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A radioreceptor binding assay for platelet-activating factor (PAF) using membranes from CHO cells expressing human PAF receptor
JOURNAL OF IYMUNOlOGICAL METHODS
EUEVIER
Journal
of Immunological
Methods
186 (1995) 225-231
A radioreceptor binding assay for platelet-activating factor (PAF) using membranes from CHO cells expressing human PAF receptor Yoshiko Aoki a,*, Motonao Nakamura a, Hisashi Kodama a, Takashi Matsumoto Takao Shimizu b, Masana Noma a
a,
a Life Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagnwa 227, Japun h Department of Biochemistry, Faculty of Medicine, The Unioersity of Tokyo, 7-3-l Hongo, Blmkyo-ku, Tokyo 113. J~prrn Received
3 October
1994; revised 22 May 1995; accepted
22 May 1995
Abstract assay (RRA) has been developed using membranes from CHO cells platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO . lF8, showed a significant intracellular Ca2+ response to PAF, and the same binding properties to [“HIWEB 2086, a PAF antagonist, as reported (K,, 13.6 * 1.9 nM; B,,,, 2.5 f 0.4 pmol/mg protein (n = 6)). A competitive binding assay was done using the CHO . lF8 cell membranes and [“HIWEB 2086. The minimum detectable dose of PAF was 0.3 nM (= 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils. A simple
which
and
reproducible
radioreccptor
can stably express human
Keywords:
Platelet-activating
factor; Radioreceptor
assay; Recombinant
human platelet-activating
factor receptor
1. Introduction
Abbreviations: PAF, platelet-activating factor; RRA, radioreceptor assay; BSA, bovine serum albumin; FMLP, formyl-methionyl-leucyl-phenylalanine; FCS, fetal calf serum; Hepes, N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid; EGTA, ethylene glycol-bis(P-aminoethyl ether)-N, N, N’, N’tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; SEM, standard error of the mean; CHO cell, Chinese hamster ovary cell. * Corresponding author. Tel.: 81-45-972-5901; Fax: 81-45972-6205. 0022-175Y/Y5/$OY.50 0 lYY.5 Elscvier SSLII 01)22-175’)(‘)5,onI47-6
Science
B.V. All rights
Platelet-activating factor (PAF), an ether phospholipid, is a mediator of cellular functions with a
wide variety of physiological and inflammatory activities (Hanahan, 1986; Braquet et al., 1987; Prescott et al., 1990; Shimizu et al., 1992). Accurate and rapid determination of PAF in biological samples is essential for a comprehensive understanding of its role and mode of action. To date, various biological (Benveniste et al., 1972; Wykle et al., 19881, mass-spectrometrical (Satouchi et reserved
Y Aoki et al. /Journal
226
of Immw~ological Methods 186 (1995) 225-231
al.,1983)
and immunological (Smal et al., 1990; Sugatani et al., 1990; Baldo et al., 1991; Cooney et al., 199la,b,1992; Karasawa et al., 1991; Sugatani et al., 1993) assays for PAF have been developed, but all have problems such as interference by endogenous PAF inhibitors, lack of specificity, data standardization or reproduction, tedious procedures for derivatization of PAF, or contamination with other membrane phospholipids which interfer with the determination (Burgers and Akkerman, 1991). Numerous actions of PAF are mediated through specific membrane receptors. Radioreceptor binding assays for PAF were developed to measure the potential of drugs to compete with [3H]PAF for binding to a receptor present on platelet plasma membranes (Hwang et al., 1983), and these assays have been used to measure PAF produced by cells or homogenates of tissues (Paulson and Nicholson, 1988; Tiberghien et al., 1991). However, these methods also have drawbacks in being tedious procedures involving the preparation of rabbit platelet membranes or unreliable due to significant deviations in binding capacity depending on the membrane batches. We have now overcome these difficulties by using membranes from CHO cells carrying a cloned human PAF receptor and [3H]WEB 2086, a PAF antagonist. We describe in this paper the characteristics of this novel assay system and its application in quantifying PAF in biological samples.
2. Materials
phosphatidylcholine and lysophosphatidic acid (chicken egg) were from Serdary Research Laboratories (Ontario). l-stearoyl-lyso-phosphatidylcholine, lyso-phosphatidylserine (bovine brain), l-palmitoyl-lyso-phosphatidylethanolamine and 1-stearoyl-lyso-phosphatidylethanolamine were from Avanti Polar Lipids (AL). Sphingomyelin (bovine erythrocytes) was from Larodan (Malmo). was from Invitrogen (CA). Fura pcDNAI.,o 2-AM (acetoxymethyl ester) was from Dojindo Laboratories (Kumamoto, Japan). BCA protein assay reagent was from Pierce (IL). 2.2. Expression of human PAF receptor in CHO cells
The coding region of the human PAF receptor cDNA on plasmid phPAF-R (Nakamura et al., 1991) was excised by Hind111 and subcloned into a eukaryotic expression vector pcDNAI,,o carrying a neomycin-resistant gene as a selection marker. The plasmid was transfected into CHO . Kl cells by electropolation, and the transformants were selected in Ham’s F-12 medium containing 10% FCS, 100 U/ml penicillin, 100 kg/ml streptomycin (culture medium), and 1 mg/ml Geneticin (Sigma, MO). Geneticin-resistant clones were isolated by limiting dilution and tested for PAF induced-intracellular Cazf mobilization and t3H]WEB 2086 binding. Clones exhibiting a high [3H]WEB 2086 binding were maintained in culture medium supplemented with 0.2 mg/ml Geneticin.
