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Inhibition by BN 52021 (Ginkgolide B) of the binding of [3h]- Platelet-activating factor to human neutrophil granulocytes
Vol. 148, No. 3, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1412-1417
November 13, 1987
INHIBITION BY BN 52021 (GINKGOLIDE B) OF THE BINDING OF [ 3H] PLATELET-ACTIVATING FACTOR TO HUMAN NEUTROPHIL GRANULOCYTES
Eva FiDLDES-FILEP 1, Pierre BRA@UET 2, and 3~nos FILEP 1 *
D e p a r t m e n t of Clinical Pharmacology, Hannover Medical S~hool, 3000 Hannover 61, FRG
Institute Henri Beau four Research Laboratories, Le Plessis Robinson, France Received October 5, 1987
The inhibitory effect of BN 52021, a specific antagonist of p l a t e l e t - a c t i v a t i n g factor (PAF) on PAF-induced acl~vation of human polymorphonuclear granulocytes (PMNL) and on the bi_q~ing of4[~H] -PAF to neutrophils were examined. BN 52021 aver the range of 10- - ]0- M inhibited PAF-induced degranulation and superoxideooroduction of PMNLs ir~ a dose-dependent manner with K . values of 0.6 + 0.1 x 10- M and 0.4 + 0.1 x 10- M, respectively. BN 52021 (up todl mM) did not show any agonistic activity and it did not affect neutrophiI responses to N - f o r m y l - m e thionyl-leucyl-phenylalanine or leukotriene B,. The K. value of. BN 52021 for the . . . . ~ . q-O specific b[ndm.g]of [ H] -PAF to neutrophds was 1.3 + ~3.5 x 1O M versus a 14. of 1.1 + 0.3 x 10- M for PAF itself. BN 52021 did n o t a f f e c t metabolism of PAF~ by PMIqL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor. ® 1987AcademicPress, Inc.
Platelet-activating factor (l-0-alkyt-2-0-acetyl-sn-glycero-3-phosphorylcholine, PAF) has been r e c e n t l y recognised as a potent mediator of allergy and i n f l a m m a tion (1,2). Stimulation of certain cells including polymorphonuclear granuloeytes (PMNL) can lead to the formation of PAF (2), which, in turn, could act itself as stimulus on tile same cells. PAF has been reported to induce degranulation, aggregation and chemotaxis of human PMNLs (3,4,5), to enhance N - f o r m y l - m e t h i o n y l leucyl-phenylalanine (fMLP)-stimulated respiratory burst (6) and to p o t e n t i a t e PMNL degranulation by leukotrine B4 (7). Further analysis of the role of PAF in neutrophil a c t i v a t i o n and in PMNL-mediated tissue injury has been hampered, however, by the lack of direct demonstration of the e f f e c t of PAF antagonists on neutrophils. In the last years numerous PAF antagonists have been developed, including BN 52021 (gH-1,7a-(epoxymethanol)-lH, 6all, cyclopenta[c ] [ 2-3-b] furo-
* To whom correspondence should be addressed at the Department of Pathophysiology, Semmelweis University Medical School, It#~5 Budapest, Hungary. Abbreviations used: PAF, p l a t e l e t - a c t i v a t i n g factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine); PMNL, polymorphonuclear leukocytes; fMLP, N-formylmethionyl-leucyt-phenylalanine;LTB#, leukotriene B4. 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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VoI. 148, No. 3, 1987
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[3;2' " 3,4] cyelopenta [],2-d I furan-5,9,12-[4H]-trione, 3-tert-butyl hexahydro-4,7b1-11 hydroxy-8 methyl; ginl
Reagents and buffers: The following were purchased from Sigma Chemical Co. ~Deisenhofen, FRG--~."PAF, fMLP, cytochalasin B, ferricytochrome C, superoxide dismutase, Micrococcus lysodeikticus, bovine serum albumin, g-glucuronidase and lactate dehydrogenase kits. Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), silicone oil (AR 200, Wack~r Chemie, Munich, FRG), Quickscint 212 (Zinsser Analytik, Frankfurt, FRG), ~H~ -PAF (80 Ci/mmol) (Amersham, UK), [~H] -fMLP (50.9 Ci/mmol) (New England Nuclear, Dreieich, FRG), LTBt~ (ONO Pharmaceuticals, Osaka, 3apan). All assays were performed in a modified Hanks' balanced salt solution (1.4 mM calcium chloride, 2501ug/ml bovine serum albumin, pH 7.4). Cell preparation. Human neutrophils were isolated from freshly drown blood according to tile method of Boyum (10). The resultant cell preparation contained more than 95% neutrophils, no erythrocytes and less than 1 platelet/100 leukocytes. Degranulation assay. PMNLs (5 x 106 cells/ml) were incubated at 37°C for 10 min, treated with cytochalasin B (Spg/ml(~ for 10 min, challenged for 5 min, placed on ice and centrifuged (400 g, # rain, ~ C) to obtain supernatant fluid that was assayed [or lysozyme (11), B-glucuronidase and lactate dehydrogenase, as measures of lysosomal enzyme release and cell integrity, respectively. Results are reported as net enzyme release, i.e. the percentage of total cellular enzyme released by challenged ceils minus that released by identically treated but unchallenged cells. Superoxide production was determind by measuring superoxide dismutase inhibitable ferricytochrome c reduction (12). 