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Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 589-595
July 31, 1986
REQUIREMENT OF FREE ARACHIDONIC ACID FOR LEUKOTRIENE B4 BIOSYNTHESIS BY 12-HYDROPEROXYEICOSATETRAENOICACID-STIMULATED NEUTROPHILS Kenji Kanaji, Minoru Okuma* Tateo Sugiyama Shigeki Sensaki Fumitaka Ushikubi and Haruto Uchino
The F i r s t Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received June 16, 1986
Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S,12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance l i q u i d chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with ]2-HPETE. In experiments using [l-14C]arachidonate labeled neutrophils, l i t t l e r a d i o a c t i v i t y was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous. ® 1986AcademicPress, lnc. Upon adequate stimulation, platelets and neutrophils release arachidonic acid from membrane phospholipids.
In p l a t e l e t s , the f a t t y acid is not only
converted to thromboxanes and prostaglandins via the cyclo-oxygenase pathway, but also converted to 12-HPETE and 12-HETE via the 12-1ipoxygenase pathway ( I ) . In neutrophils, the major pathway of arachidonic acid metabolism involves 5lipoxygenation, leading to formation of LTB4 and other hydroxy acids (2). Although the ionophore A23187 strongly stimulates the 5-1ipoxygenase metabolism in v i t r o (3), i t remains unclear what substance may act as an ionophore in vivo.
Recently i t has been demonstrated that platelet-derived 12-HPETE
stimulates LTB4 biosynthesis in human leukocytes (4).
We have also reported
that the synthesis of LTB4 is greatly increased by the addition of platelets * To whom correspondence should be addressed. Abbreviations used: FMLP, N-formylmethionylleucylphenylalanine; BSA, bovine serum albumin; HPLC, high-performance l i q u i d chromatography, LTB4, leukotriene B4; DiHETE, 5S,12S-dihydroxy-6,8,10,14-eicosatetraenoic acid; 5-HETE, 5Shydroxy-6,8,11,14-eicosatetraenoic acid; 12-HPETE, 12S-hydroperoxy-5,8,lO,14eicosatetraenoic acid; 12-HETE, 12S-hydroxy-5,8,10,14-eicosatetraenoic acid. 0006-291 X/86 $1.50 589
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in atLv ./orm reserved.
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to incubation mixtures containing neutrophils, FMLP, cytochalasin B and arachidonic acid, and that p l a t e l e t s can be substituted by 12-HPETE (5).
In
these reports, however, neutrophils are incubated with 12-HPETE and other stimuli
including arachidonic acid,FMLP and cytochalasin B, and i t has not yet
been investigated whether such stimuli
besides 12-HPETE are prerequisite to
the formation of LTB4 in 12-HPETE-stimulated neutrophils. To elucidate this question, we f i r s t
evaluated the e f f e c t of 12-HPETE on
the synthesis of 5-1ipoxygenase metabolites by human neutrophils in the presence or absence of FMLP or arachidonic acid.
Furthermore, we compared the e f -
f e c t of 12-HPETE with that of A23187 on the l i b e r a t i o n of endogenous arachidonic acid from neutrophils. MATERIALS AND METHODS Materials - [l-14C]arachidonic acid (55.5 mCi/mmol) was purchased from the Radiochemical Centre of Amersham (U.K.), unlabeled arachidonic acid from NuChek Prep. (Elysian, MN), FMLP and f a t t y acid-free BSA from Sigma Chemical Co. (St. Louis, MO), and A23187 from Calbiochem-Behring (La J o l l a , CA). 12-HPETE was synthesized from arachidonic acid by human p l a t e l e t lysates in the presence of indomethacin and p u r i f i e d by straight-phase HPLC as described previously (5). Analytical grade and HPLC grade solvents were obtained from Nakarai Chemicals (Kyoto, Japan). The other agents were the same as described previously (6). Preparation o__ffneutrophil suspensions - Human neutrophils isolated from c i t r a t e d venous blood of normal subjects (6) were suspended in the incubation buffer (50 mM Tris-HCl, I0~ mM NaCl, 5 mM glucose, pH 7.4) at a concentration of 3 x 107 c e l l s per ml (5). In some experiments, 1 ml of t h i s suspension was incubated with 0.14 ~Ci [l-14C]arachidonic acid (2.5 nmols) in the presence of 0.I % BSA for 60 min at 37°C (7, 8). Neutrophils were washed two times with the incubation buffer containing 0.I % BSA, once with BSA-free b u f f e r , and f i n a l l y suspended in the same BSA-free