Vol. 91, No. 1, 1979 November
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14, 1979
Pages
222-226
SUPEROXIDE PRODUCTION BY PHAGOCYTES Another
look
at the effect
of cytochrome
stimulated
Research
Laboratory
New England Received
September
by
M. Babior and Department
Medical
uptake
neutrophils
Bernard Blood
c on oxygen
Center,
of Medicine,
Boston,
Mass.
Tufts-
02111
27, 1979 Summary
The widely held view that stimulated phagocytes liberate 02- into the extracellular medium is supported by the alterations in oxygen uptake which occur when ferricytochrome 2 is added to a suspension of zymosan-treated neutrophils. An explanation consistent with this view is provided for some previously reported results (FEBS Lett. 100, 27) which initially appeared to conflict with the notion that 02- is released by phagocytes. The exposure
of neutrophils
of an enzyme which NADP (1).
This
catalyzes
reaction
or 0 2- production. 02- production ly observed the
cell
taken
by 02-:
the
of such a secondary
@ 1979
all
because
uptake
equivalence
the 02-
uptake
is
produced
and rareby
some of the oxygen
the course
of secondary
re-
is
reduction
of cytochrome
c
5 + 02- +ferrocytochrome is
liberated
Because
cytochrome.
suspension
by Academic Press. Inc. in any form reserved.
of reproduction
not
oxygen
oxygen
but
02- +
during
reaction
for
every
of this
reaction,
in which
oxygen
0006-291X/79/210222-05$01.00/0 Copyright All rights
basis,
and also
the activation
02-.
of oxygen
2 to a neutrophil
mole
be returned
either
stoichiometry,
is because
in the assay, will
ferricytochrome
one molecule
This
for
in
2 O2 + NADPH-+2
by measuring
reaction
on a mole
(2,3).
be detected
An example
with
occur
in practice
involving
to the
results
reaction:
can be demonstrated
up by the neutrophil
actions
stimuli
the following
According should
will
to suitable
222
the
2 + 02. molecule
In this
of 02- which
the addition is being
consumed
reduction, reacts
of ferricytochrome to form 02-
BIOCHEMICAL
Vol. 91, No. 1, 1979
should
lead
which
to a diminution
has been
in net
converted
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
oxygen
to 02- will
uptake,
because
be regenerated
some of the oxygen
by the reaction
between
the
- and the cytochrome. O2 Recently, 02- is
a report
in fact
was based
not
released
from
on the observation
that
pension
of activated
uptake.
Because
many other decided
neutrophils this
workers
to restudy
Materials
has appeared
suggestion
concerning this
question
from
Segal's
stimulated the
is
neutrophils
addition
did
not
suggesting
(4).
This
of ferricytochrome
lead
to the expected
at variance
the production in
laboratory
with
suggestion -c to a sus-
fall
the conclusions
of 02- by neutrophils
the hope of reconciling
that
in oxygen drawn (5,6),
by I
the differences.
and methods
Neutrophils (7) and opsonized zymosan (8) were prepared as previously described and suspended at appropriate concentrations in Hanks buffered saline solution containing 1.5 mM HEPES buffer (pH 7.4) (HanksiHEPES). Ferricytochrome c (Sigma type VI) and bovine erythrocyte superoxide dismutase (Sigma) were dissolved in Hanks/HEPES. Oxygen uptake and 0 - production were both measured at 37' in a Gilson oxygen uptake chamber (e 3 fective volume 1.2 ml) fitted with a Yellow Springs oxygen electrode. To measure oxygen uptake, neutrophils plus enzymes as indicated, in a total volume of 1.3 ml, were placed in the chamber of the oxygen electrode and incubated for 5 minutes with vigorous stirring to permit tegperature equilibration. Opsonized zymosan (2.5 mg in 0.1 ml, prewarmed to 37 ) was then-added, and oxygen uptake was followed for the next 5 minutes. To measure 0 production, neutrophils and cytochrome 5 (total volumeo 1.3 ml) were placed z n the chamber of the oxygen electrode and brought to 37 as described above. Opsonized zymosan (2.5 mg in 0.1 ml, prewarmed to 37O) was then added. 02--dependent cytochrome 5 reduction was terminated by the addition of 10 ug superoxide dismutase either immediately (reference) or after 2 minutes (sample). TWO and a half minutes after the addition of the zymosan, the reaction mixture was transferred from the oxygen electrode into a test tube placed in melting ice, the cells and zymosan were removed by centrifugation at o", and 0 -dependent cytochrome c reduction was determined by difference spectroscopy fsample vs reference) as-described elsewhere (9). Results
and discussion Experiments
concentration
were
used in
by zymosan-stimulated Uptake
at the higher The effect
by neutrophils
done at two cell Segal's
neutrophils cell
experiments,
was determined.