7 Quantification of PAF binding. 350 pl of ne.jJtrophil suspension (I0 cells/ml) was added to microfuge tubes which contained [ J H ] -PAF (150 pM) without and with excess unlabeled PAF, BN 52021 or the appropriate buffer. Following incubation for indicated periods at #° C, free and bound ligand were separated using a silicone oil centrifugation method (13). Supernatants and pellets were transferred into 20 ml scintillation vials, overlaid with 350 tul of 2% Triton X-100 for 3 hours and mixed with I0 ml of Quickscint 212. Vials were counted for 5 rain with an LKB 1211 scintillation counter (Turku, Finland). The system was programmed to measure each sample's quench and extrapolate from counts per minute to disintegrations per minute using tritium standards. Metabolism of [3H-]-PAF. 107 c e l l s / m l were incubated with 13 nM E3H] - P A F in the absence and presence of 0.2 mM BN 52021 for 100 minutes. P e l l e t e d c e l l s and s u p e r n a t a n t s were s e p a r a t e l y e x t r a c t e d with two volumes of c h l o r o p h o r m ; m e t h a n o l (2:1, vol:vol). The procedure always r e c o v e r e d m o r e than 90% of t h e r a d i o a c t i v i t y in the lower chlorophorm phase. M a t e r i a l was s p o t t e d on silicagel TLC p l a t e s and developed with c h l o r o f o r m : m e t h a n o l = w a t e r (65:35:6 vol:vol). Strips (0.5 cm) of silica gel were s e q u e n t i a l l y scraped from the p l a t e s , o v e r l a i d with 0,5 ml m e t h a n o l and measured for r a d i o a c t i v i t y as d e s c r i b e d above. D a t a analysis. The equilibrium dissociation c o n s t a n t s of BN 52021 for r e s p o n s e m e a s u r e m e n t s were d e t e r m i n e d by Schild analysis (14) using t h e e q u a t i o n of Kd=EBN 5 2 0 2 1 ] / C R - I , where CR i s t h e r a t i o of e q u i a c t i v e c o n c e n t r a t i o n s of P A F in the presence and a b s e n c e of a given c o n c e n t r a t i o n of BN 52021. K d v a l u e s for 1413
V o l 148, N o 3, 1 9 8 7
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
degranulation and superoxide production were calculated at 3-4 different concentrations of BN 52021 using several c o n c e n t r a t i o n s of PAF. The slope of the Schild regression (CR versus BN 52021 ) was also determined. Values are expressed as means + SEM. Krl values from binding and response m e a s u r e m e n t s were compared by Mann-Whitney~s U t e s t . A 0.05 level was considered significant.
RESULTS AND DISCUSSION Initial studies d e m o n s t r a t e d that BN 52021 inhibits degranulation (Fig. I) and superoxide production (Fig. 2) of PMNLs in a dose-related manner. F o u r - t o - e i g h t fold molar excess of BN 52021 over the c o n c e n t r a t i o n of PAF used was required to cause 50% inhibition, whereas BN 52021 at about 100 fold molar excess practically completely abolished neutrophil responses to PAF. Schild analysis of the data revealed K d values of BN 52021 of 0.6 + 0.1 x 10-6 M (n = 20) and 0.t+ + 0.1 x 10-6 M (n = 7, p > 0 . 1 ) for degranulation and superoxide production, respectively. The slope of the Schild regression was 0.7 x 107 and 1.2 x 107, respectively. BN 52021 in itself, even at a c o n c e n t r a t i o n as high as 10-3 M had no PMNL stimulating activity and did not a f f e c t cellular i n t e g r i t y . In addition, BN 52021 at a dose of 10-4 M, which completely inhibits neutrophil responses to 10-6 M PAF, had no effects on degranulation and superoxide production induced by fMLP and LTBt~. In order to examine the mechanism by which BN 52021 inhibits neutrophil activation by PAF, the capacity of BN 52021 and unlabeled PAF to compete with
~z
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z.~
ch
kM
5
LSO,ME
BN 52021 0
.N,,02,
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o
.
