Clfnical Science and Molecular Medicine (1978) 55, 4 1 3 4 1 5
SHORT COMMUNICATION
Tracing the fate of oxygen consumed during phagocytosis
by human neutrophils with lsOz
A. W . S E G A L , J . C L A R K * A N D A . C . A L L I S O N
Clinical Research Centre, Harrow, Middlesex, U.K. and
'MRC Cyclotron Unit, Hammersmith Hospital, London
(Received 3 March 1978; accepted 22 June 1978)
Summary
1. The metabolism of oxygen by phagocytosing
neutrophils was traced by using 150,.
2. The isotope did not exchange with the
incubation medium or cells to an appreciable extent
and unmetabolized oxygen was readily eluted by
gassing the cell suspension.
3. The polarographic measurements of oxygen
consumption closely paralleled the recovery of
metabolized 1 5 0 , .
4. Almost all the metabolized 150,was converted into water, both in the presence and absence
of KCN, supporting the concept that the oxygen
consumed by neutrophils is converted into H,O,. It
is unlikely that an appreciable proportion of this
oxygen is incorporated into the organic composition of the cell or of the ingested microorganism.
Key words: granulocytes, hydrogen peroxide,
leucocytes, oxygen, phagocytosis, radioactive
gases.
Introduction
The oxygen consumption of neutrophil polymorphonuclear leucocytes (neutrophils) increases
with phagocytosis (Baldridge & Gerard, 1933).
This is not due to mitochondria1 respiration, as it
is not inhibited by either cyanide (Sbarra &
Karnovsky, 1959) or other inhibitors of mitochondrial cytochromes, and it is important for the
bactericidal function of these cells (Mandell, 1974).
Correspondence: Dr A. W. Segal, Clinical Research
Centre, Watford Road, Harrow, Middlesex, HA1 3UJ,
Hydrogen peroxide, a possible substrate for
the myeloperoxide-halide microbicidal system
(Klebanoff, 1975) is produced as this oxygen is
consumed, but (Iyer, Islam & Quastel, 1961;
Homan-Muller, Weening & ROOS,1975) it is not
known what proportion of the oxygen is reduced to
H,O, and how much enters the organic composition of the ingested organism or of the cell
itself. The methods for the measurement of H,O,,
including 14C02 release from [14Clformate and
peroxidation of various substrates, are non-specific
and may require high concentrations of cyanide or
azide to prevent endogenous degradation of H,O,.
The effect of these inhibitors on other cell functions
and enzyme systems is unknown. Specific analysis
of H,O, by measuring the 0, regenerated by an
excess of catalase (EC 1.11.1.6) gives only a small
percentage recovery of the oxygen, and the fate of
most of the oxygen remains unexplained (HomanMuller et al., 1975; Zatti, Rossi & Patriarca,
1968). Aldehydes (Jacobs, Paul, Strauss & Sbarra,
1970) and oxidized lipids (Gutteridge, Lamport &
Dormandy, 1976) have antibacterial activity, and
the ingestion by neutrophils of particles containing
linoleic acid generates malonaldehyde (Stossel,
Mason & Smith, 1974). This suggests that the
oxygen not otherwise accounted for might be
incorporated into lipids in the cell, possibly
producing a bactericidal agent, or into the ingested
micro-organism.
We have studied the extent to which the
metabolized oxygen is incorporated into the
organic composition of the engulfing cell, or
engulfed micro-organism, and how much eventually forms water, by using the short-lived isotope
150,.
U.K.
413
A . W. Segal, J. Clark andA. C . Allison
414
was assayed (Davies, Page & Allison, 1974) in
the cell pellet and supernatant medium of cells
processed in an identical manner.
In four other experiments (four studies) 5 x lo7
neutrophils in 4 ml of Hanks solution were
incubated at 37OC with latex particles and 1502
for
2 min. The cell suspension was then gently heated
under vacuum and a sample of distilled water was
collected in a liquid nitrogen trap. Aliquots of the
incubation mixture and distillate were weighed and
the radioactivity was measured.