and methods
2.3. Intracellular Ca2 f measurements 2.1. Materials
l-0-hexadecyl-2-acetyl sn-glycero-3-phosphocholine (PAF) and 2-0-ethyl-PAF C-16 were purchased from Cayman (MI). Enantio-C16-PAF and 1-palmitoyl-lyso PAF were from Bachem (Bubendorf). 2-0-methyl-PAF C-18 was from Biomol Research Lab. (PA). SEP-PAK silica column was from Millipore Waters chromatography (MA). [“HIWEB 2086 (521.7 GBq/mmol) was from Du Pont Japan (Tokyo). I-0-[‘Hlalkyl-2acetyl-sn-glycero-3-phophocholine ([3H]PAF) was from Amersham (Bucks, UK). Diarachidonoyl-
Cells at subconfluence were scraped by a rubber policeman, centrifuged (200 x g, 5 min), and resuspended in a fresh culture medium at a concentration of 3 x 10h cells/ml. Fura 2-AM was added to produce a final concentration of 3 PM from a stock solution of 1 mM in DMSO. After incubation in the dark for 1 h at room temperature, the cells were centrifuged, resuspended in Hepes-Tyrode buffer (140 mM NaCl, 2.7 mM KCl, 12 mM NaHCO,, 5.6 mM D-glucose,0.49 mM MgCl,, 0.37 mM NaH,PO,, 25 mM Hepes/NaOH pH 7.4, 0.1% BSA) at 3 x 10”
Y Aoki el al. /Jo~mtal of Immunological Merhods 156 (1995) 225-231
cells/ml and used for fluorescence measurements within the next hour. Fluorescence was measured at 37°C using a CAFllO (Jasco, Tokyo) with dual excitation at 340 nm and 380 nm and emission recording at 510 nm. Measurements were made before and after the cells were exposed to PAF (10 pM-100 nM). Each cuvette contained 0.5 ml of the cell suspension (1.5 X lo6 cells) with a constant stirring at 700 rpm. The intracellular Ca2+ concentration was calculated from the ratio of the fluorescence at 340 nm and 380 nm, using the following formula: [Ca”] = K(F F,,,)/(F,,, -F). K is the apparent dissociation constant for fura-2-Ca2+, set at 224 nM, as reported by Grynkiewicz et al. (1985). F,,,, was obtained by lysis of the cells by the addition of 0.2% Triton X-100 and Fmin by the subsequent addition of 10 mM EGTA. 2.4. Preparation of CHO cell membranes Cells at subconfluence were scraped into icecold buffer A (25 mM Hepes/NaOH, pH 7.4, 0.25 M sucrose, 10 mM MgCl?) and lysed with a Potter-type homogenizer, A 800 x g supernatant of the homogenate was centrifuged at 100000 x g at 4°C for 1 h, and the pellet was suspended in buffer A and stored at -80°C until use. The protein concentration was measured by the BCA method, using BSA as a standard. 2.5. Competitive binding assay Aliquots of CHO cell membranes (100 ~1 containing 50 ,ug of protein) were incubated at 25°C for 90 min with 2 pmol of [3H]WEB 2086 in 50 ~1 of incubation buffer (25 mM Hepes/NaOH, pH 7.4, 10 mM MgCl,, 0.1% BSA), and with 10 pg-10 ng unlabeled PAF or diluted sample in 50 ~1 of incubation buffer in a 96-well microplate. At the end of the incubation, bound [3H]WEB 2086 was separated from unbound [3HlWEB 2086 by filtration through a glass fiber self-aligning filter (6.3 in. X 3.8 in.; Packard, CT> placed on a MicroMate 196 simultaneous 96-well harvester (Packard), and the filter was washed 12 times with the cold incubation buffer. Filters were placed in Matrix 9600 direct beta counters
227
(Packard), and the radioactivity was determined. The amount of PAF in each sample was quantified from a 13HlWEB 2086 displacement curve generated using different amounts of authentic unlabeled PAF. 2.6. Isolation and incubation of human neutrophils Human blood was obtained from healthy volunteers among our staff. Neutrophils were isolated by dextran sedimentation and centrifugation over Lymphoprep (Nycomed, Oslo). Briefly, heparinized (10 U/ml> blood was mixed with l/3 vol. of 6% Dextran T-500 in saline and then allowed to sediment for 30 min at room temperature. The supernatant plasma was underlayered with Lymphoprep and then centrifuged at 500 X g for 30 min at room temperature. Neutrophils which had sedimented at the bottom of the tube were resuspended in erythrocyte lysing solution containing 154 mM NH,Cl, 10 mM KHCO,, 0.1 mM EDTA. 2Na, pH 7.3, and centrifuged at 275 X g for 10 min. The sediment was washed with PBS and neutrophils were suspended in Krebs-Ringer phosphate buffer-dextrose (KRPD), containing 15.6 mM NaH,PO,, pH 7.23, 121 mM NaCl, 5 mM KCl, 1.3 mM CaC12, 1.2 mM MgSO, .7H,O, 11 mM glucose and 2% BSA at concentrations of 2.5 X 10’ cells/ml. Neutrophils were at least 95% pure by light microscopy. 1 ml of the neutrophil suspension was placed in a 2 ml Eppendorf tube and treated with 5 yg cytochalasin B for 2 min, followed by the addition of designated doses of FMLP. Incubations were carried out for 10 min at 37°C then the cells and the supernatant were separated by centrifugation at 400 X g for 5 min at 4°C. 2.7. Extraction of lipids from neutrophils The aliquot (0.2 ml) of supernatant and cell pellets in 0.95 ml of chloroform:methanol:water (1:2:0.8, v/v/v) were vortexed and centrifuged at 750 X g for 10 min, respectively. Total lipids were extracted from the samples using the method of Bligh and Dyer (1959). The supernatant was removed, and chloroform and water were added to bring the mixture ratio to 1:1:0.9
Y. Aoki et al. /Journal
228
of Immunological Methods 186 (199.5) 225-231
moved and combined with the first fraction. The extract was evaporated using a centrifugal vaporizer CVE-100D (Tokyo Rikakikai, Tokyo) and the residue was reconstituted in 0.2 ml of incubation buffer. The recovery of PAF through the extraction procedures was 77.6% + 2.2% (mean + SEM, n = 3) as determined by radiotracer studies with the internal standard L3H]PAF (= 3000 dpm).
