BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 292-299

March 15.1979

ARACHIDONIC ACID INDUCED DEGRANULATION OF RABBIT PERITONEAL NEUTROPHILS P.H. Departments

Naccache,

H.J.

of Pathology

Received

February

Showell,

E.L.

Becker

and Physiology, University Farmington, Connecticut

and R.I.

Sha'afi

of Connecticut 06032

Health

Center

6,1979

SUMMARY: We have found that arachidonic acid rapidly and selectively induces the release of lysosomal enzymes from cytochalasin B treated rabbit peritoneal 5, 8, 11, 14-eicosatetraynoic acid inhibits the arachidonate induced neutrophils. release wi h an apparent KD of 1.5 x lo-6M. 5,8,11,14-eicosatetraynoic acid (2.5 x lo- s M) also inhibits the chemotactic factors and the A23187 induced release in the presence of cytochalasin B but does not affect the degranulation induced by A23187 alone. These observations strongly suggest a role far arachidon ate metabolites in rabbit neutrophil physiology. INTRODUCTION Interrelationships clic

nucleotides

cell

activation

pholipid

are

cells

reported

A23187.

been reported

fluxes,

gating

have,

concerning

and cy-

non-muscle

stimuli

(1).

Phos-

been

of a number

(4,

5, 6) and unsaturated

PGE1, thromboxane to possess

to phagocytic

activity

shown that a close those

of Ca2+,

hydroxy

stimuli

B2 and hydroxy

chemotactic

especially

metabolism

pos-

of hormonally

fatty for

correlation

Formyl-Methionyl-Leucyl-Phenylalanine

acids

5,8,11,14-eicosatetraynoic

acid.=

ETYA.

El = PGEl.

0006-291X/79/050292-08.$01.00/0 Copyright @I I979 by Academic Press. Inc. All rights of reproduction in any form reserved.

292

fatty

have,

the neutrophils exists

and the secretory

= F-Met-Leu-Phe

(3)

and in the acids

.

iono-

in addition, (8,

between activity

gen-

(7)

and to the calcium

Abbreviations:

Prostaglandin

calcium

in particular,

mechanisms

in phospholipid

upon exposure

We have previously cation

ionic

ideas

and non-hormonal

metabolisms

changes

of prostaglandins

have been phore

in the

metabolites,

(2).

In the neutrophils, eration

acid

acid

of the present

of hormonal

arachidonic

to be involved

responsive

arachidonic

at the center

by a variety

and/or

tulated

between

9, 10). stimulated of neutro-

Vol. 87, No. 1, 1979

phils

(11).

implicating tion

BIOCHEMICAL

We wish

now to report

arachidonic

of neutrophils

acid

the

and/or

by secretory

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

results

its

of preliminary

metabolites

experiments

in the mechanism

strongly

of activa-

stimuli.

MATERIALS AND METHODS Rabbit peritoneal neutrophils obtained and treated as previously described (12) were used throughout these studies. The cells were resuspended in complete Hanks' balanced salt solution (12) in the absence of albumin. Lysosomal enzyme release was measured as described in Showell et al. (12) with particular care taken to ensure thermal equilibration. The lysosomal enzymes determined were lysozyme and B glucuronidase. Only the results with lysozyme are shown since the release ?jf the two enzymes followed essentially the same course in the various experiments. Degranulation was induced by the addition of the cells to thermally equilibrated test tubes containing the desired fatty acids and cytochalasin B. The incubations were terminated after 5 minutes by transfer to an ice cold water bath. ETYA (a generous gift of Dr. W.E. Scott, Hoffman-La Roche) was added to the cells within the minute preceeding their transfer to the test tubes. Longer preincubation with ETYA were not found to be necessary. Lactate dehydrogenase leakage, a measure of cell death, was checked routinely and never found to be significant. Stock solutions of the various fatty acids were made up at 1O-lM in dimethyl sulfoxide in the presence of 1.4 x lo-4M butylated hydroxy toluene and stored at -700C under nitrogen. Butylated hydroxy toluene was present to ensure the stability of the fatty acid solutions and did not in any detectable manner, affect the results described below. Routine control experiments with fresh batches of fatty acids were also performed. The synthetic Dr. R.J. Freer, purified C5 was nandez and ?lugli by Robert Hamil

chemotactic peptide F-Met-Leu-Phe was kindly supplied by Medical College of Virginia, Richmond, Virginia. Partially isolated from whole serum according to the method of Fer(14). The calcium ionophore A23187 was generously provided of Eli Lilly Co. Indianapolis.

