O-Alkylglycerols (alkyl-Gro), naturally occurring compounds abundant in shark liver oil, protect patients from radiotherapy side-effects. However, the protection mechanism is not well understood. Itmight be mediated by alkyl-Gro incorporation into pools of platelet-activating factor (PAF) precursor andsubsequent modification of PAF biosynthesis. Using a 3H-labelled or unlabelled natural alkyl-Gro mixture,in which prominent alkyl chains were C18 :1(9) (54Ϫ65%), C16:1(7) (5Ϫ15.5%), and C16:0 (5Ϫ10%),we investigated the incorporation of alkyl-Gro into phospholipids of human leukemic monocyte-likeTHP-1 cells. Incubation of cells for 24 h with [3H]alkyl-Gro (10 µM) resulted in their incorporation into1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1097 Ϯ25.1 pmol/2ϫ106 cells) and into 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (640.4Ϯ 12.5 pmol/2ϫ106 cells) with a total yield of 6.5 %. Such in-corporation induced production of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), whichwas increased after stimulation by the calcium ionophore A23187. HPLC analysis of the [3H]PAF molecu-lar species indicated that the three major [3H]alkyl-Gro were used for [3H]PAF synthesis in ratios similarto that of the mixture. Total production of biologically active PAF, as measured by the platelet-aggregationbioassay, was also increased by alkyl-Gro incorporation in resting (ϩ20%) and in A23187-stimulated(ϩ59%) THP-1 cells. HPLC analysis of the [3H]PAF produced in the presence of [3H]acetate, confirmedthat levels of PAF, but not of its 1-acyl analog, were increased by alkyl-Gro incorporation in resting andstimulated cells. However, the rise in [3H]acetyl-PAF, which resulted mainly from C16 :0 PAF, was re-duced by about 50 % in the presence of the PAF-receptor antagonist SR 27417, providing evidence thatstimulation of total PAF synthesis was caused by the increase in the precursor pool and autocrine amplifi-cation of PAF-induced PAF production. Thus, the supplementation of THP-1 cells in culture with naturallyoccurring alkyl-Gro led to the incorporation of alkyl-Gro into ether-containing phospholipids, which weresubsequently used for PAF synthesis. Furthermore, alkyl-Gro incorporation resulted in a significant risein PAF production by THP-1 cells under resting and stimulated conditions. These results may be ofimportance for modulating PAF production in several pathophysiological conditions, such as peroxysomedeficiencies, that are associated with a lack of ether lipid synthesis.
Keywords : alkylglycerol ; platelet-activating factor ; platelet aggregation; phospholipid.
1-O-Alkylglycerols (alkyl-Gro), naturally occurring lipids, as much as 50 % alkyl-Gro [1, 2]. Shark liver oil has been tradi- are found in notable quantities in hematopoietic organs, such as tionally used in Scandinavian medicine against debility and for bone marrow, and in milk. They are especially abundant in the wound healing. Studies have been performed to confirm and liver of several species of sharks, whose liver oil may contain establish the therapeutic properties of these compounds. Benefi- Correspondence to A. Legrand, Laboratoire de Pharmacologie cial effects in cancer treatment, such as preventive action of Mole´culaire, UFR des Sciences Pharmaceutiques et Biologiques, 2 Av.
alkyl-Gro on radiotherapy side effects, including leukopenia and thrombocytopenia, have been reported [2Ϫ4]. Furthermore, syn- Abbreviations. Alkyl-Gro, 1-O-alkylglycerol ; PAF, platelet-activa- thetic ether lipids possess anti-cancer properties [5]. The molec- ting factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine); PtdCho, ular basis of such effects are poorly understood.