f
3 t; .f
3. Results and discussion 0.01
1
0.1 PAF
10
100
(nM)
Fig. 1. PAF-induced mobilization of intracellular Ca’+. Fura2-loaded CHO’lF8 cells (0) and CHO.mock cells (0) were stimulated with various concentrations of PAF, and the Ca*+ concentration was calculated from the ratio of fluorescence at 340/380 nm, as described in Materials and methods. Results are means* half range of duplicate experiments. The basal level of intracellular Ca’+ in CHO cells ranged between 60-80 nM.
(chloroform:methanol:water). The chloroform phase was removed and another 0.5 ml of chloroform was added to the aqueous phase. After vigorous mixing, the chloroform phase was re-
m
0
[3H]WEB 2066 added (nM)
3.1. Expression of human PAF receptor in CHO cells
We obtained 15 CHO cell lines in which PAF significantly induced intracellular Ca2+ mobilization. From amongst these clones, the cell line showing the highest response, which was termed CHO . lF8, was used for the following experiments. Fig. 1 shows the concentration-response curve of PAF in eliciting CaZf response in the CHO . lF8 cells or mock-transfected cells. In CHO . lF8 cells, concentrations of PAF as low as 10 pM elicited a significant Ca2+ increase. However, in mock-transfected cells, even 100 nM PAF caused little elevation. Parental CHO +Kl cells
:
n
o-
0
0.5
1
1.5
2
2.5
bound (pmollmg protein)
Fig. 2. Binding characteristics of [“HIWEB 2086 to membrane from CHO. lF8 cells. A: saturation specific ( W) and non-specific (A) bindings is presented, Each point represents the mean of triplicate from one of six independent experiments, which gave the similar results. B: Scatchard plot analysis. B,,,,,, values were 14.0 nM and 2.53 pmol/mg protein, respectively.
isotherm of the total (O), determinations. Results are In this experiment, K,, and
Y. Aoki et al. /Jo~nal
of Immwzological Melhods 186 (1995) 225-231
exhibited the same response for PAF as seen in the CHO * mock cells (data not shown). Since nonspecific binding of PAF to the membranes was extremely high (70-80%) (Nakamura et al., 1991; Hwang et al., 1983; Paulson and Nicholson, 1988), kinetic analyses were done using [3HlWEB 2086. As shown in Fig. 2A, the membranes of CHO * lF8 cells specifically bound [3H]WEB 2086, in a saturable manner. Scatchard plot analysis revealed the presence of a single entity of the binding site with the dissociation constant (K,) value of 13.6 11.9 nM and the maximal binding (B,,,) value of 2.5 + 0.4 pmol/mg protein (mean _t SEM, n = 6) (Fig. 2B). These data showed good agreement with those already reported (Paulson and Nicholson, 1988; Tiberghien et al., 1991; Honda et al., 1991; Nakamura et al., 1991,1993). 3.2. Radioreceptor
assay
The standard curve of PAF was generated by incubating 50 pg of membrane protein and 10 nM [“HIWEB 2086 in the presence of varying concentrations of unlabeled PAF at 25°C for 90 min. The radioactivity of tracer bound to the receptor preparations in the absence of unlabeled PAF (B,) was 2234 f 57 dpm and non-specific binding was relatively low (40 & 4 dpm). Markedly decrease of the non-specific binding by the use of [‘HIWEB 2086 gave the excellent standard curve as shown in Fig. 3. The concentration of PAF required to inhibit specific binding by 50% (IC,,) was 7.1 nM or 744 pg/well. The minimum detectable dose (the dose producing a response statistically significantly different from the zerodose response) was 0.3 nM or 30 pg/well (triplicate determinations in ten independent experiments; Student’s t test, P < 0.05). The working range of the assay, giving an acceptable measurement error of 15% (Tiberghien et al., 1991), was 0.3-100 nM (data not shown). Several compounds were tested for cross-reactivity (Table 1). No significant cross-reactivity was found with lyso-PAF, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylcholine, sphingomyelin or enantio
7 6 go
80
-
60
-
m
40
-
20
-
01
I
0.001
I
0.01
329
I
I
I
1
0.1
1
IO
100
I 1000
PAF (nM) Fig. 3. A typical standard curve for PAF. The total binding (!I,) and non-specific binding were 2234 +57 dpm and 40+4 dpm, respectively. Results are means+_SEM for three determinations.
PAF C-16. On the other hand, synthetic PAF analogs Z-0-ethyl-PAF C-16 and 2-0-methyl-PAF C-18 had 33.8% and 0.4% cross-reactivity with PAF, respectively. 3.3. Determination
of PAF in biological samples
by
RRA
Human neutrophils were suspended in KRPD at 2.5 X IO6 cells/ml and lipids were extracted by the method of Bligh and Dyer (1959). The lipids from neutrophils were reconstituted in the incuTable I Specificity
of the PAF RRA
Compounds
Concentration for 50% inhibition of [ ‘HIWEB 2086 binding, IC,,, (M)
PAF C-16 Lyso-PAF Lyso-phosphatidic acid Lyso-phosphatidylcholine Lyso-phosphatidylethanolamine Lyso-phosphatidylserine Phosphatidylcholine Sphingomyelin Enantio PAF C-16 2-O-ethyl-PAF C-16 2-0-methyl-PAF C-18
7.1 x lo-” >lXlO.’ >1x10-5 11 x lo-’ > 1 x IF ' > 1x10~’ > 1x10-i > I x lori 9.9x 10 -(I 2.1 x 10 -s 1.7x 10 ”
Results were obtained different concentrations.
