Organic

inhibit

peroxides

neutrophil

leukotriene

B4

biosynthesis Ian J. Okazaki, Department

Lisa

M. Newman,

of Medicine,

Veterans

and

Administration

David Medical

W. Allen Center,

and

University

of Minnesota

Medical

School,

Minneapolis

burst

Abstract: Leukotriene B4, an autacoid metabolite of arachidonic acid produced by polymorphonuclear neutrophils, induces chemokinesis, chemotaxis, and adhesion of these cells at sites of inflammation. Because neutrophil infiltration is a self-limited process, we hypothesized that oxidized lipid products of neutrophil-damaged tissue might inhibit leukotriene B4 biosynthesis, thereby preventing additional neutrophil infiltration and limiting

[6-9]

inhibitors

and

thus

acid

.

organic

peroxides

lipid

be

as

inhibitors

of

generated

ideal

To explain

in

negative

this

penoxides

has

dependent

LTB4

biosynthesis.

parallel

with

feedback

negative

been

on

sumably

Such

tissue

control

damage

elements

in

feedback

observed

thiol

because

in

levels the

control

in

vitro

the

[10],

per-

this

incubation

hydropenoxide

infiltra-

activation

was

mixture,

pre-

stimulates

the

5-lipoxy-

(tBHP), penacetic acid (PA), and linoleic hydnopenoxide (LHP). tert-Butylhydnoperoxide is a stable organic peroxide commonly used to induce lipid penoxidation through formation of alkoxyand penoxyradicals [12]. Peracetic acid is of interest

as

radicals

related

hyde

an

oxidative

product

of

found

during

to those

metabolism

[13,

14].

in

tissue

and

ethanol

Peracetic

acid incorporation into RBC acyltransferase [15]. Linoleic penoxidized product of linoleic acid

ethanol acid

membranes.

releases

and

inhibits

phospholipids hydroperoxide acid [16], We

effects of these peroxides on free arachidonate into phospholipids, from phospholipids, the 5-lipoxygenase, (LTA4) hydnolase.

oxidation

of PMN

explored which of the steps in be inhibited by lipid-soluble of 5-lipoxygenase by hydro-

genase by reversal ofthe thiol inactivation ofthe enzyme [11]. The relation of these observations with the purified enzyme in vitro to possible in vivo activity of inhibitors remains to be determined, since the status ofcytoplasmic soluble constituents including the oxidation-reduction status is not known. We used the organic peroxides tert-butylhydroperoxide

fatty

arachidonic

be

tion in inflammatory sites, we the biosynthesis of LTB4 might peroxides. Although stimulation

sue membrane peroxidation in lipid solubility and catalase resistance, inhibited leukotriene B4 biosynthesis in a dose-dependent manner (50 % inhibitory concentration of 3.9 tM compared to 530 tM for H202). Biosynthetic steps prior to the 5-lipoxygenase did not appear to be the site of inhibition. Likewise, the step after the 5-lipoxygenase, the leukotriene A4 hydrolase, was not primarily involved. Thus a possible mechanism for controlling the influx of neutrophils and their oxidative damage during inflammation may be inhibition of the 5-lipoxygenase by catalase-resistant lipid peroxides released by tissue membranes.J. Leukoc. Biol. 52: 645-651; 1992.

Words:

act

inflammation.

peroxidative tissue damage. Erythrocyte ghosts exposed to a hydrogen peroxide-generating system served as a model of peroxidized tissue in inflammation and inhibited neutrophil leukotriene B4 production by 50% compared with unoxidized ghosts. Organic peroxides, including tert-butylhydroperoxide, peracetic acid, and linoleic hydroperoxide, resembling the product(s) of tis-

Key

and would

free

acetalde-

anachidonic

at the site of an (LHP) is the a polyunsaturated

examined

in

turn

the

arachidonate, uptake release of arachidonate and leukotniene

of A4

INTRODUCTION Polymonphonuclear matony

loci

neutnophils in

part

due

to

(PMNs)

chemotaxis

and

infiltrate

inflam-

adherence

stimu-

MATERIALS

lated by leukotniene B4 (55,12R-dihydnoxy-6,14-cis-8,iO-transeicosatetraenOic acid) (LTB4) [1, 2]. A key property of PMNs in inflammation is the control and localization of their function by a self-limited process [3]. Although there are probably multiple physiologic mechanisms for limiting tissue damage in inflammation, one site of feedback control of inflammation may be inhibition of LTB4 biosynthesis by an autoxidative

function

[4].

