Agents Actions37 (1992)
0065-4299/92/020121-06 $1.50+0.20/0 9 1992Birkhfiuser Verlag,Basel
The role of 5-hydroperoxyeicosatetraenoic acid in neutrophil activation A. Seya, T. Terano, Y. Tamura and S. Yoshida The Second Department of Internal Medicine,Chiba University, Medical School, 1-8-1 lnohana, Chiba City, Chiba 260, Japan
Abstract
5-Hydroperoxyeicosatetraenoic acid (5HPETE) has been recently reported to play an important role in regulating and modulating neutrophil function. In order to clarify the mechanism of neutrophil activation by 5HPETE, we have measured the cytosolic free calcium, which is thought to be necessary for neutrophil activation using fura-2-1oaded human neutrophils. Low concentration of 5HPETE, which is thought to be produced during cell activation, had minimal effect on cytosolic free calcium by itself but dose-dependently augmented FMLP-stimulated increase in cytosolic free calcium in the presence or absence of extracellular calcium without converting to LTB 4. 5HPETE had no effect on 3H-FMLP binding to human neutrophils. The present data suggested that 5HPETE would augment FMLP-stimulated increase in cytosolic free calcium by enhancing the influx of extracellular calcium and/or the release of calcium from intercellular pool, which resulted in augmentation of neutrophil activation by primary agonist such as FMLP. Introduction
Recently, hydroperoxy intermediates of arachidonic acid have been reported to play an important role in regulating cell functions [1, 2]. Neutrophils have quite active 5-1ipoxygenase (5-LO) pathway and it metabolizes arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5HPETE), 5-hydroxyeicosatetraenoic acid (5HETE) and leukotriene B4 (LTB4). According to O'.Flaherty's report, these metabolites might be classified into potentiators (5HPETE and 5HETE) and an agonist (LTB4) [3]. By their report, 5HPETE and 5HETE could potentiate PAF- and LTB4induced neutrophil degranulation. Furthermore, 5HPETE, and not 5HETE, was reported to enhance FMLP-stimulated neutrophil aggregation [4]. However, the exact mechanism of action of 5HPETE as a potentiator of neutrophil activation
has not been fully understood. The rise in cytosolic free calcium is known to be necessary for FMLPstimulated neutrophil activation [5]. Naccache et al. have already demonstrated that 5HPETE induced calcium uptake in neutrophils [6]. In the present study, in order to clarify the possible mechanism of the enhancing effect of 5HPETE on neutrophil activation, we examined whether 5HPETE enhanced FMLP-stimulated increase in cytosolic free calcium. At the same time, to evaluate the site of action of 5-HPETE, we studied the effect of 5-HPETE on the binding of F M L P to the specific receptor on neutrophils. Materials and methods Materials
Cytocharasin B, N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES), ethylene glycol
122 bis-(-/3-aminoethyl ether) N,N,N',N-tetraacetic acid (EGTA), phenolphthalein-fl-o-glucuronic acid, essential fatty-acid-free bovine serum albumin (fraction V) and formyl-methyonyl-leucyl-phenylalanine (FMLP) were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other materials were obtained from supplies indicated; FicollPaque from Pharmacia Fine Chemicals (Uppsala, Sweden), Hanks balanced salt solution (HBSS) from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Fura-2-acetoxymethylester (fura-2-AM) from Dojindo Laboratory Co. Ltd. (Kumamoto, Japan). Formyl-L-methyonyl-L-leucyl-L-phenylalanine, N-phenylalanine-ring-2,6-3H(N)-(aH-FMLP) (specific activity 56.4 ~tCi/mmol) and 14C-arachidonic acid (specific activity 55 laCi/mmol) from NEN Research Products (USA), LTB 4 RIA kit from Amersham Co. Ltd. (USA). Membrane filter (cellulose nitrate, pore size 0.8 lain) from Toyo Roshi Co., Ltd. (Tokyo, Japan). 5-hydroperoxyeicosatetraenoic acid (5HPETE) and 14C-5HPETE were synthesized from arachidonic acid (Funakoshi Pharmaceuticals Co. Ltd., Tokyo, Japan) and 14Carachidonic acid, respectively, using potato 5-1ipoxygenase as previously described by Shimizu et al. [7]. Methods
Preparation of human neutrophils. Human neutrophil suspensions were obtained by the method previously reported [8]. Briefly, citrated venous blood was obtained from healthy volunteers. Blood samples were centrifuged at 150 x # for 10 rain at room temperature and then platelet-rich plasma was removed to minimize the platelet contamination in purified neutrophils. Then, highly purified human neutrophils were prepared using dextran sedimentation, followed by centrifugation on Ficoll-Paque cushion and ammonium chloride lysis of erythrocytes according to the methods of Boyum and Palmar et al. [9, 10]. Finally, neutrophils (purity >95%) were suspended in HEPESbuffered Hanks balanced salt solution (HBSS), containing 1.2 mM Ca 2 + Measurement of cytosolic free calcium concentration by fura-2. Fura-2 loading was carried out by the method as previously reported [8]. In brief, the purified neutrophils were suspended at 2 x 107 cells/ml in HBSS containing 0.1% bovine serum albumin (HBSS-BSA) and incubated at
Agents Actions37 (1992) 37~ with 2 p,M of fura-2-AM for 15 min and then the neutrophil suspensions were incubated for a further 20 rain at room temperature. After washing the loaded neutrophils three times with HBSS-BSA, the cell pellets were resuspended at 5 • 106 neutrophils/ml of HBSS BSA and kept on ice until use for fluorescent measurement. The fluorescence of fura-2-1oaded neutrophils was measured using calcium analyzer, model CAF-100 (JASCO, Tokyo, Japan) which generated dual wavelength excitation beams (340 and 380 nm) by using a pair of interference filters, and was equipped with a magnetic stirrer under the thermostatted cuvette holder. 1 ml of fura-2-1oaded neutrophils in a cuvette was placed on the cuvette holder. After the cell were preincubated for 5 min at 37~ with cytochalasin B (5 ~tg/ml) under constant stirring of 200 rpm, F M L P was added to the cells through rubber septum. The ratios of emitted fluorescence at 500 nm to the 340 and 380 nm excitation wavelengths were ~ continuously monitored and recorded. Cytosolic free calcium concentration was calculated as described by Grynkiewicz et al. [11].
