Vol. 181, No. 2, 1991 December 16, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 714-721
Group II Phospholipase A2 Inhibitors Suppressed LysophosphatidylserineDependent Degranulation of Rat Peritoneal Mast Cells Makoto Murakami, Ichiro Kudo, Yumi Fujimori, Hinako Suga and Keizo Inouel Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received
October
18,
1991
Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase AZ, at concentrations below 10m5M and anti-rat 1CkDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of 1ysoPS. These results indicate that 14-kDa group II phospholipase AZ may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast ceils and that the mechanism whereby it participates may not be due to the production of 1ysoPS from PS in mast cell membranes. 0 1991Academic Press, Inc.
Mast cells are widely distributed throughout vascular&d tissues and certain epithelia, playing an important role in allergic reactions of the immediate type by releasing histamine from their secretory granules in response to cross-linking of IgE-receptors by IgE and antigen (1,2). Activated mast cells also elaborate newly synthesized lipid chemical mediators, such as prostaglandin D2 (FGD2), leukotriene c4 and platelet-activating
factor. Production of such lipid chemical mediators is induced
by activation of cellular phospholipase A2, which liberates free fatty acids at the sn-2 position of membrane glycerophospholipids.
As for rat peritoneal connective tissue mast cells, 1ysoPS has been
shown to act as an essential co-factor for degranulation by several stimuli, including IgE-antigen system (3). Several investigators have postulated the importance of phospholipase A2 in degranulation process, such as histamine release from mast cells (4,5), neurotransmitter release from neuronal cells (6,7) or ‘To whom all correspondence should be addressed. 0006-291x/91 Copyright All rights
$1.50
0 1991 by Academic Press. Inc. of reproduction in any form reserved.
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chromaffin cells (8), and hormone release from pituitary cells (9). However, the precise molecular mechanisms whereby phospholipase A2 regulates degranulation process have yet to be elucidated. In addition, no information
has accumulated what kinds of phospholipase Az are involved in
The molecular natures of mammalian phospholipases A2 have recently been clarified. One is the 14 kDa secretory phospholipases A2, which have been classified into two groups, designated as group I (pancreas type) and group II (inflammation type), based on their primary structures (10-15). Another is the 85kDa cytosolic phospholipase Aa, which preferentially hydrolyzes arachidonate-containing glycerophospholipids
in the presence of submicromolar calcium concentrations (16-20). We found
that mast cells expressed at least three discrete phospholipases A2, namely 14-kDa group II phospholipase AZ, 85-kDa cytosolic arachidonoyl-preferential phospholipase A2 which preferentially
phospholipase AZ and a novel
hydrolyzed phosphatidylserine
study, we have reported that compounds which inhibited
l&kDa
(PS) (21). In the present group II phospholipase AZ
suppressed degranulation of mast cells. MATERIALS
AND METHODS
Mast Cklls: Mast cells (purity; more than 90%) were isolated from peritoneal cavity of Wistar rats (Nippon Bio-Supply Center, Tokyo, Japan) as described previously (22). Phospholipases AZ and 7hei.r h&ibitors: The 14-kDa group II phospholipase A2, 85kDa cytosolic phospholipase AZ and PS-hydrolyzing phospholipase AZ were purified from rat platelets (1 l), rabbit platelets (16) and rat mastocytoma RBL-2H3 cells (21), respectively. Phospholipase A2 activity was measured using radiolabeled glycerophospholipids as substrates, as described previously (11). l-Acyl-211-‘4C]arachidonoyl-snglycerophosphocholine was purchased from Amersham, Buckinghamshim, UK. I-Acyl-2-[ 1-14C] linoleoyl-snglycero-3-phospho-L-serine was prepared as described previously (11). Mepacrine and p-bromophenacylbromide (p-BPB) were purchased from Wako, Osaka, Japan. Preparation of rabbit anti-rat 1CkDa group II phospholipase AZ polyclonal antibody R377 was described previously (23). Activation of Mast Cells: Mast cells were suspended in 10 mM Tris-HCl buffer (pH 7.4). which contained 150 mM NaCl, 3.7 mM KC!