Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 282-287

Vol. 147, No. 1, 1987 August 31, 1987

CONVERSION

OF

LEUKOTRIENE

C4 TO

LEUKOTRIENE

D 4 BY A CELL-SURFACE

ENZYME

OF

RAT MACROPHAGES

Isao Nagaoka and Tatsuhisa Yamashita

D e p a r t m e n t of Juntendo

Physiological Chemistry, S c h o o l of M e d i c i n e , University, Hongo, Bunkyo-ku, Tokyo 113, Japan

Received July 8, 1987

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. N e u t r o p h i l s and l y m p h o c y t e s h a r d l y m e t a b o l i z e d LTC4, w h e r e a s m a c r o p h a g e s rapidly converted LTC 4 to LTD 4. The LTC4-metabolizing enzyme of macrophages was p r e s e n t in the m e m b r a n e f r a c t i o n but not in the n u c l e a r , g r a n u l a r a n d cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates c e l l surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (>90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC 4 to LTD4, on the cell surface membrane. © 1987 Academic Press, Inc.

Sulfidopeptide biologically acid

via

leukotrienes,

active

the

cysteinyl

lipoxygenase

inflmmation and a l l e r g y transformed

by Y - g l u t a m y l by d i p e p t i d a s e

and

Using renal

are

cell-surface neutrophils

membrane, and

D 4 and

E 4 are

from arachidonic as m e d i a t o r s

of

LTC 4 has been studied as

(2) into LTD4, w h i c h is f u r t h e r

(3) into LTE 4.

In i n f l a m m e d

tissues,

large

and they are assumed to be i n v o l v e d in the

metabolism of LTs at the site of inflammation. possess

C4,

considered

enzymes,

transpeptidase

numbers of leukocytes are present,

that macrophages

(LT)

compounds which are produced

pathway,

(I).

metabolized

leukotriene

It has been recently

found

the L T D 4 - m e t a b o l i z i n g dipeptidase activity on the

which

lymphocytes

is the h i g h e s t

(4).

However,

among

leukocytes

there is little

including

information

on

the LTC4-metabolizing enzyme of leukocytes which catalyzes the conversion of LTC 4

to

LTD 4.

involvement

In

this

of l e u k o c y t e s

LTC4-metabolizing

study,

therefore,

in the m e t a b o l i s m

in

order

to

elucidate

the

of LTC 4, we h a v e s t u d i e d the

enzyme using rat leukocytes.

MATERIALS AND METHODS

Preparation of leukocytes R e s i d e n t p e r i t o n e a l m a c r o p h a g e s , glycogen-induced peritoneal neutrophils and blood lymphocytes were p r e p a r e d f r o m S p r a g u e - D a w l e y rats as d e s c r i b e d A b b ± e v i a t i o n s : LT, l e u k o t r i e n e ; HPLC, high-pressure liquid chromatography; DSA, d i a z o t i z e d s u l f a n i l i c acid; b u f f e r A, 137 m M N a C I / 2 . 7 m M K C I / 8 . 1 m M Na2HP04/I.5 mM KH2P04, pH 7.4; buffer B, 120 m M NaCI/4 m M KCI/25 mM Tris-HCl, pH 7.4.

0006-291X/87 $1.50 Copyright (© 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

282

Vol. 147, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

p r e v i o u s l y (5). The p u r i t y of i s o l a t e d l e u k o c y t e s e x a m i n e d by W r i g h t - G i e m s a s t a i n i n g was 90-95%. P r e p a r a t i o n of s u b c e l l u l a r f r a c t i o n s from m a c r o p h a g e s M a c r o p h a g e s (4 x ~ c e l l s / m l ) s u s p e n d e d in 0.34 M s u c r o s e w e r e h o m o g e n i z e d in a T e f l o n - g l a s s homogenizer at 0 ° C f o r 20 m i n (6). The homogenate was centrifuged at 5 0 0 g f o r 12 min, a n d t h e s e d i m e n t e d fraction was termed the n u c l e a r fraction. The s u p e r n a t a n t was c e n t r i f u g e d at 8,200g for 15 min, and the r e s u l t a n t p e l l e t was t e r m e d the g r a n u l a r fraction. The s u p e r n a t a n t was further centrifuged at 2 6 0 , 0 0 0 g f o r 1 hr to y i e l d t h e m e m b r a n e fraction. The r e s u l t a n t s u p e r n a t a n t was t e r m e d the c y t o s o l fraction. All pellets were w a s h e d once with 0.34 M s u c r o s e and r e s u s p e n d e d in 0.34 M s u c r o s e at 4 x 107 cell equivalents. The g r a n u l a r f r a c t i o n was s o n i c a t e d in ice for 2 min at 168 W ( S u p e r s o n i c vibrator, m o d e l UR-150P, T o m i n a g a Works, Ltd., Tokyo) b e f o r e use.

