Vol. 68, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFECT OF CYTOCHALASIN B ON THE OXIDATIVE

METABOLISM OF BUMAN PERIPHERAL

BLOOD GRANULOCYTES.

Dirk

Roes*,

Joke W.T. Homan-Miiller*

and Ron S. Weening+

f

Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, +P.O.B. 9190, Amsterdam; and Pediatric Clinic, Binnengasthuis, University of Amsterdam, Amsterdam, The Netherlands. Received

October

27, 1975

SUMMARY: Pretreatment of human granulocytes with cytochalasin B before addition of opsonized xymosan particles resulted in strong inhibition of the oxygen consumpand the hexose monophosphate shunt activition, the hydrogen peroxide production, In contrast, however, no effect of ty as compared to normal phagocytosing cells. These seemingly cytochalasin B was found on the generation of superoxide anions. controversial results can be explained by the action of cytochalasin B on the cell membrane.

INTRODUCTION Cytochalasin $=yJ$ bits

interferes

with

membrane

granulocytes, biting

the

cyto

B-treated

tion

with

of sugars

and nucleosides

this

suppresses

the capacity

drug

of the

granulocytes

immune it

(8)

activate B on several

particles

or non-immune

anions

that

(OZ-)

superoxide

of hydrogen -all

cyto

is

peroxide

oxidative

of these

is

B = Cytochalasin

in cultured

cell

dematioi-

(1,2) cells

particles

surface

lysosomal

(6,7).

and inhi-

(3-5).

In

without

inhi-

Nevertheless,

enzymes

after

stimula-

stimuli.

under

exactly

also

stimulated

the

(ll),

it

is

still

subject

unknown

B-treated of this

B, STZ = Serum-treated

the

Although

important

in cyto

is the

same conditions (10).

a quantitatively

processes

reactions

microfilaments

release (9)

mold Helminthosporium

to ingest

to the

selectively

has been shown that

of superoxide

*Abbreviations:

of the

transport

the production

cyto

the

a metabolite

of cytoplasmic

shown previously

stimuli

B)*,

function

attachment

Recently ration

B (cyto

we have

intermediate whether

cells.

gene-

in

granulocyte The action

report. xymosan.

of

Vol. 68, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Cytochalasin B (I.C.I. Research Laboratories, Alderley Park, Cheshire, England) was prepared as a 50 ug/ml stock solution in 1% dimethylsulfoxide (J.T. Baker Chemicals N.V., Deventer, The Netherlands). The granulocytes were preincubated during 10 min. at 37OC in a 1:lO dilution of the drug before addition of other compounds and stimuli. Zymosan (kindly provided by Dr. K.W. Pondman) was suspended in fresh human serum at a concentration of 4 mg/ml, homogenized in a Potter homogenizer and incubated for 30 min. at 37OC. After centrifugation and washing twice, this preparation of serum-treated aymosan (STZ) was suspended in 154 mM NaCl at a concentration of 5 mg/ml. Granulocytes were incubated with a 1:lO dilution of this preparation. Granulocytes were isolated from fresh, defibrinated human blood, as described elsewhere (12) and suspended in a buffer consisting of 138 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 0.6 mM CaC12, 1.0 mM MgC12, 5.5 mM glucose and 0.5% (w/v) human albumin, pH 7.4. The final cell suspensions, containing more than 95% granulocytes, were split into several aliquots for the assay of the metabolic parameters. It must be emphasized, however, that the different parameters were not determined under identical conditions, since addition of cytochrome 2 (for the 02- assay) resulted in strong inhibition of the other parameters (unpublished results). A theoretical explanation is shown in fig.1. Oxygen consumption was measured polarographically with an oxygen electrode Superoxide generation was determined by measuring as described before (12). the superoxide dismutase-inhibitable reduction of cytochrome 2, as previously described (11). Hydrogen peroxide production was measured with a method based on the oxidation of leucodiacetyl-2,7-dichlorofluorescein to a fluorescent compound by H202 in the preseny; of peroxidase (13). Hexose menophosphate shunt activity was assayed as CO2 production from [l-lucose by the method of Pachman (14,15). First, the cells were preincubated with [I-14C]glucase in the presence of 5.5 mM glucose for 15 min. at 37OC. Next, cyto B was added while the cells were kept at 370C for another 10 min. Finally, the experiment was started with the addition of STZ or NaCl (154 mM). --Release of lysozyme was determined as previously described (10). Extracellular lactate dehyNo significant release drogenase was measured as a control for cell integrity. of lactate dehydrogenase was found under any condition.

