102

Biochimica et Biophynica Acta. 1133( 1991) 102-106 ~ 1991 ElsevierScience Publishers B.V. All rights reserved 0167.4889/91/$113.511

BBAMCR 13057

Calcium mobilization and right-angle light scatter responses to 12-oxo-derivatives of arachidonic acid in neutrophiis: evidence for the involvement of the leukotriene B 4 receptor Paul H. Naccache

i, Y v e s L e b l a n c 2, J o s h u a R o k a c h 2.., P a o l a P a t r i g n a n i Fruteau de Laclos 1 and Pierre Borgeat J

I, B e r n a r d

i Centre de Recherche en Inflammation, Immunologic et Rhnmatologie, Centre de Recherche du CHUL. Sainte-Foy (Canada) and 2 Centre de Recherche Th~rapeutique Merck Frosst, Merck Frosst Canada btc., Pointe.Claire. Dort'al (Canada)

(Received 21 May 19911

Key words: 12-Lipoxygenase:Eicosanoid;Inflammation;Chemotaxis:Carbonylderivative The biological activities of two carbonyl compounds derived from arachidonic acid, (5Z,8Z,lOE,14Z).12.keto. 5,8,10,14.eicosatetraenoic acid (12-OxoETE) and (SZ,8Z,lOE).12-oxo.5,8,10.dodecatrienoic acid (12-OxoDTrE) were investigated. The ability of these compounds to induce a mobilization of calcium and to trigger a right-angle scatter response in isolated peripheral blood human neutrophils was determined. The W,o compounds induced a rapid and dose-dependent increase in the concentration of cytoplasmic free calcium; these effects were clearly detectable at concentrations >_ 1 0 - a M. Pre-exposure of neutrophils to leukotriene B 4 c,~mpletely abolished the calcium mobilization induced by 12-OxoDTre and 12.OxoETE, while pre.exposure of ~,'~e cells to the carbonyl compounds only slightly reduced the response to subsequent stimulation of neutrophils by leukotriene B 4. The carbon),l compounds also induced a decrease in right-angle light scatter and these effects were abolished by pretreatmcnt of neutrophils with leukotriene B 4. These data demonstrate that 12-OxoETE and 12-OxoDTrE show significant agonist activities towards human neutrophils and strongly suggest that their mechanisms of action involve the leukotriene B 4 binding sites or a common activation sequence.

Introduction The carbonyl c~mpounds ( 5 Z , 8 Z , I O E , 1 4 Z ) - 1 2 - k e t o 5,8,10,14-eicosatetraenoic acid (12-OxoETE) and ( 5 Z , S Z , lOE)-12-o×o-5,8,10-dodecatrienoic acid (12OxoDTrE) are products of the heme-catalyzed transformation of ( 5 Z , 8 Z , l O E , 1 4 Z ) - ( 1 2 S ) - h y d r o p e r o x y 5,8,10,14-eicosatetraenoic acid (12S-HpETE), itself a product of the metabolism of arachidonic acid by the 12-1ipoxygenase. The short chain aldehyde 12OxoDTrE was first identified in porcine neutrophils

* Present address: Institute of MedicinalChemistry,Melbourne. FL, U.S.A. Abbreviations: 12-OxoETE. (5Z, 8Z, 10E. 14Z)-12-keto-5.8.10.14elcosatetraenolc acid; 12-OxoDTrE. (5Z.8Z,lOE)-12-oxo-5,8,lOdodecatrienoic acid; L'I'B4, leukotnene B4. Correspondence: P. Borgeat. Centre de Recherche en Inflammation. Immunologic et Rhumatologie. Centre de Recherche du CHUL, 2705 noul. Laurier, Sainte Foy, QC, Canada GIV aG2.

