18

BBA 51284

Biochimrcu rt Biop~.vs~co Actu. 750 (1983) 78-90

Elsevier Biomedical Press

IDENTIFICATION OF LIPOXYGENASE PRODUCTS FROM ARACHIDONIC ACID METABOLISM IN STIMULATED MURINE EOSINOPHILS

JOHN TURK *, THOMAS H. RAND, RICHARD L. MAAS. JOHN A. LAWSON. ALAN R. BRASH, L. JACKSON ROBERTS,

II. DANIEL G. COLLEY and JOHN A. OATES

Depurtment of Medicine, Pharmacology tind Microbiology, Vanderbilt lJniversrt.v School of Medicine. Nushorlle, TN 37232 (U.S.A.)

(Received July 13th, 1982)

Key words: Arachidonrc acid metabohsm; Llpoxygenase; (Munne eosinophll)

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A,, and the other included products derived from the 14,15-oxido

analog of leukotriene A 4 (14,15Jeukotriene A 4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,19dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihy- droxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which

enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation

promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of

lipoxygenase products which modulate the migratory activity of the cells.

Introduction

Arachidonic acid may be transformed to bio- logically active substances after initial oxygenation

by two classes of enzymes. Cyclooxygenase action produces a hydroperoxyendoperoxide with a sub- stituted cyclopentane ring structure (prostaglandin

* Present address: Departments of Medicine and Pharmacol- ogy. Washington University School of Medicine, St. Louis,

MO 63110. U.S.A. Abbreviations: HETE. hydroxyeicosatetraenoic acid; diHETE. dlhydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosa-

tetraenoic acid: DIOL, dihydroxylated derivatives of

arachidonic acid.

OOOS-2760/83/0000-0000/$03.00 $1 1983 Elsevier Biomedical Press

G2) which is the precursor of the prostaglandins

and thromboxanes [1,2]. A family of lipoxygenase

enzymes [3-51 convert arachidonate to molecules

bearing a hydroperoxy group alpha to a con- jugated diene system (HPETE). Enzymatic reduc- tion of the hydroperoxide produces the hydroxyl- ated form of these molecules (HETE).

Arachidonic acid metabolism may play an im- portant role in the physiology of mammalian leukocytes. A variety of HETE species exert a chemotactic influence on granulocytes [6,7]. In addition, in many types of leukocytes, S-HPETE may be converted to a 5,6-oxido compound con- taining a conjugated triene system [8]. In view of

79

this structural feature and the cells of origin, the

oxido precursor and the compounds to which it is transformed have been named leukotrienes [9].

Leukotrienes C, and D4 may be important media- tors of immediate hypersensitivity reactions [lo], and leukotriene B4 exerts a chemotactic influence

on granulocytes [ 111. It has recently been demonstrated that human

eosinophils [12] express a second pathway of leukotriene biosynthesis, resulting from initial

oxygenation of arachidonate at carbon-15 rather

than at carbon-5 [13]. Among human peripheral

blood leukocytes, eosinophils were the dominant

source of these 15-series leukotrienes. Porcine

eosinophils are also capable of synthesizing 15-

series leukotrienes and do not synthesize 5-series

leukotrienes in comparable amounts [14]. To ex-

plore the possibility that the ability to synthesize

15-series leukotrienes might be a general property

of mammalian eosinophils, the products of incuba-

tion of arachidonic acid and murine eosinophils

were identified in this investigation.

The possibility that murine eosinophils syn-

thesize arachidonate lipoxygenase products also relates to recent observations about the response

to eosinophil stimulation promoter. Eosinophil

stimulation promoter is a murine lymphokine gen-

erated from lymphoid cells by specific antigen or

mitogen stimulation, and it enhances migration of murine eosinophils [ 151. This migration response is

potentiated by arachidonic acid and suppressed by

inhibitors of arachidonate lipoxygenases [ 161. The

response is little affected by cyclooxygenase in-

hibitors [16]. Moreover, arachidonic acid lip-

oxygenase products in including leukotriene B4 and 12-HETE themselves enhance the migration

of murine eosinophils [ 161. These observations sug-

gest that eosinophil stimulation promoter stimula- tion of eosinophils results in the generation of lipoxygenase products which modulate the migra-

tory activity of these cells. To explore this hy-

pothesis further we sought to determine whether murine eosinophils can synthesize lipoxygenase

products which enhance migration and whether exposure to eosinophil stimulation promoter stimulates the synthesis of any such product.

Materials and Methods

Materials

Arachidonic acid ethyl ester was obtained from

Nucheck Prep (Elysian, MN) and saponified [27].

[5,6,8,9,11,12,14,15-3H,]Arachidonic acid (78.2

Ci/mmol) and [l-‘4C]prostaglandin F,, were ob-

tained from New England Nuclear (Boston, MA).

Standard 12-HETE and 12-[5,6,8,9,11,12,14,15-

‘H,]HETE generated from arachidonic acid or

[5,6,8,9,1 1,12,14,15-2H,]arachidonic acid and

H,O, in the presence of Cu2+ [17] was the gift of

Dr. Walter Hubbard. All organic solvents were

obtained from Burdick and Jackson (Muskegon

MI). Male CBA/J mice were obtained from Jack-

son Memorial Laboratories (Bar Harbor, ME).

Eosinophil stimulation promoter was prepared and

standardized as described elsewhere [ 181.

