Br. J. Pharmacol. (1992), 105, 87-92
C Macmillan Press Ltd, 1991
Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds Cory M. Hogaboam, *Donna Donigi-Gale, *T. Scott Shoupe, Elyse Y. Bissonnette, A. Dean Befus & 'John L. Wallace Gastrointestinal and Immunological Sciences Research Groups, University of Calgary, Calgary, Alberta, Canada and *International Molecular Discovery, Purdue Frederick Company, Norwalk, Connecticut, U.S.A. 1 Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 ,UM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2 This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10M). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20,ugml-1) or a specific antibody directed against PAF. 3 The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.91M) or Wy-50,295 (IC50 of 1.21uM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties.
4 Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100pM. 5 Neither PF-5901 nor Wy-50,295 (1 or 10pM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation. 6 These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties. Keywords: PAF; mast cells; leukotrienes; inflammation; anti-inflammatory; allergy; quinolines
Introduction Inflammatory mediator release from activated mast cells represents an important early event in many disease conditions associated with chronic inflammation (Gordon et al., 1990). Because of this, the mast cell and the mediators these cells release have been identified as potential targets for the treatment of inflammatory conditions and allergic reactions. One mediator which has been implicated in a variety of inflammatory disorders is platelet-activating factor, or PAF (reviewed by Braquet et al., 1987). Such a role for PAF has been supported by numerous studies demonstrating that treatment with PAF antagonists is beneficial in a variety of animal models of inflammation and allergy. While the biosynthesis of PAF has been studied extensively, potent and specific inhibitors have not been identified. Ketotifen has been shown to inhibit PAF release from mouse mast cells, but similar concentrations of this compound also inhibited calcium influx and release of fi-hexosaminidase (Joly et al., 1987). Mast cells derived from a variety of sources synthesize PAF (Mencia-Heurta et al., 1983; Schleimer et al., 1986; Musch et al., 1987; Triggiani et al., 1990), but little is known of the regulation of PAF synthesis and release by these cells. Products of the lipoxygenase pathway of arachidonic acid metabolism appear to play a role in modulating histamine release by rat peritoneal mast cells (Chand et al., 1987; Mansini et al., 1987), although some caution must be exercised in accepting these 1 Author for correspondence at: Department of Medical Physiology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada.
results, since the lipoxygenase inhibitors used in these studies (e.g. nordihydroguaretic acid) are known to affect other enzyme systems. Furthermore, there is no direct evidence that rat peritoneal mast cells are capable of producing 5lipoxygenase products of arachidonic acid. Heavy et al. (1988) were unable to detect leukotriene B4 (LTB4) or LTC4 release from rat peritoneal mast cells stimulated with anti-IgE antibody, while intestinal mucosal mast cells released significant quantities of LTB4 and LTC4 in response to similar stimulation. The present study was performed to determine if quinolinebased inhibitors of leukotriene synthesis affect PAF synthesis by rat peritoneal (connective tissue-type) mast cells. In order to do these studies, we first had to establish a model in which PAF synthesis by these cells could be detected. An in vitro assay was established, based on that of Salvemini et al. (1990), in which peritoneal mast cells were co-incubated with rabbit platelets. Stimulation of the mast cells with calcium ionophore caused release of a pro-aggregatory substance which comigrated with PAF on thinlayer chromatography, was inactivated by preincubation with phospholipase A2 or an antibody directed against PAF, and the effects of which could be inhibited by a specific PAF receptor antagonist.
Methods Animals Male, Sprague Dawley rats (300-400 g; Charles River Canada Inc., St. Constant, Canada) and male, New Zealand white
88
C.M. HOGABOAM et al.
rabbits (2-3 kg; Reimans Fur Ranches Ltd., St. Agatha, Canada) were used in this study. Rats were housed in groups of four at a temperature of 250C on a 12 h light/dark cycle. Rabbits were individually housed in standard cages under similar conditions. All animals had free access to both food and water prior to the beginning of the study. The following experimental procedures were approved by the Animal Care Committee of the University of Calgary and are in accordance with the guidelines of the Canadian Council on Animal Care.
Isolation of rat peritoneal mast cells Rats were anaesthetized with ether, then killed by cervical dislocation prior to intraperitoneal injection of 15 ml cold (40C) HEPES-buffered Tyrode solution (pH 7.2) containing 0.1% bovine serum albumin (BSA). The abdomen of the animal was massaged for 30s and then the peritoneal cavity was exposed through a ventral incision. The peritoneal fluid was aspirated and layered upon a two-step discontinuous Percoll gradient as described previously (Lee et al., 1985). The peritoneal mast cells (PMC; 97-99% pure, 97% viability) were resuspended in RPMI-HEPES solution (1 x 106ml-1) and kept on ice until used in the platelet aggregation assay.
Platelet aggregation bioassay Platelets were isolated from rabbit arterial blood (collected under pentobarbitone anaesthesia) and 'washed' in the presence of prostacyclin (300 ng ml- 1), as previously described by Radomski & Moncada (1983). Aliquots (500lu) of the plateletrich solution (PRS) were incubated in a cuvette at 370C for 1 min in a Payton dual-channel aggregometer while continuously stirred at 800 r.p.m. Light transmission through the suspension of platelets was recorded on a chart recorder. Aggregation of the platelets leads to an increase in light transmission through the cuvette. Prior to the start of the study, the PRS was tested for its responsiveness to synthetic PAF and a standard curve was established. PMC (1 x 103-5 x 104) were then added to the PRS and the cell suspension was allowed to equilibrate for 1 min. Calcium ionophore (A23187) was then added to the cells and the resulting aggregation was recorded on a Linear dual channel aggrecorder for 2 min. When added to a suspension of platelets in the absence of PMC, the concentrations of A23187 used (1-5 gM) failed to cause platelet aggregation and did not significantly affect the responsiveness of the platelets to thrombin (50-175 mu ml-1) or PAF (10-50 pg ml 1). Experiments of this type were repeated in 7 separate preparations of platelets. A number of experiments were performed to verify that the platelet aggregation was a consequence of release of PAF by the PMC. Firstly, in each assay, PMC (1 x 103 to 5 x 104) were added to platelets which had been pre-incubated for 2min with a specific PAF receptor antagonist (WEB 2086; 10-20 pM; Casals-Stenzel et al., 1987) and then stimulated as above. These concentrations of WEB 2086 did not significantly affect the aggregatory response of the platelets to thrombin, and totally prevented platelet aggregation induced by 100pgml1' of PAF. In a second series of experiments (n = 3), PMC (1 x 1031.25 x 105) were added to 0.5ml of 0.25% BSA/0.9% saline and stimulated with 5 pM A23187. One minute later, 1.0-ml of -20°C acetone was added to the PMC suspension. After a 5min centrifugation at 1000g, PAF was extracted in equal volumes of chloroform and methanol from both the supernatant and pellet and the organic phase from each sample was applied to thin-layer chromatography (t.l.c.) plates (Whatman silica gel 60A). TLC was then performed as described previously (Parente & Flower, 1985). The portion of the chromatograph co-migrating with authentic PAF was scraped and resuspended in 0.25% BSA/0.9% saline. Each sample was tested for platelet aggregating activity by the assay described above. Duplicate samples were incubated in the presence of phospholipase A2 (bovine pancreatic; 20,ugml-1) for 5min prior to testing in the platelet aggregation assay. Finally,
experiments (n = 2) were performed in which PMC (1 x 1035 x 104) were stimulated with A23187 (1-5pM). An aliquot of the PMC suspension was then added to a suspension of platelets in the platelet aggregometer and the response of the platelets was monitored. The experiments were then repeated, but immediately after challenging the PMC with A23187, lOl of an antibody directed against PAF was added to the suspension of PMC. The antibody was used at a dilution of 1:2000. Appropriate controls were performed to determine if the antibody itself or non-immune, heat-inactivated rabbit serum modified the responsiveness of the platelets to PAF.