phosphatidylcholine ; PtdEtn, phosphatidylethanolamine ; Ste[14C]∆ - In mammals, alkyl-Gro from dietary sources are absorbed 1-stearoyl-2-[14C]arachidonoyl-sn-glycero-3-phospho- choline ; RAcylGroPCho, 1-O-alkyl-2-acyl-sn-glycero-3-phosphochol- without cleavage of their ether bond, and are used as precursors ine; RAcylGroPEtn, 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine; of membrane phospholipids in different tissues [6]. Dietary 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine ; alkyl-Gro are incorporated into 1-O-alkyl-2-acyl-sn-glycero-3- C16:1 PAF, 1-O-hexadecenyl-2-acetyl-sn-glycero-3-phosphocholine ; phosphoethanolamine (RAcylGroPEtn) and 1-O-alkyl-2-acyl- 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine ; sn-glycero-3-phosphocholine (RAcylGroPCho) in rat intestinal 1-O-octadecenyl-2-acetyl-sn-glycero-3-phosphocholine; mucosal cells [7], and in various other organs [6]. This incorpo- PhMeSO F, phenylmethylsulfonyl fluoride ; ANOVA, analysis of vari- ration into 1-alkyl-phospholipids is of particular interest because RAcylGroPCho represent the pool of precursors for biosynthesis Hichami et al. (Eur. J. Biochem. 250) of platelet-activating factor (PAF), a synthesized mediator with (20 µM). Cells were routinely cultured from 2ϫ105 cells/ml to potent biological activities on various cell types and systems, 106 cells/ml, then were centrifuged (400ϫg, 5 min) and sus- including circulation, inflammation, reproduction and develop- pended in fresh medium at 2ϫ105 cells/ml. Cell viability was ment [8Ϫ9]. 1-O-Alkyl-2-arachidonoyl-sn-glycero-3-phospho- assessed by the trypan-blue-exclusion test. Untreated cells choline also serves as an important source of arachidonic acid, showed 94 Ϯ 0.9 % viability. After 24 h treatment with 20 µM the major precursor of several families of mediators. The struc- and 100 µM alkyl-Gro, the cell viability was 94.3 Ϯ0.9 % and ture of PAF was identified as 1-O-alkyl-2-acetyl-sn-glycero-3- phophocholine. Naturally occurring PAF includes several molec- Incorporation
[3H]alkyl-Gro
phospholipids.
ular species, differing by their hydrocarbon chain length and/or [3H]alkyl-Gro were dissolved in medium supplemented with10 mM Hepes and 0.2 % BSA, pH 7.4. THP-1 cells (5ϫ105 unsaturation at the sn-1 position of the glycerol. A 1-acyl analogof PAF with low biological activity may be produced simulta- cells/ml) were seeded into Petri dishes and incubated with neously with PAF; however. The ratio between PAF and its acyl- [3H]alkyl-Gro (10 µM, 12.31 mCi/mmol) for the indicated analog may vary depending on the cell type [10].
periods of time. Cells were centrifuged, the supernatants were The molecular basis of alkyl-Gro biological activities may removed, and total lipids were extracted by the method of Bligh include modifications of the pool of precursors for PAF, result- and Dyer [13]. Lipid extract was dried under a nitrogen stream ing in an alteration of its biosynthesis. To test this hypothesis, and analyzed by TLC on silica-gel plates, using a mixture of the human promonocyte leukemia cell line THP-1 [11] was used chloroform/methanol/acetic acid (35 :14:2.7, by vol.) as the to study the incorporation of natural alkyl-Gro isolated and puri- mobile phase. Radioactive material was visualized on a radio- fied from shark liver oil into phospholipids, and its influence on chromatogram scanner (Bioscan), and the phospholipid classes PAF synthesis. Our results show that alkyl-Gro incorporated into were identified by their retention factor (R ). The zones on silica- RAcylGroPEtn and RAcylGroPCho, and therefore may account gel plates corresponding to radioactive phospholipids were for an increase in PAF precursor pools, resulting in increased scraped off, and radioactivity was measured in a liquid scintilla- production of PAF under resting and stimulated conditions.
In selected experiments, THP-1 cells (5ϫ105 cells/ml) were incubated for 48 h with [3H]alkyl-Gro (10 µM, 24.82 mCi/mmol). The cells were centrifuged, washed three times with EXPERIMENTAL PROCEDURES
NaCl/Pi (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.2 g/l Materials. RPMI medium, fetal calf serum, penicillin, kana-
KH PO , pH 7.4) ϩ 0.2 % BSA, suspended in fresh medium mycin, Hepes and glutamine were obtained from Eurobio. Fatty- (5ϫ105 cells/ml) and seeded into Petri dishes. At indicated acid-free BSA, Phenylmethylsulfonyl fluoride (PhMeSO2F) and times, the cells were centrifuged, the supernatants were re- 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine moved, and the total lipids were extracted, analyzed by TLC, purchased from Sigma Chemical Co. Deoxycholate and A23187 were obtained from Calbiochem. [3H]acetate, sodium salt Production of [3H]PAF after [3H]alkyl-Gro incorpora-