Arachidic acid, linolenic acid, linoleic acid, lignoceric acid, stearic acid, homogamma linolenic acid, 11,14,17-eicosatrienoic acid and decosatetranoic acid were obtained from Nu-check Co. Elysian, Minn. Arachidonic acid was obtained from both Nu-check Co. and Sigma Chemical Co., St. Louis, MO. and found to react indistinguishably. The purity of all of the fatty acids was greater than 99%. RESULTS AND DISCUSSION Three sidering

conditions arachidonic

represent acid

the minimal

metabolites

necessary

to be involved

requirements in the

for

secretory

activity

of the neutrophils: 1)

Exogenously natural

added

arachidonic

acid

stimuli.

293

should

mimic

the effect

con-

of

Vol. 87, No. 1, 1979

Figure

1:

2)

3)

The effects

of exogeneously

arachidonic

acid.

Inhibitors

A direct

This

reached

calcium

shows

that

induced

granule

at micromolar

potentiates does the

towards

the left

is complete

arachidonic

within

(results less

factors, not than

should

should

listed

from

cyto-

be specific

to

inhibit

the response

above conditions,

stimulus, acid

peritoneal

the arachidonic

(12)

three

as a secretory

release

acids

release

stimuli.

is 2.3k

0.2 x lo-7M.

induced

i.e.,

presented

by shifting Arachidonic

2 minutes.

Under

Maximal

release

our

for ara-

activity

Extracellular in the same

the dose-response acid

the

in Figure

secretagogue

acid.

enzyme

shown)).

294

i.e.

The mean ED50 for

of arachidonic acid

is

is a potent

neutrophils.

concentrations

chemotactic

fatty

metabolism

of the

to act

rabbit

way it

tion

first

B treated acid

acid

to secretory

of the

clearly

cytochalasin

is

neutrophils

test

figure

added

of arachidonic

of arachidonate

chidonic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Effect of arachidonic acid and ETYA on lysozyme chalasin B treated rabbit neutrophils.

of the

ability

BIOCHEMICAL

induced

experimental

curve degranulaconditions,

1.

BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CONTROL +ETYA 2.5 X 10-5,

/7

qyL, dy&J

2.sT

,

0

0

-Lb

CSa Figure

the

2:

Effect of ETYA on the chemotactic factors from cytochalasin 8 treated neutrophils.

presence

chidonate

of cytochalasin

induced

ETYA is enzymatic

potent

an analog

of both

inhibitor

are

than

such as the one shown inhibitor

by the

nature

enzyme release

(Figures

The average acid

induced

with

ETYA for

responsiveness cells

release

requirement

of lysosome

for

the ara-

experiments release

10 minuteswashed to arachidonic (results

acid

Figure

in rabbit

the arachidonic

acid

the other

of Figures

1, 2 and 3. that

apparent

KB for

was 1.5 x 10W6M. reversible,

of the inhibitor behave

not shown).

29.5

no differently

seminal 1 is a neutrophils. induced

From experiments

ETYA is

in the dose-response

the

secretagogues

by some published

by ETYA is free

sheep

to inhibit

supported

1, 2 and 3)),an

granule

degranulation

1, the assumption

of the shifts

from

ETYA as shown in

to inhibit

a comparison

has been shown to inhibit synthetase

induced

required

in Figure

KB of four

onic

treated

those

(an assumption

parallel

which

(14).

acid

of ETYA required

smaller

acid

lypoxygenase

as can be seen from

petitive

induced

to be an absolute

the prostaglandin

of arachidonic

The concentrations

tested

of arachidonic

and the soybean

release

B appears

PHE] (AA)

release.