To

simulate

tissue

Thus dation

and,

unlike

hydrogen

lipid penoxides as a consequence

peroxide,

[5] may result of the PMN

was

Materials Hanks’ 1077,

balanced

salt solution (HBSS), Histopaque 1119 and ionophone A23187, N-formyl-Met-Leu-Phe cytochalasin B, hydrogen peroxide 30%, sodium soybean lipoxygenase, glucose oxidase, superoxide

calcium

(fMLP), linoleate,

peroxidation

and explore the role of peroxides generated during inflammation [4-7], we exposed red blood cell (RBC) membranes to a hydrogen peroxide-generating system analogous to the respiratory burst of PMNs. We then incubated PMNs with the peroxidized RBC membranes and found LTB4 biosynthesis inhibited. A lipid extract of the oxidized membranes was equally effective at suppressing LTB4 biosynthesis

AND METHODS

catalase

from membrane antimicrobial

Abbreviations:

leukotriene

phonuclear retention

coenzyme

HBSS,

performance LHP, linoleic

liquid

Hanks’

chromatography;

hydroperoxide; B4; PA,

LPC,

peracetic

neutrophil; time;

tBHP,

acid;

RBC,

fMLP,

A; balanced

red

IC50,

N-formylmethionyl-leucylsalt

solution;

HPLC,

50%

inhibitory

concentration;

lysopalmitoylphosphatidylcholine; PGB1,

blood

prostaglandin

cell;

RIA,

ieri-butylhydroperoxide;

TLC,

highLTB4,

B1; PMN, radioimmunoassay; thin-layer

polymor-

RT, chromatog-

raphy.

resistant.

peroxioxidative

CoA,

phenylalanine;

Reprint 10,

Room Received

Journal

Requests:

Ian

5N-307, June

of Leukocyte

Okazaki,

Bethesda, 5,

1992;

MD accepted

Biology

National

Institutes

of Health,

Building

20892. July

Volume

30,

1992.

52,

December

1992

645

dismutase,

anachidonoyl

coenzyme

A (CoA)

and

LTB4,

LTA4

addition

of

5 ml

of

ethyl

acetate

methyl ester, and prostaglandin B1 (PGB1) standards were purchased from Sigma, St. Louis, MO. EDTA and Tnis was obtained from Fisher, Fain Lawn, NJ. HEPES was from Grand Island Biological Co., Grand Island, NY. [‘4C]Anachidonic acid and [14C]lysophosphatidylcholine were from New England Nuclear, Boston, MA. Penacetic acid and Tween-20 were supplied by Aldrich, Milwaukee, WI. Anti-CR3 (Leu-15/CD11) was from Becton Dickinson, Mountainview, CA, and goat F(ab’)2 antimouse immunoglobulins, fluonescein conjugated, were from TAGO Inc., Bunlingame, CA. The silica t-Porasil column, chromatographic pumps, and gradient controller were from Waters, Milford, MA. The

internal standard. with 0.01 N acetic

immunoassay

(LTB4

C18 reverse-phase high-performance liquid chromatography (HPLC) column was obtained from Rainin, Woburn, HPLC solvents were from Fisher, Springfield, NJ, and layer chromatography (TLC) plates silica gel H were Analtech, Newark, DE.

With LTB4

methods of same retention

Isolation

of human

of ethyl acetate under a stream

of Human

The RBC pellet centnifugation, HBSS, pH 7.4,

acid

MA. thinfrom

to

1 x

10

precursor

into

all three had the

were

ghosts

was were

in

determined incubated

[19]. Ghosts containing 10 mM glucose, 200

tM

250 jg ferrous

protein/ml sulfate

(25:30:45,

v/v).