RESULTS The inhibitory this

compound

Fig.

2 shows

obtained

action

to a suspension that

in about v/v)

experiments

we preincubated action

completely aymosan

of this

normal (fig.

alone

release

inhibition

had no effect the drug.

consumption

or inhibited

The cyto

with

cyto

B-treated enzymes

3).

44

only B for cells

by adding

in

an oxygraph.

of these

was dose-dependent

cells

of lysosomal

be demonstrated

granulocytes

of the oxygen

This

5 min.

B could

of STZ-stimulated

80% inhibition

DMSO (O.l$,

complete

of cytochalasin

(not

slightly. 10 min. were

when challenged

cells shown)

was while

In subsequent to be certain still with

capable opsonized

of of

BIOCHEMICAL

Vol. 68, No. 1, 1976

receptor

binding

membrane

or

perturbation

red&d cytochrome

Fig. also

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 Schematic showing the

cytochrome

4

&

representation of the oxidative detection method of superoxide

metabolism anions (02-).

of granulocytes,

Fig. 2 Inhibition of oxygen consumption by cyto B. The curve shown was obtained by measuring the rate of oxygen consumption at different time intervals after addition of cyto B (5 ug/ml) to a granulocyte suspension stimulated with STZ (0.5 mg/ml). Cyto B was added 7 min. after addition of STZ. The effect the oxidative was not

of 10 min. metabolism

affected

To investigate

neration

more closely,

and the cytochrome

were Since

B, whereas the

1.

3 parameters of cyto

both

(fig.

the cyto 4).

differences

parameters

The stimulated

of inhibition varied

B on several

generation were

of 02-

inhibited

B on the

for

superoxide

B concentration

Except

of

ge-

(fig.

at very high

3)

cytochrome

5

between

cyto

B-treated

and non-treated

to inhibit

the

transport

of sugars

found. cyto

B has also

before

addition

cose:

cyto

the other

c concentration

we have preincubated of cyto

by the addition

the cyto

lack

we have

cells,

started

with

shown in Table

no substantial

concentrations, cells

is

by cyto

70-90%,

preincubation

B inhibition

67% inhibition

been

reported

granulocytes B.

After

of STZ.

another This

on the oxygen by cyto

with

B, with

5.5 mM glucose

10 min.

pretreatment consumption preincubation:

45

at 37OC for

at 37OC, the did (without

not,

preincubation

66% inhibition).

15 min.

experiment

however,

into

was

prevent with

glu-

Vol. 68, No. 1, 1976

BIOCHEMICAL

lysozyme

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cyt4: reduction

release(%)

--

-STZ +STZ

40

25

(lOGa5min)

30

20

w H

-STZ +STZ

15 20

0-o 0

1

2

3

4

5 [cytoB]hg/ml)

Fig. 3 Effect of cyto B on lysozyme release and cytochrome 2 reduction by huReaction time: 15 min. Lysozyme release expressed as perman granulocytes. cent of total activity (100% = 1.4 + 0.5 ug lysozyme/106 cells, n = 7) released by Triton-X-100. Lysozyme release: o-o, -STZ; e-e, +STZ. Cytochrome 2 reduction: O-D , -STZ; 8-8 , +STZ.

TABLE I

-

EFFECT OF CYTOCHALASIN B ON SEVERAL PARAMETERS OF OXIDATIVE MRTABOLISM.

Parameter

Additions

Response granulocytes (Mean + S.E.M.)

(nmoles/106 (n) Oxygen consumption Superoxide

10 9

STZ

7 7

2.4 + 46.6 +1.6 2 14.8 -+

STZ

4 4

36.9

STZ

3 3

H202 production oxidation

cells

STZ generation

[1-14C)glucose

Normal

x 15 min.) Cyto B-cells

0.5 4.9

1.9 + 0.4 12.5 -+ 1.0 1.4 + 0.2 14.4 -+ 1.9 0 8.7 -+ 0.5 0.2 + 0.1 0.8 T- 0.1

0.3 0.7

0 -+ 2.6 0.3 + 0.1 7.4 -+ 1.2

DISCUSSION Our results tion

of the

confirm

earlier

[l-14C]glucose

and Schell-Frederick in cyto

B-treated,

cells.