[1], and both 12-OxoETE and 12-OxoDTrE were isolated from incubates of human platelets [2,3], two cell types rich in 12-1ipoxygenase acitivity [4,5]. The biological activities of the two compounds are still largely unknown. Although Glascow et al. [1] reported that the short chain aldehyde showed chemotactic activity towards human neutrophils, no data are presently available concerning 12-OxoETE. A variety of lipoxygenase products have been shown to stimulate neutrophils [6], the most active compound being ( 6 Z , 8 E , 1 0 E , 1 4 Z ) - ( 5 S , 1 2 R ) - d i h y d r o x y - 6 , 8 , 1 0 , 1 4 eicosatetraenoic acid (leukotriene B~) [7], a product of the 5-1ipoxygenase pathway [8]. Monohydroxyeicosatetraenoic acids such as ( 6 E , 8 Z , 1 1 Z , 1 4 Z ) - ( 5 S ) - h y d r o x y 6,8,11,14-eicosatetraenoic acid (5S-HETE) and (5Z, 8 Z , l O E , 1 4 Z )-(12 S )-hydrox,y-5,8,l O,14-eicosatetrae noic

acid (12S-HETE), derived respectively from the 5- and 12-1ipoxygenases, also activate neutrophils, though with a potency more than two orders of magnitude lower than that of leukotrielte B 4 [9,10]; 12R-HETE, a compound initially isolated from psoriatic scales and re-

103 cently described as a product of the metabolism of arachidonic acid by a cytochrome P-450-type enzyme was also found to be an agonist of human neutrophils [11]. Interestingly, binding and cross-desensitization experiments strongly suggested that 12S-HETE and 12R-HETE (which are structurally related to the two 12-Oxo fatty acids investigated in the present study) and leukotriene [34 share the same binding sites and a common activation sequence [12,13] Tl~e present investigation was undertaken to further characterize the effect of 12-OxoDTrE on human neutrophils and to examine the activity of 12-OxoETE on these cells. The emphasis was placecl on the measurement of cytoplasmic calcium levels and cytoskeletal reorganization, two in vitro tests of neutrophil function that may be directly relevant to cell locomotion and thus to neutrophil accumulation at inflammatory sites. The results to be described demonstrate that 12OxoETE and 12-OxoDTrE activate ncutrophils at submicromolar concentrations and suggest the involvement of the leukotriene B 4 binding sites or of a common activation sequence.

Materials and Methods Fura-2/AM was purchased from Molecular Probes (Junction City, OR, U.S.A.). Leukotriene B 4 (LTB 4) was a generous gift of Dr. R. Young (Merck-Frosst, Pointe-Claire, Quebec, Canada). The 12-OxoETE and 12-OxoDTrE were obtained by chemical synthesis as described below. The concentrations of the stock solutions of LTB 4 and the two carbonyl compounds were determined by ultraviolet photometry.

Preparation of neutrophil suspensions Human neutrophils were isolated following dextran sedimentation of erythrocytes and centrifugation on Hypaque-Ficoli cushions. The remaining erythrocytes were lysed by hypotonic shock (20 s at room temperature). Neutrophils represented at least 97% of the cell suspensions. Cell viability, as estimated by trypan blue exclusion, was better than 98%. The cells were resuspended in Hanks' balanced salt solution (HBSS) (Gibco, Burlington, Ontario, Canada) containing 10 mM Hepes (pH 7.4). Measurements of intracellular free calcium concentration and right-angle liyl~t scatter lntracellular free calcium was monitored using the fluorescent probe fura-2 as described in Grynkievicz et al. [14] and Faucher and Naccaehe [15]. Briefly, neutrophil suspensions (1-107 ceUs/ml) were incubated with 1 /zM fura-2 AM for 30 rain at 37°C. The cells were then washed free of the extracellular probe, resuspended at 5" 106 cells/ml and allowed to re-equil':brate for 5-10 rain at 37°C. They were then trans-

1

2

3

4

s

Fig. I. Synthesisof 12-OxoDTrE.(a) t-BuPH,SiCI/Et~N/DMAP/ CH,CI:; (b) cqric acid/THF/H,:D/AcOH/room temperature;tc) phosphonium salt/Li'rlMDS/THF; (d) n-BuaNF/THF/O°-25 ° C; (e) MnO2/AcOEt/roomtemperature.

ferred to the thermostatted (37°C) cuvette compartment of the fluorimeter (SLM 8000 C, Urbana, IL, U.S.A.) and the Huorescence monitored (excitation and emission wavelengths, 340 and 510 nm, respectively) under continuous stirring. The agonists were added to tile cell suspeo.sio,l~ of solutions in DMSO; the final concentration of DMSO in the cell suspensions was 0.05%. The internal calcium concentrations were calculated as described in Tsien et al. [16] using 224 nM as the dissociation constant of calcium and fura-2. The right-angle light scatter response was monitored simultaneously with the calcium measurements by taking advantage of the 'T format' layout of the SLM 8000 as previously described [12,15]. For these measurements the excitation and emission monochromators were set to 340 nm.