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t
~0, i0-8 1o-7 10-6 10-s 10-, mo[li PAF
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~
Fig. i .
IOegranulation response of neutrophils to PAF in the presence of various concentrtions of BN 52021. Cells were t r e a t e d w i t h cytochalasine B for 10 rnin and w i t h 8N 52021 for 2 rain, then ehallanged w i t h PAF- for 5 rain. Background values of net enzyme release by i d e n t i c a l l y t r e a t e d but unchallanged ceils were substracted. Each point represents the mean of duplicate determinations of 4-9 separate experiments.
Fig. 2.
PMNL superoxide production caused by PAF io the presence of various concentrations of BN 52021. Neutrophils (5 x tO ~ cells/ml) were incubated w i t h BN 521321 for 2 rain, then challanged w i t h PAF for 113 min. F e r r i c y t o e h r o m e C reduction was 91-98% attenuated at each PAF concentration by 30)Jg/ml superoxide dismutase. Results represent means for duplicate determinations of b, separate experiments,
1414
10s moll[ PAF
Vol. 148, No. 3, 1987
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100
u._ I
-r-
x 103dpm 2
5c
BN52021
o
o
o
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PAF
/
_
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Fig, 4.
0
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Inhibition of [3H 1 -PAF binding to human neutrophils with unlabeled PAF and BN 52022. Cells were preincub~ted with unlabeled PAF or BN 521321 for 2 rain and then incubated with [ H] -PAF (2513pM) for 413 min at 4° C. Values are means + SEM of 4 experiments. Reversibility of the specifire3binding of t 3 H ] -PAF to human neutrophils. Cells were incubated with L _HTJ-PAF (1513 pM) for 413~min at 4u C, and then unlabeled PAF (1.5 x 10- M), BN 52021 (3 x 113-u M) or buffer was added to the medium. 3513)ul aliquots were removed for quantitation of EJH]-PAF binding at the indicated times. The non-specific bind~4ng was defined as the amount of binding that was not inhibited with 213- ivI unlabeled PAl= . The results shown from duplicate determinations are typical for ]3 similar experiments.
3H] -PAF for binding to PMNL was compared. Concentrations of unlabeled PAF and BN 52021 between 10-9 and 10-4 M dose-dependently inhibited the specific binding of [3H]_ -PAF to neutrophils. The ICs0 values were 2.0 _+ 0.5 x 10 -7 M (n = 4) and 2.4 _+ 0.9 x 10-6 M (n = 4), respectively (Fig. 3), suggesting that BN 52021 has an approximately 10 fold lower affinity for the PAF receptor then does PAF itself. The non-specific binding (defined as the amount of binding that was not inhibited by 10 -# M of unlabeled PAF) was barely inhibited even by the highest concentration of BN 52021 (inhibition by 10-4 M BN 52021 was 2 + 6%, n = 3). The equilibrium dissociation constant (K d) for [ 3 H ] - P A F binding to its specific high affinity r e c e p tor site under the same experimental conditions is 0.2 nM (16). By assuming c o m petitive inhibition, the inhibition coefficient (K.) of BN 52021 is 1.3 + 0.5 x 10 -6 M according to the equation K i = ICs0 / ( I + [ 3 H ] JPAF / Kd), where [ 3 f i ] - P A F is the c o n c e n t r a t i o n (150 pM) used in the receptor binding i n h i b i t i o n . The s p e c i f i c binding of [ 3 H ] - P A F to PMNL was reversible (Fig. 4). Unlabeled PAF, 1.5 x 10-7 M and BN 52021, 3 x 10-6 M reversed the 13HI - P A F binding by 42 + 4% and 45 + 4% (n = 3), respectively. H a l f - m a x i m a l dissociation of bound [ 3 H I - P
F occured
7.7 +
1.0 rain
and 10.7 + 1.4 rain a f t e r addition of PAF and BN 52021, respectively. The i n h i b i t o r y
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a c t i o n of BN 52021 on the r7L3H] - P A F binding was not due to the nonspecific lipophylic a c t i o n of BN 52021, since BN 52021 (10 -4 M) did not inhibit the specific