Immunological stimulation of PAF synthesis by peritoneal mast cells In order to determine if PAF synthesis by PMC could be stimulated through immunological activation of the cells, PMC were harvested from rats infected > 15 days previously with Nippostrongylus brasiliensis, as described in detail previously (Befus & Bienenstock, 1979). Experiments were then performed in which the PMC were co-incubated with platelets, as described above, except that the mast cells were stimulated with either antigen (supernatant of the homogenate of 20 adult N. brasiliensis worms in saline) or anti-IgE (1/40 dilution of rabbit anti-rat IgE; Befus & Bienenstock, 1979). Control experiments were performed to determine if either of these stimuli affected the ability of the platelets to aggregate in response to exogenous PAF or thrombin. In addition, the ability of PMC from infected rats to release PAF in response to stimulation with A23187 (1-5juM) was compared to that from PMC from non-infected rats. The ability of the immunological stimuli to elicit histamine release from PMC was also assessed (see below).
Effects of various drugs on ionophore-stimulated PAF release by peritoneal mast cells Peritoneal mast cells (4 x 104) were incubated with various concentrations (0.1-1001uM) of one of the following compounds for 5min at 20'C: PF-5901, Wy-20,595, MK-571, A-64077 or MK-886 (Figure 1). PF-5901 and Wy-50,295 are inhibitors of 5-lipoxygenase activating protein (FLAP) and have LTD4 receptor antagonist properties (Van Inwegen et al., 1987; Kreft et al., 1990; Evans et al., 1991). MK-886 is also an inhibitor of FLAP (Rouzer et al., 1990). MK-571 is a potent LTD4 receptor antagonist (Jones et al., 1989). A-64077 is a 5-lipoxygenase inhibitor (Laursen et al., 1990). The cells were then added to fresh suspensions of washed platelets which had been incubated at 370C in the aggregometer for 1 min. The cells were then stimulated with an appropriate concentration of A23187, as above, and the response to this stimulation was recorded. The 5min incubation of PMC did not interfere with the ability of these cells to produce PAF, nor did the vehicles used for the various drugs (dimethylsulphoxide (DMSO) and methanol; final concentrations of 1%) alter PAF release by the PMC or the ability of platelets to aggregate in response to thrombin or PAF.
Histamine and prostaglandin D2 assays In order to determine whether compounds exhibiting inhibitory effect on PAF release by PMC also affected other responses of PMC, the effects of these compounds on histamine and prostaglandin D2 (PGD2) release were assessed. The PMC were isolated as outlined above and histamine release was assessed as described previously (Bissonnette & Befus, 1990). Briefly, PMC (5 x 104) were incubated for 5min at 20'C with PF-5901 or Wy-50,295 (1-10uM) or the appropriate vehicle. The PMC were then stimulated with A23187 (1 or 5 pM) and incubated for 1min. After incubation, the PMC were transferred to a tube containing 3 ml of HEPES-buffered Tyrode solution (pH 7.2) containing 0.1% BSA and centrifuged at 150 g for 3 min. The fractions were separated, and the
INHIBITION OF PAF SYNTHESIS BY QUINOLINES
a
89
Materials
b 0
CH3
C 0
/1 s~~~~~s
~sX"
cl
CO2Na
PF-5901 (a-pentyl-3-(2-quinolinylmethoxy)-benzene-methanol) was supplied by the Purdue Frederick Company (Norwalk, CT, U.S.A.). It should be noted that PF-5901 has previously been named 'REV-5901' and 'RG-5901'. The tromethamine salt of Wy-50,295 (S-(+)-a-methyl-6-(2-quinolinylmethoxy)-2-naphthaleneacetic acid was a gift from Wyeth-Ayerst Research (Princeton, NJ, U.S.A.). A-64077 (N(1-benzo(b)thien-2-ylethyl)-N-hydroxy-urea) was obtained from the R.W. Johnson Pharmaceutical Research Institute (Raritan, NJ, U.S.A.). MK-571 (3-(3-(2-(7-chloro-2quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxopropyl)thio)methyl)thio propanoic acid and MK-886 (3-[144chlorobenzyl)-3- t-butyl-thio-5- isopropylindol-2- yl]-2,2dimethyl propanoic acid) were supplied by Merck-Frosst Research Laboratories (Kirkland, Canada). Sodium prostacyclin was a gift from Burroughs-Wellcome Canada (Montreal, Canada). The prostacyclin was dissolved in 0.25 M Tris buffer (pH 8.4) and was stored in aliquots at -20'C. Solutions of PF-5901, Wy-50,295 and A-64077 were prepared in either DMSO and methanol for these studies. MK-571 was dissolved in 0.9% saline. WEB 2086 was a gift from Boehringer Ingelheim (Germany). The rabbit anti-PAF antibody was obtained from Amersham Canada (TRK 990; Oakville, Canada) and was used at a dilution of 1:2000 in phosphate buffered saline (pH 7.4). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.) or Fisher Scientific (Edmonton, Canada).
d
Results
Characterization of PAF release by peritoneal mast cells Nr-C KeNH2
Co2H F
C'
Figure 1 Chemical structures of the pharmacological compounds used in this study: (a) PF-5901; (b) Wy-50,295, (c) MK-571, (d) A64077, and (e) MK-886.
pellet fractions were boiled for 10 min to release the remaining cell-associated histamine. Following protein precipitation with 0.25% trichloroacetic acid, histamine concentrations were
determined in the supernatants and pellets by a fluorometric assay (Shore et al., 1959). Histamine release into the supernatant was expressed as a percentage of total cellular histamine content less the spontaneous background histamine release ( < 2% of total histamine). For PGD2,I100ul aliquots containing 3 x 105 PMC were added to 400 pl of HEPES-buffered Tyrode solution (pH 7.2). The cells were incubated for 5min with vehicle, PF-5901 (1 or 10pM) or Wy-50,295 (1 or 10pM) and were then stimulated with 4 fUM A23187. After a 10min incubation in a shaking water bath (370C), the samples were centrifuged at 200 g (8min; 40C). The supernatants were aspirated and the pellets resuspended in 500pl of the HEPES-buffered Tyrode solution and sonicated for 1 min. PGD2 concentrations in the samples of supernatants and pellets were determined with a commercially available ELISA (Caymen).
PMC co-incubated with washed rabbit platelets and stimulated with A23187 released a pro-aggregatory substance within 10-20 s (Figure 2). Maximal platelet aggregation was normally seen within 2-3 min following stimulation of the PMC, but the amount of platelet aggregation was dependent upon the number of PMC added to the platelet suspension. When compared to a standard curve constructed by testing the effects of synthetic PAF on platelets, the PMC produced the equivalent of 20.4 + 9.2 pg PAF per 1 x 104 cells (mean of 7 separate aggregation assays). The pro-aggregatory activity of ionophore-stimulated PMC was not the result of effects of A23187 on the platelets, since the ionophore did not stimulate platelet aggregation in the absence of PMC, nor did it affect the responsiveness of platelets to PAF or thrombin. There are several lines of evidence to support the hypothesis that the aggregation of platelets observed following stimulation of the PMC/platelet suspension with ionophore was attributable to the release of PAF by the PMC. Firstly, in each assay, this activity could be completely inhibited by preincubating the platelets with WEB 2086. Secondly, the proaggregatory activity released from PMC in response to ionophore was found to co-migrate with synthetic PAF on thin-layer chromatography and could be eliminated by preincubating the samples with 20jugml1 phospholipase A2 or excess amounts of antibody directed against PAF (n = 3). Interestingly, all of the platelet activating substance produced by the PMC could be recovered in the supernatant (i.e. extracts of the pelleted PMC contained no detectable platelet activating activity). PAF synthesis in response to immunological stimulation
Tonophore-stimulated PAF release from PMC harvested from previously infected with N. brasiliensis (24.4 + 3.5 pg/104 cells) did not significantly differ from that observed from PMC harvested from non-infected rats (20.4 + 9.2 pg/104 cells). Stimulation of sensitized PMC with antigen resulted in a mean release of 2.6 + 1.1 pg/104 cells, markedly less than was observed with ionophore stimulation. However, this amount of antigen also stimulated markedly less histamine release by rats
C.M. HOGABOAM et al.
