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

Superoxide production by phagocytes. Another look at the effect of cytochrome c on oxygen uptake by stimulated neutrophils.

Vol. 91, No. 1, 1979 November BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 14, 1979 Pages 222-226 SUPEROXIDE PRODUCTION BY PHAGOCYTES An...
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