binding of-L 3H "1-fMLP (data not shown)and the non-specific binding of "[3H'J _pAF to PMNLs. That inhibition of binding is the principial mechanism by which BN 52021 blocks the a c t i v a t i o n of PMNLs by PAF is further suggested by the close c o r r e l a t i o n b e t w e e n the K. value for BN 52021 in competing for the binding sites 1
(1.3 + 0.5 x 10 -6 M) and the K d values for BN 52021 in blocking degranulation (0.6 + 0.1 x 10-6 M, p ~ 0 . 0 5 c o m p a r e d to K i) and superoxide production (0.# z 0.1 x 10 -6 M, p ~ 0 . 0 5 c o m p a r e d to Ki). The K d value of BN 52021 in human neutrophils is similar to t h a t found in a r a b b i t p l a t e l e t m e m b r a n e p r e p a r a t i o n (1.# x 10-7 M) (15). A single peak of r a d i o a c t i v i t y was i d e n t i f i e d for supernatants and pellets obt a i n e d for PMNL t r e a t e d with BN 52021 or its vehicle. This peak coincided with t h e
~3H]-PAF m a r k e r , indicating that no substantial metabolism of PAF occured under the present incubation conditions. These findings a r e in good a g r e e m e n t with those made by O ' F l a h e r t y et al. (16) who r e p o r t e d t h a t PMNLs do not m e t a b o l i z e -"L3HJP A F a t #o C, but r a t h e r gradually a c c u m u l a t e it. In summary, the p r e s e n t d a t a suggest t h a t BN 52021 is a potent, s p e c i f i c a n t a g o n i s t of P A F - i n d u c e d neutrophil a c t i v a t i o n through the inhibition of P A F -
binding to its neutrophil receptor.
ACKNOWLEDGEMENTS We thank the nursing staff of the Blood Bank for aid in taking care of blood donors. The authors are indebted to Dr. M. Tsuboshima,(ONO Pharmaceuticals, Osaka, Japan) for generously supplying us with LTBa. E. F.-F. and 3. F. were visiting scientists from the Semmelweis University Medical School, Budapest, Hungary and were supported by the Alexander von Humboldt Foundation, Bonn, FRG and by the Boehringer-Ingelheim Foundation, Stuttgart, FRG, respectively. Thanks are also due to Mrs. Marlis Aschoff for secretarial assistance.
REFERENCES
1. 2. 3.
4. 5. d. 7. 8. 9.
Braquet, P., Touqui, L., Shen, T.Y., and Vargaftig, B.B. (1987) Pharmacol. Rev. 2;9, 97-145. Hanahan,D.3. (1986) Ann. Rev. Biochem. 55, 483-509. Shaw, 3.D., Pinckard, R.N., Ferrigni, K.S., McNanus, L.M., and Hanahan, D.3. (1981) 3. Imrnunol. 127, 1250-1255. O'Flaherty, 3.T., Wykle, R.L., Miller~ C.H., Lewis, 3.C, Waite, M., Bass, D.A., McCall, C.E., and DeChatelet, L.R. (1981) Am. 3. Pathol. 103, 70-78. O'Flaherty, 3.T., Lees, C.J., Miller, C.H., McCall, C.E., Lewis, 3.C, Love, S.H., and Wykle, R.L. (1981) 3. lmmunol. 127, 731-737. Dewald, B., and Baggiolini, M. (.1985) Biochem. Biophys. Res. Commun. 128, 297-304. O'Flaherty, 3.T. (1985) 3. Cell. Physiol. 122,229-239. Braquet, P. (1984) Treatrnent and prevention of PAF-aeether-induced illness by a new series of highly specific inhibitors. G B Patent 8, 0,18-424. Nunez, D., Chignard, M., Korth, R., LeCouedie, 3.P., Norel, X., Spinnewyn, [3.,Braquet, P., and Benveniste, P. (1986) Eur. 3. Pharmacol. 123, 197-205. 1416
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10. 11. 12. 13.
14. 15. 16.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Boyum, A. (1976) Seand. J. Immunol. 5 (Suppl. 5), 9-15. Smith, R.3., Bowman, B.3., and Iden, S.S. (1983) Clin. Immunol. Immonapathol. 28, 13-28. Metcaif, J.A., Gallin, J.A., Neuseef, W.M., and Root, R.K. (1986) In: Laboratory Manual of Neutrophil Function, pp 109-155, Raven Press, New York. Naccache, P.H., Sbowell, H.3., Becket, E.L., and Sha'afi, R.I. (1977) J. Cell. Biol. 73, 428-444 Schild, H.O. (19/49) By. 3. Pharmacol., 4, 227-280. Braquet, P., Spinnewyn, B., Braquet, M., Bouvgain, R.H., Taylor, J.E., Nunez, D., and Drieu, I<. (1985) Blood Vessels 16, 559-572. O'Flaherty, J.T., Surles, j.fv., Redman, J., Jacobsen, ID., Piantadosi, C., and Wykle, R.L. (1986) J. Clin. Invest. 78, 381-388.