(100 mCi/mmol) was from Isotopchim. 1-Stearoyl-2-[14C]-arachi- tion. THP-1 cells were incubated for 24 h with [3H]alkyl-Gro
donoyl-sn-glycero-3-phosphocholine 10 µM, 92.13 mCi/mmol), and were washed three times in 1-octadecyl-2-acetyl-sn-glycero-3-phos- buffer A [5 mM Hepes, 140 mM NaCl, 5 mM KCl, 1 mM pho[14C]choline (C18:0 [14C]PAF; 55 mCi/mmol) were from Amersham International. Standards used in reverse-phase HPLC rsuspended in the same buffer supplemented with PhMeSO F were 1-hexadecyl-2-[3H]acetyl-PAF (10Ϫ30 Ci/mmol) from Du (2 mM, 30 min). The cells were stimulated with the calcium Pont-New England Nuclear, 1-[3H]octadecyl-PAF (10Ϫ30 Ci/ ionophore A23187 (5 µM, 10 min) or with vehicle for controls, mmol) from Amersham International, and 1-[3H]octadecenyl- and the stimulation was stopped with methanol. C18 :0 [14C]PAF PAF, which was prepared as described previously [12]. Lipase (10 000 dpm, 55 mCi/mmol) and 50 µg PAF were added as from Rhizopus arrhizus was obtained from Boehringer Mann- internal standard and carrier, respectively. Total lipids were heim. Silica gel 60 CF 254 plates were from Merck. All solvents extracted according to Bligh and Dyer [13] and [3H]PAF were obtained from Prolabo. The specific PAF-receptor antago- samples were purified by using straight-phase HPLC on a nist SR 27417 was kindly provided by Dr J. M. Herbert, Sanofi 30-cm Intersil 10-µm Interchrom column (Interchim) with Preparation of unlabelled and tritiated alkyl-Gro. Alkyl-
Gro from crude shark liver oil were prepared as follows. After 63.1:33.6:3.3 chloroform/methanol/water over 40 min and to separation of squalene (55% of total mass) by short path distilla- 61.4:33.6:5 chloroform/methanol/water over 25 min. The reten- tion, the residue was transesterified with methanol, then sepa- tion time of PAF was determined using an authentic standard of rated by chromatography on silica using a gradient of pentane/ C16:0 PAF. The peak corresponding to PAF was collected, dried ethyl ether as solvent. The glycerol ether fraction was eluted under a nitrogen stream, and further analyzed by reverse-phase with ethyl ether and analyzed by gas chromatography after sily- HPLC using a Spherisorb S5C6-30R HPLC column (Interchim) lation. Alkyl-chain composition depending on the batch varied and the following solvent: 13.75% (by vol.) methanol, 41.25% as follows: 18:1(9) ϭ 54Ϫ65%, 16:1(7) ϭ 5Ϫ15.5%, 16:0 ϭ 10 mM ammonium acetate, pH ϭ 6.1, 45% acetonitrile (1 ml/ 5Ϫ10%, 14:0 ϭ 3%, 18:0 ϭ 3 %, and 17:1(9) ϭ 1.5%. Tritia- min). Fractions of 1 ml were collected, dried, and radioactivity tion was performed by 3H-labeling on the sn-3 C position of of 3H and/or 14C was measured in a liquid scintillation counter.
glycerol. Specific radioactivity was established by measuring The retention times of PAF molecular species were determined radioactivity of a weighed sample converted into moles using a using 3H-labelled C16 :0, C18:1 and C18 :0 PAF standards.
molecular mass of ϭ 344 (forthe C18 :1 prominent alkyl-Gro).
Bioassay estimation of PAF formation by THP-1 cells.
Cell culture. THP-1 cells, a promonocyte leukemia cell line
THP-1 cells were incubated for 24 h in the presence or absence [11], were cultured at 37°C, in a humidified atmosphere of air ϩ of alkyl-Gro (10 µM). They were washed three times and sus- 5% CO2, in RPMI 1640 medium supplemented with glutamine pended (2ϫ106 cells/ml) in buffer A. After a 30-min resting (2 mM), penicillin (50 UI/ml), streptomycin (50 µg/ml), kana- time, the cells were incubated with calcium ionophore A23187 mycin (50 µg/ml), 10% fetal calf serum and 2-mercaptoethanol (5 µM) or with vehicle for controls [ethanol concentration 0.2% Hichami et al. (Eur. J. Biochem. 250) (by vol.)]. At indicated times, stimulation was stopped by adding4 ml ethanol. After 1 h at room temperature, the tubes werecentrifuged (200ϫg, 20 min) and the supernatant was driedunder a nitrogen stream, and kept at Ϫ20°C until PAF titration.
PAF extracted from unstimulated or A23187-stimulated THP-1was dissolved in ethanol/water (60:40, by vol.) and biologicalactivity was assayed by measuring its ability to aggregatewashed rabbit platelets in an aggregometer as described pre-viously [14]. PAF concentrations were calculated from a calibra-tion curve constructed with C16 :0 PAF standard.