Prepurution of cells

Eosinophil-rich (85-95%) murine peritoneal

leukocyte populations were prepared as described

in detail elsewhere [ 181. Briefly, peritoneal ex-

udates were harvested by lavage from Toxocaru

can&-infected mice and enriched for eosinophils by sedimentation over continuous Percoll gradi-

ents. An aliquot of the cell population was then

counted (Coulter counter) and differentially stained (Wright’s). Virtually all contaminating cells were mononuclear. Platelet contamination was less

than one per 200 leukocytes by phase contrast

microscopy. Populations of peritoneal leukocytes

containing more than 85% mononuclear cells were

prepared as above from uninfected mice that had

received an intraperitoneal injection of proteose

peptone (1.5 ml) 60 h before the peritoneal ex-

udates were harvested. Murine platelets were iso-

lated from cardiac blood by the methods of Tim- mons and Hawiger [ 191. Briefly, blood was drawn

into titrated saline. Platelet-rich plasma was ob-

tained by centrifugation and applied to discon-

tinuous albumin gradients which were then centri-

fuged. Platelets were harvested from the interface between 25 and 50% albumin, passed over a Sep- harose 2B gel filtration column, recovered in the void volume and counted (Coulter counter).

Cell incubations

Incubations were performed in Tyrode’s gel

X0

buffer [20] supplemented with M&l, (1.2 mM)

and CaCl, (1.8 mM) and saturated with 95%

O/5% CO, just before use (pH 7.25). Cells (5 . lo6

per ml) were pre-incubated in this buffer (5 min.

37’C). Incubations were initiated by the simulta-

neous addition of arachidonic acid (100 PM) and

ionophore A23187 (10 PM) in dimethylsulfoxide

(0.2% of incubation volume), allowed to proceed

for 5 min, and terminated by the addition of 1.5

vol. of methanol and centrifugation to remove cell

debris. Parallel incubations were performed with [3H,]arachidonic acid (5 FCi per 10’ cells) and

ionophore A23 187 (10 FM). Preparations contain-

ing tritiated and unlabelled arachidonic acid were

combined just before termination of the incuba-

tions. Five separate incubations were performed in

this way. Total cell number ranged from 180 . 10h

to 460. 10h cells, and the percentage of eosinophils

ranged from 85 to 96. (Substantially the same

profile of arachidonate metabolites was recovered

from each of the five incubations, with only minor

variations in the relative abundance of the various

products.)

Product recovery and analysis

The metanolic solution was diluted with 2 vol.

of water, adjusted to pH 3.5 with 1 N HCl, and

extracted three times with 1 vol. of ethyl acetate. The extract was washed with one-fifth vol. of

water, dried over sodium sulfate, concentrated in

vacua, reconstituted in methanol, and subjected to

chromatography.

High-performance liquid chromatography

(HPLC) was performed in the reversed-phase (RP) or straight-phase (SP) mode on the following

Waters (Milford, MA) columns: I (PBondapak

C-18, 7.8 mmX 30 cm), II (PBondapak C-18, 3.9

mm x 30 cm). III (PPorasil, 3.9 mm x 30 cm), and

IV (Fatty Acid Analysis, 3.9 mm X 30 cm). Elution was performed with the following solvents at a flow of 2 ml per min: A (methanol/water/acetic acid, 65 : 35 : 0.01, v/v), B (methanol/water/acetic acid, 75 : 25 : 0.01). C (methanol), D (methanol/water/acetic acid, 70 : 30 : 0.01) E (hexane/isopropanol, 100 : 3) F (hexane/isopro- panol, 100: 0.45), G (a linear gradient over 120 min from CHCl,/acetic acid, 500 : 2, to

CHCl,/methanol/acetic acid, 500 : 50 : 2) H (methanol/ water, 93 : 7). and I (ace-

tonitrile/water/acetic acid, 23 : 77 : 0.1). Column effluent was continuously monitored for ultra-

violet absorbance at 280 or 235 nm.

Products from incubations were first subjected

to RP-HPLC on column I, eluting successively

with solvent A (180 ml), then B (60 ml), then C (60

ml). Dihydroxylated arachidonate metabolites and

more polar products (e.g., prostaglandins) eluted

from the column in solvent A and were collected

separately. Monohydroxylated derivatives eluted

in solvent B, and unmetabolized arachidonate

eluted in solvent C (Fig. 1).

Monohydroxylated metabolites were further purified by RP-HPLC (column II, solvent D).

Recovered materials were converted to the methyl

esters with ethereal diazomethane and subjected to

SP-HPLC (column III, solvent F). Dihydroxylated

metabolites were analyzed by RP-HPLC (column

II, solvent A), converted to the methyl esters, then

subjected to SP-HPLC (column III, solvent E). Prostaglandins were purified by SP-HPLC (col-

umn III, solvent G), then RP-HPLC (column IV,

solvent I) and were then converted to the methyl

esters. The ultraviolet absorption spectra of all

materials so isolated was recorded on a Beckman

(Fullerton, CA) Model 24 Spectrophotometer.

Derivatization and identification

The methyl esters (Me) of some compounds

were subjected to catalytic hydrogenation over

platinum oxide (100 pg) in ethanol (100 ~1) through

which hydrogen gas was bubbled (5 min). Water

(350 ~1) was added and extraction performed twice

with ethyl acetate. The methyl esters of some compounds were converted to the methoxime (MO) derivatives by treatment with 3% methoxyamine hydrochloride in pyridine at room temperature for

18 h, followed by drying, dilution with water, and

extraction with ethyl acetate. The methyl esters of

all compounds were converted to trimethylsilyl ether (Me,Si) derivatives by treatment with 10 ~1 N,O-bistrimethylsilyltrifluoracetamide in pyridine (10 ~1) for 30 min. at room temperature.

