Post on 12-Sep-2021
714 Blood, Vol. 63, No. 3 (March), 1984: pp. 7 14-720
Deferoxamine Enhances Phagocytic Function of Human
Polymorphonuclear Leukocytes
By B. Sweder van Asbeck, Jo J. M. Marx, Albert Struyvenberg, J. Henny van Kats, and Jan Verhoef
Inhibition of the iron-mediated generation of toxic oxygen
species by polymorphonuclear leukocytes (PMN) might
prevent oxidative damage and thus enhance phagocytic
function of PMN. To investigate this point. we studied theeffect of the specific iron chelator. deferoxamine. on theantibacterial function of PMN. PMN were incubated for 20hr with various concentrations of deferoxamine at 37’C in
medium containing 0.54 �iM endogenous iron. The cellswere then washed, and the phagocytic cell function wasassessed. The results were compared with those forcontrol PMN preincubated for 20 hr without deferoxamine,and those of nonincubated PMN. Compared with that ofcontrol PMN, the uptake of radiolabeled Staphylococcus
aureus by PMN treated with 1 MM-i mM deferoxamine
was, on average. 1 0%-20% higher. This effect was not
H UMAN POLYMORPHONUCLEAR leuko-
cytes (PMN) play a crucial role in the host
defense against invading organisms,”2 the essential
factor being their ability to initiate the sequential
reduction of oxygen to superoxide (02)� and hydrogen
peroxide (H2O2).4 O2 and H202 can react together
(Haber-Weiss reaction)5 to form even more deleterious
oxygen species, such as the hydroxyl radical (.OH)6’7
and, possibly, singlet oxygen (b02).8 Ferric compounds
have been shown to catalyze the reduction of H202 by
02� 6,9 Oxygen-derived products of PMN not only
contribute to the killing of microbes,’#{176} but are also
potentially autotoxic. PMN can be killed by exposure
to a particulate stimulus’ ‘ or a soluble activator, such
as phorbol myristate acetate,’2 because these agents
induce the production of toxic oxygen species. As
previously reported, incubation of phagocytes in a
medium with excess iron led to reduced phagocytic
function,’3 and this has been attributed to the action of
iron-catalyzed free radical oxidation. Because traces of
iron are usually present in biologic systems’4 and 02 is
also formed by PMN at rest, though in much smaller
quantities than during phagocytosis,3”5”6 iron might
contribute to the decrease in phagocytic capacity dur-
ing prolonged preincubation because of its catalytic
effect on the 02�-mediated generation of .OH.9 Some
chelating agents, for example, ethylenediaminetetra-
acetic acid (EDTA), can stimulate .OH forma-
tion,9”72#{176}whereas others, such as diethylenetriamine-
From the Departments of Medicine. Hematology, and Microbi-
ology. University Hospital. Utrecht, The Netherlands.
Submitted June 16. 1983; accepted September 19. 1983.
Address reprint requests to Dr. J. J. M. Marx, Department of
Hematology, University Hospital Utrecht. Catharijnesingel 101,
351 1 GV Utrecht, The Netherlands.
(C) 1984 by Grune & Stratton. Inc.
000tS-4971/84/6303-0033$01.00/0
observed when iron-saturated deferoxamine (DFO) wasused. Bacterial uptake was similarly increased in nonprein-cubated PMN or PMN preincubated for 20 hr at 4#{176}Cinsteadof 3TC. The intracellular killing capacity of both deferox-amine-treated and control PMN exceeded 90%. PMN incu-bated for 20 hr at 37#{176}Cwith DFO not only phagocytosedmore bacteria than control cells, but were also capable ofkilling the greater number of bacteria ingested. Thisincreased activity of deferoxamine-treated PMN wasaccompanied by enhanced generation of chemilumines-cence and production of superoxide during phagocytosis ofS. aureus. These findings indicate that deferoxamine mayenhance the antibacterial activity of PMN by protecting thecells against damage by iron-mediated generation of toxicoxygen metabolites in resting PMN.
pentaacetic acid (DTPA)’7”9 and deferoxamine,2#{176} can
be inhibitory.