[3H]acetate incorporation into PAF. THP-1 cells were in-
cubated for 24 h with or without alkyl-Gro (10 µM), then thecells were harvested, washed three times, suspended in bufferA (5ϫ106 cells/ml), and incubated for 20 min with PhMeSO F (2 mM) and [3H]acetate (20 µCi, 100 mCi/mmol). In selectedexperiments the cells were treated for 30 min with the specificPAF receptor antagonist SR 27417 (0.1 µM). They werestimulated with calcium inophore A23187 (5 µM), or withvehicle for controls. At indicated times after stimulation, incuba-tion was stopped with cold methanol, and [14C]octadecyl-PAF(10 000 dpm, 55 mCi/mmol) and 50 µg PAF were added asinternal standard and carrier, respectively. Total lipids were ex-tracted according to Bligh and Dyer [13] and dried under a nitro- Fig. 1. Incorporation and persistence of alkylglycerols into phospho-
gen stream, then 1-alkyl PAF and 1-acyl PAF were purified lipids. (a) Incorporation. THP-1 cells were incubated with [3H]alkyl-Gro
using straight-phase HPLC as described above. The peak of (10 µM, 12.31 mCi/mmol) for the indicated times. Cells were washed, mixed radiolabelled 1-alkyl PAF and 1-acyl PAF was detected total lipids were extracted, and radioactive phospholipids were separated with a solid scintillation flow detector Radiomatic Flo-One by silica-gel TLC and visualized with a radiochromatogram scanner as (Packard), collected, dried under a nitrogen stream and dissolved described in Materials and Methods. Peaks migrating with standards of in chloroform/methanol (60:40, by vol.). An aliquot was used PtdEtn (᭹) and PtdCho (᭺) were scraped and quantified in a scintillationcounter. Results are expressed as meansϮSEM of 12 observations ob- for quantitative measurement of radioactivity. The sn-1-acyl ana- tained from 3 experiments. (b) Persistence. THP-1 cells (5ϫ105 cells/ml) log of PAF was hydrolysed with lipase A1 (2000 U) from Rhizo- were incubated for 48 h with [3H]alkyl-Gro (10 µM, 24.82 mCi/mmol), pus arrhizus [15] in the presence of 1-stearoyl-2-[14C]arachi- washed three times with NaCl/P containing 0.2 % BSA, and suspended in alkyl-Gro-free medium (5ϫ105 cells/ml). At indicated times cells (5000 dpm, 55.6 mCi/mmol) as internal probe for lipase activity.
were centrifuged, the supernatant was removed, and total lipids were The incubation with lipase A was performed at room temper- extracted, analyzed and quantified as described in Fig. 1. PtdCho (᭹), 1 ml buffer containing 0.1 M sodium borate, 10 mM ᭿), and PtdChoϩPtdEtn (᭜). Results are expressed as CaCl2 1 mg/ml sodium deoxycholate, 0.04 % BSA, pH 6.5, and2 ml diethyl ether, for 30 h with constant stirring. Lipids wereextracted according to Bligh and Dyer [13], and dried under a nitrogen stream. The extract was resolved on straight-phase Alkyl-Gro incorporation and persistence in membrane
HPLC as described above. The effluent was collected as 1-ml phospholipids. When THP-1 cells were incubated with [3H]-
fractions, which were dried under vacuum/centrifuge (Jouan) alkyl-Gro (10 µM, 12.31 mCi/mmol), a significant amount was and the ratio of 14C/3H radioactivity in the peak of PAF fraction incorporated into RAcylGroPEtn) and RAcylGroPCho). Alkyl- was quantified in a liquid scintillation counter. The efficiency of Gro incorporation into RAcylGroPCho reached a plateau after lipase A1 (usually Ͼ 80%) was estimated by the ratio of 1-lyso- 24 h, while incorporation into RAcylGroPEtn increased for [14C]PtdCho/Ste[14C]∆4AchGroPCho (retention times of 52 min another 24 h (Fig. 1a). After 24 h, alkyl-Gro were readily in- and 17 min, respectively), prior to the calculation of 1-alkyl-2- corporated to a greater extent into RAcylGroPCho than into [3H]acetyl-PAF and 1-acyl-2-[3H]acetyl PAF analog formation.