& 1 mM CaCla, 0.1% (w/v) glucose and 0.5% gelatin (Sigma, St. Louis, MO), (Trisgelatin buffer) and the concentration was adjusted to 1 x lo6 cells/ml. The cells wete sensitized with 1 pg/ml mouse monoclonal anti-dinitrophenyl (DNP) IgE (Seikagalcu Kogyo, Tokyo, Japan) for 30 min at 37 “C. The sensitized cells were washed, suspended in T&-gelatin buffer (1 x lo6 cells/ml) and incubated with DNP-conjugated Ascaris suum (DNP-Ascar&) (donated by Kissei Pharmaceutical, Matsumoto, Japan) in the presence of 1ysoPS (Funakoshi, Tokyo, Japan) at 37 “C for 10 min. The activation was stopped by adding ice-cold EDTA (2 mM) to the cell suspension, which was followed immediately by centrifugation at 750 x g for 3 min at 4 “C. The amount of histamine released into the supematant was determined by a radioenzymatic assay using r3HJmethyl715
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S-adenosyl-L-methionine (New England Nuclear, Boston, MA) and a crude preparation of rat kidney histamine methyltransferase (24). The percentage release of histamine was calculated by dividing the number in each supematant by the amount of sonicated cells (Branson Sonifier, 20 pulses, setting 4, 50% pulse cycle). The amount of FGD2 released into the supematant was measured using a PGD2 assay kit (Amersham). RESULTS LysoPS-Lkpendent Activation of Mast cells Rat peritoneal mast cells sensitized with IgE were stimulated with antigen (1 p&ml) in the presence of various concentrations of 1ysoPS. Not only histamine release (Fig. 1A) as was already published (22), but also PGD2 generation (Fig. 2B) were potentiated in a lysoPS-dependent manner. An enantiomer of natural lysoPS, which had D-serine configuration, showed much weaker activity on mast cells than the corresponding 1ysoPS having L-serine. PS was much less effective to both histamine release and PGD2 generation as compared with 1ysoPS. Thus, 1ysoPS may act on rat peritoneal mast cells as a lipid chemical mediator, which induces eicosanoid generation as well as degranulation. The ‘receptor-like’
molecule, which could recognize the configuration
of serine
moiety, might be present on connective tissue mast cell surfaces. Effkct of pBPB and Mepacrine on Mast Cell Activation and Phospholipase AZ Activities: We investigated the effect of p-BPB and mepacrine on IgE-mediated activation of rat peritoneal mast cells. Mast cells sensitized with IgE were preincubated with either pBPB or mepacrine and then
B
A
?
LysoPSdependent release of histamine (left) or PGDZ (right) from IgE- and antigen-challenged rat peritoneal mast cells. Mast cells were stimulated in the presence of the indicated concentrations of l1 -acyl-sn-glycero-3-phospho-D-serine acyl-sn-glycero-3-phospho-L-serine (lysoPS(L);@), (lysoPS@);o) or bovine brain PS (A). The procedure is described in MATERIALS AND METHODS in detail. Preparation of lysoPS(D) was described previously (22). 716
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A c
Fig. 2 Effect of p-BPB on phospholipase AZ activities and mast cell activation. (A) One nanogram of purified 14-kDa group II phospholipase A2 (e), 85kDa cytosolic phospholipase A2 (0) or PShydrolyzing phospholipase A2 (A) was preincubated with the indicated concentrations of p-BPB for 30 min at room temperature, and then the remaining phospholipase AZ activities were measured. (B) & (C). Rat peritoneal mast cells (1 x 106 cells/ml) sensitized with IgE were incubated in the presence (0) or absence (0) of p-BPB (10.’ M) for 30 min at 37 “C, washed, and then treated with antigen (1 &ml) and the indicated concentrations of 1ysoPSfor 10 min at 37 “C. Histamine (B) and PCD2 (C) released into the supematants were quantified. A representative result of three independent experiments is shown. The values indicate averages Z!ZS.D. (n=3).