Gas chromatographic mass spectrometric (GC- MS) analyses were performed on a 3% SP2100 column (carrier gas flow, 25 ml/min; temperature, 215 or 235°C) interfaced with either an LKB 9000 (LKB. Bromma, Sweden) or a Ribermag RIO-1OB (Nermag Inc., Santa Clara, CA) mass spectrome-

ter. The GC retention time of each compound was

determined relative to a series of saturated fatty

acid methyl ester standards and was expressed as a

carbon value (C,,).

Stereochemical analysis

About 10 pg of the methyl ester of standard

racemic 12-HETE or cell-derived material was

converted to the menthoxycarbonyl derivative with

( - )-menthoxycarbonylchloroformate by the meth- od of Hamberg [21]. The derivative was purified

by RP-HPLC (column II, solvent H, retention volume, 15 ml) and then subjected to oxidative

ozonolysis as described by Green [22]. The prod-

ucts were treated with an excess of ethereal di- azomethane. The resultant menthoxycarbonyl de-

rivative of 2-hydroxy-dimethyl-malate was sub-

jected to GC-MS on a 6 foot 3% QFl on Gas

Chrom Q column (185°C) with selected ion moni-

toring (m/z 123, 131, 138, 163, 175, 189 and 207).

Results

The majority (70%) of radioactively labelled

arachidonate incubated with murine eosinophils

was recovered as products other than arachidonic

acid itself (Fig. 1). Of this metabolized material, species with the RP-HPLC behavior of monohy-

droxylated arachidonate derivatives (HETE) were more abundant than dihydroxylated derivatives

(DIOL) or than more polar materials such as prostaglandins (PG), as is apparent from Fig. 1. Materials from each of the three major regions of

the chromatogram (HETE, DIOL and PG) in

Fig. 1 were collected separately and subjected to

further chromatographic analysis.

Further RP-HPLC analysis of the materials be-

having as monohydroxylated products demon- strated two components, designated peak A and

peak B in Fig. 2. Each component behaved as a homogeneous material on SP-HPLC. The ultra-

violet absorption spectra of the two materials were identical to each other. The spectrum of the

material obtained from peak B is shown in Fig. 3 and suggests the presence of a cis-tram conjugated diene chromophore [23].

The mass spectrum of the Me,Me,Si derivative of the material obtained from peak B is shown in Fig. 4 and is compatible with the interpretation

35

SOLVENTS:

HETE AA

.-.J1 h

I 0 15-

IO -

5:lLl - 40 80 120 160 200 240 280 ELUTION VOLUME (ML)

Fig. 1. Chromatogram of the products of an incubation of

[ 3 Hlarachidonic acid and murine eosinophils. Products were

recovered from the incubation as described in Materials and

Methods and subjected to RP-HPLC on column I with succes-

sive elution with the indicated solvents. An aliquot (0.25%) of

each fraction was subjected to liquid scintillation counting. The

regions of elution of arachidonic acid, monohydroxyeico-

satetraenoic acids, and of polar species including prostaglan-

dins are denoted AA, HETE, DIOL and PC respectively.

14 r B

12

IO t

I

I ; “le ? P I

Fig. 2. Chromatogram of HETE species recovered from murine

eosinophils. Monohydroxylated species recovered from HPLC

solvent from the first RP-HPLC analysis (Fig. 1, HETE) were subjected to a second RP-HPLC analysis (column II. solvent

D). An aliquot (0.5%) of each fraction was subjected to liquid

scintillation counting. Peaks A and B were collected separately.

converted to the methyl esters, and subjected to SP-HPLC

(column III, solvent F, retention volume 22 ml for peak A and

21 ml for peak B).

Xmax 235

I 220 240 260 260

WAVELENGTH hm)

Fig. 3. Ultraviolet absorption spectrum of the most abundant

HETE species from murine eosinophils. The spectrum is that of

the material from peak B (Fig. 2) after conversion to the methyl

ester, SP-HPLC purification, and dissolution in methanol. The

observed wavelength of maximum absorption excludes a

trans,trcrns configuration of the conjugated diene [25].

that the parent compound was 12-hydroxyeicosa-

tetraenoic acid (12-HETE). The mass spectrum of

the Me,Me,Si derivative of the catalytic hydro-

genation product of the compound is shown in

Fig. 5 and confirms the location of the hydroxyl

group while excluding the possibility of a ring

structure. Determination of the stereochemical

composition of the optical center at carbon 12 as

100 0 2% i-x20

1 80 o-,

60 0-i

400

200 1 316

37E ,91 & 173 20, 229 pi

0 I,, 11 ., ., 150 200 250 300 350 400

Fig. 4. Mass spectrum of the Me,Me,Si derivative of the most

abundant HETE species from murine eosinophils. The material

was obtained from HETE peak B (Fig. 2). The carbon value (3% SP2100, 215°C) of the indicated derivative was 21.3.

Fragmentation was induced by electron impact. Ionization

energy was 20 eV.

200- i

0 ;

I 367 383 yz

190 200 250 300 350 400

Fig. 5. Mass spectrum of the Me,Me,Si derivative of the hydro-

genation product of the most abundant HETE species from

murine eosinophils. Material from HETE peak B (Fig. 2) was

subjected to catalytic hydrogenation, and the mass spectrum of

the indicated derivative (carbon value 22.0, 3% SP 2100, 215°C)

was obtained under the same conditions as in Fig. 4.

in Fig. 6 revealed that the compound was enanti-

opure and of the S configuration. This established

an enzymatic origin of the compound, since non-

enzymatic formation would have resulted in a racemate. The 12-HETE from human platelets [3]

and neutrophils [4] is also of the S configuration. Nearly homogeneous preparations of murine

peritoneal mononuclear leukocytes and of blood

platelets were also found to produce 12-HETE.