M o d i f i c a t i o n of m a c r o p h a g e s M a c r o p h a g e s (i0 ! c e l l s / m l ) w e r e i n c u b a t e d w i t h 2 m M D S A at 37°C for 5 min in b u f f e r A as d e s c r i b e d e a r i l e r (6). Then, t h e c e l l s w e r e w a s h e d t w i c e w i t h i c e - c o l d b u f f e r B to stop the r e a c t i o n and f i n a l l y s u s p e n d e d in b u f f e r B at 2 x 107 c e l l s / m l . After s o n i c a t i o n at 168 W for 2 min, the c e l l s o n i c a t e was a s s a y e d for e n z y m e a c t i v i t i e s . The c e l l v i a b i l i t y e x a m i n e d by a T r y p a n b l u e dye e x c l u s i o n test did not d e c r e a s e by the m o d i f i c a t i o n and was >95%. E n z y m e assay s L T C 4 - m e t a b o l i z i n g a c t i v i t y was d e t e r m i n e d by i n c u b a t i n g the c e l l s o n i c a t e s w i t h s y n t h e t i c LTC 4 (240 pmol) at 37°C for up to 40 min in a t o t a l v o l u m e of 0.12 m l b u f f e r B in t h e p r e s e n c e of 5 m M L - c y s t e i n e which inhibits the c o n v e r s i o n of LTD 4 to LTE 4 by the L T D 4 - m e t a b o l i z i n g d i p e p t i d a s e p r e s e n t in the leukocyte preparations, unless otherwise noted. The reaction was t e r m i n a t e d by t h e a d d i t i o n of 0.18 m l a c i d i f i e d m e t h a n o l (methanol:acetic acid=1000:l), followed by c e n t r i f u g a t i o n at 8 , 3 0 0 g f o r i0 min. Aliquots (i00 y l ) of the r e s u l t i n g s u p e r n a t a n t s were s u b j e c t e d to r e v e r s e p h a s e HPLC for the analysis of L T C 4 a n d i t s m e t a b o l i t e ( s ) as d e s c r i b e d eariler (7), u s i n g a F i n e p a k SIL C18 c o l u m n (5 ym, 4.6 x 250 ram, J a s c o - J a p a n S p e c t r o s c o p i c Co., Ltd., T o k y o ) a n d a TRI R O T O R - V I p u m p (Jasco). The absorbance of t h e c o l u m n e f f l u e n t was m o n i t o r e d using a U V I D E C - 1 0 0 - V I s p e c t r o p h o t o m e t e r (Jasco) a d j u s t e d to 280 nm. The p e a k area was c a l c u l a t e d u s i n g a C h r o m a t o c o r d e r ii (System Instruments Corp., Tokyo). The solvent system consisted of methanol/water/acetic a c i d (65:35:0.1, v / v ) c o n t a i n i n g 0.05% E D T A , a n d w a s a d j u s t e d to pH 5.6 w i t h ammonia. The f l o w rate was m a i n t a i n e d at 1 ml/min. In s o m e e x p e r i m e n t s , the b i o a c t i v i t i e s of t h e r e s u l t i n g supernatants were a s s a y e d u s i n g the i s o l a t e d g u i n e a - p i g i l e u m ( 6 ) . Alkaline phosphodiesterase I, g l u c o s e - 6 - p h o s p h a t a s e , ~-N-acetyl-D-glucosaminidase, cytochrome oxidase and prolyl endopeptidase activities w e r e a s s a y e d as d e s c r i b e d p r e v i o u s l y (6,8). One unit of a c t i v i t y is d e f i n e d as the a m o u n t of e n z y m e n e c e s s a r y to split 1 ~mol of s u b s t r a t e in 1 m i n u n d e r t h e c o n d i t i o n used. Protein concentration w a s m e a s u r e d by t h e m e t h o d of L o w r y et al. (9), u s i n g b o v i n e s e r u m a l b u m i n as a standard. Reagents LTD 4 was p u r c h a s e d from Wako Pure C h e m i c a l Industries, LTd., Osaka. LTC 4 was a gift from Ono Pharmaceutical Co., Ltd., O s a k a . DSA was prepared as o u t l i n e d p r e v i o u s l y (6). O t h e r r e a g e n t s w e r e of a n a l y t i c a l grade.