buffer at the same cell concentration. Incubation of unlabeled neutrophils - A f t e r the addition of 1 mM CaCI2, neutrophilsuspensions were preincubated f o r 5 min at 37°C. 12-HPETE dissolved in acetone was transferred to each assay tube and the solvent was evaporated under nitrogen. The reaction was i n i t i a t e d by the t r a n s f e r of 1 ml of neutrophil suspension to the assay tube and, i f needed,l ~M FMLP (in 5 ~I of the incubation buffer) or I0 - 80 ~M arachidonic acid (in 2 ~I of 0.I M Na2CO3 solution) was added without delay. A f t e r 5 min at 37°C, the reaction was stopped by the addition of 3 volumes of ethyl acetate and 450 ng of prostaglandin B2. Then the mixture was a c i d i f i e d to pH 3.0 with HCI. Extraction and evaporation were performed as described previously (6) and the residue was dissolved in 30 ~I methanol. Reversed-phaseHPLC analysis - Lipoxygenasc metabolites of arachidonic acid by unlabeled neutrophils were measured by reversed-phase HPLC as described previously (5). Release of labeled arachidonic acid - One ml of labeled neutrophil suspension was incubated ~ 12-HPETE (2.5 ~M or I0 ~M) in the presence or absence of 590
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FMLP (1 pM), or with A23187 (5 ~M) f o r 5 min at 370C as described above. A l l incubations were performed in polypropylene tubes and A23187 was dissolved as p r e v i o u s l y described (9). The reaction was stopped by rapid cooling a t 4°C and c e n t r i f u g a t i o n at 1,700 g f o r 5 min at the same temperature. An a l i q u o t (I00 ~I) of the supernatant from each sample was added to 5 ml s c i n t i l l a t i o n f l u i d and the r a d i o a c t i v i t y was measured. The r a d i o a c t i v i t y of I00 pl o f neutrophil suspension was also measured. The percent release o f incorporated r a d i o l a b e l in each reaction mixture was calculated as: [(counts per minute in supernatant)/(counts per minute in c e l l suspension)] x I00. The remainder of the supernatant was extracted three times with 3 volumes of ethyl acetate at pH 3.0. The combined e x t r a c t s were evaporated to dryness under nitrogen and the residue dissolved in small amounts of d i e t h y l ether/methanol (I : I ) was analysed by t h i n - l a y e r chromatography. T h i n - l a y e r chromatography - L i p i d e x t r a c t s obtained from incubations of labeled n e u t r o p h i l s were chromatographed on a s i l i c a gel p l a t e (E. Merck, Darmstadt, West Germany) along with standards, using the organic phase of an isooctane/ ethyl a c e t a t e / a c e t i c a c i d / w a t e r mixture (50 : I I 0 : 20 : I00, v / v ) as a solvent (I0). The 14C r a d i o a c t i v i t y was detected with a radiochromatogram scanner (JTC-501, Aloka, Mitaka, Japan). RESULTS AND DISCUSSION As shown in Fig. IA, s t i m u l a t i o n of n e u t r o p h i l s with 12-HPETE led to f o r mation of DiHETE, and the amount of t h i s metabolite was increased with i n creasing 12-HPETE concentration up to I0 pM.
However, no detectable amounts
of LTB4 and 5-HETE were obtained even when n e u t r o p h i l s were stimulated with
80 A
_~ .¢:
B
C
200
60
150
40
lOO
== v
W I
t--
20
50
_/
~
o 0
5
10
0
5
10
i
,
i
0
5
10
12-HPETE (pM)
Figure I.
Effects of 12-HPETE on the synthesis of 5-1ipoxygenase metabolites by neutrophils in the presence or absence of another stimulus. Unlabeled neutrophils (3 x 107 cells/ml) were incubated with varying concentrations of 12-HPETE as indicated in the absence of another stimulus (A), or in the presence of 1 pM FMLP (B) or 40 pM arachidonic acid (C) for 5 min at 37°C. Extracted metabolites were analysed by reversed-phase HPLC (5). Values are mean ± SD of 3 or 4 experiments. • , LTB4; 0 , 5-HETE; ZS , DiHETE. 591
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400
~=100
300
~
200
50
i'~ 2 5
~
0
o
100 ~
0
20
40
60
80
Arachidonic acid (~M)
Figure 2.
Effects of concentrations of arachidonic acid added on the synthesis of 5-1ipoxygenase metabolites by 12-HPETE-stimulated neutrophils. Symbols and experimental conditions are the same as those in Fig. l except that neutrophils were incubated with varying concentrations of arachidonic acid as indicated in the presence of lO ~M 12-HPETE. The data are representatives of 2 separate experiments.
I0 ~M 12-HPETE.