The amount of 12-HETE recovered from these preparations was quantified by ultraviolet spec-

troscopy and normalized to cell number. Using the

incubation and isolation procedures described

above, 3.0 pg of 12-HETE was recovered per 10’

murine peritoneal mononuclear leukocytes and 1.3

I-18 was recovered per 10” blood platelets. A total

of 13.4 pg of 12-HETE per 10’ cells was recovered

from murine eosinophil preparations (containing

85595% eosinophils, 5- 15% mononuclear leuko- cytes, and less than 1 platelet per 200 leukocytes). These data suggest that more than 95% of the 12-HETE recovered from the eosinophil-rich leukocyte preparations originated from eosinophils and less than 5% from mononuclear cells and platelets.

Peak A of Fig. 2 contained the second major eosinophil-derived HETE species. The mass spec- trum of the Me,Me,Si derivative of that com- pound is shown in Fig. 7 and indicates that the

83

2- MC-Me2 MALATE

I

s R

L

I I I I I I ,

I 2 3 4 5 6 7 6

ELUTION TIME (MINI

Fig. 6. Stereochemical analysis of I2-HETE isolated from

murine eosinophils. Standard racemic 12-HETE and 12-HETE

derived from murine eosinophils were converted to the (-)-

menthoxycarbonyl derivatives and subjected to oxidative

oronolysis. The resultant menthoxy carbonyl derivatives of

2-hydroxy-malic acid were converted to the methyl esters (2

MC-Me,-malate) and subjected to gas chromatography (under

conditions which separate the S and R stereoisomers) with

mass spectrometric monitoring of selected fragment ions. The

upper tracing was generated by material derived from racemic

12-HETE and the lower tracing by that from 12-HETE from

murine eosinophils. Further details are provided in Materials

and Methods.

parent compound was 15-HETE. About 1.5 pg of

this material was recovered per 10’ cells, and more

than 95% originated from eosinophils. On one of

five occasions a small amount of 5-HETE (0.5 p-18

per lo8 cells) was recovered from an incubation of murine eosinophils. On no occasion were signifi-

cant amounts of 8-, 9- or 1 I-HETE recovered.

Dihydroxylated metabolites from the murine eosinophils were recovered from the DIOL region of the chromatogram in Fig. 1. Further RP-HPLC analysis of these materials produced a pattern of six peaks with ultraviolet absorption at 280 nm, as shown in Fig. 8. Materials from each peak were collected separately, converted to the methyl es- ters, and further analyzed by SP-HPLC. A total of

,jr,.ll.... 150 200 250 300 350 400

Fig. 7. Mass spectrum of the Me,Me,Si derivative of the second

most abundant HETE species from murine eosinophils. The

material was obtained from HETE peak A (Fig. 2). The carbon

value (3% SP2100, 215°C) of the indicated derivative was 21.3.

Fragmentation was induced by electron impact. Ionization

energy was 20eV.

11 dihydroxylated derivatives of arachidonic acid

were distinguished in this way. The properties of these compounds are summarized in Table I.

Peak 1 from Fig. 8 consisted of a single compo-

nent. The ultraviolet absorption spectrum of this

compound is shown in Fig. 9 and indicates the

presence of a conjugated triene chromophore. The

mass spectrum of the Me,Me,Si derivative of the

compound is shown in Fig. 10 and indicates that the parent compound was an 8,15-dihydroxylated

derivative of arachidonic acid. This compound is

Fig. 8. Chromatogram of dihydroxylated derivatives of

arachidonic acid from murine eosinophils. Dihydroxylated

species recovered from LC solvent from the first RP-HPLC

analysis (Fig. 1, DIOL) were subjected to a second RP-HPLC

analysis (column II, solvent A). The eluant was continuously

monitored for ultraviolet absorbance at 280 nm (A,,,).

84

TABLE 1

DIHYDROXYLATED ARACHIDONATE METABOLITES FROM MURINE EOSINOPHILS

RP-HPLC designations correspond to those in Fig. 8. The components in each RP-HPLC peak were separated from each other by

SP-HPLC as described in Materials and Methods. SP-HPLC retention volume (column III, solvent E) of each compound is expressed

in ml. Ultraviolet (A,,,,,) is the wav I e ength of maximal absorption in nm. Amount is the quantity of compound recovered as

determined by ultraviolet absorption and is expressed as ng per 10’ cells. GC (C,) refers to the elution time of the Me,Me,Si

derivative relative to a series of standard saturated fatty acid methyl esters. MS ions are major fragment ions in the mass spectrum

which locate the position of the hydroxyl groups. Designation of compounds is described further in the text.