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Pages 1412-1417
November 13, 1987
INHIBITION BY BN 52021 (GINKGOLIDE B) OF THE BINDING OF [ 3H] PLATELET-ACTIVATING FACTOR TO HUMAN NEUTROPHIL GRANULOCYTES
Eva FiDLDES-FILEP 1, Pierre BRA@UET 2, and 3~nos FILEP 1 *
D e p a r t m e n t of Clinical Pharmacology, Hannover Medical S~hool, 3000 Hannover 61, FRG
Institute Henri Beau four Research Laboratories, Le Plessis Robinson, France Received October 5, 1987
The inhibitory effect of BN 52021, a specific antagonist of p l a t e l e t - a c t i v a t i n g factor (PAF) on PAF-induced acl~vation of human polymorphonuclear granulocytes (PMNL) and on the bi_q~ing of4[~H] -PAF to neutrophils were examined. BN 52021 aver the range of 10- - ]0- M inhibited PAF-induced degranulation and superoxideooroduction of PMNLs ir~ a dose-dependent manner with K . values of 0.6 + 0.1 x 10- M and 0.4 + 0.1 x 10- M, respectively. BN 52021 (up todl mM) did not show any agonistic activity and it did not affect neutrophiI responses to N - f o r m y l - m e thionyl-leucyl-phenylalanine or leukotriene B,. The K. value of. BN 52021 for the . . . . ~ . q-O specific b[ndm.g]of [ H] -PAF to neutrophds was 1.3 + ~3.5 x 1O M versus a 14. of 1.1 + 0.3 x 10- M for PAF itself. BN 52021 did n o t a f f e c t metabolism of PAF~ by PMIqL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor. ® 1987AcademicPress, Inc.
Platelet-activating factor (l-0-alkyt-2-0-acetyl-sn-glycero-3-phosphorylcholine, PAF) has been r e c e n t l y recognised as a potent mediator of allergy and i n f l a m m a tion (1,2). Stimulation of certain cells including polymorphonuclear granuloeytes (PMNL) can lead to the formation of PAF (2), which, in turn, could act itself as stimulus on tile same cells. PAF has been reported to induce degranulation, aggregation and chemotaxis of human PMNLs (3,4,5), to enhance N - f o r m y l - m e t h i o n y l leucyl-phenylalanine (fMLP)-stimulated respiratory burst (6) and to p o t e n t i a t e PMNL degranulation by leukotrine B4 (7). Further analysis of the role of PAF in neutrophil a c t i v a t i o n and in PMNL-mediated tissue injury has been hampered, however, by the lack of direct demonstration of the e f f e c t of PAF antagonists on neutrophils. In the last years numerous PAF antagonists have been developed, including BN 52021 (gH-1,7a-(epoxymethanol)-lH, 6all, cyclopenta[c ] [ 2-3-b] furo-
* To whom correspondence should be addressed at the Department of Pathophysiology, Semmelweis University Medical School, It#~5 Budapest, Hungary. Abbreviations used: PAF, p l a t e l e t - a c t i v a t i n g factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine); PMNL, polymorphonuclear leukocytes; fMLP, N-formylmethionyl-leucyt-phenylalanine;LTB#, leukotriene B4. 0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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VoI. 148, No. 3, 1987
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[3;2' " 3,4] cyelopenta [],2-d I furan-5,9,12-[4H]-trione, 3-tert-butyl hexahydro-4,7b1-11 hydroxy-8 methyl; ginl
Reagents and buffers: The following were purchased from Sigma Chemical Co. ~Deisenhofen, FRG--~."PAF, fMLP, cytochalasin B, ferricytochrome C, superoxide dismutase, Micrococcus lysodeikticus, bovine serum albumin, g-glucuronidase and lactate dehydrogenase kits. Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), silicone oil (AR 200, Wack~r Chemie, Munich, FRG), Quickscint 212 (Zinsser Analytik, Frankfurt, FRG), ~H~ -PAF (80 Ci/mmol) (Amersham, UK), [~H] -fMLP (50.9 Ci/mmol) (New England Nuclear, Dreieich, FRG), LTBt~ (ONO Pharmaceuticals, Osaka, 3apan). All assays were performed in a modified Hanks' balanced salt solution (1.4 mM calcium chloride, 2501ug/ml bovine serum albumin, pH 7.4). Cell preparation. Human neutrophils were isolated from freshly drown blood according to tile method of Boyum (10). The resultant cell preparation contained more than 95% neutrophils, no erythrocytes and less than 1 platelet/100 leukocytes. Degranulation assay. PMNLs (5 x 106 cells/ml) were incubated at 37°C for 10 min, treated with cytochalasin B (Spg/ml(~ for 10 min, challenged for 5 min, placed on ice and centrifuged (400 g, # rain, ~ C) to obtain supernatant fluid that was assayed [or lysozyme (11), B-glucuronidase and lactate dehydrogenase, as measures of lysosomal enzyme release and cell integrity, respectively. Results are reported as net enzyme release, i.e. the percentage of total cellular enzyme released by challenged ceils minus that released by identically treated but unchallenged cells. Superoxide production was determind by measuring superoxide dismutase inhibitable ferricytochrome c reduction (12). 