stimulated with antigen and IysoPS. p-BPB ( 10e5M) inhibited histamine release and PGDz generation
almost completely (Fig. 2B and 2C). Mepacrine (10m5M) also inhibited histamine release (Fig. 3B), but did not affect PGD2 generation (Fig. 3C). Inhibitory
effects of p-BPB and mepacrine were
observed in a dose-dependent manner (Table I). The effects of p-BPB and mepacrine on the activities of three types of phospholipase A2 expressed in mast cells were then investigated. p-BPB inhibited all three types of phospholipase A2 (Fig. 2A), whereas mepacrine inhibited the 1CkDa group II phospholipase A2 and PS-hydrolyzing phospholipase A2 but not the 85-kDa cytosolic phospholipase A2 (Fig. 3A). These results suggest that degranulation and eicosanoid generation in mast cells are mediated by discrete phospholipases A2; the former by a mepacrine-sensitive and the latter by a mepacrine-insensitive enzyme. It has been postulated that 85-kDa cytosolic phospholipase AZ plays an essential role in stimulus-coupled
eicosanoid generation, as it preferentially
hydrolyzes
arachidonoyl residues at the sn-2 position of glycerophospholipids in the presence of less than 10m6M Ca2’ (16-20). We hypothesized that a mepacrine-sensitive phospholipase AZ, either the 14-kDa group 717
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40.
z
? 2 F 3o D d 2o fY P
30.
3 % i E m .z
20.
10 .
10.
0
0 lri4
163
Melxwine
. 0
(M)
0 18
166
0
18'
lob
LYSCPS (M)
LysoPS(M)
Fig. 3 Effect of mepacrine on phospholipase AZ activities(A) andmastcell activation(histamine release (B); PGDz generation (C)). Experimentswere performedas describedin Fig. 2 except that mepacrine (lo5 M) wasusedasaninhibitor insteadof p-BPB. Symbolsareidenticalwith thosein Fig. 2. A representativeresultof threeindependentexperimentsis shown.The valuesindicate averages+ S.D. (n=3).
TableI Effects of variousconcentrations of p-BPB andmepacrineon mastcell activation. Mast cells sensitizedwith IgE werepretreatedfor 30 min at 37 “C with the indicatedconcentrations of each compoundrespectively,washed,andthenstimulatedwith the antigen(1 @ml) in the presence of 1ysoPS(KY6M). HistamineandPGDZreleased into thesupematants weremeasured asdescribed in MATERIALS AND METHODS.Thevaluesindicateinhibition(averages (8) f S.D. (n=3)) of mast cell activation.A representative resultof threeindependent experiments isshown.
Compounds (M)
pBPB
lo.* M lo-’ M 1O-6M lo-’ M 1O-4M
Mepacrlne 10-6M lo-’ M 1O-4M
Inhibitionof histamine release(8)
0 0
Inhibitionof PGD2generation(%)
0 20.4+ 1.6
63.8 + 3.7 77.4k2.5
82.8f4.1 0
89.6f5.2 94.2+ 1.2
100 0
54.8zt8.7 89.8f5.4
718
0 7.8f2.6
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-t
80.
a
40.
50.
2 ‘g :
30.
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1
D 40.
3 3
20.
20.
> g
10.
OC 0
1tP LWS
16 (M)
Fig. 4 Effect of anti-rat14kDa groupII phospholipase A2 antibodyR377onphospholipase A2 activities (A) andmastcell activation(histamine release(B); PGDzgeneration(C)). Ratperitonealmastcells sensitized with IgE werestimulated with antigen(1 pg/ml)andthe indicatedconcentrations of 1ysoPS for 10 min at 37 “C in the presenceor absenceof the antibody R377(50 lg/ml). Symbolsare identicalwith thosein Fig. 2. Trianglein (B) indicatestheresultusingconttol rabbitIgG insteadof R377.A representativeresult of threeindependentexperimentsis shown.The valuesindicate averages * S.D. (n=3).