90
b
a 1
c
1 2
d
e
12
1 2
44i ° 80 U.
±
60-
00
C40-
nc 0
3 min Figure 2 Representative platelet aggregometry tracings. Light transmission through a suspension of platelets was recorded. A downward deflection of the pen is indicative of increased light transmission, which occurs when the platelets aggregate. In (a) stimulation (arrow 1) of platelets with A23187 (1.5 pM) failed to elicit aggregation. In (b) and (c), suspensions of peritoneal mast cells (2 x 104 and 5 x 104, respectively) were added to the platelet suspension at arrow 1. Stimulation (arrow 2) with A23187 (1.5 UM) resulted in platelet aggregation, the magnitude of which was related to the number of mast cells added. In (d), the protocol was identical to that in (b), except that the mast cells were preincubated with PF-5901 (10pM) for 5min before being added to the platelet suspension (mast cells added at arrow 1, A23187 added at arrow 2). In (e) the protocol was identical to that of panel (b), except that the platelets were preincubated with WEB 2086 (10pM) for 1 min before the addition of mast cells (arrow 1); A23187 (1.5 pM) was added at arrow 2.
PMC than did ionophore. Antigen stimulation (supernatant from 20 worm equivalents) resulted in a specific release of histamine of 9 + 2%, while a 1 fM concentration of A23187 stimulated specific histamine release of 34 + 3%. Unfortunately, higher concentrations of the antigen (i.e. > 20 worm equivalents), as well as anti-IgE, interfered with the responsiveness of the platelets to exogenous PAF.
Pharmacological modulation of PAF synthesis by peritoneal mast cells Preincubation of PMC with either Wy-50,295 or PF-5901, both of which are quinoline-based FLAP inhibitors, resulted in a concentration-dependent inhibition of PAF synthesis by ionophore-stimulated PMC (Figure 3). The IC50 of Wy-50,295 was 1.2 uM, while that of PF-5901 was 3.9gM. Conversely, a structurally dissimilar 5-lipoxygenase inhibitor, A-64077, had no significant effect on PAF synthesis by PMC at concentrations of up to 100puM (Figure 3). MK-571, a quinoline-based LTD4 antagonist, also did not affect PAF release by PMC when tested at concentrations of up to 1000,M (Figure 3). Furthermore, MK-886, an inhibitor of FLAP which is not quinoline-based, failed to affect significantly synthesis of PAF by PMC at concentrations of up to 100pM (data not shown). None of the compounds tested significantly affected the responsiveness of platelets to stimulation by synthetic PAF or thrombin.
Histamine and prostaglandin D2 releasefrom stimulated peritoneal mast cells Stimulation of 5 x 104 PMC with A23187 (1 or 5pM) resulted in the specific release of 34 + 3% and 42 + 3% of total cellular histamine content, respectively. Preincubation of PMC with PF-5901 or Wy-50,295 at concentrations of 1 or 1OpM
-7
*TS I. .0-#95%. The effects of PF-5901 and Wy-50,295 on PAF release by rat peritoneal mast cells may have occurred as a consequence of a non-specific inhibition of mast cell activation. To test this hypothesis, the effects of these compounds on ionophorestimulated histamine and PGD2 release were assessed. The observation that concentrations of these two compounds which markedly inhibited PAF release failed to affect significantly the release of these two mediators suggests a more specific mechanism of action. The precise nature of that
INHIBITION OF PAF SYNTHESIS BY QUINOLINES
mechanism has yet to be established. The present results are in agreement with those of Warner et al. (1988) who, using human lung mast cells, demonstrated that a 5-lipoxygenase inhibitor (L651,392) failed to modify histamine release. The ability of PF-5901 and Wy-50,295 to inhibit PAF release by mast cells can also be distinguished from the previously reported ability of ketotifen to inhibit PAF synthesis by mouse bone marrow-derived mast cells (Joly et al., 1987). In that study, ketotifen was also found to inhibit the release of fi-hexosaminidase, a preformed granular enzyme. Release of fl-hexosaminidase has been shown to correlate well with histamine release (Mencia-Huerta et al., 1983). PAF synthesis by mast cells from various sources has been demonstrated previously (Mencia-Huerta et al., 1983; Schleimer et al., 1986; Musch et al., 1987; Triggiani et al., 1990). A striking feature of the present study was the rapidity with which PAF is released following stimulation of peritoneal mast cells. PAF release was detectable within seconds of stimulation with ionophore. The quantity of PAF released in response to ionophore challenge was also significant. Indeed, the amount of PAF released from rat peritoneal mast cells (-2ng per million cells) was an order of magnitude lower than the levels of prostaglandin D2 released by these cells in response to stimulation with ionophore. The concentrations of ionophore required to elicit this amount of PAF release from PMC was sufficient to induce specific histamine release in the 34-42% range. The mechanism through which PF-5901 and Wy-50,295 inhibit PAF synthesis is not known. Since these compounds do not interfere with ionophore-stimulated histamine or PGD2 release, it is unlikely that they significantly modify the calcium influx in response to this challenge. It is conceivable
91
that these compounds inhibit the activity of one of the two enzymes involved in the biosynthesis of PAF, namely phospholipase A2 and acetyl CoA transferase. However, since neither of these compounds affected PGD2 release in the present study, or exerted effects on prostaglandin synthesis in various other systems (Van Inwegen et al., 1987; Kreft et al., 1990), effects on phospholipase A2 seem unlikely. The possibility that PF-5901 and Wy-50,295 alter acetyl CoA transferase activity requires further investigation. Since these two compounds inhibit leukotriene synthesis by binding to FLAP (Evans et al., 1991), the extent to which inhibition of PAF synthesis can be extended to other FLAP inhibitors is of interest. However, MK-886, the prototype FLAP inhibitor, failed to affect significantly PAF synthesis by PMC in the present study. In a preliminary report, however, Lock et al. (1990) showed that MK-886 inhibited PAF synthesis by human neutrophils, albeit at concentrations well above those required for inhibition of leukotriene synthesis. In summary, two quinoline-based compounds were found to inhibit PAF release by peritoneal mast cells at concentrations in the micromolar range. These compounds may therefore be useful pharmacological probes for investigations of the role of PAF in various inflammatory and allergic conditions. Moreover, inhibition of PAF release may contribute to the anti-inflammatory effects of these compounds. This work was supported by grants from the Medical Research Council of Canada (MRC), Purdue Frederick Inc. and the Alberta Heritage Foundation for Medical Research (AHFMR). C.M.H. is supported by an AHFMR Studentship and E.Y.B. by an AHFMR Fellowship. A.D.B. is an AHFMR Medical Scholar. J.L.W. is an AHFMR Medical Scholar and MRC Scientist.
References BEFUS, A.D. & BIENENSTOCK, J. (1979). Immunologically mediated
LAURSEN, L.S., NAESDAL, J., BUKHAVE, K., LAURITSEN, K. & RASK-
intestinal mastocytosis in Nippostrongulus brasiliensis-infected rats. Immunol., 38, 95-103. BISSONNETTE, E.Y. & BEFUS, A.D. (1990). Inhibition of mast cellmediated cytotoxicity by IFN-ax/, and Y. J. Immunol., 145, 33853390. BRAQUET, P., TOUQUI, L., SHEN, T.Y. & VARGAFTIG, B. (1987). Perspectives in platelet-activating factor research. Pharmacol. Rev., 39, 97-145. CASALS-STENZEL, J., MUACEVIC, G. & WEBER, K-H. (1987). Pharmacological actions of WEB 2086, a specific antagonist of plateletactivating factor. J. Pharmacol. Exp. Ther., 241, 971-981. CHAND, N., DIAMANTIS, W., PILLAR, J. & SOFIA, R.D. (1987). Modulation of allergic and nonallergic histamine secretion by lipoxygenase inhibitors. Res. Commun. Chem. Pathol. Pharmacol., 55, 49-57.