7 Quantification of PAF binding. 350 pl of ne.jJtrophil suspension (I0 cells/ml) was added to microfuge tubes which contained [ J H ] -PAF (150 pM) without and with excess unlabeled PAF, BN 52021 or the appropriate buffer. Following incubation for indicated periods at #° C, free and bound ligand were separated using a silicone oil centrifugation method (13). Supernatants and pellets were transferred into 20 ml scintillation vials, overlaid with 350 tul of 2% Triton X-100 for 3 hours and mixed with I0 ml of Quickscint 212. Vials were counted for 5 rain with an LKB 1211 scintillation counter (Turku, Finland). The system was programmed to measure each sample's quench and extrapolate from counts per minute to disintegrations per minute using tritium standards. Metabolism of [3H-]-PAF. 107 c e l l s / m l were incubated with 13 nM E3H] - P A F in the absence and presence of 0.2 mM BN 52021 for 100 minutes. P e l l e t e d c e l l s and s u p e r n a t a n t s were s e p a r a t e l y e x t r a c t e d with two volumes of c h l o r o p h o r m ; m e t h a n o l (2:1, vol:vol). The procedure always r e c o v e r e d m o r e than 90% of t h e r a d i o a c t i v i t y in the lower chlorophorm phase. M a t e r i a l was s p o t t e d on silicagel TLC p l a t e s and developed with c h l o r o f o r m : m e t h a n o l = w a t e r (65:35:6 vol:vol). Strips (0.5 cm) of silica gel were s e q u e n t i a l l y scraped from the p l a t e s , o v e r l a i d with 0,5 ml m e t h a n o l and measured for r a d i o a c t i v i t y as d e s c r i b e d above. D a t a analysis. The equilibrium dissociation c o n s t a n t s of BN 52021 for r e s p o n s e m e a s u r e m e n t s were d e t e r m i n e d by Schild analysis (14) using t h e e q u a t i o n of Kd=EBN 5 2 0 2 1 ] / C R - I , where CR i s t h e r a t i o of e q u i a c t i v e c o n c e n t r a t i o n s of P A F in the presence and a b s e n c e of a given c o n c e n t r a t i o n of BN 52021. K d v a l u e s for 1413
V o l 148, N o 3, 1 9 8 7
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
degranulation and superoxide production were calculated at 3-4 different concentrations of BN 52021 using several c o n c e n t r a t i o n s of PAF. The slope of the Schild regression (CR versus BN 52021 ) was also determined. Values are expressed as means + SEM. Krl values from binding and response m e a s u r e m e n t s were compared by Mann-Whitney~s U t e s t . A 0.05 level was considered significant.
RESULTS AND DISCUSSION Initial studies d e m o n s t r a t e d that BN 52021 inhibits degranulation (Fig. I) and superoxide production (Fig. 2) of PMNLs in a dose-related manner. F o u r - t o - e i g h t fold molar excess of BN 52021 over the c o n c e n t r a t i o n of PAF used was required to cause 50% inhibition, whereas BN 52021 at about 100 fold molar excess practically completely abolished neutrophil responses to PAF. Schild analysis of the data revealed K d values of BN 52021 of 0.6 + 0.1 x 10-6 M (n = 20) and 0.t+ + 0.1 x 10-6 M (n = 7, p > 0 . 1 ) for degranulation and superoxide production, respectively. The slope of the Schild regression was 0.7 x 107 and 1.2 x 107, respectively. BN 52021 in itself, even at a c o n c e n t r a t i o n as high as 10-3 M had no PMNL stimulating activity and did not a f f e c t cellular i n t e g r i t y . In addition, BN 52021 at a dose of 10-4 M, which completely inhibits neutrophil responses to 10-6 M PAF, had no effects on degranulation and superoxide production induced by fMLP and LTBt~. In order to examine the mechanism by which BN 52021 inhibits neutrophil activation by PAF, the capacity of BN 52021 and unlabeled PAF to compete with
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Fig. i .