3H-FMLP bindin9 assay. 3H-FMLP binding to neutrophils was performed according to the method previously described by Williams et al. with slight modifications [12]. Human neutrophil suspensions (2.5 x 106 cells/0.5 ml of HBSS) were preincubated for 10min at 37~ Then 3H-FMLP (final concentration 1-20 nM) plus 5HPETE solution (10 7 M) were added to cell suspensions and further incubated for 12 min at 37~ Incubation was terminated by the addition of 5 ml of ice-cold incubation buffer, followed by rapid filtration of mixture on a cellulose nitrate filter. After the filter was rapidly washed with 5 ml of ice-cold incubation buffer three times, it was dried and placed in 5 ml of liquid scintillation mixture, and its radioactivity was measured in a liquid scintillation counter. Nonspecific binding was defined as the total amount of binding not inhibited by 1000-fold of unlabeled F M L P (usually 5-10% in our experimental condition). Specific binding was defined as the total amount of F M L P bound to cells minus nonspecific binding. Analysis of metabolism of exogeneous 5HPETE in human neutrophils. Neutrophil suspension (1 x 10 v cells/ml) was preincubated at 37 ~ for 5 min in the presence of cytochalasin B (5 lag/ml), then 14C-5HPETE (105 cpm) was added to the neutro-
Agents Actions37 (I992)
123
phil suspension in the presence or absence of F M L P (10 -9 M) and further incubated at 37 ~ for 1 min. Incubation was terminated by the centrifugation at 10000 x g for 30 s and supernatant was (a)
-f 1O0
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.E o
.E
l
0
F M L P ( 1 0 '*M)
-
+
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5HPETE ( M )
-
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10910 a
(b)
+
Results
5X
10810
o
10 ~ 10 9
Enhancement o[' F M LP-induced increase in cytosolic.[kee calcium by 5 H P E T E
1O0
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taken, pellets were extracted with ethanol. Eicosanoids in the supernatant (60% of total counts added) and those in extracts from pellets (38% of total counts) were analyzed by reverse-phase (RP)HPLC (JASCO, Tokyo, Japan) on an ODS column (Develosil 5 lam, 4.6 x 150 mm. N o m u r a chemical Co. Ltd., Aichi Japan) equipped with UV detector (JASCO) and radioisotope detector (Beckman 171, Beckman Instruments Inc., CA, USA). The solvent (acetonitrile/methanol/water/acetic acid = 32.5/ 32.5/35/0.01 by volume) was pumped isocratically with a tri-rotor V (JASCO, Tokyo, Japan) at a flow rate of 1 ml/min for 60 min. Furthermore, 5HPETE ( 1 0 - S M ) and F M L P (10-SM) were added to neutrophil suspensions (5 • 106/ml HBSS) in the presence of cytochalasin B (5 pg/ml). After incubation for 1 rain at 37~ ethanol was added to cell suspension and supernatant was collected by centrifugation. The supernatant was diluted with water to make 15% ethanol solution and was applied on ODS mini column (Bond elute), and then the eicosanoids were eluted by ethyl acetate as previously reported [13]. Amount of LTB 4 in eluted solution with or without R P - H P L C separation was measured using R1A kit for LTB 4 (sensitivity 10 pg/tube).
I
F M L P (10 -9 M) stimulated increase in cytosolic calcium was enhanced by the addition of 5HPETE (2x 10 9 10-8 M), which had minimal effect on calcium mobilization (less than 10 nM) in fura-2loaded human neutrophils. These results were summarized in Fig. l a, b. Dose-dependent augmentation of F M L P (10 -9 M) induced cytosolic free calcium in neutrophils by the addition of
Z
m
Figure 1
0 FMLP
( 1 0 SM)
5HPETE (M)
-
2X
+
2X
10 ~ I 0 9
-
5x
+
5x
1 0 " 10 9
+
10 a 10 a
The effectof 5HPETE on FMLP-stimulatedincreasein cytosolic free calciumby fura-2-1oadedhuman neutrophilsin the presence (a) or absence(b) of extracellularcalcium.Verticalbar represents the increase in cytosolic free calcium (A in nM), which means peak calcium concentration in stimulated cells (nM) minus calcium concentrationin restingcells prior to stimulation(nM). Data represents mean+SE [n=7-8 (a); n=3 (b)]. Means for FMLP-stimulated and FMLP-plus-SHPETE-stimulatedincrease in cytosoliccalcium(136 nM) werecompared usingpaired sample t-test *P < 0.05.