MASDEN, J. (1990). Selective 5-lipoxygenase inhibition in ulcerative colitis. Lancet, 335, 683-685.
EVANS, J.F., LEVEILLE, C., MANCINI, J.A., PRASIT, P., THERIEN, T., ZAMBONI, R., GAUTHIER, J.Y., FORTIN, R., CHARLESON, P., MACINTYRE, D.E., LUELL, S., BACH, T.J., MEURER, R., GUAY, J., VICKERS, P.J., ROUZER, C.A., GILLARD, J.W. & MILLER, D.K.
(1991). 5-lipoxygenase-activating protein is the target of a quinoline class of leukotriene synthesis inhibitors. Mol. Pharmacol., 40, 22-27. GORDON, J.R., BURD, P.R. & GALLI, S.J. (1990). Mast cells as a source of multifunctional cytokines. Immunol. Today, 11, 458-464. HEAVEY, D.J., ERNST, P.B., STEVENS, R.L., BEFUS, A.D., BIENENSTOCK, J. & AUSTEN, K.F. (1988). Generation of leukotriene C4,
leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells. J. Immunol., 140, 1953-1957. JOLY, F., BESSOU, G., BENVENISTE, J. & NINIO, E. (1987). Ketotifen inhibits paf-acether biosynthesis and fl-hexosaminidase release in mouse mast cells stimulated with antigen. Eur. J. Pharmacol., 144, 133-139. JONES, T.R., ZAMBONI, R., BELLEY, M., CHAMPION, E., CHARETTE, L., FORD-HUTCHINSON, A.W., FRENETTE, R., GAUTHIER, J.-Y., LEGER, S., MASSON, P., McFARLANE, C.S., PIECHUTA, H., ROKACH, J., WILLIAMS, H., YOUNG, R.N., DeHAVEN, R.N. & PONG, S.S. (1989). Pharmacology of L-660,711 (MK-571): a novel
potent and selective leukotriene D4 receptor antagonist. Can. J. Physiol. Pharmacol., 67, 17-28. KREFT, A., MUSSER, J., MARSHALL, L. & GRIMES, D. (1990). Wy-50,295 tromethamine. Drugs Future, 15, 805-807.
LEE, T.D.G., SHANAHAN, F., MILLER, H.R.P., BIENENSTOCK, J. &
BEFUS, A.D. (1985). Intestinal mucosal mast cells: isolation from rat lamina propria and purification using unit gravity velocity sedimentation. Immunology, 55, 721-728. LOCK, T.Y., CHANG, J.Y. & GLASER, K.B. (1990). Inhibition of PAF and LTB4 biosynthesis in human neutrophils by putative PLA2 and 5-lipoxygenase inhibitors. Proceedings of 5th Inflammation Research Association Meeting, 75. LYNCH, J.M. & HENSON, P.M. (1986). The intracellular retention of newly-synthesized platelet-activating factor. J. Immunol., 137, 2653-2661. MANSINI, E., GIANNELLA, E., BANI-SACCHI, T., FANTOZZI, R., PAL-
MERANI, B. & MANNAIONI, P.F. (1987). Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism. Agents Actions, 20, 202-205. MENCIA-HUERTA, J-M., LEWIS, R.A., RAZIN, E. & AUSTEN, K.F.
(1983). Antigen-initiated release of platelet-activating factor (PAFacether) from mouse bone marrow-derived mast cells sensitized with monoclonal Ige. J. Immunol., 131, 2958-2964. MUSCH, M.W., BILLAH, M.M. & SIEGEL, M.I. (1987). Antigen- and ionophore-stimulated synthesis of platelet-activating factor by the cloned mast cell line, MC9. Biochem. Biophys. Res. Commun., 144, 1243-1250. PARENTE, L. & FLOWER, R.J. (1985). Hydrocortisone and macrocortin inhibit the zymosan-induced release of lyso-PAF from rat peritoneal leukocytes. Life Sci., 36, 1225-1231. RADOMSKI, M. & MONCADA, S. (1983). An improved method for washing human platelets with prostacyclin. Thromb. Res., 30, 383389. ROUZER, C.A., FORD-HUTCHINSON, A.W.,
MORTON, H.E. &
GILLARD, J.W. (1990). MK-886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes. J. Biol. Chem., 265, 1436-1442. SALVEMINI, D., MASINI, E., ANGGARD, E., MANNAIONI, P.F. &
VANE, J.R. (1990). Synthesis of a nitric oxide-like factor from Larginine by rat serosal mast cells: stimulation of guanylate cyclase an inhibition of platelet aggregation. Biochem. Biophys. Res. Commun., 169, 596-601.
92
C.M. HOGABOAM et al.
SCHLEIMER, R.P., MacGLASHAN Jr., D.W., PETERS, S.P., PINCKARD, R.N., ADKINSON Jr., N.F. & LICHTENSTEIN, L.M. (1986). Charac-
terization of inflammatory mediator release from purified human lung mast cells. Am. Rev. Respir. Dis., 133, 614-617. SHORE, P.A., BURKALTER, A. & COHN, V.H. (1959). A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther., 127, 182-186. TRIGGIANI, M., HUBBARD, W.C. & CHILTON, F.H. (1990). Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by an enriched preparation of the human lung mast cell. J. Immunol., 144, 4773-4780. VAN INWEGEN, R.G., KHANDWALA, A., GORDON, R., SONNINO, P.,
COUTTS, S. & JOLLY, S. (1987). REV-5901: an orally effective peptidoleukotriene antagonist, detailed biochemical/pharmacological profile. J. Pharmacol. Exp. Ther., 241, 118-124. WARNER, J.A., LICHTENSTEIN, L.M. & MAGGLASHAN, W. (1988). Effects of specific inhibitor of 5-lipoxygenase pathway on mediator release from human basophils and mast cells. J. Pharmacol. Exp. Ther., 147, 218-222. ZIMMERMAN, G.A., McINTYRE, T.M., MEHRA, M. & PRESCOTT, S.M.
(1990). Endothelial cell-associated platelet-activating factor: a novel mechanism for signalling intercellular adhesion. J. Cell Biol., 110, 529-540.
(Received May 17, 1991 Revised September 13, 1991 Accepted September 17, 1991)
C Macmillan Press Ltd, 1991
Platelet-activating factor synthesis by peritoneal mast cells and its inhibition by two quinoline-based compounds Cory M. Hogaboam, *Donna Donigi-Gale, *T. Scott Shoupe, Elyse Y. Bissonnette, A. Dean Befus & 'John L. Wallace Gastrointestinal and Immunological Sciences Research Groups, University of Calgary, Calgary, Alberta, Canada and *International Molecular Discovery, Purdue Frederick Company, Norwalk, Connecticut, U.S.A. 1 Peritoneal mast cells from rat were co-incubated in vitro in a platelet aggregometer cuvette with washed rabbit platelets. In response to stimulation with calcium ionophore (A23187; 1-5 ,UM), the mast cells released a substance which stimulated the platelets to aggregate. These concentrations of ionophore did not stimulate platelet aggregation in the absence of mast cells, nor affect the responsiveness of the platelets to aggregation induced by thrombin or PAF. Release of a PAF-like substance was also observed in response to stimulation of the mast cells with antigen. 2 This pro-aggregatory activity is attributable to the release of PAF by the mast cells, since the activity could be abolished by preincubating the platelets with a specific PAF receptor antagonist (WEB 2086; 10M). Furthermore, the platelet-aggregating factor co-migrated with PAF on thin-layer chromatographs and could be abolished by incubation with phospholipase A2 (20,ugml-1) or a specific antibody directed against PAF. 3 The release of PAF by peritoneal mast cells could be inhibited, in a concentration-dependent manner, by PF-5901 (IC50 of 3.91M) or Wy-50,295 (IC50 of 1.21uM), two structurally similar compounds with inhibitory effects on leukotriene synthesis, as well as leukotriene D4 (LTD4) receptor antagonist properties.