HPLC

The

using

an

methanol, and 0.1% buffer, pH 5.5 (25:30:45, were collected. Leukotniene of the [14C]arachidonic absorbance,

Dupont,

identification, time as

and

radio-

Wilmington,

the

DE).

biosynthetic LTB4.

standard

hydroperoxides

of LTB4 production

A23187.

were washed three times with 172 mM Tnis Washed cells were lysed with 11 mM Tnis until the hemoglobin was completely rethe protein concentration ofthe white ghosts

kit,

was dried in 1 ml of

by organic

peroxides

Polymonphonuclear neutnophils (5 x 10’ cells/mi) alone or PMNs and RBCs (1 x 10 cells/ml) were incubated in HBSS-0.02 M HEPES, pH 7.2, 1.4 mM CaCI2, 0.8 mM MgCl2 for 5 mm at 37#{176}Cbefore adding 5 M ionophone

in

Red blood cells buffer, pH 7.4. buffer, pH 7.4, moved [18], and

H2O

as an

to pH 3.5 with 5 ml

lipid extract reconstituted

reverse-phase

ultraviolet

RIA

of linoleic

Inhibition

cells/ml.

erythrocyte

LTB4,

PGB1

of

dark after adjusting the pH to 6.9 [24]. The pH was then brought to 3.0 and the linoleic hydnoperoxides were cxtracted according to Bligh and Dyer [21]. The organic layers were evaporated under nitrogen and the residue was resuspended in phosphate-buffered saline and 0.1% bovine serum albumin, pH 7.4.

In

trations

of peroxidized

by

system

other

(1 x 109/ml)

Generation

and

analyzed

ng

adjusted three times

Linoleic hydroperoxides were generated from linoleic acid by autoxidation. Linoleic acid (2.5 mM) in 0.025 M sodium phosphate buffer with 0.5% Tween-20 at pH 9.0 (final volume 12 ml) was exposed to air for 5 days at 25#{176}Cin the

ABCs from the Histopaque gradient times, and resuspended

was

Generation

PMNs

was obtained washed three

combined and then

of tetrahydrofunan, EDTA in 0.01 M sodium acetate v/v) [23]. One-minute fractions B4 was identified by incorporation isocratic

Whole blood (20-30 ml) was obtained from normal volunteens under a protocol approved by the institutional review board. Neutnophils (containing fewer than 1% mononuclear cells) were isolated by centnifugation ofthe blood oven Histopaque 1077 and 1119 density gradients [17]. Remaining erythnocytes were removed by hypotonic lysis, yielding 3-14 x 108 PMNs total. The PMNs were resuspended in HBSS, pH 7.4, at 5 x 10 cells/ml and were more than 95% viable by trypan blue exclusion.

Isolation

[22]. The of nitrogen methanol,

extract

50

aqueous layers, were extracted

tetnahydnofuran, PMN

with

The acid,

of

then

were tBHP,

experiments,

PMNs

incubated PA,

washed

for

or

H2O2

twice

15 mm at

with

(5

x

with

107/ml)

varying

25#{176}C.Both HBSS before

or

RBCs

concen-

types of cells combining

treated PMNs with untreated RBCs and untreated PMNs with treated RBCs. Cell mixtures were incubated for 5 mm in the HBSS-HEPES, calcium, magnesium solution at 37#{176}C and then stimulated with 5 M ionophone. Leukotniene B4 was extracted with ethyl acetate and analyzed by reversephase HPLC as above. In other experiments RBCs were incubated in the pres-

in

1 mM EDTA, and 25 g/ml glucose oxidase (1 ml final volume) [20] for 2 h at 37#{176}Cto generate oxidized membnanes. Control membranes were incubated without glucose oxidase. The treated membranes were washed three times with phosphate-buffered saline, pH 7.4. In other expeniments 50 g/ml catalase or 20 g/ml supenoxide dismutase was added to RBC ghosts after incubation with glucose and glucose oxidase. The treated RBC membranes (250 g protein/ml) on their lipid extracts [21] were combined with PMNs (7.5 x 10 in 1.5 ml).

ence or absence of tBHP, PA, on H2O2 for 15 mm, washed twice with HBSS, and then resuspended in the HBSSHEPES, calcium, magnesium solution before adding 5 g LTA4. The RBCs were incubated for 5 mm at 37#{176}C,followed by ethyl acetate extraction. Conversion of LTA4 to LTB4 was determined by RIA.

Leukotriene

cording to Bligh and Dyer [21]. Extracts were separated on an HPLC silica column using a linear gradient system from 100% solvent A, hexane, 2-propanol, H20 (6:8:0.75, v/v) to 100% solvent B, hexane, 2-propanol, H2O (6:8:1.4, v/v) [25].