Synthesis of 12-OxoDTrE and 12-OxoETE 12-OxoDTrE was prepared using a procedure previously described [17], with some modifications as shown in Fig. 1. The aUylic alcohol 1, obtainable in five steps from acrolein [18-20] was silylated followed by hydrolysis of the diethylaeetal to provide the desired aldehyde 2. Condensation of the aldehyde with the phosphorane 3 gave after deprotection, the alcohol 4, which was then oxidized to the diene aldehyde 5 with fresly prepared manganese dioxide in ethylacetate as solvent. The 12-OxoDTrE was purified on silica gel chromatography (80% ethylacetate, 20% aeetnae, 10% hexane) followed by reverse phase HPLC on a Novapak C~s column (60% methanol, 40% H20, P.I% AcOH). ~HNMR 250 MHz, aeetone-d 6) 8 0.61 (quintet, 2H, J = 7.4 Hz, CHz), 2.15 (quintet, 21-t, J =: 7.4 Hz, Cl-Iz), 2.28 (t, 2H, J = 7.4 Hz, CHz), 3.15 (t, 2H, I = 6.2 Hz, CH2), 5.36 (m, 2H, H-5 and H-6), 6.00 (m, ~H, I-I-8), 6.12 (dd, 1H, J = 7.4 and 14.6 Hz,'H-I1), 6.35 (t, 1H, J = 10.8 Hz, H-9), /.71 (dd, 1H, J = 10.8 and 14.6 Hz). The ultraviolet spectrum of the synthetic material recorded

104 in m e t h a n o l a n d hexane, as well as the mass s p e c t r u m of the methyl e s t e r derivative w e r e identical to those o f natural 1 2 - O x o D T r E [2]. 1 2 - O x o E T E was o b t a i n e d by oxidation o f 1 2 - H E T E as d e s c r i b e d previously [21]. Results

Calcium mobilization T h e addition o f 1 2 - O x o D T r E or 1 2 - O x o E T E to a suspension of h u m a n n e u t r o p h i l s resulted in rapid a n d c o n c e n t r a t i o n - d c p e n d e n t increases in levels of cytoplasmic free calcium (Fig. 2). T h e r e s p o n s e s w e r e elicited within seconds of the addition o f t h e fatty acids. T h e t h r e s h o l d c o n c e n t r a t i o n s r e q u i r e d to elicit an increase in cytoplasmic free calcium w e r e o f the o r d e r o f 1 to 10 n M a n d a s a t u r a t i o n was r e a c h e d at c o n c e n t r a t i o n s of the o r d e r of 0.1 to 1.0 p,M.

350 ]

T h e effects o f the sequential additions of 12O x o D T r E or 1 2 - O x o E T E and of l e u k o t r i c n e B4 w e r e investigated next. A s shown in Fig. 3, cells first exposed to l e u k o t r i e n e B4 (10 -7 M) r e s p o n d to a m u c h s m a l l e r extent, if at all, to 1 2 - O x o D T r E o r 1 2 - O x o E T E (10 -t' M ) e v e n t h o u g h e n o u g h t i m e was allowed for the r e s p o n s e to l e u k o t r i e n e 14 to d e c a y back to the control values. O n the o t h e r h a n d , t h e p r e - c x p o s u r e to 12O x o E T E o r 1 2 - O x o D T r E (10 -6 M) did not affect in a significant m a n n e r the calcium mobilizing ability o f l e u k o t r i e n e B 4 (10 -7 M).