II phospholipase A2 or PS-hydrolyzing phospholipase AZ, might be involved in the degranulation process. Antibodies against 14-kDa Group II PhospholipaseAZ Blacked Degranulation but not PGDz Generationof Mast cells:
In a previous study, we established a rabbit antibody, R377, which reacted specifically with rat 14kDa group II phospholipase AZ (23). This antibody inhibited the enzymatic activity of rat 1CkDa group II phospholipase AZ, but not the other two phospholipases A2 (Pig. 4A). When rat peritoneal mast cells were activated in the presence of 50 p&ml antibody R377, inhibition of histamine, but not PGD2, release was observed (Fig. 4B and 4C). No inhibitory
effect on degranulation was observed
with control antibody (Fig. 4B). These results indicate that 16kDa group II phospholipase A2 may be involved in the regulation of rat peritoneal mast cell degranulation. DISCUSSION In this study, we found that inhibitors of mammalian
16kDa group II phospholipase A2 blocked
histamine release from mast cells. It is noteworthy that little inhibition of eicosanoid generation was 719
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observed with mepacrine and anti-rat 14-kDa group II phospholipase AZ-specific antibody. Only p BPB prevented PGDz generation, which suggests that eicosanoid generation may be regulated mainly by 85-kDa cytosolic phospholipase AZ, which is inhibited by p-BPB and hydrolyzes arachidonatecontaining phospholipids preferentially. Thus, mast ccl1 activation may be promoted by at least two discrete phospholipases AZ independently;
degranulation by the 16kDa group II enzyme and
eicosanoid generation by the 85-kDa cytosolic enzyme. This speculation is consistent with the previous conclusion that degranulation
and eicosanoid generation progressed independently
in
activated mast cells; suppression of cyclooxygenase or lipoxygenase pathways did not affect stimulus-mediated granule secretion (25). Several reports have indicated that mepacrine inhibited not only histamine release but also FGD2 generation from mast cells (4). This discrepancy might be explained by the concentration of mepacrine used in the experiments. In this study, we used 10m5M mepacrine and observed that higher concentrations inhibited PGDz generation (data not shown), may be due to non-specific effects, other than inhibition of phospholipase AZ. Little information has accumulated how phospholipase AZ participated in degranulation process, although several investigators have postulated the importance of either lysophospholipids (2627) or arachidonic acid metabolites (8,28), both of which were produced via phospholipase A2 action, as potential fusogenic candidates. LysoPS potentiates degranulation of connective tissue mast cells (3,22,29), which raises the possibility
that one of the mechanisms whereby 14-kDa group II
phospholipase A2 may function in &granulation
is to generate 1ysoPS by hydrolyzing membrane PS.
However, this could be eliminated since PGD2 generation from rat peritoneal mast cells, which was also 1ysoPSdependent (Pig. l), was not affected by the antibody R377 or mepacrine. We recently found that mast cells released 1bkDa group II phospholipase A2 upon stimulation, indicating that the enzyme might be stored in secretory granules (Murakami, M. et al. manuscript in preparation). Although the mechanism how 1CkDa group II phospholipase AZ regulates the process of degranulation remains unclear, our findings that 16kDa group II phospholipase A2 inhibitors blocked histamine release from mast cells may contribute to the development of novel therapeutic and/or prophylatic drugs for allergic diseases.
This work was supported by Grants-in-Aid for Scientific Research (Nos. 01639509,02557090 and 03680163) from the Minisny of Education, Science and Culture of Japan. 720
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REFERENCES
1. Stevens, R.L. and Austen, K.F. (1989) Immunol. Today 10: 381-386 2. Galli, S.J. (1990) Lab. Invest. 62: 626-634 3. Martin, T.W. and Lagunoff, D. (1979) Nature 279,250-252 4. McGivney, A., Morita, V., Crews, F.T., Hirata, F., Axwlrod, J-and Siraganian, R.P. (1981) Arch. Biochem. Biophys. 212: 572-580 5. Bronner, C., Cothenet, V., Monte, D., Joseph, M., La&y, Y. and Capron, A. (1990) Agents Actions 30: 95-97