RAcylGroPEtn, and incorporation into RAcylGroPEtn plus In selected experiments, performed in the absence of internal RAcylGroPCho was 6.5 % of the added radioactive alkyl-Gro.
standard, the peak of PAF fraction obtained upon 10-min stim- We did not detect any significant incorporation of alkyl-Gro into ulation was further separated (before and after lipase A1) on phosphatidylinositol. After incubation of THP-1 cells under reverse-phase HPLC as described above, and the distribution of similar conditions for 48 h, [3H]alkyl-Gro were removed by 1-alkyl-2-[3H]acetyl-PAF species was analysed.
washing, and the time-dependent distribution of radioactivity Statistical analysis. Results are expressed as mean values
into phospholipid classes was analyzed. We observed a substan- ϮSEM of the indicated number of observations. The computing tial decrease in RAcylGroPCho-associated radioactivity and program for statistics, STATPAK 4.1, was from Northwest Ana- conversely an increase in RAcylGroPEtn-associated radio- lytical, Inc. Significance of the difference between untreated and activity (Fig. 1b). After 48 h, the amount of [3H]RAcylGroPCho alkyl-Gro-treated cells was assessed by three-way analysis of 32 %, whereas that of [3H]RAcylGroPEtn variance (ANOVA). The three variables were (a) treatment by increased by 15%. This suggested a remodelling of 1-[3H]alkyl- alkyl-Gro or not, (b) different times or chemical species of PAF, PC to [3H]RAcylGroPEtn by phospholipases C and D and polar- and (c) separate experiments performed in triplicate. ANOVA head-group transferases. 48 h after the end of the incubation was followed in indicated cases by Student’s paired t-tests.
with [3H]alkyl-Gro, 88 % of the initial radioactivity remained in Significance of the treatment by SR 27417 on stimulated PAF cell phospholipids, indicating a slow turnover of alkyl-Gro in release was tested by paired t-tests.
Hichami et al. (Eur. J. Biochem. 250) Fig. 2. Participation of incorporated alkylglycerols to PAF produc-
tion. THP1 cells were incubated for 24 h in the presence of [3H]alkyl-
Fig. 3. Influence of alkyl-Gro on the production of biologically active
Gro (10 µM, 92.13 mCi/mmol). The cells were centrifuged, washed and PAF. THP-1 cells were cultured for 24 h in the presence (solid column)
suspended in buffer A. Cells (2ϫ107 cells/ml) were stimulated for or absence (open column) of alkyl-Gro (10 µM). The cells were washed 10 min with calcium ionophore A23187 (5 µM) (solid column) or with and suspended in buffer A (2ϫ106 cells/ml, 1 ml/tube) in glass tubes vehicle for controls (open column). Incubation was stopped with metha- and incubated for 30 min at 37 °C prior to stimulation with calcium iono- nol, and [14C]octadecyl-PAF was added as an internal standard. Total phore A23187 (5 µM) or with vehicle for controls. At indicated times, lipids were extracted and PAF was purified on straight-phase HPLC.
stimulation was stopped by adding 4 ml of ethanol. After 1 h at room Molecular species were separated on reverse-phase HPLC and collected, temperature, the tubes were centrifuged (200ϫg, 20 min), the superna- and 3H/14C radioactivity was quantified in a liquid scintillation counter tant was dried under a nitrogen stream, and the remaining residue was as described in Materials and Methods. Results are expressed as resuspended in ethanol 60 % and tested for its ability to aggregate rabbit meansϮ SEM of five experiments. The significance of the difference platelets. Results are expressed as meansϮ SEM of five experiments.
between untreated and alkyl-Gro-treated cells was tested by three-way The significance of the difference between untreated and alkyl-Gro- ANOVA (P Ͻ 0.001) followed by individual paired t-tests for each alkyl treated cells was tested by three-way ANOVA (P Ͻ 0.01) followed by species : *, P Ͻ 0.05; **, PϽ 0.01.
individual paired t tests for each time-point: *, P Ͻ 0.02.
A23187 stimulation, we measured the production of [3H]PAF [3H]PAF formation after [3H]alkyl-Gro incorporation. To
after incubation with [3H]acetate. Stimulation of untreated cells demonstrate that the alkyl-Gro incorporated into phospholipids with the calcium ionophore induced increases in the [3H]acetate was used for PAF synthesis, we measured [3H]PAF formation incorporation into PAF of 130 % and 134% after 10 min and after [3H]alkyl-Gro incorporation into phospholipids of THP-1 20 min stimulation, respectively, as shown after separation on cells. After [3H]alkyl-Gro incubation (10 µM, 24 h) and in- straight-phase HPLC (Fig. 4 a). Alkyl-Gro treatment enhanced corporation into phospholipids, the THP-1 cells produced significantly (P Ͻ 0.001) 1-alkyl-2-[3H]acetyl-PAF production, 1.85Ϯ 0.54 pmol [3H]PAF/2ϫ106 cells under resting conditions.
which was raised by 64.3% in resting cells, and by 24.5% and After a 10-min stimulation by calcium ionophore A23187, 36.4% after 10 min and 20 min stimulation, respectively, com- [3H]PAF raised significantly (P Ͻ 0.001) to 3.58Ϯ 0.7 pmol pared with untreated cells. By contrast, alkyl-Gro had no effect [3H]PAF/2ϫ106 cells. [3H]alkyl-Gro incorporation induced the on 1-acyl-2-[3H]acetyl PAF analog production in resting or in formation of three distinct [3H]PAF molecular species, namely A23187-stimulated THP-1 cells (Fig. 4 b).
