0192-0561/92 $5.00 + .00 Pergamon Press Ltd. ~)1992 [nternalionalSocietyfor lmmunopharmacology.

Int. J. Immunopharmac., Vol. 14, No. 4, pp. 5 1 5 - 5 2 3 , 1992. Printed in Great Britain.

B I N D I N G OF C A R B A M Y L - P L A T E L E T - A C T I V A T I N G FACTOR TO THE RAJI L Y M P H O B L A S T P L A T E L E T - A C T I V A T I N G FACTOR RECEPTOR JEFFREY B. TRAVERS,* QIAN LI, t HOWARD SPRECHER t and RICHARD H. FERTEL *t Departments of *Pharmacology and ~Physiological Chemistry, The Ohio State University College of Medicine, Columbus, OH 43210, U.S.A.

(Received 12 April 1991 and in final form 27 November 1991) -Carbamyl-platelet-activating factor (1-hexadecyl-2-N-methylcarbamyl-glycero-3-phosphocholine; CPAF) is an analog of platelet-activating factor (PAF) containing an N-methylcarbamyl moiety at the sn-2 position. CPAF was tested for effects on the Raji lymphoblast PAF receptor. Binding studies conducted at 4°C demonstrated specific binding that reached saturation within 6 0 - 8 0 min. Scatchard analysis of CPAF binding data revealed a single class of CPAF binding sites (14,800/ce11) with a K = 2.9 +_ 0.9 nM. Competition binding studies with PAF indicated that CPAF has about one-third the potency of native PAF. Unlike PAF, however, CPAF was not significantly metabolized by Raji lymphoblasts at 37°C. CPAF was shown to have PAF-agonistic qualities, since 100 pM to 1 taM CPAF increased free intracellular calcium in a dose-dependent manner. The structurally dissimilar PAF receptor antagonists CV-6209 and alprazolam inhibited the CPAF-induced calcium changes at doses that competed with CPAF binding. Treatment of Raji lymphoblasts with PAF or CPAF (10 p M - 1 taM) did not affect spontaneous proliferation, suggesting that the PAF receptor is not involved in the proliferative process in this cell line. These studies demonstrate that CPAF is a metabolically stable lymphoblast PAF receptor agonist that may provide a useful tool in the further elucidation of the role of PAF in lymphocyte function. Abslracl

Platelet-activating factor (l-O-alkyl-2-acetyl-snglycero-3-phosphocholine; PAF) is a potent mediator of inflammatory responses (Braquet, Touqui, Shen & Vargaftig, 1987). Evidence from radioligand binding studies (Valone, 1987) and the development of potent and specific P A F receptor antagonists (Saunders & Handley, 1987) indicates that P A F exerts its effects by interaction with a specific receptor. The P A F receptor is functionally linked to the activation of a phosphoinositol-specific phospholipase C (Shukla, 1985) a n d / o r inhibition of adenylate cyclase activity (Haslam & Vanderwel, 1982). P A F has also been shown to elicit arachidonic acid liberation, stimulate eicosanoid generation, and induce transcription of the nuclear proto-oncogenes c-fos and c-jun in a B-lymphoblastoid cell line (SKW6.4) (Schulam, Kuruvilla, Putcha, Mangus, Franklin-Johnson & Shearer, 1991). Recent evidence from studies using fluorescent calcium probes, such as fura-2, indicate that P A F causes an increase in

intracellular calcium levels (Barzaghi, Sarau & Mong, 1989; Selak & Smith, 1989). P A F is a readily metabolized by many cell types including platelets (Pieroni & H a n a h a n , 1983; Malone, Lee & Snyder, 1985), neutrophils (Chilton, O'Flaherty, Ellis, Swendsen & Wykle, 1983), monocytes (Robinson & Snyder, 1985) and lymphocytes (Travers, Sprecher & Fertel, 1990). These studies have indicated that l-alkyl-2-acetylG P C is first converted to 1-alkyl-2-1yso-GPC (lysoPAF) by acetyl hydrolase, an enzyme found in both cytosolic and extracellular forms (Blank, Lee, Fitzgerald & Snyder, 1981; Farr, Cox, Wardlow & Jorgenson, 1980). The l-alkyl-2-1yso-GPC which is formed is then converted to 1-alkyl-2-acyl-GPC by the addition of a long-chain fatty acid (often arachidonic acid) in a reaction catalyzed by a transacylase. Since P A F is quickly metabolized to inactive molecules in biological systems, metabolically stable P A F receptor antagonists have been

*Author to whom correspondence should be addressed: Department of Pharmacology, The Ohio State University, 5018 Graves Hall, 333 West 10th Ave, Columbus, OH 43210, U.S.A. 515

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PAF binding sites with a dissociation constant (KD) of 2.3 _+ 0.3 nM (Travers, Li, Kniss & Fertel, 1989). These binding sites are thought to be functional PAF receptors, since PAF treatment resulted in an intracellular calcium mobilization. The objective of the present study was to test the stable neutrophil PAF agonist CPAF in the Raji lymphoblast model system. First, the ability of Raji lymphoblasts to metabolize CPAF was assessed. Second, binding studies were conducted to compare CPAF with PAF. Third, the ability of CPAF to mobilize Raji lymphoblast intracellular calcium was tested. Finally, PAF and CPAF were tested for possible effects on Raji spontaneous proliferation.