The present report concerns the effect of deferox-
amine, a naturally occurring sideramine,2’ on the
antibacterial activity of PMN during overnight incu-
bation. This effect was evaluated on the basis of the
capacity of the PMN to phagocytose and kill radiola-
beled, opsonized Staphylococcus aureus as well as the
capacity to generate chemiluminescence and O2 upon
stimulation. Evidence is presented for a deferoxamine-
mediated enhancement of these PMN functions, and it
is suggested that this is the result of protection against
injury by an iron-dependent system that requires an
active metabolism.
Reagents
MATERIALS AND METHODS
Solutions of deferoxamine methanesulfonate (Ciba-Geigy, Basel,
Switzerland) (pH 5.0) and FeC13 - 6H20 (Merck, Darmstadt,
Germany) (pH 1.0) were prepared, immediately prior to use, in
concentrations of 100 mM and 2 M, respectively, in double glass-
distilled water. Mannitol (OPG, Utrecht, The Netherlands) was
dissolved in medium 1640 of the Roswell-Park Memorial Institute
(RPMI) (GIBCO Europe, Paisley, U.K.) in a concentration of 500
mM. Luminol (5-amino-2,3-hydro-l,4-phthalazinedione, Eastman
Kodak Co., Rochester, NY) was prepared as a 1.5 mM stock
solution in dimethyl sulfoxide and diluted in Hanks’ balanced salt
solution (HBSS; GIBCO) containing 0.1% gelatin (GHBSS) to a
final concentration of I .4 nM/scintillation vial. Ferricytochrome c
(horse heart type I) and superoxide dismutase (SOD; E.C.1.l 5. 1.1;
bovine blood type I) were obtained from Sigma Chemical Co., St.
Louis, MO, and were solubilized in GHBSS at concentrations of
1 1.2 mg/mI and 1 . I mg/3 ml, respectively, just before use.
Isolation of PMN
PMN were recovered from venous donor blood drawn into hepa-
rinized syringes (10 U heparin/ml blood) by a modification of a
method developed by Boyum,22 as described elsewhere.23 Purity of
the final PMN suspension was evaluated by Wright’s stained smears
and always exceeded 90%. Viability, as assessed by trypan blue
exclusion, was 90%-93%.
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DEFEROXAMINE ENHANCES FUNCTION OF PMN 715
Pretreatment ofPMN With Deferoxamine
PMN were incubated for 20 hr at 37#{176}Cin air containing 5% CO2
in polypropylene vials (Bio-vials, Beckman, Chicago, IL) at a
concentration of 2.5 x 106 cells/mI. The vials were stationary to
minimize activation of PMN. RPMI 1640, a chemically defined
medium supplemented with penicillin (100 U/liter) and streptomy-
cm ( I 00 �g/ml), was used as incubation medium. Deferoxamine was
added to obtain the final concentrations indicated below. The final
volume of the suspension was 2 ml; the pH was 7.4. After preincuba-
tion, the cells were washed twice with RPM! 1640, resuspended in
GHBSS, and tested immediately. Viability of the PMN, assessed by
trypan blue exclusion, was between 87% and 93% for both deferox-
amine-treated and control cells, respectively. In some experiments,
PMN were incubated at 4#{176}C.In other control experiments, use was
made of iron-saturated deferoxamine, which was prepared by adding
100 �l FeCl3 (2M) to 4 ml deferoxamine (50 mM) brought to pH 2
with 6 N HCI. The pH was then slowly adjusted to 7.4 with solid
NaHCO3 under continuous stirring, and the volume made up to 5.0
ml. The concentration of iron in the basic incubation medium,
determined by atomic emission spectrophotometry, was 0.54 �M. At
neutral pH and atmospheric oxygen tension, iron in aqueous solution