B4 biosynthesis

Journal

of Leukocyte

Lipids from PMNs 1 h and treated with

and analysis

Neutrophils (7.5 x 10 cells in 1.5 ml) HBSS with [i4C]arachidonic acid for PMNs were washed twice with HBSS alone on with varying concentrations of LHP for 15 mm at 25#{176}C.The samples with HBSS and incubated for 5 mm HEPES buffer, pH 7.2, with 1.4 mM MgCl2 at 37#{176}Cbefore stimulation with phone A23187 or 5 g cytochalasin B fMLP. After 2 mm at 37#{176}C,the reaction

646

[14C]Arachidonic

Biology

were incubated in 1 h at 25#{176}C. The and then incubated tBHP, PA, H202, on were washed twice in HBSS-0.02 M CaC12 and 0.8 mM 5 M calcium ionofollowed by 10 M was stopped by the

Volume

52,

December

acid

recovery

labeled tBHP,

with [‘4C]anachidonic PA, or H202 were

acid extracted

for ac-

The 6-mm free fatty acid fraction was collected and [‘4C]anachidonic acid detected with a beta scintillation counten. The phosphatidylethanolamine, phosphatidylinositol, and phosphatidylcholine fractions were also collected and counted. Cell extracts were subjected to silica plate TLC using hexane, 2-propanol, acetic acid (137.4:12:0.6, v/v). The lanes by

1992

were beta

scraped

counting.

and

[‘4C]arachidonic

acid

was

detected

a. Whole

b. Lipid Extracts of Membranes

Membrane

30

c.

.

LII 30

ghosts

25

.;

20

E 15

- Oxidized

Control

Oxidized

Control

20

whole

membranes

was

51.4

±

(mean

of PMN acyltransferase

tBHP,

in 0.1 50

iM

M PA,

phosphate or

1 mM

buffer H2O2

with in

the

or

without

± SE)

absence

of 1 tg/ml arachidonoyl CoA and 1 nmol [‘4C]lysopalmitoylphosphatidylchoiine (LPC), final volume 1 ml. The mixture was incubated at 37#{176}Cfor 10 mm. The reaction was stopped with 5 ml of methanol. After 30 mm, 5 ml of chloroform and 50 tg of [12C]LPC were added and the mixture was incubated for 10 mm at 25#{176}C.The suspension was filtered, evaporated under nitrogen, resuspended in chloroform, and run on TLC plates, which were developed with chloroform, methanol, 27% ammonia solution, H2O (90:54:4.8:6.2, v/v). The lanes were scraped and the percent conversion of LPC to [‘4C]phosphatidylcholine was determined [26].

Assessment Neutrophil was assessed

of PMN viability viability after treatment by trypan blue exclusion,

with

organic the ability

peroxides of PMNs

to increase surface expression of the CD11b/18 (Mac-i) adhesion molecule utilizing fluorescein-activated cell sorting [27], and the capacity of PMNs to phagocytose 1-zm polystyrene beads [28].

RESULTS

We examined produced at

hydrogen peroxide lipid extract still of

the control different

the possibility inflammatory

membranes that sites

oxidized could

inhibit tissue inhibit

and

HPLC. ± 26.0% membranes

LTB4

The ofthat (P

production

resulting ofcontrol < .001)

was

PMN PMNs (Fig. la).

neutrophil membranes PMN LTB4

and was caused

.

of

followed by lipid extraction. PMN LTB4 to be 62.05

value, a degree of inhibition not from that of peroxidized membranes

±

sig-

.

without effects

catalase (P of peroxidized

=

by PMNs. Incubating RBC membranes in effect on the inhibition shown).

Effect of inhibitors

721) RBC .

(Fig. ic). membranes

Figure 1 shows the on LTB4 produced

20 ig/ml superoxide glucose and glucose of neutrophil LTB4

on LTB4 production

dismutase oxidase biosynthesis

had

with no (not

by PMNs

The effect of lipid peroxides on PMN LTB4 production was studied further using the organic peroxides tBHP, PA, and LHP as well as H202. Treating PMNs for 15 mm with these peroxides before stimulating the cells with ionophore A23187 resulted in a dose-dependent inhibition of LTB4 production. Figure 2 is the dose-response curve for inhibition of LTB4 biosynthesis by H202 compared with the inhibition by the organic penoxides expressed as percent control versus log concentration of inhibitor. No significant difference was found between the activities of the organic peroxide inhibitons, so the results for these inhibitors were pooled and analyzed together. For H2O2 the concentration at 50% inhibition (IC50) = 530 M, and for organic peroxides IC50 = 3.9 tM (P < .001). Catalase (20 g/ml) could totally prevent inhibition of LTB4 production when present with H2O2 but not when added or LHP. Neutrophils