124
Agents
5HPETE was observed not only in calcium containing medium (HBSS-BSA) (Fig. la) but also calcium-free medium (HBSS BSA containing EGTA) (Fig. lb). However, the extent of enhancement was less prominent in the absence of calcium than that in the presence of extracellular calcium.
In order to examine whether exogenous 5HPETE enhances F M L P binding to h u m a n neutrophils, we compared the specific binding of 3H-FMLP to its receptor on h u m a n neutrophils in the presence or absence of 5HPETE. As shown in Fig. 2, no significant change in specific binding to its receptor on neutrophils was demonstrated by the addition of 5HPETE.
Metabolism of exoyenously added 14C-5HPETE and 5HPETE in human neutrophils In order to determine the conversion of exogenous 5HPETE to LTB4, a potent bioactive substance for neutrophils, 14C-5HPETE was added to neutrophil suspensions in the presence or absence of F M L P and incubated for 1 rain at 37 ~ then analyzed as described in Materials and Methods section, using reverse-phase H P L C with radiodetector. As shown in Fig. 3, one radioactive peak appeared at the retention time of 38 rain in accordance with authentic 5HETE standard and detectable amount of
"o
10"
w
9
,'2 _i 1
9.r
i
I
i
t
I
5
10
15
20
FMLP (nM)
Figure 2
Effects of the addition of 5HPETE on 3H-FMLP binding to human neutrophils. Specificbinding(total bindingminus nonspecific binding) is plotted against the concentration of 3HFMLP; (O) buffer-treatedcells, (e) 10_7 M 5HPETE-treated cells. Data represent mean+ SE (n= 3).
37
(1992)
5HETE 5HPETE
LTB4
ceit ( - )
celt (+)
Effect of 5HPETE on 3H-FMLP bindin.q to human neutrophils
Actions
lo
io
3'o
k i0
retention time (mini
Figure 3
Metabolism of exogenously added 14C-5HPETE by human neutrophils analyzed by reverse-phase HPLC equipped with radiodetector. Vertical bar represents radioactivityof ~4C and horizontal bar represents retention time (min): (upper panel) sample from HBSS incubatedwith laC-5HPETE;(lower panel) sample extracted by methanolfrom pelletsof neutrophilsuspension incubatedwith IaC-5HPETE.
radioactivity was not observed in fractions corresponding to LTB 4 and 5HPETE both in the supernatant and pellet of incubation mixture. Furthermore, conversion of 5HPETE to LTB 4 was not observed when 5HPETE (10 -8 M) was incubated for 1 min at 37~ in neutrophil suspension; the formed LTB4 was measured by RIA for LTB4 (sensitivity 10 pg). Discussion
The present study demonstrated that 5HPETE, at the concentration of 2 x 10-9-10 -8 M, which had minimal effect on calcium mobilization by itself, augmented 10-9 M FMLP-induced increase in cytosolic free calcium in fura-2-1oaded human neutrophils. According to Beckman's report, 1-4 gM of 5HPETE enhanced 1 0 - T M FMLP-induced neutrophil aggregation [4]. This discrepancy concerning about concentrations of primary stimulus F M L P between these two experiments might be explained as follows: first, FMLP-stimulated calcium mobilization using fura-2 could be observed at lower concentrations of F M L P than that required for the stimulation of biological functions such as aggregation. Second, calcium mobilization is thought to be obligatory but not a sufficient signal for the stimulation of functional reponses to physiological agonists [4]. In our experimental system, 5 H P E T E (2 x 10-9-10 8 M) could not en-
125
Agents Actions 37 (1992) hance F M L P - i n d u c e d calcium mobilization when higher concentrations ( > 10 -8 M) of F M L P were used as a primary agonist. This might be due to the fact that a higher concentration of F M L P could stimulate the formation of endogenous 5-1ipoxygenase (LO) metabolites including 5HPETE, which might mask the effect of exogenously added 5HPETE. As described in Materials and Methods section, we employed 10 9 M F M L P as the primary stimulus. Previous reports indicated that LTB4 formation could not be detected in neutrophil suspensions stimulated by 1 0 - 9 M of F M L P , suggesting that this concentrations of F M L P did not fully activate endogenous 5LO and/or LTA4 synthase [14, 15]. Also under our experimental condition, conversion of 5 H P E T E to LTB 4 was not observed. 