6. Moskowitz, N., Schook, W. and Puszkin, S. (1982) science 216: 305-307 7. Bradfod, P.G., Marinetti, G.V. and Abood, L.G. (1983) .J. Neurochem.41: 1684-1693 8. Frye, R.A. and Holz, R.W. (1985) J. Neuruchem.44: 265-273 9. Cowell, M., Flower, R.J. and Bucjingham, J.C. (1991) f. En&ctin. 130: 21-32 10. Sakata, T., Nakamura, E., Tsuruta, Y., Tamaki, M.. Teraoka, H., Tojo, H., Ono, T. and Okamoto, M. (1989) B&him. Biophys. Acta 1007: 124- 126 11. Horigome, K., Hayakawa, M., Inoue, K. and Nojima, S. (1987) 1 Biochem. 101: 53-61 12. Komada, M., Kudo, I., Mizushima, H., Kitamura, N. and Inoue, K. (1989) J. B&hem. 106: 545-547 13. Kramer, R.M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E.P., Tizard, R. and Pepinski, R.B. (1989) I. Biol. Chem. 264: 5768-5775 14. Hara, S., Kudo, I., Chang, H.W., Matsuta, K., Miyamoto, T. and Inoue, K. (1989) J. Biochem. 105: 395-399 15. Nakano, T. and Arita, H. (1990) FEBSLett. 273.23-26 16. Kim. D.K., Kudo, I. and Inoue, K. (1991) B&him. Biophys. Acta 1083: 80-88 17. Takayama, K., Kudo, I., Kim, D.K., Nagata, K., Nozawa, Y. and Inoue, K. (1991) FEBS L.&t 282: 326-330 18. Leslie, C.C. (1991) J. Biol. Chem. 266: 11366-11371 19. Sharp, J.D., White, D.L., Chiou, X.G., Goodson, T., Gamboa, G.C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P.L., Sportsman, J.R., Becker, G-W., Kang, L.H., Roberts, E.F. and Kramer, R.M. (1991) J. Biol. Chem.266: 14850-14853 20. Clark, J.D., Lin, L-L., Kriz, R.W., Ramesha, C.S., Sultzman, L-A., Lin, A.Y. Milona, N. and Knopf, J.L. (1991) Cefl 65, 1043-1051 21. Murakami, M., Kudo, I., Umeda, M., Matsuzawa, A., Takeda, M., Komada, M., Fujimori, Y., Takahashi, K. and Inoue, K. L B&hem. submitted 22. Horigome, K., Tamori-Natori, Y., Inoue, K. and Nojima, S. (1986) J. B&hem. 100: 571-579 23. Murakami, M., Kudo, I., Natori, Y. and Inoue, K. (1990) Biochim. Biophys. Acta 1043: 34-42 24. Schaff, R.E. and Beaven, M.A. (1979) Anal. B&hem. 94,425-429 25. Lewis, R.A., Holgate, ST., Roberts, L.J., Maguire, J.F., Otes, J.A. and Austen, K.F. (1979) J. Immunol. 123: 1663-1668 26. Poole, A.R., Howell, J-1. and Lucy, J.A. (1970) Nature227, 810-814 27. Metz, A. (1986) J. &%nzracol. Exp. Ther. 238: 819-832 28. Karli, U.O., Schafer, T. and Burger, M.M. (1990) Proc. Natl. Acad. Sci. USA 87: 5912-5915 29. Murakami, M., Umeda, M., Kudo, I. and Inoue, K. (1991) Int Archs. Allergy appl. fmmunol. in press
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Group II Phospholipase A2 Inhibitors Suppressed LysophosphatidylserineDependent Degranulation of Rat Peritoneal Mast Cells Makoto Murakami, Ichiro Kudo, Yumi Fujimori, Hinako Suga and Keizo Inouel Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received
October
18,
1991
Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase AZ, at concentrations below 10m5M and anti-rat 1CkDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of 1ysoPS. These results indicate that 14-kDa group II phospholipase AZ may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast ceils and that the mechanism whereby it participates may not be due to the production of 1ysoPS from PS in mast cell membranes. 0 1991Academic Press, Inc.
Mast cells are widely distributed throughout vascular&d tissues and certain epithelia, playing an important role in allergic reactions of the immediate type by releasing histamine from their secretory granules in response to cross-linking of IgE-receptors by IgE and antigen (1,2). Activated mast cells also elaborate newly synthesized lipid chemical mediators, such as prostaglandin D2 (FGD2), leukotriene c4 and platelet-activating
factor. Production of such lipid chemical mediators is induced
by activation of cellular phospholipase A2, which liberates free fatty acids at the sn-2 position of membrane glycerophospholipids.
As for rat peritoneal connective tissue mast cells, 1ysoPS has been
shown to act as an essential co-factor for degranulation by several stimuli, including IgE-antigen system (3). Several investigators have postulated the importance of phospholipase A2 in degranulation process, such as histamine release from mast cells (4,5), neurotransmitter release from neuronal cells (6,7) or ‘To whom all correspondence should be addressed. 0006-291x/91 Copyright All rights
$1.50
0 1991 by Academic Press. Inc. of reproduction in any form reserved.