to the incubation were >95%

mixture with viable by trypan

tBHP, blue

PA, after

treatment by organic peroxides at concentrations that inhibited LTB4 biosynthesis. Neutnophils also maintained the ability to increase surface expression of CD11b/18 adhesion molecule after exposure to peroxides followed by stimulation with fMLP or A23187. Phagocytosis of 1-tm polystyrene beads was not inhibited by organic penoxides. After treatment with 100 tzM tBHP, PMNs had 98.7 ± 4.8% of control phagocytic activity (P = .587). Likewise, PMNs treated with 100 M PA had 107.5 ± 11.9% of control phagocytic activity (P = .295) and those treated with 5 mM H202 had 96.1 ± 18.1% of control phagocytic activity (P = .658).

Lack of effect precursor

of peroxides

on [14C]arachidonic

acid

Initially, we ruled out alteration by the peroxide inhibitors the [‘4C]arachidonic acid used as a precursor for LTB4 synthesis. Under the conditions of the experiments treated [‘4C]arachidonic acid-labeled PMNs with 100 tBHP,

100

sm

PA,

or

polyunsaturated fatty recovered from treated from control PMNs

Peroxidized erythrocyte LTB4 production

A23187

reverse-phase was 51.4 nonoxidized

I

50 jM or

with

for

27.3% nificantly

activity

presence

stimulated

107/ml)

tent This

Neutrophils, 10-20 x 108, in 0.5 ml of0.1 M phosphate solution, pH 7.4, were sonicated and then centrifuged at 1500g x 10 mm at 4#{176}C.The supernatant was analyzed for protein concentration. Supernatant protein (100 jsg) was incubated

x

5

control, n = 12, P < .001. (b) Treated membranes were also extracted per Bligh and Dyer [211 and the resulting lipid extract was then incubated with PMNs followed by stimulation with A23l87. In PMNs exposed to lipid cxtracts of oxidized membranes, LTB4 was 56.8 ± 20.4% of control, n = 6, P = .001. (c) Neutrophils incubated with extracts from membranes previously treated with glucose, glucose oxidase, and 50 g catalase still had inhibited LTB4 production, which was 62.05 ± 27.3% of control, not significantly different from peroxidized membranes without catalase, n = 4, P =721.

Determination

(5

peroxiuizea erytnrocyte 1 h at 25#{176}C.The neutro-

10

Oxidized

26.0%

were

.

we incuDatea

A lipid extract of the peroxidized RBC ghosts also resulted in PMN LTB4 biosynthesis at 56.8 ± 20.4% of the control value (P = .001) (Fig. ib). Catalase (50 tg) was added to the washed oxidized membranes to ensure the absence of persis-

1. Effect of peroxidized RBC membranes on LTB4 biosynthesis. (a) Erythrocyte membranes (250 g protein/ml) were oxidized by 25 sg of glucose oxidase in 10 mM glucose, 200 iM ferrous sulfate in EDTA, in the presence or absence of 50 g of catalase. Control membranes were similarly incubated without glucose oxidase. The membranes were then incubated with PMNs, stimulated with ionophore A23l87, and LTB4 production was assayed by reverse-phase HPLC. Leukotriene B4 produced by PMNs after to oxidized

nereiore

PMNs

phils

Fig.

exposure

with

quantified by LTB4 production incubated with

10

Control

i

25

15

5

.

proauction.

of biowe M

5 mM H202 and then analyzed the acids by TLC. The arachidonic acid PMNs was identical to that recovered in chromatographic properties and in

percent of total counts pen minute: control, 30.7 ± 2.0%; 100 tM tBHP, 33.8 ± 3.2%; 100 jM PA, 26.6 ± 2.7%; 5 mM H202, 27.2 ± 1.0%. No differences were significant at the 10% level. There were also no significant differences in total lipid radioactivity.

Ok.aza/ci

ci al.

Organic

peroxides

inhibit

PMN

LTB4

647

4.0 P1 3.5

C.)