C16 :0, 16:1, and C18 :1 PAF. Their respective amounts were Analysis of PAF on reverse-phase HPLC allowed separation 15.2, 12.5 and 72.3 % in resting cells, and 16, 15.4 and 68.6% of 1-acyl-2-[3H]acetyl PAF analog from different species of 1-alkyl-2-[3H]acetyl-PAF. Untreated THP-1 cells producedmainly C16 :0 1-alkyl-PAF (97.12%) after a 10-min stimulation.
Influence of alkyl-Gro incorporation on PAF production. To
When the cells were incubated with alkyl-Gro containing promi- study the effect of alkyl-Gro incorporation into RAcylGroPCho nently the C18:1 hydrocarbon chain, a 15-fold increase in on PAF production, the production of PAF was measured by a C18:1 PAF occurred, representing 9.07 % of total PAF, and the bioassay based on aggregation of washed rabbit platelets, and fraction of C16:0 PAF dropped to 88.3% (Table 1).
by biochemical quantitation of [3H]PAF and the acyl analog of[3H]PAF produced during incubation with [3H]acetate.
Effect of PAF receptor antagonist on [3H]PAF production.
After supplementation with alkyl-Gro, which contained pre-
PAF measured by platelet aggregation. THP-1 cells were in-
dominantly the C18 :1 alkyl chain, the THP-1 cells produced cubated for 24 h with or without alkyl-Gro (10 µM), washed and mainly C16:0 PAF. Therefore we proposed that endogenously stimulated with calcium ionophore A23187 (5 µM) for 10 min produced PAF may increase its own production, as suggested by and 20 min, and PAF was measured. The A23187 addition our earlier study [16]. After treatment with alkyl-Gro, THP-1 strongly stimulated PAF production with a maximum at 10 min cells were incubated with the specific PAF-receptor antagonist in treated or untreated cells (Fig. 3). The treatment with alkyl- SR 27417, before measurement of PAF in resting and ionophore- Gro induced a significant increase in PAF production, compared stimulated cells. In stimulated THP-1 cells, the A23187-induced withuntreated cells (P Ͻ 0.01). Furthermore, the rate of decrease increase in PAF production was significantly reduced, by about in PAF after a 20-min stimulation dropped less in treated cells 50 %, in the presence of SR 27417, whereas this PAF-receptor (59% and 24 % decreases compared with that after a 10-min inhibitor did not change PAF production in unstimulated THP-1 stimulation, in untreated and alkyl-Gro-treated cells, respective- ly). Alkyl-Gro treatment induced about 20% increase in PAFproduction under resting conditions.
DISCUSSION
[3H]PAF and acyl analog of [3H]PAF production. Using un-
In this study, we report that THP-1 cells incubated with natu- labelled alkyl-Gro under the same conditions of incubation and rally occurring alkyl-Gro incorporated such ether lipids into al- Hichami et al. (Eur. J. Biochem. 250) Fig. 5. Effect of PAF-receptor antagonist on PAF production. THP-1
cells were cultured for 24 h with alkyl-Gro (10 µM). Cells were washed
and incubated with [3H]acetate (20 µCi/assay), and with or without PAF-
receptor antagonist SR 27417 (0.1 µM) for 30 min. Cells were stimulated
with calcium ionophore A23187 (5 µM) or with vehicle for controls.
After 10 min, cell stimulation was stopped with methanol and [14C]octa-
decyl-PAF was added as internal standard. Total lipids were extracted,
total (alkyl ϩ acyl analog) PAF was separated using straight-phase
HPLC, and radioactivity was measured. Results are expressed as
meansϮSEM of five experiments. The significance of the difference
Fig. 4. Influence of alkyl-Gro on PAF (a) and 1-acyl-PAF (b) produc-
between SR-27417-treated and untreated cells was assessed by paired tion. THP-1 cells were cultured for 24 h without (᭹) or with (᭺) alkyl-
t-tests: *, P ϭ 0.025.