Materials and methods
Neutrophils were purified from human blood by the
technique of Boyum (1968) and suspended in
Hanks balanced salt solution (2.5 x 1O8/ml). An
aliquot of the cell suspension (1.0 ml) was stirred
rapidly in the chamber of a platinum oxygen
electrode (Rank) at 37OC and to this was added
1.0 ml of Hanks solution which had been gassed
for 4 min with a mixture of 150,+ N, (20 :80, v/v;
1-2 mCi) (Clark & Buckingham, 1975).
Phagocytosis was stimulated by the addition of 1 x
1OIo latex particles coated with human IgG (0.81
p m diameter) or 1 x 1O'O serum opsonized, heatkilled staphylococci (Oxford strain) in 100 pl of
Hanks solution. Oxygen consumption was
measured polarographically for 4-6 min, after
which the cell suspension was removed, the
incubation chamber washed with 1.0 ml of
phosphate-buffered saline [sodium phosphate (8
mmol/l of sodium chloride solution (154 mmol/l),
pH 7.21 and the mixture was centrifuged at 8000 g
for 30 s in an Eppendorf 3200 centrifuge. The
pellets were washed twice with 1.5 ml of phosphatebuffered saline. The supernatants were pooled
and gassed for 2 min with N, to remove unmetabolized "0,. Radioactivity in the cell pellets and
gassed supernatants was measured in a well-type
gamma counter and compared with that in an
aliquot of the I50,-gassed medium. Experiments
were also performed with heat-killed cells (95OC for
4 min) and in the presence of KCN (5 mmol/l).
Lactate dehydrogenase (EC 1.1.1.27) activity
Results
Unmetabolized I5O, was completely removed from
the medium by gassing it with N,. The neutrophils
demonstrated a burst of oxygen consumption after
the addition of particles to the cell suspension,
which was enhanced by the presence of K C N
(Table 1). The proportion of the oxygen in the
chamber that was consumed as measured by
polarography was very similar to the proportion of
radioactivity that was recovered. Almost all the
radioactivity was in the aqueous medium and very
little remained in the cells. In two control studies,
16 and 17% of the cellular lactate dehydrogenase,
a marker of cell viability (Davies et al., 1974), was
released into the medium. The specific radioactivity of the water distilled from an incubation
mixture of phagocytosing cells was 91.6, 101.4,
87.6 and 113.4% in four studies (mean value
98.5%).
TABLE 1. Oxygen consumption by human neutrophils and the subsequent
distribution of
in the cells and incubation medium
Oxygen was consumed by neutrophils at a rate of 0.05 fmol min-' cell-' before
the addition of bacteria and latex particles, which did not consume oxygen by
themselves. Results are shown for two studies in each category except for the
single study on the cells that were killed by heating at 95OC for 4 min.
Incubation
conditions
Oxygen consumption
(polarographic measurement)
Rate
(fmol min-1 cell-')
Proportion
consumed
'$0,
radioactivity
recovered (%)
Cell
Supernatant
Total
pellet
(96)
Viable cells +
latex particles
Viable cells +
latex particles +
KCN (4 mmol/l)
Viable cells +
dead bacteria
Dead cells +
latex particles
54
51
3.5
0.51
0.59
2.5
52.3
46.5
49.0
1.04
1.oo
76
76
1.3
4.1
78.0
68.4
79.3
12.5
0.86
0.66
57
26
3.5
1.7
51.1
25.8
54.6
27.5
0
0
1.3
3.3
4.6
55.8
150consumption
2
by phagocytosing granulocytes
415
Discussion
References
1502
does not rapidly exchange with oxygen of
water and could therefore be used to determine the
fate of the oxygen in the striking burst of oxygen
consumption that accompanies phagocytosis. Almost all the metabolized oxygen was found to enter
the suspending medium. This was not due to
damage to the cells as only a small proportion of
the cytoplasmic marker enzyme lactate dehydrogenase (Segal & Peters, 1977) was released from
the cells during the studies. The I5O2
was shown to
enter the water itself, as opposed to being present
as a soluble metabolite, as its specific radioactivity
in water distilled from the incubation mixture was
very similar to that in the original cell suspension.
The 1 5 0 , was not metabolized to water by mitochondrial respiration, as its consumption was not
inhibited by KCN, nor was its exchange with the
0, simply accelerated by cellular components, as it
did not occur in the -resence of dead cells. Very
little (less than 4%) if any, of the oxygen that is
consumed becomes incorporated into the organic
structure of neutrophils or ingested bacteria.