5 H P E T E (10 7 M) at higher concentration by itself could mobilize cytosolic calcium, so the enhancing effect of 5 H P E T E in F M L P - s t i m u lated neutrophils was not recognized. In order to clarify the mechanism of enhancing effect of 5HPETE in calcium mobilization, we firstly checked the possibility that 5 H P E T E might augment F M L P binding to its specific receptor. As described in Materials and Methods section, our experiment of F M L P binding to neutrophils was carried out by the methods reported by Williams et al. [12]. According to their report, 12 min incubation time at 37 ~ was necessary to reach equilibrium for 3 H - F M L P binding to neutrophils. However, Vitkausas et al. reported that internalization of chemotactic peptide occurred by the incubation of neutrophils with 3 H - F M L P at 37~ [16]. We further confirmed that 5 H P E T E had no effect on 3 H - F M L P binding to its specific sites at shorter incubation time for 30 s to 5 min at 37 ~ (data not shown), in order to minimize the internalization of F M L P receptor complex and to compare the time course of binding assay with that of calcium mobilization using fura-2 which occurred within 30 s after stimulation. This data indicated that site of action of 5 H P E T E was not F M L P receptor, but possibly post-receptor sites. As shown in Fig. lb, the enhancement of calcium mobilization by 5 H P E T E was also observed in calcium-free medium, indicating that 5 H P E T E might mobilize cytosolic calcium of intracellular calcium store as well as calcium influx from extracellular medium. Naccache et al. [6] suggested that calcium influx in neutrophils induced by 5 H P E T E was due to its conversion to LTB4. However, following evidence would rule out the possibility that exogenously
added 5 H P E T E acted on intracellular calcium mobilization after conversion to LTB 4. First, exogenously added 5 H P E T E is reported to be p o o r substrate for 5LO and LTA 4 synthase activity [17]. Second, under our experimental conditions, exogenously added 5 H P E T E was converted to 5HETE but not to LTB 4. These d a t a demonstrated that 5 H P E T E by itself, without converting to LTB4, might play an important role in calcium mobilization in the process of F M L P - s t i m u l a t e d neutrophil activation. As previously reported, 5HETE, a reduced form of 5HPETE, was weaker in biological potencies than 5 H P E T E such as in the enhancement of F M L P stimulated neutrophil aggregation [4] and that of IgE-induced histamine releases in basophils [17]. In our data, 5HETE was less potent than 5 H P E T E in enhancing F M L P - s t i m u l a t e d calcium mobilization (data not shown). Accordingly, the enhancing effect of 5 H P E T E would be induced by 5 H P E T E itself, and not by 5HETE, produced from the reduction of 5 H P E T E (for example, by glutathione peroxidase). In summary, we have demonstrated that the possibility that 5 H P E T E by itself might play an important role in the regulation of cytosolic calcium in F M L P - s t i m u l a t e d neutrophil activation. Received 4 January 1992; accepted by I.G. Otterness 24 February 1992
References [1] S. Metz, M. VanRollins, R. Strife, M. Fujimoto and R. P. Robertson, Lipoxygenase pathway in islet endocrine cells, oxidative metabolism of arachidonic acid promotes insulin release. J. Clin. Invest. 71, 1191-1205 (1983).
[2] A. Hirai, K. Tahara, Y. Tamura, H. Saito, T. Terano and S. Yoshida, Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands. Prostaglandins 31, 74%767 (1985).
t-3] J. T. O'Flaherty, R. L. Wykle, N. J. Thomas and C. E. McCall, Neutrophil degranulation responses to combination of arachidonate metabolites and platelet-activatin 9 factor. Res. Commun. Chem. Pathol. Pharmacol. 43, 3-23 (1984).
[4] J. K. Beckman,J. C. Gay, A. R. Brash, J. H. Luken and J. A. Oates, Differential effects of lipoxygenase products on FM LP and L TB 4 evoked neutrophil aygregation. Lipids 20, 357 360 (1985). 1-5] H. M. Korchak, K. Vienne,L. E. Rutherford, C. Wilkenfeld, M. C. Finkelstein and G. Weissman, Stimulus response couplin 0 in the human neutrophil; 2. Temporal analysis of chan.qes in cytosolic calcium and calcium effiux. J. Biol. Chem. 259, 4076-4082 (1984).