RESULTS

AND

LTC 4 was and the

DISCUSSION

incubated

supernatants

HPLC

for LTC 4 and

the

peak

with

rat

sonicated

of the r e a c t i o n

mixture

its m e t a b o l i t e ( s ) .

at

280

nm

of

LTC 4 with

decreased

and

was

quantifiably

retention

time

leukocytes

37°C

analyzed

By t h e i n c u b a t i o n the

retention

converted

of 16.0 ± 0.i m i n a n d

were

at

the

283

to

time

a single

absorption

for

up to 40 min,

by r e v e r s e

phase

with macrophages, of new

maximum

9.8

±

peak at 2 8 0

0.i

min

with

the

nm with

Vol. 147, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

86

6

6

4

4

2

21

vx

E ok

<

0

10

'

i

'o

15 '

2

10

0

15

20

0

10

15

20

E l u t i o n time (rain) Fig. i. R e v e r s e p h a s e H P L C e l u t i o n p r o f i l e of L T C 4 a f t e r i n c u b a t i o n w i t h macropba~es. LTC 4 (240 pmol) was incubated with rat sonicated macrophages (1.2 x I0 c e l l s ) f o r 0 m i n (A) or 40 m i n (B) at 3 7 ° C in 0.12 ml b u f f e r B. A f t e r the t e r m i n a t i o n of the r e a c t i o n and s u b s e q u e n t c e n t r i f u g a t i o n , an a l i q u o t of the r e s u l t i n g s u p e r n a t a n t was s u b j e c t e d to HPLC. (C) a u t h e n t i c LTD 4 .

the

shoulders

(Fig. almost

i).

at

The

270

peak

completely

290

converted

(Fig.

2A).

The

(n=3)

of

initial

the

and

to

spasmogenic activity

corresponding

the

activity after

to

in a time

peak

that

LTC 4

to

increased

incubation

of

synthetic

dependent

corresponding of

an

~ , ~

A

~I00

nm,

of LTC 4 disappeared

with

manner

LTD 4 to

was

40

min

after 357.5

macrophages

LTD 4

and

±

29.8% for

I00- B

5

o so u

J 0

5

10

20

40 Incubation

5 10 20 time (rain)

,40

Fig. 2. Conversion of L T C 4 t o L T D 4 b y r a t l e u k o c y t e s or macrophage subcellular fractions. (A) LTC 4 (240 p m o l ) w a s incubated with each sonicated + 8 ~ g p r o t e i n for leukocyte fraction (1.2 x i0 6 c e l l s ) c o n t a i n i n g 84 _ n e u t r o p h i l s (O), 65 ± 7 D g p r o t e i n for l y m p h o c y t e s (D) and 96 + 8 ~ g p r o t e i n for m a c r o p h a g e s (O), at 37°C f o r i n d i c a t e d t i m e p e r i o d s in 0.12 m l b u f f e r B. (B) LTC~ (240 pmol) was incubated with each macrophage s u b c e l l u l a r fraction ~ 6 + (1.2 x i0 cell equivalents) eontalnlng 15 - 2 y g p r o t e l n for n u c l e a r f r a c t i o n {m), 19 ± l y g p r o t e i n for g r a n u l a r f r a c t i o n (e), 25 ± 4 ~ g p r o t e i n for c y t o s o l f r a c t i o n (D) a n d 25 ± 7 ~ g p r o t e i n for m e m b r a n e f r a c t i o n Co). After the termination of the reaction and subsequent centrifugation, aliquots of the r e s u l t i n g supernatants were subjected to reverse phase HPLC and the amount of LTC 4 and LTD 4 was measured. The amount of LTC 4 c o n v e r t e d to LTD 4 is e x p r e s s e d in t e r m s of p e r c e n t of the t o t a l L T C 4 a n d L T D 4 r e c o v e r e d , a n d g i v e n as the mean ± S.D. of 3 experiments. Total r e c o v e r y of LTC 4 and LTD 4 after incubation with leukocytes was 93-100% relative to L T C 4 w i t h o u t incubation.