0

3.0

2.5 C

2.0 Q.

1.5 C 0

U

PC

20

10

LOG Fig.

2. Inhibition

tion

by

of LTB4

PMNs:

biosynthesis.

i-butyl

Effect

hydroperoxide

analyzed

using

Newton-Gauss

Asystant

software

pany)

Y

and

a user-fit

percent

-

lion

of

0.888

control

the 0.21,

±

tween

the M

points

and

for

[14C]Arachidonic and free fatty Arachidonic was

PMN

LTB4

ganic

peroxides

the

organic

was

organic left

peroxides

H202

was

as

production. then

=

The

=

for

[14CjArachidonic

the

peroxides

is

phospholipids.

at

Thus

inhibiting

PA

phospho-

of

of or-

inhibition

treated

with

-phosphocholine

was

selectively

also

to

with

significantly

[14C]arachidonic

inhibition

by

conversion

648

for

to

several

Journal

of

especially

the

fMLP fMLP decrease treating

peroxides

to be

of

that

a likely

a pattern

acid

of

uptake

of Leukocyte

radioactivity

than

a control

not

treated

with

ratio of radioactivity = 1, n = 9, P = .026). with fMLP was inhibited with 50 m tBHP before

with This by cx-

120 0

100

C

0

U

80

C

a)

effective

in

less

(i-test with hypothesized divided by control in the radioactivity the labeled PMNs

140

three

C.)

PMN

60

a)

0.

H2O2. (acyltransferase) by

PA

[15].

There-

peroxides on the Table 1 compares

this

PMN

enzyme

acylthe for

this

enzyme

candidate

site

for

PMNs,

the

Biology

to

intact

as

inhibition

PMN phospholipids extract with defined

sensitivity

by

in

inhibitors

Phosphatldylethanolamlne

to

Fig.

in

(‘4CjArachidonic

of

phatidylinositol,

before control

by PA, pre-

similar

to

cell.

Volume

52,

December

PhosphatidylInosltol

Phosphatldylcholine

the

[ ‘4C]palmitoyllysophosphatidylcholine

incorporation into enzyme in a cell-free fatty

be

inhibition

of organic sonicates.

Note

shows

each

acyltransferase

phosphatidylcholine.

that

and

stores

arachidonoyl-CoA-palmitoyl-sn-

the effect in PMN

of

appears

to

into

[‘4C]arachidonate

tBHP

sensitive

fore we examined transfenase assayed

arachidonate since the

found

from phospholipid

acid from the membrane phossite of inhibition of LTB4 biosynthesis by organic peroxides. When PMNs were first labeled with [ 14C]arachidonic acid and then stimulated for LTB4 production with fMLP, there was a decrease in the radioactivity of the phosphatidylethanolamine (4%), phosphatidylinositol (6%), and phosphatidylcholine (17%). Of these three phospholipids, only phosphatidylcholine showed

phospholipid

were

uptake

of

enzyme

glycero-3

cursors

was

compared

enythnocyte

RBCs,

for

incorporation

phospholipids

The

similar

acid release

H202

acid. Neutrophil phospholipid extracts were then subjected to normal-phase HPLC. Figure 3 is an HPLC chromatogram of an experiment in which PMNs were treated with 100 tM PA and results expressed as total counts recovered from fractions collected from the HPLC column versus retention time (RT) in minutes. There is decreased uptake of [‘4C]arachidonic acid into membrane phosphatidylethanolamine (RT = 11 mm), phosphatidylinositol (RT = 18 mm), and phosphatidylcholine (RT = 33 mm) by cells treated with PA. This was not observed in PMNs treated with 100 iM tBHP. With 50 times the concentration, 5 mM H2O2 had a smaller effect (Fig. 4). Note that the inhibition by

line).

The release of arachidonic pholipids is another potential

site

incubated

(solid

be-

by the

IC50

40

(minutes)

=

difference

membrane

possible

Neutnophils

and

n

PMNs

B 18, B 7,

=

M.

into

one

form

demonstrated

into precursor

incorporation

examined

n

significant

curve.