Finally,

et al.

reports

oxidation (19)

by cyto reported

zymosan-stimulated Curnutte

(6,16,17)

and Babior

B.

inhibition

granulocytes (20)

46

found

about

a dose-dependent

Recently,

both

of the

oxygen

as compared 60% stimulation

Rossi

inhibiet al.

(18)

consumption

to non-treated of the

superoxide

BIOCHEMICAL

Vol. 68, No. 1, 1976

0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

200

100

300

[cyt c] added Fig. 4 Influence of cytochrome 2 concentration on cytochrome 2 reduction granulocytes incubated for 15 min. with STZ, with (o-o) or without (o-o)

generation

in suspensions

No attention, oxygen tion

however,

consumption of its

product,

(fig.

Therefore,

1).

a hypothetical

reaction it

direct

B is ruled More

attachment impaired it cell

discrepancy

stimulation)

also

of cyto

with

--E.Coli

of inhibition

(10,ZO)

of the

of genera-

superoxide

is

peroxide that

must be sought

particles

like

granulocytes.

by binding

of stimuli

47

cyto

to normal

zymosan

level

an important

B only

in-

inhibits

interfering

with

cytochrome

of the

B-treated cells

bind

Since

by superoxide

a

-c and

3 and 4.

at the

It has been shown that as compared

cyto

between

in figs.

5,

in granulocytes

II202 without

competition shown

of cytochrome

reaction

imaginable into

reduction

of this

to hydrogen

Finally,

experiments

B-treated

are initiated

that

of oxygen

of particles that

(11)

hardly

the explanation

probable

(90-95s)

shown

conversion

out by the

engulfment

membrane

granulocytes

is

to the membrane.

is highly

incubated

by a non-specific

from oxygen

pathway.

likely,

to the

(even

inhibition

we have

superoxide-containing cyto

granulocytes

so far

inhibition

strong

Previously, in the

paid

be explained

found

termediate

B-treated

B.

superoxide.

cannot

we always

dismutase.

has been

without

Our results since

of cyto

by cyto

to less

cell

cells

(6,8,21).

surface

show Thus,

receptors

the metabolic

to the

particle

on the

reactions receptors

in

Vol. 68, No. 1, 1976

(lo),

this

lic

inhibition

inhibition.

that

cyto

B should

method

we have the

les

be able

used for

membrane

is produced originate

enzyme superoxide

part

of their

efficient less

the outer the

do not we think

resulting

the medium

cytochrome for

that

in

cyto

B-treated

of this

cells.

organelfrom

rapidly

by the

completely

a much larger a much more

Thus,

cells,

this

diffuses

are

release

electron

B treatment

stimulation

reaction

normal

in

since

there

acceptors

for

is

02-.

parameters. leads

of all

seems to be excluded

by a more efficient

will

than

other

superoxide

granulocytes

cells

located

these

be inactivated

c and other

the

cyto

will

since

by

Previously,

phagocytosis,

phagosomes,

these

2).

of normal

B-treated

(8),

be explained

by a system

Once the it

cyto

is possible

in impaired

generation

conditions

however, Since

exist

may simply

generated

of the

of the metabo-

may even be derived

of cytochrome

is

membrane.

cause

bonds.

production

under

particles

between

2 it

(reduction

cell

into

in fig.

superoxide

cytosol,

be the

particle-cell

in the membranes

of 02-

competition

02-

Thus,

zymosan

trapping

In short,

that

(9,lO).

superoxide

Such arguments

ding,

that

dismutase.

to ingest

shown

determination

evidence

into

may well

of superoxide

its

also

from

the phagosomes

unable

binding

to disconnect

of inhibition

presented

cell

agent

of receptor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Prom the experiment

The lack the

BIOCHEMICAL

oxidative

from this

agent

to less

with

cell-particle

bin-

reactions.

inhibition

cytochrome

The finding

can be explained 2 under

these

condi-

tions. This tions

and those

bolism that

concept

cyto

B only

that

It with

that

the

oxygen

B-treated

proportional

cells

threshold

the

consumption

that

and that

the rate

of bonds amount

48

little

with

our

effect

observa-

on the meta-

finding

of Rossi

et al.