714
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chromaffin cells (8), and hormone release from pituitary cells (9). However, the precise molecular mechanisms whereby phospholipase A2 regulates degranulation process have yet to be elucidated. In addition, no information
has accumulated what kinds of phospholipase Az are involved in
The molecular natures of mammalian phospholipases A2 have recently been clarified. One is the 14 kDa secretory phospholipases A2, which have been classified into two groups, designated as group I (pancreas type) and group II (inflammation type), based on their primary structures (10-15). Another is the 85kDa cytosolic phospholipase Aa, which preferentially hydrolyzes arachidonate-containing glycerophospholipids
in the presence of submicromolar calcium concentrations (16-20). We found
that mast cells expressed at least three discrete phospholipases A2, namely 14-kDa group II phospholipase AZ, 85-kDa cytosolic arachidonoyl-preferential phospholipase A2 which preferentially
phospholipase AZ and a novel
hydrolyzed phosphatidylserine
study, we have reported that compounds which inhibited
l&kDa
(PS) (21). In the present group II phospholipase AZ
suppressed degranulation of mast cells. MATERIALS
AND METHODS
Mast Cklls: Mast cells (purity; more than 90%) were isolated from peritoneal cavity of Wistar rats (Nippon Bio-Supply Center, Tokyo, Japan) as described previously (22). Phospholipases AZ and 7hei.r h&ibitors: The 14-kDa group II phospholipase A2, 85kDa cytosolic phospholipase AZ and PS-hydrolyzing phospholipase AZ were purified from rat platelets (1 l), rabbit platelets (16) and rat mastocytoma RBL-2H3 cells (21), respectively. Phospholipase A2 activity was measured using radiolabeled glycerophospholipids as substrates, as described previously (11). l-Acyl-211-‘4C]arachidonoyl-snglycerophosphocholine was purchased from Amersham, Buckinghamshim, UK. I-Acyl-2-[ 1-14C] linoleoyl-snglycero-3-phospho-L-serine was prepared as described previously (11). Mepacrine and p-bromophenacylbromide (p-BPB) were purchased from Wako, Osaka, Japan. Preparation of rabbit anti-rat 1CkDa group II phospholipase AZ polyclonal antibody R377 was described previously (23). Activation of Mast Cells: Mast cells were suspended in 10 mM Tris-HCl buffer (pH 7.4). which contained 150 mM NaCl, 3.7 mM KC!& 1 mM CaCla, 0.1% (w/v) glucose and 0.5% gelatin (Sigma, St. Louis, MO), (Trisgelatin buffer) and the concentration was adjusted to 1 x lo6 cells/ml. The cells wete sensitized with 1 pg/ml mouse monoclonal anti-dinitrophenyl (DNP) IgE (Seikagalcu Kogyo, Tokyo, Japan) for 30 min at 37 “C. The sensitized cells were washed, suspended in T&-gelatin buffer (1 x lo6 cells/ml) and incubated with DNP-conjugated Ascaris suum (DNP-Ascar&) (donated by Kissei Pharmaceutical, Matsumoto, Japan) in the presence of 1ysoPS (Funakoshi, Tokyo, Japan) at 37 “C for 10 min. The activation was stopped by adding ice-cold EDTA (2 mM) to the cell suspension, which was followed immediately by centrifugation at 750 x g for 3 min at 4 “C. The amount of histamine released into the supematant was determined by a radioenzymatic assay using r3HJmethyl715
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S-adenosyl-L-methionine (New England Nuclear, Boston, MA) and a crude preparation of rat kidney histamine methyltransferase (24). The percentage release of histamine was calculated by dividing the number in each supematant by the amount of sonicated cells (Branson Sonifier, 20 pulses, setting 4, 50% pulse cycle). The amount of FGD2 released into the supematant was measured using a PGD2 assay kit (Amersham). RESULTS LysoPS-Lkpendent Activation of Mast cells Rat peritoneal mast cells sensitized with IgE were stimulated with antigen (1 p&ml) in the presence of various concentrations of 1ysoPS. Not only histamine release (Fig. 1A) as was already published (22), but also PGD2 generation (Fig. 2B) were potentiated in a lysoPS-dependent manner. An enantiomer of natural lysoPS, which had D-serine configuration, showed much weaker activity on mast cells than the corresponding 1ysoPS having L-serine. PS was much less effective to both histamine release and PGD2 generation as compared with 1ysoPS. Thus, 1ysoPS may act on rat peritoneal mast cells as a lipid chemical mediator, which induces eicosanoid generation as well as degranulation. The ‘receptor-like’
molecule, which could recognize the configuration
of serine
moiety, might be present on connective tissue mast cell surfaces. Effkct of pBPB and Mepacrine on Mast Cell Activation and Phospholipase AZ Activities: We investigated the effect of p-BPB and mepacrine on IgE-mediated activation of rat peritoneal mast cells. Mast cells sensitized with IgE were preincubated with either pBPB or mepacrine and then
B
A
?