RP-HPLC

peak

SP-HPLC

retention

volume

Ultraviolet Amount

(A,,) (ng/lOs cells)

GC

(C”)

MS ions

(m/z)

Designation

1 28 269 90 24.9 173,353 compound 1

2 23 268 140 23.6 173,353 compound 11

3 44 269 190 24.9 203,383 5s 12 R-6-trans-leukotriene B4

3 14 243 620 24.2 a 173,203 5,15-diHETE

4 30 268 110 23.6 173.353 compound III

4 31 269 80 24.9 173,353 compound IV

4 48 269 140 24.9 203,383 5S,12S-6-rrans-leukotriene B4

5 39 268 430 23.6 203,383 5,12-diHETE

5 60 270 60 23.6 203,383 leukotriene B4

6 23 272 180 23.9 173,321 compound V

6 24 270 30 24.9 173,321 compound VI

a Refers to the Me,Me,Si derivative of the catalytic hydrogenization product.

designated compound I in Table I. The properties exhibited by compound I on RP-HPLC, SP-HPLC,

ultraviolet, GC and MS indicated that it was iden- tical to a product obtained from porcine leuko-

cytes [13] whose structure has been established as

(8S, 1 SS)-dihydroxy-5-c&9,11,13- tram- eicosate- traenoic acid. That compound is one of the hy-

drolysis products of (14S, 1 SS)-trans-oxido-5,8-cis-

10,12-truns-eicosatetraenoic acid [ 131. The trivial

name 14,15-leukotriene A, has been suggested for

Xmm 269

240 260 280 330

WAVELENGTH

Fig. 9. Ultraviolet absorption spectrum of a conjugated triene derivative of arachidonic acid from murine eosinophils. The spectrum is that of compound I (Table I) from peak 1 (Fig. 8) after conversion to the methyl ester, SP-HPLC purification, and dissolution in

methanol.

Fig. 10. Mass spectrum of the Me,Me,Si derivative of an 815dihydroxylated conjugated triene metabolite of arachidonic acid from murine eosinophils. The material was obtained from DIOL peak 1 (Fig. 8) and corresponds to compound I of Table 1. Fragmentation

was induced by electron impact with an ionization energy of 70 eV.

this precursor epoxide 1241 which is believed to arise from enzyme action on 15-HPETE [13,25]. (The double bond geometry of the 14,15-epoxide generated enzymatically remains uncertain.) No 8,15-dihydroxylated arachidonate derivatives were recovered from incubations with murine mono- nuclear cells or platelets.

Peak 2 of Fig. 8 consisted of a second 8,15-d& hydroxylated conjugated triene derivative of arachidonic acid designated compound II in Table I. The structure of this compound has been established [ 131 as (8S, 15~)-dihydroxy-5,1 l-cis- 9,13-trans-eicosatetraenoic acid by comparison with reference material generated from the action of soybean lipoxygenase-I on arachidonic acid

1261. Peak 3 of Fig. 8 contained two components.

The less abundant component exhibited the ultra- violet absorption spectrum of a conjugated triene. The mass spectrum of the Me,Me,Si derivative of this compound is shown in Fig. 11 and indicates that the parent compound was a 5,12-dihydroxyl- ated arachidonate derivative. This compound is believed to arise from hydrolysis of leukotriene A, [S] and is designated 5S,12R,6-truns-leukotriene B4

85

in Table I, Calculations similar to those described above indicated that more than 90% of the 5,12-di- hydroxylated arachidonate metabolites recovered from eosinophil-rich murine leukocyte populations originated from eosinophils and less than 10% from mononuclear cells.

The second component of peak 3 of Fig. 8 was the most abundant of the dihydroxylated arachi- donate derivatives recovered from the murine eosinophil preparations. The ultraviolet absorption spectrum of this material is shown in Fig. 12 and is identical to that of a double dioxygenation product of arachidonic acid recently isolated from rat and human leukocytes [27]. The unfavorable vapor phase properties of the Me,Me,Si derivative of this molecule necessitated catalytic hydrogena- tion before W-MS analysis. The mass spectrum of the Me,Me,Si derivative of the hydrogenation product is shown in Fig. 13 and indicates that the parent compound was a 5,l%dihydroxylated arachidonate derivative. This compound is desig- nated 5,15-diHETE in Table I.

Peak 4 of Fig. 8 contained three components. Two of them were 8,15_dihydroxylated conjugated triene arachidonate derivatives. designated com-

1 X rnox 243

Fig. 11. Mass spectrum of the Me,Me,Si derivative of a 5,12_dihydroxylated conjugated triene metabolite of arachidonic acid from murine eosinophils. The spectrum is that of one of the components (5SJ2R. 6-trans-leukotriene B4, Table I) of DIOL peak 3 (Fig. 8)

and was obtained under the same conditions as in Fig. 10.

Fig. 12. Ultraviolet absorption spectrum of a 5,15-dihydroxylated derivative of arachidonic acid from murine eosinophils. The

spectrum is that of one of the components (5,15-diHETE, Table I) of DIOL peak 3 (Fig. 8) after conversion to the methyl ester,

SP-HPLC purification and dissolution in methanol.

/* 203

110 130 150 170 190 210 ?30 250 2-m 290 310 330 350 370 390 410 430 450 17; 493 515

Fig. 13. Mass spectrum of the Me,Me,Si derivative of the hydrogenation product of a 5,15-dihydroxylated metabolite of arachidonic

acid from murine eosinophils. One of the components (5,15-diHETE, Table I) of DIOL peak 3 (Fig. 8) was subjected to catalytic

hydrogenation and the mass spectrum of the indicated derivative was obtained under the same conditions as in Fig. 10.

pounds III and IV in Table I. On the basis of the

similarities of these materials with porcine leuko-

cyte products [ 131 it is believed that compound III is an epimer at C8 of compound II and that compound IV is an epimer at C8 of compound I.

The third component of peak 3 of Fig. 8 was a

5,12-dihydroxylated conjugated triene with the properties of one of the hydrolysis products of

leukotriene A, [8] and is designated 5S,12S-6- tvans-leukotriene B, in Table I.

Peak 5 of Fig. 8 contained two 5,12-dihydroxyl-

ated conjugated triene arachidonate derivatives.