Gro (10 µM). Cells were washed and incubated with [3H]acetate (20 µCi/assay) for 20 min, then stimulated with calcium ionophore A23187(5 µM) or with vehicle for controls. At indicated times, cell stimulation Table 1. Influence of alkyl-Grp on PAF species production. THP-
was stopped with methanol and [14C]octadecyl-PAF was added as in- cells were cultured for 24 h with or without alkyl-Gro (10 µM). Cells ternal standard. Total lipids were extracted, total (alkyl ϩ acyl analog) were washed and suspended in buffer A, then 5 ml cell suspension PAF was separated using straight-phase HPLC, and radioactivity was (5ϫ106 cells/ml) were incubated for 20 min with [3H]acetate (20 µCi/ counted in an aliquot. The acyl analog of PAF was hydrolyzed with tubeThe cells were stimulated with calcium inophore A23 lipase A1 (from R. arrhizus) in the presence of Ste[14C]∆4 for 10 min. Total lipids were extracted, and total PAF was separated as as probe for lipase activity. Non-hydrolyzed PAF was extracted, purified in Fig. 6. Molecular species of 1-[3H]alkyl-PAF were separated using by straight-phase HPLC, and its radioactivity measured in a liquid scin- a reverse-phase HPLC, quantified in a liquid scintillation counter, tillation counter (a). The yield of lipase activity was established from and their distributions were calculated. Results are expressed as the ratio lyso[14C]PtdCho/Ste[14C]∆ AchGroPCho radioactivity, and the meansϮSEM of data from three experiments performed in duplicate.
initial quantity of the acyl analog of PAF was calculated (b). Results The significance of the difference between untreated and treated cells are expressed as means*SEM of six experiments. Significance of the was tested by three-way ANOVA followed by paired t-tests. n.d., not difference between untreated and alkyl-Gro-treated cells in (a) was tested by three-way ANOVA (P Ͻ 0.001) followed by individual paired t-testsfor each time-point : *, P Ͻ 0.05; **, P Ͻ 0.001.
kyl-phospholipid precursors of PAF, contributing to an increasedPAF production in resting and stimulated cells. This incorpora- tion into alkyl-phospholipids is in agreement with previous data showing that dietary alkyl-Gro or diacetyl derivatives may par- ticipate in the elevation of alkylacylglycerophospholipid content in several rat tissues, without modifying the amount of totalphospholipids in the cells, or the ratio of phospholipid classes Alkyl-Gro are phosphorylated by an ATP:alkylglycerol 1-O-alkyl-2-lyso-sn-glycerol-3- phosphate. Via this pathway, the alkyl-Gro from dietary intakeor resulting from metabolism enter the biosynthetic pathwaysresponsible for the production of structural lipids that are com-ponents of membrane bilayers, and precursors of lipid mediators polar groups such as choline and ethanolamine in ether phospho- including PAF [17]. In THP-1 cells, the incorporation of alkyl- Gro occurred predominantly into PtdCho, and to a lesser extent PAF is a potent mediator involved in human pathophysiol- and at a slower rate, into PtdEtn. The study of the turnover of ogy including septic shock, asthma and allergy. PAF plays a role [3H]alkyl-Gro after pulse labelling showed that after 48 h, only in neuronal functions, reproduction and fetal development [8, 9, 12% of the initial labelled phospholipids were lost, suggesting 19]. Many PAF activities are mediated through transmembrane a slow turnover of 1-alkyl-phospholipids in which [3H]alkyl- receptors ; however, PAF has been described recently as an intra- Gro were incorporated. We noted a substantial decrease in cellular messenger with PAF-binding sites in subcellular frac- [3H]RAcylGroPCho which paralleled the increase in [3H]- tions of rat cerebral cortex [20]. PAF induces early-gene expres- RAcylGroPEtn, which suggests a conversion of RAcylGroPCho sion, such as c-fos and zif-268 in rat hippocampus [21] or c-fos into RAcylGroPEtn. Such a reaction involving the transfer of and c-jun in human neuroblastoma cells [22]. This makes PAF Hichami et al. (Eur. J. Biochem. 250) an almost unique type of molecule with autacoid and second- cies, which represented initially 97.1% of PAF in alkyl-Gro-free messenger properties. Previous reports have indicated that the cells, rose by 57.3% after alkyl-Gro incorporation. On the other exogenous PAF precursor 1-O-alkyl-sn-glycero-3-phosphocho- hand, C18 :1 PAF represented only 1% of PAF in alkyl-Gro-free line increases PAF production in human neutrophils [23] and cells and was increased 15-fold after alkyl-Gro incorporation, macrophages [12]. As alkyl-Gro was incorporated predomi- rising to 9.1 % its level produced by stimulated cells. Thus, after nantly into the PAF precursor RAcylGroPCho, it was of interest alkyl-Gro incorporation followed by stimulation, the ratio of to establish whether the incorporated alkyl-Gro could account