As 12-HETE formed from 12-HPETE i s converted to DiHETE by 5-
lipoxygenase ( l l ,
12), !2-HPETE i s assumed to serve as a substrate f o r the
synthesis of DiHETE (13).
However, 5-1ipoxygenase of n e u t r o p h i l s needs to be
a c t i v a t e d by a strong s t i m u l u s , such as A23187, f o r the conversion from 12-HETE to DiHETE ( l l ,
12).
!n our experiments, t h e r e f o r e , 12-HPETE was considered to
s t i m u l a t e the 5-1ipoxygenase a c t i v i t y
and to be converted to DiHETE.
12-HPETE induced the synthesis of small amounts of LTB4 in FMLP-stimulated n e u t r o p h i l s (Fig. I B ) .
DiHETE synthesis was greater than t h a t obtained
w i t h o u t FMLP, and 5-HETE was not produced in t h i s incubation. When n e u t r o p h i l s were incubated with 40 ~M arachidonic acid, the a d d i t i o n of 12-HPETE g r e a t l y increased the synthesis of LTB4 and 5-HETE, but DiHETE formation was not so remarkably increased as t h a t obtained from the incubation with 12-HPETE alone or with 12-HPETE plus FMLP (Fig. IC).
These e f f e c t s of
exogenous arachidonic acid were f u r t h e r studied by using neutrophil suspensions incubated with lO ~M 12-HPETE in the absence or presence of varying concentrat i o n s of arachidonic acid up to 80 ~M (Fig. 2).
The formation of LTB4 was
l e v e l e d o f f at the concentration of 20 ~M, and 5-HETE synthesis was increased almost l i n e a r l y up to the highest concentration employed, whereas DiHETE f o r mation was decreased with increasing concentrations of arachidonic acid added. 592
Vol. 138, No. 2, 1986 Table I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Release of radioactivity from [l-14C]arachidonate-labeled neutrophils
Sti mul us
Radioacti vi ty (%) 1.5±0.2 1.6±0.2 1.7±0.3 1.7±0.2 1.8±0.2 5.0±0.5
None
12-HPETE (2.5 IJM) 12-HPETE (I0 HM) FMLP (I ~M) + 12-HPETE (2.5 !JM) FMLP (I ~M) + 12-HPETE (I0 ~M) A23187 (5 IJM)
* [l-14C]arachidonate-labeled neutrophils (3 x 107 cells/ml) were incubated with various stimuli as indicated for 5 min at 37°C. ** Values were calculated as described in MATERIALSAND METHODS(mean ± SD, n = 4).
The above data indicate that the synthesis of LTB4 by 12-HPETE~stimulated neutrophils requires another stimulus, especially arachidonic acid, even i f 5-1ipoxygenase is activated by 12-HPETE. The data in Fig. 2 also suggest that 12-HPETE or 12-HETE derived from i t may compete with arachidonic acid for 5lipoxygenation.
I t has been demonstrated that FMLP l i b e r a t e s arachidonic acid
and stimulates the 5-1ipoxygenase pathway in human neutrophils although not so e f f e c t i v e l y as A23187 (14 - 16).
Exogenous arachidonic acid is converted to
LTB4 and 5-HETE i f 5-1ipoxygenase is activated (2, 3).
Indeed radiolabeled
compounds co-migrating with LTB4 and 5-HETE by t h i n - l a y e r chromatography were obtained from incubation of unlabeled neutrophils with I0 ~M 12-HPETE and 40 ~M [l-14C]arachidonic acid (5 mCi/mmol) (data not shown).
Therefore, i t is
assumed that liberated arachidonic acid is i n s u f f i c i e n t to LTB4 formation in the incubation of neutrophils with 12-HPETE alone.
This is probably because
12-HPETE is unable to release arachidonic acid e f f e c t i v e l y in neutrophils. Accordingly we investigated e f f e c t s of 12-HPETE on the release of radiolabel from [l-14C]arachidonate-labeled neutrophils (Table I ) .
With ionophore
a c t i v a t i o n , about 5 % o f the incorporated r a d i o a c t i v i t y was released, and t h i n - l a y e r chromatography showed that radioactive components extracted from the supernatant co-migrated with arachidonic acid, 5-HETE and LTB4 (Fig. 3A).
Al-
though a peak between phospholipids and LTB4 on the t h i n - l a y e r radiochromatogram was not i d e n t i f i e d ,
this compound was assumed to be 5,12,20-trihydroxy593
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A o
g
'S_
_~
.z2_
.i
o
~E
B
i
0
i
i
i
i
4
8
12
16
D i s t a n c e from origin (cm)
Figure 3.