4 Inhibition of PAF synthesis was not observed when the mast cells were incubated with a structurally unrelated 5-lipoxygenase inhibitor (A-64077), a structurally dissimilar inhibitor of 5-lipoxygenase activating protein (MK-886) or with a structurally related LTD4 receptor antagonist (MK-571) which lacks inhibitory effects on leukotriene synthesis, each at concentrations of up to 100pM. 5 Neither PF-5901 nor Wy-50,295 (1 or 10pM) significantly affected histamine release or prostaglandin D2 synthesis by peritoneal mast cells in response to calcium ionophore stimulation. 6 These results demonstrate the ability of a class of quinoline-based compounds to inhibit PAF synthesis by peritoneal mast cells. This activity does not appear to be related to effects of these compounds on leukotriene synthesis or LTD4 receptors. The ability of these compounds to inhibit PAF synthesis may contribute to their anti-inflammatory properties. Keywords: PAF; mast cells; leukotrienes; inflammation; anti-inflammatory; allergy; quinolines
Introduction Inflammatory mediator release from activated mast cells represents an important early event in many disease conditions associated with chronic inflammation (Gordon et al., 1990). Because of this, the mast cell and the mediators these cells release have been identified as potential targets for the treatment of inflammatory conditions and allergic reactions. One mediator which has been implicated in a variety of inflammatory disorders is platelet-activating factor, or PAF (reviewed by Braquet et al., 1987). Such a role for PAF has been supported by numerous studies demonstrating that treatment with PAF antagonists is beneficial in a variety of animal models of inflammation and allergy. While the biosynthesis of PAF has been studied extensively, potent and specific inhibitors have not been identified. Ketotifen has been shown to inhibit PAF release from mouse mast cells, but similar concentrations of this compound also inhibited calcium influx and release of fi-hexosaminidase (Joly et al., 1987). Mast cells derived from a variety of sources synthesize PAF (Mencia-Heurta et al., 1983; Schleimer et al., 1986; Musch et al., 1987; Triggiani et al., 1990), but little is known of the regulation of PAF synthesis and release by these cells. Products of the lipoxygenase pathway of arachidonic acid metabolism appear to play a role in modulating histamine release by rat peritoneal mast cells (Chand et al., 1987; Mansini et al., 1987), although some caution must be exercised in accepting these 1 Author for correspondence at: Department of Medical Physiology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada.
results, since the lipoxygenase inhibitors used in these studies (e.g. nordihydroguaretic acid) are known to affect other enzyme systems. Furthermore, there is no direct evidence that rat peritoneal mast cells are capable of producing 5lipoxygenase products of arachidonic acid. Heavy et al. (1988) were unable to detect leukotriene B4 (LTB4) or LTC4 release from rat peritoneal mast cells stimulated with anti-IgE antibody, while intestinal mucosal mast cells released significant quantities of LTB4 and LTC4 in response to similar stimulation. The present study was performed to determine if quinolinebased inhibitors of leukotriene synthesis affect PAF synthesis by rat peritoneal (connective tissue-type) mast cells. In order to do these studies, we first had to establish a model in which PAF synthesis by these cells could be detected. An in vitro assay was established, based on that of Salvemini et al. (1990), in which peritoneal mast cells were co-incubated with rabbit platelets. Stimulation of the mast cells with calcium ionophore caused release of a pro-aggregatory substance which comigrated with PAF on thinlayer chromatography, was inactivated by preincubation with phospholipase A2 or an antibody directed against PAF, and the effects of which could be inhibited by a specific PAF receptor antagonist.
Methods Animals Male, Sprague Dawley rats (300-400 g; Charles River Canada Inc., St. Constant, Canada) and male, New Zealand white
88
C.M. HOGABOAM et al.
rabbits (2-3 kg; Reimans Fur Ranches Ltd., St. Agatha, Canada) were used in this study. Rats were housed in groups of four at a temperature of 250C on a 12 h light/dark cycle. Rabbits were individually housed in standard cages under similar conditions. All animals had free access to both food and water prior to the beginning of the study. The following experimental procedures were approved by the Animal Care Committee of the University of Calgary and are in accordance with the guidelines of the Canadian Council on Animal Care.
Isolation of rat peritoneal mast cells Rats were anaesthetized with ether, then killed by cervical dislocation prior to intraperitoneal injection of 15 ml cold (40C) HEPES-buffered Tyrode solution (pH 7.2) containing 0.1% bovine serum albumin (BSA). The abdomen of the animal was massaged for 30s and then the peritoneal cavity was exposed through a ventral incision. The peritoneal fluid was aspirated and layered upon a two-step discontinuous Percoll gradient as described previously (Lee et al., 1985). The peritoneal mast cells (PMC; 97-99% pure, 97% viability) were resuspended in RPMI-HEPES solution (1 x 106ml-1) and kept on ice until used in the platelet aggregation assay.
Platelet aggregation bioassay Platelets were isolated from rabbit arterial blood (collected under pentobarbitone anaesthesia) and 'washed' in the presence of prostacyclin (300 ng ml- 1), as previously described by Radomski & Moncada (1983). Aliquots (500lu) of the plateletrich solution (PRS) were incubated in a cuvette at 370C for 1 min in a Payton dual-channel aggregometer while continuously stirred at 800 r.p.m. Light transmission through the suspension of platelets was recorded on a chart recorder. Aggregation of the platelets leads to an increase in light transmission through the cuvette. Prior to the start of the study, the PRS was tested for its responsiveness to synthetic PAF and a standard curve was established. PMC (1 x 103-5 x 104) were then added to the PRS and the cell suspension was allowed to equilibrate for 1 min. Calcium ionophore (A23187) was then added to the cells and the resulting aggregation was recorded on a Linear dual channel aggrecorder for 2 min. When added to a suspension of platelets in the absence of PMC, the concentrations of A23187 used (1-5 gM) failed to cause platelet aggregation and did not significantly affect the responsiveness of the platelets to thrombin (50-175 mu ml-1) or PAF (10-50 pg ml 1). Experiments of this type were repeated in 7 separate preparations of platelets. A number of experiments were performed to verify that the platelet aggregation was a consequence of release of PAF by the PMC. Firstly, in each assay, PMC (1 x 103 to 5 x 104) were added to platelets which had been pre-incubated for 2min with a specific PAF receptor antagonist (WEB 2086; 10-20 pM; Casals-Stenzel et al., 1987) and then stimulated as above. These concentrations of WEB 2086 did not significantly affect the aggregatory response of the platelets to thrombin, and totally prevented platelet aggregation induced by 100pgml1' of PAF. In a second series of experiments (n = 3), PMC (1 x 1031.25 x 105) were added to 0.5ml of 0.25% BSA/0.9% saline and stimulated with 5 pM A23187. One minute later, 1.0-ml of -20°C acetone was added to the PMC suspension. After a 5min centrifugation at 1000g, PAF was extracted in equal volumes of chloroform and methanol from both the supernatant and pellet and the organic phase from each sample was applied to thin-layer chromatography (t.l.c.) plates (Whatman silica gel 60A). TLC was then performed as described previously (Parente & Flower, 1985). The portion of the chromatograph co-migrating with authentic PAF was scraped and resuspended in 0.25% BSA/0.9% saline. Each sample was tested for platelet aggregating activity by the assay described above. Duplicate samples were incubated in the presence of phospholipase A2 (bovine pancreatic; 20,ugml-1) for 5min prior to testing in the platelet aggregation assay. Finally,
experiments (n = 2) were performed in which PMC (1 x 1035 x 104) were stimulated with A23187 (1-5pM). An aliquot of the PMC suspension was then added to a suspension of platelets in the platelet aggregometer and the response of the platelets was monitored. The experiments were then repeated, but immediately after challenging the PMC with A23187, lOl of an antibody directed against PAF was added to the suspension of PMC. The antibody was used at a dilution of 1:2000. Appropriate controls were performed to determine if the antibody itself or non-immune, heat-inactivated rabbit serum modified the responsiveness of the platelets to PAF.