LysoPSdependent release of histamine (left) or PGDZ (right) from IgE- and antigen-challenged rat peritoneal mast cells. Mast cells were stimulated in the presence of the indicated concentrations of l1 -acyl-sn-glycero-3-phospho-D-serine acyl-sn-glycero-3-phospho-L-serine (lysoPS(L);@), (lysoPS@);o) or bovine brain PS (A). The procedure is described in MATERIALS AND METHODS in detail. Preparation of lysoPS(D) was described previously (22). 716
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A c
Fig. 2 Effect of p-BPB on phospholipase AZ activities and mast cell activation. (A) One nanogram of purified 14-kDa group II phospholipase A2 (e), 85kDa cytosolic phospholipase A2 (0) or PShydrolyzing phospholipase A2 (A) was preincubated with the indicated concentrations of p-BPB for 30 min at room temperature, and then the remaining phospholipase AZ activities were measured. (B) & (C). Rat peritoneal mast cells (1 x 106 cells/ml) sensitized with IgE were incubated in the presence (0) or absence (0) of p-BPB (10.’ M) for 30 min at 37 “C, washed, and then treated with antigen (1 &ml) and the indicated concentrations of 1ysoPSfor 10 min at 37 “C. Histamine (B) and PCD2 (C) released into the supematants were quantified. A representative result of three independent experiments is shown. The values indicate averages Z!ZS.D. (n=3).
stimulated with antigen and IysoPS. p-BPB ( 10e5M) inhibited histamine release and PGDz generation
almost completely (Fig. 2B and 2C). Mepacrine (10m5M) also inhibited histamine release (Fig. 3B), but did not affect PGD2 generation (Fig. 3C). Inhibitory
effects of p-BPB and mepacrine were
observed in a dose-dependent manner (Table I). The effects of p-BPB and mepacrine on the activities of three types of phospholipase A2 expressed in mast cells were then investigated. p-BPB inhibited all three types of phospholipase A2 (Fig. 2A), whereas mepacrine inhibited the 1CkDa group II phospholipase A2 and PS-hydrolyzing phospholipase A2 but not the 85-kDa cytosolic phospholipase A2 (Fig. 3A). These results suggest that degranulation and eicosanoid generation in mast cells are mediated by discrete phospholipases A2; the former by a mepacrine-sensitive and the latter by a mepacrine-insensitive enzyme. It has been postulated that 85-kDa cytosolic phospholipase AZ plays an essential role in stimulus-coupled
eicosanoid generation, as it preferentially
hydrolyzes
arachidonoyl residues at the sn-2 position of glycerophospholipids in the presence of less than 10m6M Ca2’ (16-20). We hypothesized that a mepacrine-sensitive phospholipase AZ, either the 14-kDa group 717
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40.
z
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30.
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20.
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10.
0
0 lri4
163
Melxwine
. 0
(M)
0 18
166
0
18'
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LYSCPS (M)
LysoPS(M)
Fig. 3 Effect of mepacrine on phospholipase AZ activities(A) andmastcell activation(histamine release (B); PGDz generation (C)). Experimentswere performedas describedin Fig. 2 except that mepacrine (lo5 M) wasusedasaninhibitor insteadof p-BPB. Symbolsareidenticalwith thosein Fig. 2. A representativeresultof threeindependentexperimentsis shown.The valuesindicate averages+ S.D. (n=3).
TableI Effects of variousconcentrations of p-BPB andmepacrineon mastcell activation. Mast cells sensitizedwith IgE werepretreatedfor 30 min at 37 “C with the indicatedconcentrations of each compoundrespectively,washed,andthenstimulatedwith the antigen(1 @ml) in the presence of 1ysoPS(KY6M). HistamineandPGDZreleased into thesupematants weremeasured asdescribed in MATERIALS AND METHODS.Thevaluesindicateinhibition(averages (8) f S.D. (n=3)) of mast cell activation.A representative resultof threeindependent experiments isshown.