Representative thin-layer radiochromatograms of arachidonate metabolites released from labeled neutrophils by 5 ~M A23187 (A) or by 1 ~M FMLP plus I0 ~M 12-HPETE (B). The incubation was the same as that in Table I, and arachidonate metabolites extracted from the supernatant were developed as described in MATERIALSAND METHODS.
eicosatetraenoic acid ( I 0 ) .
in contrast, neutrophils stimulated with I0 ~M
12-HPETE released only 1.8 % and Io7 % of the incorporated r a d i o a c t i v i t y in the presence of 1 ~M FMLP and in i t s absence,respectively (Table I ) . the r a d i o a c t i v i t y
Most of
in the supernatant appeared to co-migrate with neutral
l i p i d s by t h i n - l a y e r chromatography when neutrophils were incubated with 12HPETE plus FMLP (Fig. 3B) or with 12-HPETE alone (data not shown).
Although
the data in Fig. IB demonstrated the synthesis of small amounts of LTB4, the peak corresponding to LTB4 was not detectable on the t h i n - l a y e r radiochromatogram shown in Fig. 3B. These findings reinforce the hypothesis that 12-HPETE can not release an enough amount of arachidonic acid to be oxygenated to LTB4 overcoming i t s competition with 12-HETE or 12-HPETE. Therefore, free arachidonic acid needs to be supplied to 12-HPETE-stimulated neutrophils for the synthesis of LTB4. This may be possible by the endogenous release of arachidonic acid in the neutrophils themselves stimulated by some agents, such as FMLP, or exogenous arachidonic acid derived from other sources, such as p l a t e l e t s , or both. Activated p l a t e l e t s release arachidonic acid as well as 12-HPETE ( I ) , 594
and
Vo1.138, No. 2,1986
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
platelet-derived arachidonic acid can be converted to LTB4 and 5-HETE by a c t i vated neutrophils (12).
Thus, platelets seem to stimulate the synthesis of
LTB4 in neutrophils by supplying substrates as well as enhancing the 5lipoxygenase a c t i v i t y . ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientic Research from the Ministry of Education, Science and Culture of Japan, and grants from the Sankyo Research Foundation for Life Sciences and from the Takeda Medical Foundation. REFERENCES I . Marcus, A.J. (1978) d. Lipid Res. 19:793-826. 2. Samuelsson, B. (1983) Science 220:568-575. 3. Borgeat, P., and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA 76: 2148-2152. 4. Maclouf, J., Fruteau de Laclos, B., and Borgeat, P. (1982) Proc. Natl. Acad. Sci. USA 79:6042-6046. 5. Kanaji, K., Okuma, M., and Uchino, H. (1986) Blood 67:903-908. 6. Takayama, H., Okuma, M., Kanaji, K., Sugiyama, T., Sensaki, S., and Uchino, H. (1983) Prostaglandins Leukotrienes Med. 12:261-272. 7. B i l l a h , M.M., Bryant, R.W., and Siegel, M.I. (1985) J. Biol. Chem. 260: 6899-6906. 8. Lee, T.H., Mencia-Huerta, J.-M., Shih, C., Corey, E.J., Lewis, R.A., and Austen, K.F. (1984) J. Clin. Invest. 74:1922-1933. 9. Okuma, M., Takayama, H., and Uchino, H. (1982) Br. J. Haematol. 51:469477. lO. Sun, F.F., and McGuire J.C. (1984) Biochim. Biophys. Acta 794:56-64. I I . Borgeat, P., Fruteau de Laclos, B., Picard, S., Drapeau, J., Vallerand, P., and Corey, E.J. (1982) Prostaglandins 23:713-724. 12. Marcus, A.J., Broekman, M.J., Safier, L.B., Ullman, H.L., Islam, N., Serhan, C.N., Rutherford, L.E., Korchak, H.M., and Weissmann, G. (1982) Biochem. Biophys. Res. Commun. I09:130-137. 13. Borgeat, P., Fruteau de Laclos, B., and Maclouf, J. (1983) Biochem. Pharmacol. 32:381-387. 14. Ham, E.A., Soderman, D.D., Zanetti, M.E., Dougherty, H.W., McCauley, E., and Kuehl, F.A., Jr. (1983) Proc. Natl. Acad. Sci. USA 80:4349~4352. 15. Jubiz, W., R~dmark, 0., Malmsten, C., Hansson, G., Lindgren, J.A., Palmblad, J., Ud~n, A.-M., and Samuelsson, B. (1982) J. Biol. Chem. 257: 6106-6110. 16. Salari, H., Braquet, P., Naccache, P., and Borgeat, P. (1985) Inflammation 9:127-138.