[6] P. H. Naccache, R. I. Sha'afi, P. Borgeat and E. J. Goetzl,
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[7]
[8]
[9] [10]
[11] [12]
[13]
Agents Actions 37 (1992) Mono- and Dihydroxyeicosatetraenoic acids alter calcium homeostasis in rabbit neutrophils. J. Clin. Invest. 67, 1584 1587 (1981). T. Shimizu, O. Radmak and B. Samuelsson, Enzyme with dual lipoxygenase activities catalyzes leukotriene A 4 Synthesis from arachidonic acid. Proc. Natl. Acad. Sci. 81, 689 693 (1984). A. Seya, T. Terano, Y. Tamura and S. Yoshida, Comparative effect of leukotriene B 4 and Leukotriene B~ on calcium mobilization in human neutrophils. Prostaglandins Leukot. Essent. Fatty Acids 34, 47-50 (1988). A. Boyum, Isolation of leukocytes from human blood." Further observations. Scand. J. Lab. Clin. Invest. 21 (Suppl.), 77 89 (1968). R. M. J. Palmar, R. J. Stepney, G. A. Higgs and K. E. Eakins, Chemokinetic activity of arachidonic lipoxygenase products on leukocytes of different species. Prostaglandins 20, 411 418 (1980). G. Grynkiewicz, M. Poenie and R. Y. Tsien, A new generation of C a 2 + indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440 3450 (1985). L. T. Williams, R. Snyderman, M. C. Pike and R. J. Lefkowitz, Specific receptor sitesfor chemotactic peptides on human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. 74, 1204-1208 (1977). T. Terano, A. Hirai, Y. Tamura, S. Yoshida, J. A. Salmon and S. Moncada, Effect of eicosapentaenoic acid on eico-
[14]
[15]
[16]
[17]
[18]
sanoid formation by stimulated human polymorphonuclear leukocytes. Prog. Lipid Res. 25, 129 137 (1986). R. M. J. Palmar and J. A. Salmon, Release ofleukotriene B4 from human neutrophils and its relationship to degranulation induced by N-formyl-methyonyl-leucyl-phenylalanine, serum-treated zymosan and the ionophore A23187. Immunology 50, 65-73 (1983). W. Jubitz, O. Radmak, C. Malmsten, G. Hansson, J. A. Lindgren, J. A. Palmblad, A.-M. Uden and B. Samuelsson, A novel leukotriene produced by stimulation of leukocytes with formyl-methyonyl-leucyl-phenylalanine. J. Biol. Chem. 257, 6106-6110 (1982). G. Vitkauskas, H. J. Showell and E. L. Becker, Specific binding of synthetic chemotactic peptides to rabbit peritoneal neutrophils; Effects on dissociability of bound peptide, receptor activity and subsequent biologic responsiveness (deactivation). Mol. Immunol. 17, 171 180 (1980) T. Puustinen, M. M. Scheffer and B. Samuelson, Endogenously generated 5-hydroperoxyeicosatetraenoic acid is preferred substrate for human leukocyte leukotriene A 4 synthase activity. FEBS Lett. 217, 265 268 (1987). S. P. Peters, R. P. Sleimer, G. Marone, A. Kagey-Sobotka, M. I. Siegel and L. M. Lichitenstein, Lipoxygenase products of arachidonic acid: Role in modulation of IgE-induced histamine release. In Leukotrienes and Other Lipoxygenase Products (Eds. B. Samuelsson and R. Paoletti) pp. 315 324, Raven Press, New York (1982).
0065-4299/92/020121-06 $1.50+0.20/0 9 1992Birkhfiuser Verlag,Basel
The role of 5-hydroperoxyeicosatetraenoic acid in neutrophil activation A. Seya, T. Terano, Y. Tamura and S. Yoshida The Second Department of Internal Medicine,Chiba University, Medical School, 1-8-1 lnohana, Chiba City, Chiba 260, Japan
Abstract
5-Hydroperoxyeicosatetraenoic acid (5HPETE) has been recently reported to play an important role in regulating and modulating neutrophil function. In order to clarify the mechanism of neutrophil activation by 5HPETE, we have measured the cytosolic free calcium, which is thought to be necessary for neutrophil activation using fura-2-1oaded human neutrophils. Low concentration of 5HPETE, which is thought to be produced during cell activation, had minimal effect on cytosolic free calcium by itself but dose-dependently augmented FMLP-stimulated increase in cytosolic free calcium in the presence or absence of extracellular calcium without converting to LTB 4. 5HPETE had no effect on 3H-FMLP binding to human neutrophils. The present data suggested that 5HPETE would augment FMLP-stimulated increase in cytosolic free calcium by enhancing the influx of extracellular calcium and/or the release of calcium from intercellular pool, which resulted in augmentation of neutrophil activation by primary agonist such as FMLP. Introduction