IOegranulation response of neutrophils to PAF in the presence of various concentrtions of BN 52021. Cells were t r e a t e d w i t h cytochalasine B for 10 rnin and w i t h 8N 52021 for 2 rain, then ehallanged w i t h PAF- for 5 rain. Background values of net enzyme release by i d e n t i c a l l y t r e a t e d but unchallanged ceils were substracted. Each point represents the mean of duplicate determinations of 4-9 separate experiments.
Fig. 2.
PMNL superoxide production caused by PAF io the presence of various concentrations of BN 52021. Neutrophils (5 x tO ~ cells/ml) were incubated w i t h BN 521321 for 2 rain, then challanged w i t h PAF for 113 min. F e r r i c y t o e h r o m e C reduction was 91-98% attenuated at each PAF concentration by 30)Jg/ml superoxide dismutase. Results represent means for duplicate determinations of b, separate experiments,
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100
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-r-
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Inhibition of [3H 1 -PAF binding to human neutrophils with unlabeled PAF and BN 52022. Cells were preincub~ted with unlabeled PAF or BN 521321 for 2 rain and then incubated with [ H] -PAF (2513pM) for 413 min at 4° C. Values are means + SEM of 4 experiments. Reversibility of the specifire3binding of t 3 H ] -PAF to human neutrophils. Cells were incubated with L _HTJ-PAF (1513 pM) for 413~min at 4u C, and then unlabeled PAF (1.5 x 10- M), BN 52021 (3 x 113-u M) or buffer was added to the medium. 3513)ul aliquots were removed for quantitation of EJH]-PAF binding at the indicated times. The non-specific bind~4ng was defined as the amount of binding that was not inhibited with 213- ivI unlabeled PAl= . The results shown from duplicate determinations are typical for ]3 similar experiments.
3H] -PAF for binding to PMNL was compared. Concentrations of unlabeled PAF and BN 52021 between 10-9 and 10-4 M dose-dependently inhibited the specific binding of [3H]_ -PAF to neutrophils. The ICs0 values were 2.0 _+ 0.5 x 10 -7 M (n = 4) and 2.4 _+ 0.9 x 10-6 M (n = 4), respectively (Fig. 3), suggesting that BN 52021 has an approximately 10 fold lower affinity for the PAF receptor then does PAF itself. The non-specific binding (defined as the amount of binding that was not inhibited by 10 -# M of unlabeled PAF) was barely inhibited even by the highest concentration of BN 52021 (inhibition by 10-4 M BN 52021 was 2 + 6%, n = 3). The equilibrium dissociation constant (K d) for [ 3 H ] - P A F binding to its specific high affinity r e c e p tor site under the same experimental conditions is 0.2 nM (16). By assuming c o m petitive inhibition, the inhibition coefficient (K.) of BN 52021 is 1.3 + 0.5 x 10 -6 M according to the equation K i = ICs0 / ( I + [ 3 H ] JPAF / Kd), where [ 3 f i ] - P A F is the c o n c e n t r a t i o n (150 pM) used in the receptor binding i n h i b i t i o n . The s p e c i f i c binding of [ 3 H ] - P A F to PMNL was reversible (Fig. 4). Unlabeled PAF, 1.5 x 10-7 M and BN 52021, 3 x 10-6 M reversed the 13HI - P A F binding by 42 + 4% and 45 + 4% (n = 3), respectively. H a l f - m a x i m a l dissociation of bound [ 3 H I - P
F occured
7.7 +
1.0 rain
and 10.7 + 1.4 rain a f t e r addition of PAF and BN 52021, respectively. The i n h i b i t o r y
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a c t i o n of BN 52021 on the r7L3H] - P A F binding was not due to the nonspecific lipophylic a c t i o n of BN 52021, since BN 52021 (10 -4 M) did not inhibit the specific
binding of-L 3H "1-fMLP (data not shown)and the non-specific binding of "[3H'J _pAF to PMNLs. That inhibition of binding is the principial mechanism by which BN 52021 blocks the a c t i v a t i o n of PMNLs by PAF is further suggested by the close c o r r e l a t i o n b e t w e e n the K. value for BN 52021 in competing for the binding sites 1