595
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS Pages 589-595
July 31, 1986
REQUIREMENT OF FREE ARACHIDONIC ACID FOR LEUKOTRIENE B4 BIOSYNTHESIS BY 12-HYDROPEROXYEICOSATETRAENOICACID-STIMULATED NEUTROPHILS Kenji Kanaji, Minoru Okuma* Tateo Sugiyama Shigeki Sensaki Fumitaka Ushikubi and Haruto Uchino
The F i r s t Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received June 16, 1986
Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S,12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance l i q u i d chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with ]2-HPETE. In experiments using [l-14C]arachidonate labeled neutrophils, l i t t l e r a d i o a c t i v i t y was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous. ® 1986AcademicPress, lnc. Upon adequate stimulation, platelets and neutrophils release arachidonic acid from membrane phospholipids.
In p l a t e l e t s , the f a t t y acid is not only
converted to thromboxanes and prostaglandins via the cyclo-oxygenase pathway, but also converted to 12-HPETE and 12-HETE via the 12-1ipoxygenase pathway ( I ) . In neutrophils, the major pathway of arachidonic acid metabolism involves 5lipoxygenation, leading to formation of LTB4 and other hydroxy acids (2). Although the ionophore A23187 strongly stimulates the 5-1ipoxygenase metabolism in v i t r o (3), i t remains unclear what substance may act as an ionophore in vivo.
Recently i t has been demonstrated that platelet-derived 12-HPETE
stimulates LTB4 biosynthesis in human leukocytes (4).
We have also reported
that the synthesis of LTB4 is greatly increased by the addition of platelets * To whom correspondence should be addressed. Abbreviations used: FMLP, N-formylmethionylleucylphenylalanine; BSA, bovine serum albumin; HPLC, high-performance l i q u i d chromatography, LTB4, leukotriene B4; DiHETE, 5S,12S-dihydroxy-6,8,10,14-eicosatetraenoic acid; 5-HETE, 5Shydroxy-6,8,11,14-eicosatetraenoic acid; 12-HPETE, 12S-hydroperoxy-5,8,lO,14eicosatetraenoic acid; 12-HETE, 12S-hydroxy-5,8,10,14-eicosatetraenoic acid. 0006-291 X/86 $1.50 589
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in atLv ./orm reserved.
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to incubation mixtures containing neutrophils, FMLP, cytochalasin B and arachidonic acid, and that p l a t e l e t s can be substituted by 12-HPETE (5).
In
these reports, however, neutrophils are incubated with 12-HPETE and other stimuli
including arachidonic acid,FMLP and cytochalasin B, and i t has not yet
been investigated whether such stimuli
besides 12-HPETE are prerequisite to
the formation of LTB4 in 12-HPETE-stimulated neutrophils. To elucidate this question, we f i r s t
evaluated the e f f e c t of 12-HPETE on
the synthesis of 5-1ipoxygenase metabolites by human neutrophils in the presence or absence of FMLP or arachidonic acid.
Furthermore, we compared the e f -
f e c t of 12-HPETE with that of A23187 on the l i b e r a t i o n of endogenous arachidonic acid from neutrophils. MATERIALS AND METHODS Materials - [l-14C]arachidonic acid (55.5 mCi/mmol) was purchased from the Radiochemical Centre of Amersham (U.K.), unlabeled arachidonic acid from NuChek Prep. (Elysian, MN), FMLP and f a t t y acid-free BSA from Sigma Chemical Co. (St. Louis, MO), and A23187 from Calbiochem-Behring (La J o l l a , CA). 12-HPETE was synthesized from arachidonic acid by human p l a t e l e t lysates in the presence of indomethacin and p u r i f i e d by straight-phase HPLC as described previously (5). Analytical grade and HPLC grade solvents were obtained from Nakarai Chemicals (Kyoto, Japan). The other agents were the same as described previously (6). Preparation o__ffneutrophil suspensions - Human neutrophils isolated from c i t r a t e d venous blood of normal subjects (6) were suspended in the incubation buffer (50 mM Tris-HCl, I0~ mM NaCl, 5 mM glucose, pH 7.4) at a concentration of 3 x 107 c e l l s per ml (5). In some experiments, 1 ml of t h i s suspension was incubated with 0.14 ~Ci [l-14C]arachidonic acid (2.5 nmols) in the presence of 0.I % BSA for 60 min at 37°C (7, 8). Neutrophils were washed two times with the incubation buffer containing 0.I % BSA, once with BSA-free b u f f e r , and f i n a l l y suspended in the same BSA-free buffer at the same cell concentration. Incubation of unlabeled