Right-angle light scatter Both 1 2 - O x o E T E a n d 1 2 - O x o D T r E i n d u c e d c h a n g e s in right-angle light s c a t t e r (top panels, Fig. 4). T h e t i m e - c o u r s e o f the c h a n g e s in right-angle light scatter w e r e rapid, initiated without any d e t e c t a b l e lag time,

= • 3so •

12-OxoDTtE

12-OxoETE

O

i

300

O

300 '

~ 2so 200 1so I

.

0

.

.

25

.

5o

75

iS0

lOO

2'0

TIME Islcl

~o

io

io

TIME lice)

Fig. 2. Mobilization of calcium in human neutrophils by 12-OxoDTrE (left) and I?-OxoETE (right). The fatty acids, at the indicated concentrations, were added at the arrows, and the concentration of cytoplasmic free calcium monitored as described in Materials and Methods. The results are from individual experiments and are representative of data obtained with three separate cell preparations.

i i

-- gO° lz-0x~tE

500

400 12,-oxoO~E

LTB4

Too

L'JB

o 300

.- .0]~,~ O

.. ,oolL~, O

500

0

40

80 TIME (sec}

120

IN

0

50

I00 TIME [.c)

|SO

200

Fig. 3. Cross-desensitization of the mobilization of calcium induced by 12-OxoDTrE, 12-OxoETE and leukotriene B4 in human neutrophils, in the top figure ,nf each panel, the indicated fatty acid was added at the first arrow followed by leukotriene B4 al the second arrow. In the bottom figure of each panel, leukotriene B4 was added first followed at the second arrow by the indicated tony acid. The concentrations of 12-OxoDTrE, 12-OxoETE and leukotriene B4 (LTB4) were ',9 -6 M, 10-~' M and 10-7 M, respectively. The results are from a single experiment representative of three similar experiments.

105

-

_

Fig. 4. Right-angle light scatter responses of human r,eutrophils to 12-OxoDTrE and 12-OxoETE. In the top figure of each panel, the indicated fatty acid was added at the first arrow followed by leukotriene B4 (LTB.D at the ~,~:condarrow. In the bottom figure of each panel, leukotriene B4 was added first followed at the second arrow by the indicated fatty acid. The concentrations of 12-OxoDTrE. 12-OxoETE and leukotriene B4 were 10-7 M, 10 7 M and 10-s M. respectively. The results are representative of two similar experiments.

and completed within less than 10-15 s. The magnitude of the responses to the two fatty acids at 10 -7 M was about half of that induced by l0 -~ M leukotriene B 4. Furthermore, as observed in the calcium assays (see Fig. 2), while the addition of leukotriene B 4 essentially abolished the subsequent response to 12-OxoETE or 12-OxoDTrE, the latter two compounds (at the concentrations tested) did not significantly affect the response to leukotriene B 4. D i s c u s s i o n

There are only a few reports on the biological activities of oxo-derivatives of unsaturated fatty acids. In plants, (lOZ)-12-oxo-lO-dodecaenoie acid or traumatine, a compound closely related to 12-OxoDTrE is believed to be a wound hormone [22]. In the marine mollusk Aplysia culifornica, 12-OxoETE generated from 12-HpETE appears to be a mediator of histaminergic synaptie response stimulation [23]. A positional isomer of 12-oxoETE, (5Z,8Z, llZ,13E)-15-keto. 5,8,11,13-eicosatetraenoic acid inhibits the neutrophil 5-1ipoxygenase in micromolar concentrations [24]. Finally, it is noteworthy that the short chain aldehyde 2,4-deeadienal derived from the peroxidation of polyunsaturated fatty acids, which has been found in micromolar concentration in inflammatory exudates, stimulates and suppresses human neutrophil random migration at sub- and supramicromolar concentrations, respectively [25]. The results of the present study clearly indicate that 12-OxoETE and 12-OxoDTrE can stimulate human