activities

vesicles

[F-MET-;“-

acting reports

curves

for

as a com(15)

and

lysosomal

ETYA can be calculated,

The inhibition i.e., and tested than

of arachidcells for

control,

pretreated secretory un-

BIOCHEMICAL

Vol. 87, No. 1, 1979

AND BIOPHYSICAL

l CVTOCHALASIN

RESEARCH COMMUNICATIONS

8

-CV~~~~IALA~IN

1

I

6

I

I

5

0

* -LOG [A231b] Figure

3:

Effect of ETYA on the A23187 neutrophils in the presence

The specificity sessing

the

other

of the effect

secretory

fatty

activity

acids

possessed

neutrophils

although

they

structurally

only

slightly

A satisfactory provided that

tion

by the results

lysosomal

either

answer

is rapid,

inhibition

10e5M.

release

is

reversible

is also

noteworthy

instead

of F-Met-Leu-Phe

of the

three 2.

for

by as-

rabbit

acid

criteria

and differ

with

ETYA induced defined

the lower

figure

above

ETYA measured

296

not

B by

required.

The

of ETYA enzyme

shown).

It

of ETYA when C5a

stimulus

(% inhibition

by the

and C5a, were experiments),

is

The inhibi-

of lysosomal

(results

by 2.5 x lo-5M

of four

of cytochalasin

at concentrations

inhibition

above

demonstrates

ETYA being

effectiveness

by F-Met-Leu-Phe

listed

by 2.5 x 10m5M ETYA.

secretory

averages

rabbit

None of these

arachidonic

in the presence

and can be observed

out

tested

activity

This

is used as the

as induced

and 37 + 6 respectively,

with

preincubation

in the manner

acids.

from B.

1),

in Figure

In addition,

enzyme release curves,

(Table third

1 minute

to point

fatty

was then

secretory

as induced

is dose dependent than

response

it-

illustrated

than

larger

lysosomal

other

or C5a can be inhibited

less

acid

many properties

to the

enzyme release,

F-Met-Leu-Phe

of arachidonic

any significant

from

6

induced release of lysozyme and absence of cytochalasin

of various

share

(M)

shifts

found

of in the dose

to be 71.k

6

Vol. 87, No. 1, 1979

oxide

BIOCHEMICAL

dismutase

ance of sharp

(30 mg/ml) signal.

added

during

or EDTA (10 mM) had little

Further,

a(-tocopherol-treated

the

microsomes

anion

same spectral

These

or lipid

on the

with

wet weight,

seem to dismiss as a possible

appear-

was observed

25 mg/g liver

results

peroxidation

effect change

(o(-tocopherol,

homogenization).

of superoxide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

was

involvement

explanation

for

the

phenomenon. After brane

aerobic

fraction

showed

incubation was recovered

an EPR spectrum

NADPH addition natant spin Little

is

only

that

the

degeneration

to that

into

treatment,

since

appearance

of the rapidly

of the original were

signal

(Fig.

3).

aqueous

phase

during

of electron

transfer

the membrane

fraction

tumbling

with

NADPH, the mem-

The membrane

the peak heights

sharp

released

microsomes

by centrifugation.

similar

except

showed label

of spin-labeled

decreased, results

appears again

before

while

the

super-

indicate

the reaction

responded component

microsomes

These

system

fraction

with

to occur

that NADPH.

during

to NADPH with

this the

in EPR spectrum.

DISCUSSION The functional known

properties

to be intimately

related

phospholipids

(3-6).

phospholipids

composing

turn,

the

the microsomal

release

of its

component anion place

during

of high

the in this

estimate field

that

the would

in

The experiments point.

transfer bilayer

The

from NADPH to resulting

in the

phase.

and lipid

peroxidation,

oxidation

of NADPH by microsomes

both

of which (9,

are lo),

do

phenomenon.

of the signal

electron

are

by membrane

enzyme system

the latter

of the lipid

the aqueous

formation

seem to be involved A quantitative

into

of this

to explore

perturbation

system

be expected

enzyme reactions.

that

a physical

peak height

during

suggest

induces

not

the microenvironment

attempted

oxygen

P-450 provided

naturally

were

clearly

known to take

will

paper

results

Superoxide

it

perturbed

in this

cytochrome

to the microenvironment

Therefore,

be physically

presented

of microsomal

released

using

304

spin

spin-labeled

moiety

was made from the

stearic

acid

in aqueous

BIOCHEMICAL

Vol. 87, No. 1, 1979

The relative

contributions

pathways

to the

Goldstein

et al.