(1.3 + 0.5 x 10 -6 M) and the K d values for BN 52021 in blocking degranulation (0.6 + 0.1 x 10-6 M, p ~ 0 . 0 5 c o m p a r e d to K i) and superoxide production (0.# z 0.1 x 10 -6 M, p ~ 0 . 0 5 c o m p a r e d to Ki). The K d value of BN 52021 in human neutrophils is similar to t h a t found in a r a b b i t p l a t e l e t m e m b r a n e p r e p a r a t i o n (1.# x 10-7 M) (15). A single peak of r a d i o a c t i v i t y was i d e n t i f i e d for supernatants and pellets obt a i n e d for PMNL t r e a t e d with BN 52021 or its vehicle. This peak coincided with t h e
~3H]-PAF m a r k e r , indicating that no substantial metabolism of PAF occured under the present incubation conditions. These findings a r e in good a g r e e m e n t with those made by O ' F l a h e r t y et al. (16) who r e p o r t e d t h a t PMNLs do not m e t a b o l i z e -"L3HJP A F a t #o C, but r a t h e r gradually a c c u m u l a t e it. In summary, the p r e s e n t d a t a suggest t h a t BN 52021 is a potent, s p e c i f i c a n t a g o n i s t of P A F - i n d u c e d neutrophil a c t i v a t i o n through the inhibition of P A F -
binding to its neutrophil receptor.
ACKNOWLEDGEMENTS We thank the nursing staff of the Blood Bank for aid in taking care of blood donors. The authors are indebted to Dr. M. Tsuboshima,(ONO Pharmaceuticals, Osaka, Japan) for generously supplying us with LTBa. E. F.-F. and 3. F. were visiting scientists from the Semmelweis University Medical School, Budapest, Hungary and were supported by the Alexander von Humboldt Foundation, Bonn, FRG and by the Boehringer-Ingelheim Foundation, Stuttgart, FRG, respectively. Thanks are also due to Mrs. Marlis Aschoff for secretarial assistance.
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Braquet, P., Touqui, L., Shen, T.Y., and Vargaftig, B.B. (1987) Pharmacol. Rev. 2;9, 97-145. Hanahan,D.3. (1986) Ann. Rev. Biochem. 55, 483-509. Shaw, 3.D., Pinckard, R.N., Ferrigni, K.S., McNanus, L.M., and Hanahan, D.3. (1981) 3. Imrnunol. 127, 1250-1255. O'Flaherty, 3.T., Wykle, R.L., Miller~ C.H., Lewis, 3.C, Waite, M., Bass, D.A., McCall, C.E., and DeChatelet, L.R. (1981) Am. 3. Pathol. 103, 70-78. O'Flaherty, 3.T., Lees, C.J., Miller, C.H., McCall, C.E., Lewis, 3.C, Love, S.H., and Wykle, R.L. (1981) 3. lmmunol. 127, 731-737. Dewald, B., and Baggiolini, M. (.1985) Biochem. Biophys. Res. Commun. 128, 297-304. O'Flaherty, 3.T. (1985) 3. Cell. Physiol. 122,229-239. Braquet, P. (1984) Treatrnent and prevention of PAF-aeether-induced illness by a new series of highly specific inhibitors. G B Patent 8, 0,18-424. Nunez, D., Chignard, M., Korth, R., LeCouedie, 3.P., Norel, X., Spinnewyn, [3.,Braquet, P., and Benveniste, P. (1986) Eur. 3. Pharmacol. 123, 197-205. 1416
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Boyum, A. (1976) Seand. J. Immunol. 5 (Suppl. 5), 9-15. Smith, R.3., Bowman, B.3., and Iden, S.S. (1983) Clin. Immunol. Immonapathol. 28, 13-28. Metcaif, J.A., Gallin, J.A., Neuseef, W.M., and Root, R.K. (1986) In: Laboratory Manual of Neutrophil Function, pp 109-155, Raven Press, New York. Naccache, P.H., Sbowell, H.3., Becket, E.L., and Sha'afi, R.I. (1977) J. Cell. Biol. 73, 428-444 Schild, H.O. (19/49) By. 3. Pharmacol., 4, 227-280. Braquet, P., Spinnewyn, B., Braquet, M., Bouvgain, R.H., Taylor, J.E., Nunez, D., and Drieu, I<. (1985) Blood Vessels 16, 559-572. O'Flaherty, J.T., Surles, j.fv., Redman, J., Jacobsen, ID., Piantadosi, C., and Wykle, R.L. (1986) J. Clin. Invest. 78, 381-388.
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