Immunological stimulation of PAF synthesis by peritoneal mast cells In order to determine if PAF synthesis by PMC could be stimulated through immunological activation of the cells, PMC were harvested from rats infected > 15 days previously with Nippostrongylus brasiliensis, as described in detail previously (Befus & Bienenstock, 1979). Experiments were then performed in which the PMC were co-incubated with platelets, as described above, except that the mast cells were stimulated with either antigen (supernatant of the homogenate of 20 adult N. brasiliensis worms in saline) or anti-IgE (1/40 dilution of rabbit anti-rat IgE; Befus & Bienenstock, 1979). Control experiments were performed to determine if either of these stimuli affected the ability of the platelets to aggregate in response to exogenous PAF or thrombin. In addition, the ability of PMC from infected rats to release PAF in response to stimulation with A23187 (1-5juM) was compared to that from PMC from non-infected rats. The ability of the immunological stimuli to elicit histamine release from PMC was also assessed (see below).
Effects of various drugs on ionophore-stimulated PAF release by peritoneal mast cells Peritoneal mast cells (4 x 104) were incubated with various concentrations (0.1-1001uM) of one of the following compounds for 5min at 20'C: PF-5901, Wy-20,595, MK-571, A-64077 or MK-886 (Figure 1). PF-5901 and Wy-50,295 are inhibitors of 5-lipoxygenase activating protein (FLAP) and have LTD4 receptor antagonist properties (Van Inwegen et al., 1987; Kreft et al., 1990; Evans et al., 1991). MK-886 is also an inhibitor of FLAP (Rouzer et al., 1990). MK-571 is a potent LTD4 receptor antagonist (Jones et al., 1989). A-64077 is a 5-lipoxygenase inhibitor (Laursen et al., 1990). The cells were then added to fresh suspensions of washed platelets which had been incubated at 370C in the aggregometer for 1 min. The cells were then stimulated with an appropriate concentration of A23187, as above, and the response to this stimulation was recorded. The 5min incubation of PMC did not interfere with the ability of these cells to produce PAF, nor did the vehicles used for the various drugs (dimethylsulphoxide (DMSO) and methanol; final concentrations of 1%) alter PAF release by the PMC or the ability of platelets to aggregate in response to thrombin or PAF.
Histamine and prostaglandin D2 assays In order to determine whether compounds exhibiting inhibitory effect on PAF release by PMC also affected other responses of PMC, the effects of these compounds on histamine and prostaglandin D2 (PGD2) release were assessed. The PMC were isolated as outlined above and histamine release was assessed as described previously (Bissonnette & Befus, 1990). Briefly, PMC (5 x 104) were incubated for 5min at 20'C with PF-5901 or Wy-50,295 (1-10uM) or the appropriate vehicle. The PMC were then stimulated with A23187 (1 or 5 pM) and incubated for 1min. After incubation, the PMC were transferred to a tube containing 3 ml of HEPES-buffered Tyrode solution (pH 7.2) containing 0.1% BSA and centrifuged at 150 g for 3 min. The fractions were separated, and the
INHIBITION OF PAF SYNTHESIS BY QUINOLINES
a
89
Materials
b 0
CH3
C 0
/1 s~~~~~s
~sX"
cl
CO2Na
PF-5901 (a-pentyl-3-(2-quinolinylmethoxy)-benzene-methanol) was supplied by the Purdue Frederick Company (Norwalk, CT, U.S.A.). It should be noted that PF-5901 has previously been named 'REV-5901' and 'RG-5901'. The tromethamine salt of Wy-50,295 (S-(+)-a-methyl-6-(2-quinolinylmethoxy)-2-naphthaleneacetic acid was a gift from Wyeth-Ayerst Research (Princeton, NJ, U.S.A.). A-64077 (N(1-benzo(b)thien-2-ylethyl)-N-hydroxy-urea) was obtained from the R.W. Johnson Pharmaceutical Research Institute (Raritan, NJ, U.S.A.). MK-571 (3-(3-(2-(7-chloro-2quinolinyl)ethenyl)phenyl) ((3-dimethyl amino-3-oxopropyl)thio)methyl)thio propanoic acid and MK-886 (3-[144chlorobenzyl)-3- t-butyl-thio-5- isopropylindol-2- yl]-2,2dimethyl propanoic acid) were supplied by Merck-Frosst Research Laboratories (Kirkland, Canada). Sodium prostacyclin was a gift from Burroughs-Wellcome Canada (Montreal, Canada). The prostacyclin was dissolved in 0.25 M Tris buffer (pH 8.4) and was stored in aliquots at -20'C. Solutions of PF-5901, Wy-50,295 and A-64077 were prepared in either DMSO and methanol for these studies. MK-571 was dissolved in 0.9% saline. WEB 2086 was a gift from Boehringer Ingelheim (Germany). The rabbit anti-PAF antibody was obtained from Amersham Canada (TRK 990; Oakville, Canada) and was used at a dilution of 1:2000 in phosphate buffered saline (pH 7.4). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.) or Fisher Scientific (Edmonton, Canada).
d
Results
Characterization of PAF release by peritoneal mast cells Nr-C KeNH2
Co2H F
C'
Figure 1 Chemical structures of the pharmacological compounds used in this study: (a) PF-5901; (b) Wy-50,295, (c) MK-571, (d) A64077, and (e) MK-886.
pellet fractions were boiled for 10 min to release the remaining cell-associated histamine. Following protein precipitation with 0.25% trichloroacetic acid, histamine concentrations were
determined in the supernatants and pellets by a fluorometric assay (Shore et al., 1959). Histamine release into the supernatant was expressed as a percentage of total cellular histamine content less the spontaneous background histamine release ( < 2% of total histamine). For PGD2,I100ul aliquots containing 3 x 105 PMC were added to 400 pl of HEPES-buffered Tyrode solution (pH 7.2). The cells were incubated for 5min with vehicle, PF-5901 (1 or 10pM) or Wy-50,295 (1 or 10pM) and were then stimulated with 4 fUM A23187. After a 10min incubation in a shaking water bath (370C), the samples were centrifuged at 200 g (8min; 40C). The supernatants were aspirated and the pellets resuspended in 500pl of the HEPES-buffered Tyrode solution and sonicated for 1 min. PGD2 concentrations in the samples of supernatants and pellets were determined with a commercially available ELISA (Caymen).