dismutase
2 x lo7
and 4 x 10
at these
concentration
of superoxide
concentrations:
is
6
cells/ml.
two concentrations similar
to that
and ferricytochrome
As reported
223
by Segal,
cells/ml,
the
Oxygen is
reported
shown in Fig.1. by Segal.
2 on oxygen
the presence
uptake
uptake
of 0.25
mM
BIOCHEMICAL
Vol. 91, No. 1, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
4x IO6 PMN/ml 2 x IO’ PMN/ml
50 nmoles oxyw 1\2 min
Fig. 1. Oxygen uptake by zymosan-stimulated neutrophils. Reaction mixtures contained neutrophils as the concentrations indicated, together with the opby the arrow. sonized zymosan. The zymosan was added at the point indicated Oxygen uptake was determined as described in the text. Table I. The effect of cytochrome 5 and superoxide dismutase on oxygen uptake by zymosan-stimulated neutrophils. Oxygen uptake was determined as described in the text. The concgntrations of selected constituents were as follows: neutrophils, 4 x 10 /ml; cytochrome 2, 0.25 mM; superoxide dismutase, 10 ug/ml. Oxygen uptake is expressed as nmoles consumed during the designated time intervals. The results represent the mean * SE of 4 experiments. Additions
Oxygen uptake
35.2 f 1.7
80.4 f 5.0
Cytochrome 5
23.2 f 2.5
57.1 f 5.7
Dismutase
31.2 + 2.1
65.3 + 3.2
Cytochrome c plus diemutase
35.5 f 4.8
72.0 f 1.7
None
ferricytochrome ied
out with
shown).
5 had little neutrophils
take
toward
on oxygen in Table phils
The further control
uptake 1, are
release
levels. in
into
consumption of 2 x lo7
of neutrophils a decrease
addition
of superoxide
Cytochrome
with
cwas
of superoxide the widely
the medium.
224
held
in experiments cells/ml
(results
was treated
c, however,
the presence
consistent
02-
on oxygen
concentration
of cytochrome
was observed.
effect
at a concentration
When a lower
centration
(nmoles)
in net
oxygen
dismutase
found
to have
dismutase. notion
that
with uptake returned
the
carrnot same con-
of over oxygen
30% up-
only
a slight
effect
These
results,
shown
stimulated
neutro-
BIOCHEMICAL
Vol. 91, No. 1, 1979
What accounts centrations? released
One possibility
is high,
erated
it
0 - will 2
dismutation return
relative
to the
to react
with
5 reduction
suits
consumed in
Table
carried
at a neutrophil
reduced
of 2.5.