Recently, hydroperoxy intermediates of arachidonic acid have been reported to play an important role in regulating cell functions [1, 2]. Neutrophils have quite active 5-1ipoxygenase (5-LO) pathway and it metabolizes arachidonic acid into 5-hydroperoxyeicosatetraenoic acid (5HPETE), 5-hydroxyeicosatetraenoic acid (5HETE) and leukotriene B4 (LTB4). According to O'.Flaherty's report, these metabolites might be classified into potentiators (5HPETE and 5HETE) and an agonist (LTB4) [3]. By their report, 5HPETE and 5HETE could potentiate PAF- and LTB4induced neutrophil degranulation. Furthermore, 5HPETE, and not 5HETE, was reported to enhance FMLP-stimulated neutrophil aggregation [4]. However, the exact mechanism of action of 5HPETE as a potentiator of neutrophil activation
has not been fully understood. The rise in cytosolic free calcium is known to be necessary for FMLPstimulated neutrophil activation [5]. Naccache et al. have already demonstrated that 5HPETE induced calcium uptake in neutrophils [6]. In the present study, in order to clarify the possible mechanism of the enhancing effect of 5HPETE on neutrophil activation, we examined whether 5HPETE enhanced FMLP-stimulated increase in cytosolic free calcium. At the same time, to evaluate the site of action of 5-HPETE, we studied the effect of 5-HPETE on the binding of F M L P to the specific receptor on neutrophils. Materials and methods Materials
Cytocharasin B, N-2-hydroxyethylpiperazine-N-2ethanesulfonic acid (HEPES), ethylene glycol
122 bis-(-/3-aminoethyl ether) N,N,N',N-tetraacetic acid (EGTA), phenolphthalein-fl-o-glucuronic acid, essential fatty-acid-free bovine serum albumin (fraction V) and formyl-methyonyl-leucyl-phenylalanine (FMLP) were all purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other materials were obtained from supplies indicated; FicollPaque from Pharmacia Fine Chemicals (Uppsala, Sweden), Hanks balanced salt solution (HBSS) from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan). Fura-2-acetoxymethylester (fura-2-AM) from Dojindo Laboratory Co. Ltd. (Kumamoto, Japan). Formyl-L-methyonyl-L-leucyl-L-phenylalanine, N-phenylalanine-ring-2,6-3H(N)-(aH-FMLP) (specific activity 56.4 ~tCi/mmol) and 14C-arachidonic acid (specific activity 55 laCi/mmol) from NEN Research Products (USA), LTB 4 RIA kit from Amersham Co. Ltd. (USA). Membrane filter (cellulose nitrate, pore size 0.8 lain) from Toyo Roshi Co., Ltd. (Tokyo, Japan). 5-hydroperoxyeicosatetraenoic acid (5HPETE) and 14C-5HPETE were synthesized from arachidonic acid (Funakoshi Pharmaceuticals Co. Ltd., Tokyo, Japan) and 14Carachidonic acid, respectively, using potato 5-1ipoxygenase as previously described by Shimizu et al. [7]. Methods
Preparation of human neutrophils. Human neutrophil suspensions were obtained by the method previously reported [8]. Briefly, citrated venous blood was obtained from healthy volunteers. Blood samples were centrifuged at 150 x # for 10 rain at room temperature and then platelet-rich plasma was removed to minimize the platelet contamination in purified neutrophils. Then, highly purified human neutrophils were prepared using dextran sedimentation, followed by centrifugation on Ficoll-Paque cushion and ammonium chloride lysis of erythrocytes according to the methods of Boyum and Palmar et al. [9, 10]. Finally, neutrophils (purity >95%) were suspended in HEPESbuffered Hanks balanced salt solution (HBSS), containing 1.2 mM Ca 2 + Measurement of cytosolic free calcium concentration by fura-2. Fura-2 loading was carried out by the method as previously reported [8]. In brief, the purified neutrophils were suspended at 2 x 107 cells/ml in HBSS containing 0.1% bovine serum albumin (HBSS-BSA) and incubated at
Agents Actions37 (1992) 37~ with 2 p,M of fura-2-AM for 15 min and then the neutrophil suspensions were incubated for a further 20 rain at room temperature. After washing the loaded neutrophils three times with HBSS-BSA, the cell pellets were resuspended at 5 • 106 neutrophils/ml of HBSS BSA and kept on ice until use for fluorescent measurement. The fluorescence of fura-2-1oaded neutrophils was measured using calcium analyzer, model CAF-100 (JASCO, Tokyo, Japan) which generated dual wavelength excitation beams (340 and 380 nm) by using a pair of interference filters, and was equipped with a magnetic stirrer under the thermostatted cuvette holder. 1 ml of fura-2-1oaded neutrophils in a cuvette was placed on the cuvette holder. After the cell were preincubated for 5 min at 37~ with cytochalasin B (5 ~tg/ml) under constant stirring of 200 rpm, F M L P was added to the cells through rubber septum. The ratios of emitted fluorescence at 500 nm to the 340 and 380 nm excitation wavelengths were ~ continuously monitored and recorded. Cytosolic free calcium concentration was calculated as described by Grynkiewicz et al. [11].