ncutrophils, as observed by measurements of calcium mobilization and changes in right-angle light scatter. The second messenger role of calcium in the initiation and control of neutrophil functions is well established [26,27]. Changes in right-angle light scatter are indices of morphological changes which have been primarily, but not exclusively [28] related, by correlative measurements under a variety of conditions, to the reorganization of the cytoskeleton (polymerization of actin) of neutrophils [29]. Transient increases in cytoplasmic calcium and reorganization of the actin network of neutrophils have been extensively described in response to chemotactic factors and other agonists including various eicosanoids [30]. The results obtained in our study using 12-OxoDTrE strongly support and extend to the biochemical level the previous finding of Iocomotory activity of this compound by Glascow et al. [1]. Indeed, calcium mobilization and actin polymerization (measured as changes in right-angle light scatter) are two cellular responses generally accepted to be relevant to the ability of neutrophils to migrate; in fact, there is an excellent correlation between the ability of various lipoxygenase products to mobilize calcium in neutrophils and their potency as ehemotactie and chemokinetic agents [6,31]. In the present study, the two oxoderivatives of arachidonie acid tested were clearly active but showed less than one tenth the activity of leukotriene B 4 on these two parameters of cell activation. The results of previous experiments with 12S-HETE and 12R-HETE indicated that these compounds had chemotactic activity towards human neutrophils, elicited rises in intraeellular calcium concentration and stimulated actin polymerization [12]. It was also shown in binding studies and desensitization experiments that 12S-HETE and 12R-HETE are likely to interact with the leukotriene B 4 receptor [12,13]. That the two carbonyl compounds investigated in the present study are structurally related (oxygenated at carbon-12) to 12H E T E s raised the possibility of a mechanism of action involving the leukotriene B 4 receptor. Accordingly, sub-micromolar concentrations of leukotriene B 4 were shown to completely desensitize neutrophils to a subsequent exposure to either 12-OxoETE and 12-OxoDTrE (at 10 times molar excess), both in term of calcium mobilization and changes in right-angle light scatter (Figs. 3 and 4]. These data support the hypothesis that 12-OxoETE and 12-OxoDTrE utilize the same set(s)of receptors as leukotriene Bn, or at least indicate the involvement of a common pathway in the activation of neutrophils by leukotriene B4 and the carbonyl compounds. ~a summary, we have reported herein that the two 12-1ipoxygenase products 12-OxoETE and 12-OxoDTrE induce neutrophil responses relevant to the motile functions of these cells and therefore to their ability to

106 a c c u m u l a t e at i n f l a m m a t o r y sites. F u r t h e r m o r e , activation o f n e u t r o p h i l s by 1 2 - O x o E T E a n d 1 2 - O x o D T r E a p p e a r s to involve t h e l e u k o t r i e n e B 4 r e c e p t o r s . 12O x o E T E a n d 1 2 - O x o D T r E a r e m e t a b o l i t e s o f t h e 12lipoxygenase [1-3] a n d t h u s are likely to b e f o r m e d in i n f l a m m a t o r y c o n d i t i o n s involving a c t i v a t e d platelets, i n c l u d i n g t h e sites o f t h r o m b u s f o r m a t i o n . In this reg a r d , it is n o t e w o r t h y t h a t d a t a s u p p o r t i n g t h e role o f platelets in i n f l a m m a t i o n as well as o f t h e i r i n t e r a c tions with n e u t r o p h i l s a r e a c c u m u l a t i n g rapidly [32]. T h e potential c o n t r i b u t i o n o f 1 2 - O x o D T r E a n d 12O x o E T E as stimuli o f n e u t r o p h i l a n d / o r o t h e r p h a g o cyte m i g r a t i o n a n d activation r e m a i n s to b e investig a t e d a n d will r e q u i r e t h e m e a s u r e m e n t o f t h e s e c o m p o u n d s at i n f l a m m a t o r y sites.

Acknowledgements T h i s w o r k w a s s u p p o r t e d by s c h o l a r s h i p s a n d fellowships f r o m ' L e F o n d s d e la R e c h e r c h e e n S a n t 6 d u Q u 6 b e c (P.B., P . H . N . a n d B.F.L.) a n d T h e A r t h r i t i s Society o f C a n a d a ( P . H . N . ) a n d by g r a n t s f r o m T h e M e d i c a l R e s e a r c h Council o f C a n a d a .

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