284

40

Vol. 147, No. 1, 1987

Table i.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Distribution of marker enzymes in subcellular fractions of macrophages Relative enzyme activity (%) G C M

N Alkaline phosphodiesterase I Glucose-6-phosphatase ~-N-Acetyl-D-glucosaminidase Cytochrome oxidase Prolyl endopeptidase Protein

7.1 ± 3.1 5.8±2.4 13.8±4.5 14.8±0.7 2.2±1.2 18.3±4.2

10.8 ± 2.1 16.2±2.8 70.2±1.6 70.4±1.5 7.2±1.8 23.1±3.1

2.7 ± 0.6 20.5±6.4 4.6±0.7 3.1±1.5 81.2±1.8 29.7±1.7

80.6 ± 4.6 57.7±1.2 ii.5±2.2 11.8±3.6 9.5±2.3 28.9±5.7

Enzyme activities and protein concentrations in subcellular fractions are expressed in terms of percent of the total enzyme activities and protein recovered, respectively. Total recovery of the enzymes and protein was 90110%. Total enzyme activities per 107 cells were 1154 ± 200 mU for alkaline phosphodiesterase I, 1.4 ± 0.2 mU for glucose-6-phosphatase, 18.6 ± 2.2 mU for 8-N-acetyl-D-glucosaminidase, 0.92 ± 0.26 A550/min for cytochrome oxidase and 5.1 ± 1.5 mU for prolyl endopeptidase. Total protein concentration was 803 ± 67 ~g/ 107 cells. + S.D. of 3-4 experiments. Values represent the mean _ N, nuclear fraction; G, granular fraction; C, cytosol fraction; M, membrane fraction.

min.

This v a l u e was a l m o s t

activity

of

L T D 4 to

results would enzyme

lymphocytes

converts hardly

As m a c r o p h a g e s localization Table

a molar

LTC 4 to LTD 4.

metabolized

possess

6-phosphatase,

fraction. cytosol

of

2B

subcellular

fractions. LTC4, whereas

These

in

membrane

the

fractions

results

i).

membrane

inactivates

However,

enzymes

were

studied, among

recovered

marker,

the

but

and

that not

a

marker

the in

was

fractions.

and g l u c o s e -

and

cytosol

rapidly

nuclear,

fraction.

marker

in t h e

enriched

in t h e in

fractions converted enzyme

granular

and

granular

activities

LTC4-metabolizing the

macroph~Qs.

lysosomal

ca. 7 0 %

marker,

fraction

using

in the m e m b r a n e

were

granular

the s u b c e l l u l a r

subeellular

LTC4-metabolizing

the membrane

fraction

These

rat n e u t r o p h i l s

I, a c e l l - s u r f a c e

The nuclear,

fraction

Then, or

macrophages

esterase

was

a cytosol

shows

indicate

n=3).

LTC4-metabolizing

is

and

these hardly

L T C 4 to present cytosol

of macrophages.