The current concept that all the oxygen consumed in association with phagocytosis forms
H,O, (Klebanoff, 1975; Homan-Muller et al.,
1975; Iyer et al., 1961) appears correct. The low
recovery of H,O, previously observed, which
accounted for less than 10% of the oxygen
consumed, probably results from further metabolism of H,O, by myeloperoxidase (EC 1.1 1.1.17) as
H,O, accumulates in myeloperoxidase-deficient
neutrophils (Klebanoff & Pincus, 1971) and as the
recovery of H,O, amounts to 5&70% of the
oxygen consumed in the presence of cyanide and
azide (Homan-Muller et al.,1975), which inhibit this
enzyme. Cyanide appears to enhance oxygen
consumption under the conditions in which the
present studies were conducted because it is basic
and buffers the acid produced by phagocytosing
cells (Sbarra & Karnovsky, 1959).
BALDRIDGE, C.W. & GERARD, R.W. (1933) The extra
respiration of phagocytosis. American Journal of Physiology,
103,235-236.
BOYUM,A. (1968) Isolation of leukocytes from human blood.
Further observations. Methylcellulose, dextran and ficoll as
erythrocyte aggregating agents. Scandinavian Journal of
Clinical and Laboratory Investigaton, 21 (Suppl. 97), 3 1-50.
CLARK,J.C. & BUCKINGHAM,
P.D. (1975) In: Short-lived
Radioactive Gasesfor Clinical Use. Butterworth, London.
DAVIES,P., PAGE, R.C. & ALLISON,A.C. (1974) Changes in
cellular enzyme levels and extracellular release of lysosomal
acid hydrolases in macrophages exposed to group A streptococcal cell wall substance. Journal of Experimental Medicine,
139,1262-1282.
GUTTERIDGE,
J.M., LAMPORT,P. & DORMANDY,
T.L. (1976)
The antibacterial effect of water-soluble compounds from
autoxidising linolenic acid. Journal of Medical Microbiology,
9,105-1 10.
HOMAN-MULLER,
J.W.T., WEENING,R.S. & Roos, D. (1975)
Production of hydrogen peroxide by phagocytizing human
granulocytes. Journal of Laboratory and Clinical Medicine,
85,198-207.
IYER, G.Y.N., ISLAM,M.F. & QUASTEL,J.H.(1961) Biochemical aspects of phagocytosis. Nature (London), 192,
535-541.
JACOBS,A.A., PAUL, B.B., STRAUSS,R.R. & SBARRA,A.J.
(1970) The role of the phagocyte in post-parasite interactions. XXIII. Relation of bactericidal activity to
peroxidase-associated decarboxylation and deamination. Biochemical and Biophysical Research Communications, 39,
284-289.
KLEBANOFP,
S.J. (1975) Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Seminars in Hematology, 12,117-160.
KLEBANOFF,S.J. & Pmcus, S.H. (1971) Hydrogen peroxide
utilisation in myeloperoxidase-deficient leukocytes: a possible
microbicidal control mechanism. Journal of Clinical
Investigation, 50,2226-2229.
MANDELL, G.L. (1974) Bactericidal activity of aerobic and
anaerobic polymorphonuclear neutrophils. Infection and
Immunity, 9,337-341.
SBARRA,A.J. & KARNOVSKY,
M.L. (1959) The biochemical
basis of phagocytosis. I. Metabolic changes during the
ingestion of particles by polymorphonuclear leukocytes.
Journal ofBiologica1 Chemistry, 234,1355-1362.
SEGAL,A.W. & PETERS,T.J. (1977) Analytical subcellular
fractionation of human granulocytes with special reference to
the localization of enzymes involved in microbicidal mechanisms. Clinical Science and Molecular Medicine, 52.429442.
STOSSEL, T.P., MASON, R.J. & SMITH,A.L. (1974) Lipid
peroxidation by human blood phagocytes. Journal of Clinical
Investigation, 54,638-645.
ZATTI,M., Rossr, F. & PATRIARCA,
P. (1968) The H,O, production by polymorphonuclear leukocytes during phagocytosis. Experientia, 24,669-670.