conditions

phenomena (5)

that

that

high

F-Met-Leu-Phe

Showell,

of effects

importance Taken

together,

these

metabolites

initiation

sential

or only

of a modulatory

coupling nature

postulated

in the neutrophils of the various

su~arized

in the

metabolism

may also

results

are above

role

is

introduction be involved

point

at present.

functions is

however

in neutrophil

(17) very

rabbit

out

however,

for

a role

enzyme release metabolites

is

of the

and of the evidence

chemotaxfs

the

of arain neutro-

of an es-

of stimulus-secretion

In view

likely

neutro-

of arachidonic

evidence

in the mechanism

the

to interpret

metabolite(s)

of arachidonate

unknown

it

clearly

of lysosomal

nature

neutrophils

difficult

strong

have shown

of the multiplicity

however

active

enzyme release

from

In view

reports

M) do inhibit

enzyme release

present

the

lysosomal

(>lO-5

results

Whether

Previous

of indomethacin

the

phils.

(experimental

experiments

presented

in the

enzyme release

Our preliminary

of defining

these

at present.

by indomethacin.

observations).

lipoxygenase

be ascertained

some circumstances,

unpublished

and necessity

cannot

of lysosomal

The results

and of the

lysosomal

inhibited

(16).

of indomethacin,

mechanistically.

that

under

release

cyclooxygenase above

concentrations

induced

(H.J.

chidonate

not

to indomethacin

relatively

phils

described

is

have however,indicated is sensitive

ofthe

have stated

unspecified)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

interrelated briefly

arachidonate

and phagocytosis.

ACKNOWLEDGMENTS We would like to thank Dr. M.B. Feinstein This study was supported and timely advice. of Health Grants AI-13734 and AI-06948.

for many stimulating in part by the National

discussions Institute

REFERENCES 1. 2. i: 5.

Rubin, R.P. and Laychock, S.G. (1978) Ann. N.Y. Acad. Sci. 307, 377-390. Michell, R.H., Jafferti, S.S., and Jones, L.H. (1977) Adv. Exp. Med. Biol. 83, 447-464. and Shohet, S.B. (1974) J. Clin. Invest. 53, 726-734. Smolen, J.E., Zurier, R.B. (1976) Adv. Prost. Throm. Res. 2, 815-818. Malmsten, C.L. Kindahl, H.,laplan, H.B., Radmard, O., Goldstein, I.M., Samuelsson, B. and Weissmann, G. (-1978) J. Exp. Med. 787-792.

acid

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6. 7. 8. 9. E: 12. ii: E: 17.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Higgs, G.A., Bunting, S., Moncada, S., and Vane, J.R. (1976). Prostanglandins 12, 749-757. J.P. Kulzycki, A., and Parker, C.W. (1978) Fed. Stenson, W.F., Atkinson, Proc. 36, 1318. Turner, S.R., Tayner, J.A., and Lynn, W.S. (1975) Nature 257, 680-681. Higgs, G.A., McCall, E., and Youlten, L.J.F. (1975) Br. J. Pharmacol. 53, 539-546. Kitchen, E.A., Boot, J.R. and Dawson, W. (1978). Prostaglandins 16, 239-244. H.J. Becker, E.L. and Sha'afi, R.I. (1977) J. Cell Naccache, P.H., Showell, Biol. 75, 635-649. ShowelK H.J., Williams, D., Becker, E.L., Naccache, P.H., and Sha'afi, R.I. (1979)J,Reticul. Endo. Sot. (.in press). Fernandez, H.N., and Hugli, T.E. (1976). J. Imnunol. 117, 1688-1694. Downing, D.T., Ahern, D.G. and Bachta, M. (1970) Biochem. Biophys. Connn, 40, 218-223. Flowers, R.J. (1974) Pharmac. Rev. 26-, 33-67. Northover, B.J. (1977) Brit. J. Pharmac. 59, 253-259. Becker, E.L. (1976) Am. J. Pathol. 85, 385-394.

299