is in the ferric oxidation state24 and is then readily hydrolyzed to
form aggregating ferric hydroxide complexes.25 Chelators, for exam-
pIe amino acids, present in RPM! I 640 prevent aggregation and thus
permit the iron to be catalytically active.26
Culturing and Radiolabeling of Bacteria
For each experiment, S. aureus Ev, a clinical isolate maintained
on blood agar plates at 4#{176}C,was allowed to proliferate in Mueller
Hinton broth (Difco Laboratories, Detroit, MI) in a 37#{176}Cshaking
incubator for I 8 hr. was washed 3 times with phosphate-buffered
saline (PBS) (pH 7.4), and then suspended at 2.5 x lO� microor-
ganisms/mI in PBS. For phagocytosis studies, the bacteria were
radiolabeled by incubation in 5 ml Mueller Hinton broth containing
20 �Ci of 3H-methyl-thymidine (specific activity 5 Ci/mmole,
Amersham, Bucks, U.K.), as described elsewhere.23
Opsonins and Opsonization Procedure
Serum from I 0 healthy donors was pooled and stored in I -ml
portions at - 70#{176}C.Just before use, samples were thawed and diluted
to 5% in GHBSS. For opsonization, 100 �l ofa suspension containing
5 x iO� S. aureus was mixed with 0.9 ml 5% serum, and the mixture
was incubated for 30 mm at 37#{176}Cand held at 4#{176}Cuntil use.
Phagocytosis and Killing Assay
The uptake and intracellular killing of opsonized S. aureus by
PMN were determined with radiolabeled bacteria in an assay
described elsewhere.23 Briefly, 200 �l of a suspension of opsonized
bacteria was mixed with 200 Ml of a suspension of control or
deferoxamine-treated PMN in 4 polypropylene vials, and phagocy-
tosis was allowed to proceed for 2, 6, and 12 mm in a 37#{176}Cshaking
incubator. The final bacteria-to-phagocyte ratio was 10: 1 . Phagocy-
tosis was stopped by adding 2.5 ml ice-cold PBS to the mixture.
Non-phagocyte-associated bacteria were removed by 3 cycles of
centrifugation (each 5 mm, 160 g, 4#{176}C),and the phagocyte-
associated radioactivity in the final pellet was determined by liquid
scintillation counting. Phagocytosis was expressed as the percent
uptake of total added radioactivity. Total added radioactivity (repre-
senting both non-leukocyte-associated and leukocyte-associated bac-
teria) was determined in the pellet of the fourth vial after centrifuga-
tion at 1 ,600 g for I 5 mm. Results are expressed as percent uptake of
S. aureus by PMN after 2, 6, and 12 mm of phagocytosis.
The percentage ofviable leukocyte-associated bacteria at 2 and I 2
mm was determined by a standard pour-plate technique in a sample
taken from the washed cell suspension. Results are expressed as
percent intracellular killing ofS. aureus ingested by PMN at 2 or 12
mm of phagocytosis.
Chemiluminescence Assay
Luminol chemiluminescence responses were monitored with an
ambient temperature-counting spectrometer (Mark II, Nuclear,
Chicago IL) as described elsewhere.2’ Assay components were dark
adapted and preincubated at 37#{176}Cin polypropylene vials for 20 mm.
Background counts were performed for each vial, as well as in the
standard assay mixture, which was prepared by combining 1 ml of a
solution containing 5 x l07/ml opsonized S. aureus. 100 �l stock
luminol, and enough GHBSS to obtain a final volume of 2.! ml. At
zero time, I ml ofa suspension containing 5 x 106 PMN was added
to start the reaction. The mixture was then agitated for 30 sec. and
chemiluminescence was monitored for 0. 1 mm at 1 5-sec intervals
over a 1 2-mm period. Vials were held at 37#{176}Cbetween counts.