3.9

the concentra-

H202,

peroxides no

Com-

of

c is the

For

peroxides

of the

acid uptake acid stores acid

lipids

various to

where

organic

There

Software

equation

constants. For

(0); was drawn for fitting by the

(Macmillan

30

Time

Fig. 3. [‘4ClArachidonic acid uptake. High-performance liquid chromatography analyses of lipid extracts from control PMNs and PMNs treated with 100 M PA. Both PMN samples were then labeled with [‘4C]arachidonic acid. Radioactivity (counts per minute) recovered from free fatty acids (6 mm), phosphatidylethanolamine (11 mm), phosphatidylinositol (18 mm) and phosphatidylcholine (33 mm) was reduced in lipids from PMNs treated with PA (dashed line, #{149}) compared to that in lipids from untreated control

produc-

hydroperoxide

Hill’s

K?1)],

LTB4

curve curve

nonlinear

+

0.35.

±

± 0.064.

of the

of data

530

1.75

=

0.168

=

activities

clustering was

K

K

linoleic

from

-

and

and and

adapted

lOO[Kc6/(l K and B are

100

=

inhibitor

± 0.202,

1.30

equation

on

( A ). A smooth

peroxide

with

of inhibitors

(+);

peracetic acid (#{149}); hydrogen H202 and the results were method

Retention

(pM)

1992

4.

Inhibition

of acid and

by

5 mM tBHP

H202 (solid

incorporation into

100 M

(light bars).

PA (dark

hatched

of

PMNs

acid.

uptake

hatched

bars)

and

into

phospholipids.

phosphatidylethanolamine,

phosphatidylcholine

with I 14C]arachidonic [‘4Clarachidonate

incubation PMNs,

inhibited

arachidonate incorporation

bars).

When

into There

no inhibition

phos-

treated

with

expressed

all three was

peroxides

as

percent

phospholipids a smaller

of uptake

effect

with

of

was with

100 M

TABLE Activity

1 . Comparison of Arachidonoyl-CoA: Acyltransferase

of

the

Effect

of

Several

Peroxides

on

the

i-palmitoyl-sn-glycero-3-phosphocholine Present in a Sonicate of PMNs Perce

nt of Control

(6930

0.04

cpm)

E Peracetic (50 &M,

Mean SE P vs.

acid n

-

t-Butylhydroperoxide 7)

(50

43.6 4.3 control,

i-test

tiM,

n

-

4)

H202 (5 mM, n

96.2 7.8

.

92.8 8.6

0.342

0.03

C

0

-

0

z

0.02

0.540 0 C’)

0.01

posure PMNs

to LMLP. tert-Butylhydroperoxide prior to fMLP resulted in

treatment of 15.9

retention

of ±

the

3.7%

(n = 15, P = .001) more radioactivity than the controls, indicating inhibition of release of the arachidonate by tBHP. In similar experiments 50 M PA and 5 mM H202 had no significant effect. Release of free arachidonate is required for activity of the 5-lipoxygenase in LTB4 biosynthesis, and of the peroxides tested only tBHP inhibited such release and then

only

0.00 0

Fig. 6. Inhibition HPLC chromatogram hydration

lated the

Inhibition of 5-lipoxygenase McGee enzyme

and Fitzpatrick [22] LTA4 hydnolase,

have shown that RBCs which, when combined

have

the with

A23l87.

from

PA

(-

reduction

hydrolase

of both

LTB4

that

(which

the

is required

by

peroxides.

LTB,

and

PMNs

or 100 M B1 was used

-),

indicating

of

control

Prostaglandin

of PA,

isomers

recovery

and

PA (- - -) as an internal and

its

nonenzymatic

PMNs

isomers

5-lipoxygenase for

Reverse-phase

two

treated

with

and then standard.

stimuNote

with

increasing

is inhibited

biosynthesis

of

and

LTB4

but

not

not its

isomers).

20

tion of both LTB4 and tion of inhibitor can 5-lipoxygenase. Similar

I

I

I

E C.) 0)

0 C

LTB4

50 iM

dose-dependent LTA4

LTB4

30

__

40

C.)