Compounds (M)
pBPB
lo.* M lo-’ M 1O-6M lo-’ M 1O-4M
Mepacrlne 10-6M lo-’ M 1O-4M
Inhibitionof histamine release(8)
0 0
Inhibitionof PGD2generation(%)
0 20.4+ 1.6
63.8 + 3.7 77.4k2.5
82.8f4.1 0
89.6f5.2 94.2+ 1.2
100 0
54.8zt8.7 89.8f5.4
718
0 7.8f2.6
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Fig. 4 Effect of anti-rat14kDa groupII phospholipase A2 antibodyR377onphospholipase A2 activities (A) andmastcell activation(histamine release(B); PGDzgeneration(C)). Ratperitonealmastcells sensitized with IgE werestimulated with antigen(1 pg/ml)andthe indicatedconcentrations of 1ysoPS for 10 min at 37 “C in the presenceor absenceof the antibody R377(50 lg/ml). Symbolsare identicalwith thosein Fig. 2. Trianglein (B) indicatestheresultusingconttol rabbitIgG insteadof R377.A representativeresult of threeindependentexperimentsis shown.The valuesindicate averages * S.D. (n=3).
II phospholipase A2 or PS-hydrolyzing phospholipase AZ, might be involved in the degranulation process. Antibodies against 14-kDa Group II PhospholipaseAZ Blacked Degranulation but not PGDz Generationof Mast cells:
In a previous study, we established a rabbit antibody, R377, which reacted specifically with rat 14kDa group II phospholipase AZ (23). This antibody inhibited the enzymatic activity of rat 1CkDa group II phospholipase AZ, but not the other two phospholipases A2 (Pig. 4A). When rat peritoneal mast cells were activated in the presence of 50 p&ml antibody R377, inhibition of histamine, but not PGD2, release was observed (Fig. 4B and 4C). No inhibitory
effect on degranulation was observed
with control antibody (Fig. 4B). These results indicate that 16kDa group II phospholipase A2 may be involved in the regulation of rat peritoneal mast cell degranulation. DISCUSSION In this study, we found that inhibitors of mammalian
16kDa group II phospholipase A2 blocked
histamine release from mast cells. It is noteworthy that little inhibition of eicosanoid generation was 719
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observed with mepacrine and anti-rat 14-kDa group II phospholipase AZ-specific antibody. Only p BPB prevented PGDz generation, which suggests that eicosanoid generation may be regulated mainly by 85-kDa cytosolic phospholipase AZ, which is inhibited by p-BPB and hydrolyzes arachidonatecontaining phospholipids preferentially. Thus, mast ccl1 activation may be promoted by at least two discrete phospholipases AZ independently;
degranulation by the 16kDa group II enzyme and
eicosanoid generation by the 85-kDa cytosolic enzyme. This speculation is consistent with the previous conclusion that degranulation
and eicosanoid generation progressed independently
in
activated mast cells; suppression of cyclooxygenase or lipoxygenase pathways did not affect stimulus-mediated granule secretion (25). Several reports have indicated that mepacrine inhibited not only histamine release but also FGD2 generation from mast cells (4). This discrepancy might be explained by the concentration of mepacrine used in the experiments. In this study, we used 10m5M mepacrine and observed that higher concentrations inhibited PGDz generation (data not shown), may be due to non-specific effects, other than inhibition of phospholipase AZ. Little information has accumulated how phospholipase AZ participated in degranulation process, although several investigators have postulated the importance of either lysophospholipids (2627) or arachidonic acid metabolites (8,28), both of which were produced via phospholipase A2 action, as potential fusogenic candidates. LysoPS potentiates degranulation of connective tissue mast cells (3,22,29), which raises the possibility
that one of the mechanisms whereby 14-kDa group II
phospholipase A2 may function in &granulation
is to generate 1ysoPS by hydrolyzing membrane PS.
However, this could be eliminated since PGD2 generation from rat peritoneal mast cells, which was also 1ysoPSdependent (Pig. l), was not affected by the antibody R377 or mepacrine. We recently found that mast cells released 1bkDa group II phospholipase A2 upon stimulation, indicating that the enzyme might be stored in secretory granules (Murakami, M. et al. manuscript in preparation). Although the mechanism how 1CkDa group II phospholipase AZ regulates the process of degranulation remains unclear, our findings that 16kDa group II phospholipase A2 inhibitors blocked histamine release from mast cells may contribute to the development of novel therapeutic and/or prophylatic drugs for allergic diseases.
This work was supported by Grants-in-Aid for Scientific Research (Nos. 01639509,02557090 and 03680163) from the Minisny of Education, Science and Culture of Japan. 720
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REFERENCES
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