3H-FMLP bindin9 assay. 3H-FMLP binding to neutrophils was performed according to the method previously described by Williams et al. with slight modifications [12]. Human neutrophil suspensions (2.5 x 106 cells/0.5 ml of HBSS) were preincubated for 10min at 37~ Then 3H-FMLP (final concentration 1-20 nM) plus 5HPETE solution (10 7 M) were added to cell suspensions and further incubated for 12 min at 37~ Incubation was terminated by the addition of 5 ml of ice-cold incubation buffer, followed by rapid filtration of mixture on a cellulose nitrate filter. After the filter was rapidly washed with 5 ml of ice-cold incubation buffer three times, it was dried and placed in 5 ml of liquid scintillation mixture, and its radioactivity was measured in a liquid scintillation counter. Nonspecific binding was defined as the total amount of binding not inhibited by 1000-fold of unlabeled F M L P (usually 5-10% in our experimental condition). Specific binding was defined as the total amount of F M L P bound to cells minus nonspecific binding. Analysis of metabolism of exogeneous 5HPETE in human neutrophils. Neutrophil suspension (1 x 10 v cells/ml) was preincubated at 37 ~ for 5 min in the presence of cytochalasin B (5 lag/ml), then 14C-5HPETE (105 cpm) was added to the neutro-
Agents Actions37 (I992)
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phil suspension in the presence or absence of F M L P (10 -9 M) and further incubated at 37 ~ for 1 min. Incubation was terminated by the centrifugation at 10000 x g for 30 s and supernatant was (a)
-f 1O0
-r
Z
% o
.E o
.E
l
0
F M L P ( 1 0 '*M)
-
+
2x
5HPETE ( M )
-
2x
10910 a
(b)
+
Results
5X
10810
o
10 ~ 10 9
Enhancement o[' F M LP-induced increase in cytosolic.[kee calcium by 5 H P E T E
1O0
,:q
8
--
+
5X
I
T
50
.c_
taken, pellets were extracted with ethanol. Eicosanoids in the supernatant (60% of total counts added) and those in extracts from pellets (38% of total counts) were analyzed by reverse-phase (RP)HPLC (JASCO, Tokyo, Japan) on an ODS column (Develosil 5 lam, 4.6 x 150 mm. N o m u r a chemical Co. Ltd., Aichi Japan) equipped with UV detector (JASCO) and radioisotope detector (Beckman 171, Beckman Instruments Inc., CA, USA). The solvent (acetonitrile/methanol/water/acetic acid = 32.5/ 32.5/35/0.01 by volume) was pumped isocratically with a tri-rotor V (JASCO, Tokyo, Japan) at a flow rate of 1 ml/min for 60 min. Furthermore, 5HPETE ( 1 0 - S M ) and F M L P (10-SM) were added to neutrophil suspensions (5 • 106/ml HBSS) in the presence of cytochalasin B (5 pg/ml). After incubation for 1 rain at 37~ ethanol was added to cell suspension and supernatant was collected by centrifugation. The supernatant was diluted with water to make 15% ethanol solution and was applied on ODS mini column (Bond elute), and then the eicosanoids were eluted by ethyl acetate as previously reported [13]. Amount of LTB 4 in eluted solution with or without R P - H P L C separation was measured using R1A kit for LTB 4 (sensitivity 10 pg/tube).
I
F M L P (10 -9 M) stimulated increase in cytosolic calcium was enhanced by the addition of 5HPETE (2x 10 9 10-8 M), which had minimal effect on calcium mobilization (less than 10 nM) in fura-2loaded human neutrophils. These results were summarized in Fig. l a, b. Dose-dependent augmentation of F M L P (10 -9 M) induced cytosolic free calcium in neutrophils by the addition of
Z
m
Figure 1
0 FMLP
( 1 0 SM)
5HPETE (M)
-
2X
+
2X
10 ~ I 0 9
-
5x
+
5x
1 0 " 10 9
+
10 a 10 a
The effectof 5HPETE on FMLP-stimulatedincreasein cytosolic free calciumby fura-2-1oadedhuman neutrophilsin the presence (a) or absence(b) of extracellularcalcium.Verticalbar represents the increase in cytosolic free calcium (A in nM), which means peak calcium concentration in stimulated cells (nM) minus calcium concentrationin restingcells prior to stimulation(nM). Data represents mean+SE [n=7-8 (a); n=3 (b)]. Means for FMLP-stimulated and FMLP-plus-SHPETE-stimulatedincrease in cytosoliccalcium(136 nM) werecompared usingpaired sample t-test *P < 0.05.
124
Agents
5HPETE was observed not only in calcium containing medium (HBSS-BSA) (Fig. la) but also calcium-free medium (HBSS BSA containing EGTA) (Fig. lb). However, the extent of enhancement was less prominent in the absence of calcium than that in the presence of extracellular calcium.
In order to examine whether exogenous 5HPETE enhances F M L P binding to h u m a n neutrophils, we compared the specific binding of 3H-FMLP to its receptor on h u m a n neutrophils in the presence or absence of 5HPETE. As shown in Fig. 2, no significant change in specific binding to its receptor on neutrophils was demonstrated by the addition of 5HPETE.
Metabolism of exoyenously added 14C-5HPETE and 5HPETE in human neutrophils In order to determine the conversion of exogenous 5HPETE to LTB4, a potent bioactive substance for neutrophils, 14C-5HPETE was added to neutrophil suspensions in the presence or absence of F M L P and incubated for 1 rain at 37 ~ then analyzed as described in Materials and Methods section, using reverse-phase H P L C with radiodetector. As shown in Fig. 3, one radioactive peak appeared at the retention time of 38 rain in accordance with authentic 5HETE standard and detectable amount of
"o
10"
w
9
,'2 _i 1
9.r
i
I
i
t
I
5
10
15
20
FMLP (nM)
Figure 2
Effects of the addition of 5HPETE on 3H-FMLP binding to human neutrophils. Specificbinding(total bindingminus nonspecific binding) is plotted against the concentration of 3HFMLP; (O) buffer-treatedcells, (e) 10_7 M 5HPETE-treated cells. Data represent mean+ SE (n= 3).