The m e m b r a n e (Table

marker,

endopeptidase,

metabolized LTD 4.

of m a r k e r

a mitochondrial

Fig.

the

activity,

~-N-acetyl-D-glucosaminidase,

Prolyl

fraction.

enzyme

phosphodiesterase

oxidase,

± 63.5%,

spasmogenic

LTC 4 (Fig. 2A).

the high L T C 4 - m e t a b o l i z i n g

a microsomal

recoveries

(377.4 possess

On the o t h e r hand,

of the L T C 4 - m e t a b o l i z i n g

of a l k a l i n e

cytochrome

basis

that rat m a c r o p h a g e s

1 shows the d i s t r i b u t i o n

The b u l k

The

L T C 4 on

indicate

which

the same as that of the r a t i o of the

the

consisted

in o r d e r

cell-surface

I was

inhibited

the

to e x a m i n e

microsome,

were m o d i f i e d

of

cell-surface which

possesses

chemically enzymes

component,

the

w i t h DSA,

selectively

the

and m i c r o s o m e cell-surface

LTC4-metabolizing

a pooly

to 15.4 ± 5.3% (n=3) of

glucose-6-phosphatase,

membrane

permeant

(i0).

reagent

Alkaline

which

phosphodi-

by the

modification.

~-N-acetyl-D-glucosaminidase,

cytochrome

285

control

enzyme,

Vol. 147, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oxidase and prolyl

endopeptidase were hardly

although these enzymes were c o m p l e t e l y was

modified

(data not

shown).

modification condition, the

inhibited

(92-100% of control),

inhibited by DSA if the cell

These

results

indicate

that

sonicate

under

our

DSA modified c e l l - s u r f a c e enzyme selectively.

LTC4-metabolizing a c t i v i t y

of m o d i f i e d

macrophages

was

When

examined,

activity was inhibited 91.1 ± 4.5% (n=3) by the modification.

the

These results

w o u l d suggest that at least 90% of the LTC4-metabolizing enzyme is located on the c e l l - s u r f a c e activity

of m a c r o p h a g e s .

In the case w h e r e m o s t of the e n z y m e

is present on the c e l l - s u r f a c e membrane,

intact c e l l s

to show the same enzyme activity as sonicated c e l l s the LTC4-metabolizing n=3) w a s a l m o s t cells, n=3).

activity

(i0,ii).

of intact macrophages

are reported In this study,

(90 ± 22 ~ U / 1 0 7

the same as t h a t of s o n i c a t e d m a c r o p h a g e s

cells,

(98 ± 24 ~ U / 1 0 7

These results seem to support the possibility that most of the

LTC4-metabolizing

enzyme

is

present

on

the

cell-surface

membrane

of

macrophages. The

property

of the LTC4-metabolizing

enzyme of macrophages

was examined.

The Km and Vmax values for the enzyme determined by Lineweaver-Burk analysis of data obtained

at 5 substrate

and i00 ± 20 ~ U / 1 0 7

cells

concentrations

(n:3),

enzyme inhibitors

on m a c r o p h a g e

~g/ml),

(100yg/ml),

captopril

fluorophosphate

(0.25-4 ~M)

respectively.

When the effect of various

e n z y m e was studied,

c-amino-n-caproic

(3 mM), N-ethylmaleimide

n e i t h e r b e s t a t i n (i00

a c i d (3 mM), d i i s o p r o p y l

(5 mM), o-phenanthroline

cysteine (5 mM) affected the LTC4-metabolizing activity. serine-borate complex remarkably

(25 mM),

decreased

the

a Y-glutamyl

enzyme

We

On the other hand,

(8.6 ± 4.9% of

control,

n=3),

enzyme of macrophages is similar to Y-

transpeptidase.

have

recently

found

that

macrophages

dipeptidase which converts LTD 4 to LTE4, study,

(2 mM) nor

transpeptidase inhibitor (12,13),

activity

suggesting that the LTC4-metabolizing glutamyl

were 1.3 ± 0.3 ~ M

we h a v e

clarified

enzyme which converts suggest that among modulation inflammatory

of

the

have

the

on the cell

that m a c r o p h a g e s

LTD4-metabolizing

surface

(4).

In this

also have the LTC4-metabolizing

LTC 4 to LTD4, on the c e l l

surface.

These findings

leukocytes macrophages may play an important role in the inflammatory

mediators

responses

through

such as s u l f i d o p e p t i d e

the

degradation

of

leukotrienes which have the

ability to increase vascular permeability and constrict airway smooth muscles (i).

REFERENCES

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286

Vol. 147, No. 1, 1987

4. 5. 6. 7. 8. 9. i0. ii. 12. 13.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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287