The more abundant component exhibited the

properties of a recently described double di-

oxygenation product [28] and is designated 5,12-

diHETE in Table I. The less abundant component

exhibited the properties of leukotriene B4 [29] and

is designated leukotriene B4 in Table I. Peak 6 of Fig. 8 contained two conjugated tri-

ene arachidonate derivatives with similar mass spectra, designated compounds V and VI in Table I. The mass spectrum of the Me,Me,Si de- rivative of compound V is shown in Fig. 14 and indicates that the parent compound was a 14,15- dihydroxylated arachidonate derivative. It has been suggested that compounds V and VI arise from 14,15-leukotriene A, [ 131, but the actual mecha- nism by which these compounds are generated is still under investigation [ 141.

Analysis of materials eluting in the prostaglan-

din region of Fig. 1 revealed the presence of multi-

ple polar species, many of which did not corre-

spond to known cyclooxygenase products. Small

amounts of prostaglandin E, and thromboxane B,

were identified among these materials but the ina-

bility to quantitate these compounds precisely pre- vented confident assignment of the eosinophil as

their cell of origin. A summary of the metabolism of arachidonic

acid by lipoxygenase enzymes in murine eosino-

phils stimulated with exogenous arachidonic acid and ionophore A12187 is provided in Fig. 15.

While the eosinophils can express these pathways under the incubation conditions employed it is not

certain that resting cells or cells responding to physiologic stimuli produce a similar profile of

arachidonate metabolites. The small quantities of

arachidonate metabolites recovered from un-

stimulated cells precluded the direct identification of these materials by the methods described above. however.

To gain some information about arachidonic acid metabolism by unstimulated cells murine eosinophils were labelled with [ “Hlarachidonic acid (see legend of Fig. 16) then washed free of non-cell-associated radioactive label, and in- cubated at 37°C. Neither ionophore A23187 nor unlabelled, exogenous arachidonate were included

87

c (CH

A SiO i OSiKH_&

A AZ‘ ‘Y

321 173

321 463 479 4M4

,,,I ,,(,,,,,,,,,,,,, / ,,,.,,,,.,,,,,,,., I/,.; ,,,, ;,l,i ,,,,,,,,, I.,,I;!, f

150 170 190 210 250 P-50 270 290 310 330 350 370 390 410 433 ‘co 470 490

Fig. 14. Mass spectrum of the Me,Me,Si derivative of a 14,15-dihydroxylated conjugated triene metabolite of arachidonic acid from

murine eosinophils. The spectrum is that of the indicated derivative of one of the components (compound V, Table I) of DIOL peak 6

(Fig. 8) and was obtained under the same conditions as in Fig. 10. The double bond structure depicted in the schematic diagram has

not been established.

in these incubations. The predominant radioac-

tively labelled material recovered from these in-

cubations that exhibited the chromatographic be-

havior of a lipoxygenase product co-migrated with

I2-HETE, as shown in the lower panel of Fig. 16.

When cells prepared in this way were incubated

Fig. 15. Summary of arachidonic acid lipoxygenase pathways in

the murine eosinophil.

TABLE II

STIMULATION OF 12-HETE PRODUCTION BY MURINE

EOSINOPHILS UPON TREATMENT WITH EOSINOPHIL

STIMULATION PROMOTER AS MEASURED BY A STA-

BLE ISOTOPE DILUTION CC-MS ASSAY

Murine eosinophils were isolated as described in Materials and

Methods. In experiment 1 the control population and the

population to be stimulated with eosinophil stimulation promo-

ter contained 48. lo6 cells (92% eosinophils, 8% mononuclear

leukocytes). In experiment 2 each population contained 70. IO6

cells (100% eosinophils). Incubations were performed in

Tyrode’s gel buffer supplemented with 0.6 mM CaCl, and 1

mM MgCl, at a concentration of 10’ cells/ml for 80 min at

37°C. The stimulated populations were exposed to partially

purified eosinophil stimulation promoter at a concentration of

5 pg/protein per ml. Incubations were terminated by addition

of 1.5 vol. of methanol, and the internal standard octadeutero-

12-HETE (500 ng/lO’ cells) was then added. The 12-HETE

was extracted and isolated as described in Materials and Meth-

ods, converted to the ethyl ester, trimethylsilyl ether derivative,

and analyzed by CC-MS (3 foot 3% SP2100 column, 215°C

carbon value 21.8) with monitoring of the ions m/z 309 (from

the derivative of endogenous 12-HETE) and m/r 315 (from

the derivative of the internal standard).

Experiment Amount of 12 HETE (ng per IO6

cells)

Ratio

Control Eosinophil stimulation-

promoter-stimulated

1 0.83 2.8 1 3.37 2 2.22 5.60 2.61

16

8

i

CONTROL x

E

u” 16

Y

1 I

30 60 90 120 150 I80 210 240 270

ELUTION VOLUME (ml1

Fig. 16. Stimulation of 12-HETE production by murine

eosinophils upon treatment with eosinophil stimulation promo-

ter demonstrated by HPLC of products from cells pre-labelled

with [ ‘Hlarachidonate. Murine eosinophils were prepared as

described in Materials and Methods. 60. IO6 cells (90%

eosinophils 10% mononuclear leukocytes) were incubated with

50 PCi of [ 3 Hlarachidonic acid for 30 min at 30°C in Tyrode’s

gel buffer. Cells were collected by centrifugation (100 x g, 10

min) and washed free of non-cell-associated radioactive label

by resuspension in fresh buffer and centrifugation. The radio-

actively labelled cells were then divided into two populations

containing equal numbers of cells. One population was in-

cubated with partially purified eosinophil stimulation promoter

(5 pg/ml) in Tyrode’s gel buffer supplemented with 0.6 mM

CaCI, and I mM MgCl, (IO’ cells/ml) at 37°C for 80 min. The other population was incubated under identical conditions

in the absence of eosinophil stimulation promoter. Incubations

were terminated by the addition of 1.5 vol. of methanol and

0.01 PCi of [‘4C]prostaglandin F2_ was added to each sample to monitor extraction efficiency and column recovery. Products

were extracted as described in Materials and Methods and

subjected to HPLC on column I, eluting successively with

solvent A (140 ml), solvent B (100 ml). and solvent C (50 ml).