by triplicate
clctcl-nlill:ltioll\
;II 6-8
Y. Aoki et al. /Jound
230
of Immunological Methods 186 (1995) 225-231
r 5.
a@ m
I
0
0.1
0.01 PAF
I
1
t
1
IO
100
(nM)
Fig. 4. RRA standard curve in the presence or absence of the extracted lipids. The lipids were extracted from neutrophils by the method of Bligh and Dyer (1959). PAF RRA standard curves were then constructed in the presence (0) or absence (0) of the extracted lipids. Data represent the means+SEM of triplicate experiments.
bation buffer and the PAF standard solution was generated in the presence or absence of the extracted lipids. Inclusion of the extracted lipid in the PAF RRA scarcely interfered with the determination of PAF levels, since total binding (B,,) radioactivity of 1989 dpm and 2036 dpm were obtaind in the presence and absence of the ex-
tracted lipid, respectively. The 50% inhibition value of the assay was only slightly changed (8.63.7 nM) (Fig. 4). Then, PAF produced by human neutrophils was quantified by RRA. When cytochalasin B-pretreated human neutrophils were stimulated with varying concentrations of FMLP, PAF was produced in a dose-dependent manner. However, as shown in Fig. 5, 60-80% of PAF remained cell-associated. No PAF was detected without stimulation. These observations are in good agreement with data in literature, and the PAF levels determined by this method are within the expected range (Lynch and Henson, 1986; Ludwig et al., 1984) (Fig. 5). In the current methods for the determination of PAF in neutrophils, it is necessary to remove the interfering lipids such as lysophosphatidylcholine and sphingomyelin by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) (Lynch and Henson, 1986; Sugiura et al., 1990; Sugatani et al., 1990; Miwa et al., 1992). Our method needs no purification of PAF produced by neutrophils, because those interfering lipids do not disturb the binding between [“HIWEB 2086 and receptor preparations. Furthermore, this method make it possible to assay the large number of samples in 96-well microplates, rapidly and simultaneously. This method is also applicable to screen the PAF antagonist.
Acknowledgements
We thank Dr. C. Sakanaka of the University of Tokyo for suggestions in obtaining CHO stable transformant cells, and M. Ohara for comments.
1
I
t
2
4
6
FMLP
References
(FM)
Fig. 5. Dose response of PAF production and release by FMLP-stimulated human neutrophils. Neutrophils (2.5 X 10h cells) were incubated at 37°C with 5 pg/ml cytochalasin B for 2 min, then with various concentration of FMLP for 10 min. Supernatant (0) and ceil-associated (0) PAF were then assayed. Data represent the means+_SEM of triplicate experiments.
Baldo, B.A., Smal, M.A. and McCaskill, SC. (1YYl) A specific, sensitive and high-capacity immunoassay for PAF. Lipids 26, 1136. Benveniste, J., Henson, P.M. and Cochrane, C.G. (1972) Leukocyte-dependent histamine release from rabbit platelets. The role of IgE, basophils and platelet-;~ctiv;ltin:: factor. J. Exp. Med. 136, 1356.
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of Immunological
Bligh, E.C. and Dyer, W.J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911. Braquet, I’., Touqui, L., Shen, T.Y. and Vargaftig, B.B. (1987) Perspective in platelet-activating factor research. Pharmacot. Rev. 39, 97. Burgers, J.A. and Akkerman, J.-W.N. (1991) Measurement of platelet-activating factor in biological specimens. J. Lipid Med. 3, 241. Cooney, S.J., Smal, M.A. and Baldo, B.A. (1991a) Quantitation by radioimmunoassay of PAF in human saliva. Lipids 26, 1140. Cooney, S.J., Smal, M.A. and Baldo, B.A. (1991b) Stability of platelet activating factor (PAF) in human saliva. Quantitation by radioimmunoassay. Clin. Chim. Acta 200, 161. Cooney, S.J., Smal, M.A. and Baldo, B.A. (1992) Measurement of PAF in blood by radioimmunoassay. Examination of interfering factors and problems involved in accurate quantification. J. Immunol. Methods 151, 131. Grynkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fIuorescence properties. J. Biol. Chem. 260, 3440. Hnnahan, D.J. (1986) Platelet activating factor: A biologically active phosphoglyceride. Annu. Rev. Biochem. 55, 483. Honda, Z.-i., Naknmura, M., Miki, I., Minami, M., Watanabe, T., Seyama, Y.. Okado, H., Toh, H., Ito, K., Miyamoto, T. ;md Shlmizu. T. (1991) Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. Nature 349, 342. Hwang, S.-B.. Lee. C.-S.C.. Cheah, M.J. and Shen, T.Y. (1983) Specific receptor sites for I-O-alkyl-2-acetyl-snglycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes. Biochemistry 22, 4756. Karasawa. K., Satoh, N., Hongo, T., Nakagawa, Y.. Setaka, M. and Nojima, S. (1991) Radioimmunoassay for plateletactivating factor. Lipids 26, 1126. Ludwig, J.C., McManus, L.M., Clark, P.O., Hanahan, D.J. and Pinckard, R.N. (1984) Modulation of platelet-activating factor (PAF) synthesis and release from human polymorphonuclear leukocytes (PMN): role of extracellular Ca’+. Arch. Biochem. Biophys. 232, 102. Lynch, J.M. and Henson, P.M. (1986) The intracellular retention of newly synthesized platelet-activating factor. J. Immunol. 137, 2653. Miwa. M., Sugatani. J., Ikemura. T., Okamoto, Y., Ino, M.,