C16:0/C18 :1 was 9.7 in PAF, whereas it was only around 0.12 for and/or modify PAF production in resting or stimulated in the alkyl-Gro mixture. Since THP-1 cells possess PAF recep- tors, we propose that the rise in PAF production after alkyl-Gro To demonstrate that alkyl-Gro incorporated into cellular incorporation results primarily from the increase in the pool of phospholipids acted as precursors for PAF synthesis, we mea- PAF precursors. This rise is amplified by PAF, which mobilizes sured [3H]PAF formation after [3H]alkyl-Gro incorporation into mainly a C16:0 precursor, thus producing prominently C16 :0 phospholipids of THP-1. The THP-1 cells that incorporated PAF. The highly specific PAF-receptor antagonist SR 27417 re- [3H]alkyl-Gro produced three major [3H]PAF molecular species, duced the ionophore-stimulated PAF production by about 50 %, C18 :1, C16 :0 and C16 :1 PAF under resting and stimulated con- indicating that such a mechanism could be operational in these ditions. The distribution of the three major molecular species of cells. This proposed mechanism is supported by recent data sug- labelled PAF produced by stimulated cells was 68.7, 15.4 and gesting autocrine amplification of PAF biosynthesis [16]. Previ- 15.9% for C18 :1, C16:0 and C16 :1 PAF, respectively. As this ous data have shown that in isolated rat, mouse and guinea pig distribution fits approximately the ratio of corresponding precur- neutrophils, the composition of PAF molecular species produced sors contained in alkyl-Gro, it indicates that different alkyl-Gro by the unstimulated or A23187-stimulated cells had a profile were used as PAF precursors without species selectivity. Resting that was different from potential precursors, indicating a high THP-1 cells produced [3H]PAF, and such formation was en- selectivity for utilization of precursor substrate [26]. Our data hanced in A23187-stimulated cells. Thus, we demonstrated that indicate that the PAF species profile could be significantly alkyl-Gro were incorporated into phospholipid pools involved in altered by the addition of an exogenous precursor, such as C18:1 PAF synthesis under resting and stimulated conditions. Dual alkyl-Gro. This modulation could occur under physiological PAF-biosynthetic pathways have been proposed [24]. One path- situations since the molecular species distribution of PAF is in- way, referred to as the remodeling pathway, involves the hydrol- dependent of the stimulus used to elicit its synthesis [27]. Since ysis of pre-existing 1-O-alkyl-2-acyl-sn-glycero-3-phosphocho- various molecular species possess distinct biological activities line by phospholipase A and/or by transacylase, and acetylation [28], this represents an interesting way to modulate such activi- of 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) by acetyl- ties. Such a modulation of PAF synthesis might represent bene- CoA :lyso-PAF acetyl transferase. The second pathway, or de fits in various pathophysiological situations in which PAF may novo route, involves acetylation of 1-O-alkyl-2-lyso-sn-glycero- have an important role, i.e. stimulation of cells involved in im- 3-phosphate with a subsequent removal of the phosphate by a munological responses, such as lymphoid cells [29].
1-O-alkyl-2-lyso-sn-glycero-3-phosphate phosphohydrolase and Our results suggest a possibility of dietary supplementation the transfer of phosphocholine from CDP-choline to 1-alkyl-2- with alkyl-Gro to prevent the consequences of a genetic deficit acetyl-sn-glycerol by a CDP-choline phosphotransferase. PAF in ether-lipid synthesis, such as in Zellweger syndrome. This formation induced by various stimuli is considered to occur disease is characterized by the absence of ultrastructurally mainly via the remodeling pathway, whereas in resting cells, the detectable peroxisomes in patients’ tissues, associated with the de novo synthetic pathway is predominantly involved in PAF absence of ether-lipid synthesis [30Ϫ32].
production. Our data suggest that alkyl-Gro may participate in In conclusion, our study demonstrates that exogenous alkyl- PAF synthesis via both pathways, since it increases PAF produc- Gro may participate in PAF synthesis and increase its produc- tion in resting and stimulated cells. However, our unpublished tion. This could be of importance in several physiological and data show that the basal activity of acetyltransferase of the re- modelling pathway is high in THP-1 cells, and thus may be The authors thank Mr Guy Boue¨r for his assistance in preparing the involved in PAF formation in resting cells.
The next step was to study the influence of alkyl-Gro incor- poration on the quantity of PAF produced by the cells. PAF titra-tion by platelet-aggregation assays showed that alkyl-Gro REFERENCES
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