An aliquot of each fraction was subjected to liquid scintillation

counting for ‘H and 14C The retention volume of standard

12-HETE is indicated by the arrow.

with the lymphokine eosinophil stimulation pro- moter in a concentration sufficient to induce a

migration response, a larger amount of radioactiv-

ity co-migrated with 12-HETE, as shown in the

upper panel of Fig. 16. After correcting for extrac-

tion efficiency and column recovery, the amount

of radioactivity co-migrating with 12-HETE from

incubations of eosinophil stimulation promoter-

treated cells divided by the amount from unstimu-

lated cells was 2.2 and 1.8 in duplicate experi-

ments. To demonstrate that eosinophil stimulation pro-

moter treatment did result in increased production of 12-HETE, the compound was quantified by a

stable isotope dilution GC-MS assay employing

octadeutero-12-HETE as internal standard [ 171. In

duplicate experiments eosinophil stimulation pro-

moter treatment resulted in a 2.6- and 3.3-fold

increase in 12-HETE levels compared to control

incubations, as shown in Table II.

Discussion

The presence of arachidonic acid lipoxygenase enzymes in murine eosinophils was demonstrated

by the isolation and identification of several lip- oxygenase products from incubation of these cells.

Three classes of lipoxygenase products were iso-

lated from these cells after stimulation with iono-

phore A23187 and exogenous arachidonic acid: (1)

Products formed via a single dioxygenation fol-

lowed by reduction included 12-S-HETE and 15

HETE, which were the two most abundant

arachidonate metabolites recovered from the cells. (2) Products formed via two successive dioxygena-

tion steps and reduction included 5,15-diHETE and 5,12-diHETE. (3) Products formed by a single

dioxygenation followed by epoxide formation and then reaction with a nucleophile (e.g., water) in-

cluded 15-series leukotrienes (e.g., compound I)

and 5-series leukotrienes (e.g., leukotriene B4).

Lipoxygenases with regional specificities for carbons 12, 15 and 5 appear to be active in these cells. The failure to isolate 5-HETE from the in- cubations on four of five occasions likely reflects that reduction of 5-HPETE to 5-HETE occurs less readily in those cells than does the conversion of 5-HPETE to leukotriene A, or to the double di- oxygenation products, 5,15-diHETE and 5,12-di- HETE.

The spectrum of lipoxygenase products from

murine and human eosinophils [ 121 is remarkably

similar, with the exception that the murine cells generate much larger amounts of 12-HETE. The

synthesis of 15series leukotrienes by eosinophils

of both species is is particularly striking in view of

the finding that among human blood leukocytes

eosinophils are the dominant source of these com-

pounds [12]. Homogenous populations of porcine eosinophils have also recently been shown to syn-

thesize 15series leukotrienes [ 141. Expression of

this novel biochemical pathway may prove to be a general property of mammalian eosinophils. Infor-

mation on what role the products of this pathway

play in the function of the eosinophil may soon be

provided by chemical synthesis [30,31] and bio-

logical testing of the 14,15-oxido analog of

leukotriene A 4.

The possibility that eosinophils play a central

role in the modulation of levels of mediators of

immediate hypersensitivity is suggested by the re-

cent observations that horse and human eosino-

phils can both synthesize [32] and inactivate [33]

leukotrienes B4, C, and D4. Peptide conjugates of arachidonate derivatives (e.g., leukotrienes C, and

D4) would not have been recovered by the isola- tion procedures used in this study. The ability of

murine eosinophils to synthesize leukotriene A 4 was demonstrated by the isolation of hydrolysis

products of that molecule including leukotriene B4. The isolation of leukotriene B4 and of 12-HETE

from the murine eosinophils is of special interest

in view of our recent demonstration that both of these molecules induce a migration response in

murine eosinophils and that inhibitors of

arachidonic acid lipoxygenases block the migra-

tion response of the cells to the lymphokine

eosinophil stimulation promoter [ 161. These ob-

servations have suggested the possibility that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipo-

xygenase products which modulate the migratory activity of these cells. Our demonstration here that

eosinophil stimulation promoter treatment stimu- lates 12-HETE production by murine eosinophils is further evidence in support of this hypothesis.

The amounts of I2-HETE recovered from the incubations were small (281-560 ng per 10’ cells) compared to the amounts of exogenous 12-HETE required to produce a maximal migration response

89

(2 pg per ml) [16]. It should be noted that up to

24 h is required to develop a detectable response in this assay system, however. Considerable

metabolism of 12-HETE (e.g., to the double di-

oxygenation product 5,12-diHETE) could occur

during this period and reduce the actual 12-HETE

levels to far below those initially added to the

incubations. Eosinophil stimulation promoter

could act by producing a small but sustained increase in 12-HETE production. It is also possi-

ble that eosinophil stimulation promoter stimu-

lates synthesis of lipoxygenase products in addi-

tion to 12-HETE. Leukotriene B4 is a more potent

stimulus to eosinophil migration than is I2-HETE

[16]. The lack of a deuterated internal standard

has so far prevented quantification of nanogram

levels of this compound.