neutrophils - A f t e r the addition of 1 mM CaCI2, neutrophilsuspensions were preincubated f o r 5 min at 37°C. 12-HPETE dissolved in acetone was transferred to each assay tube and the solvent was evaporated under nitrogen. The reaction was i n i t i a t e d by the t r a n s f e r of 1 ml of neutrophil suspension to the assay tube and, i f needed,l ~M FMLP (in 5 ~I of the incubation buffer) or I0 - 80 ~M arachidonic acid (in 2 ~I of 0.I M Na2CO3 solution) was added without delay. A f t e r 5 min at 37°C, the reaction was stopped by the addition of 3 volumes of ethyl acetate and 450 ng of prostaglandin B2. Then the mixture was a c i d i f i e d to pH 3.0 with HCI. Extraction and evaporation were performed as described previously (6) and the residue was dissolved in 30 ~I methanol. Reversed-phaseHPLC analysis - Lipoxygenasc metabolites of arachidonic acid by unlabeled neutrophils were measured by reversed-phase HPLC as described previously (5). Release of labeled arachidonic acid - One ml of labeled neutrophil suspension was incubated ~ 12-HPETE (2.5 ~M or I0 ~M) in the presence or absence of 590
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FMLP (1 pM), or with A23187 (5 ~M) f o r 5 min at 370C as described above. A l l incubations were performed in polypropylene tubes and A23187 was dissolved as p r e v i o u s l y described (9). The reaction was stopped by rapid cooling a t 4°C and c e n t r i f u g a t i o n at 1,700 g f o r 5 min at the same temperature. An a l i q u o t (I00 ~I) of the supernatant from each sample was added to 5 ml s c i n t i l l a t i o n f l u i d and the r a d i o a c t i v i t y was measured. The r a d i o a c t i v i t y of I00 pl o f neutrophil suspension was also measured. The percent release o f incorporated r a d i o l a b e l in each reaction mixture was calculated as: [(counts per minute in supernatant)/(counts per minute in c e l l suspension)] x I00. The remainder of the supernatant was extracted three times with 3 volumes of ethyl acetate at pH 3.0. The combined e x t r a c t s were evaporated to dryness under nitrogen and the residue dissolved in small amounts of d i e t h y l ether/methanol (I : I ) was analysed by t h i n - l a y e r chromatography. T h i n - l a y e r chromatography - L i p i d e x t r a c t s obtained from incubations of labeled n e u t r o p h i l s were chromatographed on a s i l i c a gel p l a t e (E. Merck, Darmstadt, West Germany) along with standards, using the organic phase of an isooctane/ ethyl a c e t a t e / a c e t i c a c i d / w a t e r mixture (50 : I I 0 : 20 : I00, v / v ) as a solvent (I0). The 14C r a d i o a c t i v i t y was detected with a radiochromatogram scanner (JTC-501, Aloka, Mitaka, Japan). RESULTS AND DISCUSSION As shown in Fig. IA, s t i m u l a t i o n of n e u t r o p h i l s with 12-HPETE led to f o r mation of DiHETE, and the amount of t h i s metabolite was increased with i n creasing 12-HPETE concentration up to I0 pM.
However, no detectable amounts
of LTB4 and 5-HETE were obtained even when n e u t r o p h i l s were stimulated with
80 A
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B
C
200
60
150
40
lOO
== v
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t--
20
50
_/
~
o 0
5
10
0
5
10
i
,
i
0
5
10
12-HPETE (pM)
Figure I.
Effects of 12-HPETE on the synthesis of 5-1ipoxygenase metabolites by neutrophils in the presence or absence of another stimulus. Unlabeled neutrophils (3 x 107 cells/ml) were incubated with varying concentrations of 12-HPETE as indicated in the absence of another stimulus (A), or in the presence of 1 pM FMLP (B) or 40 pM arachidonic acid (C) for 5 min at 37°C. Extracted metabolites were analysed by reversed-phase HPLC (5). Values are mean ± SD of 3 or 4 experiments. • , LTB4; 0 , 5-HETE; ZS , DiHETE. 591
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
400
~=100
300
~
200
50
i'~ 2 5
~
0
o
100 ~
0
20
40
60
80
Arachidonic acid (~M)
Figure 2.
Effects of concentrations of arachidonic acid added on the synthesis of 5-1ipoxygenase metabolites by 12-HPETE-stimulated neutrophils. Symbols and experimental conditions are the same as those in Fig. l except that neutrophils were incubated with varying concentrations of arachidonic acid as indicated in the presence of lO ~M 12-HPETE. The data are representatives of 2 separate experiments.
I0 ~M 12-HPETE.