Fig. 1. Structures of PAF and CPAF. useful tools in the elucidation of the effects of endogenous PAF and PAF receptors. The recent description of 1-alkyl-2-N-methylcarbamyl-GPC (carbamyl-PAF; CPAF; structure shown in Fig. 1) as a nonmetabolizable PAF agonist in neutrophils (O'Flaherty et al., 1987) potentially allows studies of PAF receptor stimulation in biological systems where metabolism is a problem. Although evidence from studies with cell lines suggests that PAF is produced by lymphocytes (Bussolino, Fou, Malavasi, Ferrando & Camussi, 1984), the role of PAF in lymphocyte function has not been firmly established (reviewed by Braquet & Rola-Pleszczynski, 1987). In 3-day cultures of lymphocytes stimulated with a mitogenic lectin, PAF has been reported to stimulate (Barrett, Lewis, Ward & Westwick, 1986), inhibit (Rola-Pleszczynski, Pignol, Pouliot & Braquet, 1987; Dulioust, Vivier, Salem, Benveniste & Thomas, 1988), or have no effect (Patrignani, Valitutti, Aiello & Musiani, 1987) on T-lymphocyte proliferation. PAF has been shown to inhibit interleukin 2 production (Rola-Pleszczynski et al., 1987; Dulioust et al., 1988) and decrease CD2 and CD3 antigen expression in T-lymphocytes (Vivier et al., 1988). Little is known about PAF effects on B-cell function (Behrens & Goodwin, 1987). Progress in understanding the role of PAF in lymphocyte biology has been hampered in part by both the rapid degradation of this phospholipid in biological systems, and the lack of a suitable lymphocyte PAF receptor model system. Unlike PAF-responsive cell types, such as platelets, neutrophils and monocytes, it is not known whether lymphocytes express PAF receptors. Previous studies in our laboratory have demonstrated that Rail, an Epstein-Barr virus (EBV)-immortalized B-cell line from a Burkitt lymphoma contains a single class of

EXPERIMENTAL PROCEDURES

Materials

Labeled CPAF (l-[3H]hexadecyl-2-N-methylcarbamyl-GPC, 54.6 Ci/mmole) and [3H]thymidine([~H]methyl-thymidine)were obtained from New England Nuclear, Boston, MA, U.S.A. Labeled PAF (1-[3H]octadecyl-2-acetyl-GPC, 132 Ci/mmole) was obtained from Amersham Corp., Arlington Heights, IL, U.S.A. PAF (1-hexadecyl-2-acetylGPC), lysoPAF (1-hexadecyl-2-1yso-GPC), dipalmitoyl-phosphatidylcholine and fatty acid-free BSA were purchased from Sigma Chemical Company, St. Louis, MO, U.S.A. CV-6209 was provided by Dr Y. Oka, Takeda Chemical Industries, Japan. Alprazolam was provided by The Up John Company, Kalamazoo, MI, U.S.A. The unlabeled natural isomer of CPAF (1-hexadecyl-2(R)-N-methylcarbamyl-GPC) was prepared from lysoPAF and methyl isocyanate (O'Flaherty et al., 1987). The purity of CPAF was assessed by TLC on LK6D silica gel plates (Whatman Inc., Clifton, N J, U.S.A.) using a basic solvent system containing c h l o r o f o r m ~ m e t h a n o l ~ a m m o n i u m hydroxide (70 : 35 : 7 v/v). The finding of a single spot with R, = 0.35 (corresponding to a CPAF standard generously provided by Dr Robert Wykle of The Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, NC, U.S.A.) after exposing the plate to iodine vapor was taken as an indication of high purity. Gas chromatography-mass spectrometry of the corresponding tertiary-butyl dimethyl silane (TBDMS) derivative demonstrated ions at m / z = 430 (M = 57); r n / z = 355 (rearrangement ion formed from the hexadecyl sn-1 group); and m / z = 132 (rearrangement ion formed from N-methylcarbamyl