37
(1992)
5HETE 5HPETE
LTB4
ceit ( - )
celt (+)
Effect of 5HPETE on 3H-FMLP bindin.q to human neutrophils
Actions
lo
io
3'o
k i0
retention time (mini
Figure 3
Metabolism of exogenously added 14C-5HPETE by human neutrophils analyzed by reverse-phase HPLC equipped with radiodetector. Vertical bar represents radioactivityof ~4C and horizontal bar represents retention time (min): (upper panel) sample from HBSS incubatedwith laC-5HPETE;(lower panel) sample extracted by methanolfrom pelletsof neutrophilsuspension incubatedwith IaC-5HPETE.
radioactivity was not observed in fractions corresponding to LTB 4 and 5HPETE both in the supernatant and pellet of incubation mixture. Furthermore, conversion of 5HPETE to LTB 4 was not observed when 5HPETE (10 -8 M) was incubated for 1 min at 37~ in neutrophil suspension; the formed LTB4 was measured by RIA for LTB4 (sensitivity 10 pg). Discussion
The present study demonstrated that 5HPETE, at the concentration of 2 x 10-9-10 -8 M, which had minimal effect on calcium mobilization by itself, augmented 10-9 M FMLP-induced increase in cytosolic free calcium in fura-2-1oaded human neutrophils. According to Beckman's report, 1-4 gM of 5HPETE enhanced 1 0 - T M FMLP-induced neutrophil aggregation [4]. This discrepancy concerning about concentrations of primary stimulus F M L P between these two experiments might be explained as follows: first, FMLP-stimulated calcium mobilization using fura-2 could be observed at lower concentrations of F M L P than that required for the stimulation of biological functions such as aggregation. Second, calcium mobilization is thought to be obligatory but not a sufficient signal for the stimulation of functional reponses to physiological agonists [4]. In our experimental system, 5 H P E T E (2 x 10-9-10 8 M) could not en-
125
Agents Actions 37 (1992) hance F M L P - i n d u c e d calcium mobilization when higher concentrations ( > 10 -8 M) of F M L P were used as a primary agonist. This might be due to the fact that a higher concentration of F M L P could stimulate the formation of endogenous 5-1ipoxygenase (LO) metabolites including 5HPETE, which might mask the effect of exogenously added 5HPETE. As described in Materials and Methods section, we employed 10 9 M F M L P as the primary stimulus. Previous reports indicated that LTB4 formation could not be detected in neutrophil suspensions stimulated by 1 0 - 9 M of F M L P , suggesting that this concentrations of F M L P did not fully activate endogenous 5LO and/or LTA4 synthase [14, 15]. Also under our experimental condition, conversion of 5 H P E T E to LTB 4 was not observed. 5 H P E T E (10 7 M) at higher concentration by itself could mobilize cytosolic calcium, so the enhancing effect of 5 H P E T E in F M L P - s t i m u lated neutrophils was not recognized. In order to clarify the mechanism of enhancing effect of 5HPETE in calcium mobilization, we firstly checked the possibility that 5 H P E T E might augment F M L P binding to its specific receptor. As described in Materials and Methods section, our experiment of F M L P binding to neutrophils was carried out by the methods reported by Williams et al. [12]. According to their report, 12 min incubation time at 37 ~ was necessary to reach equilibrium for 3 H - F M L P binding to neutrophils. However, Vitkausas et al. reported that internalization of chemotactic peptide occurred by the incubation of neutrophils with 3 H - F M L P at 37~ [16]. We further confirmed that 5 H P E T E had no effect on 3 H - F M L P binding to its specific sites at shorter incubation time for 30 s to 5 min at 37 ~ (data not shown), in order to minimize the internalization of F M L P receptor complex and to compare the time course of binding assay with that of calcium mobilization using fura-2 which occurred within 30 s after stimulation. This data indicated that site of action of 5 H P E T E was not F M L P receptor, but possibly post-receptor sites. As shown in Fig. lb, the enhancement of calcium mobilization by 5 H P E T E was also observed in calcium-free medium, indicating that 5 H P E T E might mobilize cytosolic calcium of intracellular calcium store as well as calcium influx from extracellular medium. Naccache et al. [6] suggested that calcium influx in neutrophils induced by 5 H P E T E was due to its conversion to LTB4. However, following evidence would rule out the possibility that exogenously
added 5 H P E T E acted on intracellular calcium mobilization after conversion to LTB 4. First, exogenously added 5 H P E T E is reported to be p o o r substrate for 5LO and LTA 4 synthase activity [17]. Second, under our experimental conditions, exogenously added 5 H P E T E was converted to 5HETE but not to LTB 4. These d a t a demonstrated that 5 H P E T E by itself, without converting to LTB4, might play an important role in calcium mobilization in the process of F M L P - s t i m u l a t e d neutrophil activation. As previously reported, 5HETE, a reduced form of 5HPETE, was weaker in biological potencies than 5 H P E T E such as in the enhancement of F M L P stimulated neutrophil aggregation [4] and that of IgE-induced histamine releases in basophils [17]. In our data, 5HETE was less potent than 5 H P E T E in enhancing F M L P - s t i m u l a t e d calcium mobilization (data not shown). Accordingly, the enhancing effect of 5 H P E T E would be induced by 5 H P E T E itself, and not by 5HETE, produced from the reduction of 5 H P E T E (for example, by glutathione peroxidase). In summary, we have demonstrated that the possibility that 5 H P E T E by itself might play an important role in the regulation of cytosolic calcium in F M L P - s t i m u l a t e d neutrophil activation. Received 4 January 1992; accepted by I.G. Otterness 24 February 1992
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