As 12-HETE formed from 12-HPETE i s converted to DiHETE by 5-
lipoxygenase ( l l ,
12), !2-HPETE i s assumed to serve as a substrate f o r the
synthesis of DiHETE (13).
However, 5-1ipoxygenase of n e u t r o p h i l s needs to be
a c t i v a t e d by a strong s t i m u l u s , such as A23187, f o r the conversion from 12-HETE to DiHETE ( l l ,
12).
!n our experiments, t h e r e f o r e , 12-HPETE was considered to
s t i m u l a t e the 5-1ipoxygenase a c t i v i t y
and to be converted to DiHETE.
12-HPETE induced the synthesis of small amounts of LTB4 in FMLP-stimulated n e u t r o p h i l s (Fig. I B ) .
DiHETE synthesis was greater than t h a t obtained
w i t h o u t FMLP, and 5-HETE was not produced in t h i s incubation. When n e u t r o p h i l s were incubated with 40 ~M arachidonic acid, the a d d i t i o n of 12-HPETE g r e a t l y increased the synthesis of LTB4 and 5-HETE, but DiHETE formation was not so remarkably increased as t h a t obtained from the incubation with 12-HPETE alone or with 12-HPETE plus FMLP (Fig. IC).
These e f f e c t s of
exogenous arachidonic acid were f u r t h e r studied by using neutrophil suspensions incubated with lO ~M 12-HPETE in the absence or presence of varying concentrat i o n s of arachidonic acid up to 80 ~M (Fig. 2).
The formation of LTB4 was
l e v e l e d o f f at the concentration of 20 ~M, and 5-HETE synthesis was increased almost l i n e a r l y up to the highest concentration employed, whereas DiHETE f o r mation was decreased with increasing concentrations of arachidonic acid added. 592
Vol. 138, No. 2, 1986 Table I.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Release of radioactivity from [l-14C]arachidonate-labeled neutrophils
Sti mul us
Radioacti vi ty (%) 1.5±0.2 1.6±0.2 1.7±0.3 1.7±0.2 1.8±0.2 5.0±0.5
None
12-HPETE (2.5 IJM) 12-HPETE (I0 HM) FMLP (I ~M) + 12-HPETE (2.5 !JM) FMLP (I ~M) + 12-HPETE (I0 ~M) A23187 (5 IJM)
* [l-14C]arachidonate-labeled neutrophils (3 x 107 cells/ml) were incubated with various stimuli as indicated for 5 min at 37°C. ** Values were calculated as described in MATERIALSAND METHODS(mean ± SD, n = 4).
The above data indicate that the synthesis of LTB4 by 12-HPETE~stimulated neutrophils requires another stimulus, especially arachidonic acid, even i f 5-1ipoxygenase is activated by 12-HPETE. The data in Fig. 2 also suggest that 12-HPETE or 12-HETE derived from i t may compete with arachidonic acid for 5lipoxygenation.
I t has been demonstrated that FMLP l i b e r a t e s arachidonic acid
and stimulates the 5-1ipoxygenase pathway in human neutrophils although not so e f f e c t i v e l y as A23187 (14 - 16).
Exogenous arachidonic acid is converted to
LTB4 and 5-HETE i f 5-1ipoxygenase is activated (2, 3).
Indeed radiolabeled
compounds co-migrating with LTB4 and 5-HETE by t h i n - l a y e r chromatography were obtained from incubation of unlabeled neutrophils with I0 ~M 12-HPETE and 40 ~M [l-14C]arachidonic acid (5 mCi/mmol) (data not shown).
Therefore, i t is
assumed that liberated arachidonic acid is i n s u f f i c i e n t to LTB4 formation in the incubation of neutrophils with 12-HPETE alone.
This is probably because
12-HPETE is unable to release arachidonic acid e f f e c t i v e l y in neutrophils. Accordingly we investigated e f f e c t s of 12-HPETE on the release of radiolabel from [l-14C]arachidonate-labeled neutrophils (Table I ) .
With ionophore
a c t i v a t i o n , about 5 % o f the incorporated r a d i o a c t i v i t y was released, and t h i n - l a y e r chromatography showed that radioactive components extracted from the supernatant co-migrated with arachidonic acid, 5-HETE and LTB4 (Fig. 3A).
Al-
though a peak between phospholipids and LTB4 on the t h i n - l a y e r radiochromatogram was not i d e n t i f i e d ,
this compound was assumed to be 5,12,20-trihydroxy593
Vol. 138, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A o
g
'S_
_~
.z2_
.i
o
~E
B
i
0
i
i
i
i
4
8
12
16
D i s t a n c e from origin (cm)
Figure 3.