This
into lower
reduced
the
of the
obtained
finding extracellular
two cytochrome
at 2 x lo7
concentration, the amount
of
the cytochrome.
the amount expected
that
concentration all
the
of 0.1 mM was was increased
the 02-
released
In contrast
cell
mM cytochrome Furthermore,
-c as was trapped even at the highest
2 reduced
of the
results
concentration,
by 02- was far obtained
by
by the
-c concentrations.
of cytocbrome
on the basis
of 4 x 106/m1,
-c concentration
to
cytochrome
by the cytochrome
At this
of
appears
medium was trapped
cells/ml. by 0.25
explanation
concentration
indicates
likely
portion
of 02- -dependent
In assays
at a cytochrome
0 2- is
Con-
reaction.
foregoing
when the cytochrome
as much 0 2 - was captured concentration
the
be too
the assay.
of a substantial
in the 02--forming
lib-
the
-c might in
concen
rapid
such circumstances, cytochrome
are half
even the reagain
by the lower cytochrome less
than
at 4 x IO6 cells/ml,
Table 2. c2--dependent cytochrome c reduction by zymosan-stimulated neutrophils. 02 -dependent cytochrome 2 reduction was determined as described in the text. The concentrations of neutrophils and cytochrome 2 are shown in the table. The results represent the mean + SE of 5 experiments! Cell concentration 02--dependent cytochrome g, reduction (nmoles/2 min) 0.1 n!M cyt 5 0.25 InM cyt g 4 x lo6 PMN/ml
22.4 + 4.2
21.6 _+ 2.7
2 x107
35.8 k 3.2
53.6 k 8.6
PMN/ml
225
con-
the 02-
of the
undergoing
of the liberated
the return
observations.
fraction
to be detected
most
2 show that
out
Under
which
When the cell
the remainder
uptake
the present
the same as that
neutrophils
with
a small
of 0 - with 2
concentration,
of cytochrome
a factor
oxygen
with
cytochrome.
only
cytochrome,
at the two cell
the efficiency
reactions.
the cytochrome,
The results
amount
total
originally
for
the
RESEARCH COMMUNICATIONS
the results
by the that
due to the reaction
at a low cell
account
be expected
secondary
versely,
the oxygen
was trapped
with
and other
between
has to do with
might
react
of oxygen
at the
the differences
by the neutrophil
tration
small
for
AND BIOPHYSICAL
BIOCHEMICAL
Vol. 91, No. 1, 1979
suggesting
that
pears
probable
lated
neutrophils
to react in oxygen
with
substantial from
the
uptake
liberate
cytochrome,
0 - into 2 with
of 0 - still 2
that
employed
and it
observed
is
for
in those
provide
this
escaped
the medium.
detection.
released
in Segal's reason
It
by the
experiments that
the
ap-
stimufailed
expected
drop
experiments.
additional
the determination
RESEARCH COMMUNICATIONS
most of the 02-
the conditions
observations
lems associated O2
results
was not
The present trophils
quantities
these
under
AND BIOPHYSICAL
evidence
They also
that
illustrate
of substances
stimulated
neu-
some of the prob-
as short-lived
as the
radical.
Acknowledgement.
I thank
Supported
by Grant
in part
and Infectious
Janet
Mitchell
AI 11827
for
from
excellent
the National
technical Institute
assistance. of Allergy
Diseases.
References
1. 2. 3. 4. 5. 6. 7. 8. 9.
M&fur-rich, B.J. (1976) J. Clin. Invest. 58, Babior, B.M., Curnutte, J.T., 989-996. Root, R.K., Metcalf, J.A. (1977) J. Clin. Invest. 60, 1266-1279. Weening, R.S., Wever, R., Roos, D. (1975) J. Lab. Clin. Med. 85, 245-252. Segal, A.W., Meshulam, T. (1979) FEBS Lett. 100, 27-32. Babior, B.M. (1978) N. Engl. J. Med. 298, 659-665. Klebanoff, S.J., Clark, R.A. (1978) The neutrophil: function and clinical disorders, North-Holland, New York, p. 294-295. Babior, B.M., Kipnes, R.S., Curnutte, J.T. (1973) J. Clin. Invest. 52, 741-744. R.I. (1975) J. Clin. Invest. 55, 707-713. Hahn, D.C., Lehrer, B.M. (1974) N. Engl. J. Med. 290, Curnutte, J.T., Whitten, D.M., Babior, 593-597.
226