(18)

of cells

stimulated

with

solid

are used,

however,

to the number

a certain

in accordance

B has only

Also,

activators

has to be proven,

is

cyto

granulocytes.

inhibits

cyto

have bound

stimulation

(6,20)

and not when soluble

planation.

directly

of others

of non-stimulated

stimuli

formed

of granulocyte

completely

less

particle-cell of oxidative

formed of ligands.

fits

into

our

bonds reactions

or to the number

ex-

are is

of cells

BIOCHEMICAL

Vol. 68, No. 1, 1976

Finally,

it

of the oxidative (10)

that

lation

metabolism

attachment

of the

superoxide

oxidative

only,

by STZ.

B-treated

in these

STZ stimulates

membrane

cells,

may also

is

based with but

membrane

receptors

initiate

parameters

conclusion

sufficient

for

stimu-

upon observations

metabolism,

granulocyte

all

Our earlier

cells.

to the cell

the oxidative

but

1 that

have now been confirmed

Thus,

recognition,

pany phagocytosis

in cyto

reactions

B inhibits

stimulation

from Table

of particles

generation

cytochalasin

particle

may be derived

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

other

does not

the biochemical

not

of

parameters: prevent

the

only

serve

in

events

which

accom-

and killing. ACKNOWLEDGEMENTS

We thank cially

Dr.

supported

Research

(Z.W.O.),

I.M.

Goldstein

for

by the Netherlands The Hague,

his

valuable

Organization

The Netherlands

advice. for

(grant

This

study

the

Advancement

nr.

91-5).

was finanof Pure

REFERENCES 1. 2. 3. 4. 5. 6. 7.

a. 9. 10. 11. 12. 13. 14.

Carter, S.B. (1967) Nature, 2&3, 261-264. Spooner, B.S., and Wessels, N.K. (1970) Proc. Nat. Acad. Sci. U.S.A., 66, 360-364. Kletzien, R.F., Perdue, J.F., and Springer, A. (1972) J. Biol. Chem., 247, 2964-2966. Estensen, R.D., and Plagemann, P.G.W. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, 1430-1434. Plagemann, P.G.W., and Estensen, R.D. (1972) J. Cell Biol., g, 179-185. Malawista, S.E., Gee, J.B.L., and Bensch, K.G. (1971) Yale J. Biol. Med., 44, 286-300. Davies, P., Fox, R.I., Polyzonis, M., Allison, A.C., and Haswell, A.D. (1973) Lab. Invest., 28, 16-22. Zurier, R.B., Hoffstein, S., and Weissmann, G. (1973) Proc. Nat. Acad. Sci. U.S.A., 7cJ, 844-848. Roos, D., Goldstein, I.M., and Weissmann, G. (1976) Proc. X Leuk. Cult. Conf., in press. Goldstein, I.M., Roos, D., Kaplan, H.B., and Weissmann, G. (1975) J. Clin. Invest., in press. Weening, R.S., Wever, R., and Roos, D. (1975) J. Lab. Clin. Med., 8&, 245-252. Weening, R.S., Roos, D., and Loos, J.A. (1974) J. Lab. Clin. Med., 83, 570-576. Homan-Mliller, J.W.T., Weening, R.S., and Roos, D. (1975) J. Lab. Clin. Med., SJ, 198-207. Pachman, L.M., Jayanetra, P., and Rothberg, R.M. (1973) Pediatrics, 52, 823-830.

49

Vol. 68, No. 1, 1976

15. 16. 17. 18.

19. 20.

21.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Weening, R.S., Roos, D., Weemaes, C.M.R., Homan-Miiller, J.W.T., and Van Schaik, M.L.J. (1975) J. Lab. Clin. Med., in press. Zigmund, S.H., and Hirsch, J.G. (1972) Exp. Cell Res., 73, 383-393. Skosey, J.L., Chow, D., Damgaard, E., and Sorensen, L.B. (1973) J. Cell Biol., 57, 237-240. Rossi, F., Patriarca, P., and Romeo, D. (1975) Proc. VII Intern. Congress R.E.S., in press. Schell-Frederick, E., Paridaens, R., and Dubois, L. (1975) Proc. VII Intern. Congress R.E.S., in press. Curnutte, J.T., and Babior, B.M. (1975) Blood, 45, 851-861. Allison, A.C., Davies, P., and de Petris, S. (1971) Nature New Biol., 232, 153-155.

50

Effect of cytochalasin B on the oxidative metabolism of human peripheral blood granulocytes.

Vol. 68, No. 1, 1976 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS EFFECT OF CYTOCHALASIN B ON THE OXIDATIVE METABOLISM OF BUMAN PERIPHERAL...
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