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Saito, K.. Suzuki, Y. and Matsumoto, M. (1992) Release of newly synthesized platelet-activating factor (PAF) from human polymorphonuclear leukocytes under in vivo conditions. Contriburion of PAF-releasing factor in serum. J. Immunol. 148, 872. Nakamura, M., Honda, Z.-i., Izumi, T., Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T., Noma, M. and Shim& T. (1991) Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J. Biol. Chem. 266, 20400. Nakamura, M., Honda, Z.-i., Matsumoto, T.. Noma, M. and Shimizu, T. (1993) Isolation and properties of plateletactivating factor receptor cDNAs. J. Lipid Med. 6, 163. Paulson, SK. and Nicholson, N.S. (1988) A radioreceptor binding assay for measurement of platelet-activating factor synthesis by human neutrophils. J. Immunol. Methods 110. 209. Prescott, S.M., Zimmerman, G.A. and McIntyre, T.M. (1990) Platelet-activating factor. J. Biol. Chem. 265, 17381. Satouchi, K., Oda, M., Yasunaga, K. and Saito, K. (1983) Application of selected ion monitoring to determination of platelet-activating factor. J. Biochem. 94. 2067. Shimizu, T. Honda, Z.-i., Nakamura, M., Biro. H. and Izumi. T. (1992) Platelet-activating factor receptor ;hnd signal transduction. Biochem. Pharmacol. 44, 1001. Smal, M.A., Baldo, B.A. and McCaskill. A. (1990) A specitic. sensitive radioimmunoassay for platelet-activ;llinl: factor (PAF). J. Immunot. Methods 1%: 183. Sugatani, J.. Lee, D.Y., Hughes. K.T. and Saito, K. (1990) Development of a novel scintillation proximity radioimmunoassay for platelet-activating factor measurement: Comparison with bioassay and CC/MS. Life Sci. 46, 1443. Sugatani, J., Miwa, M., Komiyama, Y. and Murakami. T. (1993) Quantitative analysis of platelet-activating factor in human plasma. J. Immunol. Methods 166. 251. Sugiura, T., Fukuda, T.. Masuzawa, Y. and Waku K. (1990) Ether lysophospholipid-induced production of platetetactivating factor in human polymorphonuclear leukocytes. Biochim. Biophys. Acts 1047, 223. Tiberghien, C., Laurent, L., Junier, M.P. and Dray, F. (1991) A competitive receptor binding assay for platelet-activating factor (PAF): Quantification of PAF in rat brain. J. Lipid Med. 3, 249. Wykle, R.L., O’Flaherty, J.T. and Thomas, M.J. (1988) Platelet-activating factor. Methods Enzymot. 163, 44.
EUEVIER
Journal
of Immunological
Methods
186 (1995) 225-231
A radioreceptor binding assay for platelet-activating factor (PAF) using membranes from CHO cells expressing human PAF receptor Yoshiko Aoki a,*, Motonao Nakamura a, Hisashi Kodama a, Takashi Matsumoto Takao Shimizu b, Masana Noma a
a,
a Life Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagnwa 227, Japun h Department of Biochemistry, Faculty of Medicine, The Unioersity of Tokyo, 7-3-l Hongo, Blmkyo-ku, Tokyo 113. J~prrn Received
3 October
1994; revised 22 May 1995; accepted
22 May 1995
Abstract assay (RRA) has been developed using membranes from CHO cells platelet-activating factor (PAF) receptor. The CHO cells expressing the PAF receptor, termed CHO . lF8, showed a significant intracellular Ca2+ response to PAF, and the same binding properties to [“HIWEB 2086, a PAF antagonist, as reported (K,, 13.6 * 1.9 nM; B,,,, 2.5 f 0.4 pmol/mg protein (n = 6)). A competitive binding assay was done using the CHO . lF8 cell membranes and [“HIWEB 2086. The minimum detectable dose of PAF was 0.3 nM (= 30 pg per well) and the assay was highly specific for PAF. This method makes it possible to handle large numbers of samples rapidly and simultaneously, since the receptor membrane is prepared in advance and the binding assay can be completed within 3 h. Using this method, we have determined the production and cell association of PAF in human neutrophils. A simple
which
and
reproducible
radioreccptor
can stably express human
Keywords:
Platelet-activating
factor; Radioreceptor
assay; Recombinant
human platelet-activating
factor receptor
1. Introduction
Abbreviations: PAF, platelet-activating factor; RRA, radioreceptor assay; BSA, bovine serum albumin; FMLP, formyl-methionyl-leucyl-phenylalanine; FCS, fetal calf serum; Hepes, N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid; EGTA, ethylene glycol-bis(P-aminoethyl ether)-N, N, N’, N’tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; SEM, standard error of the mean; CHO cell, Chinese hamster ovary cell. * Corresponding author. Tel.: 81-45-972-5901; Fax: 81-45972-6205. 0022-175Y/Y5/$OY.50 0 lYY.5 Elscvier SSLII 01)22-175’)(‘)5,onI47-6
Science
B.V. All rights
Platelet-activating factor (PAF), an ether phospholipid, is a mediator of cellular functions with a
wide variety of physiological and inflammatory activities (Hanahan, 1986; Braquet et al., 1987; Prescott et al., 1990; Shimizu et al., 1992). Accurate and rapid determination of PAF in biological samples is essential for a comprehensive understanding of its role and mode of action. To date, various biological (Benveniste et al., 1972; Wykle et al., 19881, mass-spectrometrical (Satouchi et reserved
Y Aoki et al. /Journal
226
of Immw~ological Methods 186 (1995) 225-231
al.,1983)
and immunological (Smal et al., 1990; Sugatani et al., 1990; Baldo et al., 1991; Cooney et al., 199la,b,1992; Karasawa et al., 1991; Sugatani et al., 1993) assays for PAF have been developed, but all have problems such as interference by endogenous PAF inhibitors, lack of specificity, data standardization or reproduction, tedious procedures for derivatization of PAF, or contamination with other membrane phospholipids which interfer with the determination (Burgers and Akkerman, 1991). Numerous actions of PAF are mediated through specific membrane receptors. Radioreceptor binding assays for PAF were developed to measure the potential of drugs to compete with [3H]PAF for binding to a receptor present on platelet plasma membranes (Hwang et al., 1983), and these assays have been used to measure PAF produced by cells or homogenates of tissues (Paulson and Nicholson, 1988; Tiberghien et al., 1991). However, these methods also have drawbacks in being tedious procedures involving the preparation of rabbit platelet membranes or unreliable due to significant deviations in binding capacity depending on the membrane batches. We have now overcome these difficulties by using membranes from CHO cells carrying a cloned human PAF receptor and [3H]WEB 2086, a PAF antagonist. We describe in this paper the characteristics of this novel assay system and its application in quantifying PAF in biological samples.