PMC co-incubated with washed rabbit platelets and stimulated with A23187 released a pro-aggregatory substance within 10-20 s (Figure 2). Maximal platelet aggregation was normally seen within 2-3 min following stimulation of the PMC, but the amount of platelet aggregation was dependent upon the number of PMC added to the platelet suspension. When compared to a standard curve constructed by testing the effects of synthetic PAF on platelets, the PMC produced the equivalent of 20.4 + 9.2 pg PAF per 1 x 104 cells (mean of 7 separate aggregation assays). The pro-aggregatory activity of ionophore-stimulated PMC was not the result of effects of A23187 on the platelets, since the ionophore did not stimulate platelet aggregation in the absence of PMC, nor did it affect the responsiveness of platelets to PAF or thrombin. There are several lines of evidence to support the hypothesis that the aggregation of platelets observed following stimulation of the PMC/platelet suspension with ionophore was attributable to the release of PAF by the PMC. Firstly, in each assay, this activity could be completely inhibited by preincubating the platelets with WEB 2086. Secondly, the proaggregatory activity released from PMC in response to ionophore was found to co-migrate with synthetic PAF on thin-layer chromatography and could be eliminated by preincubating the samples with 20jugml1 phospholipase A2 or excess amounts of antibody directed against PAF (n = 3). Interestingly, all of the platelet activating substance produced by the PMC could be recovered in the supernatant (i.e. extracts of the pelleted PMC contained no detectable platelet activating activity). PAF synthesis in response to immunological stimulation
Tonophore-stimulated PAF release from PMC harvested from previously infected with N. brasiliensis (24.4 + 3.5 pg/104 cells) did not significantly differ from that observed from PMC harvested from non-infected rats (20.4 + 9.2 pg/104 cells). Stimulation of sensitized PMC with antigen resulted in a mean release of 2.6 + 1.1 pg/104 cells, markedly less than was observed with ionophore stimulation. However, this amount of antigen also stimulated markedly less histamine release by rats
C.M. HOGABOAM et al.
90
b
a 1
c
1 2
d
e
12
1 2
44i ° 80 U.
±
60-
00
C40-
nc 0
3 min Figure 2 Representative platelet aggregometry tracings. Light transmission through a suspension of platelets was recorded. A downward deflection of the pen is indicative of increased light transmission, which occurs when the platelets aggregate. In (a) stimulation (arrow 1) of platelets with A23187 (1.5 pM) failed to elicit aggregation. In (b) and (c), suspensions of peritoneal mast cells (2 x 104 and 5 x 104, respectively) were added to the platelet suspension at arrow 1. Stimulation (arrow 2) with A23187 (1.5 UM) resulted in platelet aggregation, the magnitude of which was related to the number of mast cells added. In (d), the protocol was identical to that in (b), except that the mast cells were preincubated with PF-5901 (10pM) for 5min before being added to the platelet suspension (mast cells added at arrow 1, A23187 added at arrow 2). In (e) the protocol was identical to that of panel (b), except that the platelets were preincubated with WEB 2086 (10pM) for 1 min before the addition of mast cells (arrow 1); A23187 (1.5 pM) was added at arrow 2.
PMC than did ionophore. Antigen stimulation (supernatant from 20 worm equivalents) resulted in a specific release of histamine of 9 + 2%, while a 1 fM concentration of A23187 stimulated specific histamine release of 34 + 3%. Unfortunately, higher concentrations of the antigen (i.e. > 20 worm equivalents), as well as anti-IgE, interfered with the responsiveness of the platelets to exogenous PAF.
Pharmacological modulation of PAF synthesis by peritoneal mast cells Preincubation of PMC with either Wy-50,295 or PF-5901, both of which are quinoline-based FLAP inhibitors, resulted in a concentration-dependent inhibition of PAF synthesis by ionophore-stimulated PMC (Figure 3). The IC50 of Wy-50,295 was 1.2 uM, while that of PF-5901 was 3.9gM. Conversely, a structurally dissimilar 5-lipoxygenase inhibitor, A-64077, had no significant effect on PAF synthesis by PMC at concentrations of up to 100puM (Figure 3). MK-571, a quinoline-based LTD4 antagonist, also did not affect PAF release by PMC when tested at concentrations of up to 1000,M (Figure 3). Furthermore, MK-886, an inhibitor of FLAP which is not quinoline-based, failed to affect significantly synthesis of PAF by PMC at concentrations of up to 100pM (data not shown). None of the compounds tested significantly affected the responsiveness of platelets to stimulation by synthetic PAF or thrombin.
Histamine and prostaglandin D2 releasefrom stimulated peritoneal mast cells Stimulation of 5 x 104 PMC with A23187 (1 or 5pM) resulted in the specific release of 34 + 3% and 42 + 3% of total cellular histamine content, respectively. Preincubation of PMC with PF-5901 or Wy-50,295 at concentrations of 1 or 1OpM
-7
*TS I. .0-#95%. The effects of PF-5901 and Wy-50,295 on PAF release by rat peritoneal mast cells may have occurred as a consequence of a non-specific inhibition of mast cell activation. To test this hypothesis, the effects of these compounds on ionophorestimulated histamine and PGD2 release were assessed. The observation that concentrations of these two compounds which markedly inhibited PAF release failed to affect significantly the release of these two mediators suggests a more specific mechanism of action. The precise nature of that
INHIBITION OF PAF SYNTHESIS BY QUINOLINES
mechanism has yet to be established. The present results are in agreement with those of Warner et al. (1988) who, using human lung mast cells, demonstrated that a 5-lipoxygenase inhibitor (L651,392) failed to modify histamine release. The ability of PF-5901 and Wy-50,295 to inhibit PAF release by mast cells can also be distinguished from the previously reported ability of ketotifen to inhibit PAF synthesis by mouse bone marrow-derived mast cells (Joly et al., 1987). In that study, ketotifen was also found to inhibit the release of fi-hexosaminidase, a preformed granular enzyme. Release of fl-hexosaminidase has been shown to correlate well with histamine release (Mencia-Huerta et al., 1983). PAF synthesis by mast cells from various sources has been demonstrated previously (Mencia-Huerta et al., 1983; Schleimer et al., 1986; Musch et al., 1987; Triggiani et al., 1990). A striking feature of the present study was the rapidity with which PAF is released following stimulation of peritoneal mast cells. PAF release was detectable within seconds of stimulation with ionophore. The quantity of PAF released in response to ionophore challenge was also significant. Indeed, the amount of PAF released from rat peritoneal mast cells (-2ng per million cells) was an order of magnitude lower than the levels of prostaglandin D2 released by these cells in response to stimulation with ionophore. The concentrations of ionophore required to elicit this amount of PAF release from PMC was sufficient to induce specific histamine release in the 34-42% range. The mechanism through which PF-5901 and Wy-50,295 inhibit PAF synthesis is not known. Since these compounds do not interfere with ionophore-stimulated histamine or PGD2 release, it is unlikely that they significantly modify the calcium influx in response to this challenge. It is conceivable
91
that these compounds inhibit the activity of one of the two enzymes involved in the biosynthesis of PAF, namely phospholipase A2 and acetyl CoA transferase. However, since neither of these compounds affected PGD2 release in the present study, or exerted effects on prostaglandin synthesis in various other systems (Van Inwegen et al., 1987; Kreft et al., 1990), effects on phospholipase A2 seem unlikely. The possibility that PF-5901 and Wy-50,295 alter acetyl CoA transferase activity requires further investigation. Since these two compounds inhibit leukotriene synthesis by binding to FLAP (Evans et al., 1991), the extent to which inhibition of PAF synthesis can be extended to other FLAP inhibitors is of interest. However, MK-886, the prototype FLAP inhibitor, failed to affect significantly PAF synthesis by PMC in the present study. In a preliminary report, however, Lock et al. (1990) showed that MK-886 inhibited PAF synthesis by human neutrophils, albeit at concentrations well above those required for inhibition of leukotriene synthesis. In summary, two quinoline-based compounds were found to inhibit PAF release by peritoneal mast cells at concentrations in the micromolar range. These compounds may therefore be useful pharmacological probes for investigations of the role of PAF in various inflammatory and allergic conditions. Moreover, inhibition of PAF release may contribute to the anti-inflammatory effects of these compounds. This work was supported by grants from the Medical Research Council of Canada (MRC), Purdue Frederick Inc. and the Alberta Heritage Foundation for Medical Research (AHFMR). C.M.H. is supported by an AHFMR Studentship and E.Y.B. by an AHFMR Fellowship. A.D.B. is an AHFMR Medical Scholar. J.L.W. is an AHFMR Medical Scholar and MRC Scientist.