the

)

and

even in the presence of untreated RBCs. Red blood cells treated with 100 M tBHP, 100 tM PA, or 5 mM H2O2 had no effect on LTB4 biosynthesis. Figure 5 shows the results of one such experiment using 100 tM PA as the inhibitor. Similan results were obtained using 100 M tBHP or 5 mM H202. These findings suggest that the 5-lipoxygenase enzyme complex of the PMNs was inhibited. The LTA4 hydrolase of the PMNs or RBCs could not be the only enzyme inhibited by the organic peroxides used, because additional LTA4 hydnolase present in untreated RBCs did not remove the inhibition. Inhibition of 5-lipoxygenase should prevent production of both LTB4 and its nonenzymatically formed isomers, whereas after inhibition of LTA4 hydrolase, the nonenzymatically formed LTB4 isomers should still be detected. Figure 6 shows an example of a reverse-phase HPLC analysis at 280 nm absonbance plotted against retention time. Prostaglandin B1 is used as the internal standard. Leukotniene B4 and two of the nonenzymatic LTB4 isomers are eluted as separate peaks. A dose-dependent reduction in the produc-

50

U)

with

of

- . .

concentration

PMNs, can potentiate LTB4 production. This transcellular biosynthesis of LTB4 can be utilized to determine whether the 5-lipoxygenase enzyme complex of the PMNs exclusively or the LTA4 hydnolase of the PMNs on RBCs is inhibited by organic peroxides. Neutrophils alone (5 x 107/ml) or PMNs plus RBCs (1 x 109/ml), untreated PMNs plus RBCs treated with peroxide on PMNs treated with peroxide plus untreated RBCs, were incubated in HBSS-0.02 M HEPES buffer, pH 7.2, with 1.4 mM CaCl2 and 0.8 mM MgC12 and then stimulated with 5 sM A23187. Leukotniene B4 production was assayed by reverse-phase HPLC. When neutrophils were incubated with 100 M tBHP, 100 tM PA, on 5 mM H2O2, production of LTB4 and its isomers was completely abolished

-j

(

PA

LTB4

showing

isomers

10 M

partially.

of

its isomers with increasing concentrabe seen, indicating inhibition of the dose-response curves were obtained

C

with

tBHP

The 10

hydrolase

___________

0

PMN

PMN

PMN

rbc

Inhibition

of the

neutrophil

PMN

+

+

Fig. 5.

and

lack

rbc 100pM

5-lipoxygenase.

PA

Leukotriene

100pM

PA

rbc

B4 produc-

tion from PMNs alone, PMNs + RBCs, PMNs + RBCs treated with PA, or PMNs treated with PA + untreated RBCs and then stimulated with A23187. Peracetic acid (100 M) totally abolished LTB4 production by PMNs despite the presence of untreated RBCs. However, there was no inhibition of LTB4 production when RBCs, treated with 100 jM PA, were then combined with untreated PMNs.

of was

which

contain

of

M

100

H202

effect also

the tBHP,

(not

of shown

LTA4 100

shown).

the

organic

by

penoxides

incubating

hydrolase, M

PA,

in the on

on

RBCs

5 mM

(1

presence H2O2.

the x

LTA4

109/ml),

or absence Leukotniene

A4 was then

added and its conversion to LTB4 determined by RIA. In two separate experiments LTB4 produced by RBCs treated with 100 tM tBHP was 137% of that produced by control untreated RBCs (range 84.6-190%). Similarly, RBCs treated with 100 tM PA had 153% ofthe control LTB4 production (range 85-202%) and 5 mM H202 treatment resulted in 135% of the control LTB4 production (range 90-174%).

Although

resulted in RBC LTA4

total inhibition of hydrolase remained

Okazaki

et al.

Organic

similar

peroxides

doses

of

PMN LTB4 unaffected.

inhibit

organic

penoxides

biosynthesis,

PMN

LTB4

the

649

production, thus confining oxidative tissue damage. Compared to hydrogen peroxide, the initial peroxide product of the PMN oxidative burst, lipid peroxides are catalase resistant and inhibit LTB4 biosynthesis at micromolar concentrations. Lipid peroxides may be effective feedback inhibitors of leukotniene formation because, being hydrophobic and lipid

DISCUSSION The results of this study show suppression of PMN biosynthesis of LTB4 by peroxidized membranes and dosedependent inhibition by such organic penoxides as tBHP, PA, and LHP. Although selective modulation of LTB4 biosynthesis

by

different

5-lipoxygenase

may

tion

at

stages

into

phospholipids,

earlier

penoxides

be the of

or

occurs

primary

site

arachidonate

release

at

several

steps,

of inhibition. uptake,

therefrom

soluble, governing hydration.

the

Inhibi-

incorporation

was

less

Supported

by

products of inflammation for catalase-resistant lipid

Journal

of Leukocyte

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ACKNOWLEDGMENTS

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