2. Materials
phosphatidylcholine and lysophosphatidic acid (chicken egg) were from Serdary Research Laboratories (Ontario). l-stearoyl-lyso-phosphatidylcholine, lyso-phosphatidylserine (bovine brain), l-palmitoyl-lyso-phosphatidylethanolamine and 1-stearoyl-lyso-phosphatidylethanolamine were from Avanti Polar Lipids (AL). Sphingomyelin (bovine erythrocytes) was from Larodan (Malmo). was from Invitrogen (CA). Fura pcDNAI.,o 2-AM (acetoxymethyl ester) was from Dojindo Laboratories (Kumamoto, Japan). BCA protein assay reagent was from Pierce (IL). 2.2. Expression of human PAF receptor in CHO cells
The coding region of the human PAF receptor cDNA on plasmid phPAF-R (Nakamura et al., 1991) was excised by Hind111 and subcloned into a eukaryotic expression vector pcDNAI,,o carrying a neomycin-resistant gene as a selection marker. The plasmid was transfected into CHO . Kl cells by electropolation, and the transformants were selected in Ham’s F-12 medium containing 10% FCS, 100 U/ml penicillin, 100 kg/ml streptomycin (culture medium), and 1 mg/ml Geneticin (Sigma, MO). Geneticin-resistant clones were isolated by limiting dilution and tested for PAF induced-intracellular Cazf mobilization and t3H]WEB 2086 binding. Clones exhibiting a high [3H]WEB 2086 binding were maintained in culture medium supplemented with 0.2 mg/ml Geneticin.
and methods
2.3. Intracellular Ca2 f measurements 2.1. Materials
l-0-hexadecyl-2-acetyl sn-glycero-3-phosphocholine (PAF) and 2-0-ethyl-PAF C-16 were purchased from Cayman (MI). Enantio-C16-PAF and 1-palmitoyl-lyso PAF were from Bachem (Bubendorf). 2-0-methyl-PAF C-18 was from Biomol Research Lab. (PA). SEP-PAK silica column was from Millipore Waters chromatography (MA). [“HIWEB 2086 (521.7 GBq/mmol) was from Du Pont Japan (Tokyo). I-0-[‘Hlalkyl-2acetyl-sn-glycero-3-phophocholine ([3H]PAF) was from Amersham (Bucks, UK). Diarachidonoyl-
Cells at subconfluence were scraped by a rubber policeman, centrifuged (200 x g, 5 min), and resuspended in a fresh culture medium at a concentration of 3 x 10h cells/ml. Fura 2-AM was added to produce a final concentration of 3 PM from a stock solution of 1 mM in DMSO. After incubation in the dark for 1 h at room temperature, the cells were centrifuged, resuspended in Hepes-Tyrode buffer (140 mM NaCl, 2.7 mM KCl, 12 mM NaHCO,, 5.6 mM D-glucose,0.49 mM MgCl,, 0.37 mM NaH,PO,, 25 mM Hepes/NaOH pH 7.4, 0.1% BSA) at 3 x 10”
Y Aoki el al. /Jo~mtal of Immunological Merhods 156 (1995) 225-231
cells/ml and used for fluorescence measurements within the next hour. Fluorescence was measured at 37°C using a CAFllO (Jasco, Tokyo) with dual excitation at 340 nm and 380 nm and emission recording at 510 nm. Measurements were made before and after the cells were exposed to PAF (10 pM-100 nM). Each cuvette contained 0.5 ml of the cell suspension (1.5 X lo6 cells) with a constant stirring at 700 rpm. The intracellular Ca2+ concentration was calculated from the ratio of the fluorescence at 340 nm and 380 nm, using the following formula: [Ca”] = K(F F,,,)/(F,,, -F). K is the apparent dissociation constant for fura-2-Ca2+, set at 224 nM, as reported by Grynkiewicz et al. (1985). F,,,, was obtained by lysis of the cells by the addition of 0.2% Triton X-100 and Fmin by the subsequent addition of 10 mM EGTA. 2.4. Preparation of CHO cell membranes Cells at subconfluence were scraped into icecold buffer A (25 mM Hepes/NaOH, pH 7.4, 0.25 M sucrose, 10 mM MgCl?) and lysed with a Potter-type homogenizer, A 800 x g supernatant of the homogenate was centrifuged at 100000 x g at 4°C for 1 h, and the pellet was suspended in buffer A and stored at -80°C until use. The protein concentration was measured by the BCA method, using BSA as a standard. 2.5. Competitive binding assay Aliquots of CHO cell membranes (100 ~1 containing 50 ,ug of protein) were incubated at 25°C for 90 min with 2 pmol of [3H]WEB 2086 in 50 ~1 of incubation buffer (25 mM Hepes/NaOH, pH 7.4, 10 mM MgCl,, 0.1% BSA), and with 10 pg-10 ng unlabeled PAF or diluted sample in 50 ~1 of incubation buffer in a 96-well microplate. At the end of the incubation, bound [3H]WEB 2086 was separated from unbound [3HlWEB 2086 by filtration through a glass fiber self-aligning filter (6.3 in. X 3.8 in.; Packard, CT> placed on a MicroMate 196 simultaneous 96-well harvester (Packard), and the filter was washed 12 times with the cold incubation buffer. Filters were placed in Matrix 9600 direct beta counters
