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
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BIOCHEMICAL
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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