In summary, evidence has been presented indi-

cating that murine eosinophils possess lip- oxygenase enzymes with regional specificities for

carbon atoms 12, 15 and 5 of arachidonic acid. These cells also convert 5-HPETE and 15-HPETE

to the corresponding oxido compounds (leuko-

triene A, and 14,15-leukotriene A4). Stimulation

of the cells with the lymphokine eosinophil stimu-

lation promoter results in increased synthesis of

12-HETE, which is known to induce migration of

these cells.

Acknowledgements

Supported by grants GM-l 543 1 and AI-l 1289 from the National Institutes of Health and the

Veteran’s Administration. T.L.R. is supported by

a Medical Scientist Training Grant 2T32 GM07397

from the National Institutes of Health. R.L.M. is

supported by a Vivian Allen M.D., Ph.D. Training

Fellowship from Vanderbilt University. J.A.O. is

the Joe and Morris Werthan Professor of Investi- gative Medicine.

References

Hamberg, M., Svenson, J., Wakabayashi, T. and Samuels- son, B. (1974) Proc. Nat]. Acad. Sci. U.S.A. 71, 345-349

Hamberg, M., Svensson, J. and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2994-2998

Hamberg, M. and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3400-3404

90

4 Borgeat, P., Hamberg, M. and Samuelsson, B. (1976) J.

Biol. Chem. 251, 7816-7820

5 Narumiya, S., Salmon, J.A., Cottee, F.H., Weatherley, B.C.

and Flower, R.J. (1981) J. Biol. Chem. 256, 9583-9592

6 Goetzl, E.J., Brash. A.R.. Tauber, A.I., Oates. J.A. and

Hubbard, W.C. (1980) Immunology 39, 491-498

7 Goetzl, E.J., Weller, P.F. and Sun, F.F. (1980) J. Immunol.

124. 926-933

8 Borgeat, P. and Samuelsson, B. (1979) Proc. Nat!. Acad.

Sci. U.S.A. 76. 3213-3217

9 Samuelsson, B., Borgeat, P., Hammarstrom, S. and Murphy,

R.C. (1979) Prostaglandins 17, 785-787

IO Borgeat, P. and Sirois, P. (1981) J. Med. Chem. 24, 121-125

I I Goetzl, E.J. and Pickett. W.C. (1980) J. Immunol. 125,

1789-1797

12 Turk, J., Maas, R.L., Brash. A.R., Roberts, L.J., 11 and

Oates, J.A. (1982) J. Biol. Chem. 257, 7068-7076

13 Maas, R.L., Brash, A.R. and Oates, J.A. (1981) Proc. Natl.

Acad. Sci. U.S.A. 78, 5523-5227

14 Turk, J., Maas, R.L., Roberts, L.J., Brash, A.R., Oates, J.A.

(1983) Adv., Prostaglandin, Thromboxane Leukotriene Res.

11, 123-132

15 Colley, D.G. (1973) J. Immunol. 110. 1419-1426

16 Rand,T.H., Turk, J., Maas, R.L. and Colley, D.G. (1982) J.

Immunol. 129, 123991244

17 Boeynaems, J.M., Brash, A.R., Oates, J.A. and Hubbard,

W.C. (1980) Anal. Biochem. 104, 259-267

18 Rand, T.H. and Colley, D.G. (1982) Cell. Immunol. 71, in

the press

19 Timmons, S. and Hawiger. J. (1978) Thromb. Res. 12.

297-301

20 Vadas, M.A., David, J.R.. Butterworth, A., Pisani, N.T. and

Siongok, T.A. (1979) J. Immunol. 122. 1228- 1236

21 Hamberg, M. (1971) Anal. Biochem. 43, 5155526

22 Green, K. (1971) Biochemistry 10, 1072-1086

23 Porter, N.A., Wolf, R.A., Yarbro, E.M. and Woenan, H.

(1979) Biochem. Biophys. Res. Commun. 89. 10588 1064

24 Jubiz, W., Radmark, O., Lindgren, J.A., Malmsten, C. and

Samuelsson, B. (1981) Biochem. Biophys. Res. Commun.

99, 976-986

25 Lundberg, U., Radmark, O., Malmsten, C. and Samuelason,

B. (1981) FEBS Lett. 126, 127-132

26 Van OS, C.P.A., Rijke-Schildre, G.P.M.. Halbeek. H.V.,

Verhagen, J. and Vliegenthart. J.F.G. (1981) Biochim. Bio-

phys. Acta 663, 177-193

27 Maas, R.L., Turk, J., Oates, J.A. and Brash, A.R. (1982) J.

Biol. Chem. 257, 705667067

28 Borgeat, P., Picard. S.. Vallerand, P. and Sirois, P. (1981)

Prostaglandins Med. 6, 557-565

29 Borgeat, P. and Samuelsson. B. (1979) J. Biol. Chem. 254,

2643-2646

30 Corey, E.J., Marafat, A. and Goto, G. (1980) J. Am. Chem.

Sot. 102, 6607-6608

31 Atrache. V., Pai, J.-K., Sok, D.E. and Sih. C.J. (1981)

Tetrahedron Lett. 22, 3443-3446

32 Jorg, A.. Henderson, W.R.. Murphy, R.C. and Klebanoff.

S.J. (1982) J. Exp. Med. 155. 390-402

33 Henderson, W.R., Jorg, A. and Klebanoff, S.J. (1982) J.

Immunol. 128. 2069-2075