Vol. 176,

No.

April

1991

15,

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

STIMULATION

OF PHAGOCYTOSIS IN RAT POLYMORPHONLJCLEARLEUKOCYTES

BY AZ3187

IS ACCOMPANIED BY ACTIVATION

EIBAI Department

of

LEE,

MITSUGU FUJITA

Pharmacology,

Kobe-Gakuin

University,

February

26,

OF MYELOPEROXIDASE

and KIM10 KARIYA*

Faculty

of

Pharmaceutical

Ikawadani-cho,

Kobe 651-21, Received

364-370

Sciences,

Nishi-ku,

Japan

1991

The incubation of polymorphonuclear leukocytes from rats with A23187 resulted in stimulation of phagocytosis with a concomitant increase in the activity of myeloperoxidase. Activation of the enzyme by A23187 reached the maximum at 2.5 min of incubation and preceded the maximum stimulation of phagocytosis. Propylthiouracil, an inhibitor of myeloperoxidase, and CaZ* chelators prevented activation of myeloperoxidase and phagocytosis by A23187. These results indicate that A23187 increases intracellular CaZ* level, followed by activation of myeloperoxidase involved in stimulation of phagocytosis. @1991 Acade"lcmess, Ire. SUMMARY:

Phagocytosis animals.

is

It

hydrogen

is

the

well

peroxide is

the

system.

opsonization

(6,7). in

in

the

However,

defense that

mechanism

bactericidal

in

function

myeloperoxidase little

in

myeloperoxidase

EC 1.11.1.7)

In addition,

is

known

the

(donor: the

mature

(l-5)

among

prevented about

the

a role

phagocytosis.

Myeloperoxidase, oxidizes

established

involved

myeloperoxidase

important

oxidereductase,

neutrophils defense

most

a variety

*To whom correspondence

a marker of

of

compounds

should

neutrophilic in

the

granulocytes, presence

of

hydrogen

be addressed.

Abbreviations used: EGTA, ethylene glycol bis(b -aminoethylether)N,N,N' ,N'-tetraacetic acid; Quin-2, 2-[(2-amino-5methylPhenoxy)methyll-6-methoxy-8-aminoquinoline-N,N,N',N'-tetraacetic acid; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'tetraacetic acid; PBS, phosphate-buffered saline; BCA, bicinchoninic acid. 0006-291X/91 Copyright All rights

$1.50

0 1991 by Academic Press, Im. of reproduction in any form reserved.

364

of

Vol.

176,

No.

peroxide

1, 1991

(8,9).

metabolism reported marrow the

BIOCHEMICAL

of that and

enzyme

is

some

compounds

(10,ll).

purification

of

this

obtained

from

pression

of

decreased

during

Thus

we have

role

in This

during

enzyme mature

that

differentiation study

was

of

that

stimulation

to

stimulated

of

the

was accompanied

the

bone

characterized

and

In

addition,

ex-

cells

was

marrow

granulocytes

of

the

(submitted).

may play

an important

cells. the to

enzyme clarify

Present

activity a possible

paper

polymorphonuclear

by activation

rat

(12).

by A23187

in

have

those

determine

phagocytosis.

we

to

bone

marrow

phagocytosis

oxidative

from

well

myeloperoxidase

in

in

similar

leukocytes

to

designed

myeloperoxidase

concerned

were

in

COMMUNICATIONS

Previously

was very

of bone

phagocytosis

rats

properties

differentiation

shown

also

RESEARCH

of myeloperoxidase

myeloperoxidase

role

of

peroxidase

enzymatic

properties

BIOPHYSICAL

This

the its

AND

describes leukocytes

of myeloperoxidase.

MATEXIAJX ANDMETHODS Materials. Microsphere cience Inc. RPM1 1640 Laboratories.

A23187 was purchased from Calbiochem Co. FITC(Fluoresbright, 2.0 ,U m diameter) was from PolysBovine serum albumin was from Sigma Chemical Co. medium and fetal calf serum were from Flow

Preparation of the cells. Polymorphonuclear leukocytes were prepared from peritoneal exudates of casein (2 g/kg body weight) treated male Wistar rats, weighing about 250-300 g. The cells were extensively washed with phosphate buffered saline (PBS) and were hypoosmolytically treated to remove erythrocytes. The resulting cells were used throughout this experiment. These cells contained more than 90% of polymorphonuclear leukocytes. Assay of phagocytosis. Phagocytosis was determined by flow cytometry according to Stewart et al. (13). Briefly, standard incubation mixture contained polymorphonuclear leukocytes (1 x 106 cells), fluorescent microspheres (1 x lo=), RPM1 1640 and 5% fetal calf serum in a total volume of 1.0 ml. Unless otherwise indicated, the mixture was incubated at 37O C for 5 min. After the incubation the mixture was centrifuged at 100 x g for 8 min to separate the cells. Resulting cells were washed with the medium containing 2% BSA. This washing procedure were repeated 2 times to remove unphagocytized microspheres. The final cell suspension (2 x lO* cells)was analyzed by using flow cytometer (Epics 750). Assay of myeloperoxidase activity. the without fluorescent microspheres, 365

cells

After

were

the

incubatjon collected by

Vol.

176,

No.