CPAF Binding to Raji Lymphoblasts

sn-2 group and dimethylsilanol moieties), which correspond to the ions reported for the TBDMS derivative of (1-hexadecyl) PAF (Satouchi, Oda, Yasunaga & Saito, 1983). Characteristics and maintenance o f Raji lymphoblasts Raji is a lymphoid leukemic cell line derived from a patient with Burkitt lymphoma (Pulvertaft, 1965). Raji was provided by The Ohio State University Cell Culture Service, Columbus, OH, U.S.A. and was originally obtained from the American Type Culture Collection, Bethesda, MD, U.S.A. Cells were grown as stationary suspension cultures in upright T-75 or T-150 plastic flasks (Corning Glass Works, Corning, NY, U.S.A.) at 37°C in an atmosphere of 507o CO:. The cell culture medium consisted of RPMI 1640 supplemented with heat (56°C, 30 min)-inactivated serum, 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ~g/ml streptomycin (Gibco, Grand Island, NY, U.S.A.). Viability of Raji lymphoblasts used in these studies was more than 9507o as measured by trypan blue exclusion. Measurement of [3H]CPAF and [3H]PAF metabolism The metabolism of [3H]CPAF and [3H]PAF was quantitated as described previously (Travers et al., 1989, 1990). Briefly, Raji lymphoblasts (2.5 x 106 cells) were resuspended with a final volume of 0.95 ml of RPMI 1640 without serum. To the above cell suspension 50/A of aqueous solution containing [3H]CPAF or [3H]PAF complexed to BSA (2.5 mg/ml) was added, bringing the final concentration of phospholipid to 1 x 10 -9 M. The incubation mixture was shaken gently at 37°C. All reactions were terminated by the addition of 3.75 ml of chloroform/methanol ( 1 : 2 v/v) to the cell suspension and the lipids were extracted by the method of Bligh & Dyer (1959). The recovered products were those present in both the cells and medium and the recovery of tritium label in the lipid fraction was >87%. Solvents were removed by a stream of nitrogen and the lipids were resuspended in chloroform/methanol (2 : 1 v/v). The phospholipids and phospholipid standards were separated on LK6D linear K silica gel TLC plates (Whatman Inc., Clifton, NJ, U.S.A.) using an acidic solvent system containing chloroform/methanol/water/glacial acetic acid (65 : 35 : 8 : 1, v/v). The distribution of label was assayed by zonal scanning (2.5 mm increments). Radioactivity was measured in a Beckman LS6800 liquid scintillation counter

(Beckman U.S.A.).

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Measurement o f [~H]CPAF binding Binding studies were conducted as described previously (Traverset al., 1989). Briefly, lymphoblasts (5 x 106 cells), were washed and resuspended to a final volume of 0.9 ml with Hank's Balanced Salt Solution (HBSS) in 15 x 85 mm glass tubes treated with Aquasil (Pierce, Rockford, IL, U.S.A.). Suspensions were then simultaneously exposed to 50/al of aqueous solution containing 1.0 pmole of [3H]CPAF complexed to BSA (10 mg/ml), and 50/al of either unlabeled CPAF, PAF, CV-6209, lysoPAF, or BSA alone. Alprazolam was added in DMSO (DMSO at the final concentration of 0.2%, did not affect PAF binding). The mixture of ceils, [3H]CPAF and inhibitors was shaken gently at 4°C for 120 min. Incubation of Raji lymphoblasts under these conditions was shown previously to result in binding saturation with negligible metabolism of [3H]PAF (Travers et al., 1989). Each sample was run in triplicate. The suspensions were harvested by suction through GF/C filters (Whatman, Clifton, N J, U.S.A.) premoistened with 10 mg/ml BSA using a Hoefer Filtration apparatus (Hoefer Scientific, San Francisco, CA, U.S.A.). The tubes were then washed three times with 5 ml cold HBSS. The filters were air dried and placed in 10 ml scintillation vials, to which 7 ml of Scintiverse (Fisher, Fairlawn, N J, U.S.A.) was added. The vials were counted in a Beckman LS6800 scintillation counter that was programmed to measure each sample's quench and determine dis/min from counts/min using tritium standards. The Aquasil-treated tubes retained essentially none of the [~H]CPAF added. The samples were corrected for filter retention of radiolabel by running control samples that lacked cells. Specifically bound CPAF was determined by incubation with excess (1 /aM) unlabeled PAF. Analysis of binding data was carried out using analytical software (GraphPAD Software, San Diego, CA, U.S.A.). Measurement o f CPAF-induced intracellular C d ÷ mobilization Fura-2 loading was achieved by incubating the cells (5 x 106 cells/ml) with 2/aM fura-2/AM (Molecular Probes, Eugene, OR, U.S.A.) for 10 min at room temperature, followed by a wash with HBSS containing 1.4 mM CaC1 and 25 mM HEPES, pH 7.4. Cell suspensions were then diluted to 1 x 106 cells/ml and post-incubated at room temperature for 2 h to facilitate hydrolysis of the

J. B. TRAVERSet al.

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fura-2 ester. P r i o r to m e a s u r e m e n t , a n aliquot (3 ml) o f cells was t r a n s f e r r e d to a fluorimeter cuvette a n d p r e w a r m e d to 37°C. Fluorescence signals were recorded in a P e r k i n - E l m e r LS-5B spectrofluorimeter ( P e r k i n - E l m e r C o r p . , P o m o n a , CA, U.S.A), at excitation a n d emission wavelengths o f 340 a n d 510 n M , respectively. All experiments were p e r f o r m e d at 37°C with c o n s t a n t mixing. C P A F a n d o t h e r drugs were i n t r o d u c e d with D M S O as the solvent. T h e final c o n c e n t r a t i o n o f solvent was