227
(Packard), and the radioactivity was determined. The amount of PAF in each sample was quantified from a 13HlWEB 2086 displacement curve generated using different amounts of authentic unlabeled PAF. 2.6. Isolation and incubation of human neutrophils Human blood was obtained from healthy volunteers among our staff. Neutrophils were isolated by dextran sedimentation and centrifugation over Lymphoprep (Nycomed, Oslo). Briefly, heparinized (10 U/ml> blood was mixed with l/3 vol. of 6% Dextran T-500 in saline and then allowed to sediment for 30 min at room temperature. The supernatant plasma was underlayered with Lymphoprep and then centrifuged at 500 X g for 30 min at room temperature. Neutrophils which had sedimented at the bottom of the tube were resuspended in erythrocyte lysing solution containing 154 mM NH,Cl, 10 mM KHCO,, 0.1 mM EDTA. 2Na, pH 7.3, and centrifuged at 275 X g for 10 min. The sediment was washed with PBS and neutrophils were suspended in Krebs-Ringer phosphate buffer-dextrose (KRPD), containing 15.6 mM NaH,PO,, pH 7.23, 121 mM NaCl, 5 mM KCl, 1.3 mM CaC12, 1.2 mM MgSO, .7H,O, 11 mM glucose and 2% BSA at concentrations of 2.5 X 10’ cells/ml. Neutrophils were at least 95% pure by light microscopy. 1 ml of the neutrophil suspension was placed in a 2 ml Eppendorf tube and treated with 5 yg cytochalasin B for 2 min, followed by the addition of designated doses of FMLP. Incubations were carried out for 10 min at 37°C then the cells and the supernatant were separated by centrifugation at 400 X g for 5 min at 4°C. 2.7. Extraction of lipids from neutrophils The aliquot (0.2 ml) of supernatant and cell pellets in 0.95 ml of chloroform:methanol:water (1:2:0.8, v/v/v) were vortexed and centrifuged at 750 X g for 10 min, respectively. Total lipids were extracted from the samples using the method of Bligh and Dyer (1959). The supernatant was removed, and chloroform and water were added to bring the mixture ratio to 1:1:0.9
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of Immunological Methods 186 (199.5) 225-231
moved and combined with the first fraction. The extract was evaporated using a centrifugal vaporizer CVE-100D (Tokyo Rikakikai, Tokyo) and the residue was reconstituted in 0.2 ml of incubation buffer. The recovery of PAF through the extraction procedures was 77.6% + 2.2% (mean + SEM, n = 3) as determined by radiotracer studies with the internal standard L3H]PAF (= 3000 dpm).
f
3 t; .f
3. Results and discussion 0.01
1
0.1 PAF
10
100
(nM)
Fig. 1. PAF-induced mobilization of intracellular Ca’+. Fura2-loaded CHO’lF8 cells (0) and CHO.mock cells (0) were stimulated with various concentrations of PAF, and the Ca*+ concentration was calculated from the ratio of fluorescence at 340/380 nm, as described in Materials and methods. Results are means* half range of duplicate experiments. The basal level of intracellular Ca’+ in CHO cells ranged between 60-80 nM.
(chloroform:methanol:water). The chloroform phase was removed and another 0.5 ml of chloroform was added to the aqueous phase. After vigorous mixing, the chloroform phase was re-
m
0
[3H]WEB 2066 added (nM)
3.1. Expression of human PAF receptor in CHO cells
We obtained 15 CHO cell lines in which PAF significantly induced intracellular Ca2+ mobilization. From amongst these clones, the cell line showing the highest response, which was termed CHO . lF8, was used for the following experiments. Fig. 1 shows the concentration-response curve of PAF in eliciting CaZf response in the CHO . lF8 cells or mock-transfected cells. In CHO . lF8 cells, concentrations of PAF as low as 10 pM elicited a significant Ca2+ increase. However, in mock-transfected cells, even 100 nM PAF caused little elevation. Parental CHO +Kl cells
:
n
o-
0
0.5
1
1.5
2
2.5
bound (pmollmg protein)
Fig. 2. Binding characteristics of [“HIWEB 2086 to membrane from CHO. lF8 cells. A: saturation specific ( W) and non-specific (A) bindings is presented, Each point represents the mean of triplicate from one of six independent experiments, which gave the similar results. B: Scatchard plot analysis. B,,,,,, values were 14.0 nM and 2.53 pmol/mg protein, respectively.