References BEFUS, A.D. & BIENENSTOCK, J. (1979). Immunologically mediated
LAURSEN, L.S., NAESDAL, J., BUKHAVE, K., LAURITSEN, K. & RASK-
intestinal mastocytosis in Nippostrongulus brasiliensis-infected rats. Immunol., 38, 95-103. BISSONNETTE, E.Y. & BEFUS, A.D. (1990). Inhibition of mast cellmediated cytotoxicity by IFN-ax/, and Y. J. Immunol., 145, 33853390. BRAQUET, P., TOUQUI, L., SHEN, T.Y. & VARGAFTIG, B. (1987). Perspectives in platelet-activating factor research. Pharmacol. Rev., 39, 97-145. CASALS-STENZEL, J., MUACEVIC, G. & WEBER, K-H. (1987). Pharmacological actions of WEB 2086, a specific antagonist of plateletactivating factor. J. Pharmacol. Exp. Ther., 241, 971-981. CHAND, N., DIAMANTIS, W., PILLAR, J. & SOFIA, R.D. (1987). Modulation of allergic and nonallergic histamine secretion by lipoxygenase inhibitors. Res. Commun. Chem. Pathol. Pharmacol., 55, 49-57.
MASDEN, J. (1990). Selective 5-lipoxygenase inhibition in ulcerative colitis. Lancet, 335, 683-685.
EVANS, J.F., LEVEILLE, C., MANCINI, J.A., PRASIT, P., THERIEN, T., ZAMBONI, R., GAUTHIER, J.Y., FORTIN, R., CHARLESON, P., MACINTYRE, D.E., LUELL, S., BACH, T.J., MEURER, R., GUAY, J., VICKERS, P.J., ROUZER, C.A., GILLARD, J.W. & MILLER, D.K.
(1991). 5-lipoxygenase-activating protein is the target of a quinoline class of leukotriene synthesis inhibitors. Mol. Pharmacol., 40, 22-27. GORDON, J.R., BURD, P.R. & GALLI, S.J. (1990). Mast cells as a source of multifunctional cytokines. Immunol. Today, 11, 458-464. HEAVEY, D.J., ERNST, P.B., STEVENS, R.L., BEFUS, A.D., BIENENSTOCK, J. & AUSTEN, K.F. (1988). Generation of leukotriene C4,
leukotriene B4, and prostaglandin D2 by immunologically activated rat intestinal mucosa mast cells. J. Immunol., 140, 1953-1957. JOLY, F., BESSOU, G., BENVENISTE, J. & NINIO, E. (1987). Ketotifen inhibits paf-acether biosynthesis and fl-hexosaminidase release in mouse mast cells stimulated with antigen. Eur. J. Pharmacol., 144, 133-139. JONES, T.R., ZAMBONI, R., BELLEY, M., CHAMPION, E., CHARETTE, L., FORD-HUTCHINSON, A.W., FRENETTE, R., GAUTHIER, J.-Y., LEGER, S., MASSON, P., McFARLANE, C.S., PIECHUTA, H., ROKACH, J., WILLIAMS, H., YOUNG, R.N., DeHAVEN, R.N. & PONG, S.S. (1989). Pharmacology of L-660,711 (MK-571): a novel
potent and selective leukotriene D4 receptor antagonist. Can. J. Physiol. Pharmacol., 67, 17-28. KREFT, A., MUSSER, J., MARSHALL, L. & GRIMES, D. (1990). Wy-50,295 tromethamine. Drugs Future, 15, 805-807.
LEE, T.D.G., SHANAHAN, F., MILLER, H.R.P., BIENENSTOCK, J. &
BEFUS, A.D. (1985). Intestinal mucosal mast cells: isolation from rat lamina propria and purification using unit gravity velocity sedimentation. Immunology, 55, 721-728. LOCK, T.Y., CHANG, J.Y. & GLASER, K.B. (1990). Inhibition of PAF and LTB4 biosynthesis in human neutrophils by putative PLA2 and 5-lipoxygenase inhibitors. Proceedings of 5th Inflammation Research Association Meeting, 75. LYNCH, J.M. & HENSON, P.M. (1986). The intracellular retention of newly-synthesized platelet-activating factor. J. Immunol., 137, 2653-2661. MANSINI, E., GIANNELLA, E., BANI-SACCHI, T., FANTOZZI, R., PAL-
MERANI, B. & MANNAIONI, P.F. (1987). Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism. Agents Actions, 20, 202-205. MENCIA-HUERTA, J-M., LEWIS, R.A., RAZIN, E. & AUSTEN, K.F.
(1983). Antigen-initiated release of platelet-activating factor (PAFacether) from mouse bone marrow-derived mast cells sensitized with monoclonal Ige. J. Immunol., 131, 2958-2964. MUSCH, M.W., BILLAH, M.M. & SIEGEL, M.I. (1987). Antigen- and ionophore-stimulated synthesis of platelet-activating factor by the cloned mast cell line, MC9. Biochem. Biophys. Res. Commun., 144, 1243-1250. PARENTE, L. & FLOWER, R.J. (1985). Hydrocortisone and macrocortin inhibit the zymosan-induced release of lyso-PAF from rat peritoneal leukocytes. Life Sci., 36, 1225-1231. RADOMSKI, M. & MONCADA, S. (1983). An improved method for washing human platelets with prostacyclin. Thromb. Res., 30, 383389. ROUZER, C.A., FORD-HUTCHINSON, A.W.,
MORTON, H.E. &
GILLARD, J.W. (1990). MK-886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes. J. Biol. Chem., 265, 1436-1442. SALVEMINI, D., MASINI, E., ANGGARD, E., MANNAIONI, P.F. &
VANE, J.R. (1990). Synthesis of a nitric oxide-like factor from Larginine by rat serosal mast cells: stimulation of guanylate cyclase an inhibition of platelet aggregation. Biochem. Biophys. Res. Commun., 169, 596-601.
92
C.M. HOGABOAM et al.
SCHLEIMER, R.P., MacGLASHAN Jr., D.W., PETERS, S.P., PINCKARD, R.N., ADKINSON Jr., N.F. & LICHTENSTEIN, L.M. (1986). Charac-
terization of inflammatory mediator release from purified human lung mast cells. Am. Rev. Respir. Dis., 133, 614-617. SHORE, P.A., BURKALTER, A. & COHN, V.H. (1959). A method for the fluorometric assay of histamine in tissues. J. Pharmacol. Exp. Ther., 127, 182-186. TRIGGIANI, M., HUBBARD, W.C. & CHILTON, F.H. (1990). Synthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine by an enriched preparation of the human lung mast cell. J. Immunol., 144, 4773-4780. VAN INWEGEN, R.G., KHANDWALA, A., GORDON, R., SONNINO, P.,
COUTTS, S. & JOLLY, S. (1987). REV-5901: an orally effective peptidoleukotriene antagonist, detailed biochemical/pharmacological profile. J. Pharmacol. Exp. Ther., 241, 118-124. WARNER, J.A., LICHTENSTEIN, L.M. & MAGGLASHAN, W. (1988). Effects of specific inhibitor of 5-lipoxygenase pathway on mediator release from human basophils and mast cells. J. Pharmacol. Exp. Ther., 147, 218-222. ZIMMERMAN, G.A., McINTYRE, T.M., MEHRA, M. & PRESCOTT, S.M.
(1990). Endothelial cell-associated platelet-activating factor: a novel mechanism for signalling intercellular adhesion. J. Cell Biol., 110, 529-540.
(Received May 17, 1991 Revised September 13, 1991 Accepted September 17, 1991)