BIOCHEMICAL

1, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

centrifugation at 100 x g for 8 min and washed with PBS for 3 Resulting cells were homogenized with 200 p 1 of PBS by times. This homogenate was used as the enzyme Polytron apparatus. The activity of myeloperoxidase of polymorphonuclear source. leukocytes was determined as described previously (12). Myeloperoxidase was homogeneously purified from rat bone marrow as described elsewhere (12). Protein was determined by using BCA protein assay reagent (14). RESULTS AND DISCUSSION Fig.

1 shows In

cytometry. cells

the

into

the

containing

1,

Stewart

to

the

microspheres,

91-255

in

number

containing

1,

3 and

(Fig.

Although

1).

possessed of

2,

a low

phagocytosis

A23187,

ingestion

spheres.

Therefore,

the

activity

analyzed

more

of

of by flow

of of

the not

this

of

the

numbers

of

than

could

41-66,

of

in

treated

one but

result

explains in

using

more that

polymorphonuclear fluorescent

cells

stimulation with

2.5 A23187

than

n M ac-

two micro-

A23187

stimulates leukocytes

microspheres.

Fig. 1. Histograms of phagocytosis in control and A23187stimulated polymorphonuclear leukocytes. The cells were incubated with fluorescent microspheres at 37-C for S min in the presence or absence of 2.5 ti M A23187. The cells were extensively washed with the medium contajning 2% BSA after the jncubation. The resulting cells (2 x 104 cells) were analyzed by flow cytometer. 366

and

leukocytes

In addition, also

by

respectively

four-fold

only

cells

phagocytic

phagocytosis,

neutrophils.

be ex-

67-90

polymorphonuclear

cells

the

as described

4 microspheres,

the

of

phagocytic

13-40,

numbers

by flow

percentage

procedure

O-12,

the

phagocytosis cytometry

analyzed

4 microspheres

unstimulated

was observed

celerated

than

shows

activity

an activator

the

Each value

channel

of this

of

3 and more

(13).

phagocytosis

analysis

determination 2,

et al. --

of

addition

containing

panded

histograms

Vol.

176,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

MPO

2.

Time

COMMUNICATIONS

Phagocytosis

Time

Fig.

RESEARCH

courses

(min)

of

Time

the

stimulation

of

Cmin)

myeloperoxith&?c?

and

phagocytosis activities by A23187. When the enzyme activity was assayed, the cells were incubated without microspheres. Ai the time indicated, the cells were collected by centrifugation and were extensively washed with PBS. The homogenates of the resulting cells were used as the enzyme source. Phagocytosis was determined as described in Fig. 1. control; l , 2.5 ,U M A23187. 0.

Fig.

2 describes

time

and myeloperoxidase was stimulated Maximum

activities

by A23187

stimulation

20 min

after

the

myeloperoxidase cubation

in

the

by the

ionophore

tion.

Since

medium

in

phagocytosis

addition

of

presence

of

was detected

tion

(data

AZ3187

activation into the

the

of

the

medium

enzyme

not

enzyme

by

(data

stimulated

by 1 p M A23187.

tion

2-3

p M A23187. of

myeloperoxidase

phagocytosis.

The

the

shown).

medium

were

the

also activities

stimulatory

A23187 effect 367

the

incubathe

enzyme

into

release

of

addiin

the

the

enzyme

phagocytosis

and

on its

were

similar

con-

significantly was observed

curve

of A23187

of

the

decrease

stimulation

was

enzyme

when

the

dependent

The concentration-response by

the

in-

The activity

of

The maximum

the

medium of

hand, the

10 min after

Therefore,

Both

after

examined.

The stimulation A23187

after of

into

release

may be due to

centration

at

not

the

shown).

by A23187.

activity

5 min

excreted

was also in

other

The activation

(15),

by A23187

min

manner.

was observed

On the 2.5

decreased

activated

Phagocytosis

compound

highest

was

phagocytosis

time-dependent

ionophore.

A23187.

was gradually

of

by A23187.

by this

the

was the

induced

stimulation

incubation

of

myeloperoxidase of

the

activity

were

of

induced

myeloperoxidase

neutrophils the

courses

for to

activathat

was disappeared

of

Vol.

176,

No.

BIOCHEMICAL

1, 1991

AND

f3lOPHYSlCAL

COMMUNICATIONS

RESEARCH

Corltrol

Colltrol

outi

Qllin2 BAPTA

BAPTA

EGTA

EGTA

Myebperoxidase

activity

wrlehlg

Phagocytosis

(%)

proteid

3. Effects of various compounds on the stimulation of the activities of myeloperoxidase and phagocytosis induced by A23187. Quin-2 (50 ,u M) and BAPTA (30 b M) were preloaded to the cells as

Fig.

their

t.etzraacetoxymet,hyl

ester

at

37~ C for

30 min.

were extensively washed after t,he preincubation, tivities of phagocyizosis and the peroxidase

The

cells

and then the acwere determined in

the presence or absence of 2.5 fi M A23187 as described in Figure 1 and 2. Phagocytosis was expressed as % of total phagocytized cells. EGTA (8 mM) was simultaneously added with A23187. PTU

(100 ,LLM) was preincubated witzh the cell then A23187 was added. PTU, propylthiouracil. EI , 2.5 ,U M A23187. III! control: l P