isotherm of the total (O), determinations. Results are In this experiment, K,, and
Y. Aoki et al. /Jo~nal
of Immwzological Melhods 186 (1995) 225-231
exhibited the same response for PAF as seen in the CHO * mock cells (data not shown). Since nonspecific binding of PAF to the membranes was extremely high (70-80%) (Nakamura et al., 1991; Hwang et al., 1983; Paulson and Nicholson, 1988), kinetic analyses were done using [3HlWEB 2086. As shown in Fig. 2A, the membranes of CHO * lF8 cells specifically bound [3H]WEB 2086, in a saturable manner. Scatchard plot analysis revealed the presence of a single entity of the binding site with the dissociation constant (K,) value of 13.6 11.9 nM and the maximal binding (B,,,) value of 2.5 + 0.4 pmol/mg protein (mean _t SEM, n = 6) (Fig. 2B). These data showed good agreement with those already reported (Paulson and Nicholson, 1988; Tiberghien et al., 1991; Honda et al., 1991; Nakamura et al., 1991,1993). 3.2. Radioreceptor
assay
The standard curve of PAF was generated by incubating 50 pg of membrane protein and 10 nM [“HIWEB 2086 in the presence of varying concentrations of unlabeled PAF at 25°C for 90 min. The radioactivity of tracer bound to the receptor preparations in the absence of unlabeled PAF (B,) was 2234 f 57 dpm and non-specific binding was relatively low (40 & 4 dpm). Markedly decrease of the non-specific binding by the use of [‘HIWEB 2086 gave the excellent standard curve as shown in Fig. 3. The concentration of PAF required to inhibit specific binding by 50% (IC,,) was 7.1 nM or 744 pg/well. The minimum detectable dose (the dose producing a response statistically significantly different from the zerodose response) was 0.3 nM or 30 pg/well (triplicate determinations in ten independent experiments; Student’s t test, P < 0.05). The working range of the assay, giving an acceptable measurement error of 15% (Tiberghien et al., 1991), was 0.3-100 nM (data not shown). Several compounds were tested for cross-reactivity (Table 1). No significant cross-reactivity was found with lyso-PAF, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylcholine, sphingomyelin or enantio
7 6 go
80
-
60
-
m
40
-
20
-
01
I
0.001
I
0.01
329
I
I
I
1
0.1
1
IO
100
I 1000
PAF (nM) Fig. 3. A typical standard curve for PAF. The total binding (!I,) and non-specific binding were 2234 +57 dpm and 40+4 dpm, respectively. Results are means+_SEM for three determinations.
PAF C-16. On the other hand, synthetic PAF analogs Z-0-ethyl-PAF C-16 and 2-0-methyl-PAF C-18 had 33.8% and 0.4% cross-reactivity with PAF, respectively. 3.3. Determination
of PAF in biological samples
by
RRA
Human neutrophils were suspended in KRPD at 2.5 X IO6 cells/ml and lipids were extracted by the method of Bligh and Dyer (1959). The lipids from neutrophils were reconstituted in the incuTable I Specificity
of the PAF RRA
Compounds
Concentration for 50% inhibition of [ ‘HIWEB 2086 binding, IC,,, (M)
PAF C-16 Lyso-PAF Lyso-phosphatidic acid Lyso-phosphatidylcholine Lyso-phosphatidylethanolamine Lyso-phosphatidylserine Phosphatidylcholine Sphingomyelin Enantio PAF C-16 2-O-ethyl-PAF C-16 2-0-methyl-PAF C-18
7.1 x lo-” >lXlO.’ >1x10-5 11 x lo-’ > 1 x IF ' > 1x10~’ > 1x10-i > I x lori 9.9x 10 -(I 2.1 x 10 -s 1.7x 10 ”
Results were obtained different concentrations.
by triplicate
clctcl-nlill:ltioll\
;II 6-8
Y. Aoki et al. /Jound
230
of Immunological Methods 186 (1995) 225-231
r 5.
a@ m
I
0
0.1
0.01 PAF
I
1
t
1
IO
100
(nM)
Fig. 4. RRA standard curve in the presence or absence of the extracted lipids. The lipids were extracted from neutrophils by the method of Bligh and Dyer (1959). PAF RRA standard curves were then constructed in the presence (0) or absence (0) of the extracted lipids. Data represent the means+SEM of triplicate experiments.
bation buffer and the PAF standard solution was generated in the presence or absence of the extracted lipids. Inclusion of the extracted lipid in the PAF RRA scarcely interfered with the determination of PAF levels, since total binding (B,,) radioactivity of 1989 dpm and 2036 dpm were obtaind in the presence and absence of the ex-
tracted lipid, respectively. The 50% inhibition value of the assay was only slightly changed (8.63.7 nM) (Fig. 4). Then, PAF produced by human neutrophils was quantified by RRA. When cytochalasin B-pretreated human neutrophils were stimulated with varying concentrations of FMLP, PAF was produced in a dose-dependent manner. However, as shown in Fig. 5, 60-80% of PAF remained cell-associated. No PAF was detected without stimulation. These observations are in good agreement with data in literature, and the PAF levels determined by this method are within the expected range (Lynch and Henson, 1986; Ludwig et al., 1984) (Fig. 5). In the current methods for the determination of PAF in neutrophils, it is necessary to remove the interfering lipids such as lysophosphatidylcholine and sphingomyelin by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC) (Lynch and Henson, 1986; Sugiura et al., 1990; Sugatani et al., 1990; Miwa et al., 1992). Our method needs no purification of PAF produced by neutrophils, because those interfering lipids do not disturb the binding between [“HIWEB 2086 and receptor preparations. Furthermore, this method make it possible to assay the large number of samples in 96-well microplates, rapidly and simultaneously. This method is also applicable to screen the PAF antagonist.
Acknowledgements
We thank Dr. C. Sakanaka of the University of Tokyo for suggestions in obtaining CHO stable transformant cells, and M. Ohara for comments.
1
I
t
2
4
6
FMLP
References
(FM)
Fig. 5. Dose response of PAF production and release by FMLP-stimulated human neutrophils. Neutrophils (2.5 X 10h cells) were incubated at 37°C with 5 pg/ml cytochalasin B for 2 min, then with various concentration of FMLP for 10 min. Supernatant (0) and ceil-associated (0) PAF were then assayed. Data represent the means+_SEM of triplicate experiments.
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