Representative thin-layer radiochromatograms of arachidonate metabolites released from labeled neutrophils by 5 ~M A23187 (A) or by 1 ~M FMLP plus I0 ~M 12-HPETE (B). The incubation was the same as that in Table I, and arachidonate metabolites extracted from the supernatant were developed as described in MATERIALSAND METHODS.
eicosatetraenoic acid ( I 0 ) .
in contrast, neutrophils stimulated with I0 ~M
12-HPETE released only 1.8 % and Io7 % of the incorporated r a d i o a c t i v i t y in the presence of 1 ~M FMLP and in i t s absence,respectively (Table I ) . the r a d i o a c t i v i t y
Most of
in the supernatant appeared to co-migrate with neutral
l i p i d s by t h i n - l a y e r chromatography when neutrophils were incubated with 12HPETE plus FMLP (Fig. 3B) or with 12-HPETE alone (data not shown).
Although
the data in Fig. IB demonstrated the synthesis of small amounts of LTB4, the peak corresponding to LTB4 was not detectable on the t h i n - l a y e r radiochromatogram shown in Fig. 3B. These findings reinforce the hypothesis that 12-HPETE can not release an enough amount of arachidonic acid to be oxygenated to LTB4 overcoming i t s competition with 12-HETE or 12-HPETE. Therefore, free arachidonic acid needs to be supplied to 12-HPETE-stimulated neutrophils for the synthesis of LTB4. This may be possible by the endogenous release of arachidonic acid in the neutrophils themselves stimulated by some agents, such as FMLP, or exogenous arachidonic acid derived from other sources, such as p l a t e l e t s , or both. Activated p l a t e l e t s release arachidonic acid as well as 12-HPETE ( I ) , 594
and
Vo1.138, No. 2,1986
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platelet-derived arachidonic acid can be converted to LTB4 and 5-HETE by a c t i vated neutrophils (12).
Thus, platelets seem to stimulate the synthesis of
LTB4 in neutrophils by supplying substrates as well as enhancing the 5lipoxygenase a c t i v i t y . ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Scientic Research from the Ministry of Education, Science and Culture of Japan, and grants from the Sankyo Research Foundation for Life Sciences and from the Takeda Medical Foundation. REFERENCES I . Marcus, A.J. (1978) d. Lipid Res. 19:793-826. 2. Samuelsson, B. (1983) Science 220:568-575. 3. Borgeat, P., and Samuelsson, B. (1979) Proc. Natl. Acad. Sci. USA 76: 2148-2152. 4. Maclouf, J., Fruteau de Laclos, B., and Borgeat, P. (1982) Proc. Natl. Acad. Sci. USA 79:6042-6046. 5. Kanaji, K., Okuma, M., and Uchino, H. (1986) Blood 67:903-908. 6. Takayama, H., Okuma, M., Kanaji, K., Sugiyama, T., Sensaki, S., and Uchino, H. (1983) Prostaglandins Leukotrienes Med. 12:261-272. 7. B i l l a h , M.M., Bryant, R.W., and Siegel, M.I. (1985) J. Biol. Chem. 260: 6899-6906. 8. Lee, T.H., Mencia-Huerta, J.-M., Shih, C., Corey, E.J., Lewis, R.A., and Austen, K.F. (1984) J. Clin. Invest. 74:1922-1933. 9. Okuma, M., Takayama, H., and Uchino, H. (1982) Br. J. Haematol. 51:469477. lO. Sun, F.F., and McGuire J.C. (1984) Biochim. Biophys. Acta 794:56-64. I I . Borgeat, P., Fruteau de Laclos, B., Picard, S., Drapeau, J., Vallerand, P., and Corey, E.J. (1982) Prostaglandins 23:713-724. 12. Marcus, A.J., Broekman, M.J., Safier, L.B., Ullman, H.L., Islam, N., Serhan, C.N., Rutherford, L.E., Korchak, H.M., and Weissmann, G. (1982) Biochem. Biophys. Res. Commun. I09:130-137. 13. Borgeat, P., Fruteau de Laclos, B., and Maclouf, J. (1983) Biochem. Pharmacol. 32:381-387. 14. Ham, E.A., Soderman, D.D., Zanetti, M.E., Dougherty, H.W., McCauley, E., and Kuehl, F.A., Jr. (1983) Proc. Natl. Acad. Sci. USA 80:4349~4352. 15. Jubiz, W., R~dmark, 0., Malmsten, C., Hansson, G., Lindgren, J.A., Palmblad, J., Ud~n, A.-M., and Samuelsson, B. (1982) J. Biol. Chem. 257: 6106-6110. 16. Salari, H., Braquet, P., Naccache, P., and Borgeat, P. (1985) Inflammation 9:127-138.
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