Results are expressed as the mean of values of counts per unit time
[counts per minute (cpm) x l0�] within 2-mm intervals corrected
for background activity.
Superoxide Production
Production of superoxide was measured as the reduction of
ferricytochrome c, according to a modification of a method already
described.’6 The standard reaction mixture contained 100 MI stock
ferricytochrome c, 500 Ml of a suspension containing l0� PMN/ml,
100 �zl of a suspension with 2.5 x !09/ml opsonized S. aureus, and
enough GHBSS to obtain a final volume of 2 ml. Paired reactions
with and without superoxide dismutase (1 10 gig/reaction mixture)
were assessed. All of the components were brought into polypropy-
lene vials, which were placed in a 37#{176}Cshaking incubator for 30 mm.
At the end of the experiment, the vials were promptly centrifuged at
4#{176}Cand 160 g for 10 mm, the supernatant fractions were removed
and centrifuged at 4#{176}Cand 1 ,600 g for 15 mm to sediment the
bacteria, and the relative difference in absorbance of the superna-
tants was determined at 550 nm in a double-beam spectrophoto-
meter (Perkin-E!mer model 1 24). Nanomoles of reduced cyto-
chrome c were determined from the increase in the absorbance at
550 nm, using the extinction coefficient E5��m 2.10 X !0� M’
cm ‘ . Results are expressed as nanomole cytochrome c reduced by
5 x 106 PMN after 30 mm of incubation.
Statistical Analysis
The standard error was taken as an estimate of variance. Statisti-
cal differences were determined by analysis of variance.28
RESULTS
Effect ofDeferoxamine on the Phagocytosis and
Killing ofS. Aureus by PMN
PMN were incubated in the presence of various
concentrations of deferoxamine at 37#{176}C.After 20 hr,
the PMN were washed and radiolabeled opsonized
staphylococci were added. Uptake and killing of S.
aureus by deferoxamine-treated and control PMN
(incubated for 20 hr at 37#{176}Cwithout deferoxamine)
were determined simultaneously. As Table 1 shows,
the percent uptake of S. aureus after 2, 6, and 1 2 mm
of incubation with PMN preincubated with 1 jsM
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Table 1 . Effect of Pretreatment of Human Polymorphonuclear Leukocytes (PMN) With Deferoxamine
on Phagocytosis of Staphylococcus aureus
bacteria ingested. As illustrated in Fig. 1, deferox-
amine had no effect on the intracellular killing capac-
ity. After 2 and 1 2 mm of incubation of staphylococci
with PMN that had been preincubated for 20 hr at
37#{176}Cin the presence or absence of deferoxamine (1
mM), more than 90% of the PMN-associated bacteria
had been killed by deferoxamine-treated PMN as well
as by control PMN.
A B100
2 12 2 12Incubation time (mm)
Fig. 1 . Effect of pretreatment with deferoxamine on theintracellular killing capacity of human polymorphonuclear leuko-cytes (PMN). PMN were incubated for 20 hr at 37#{176}C(5% CO2 in air)in RPMI 1640. with or without deferoxamine (1 mM). Next. 10’washed deferoxamine-treated (A) or untreated (B) PMN wereincubated for 2 and 1 2 mm at 37#{176}C,together with 1 0� radiolabeledopsonized Staphylococcus aureus in GHBSS (final volume 0.4 ml.pH 7.4). and phagocytosis was stopped by adding ice-cold PBS.After 3 washes to remove the non-leukocyte-associated bacteria.the PMN were disrupted by mixing in sterile distilled water. Afterappropriate dilution. samples were pour-plated in nutrient agarand colonies were counted after 48 hr of incubation at 37#{176}C.Theinitial number of bacteria added to the vials at time zero waschecked spectrophotometrically. The number of leukocyte-asso-ciated bacteria viable after 2 and 1 2 mm of incubation was thencalculated on the basis of the percentage of staphylococci takenup by the PMN at those times. Data are expressed as the mean ±
SEM of the percentage of intracellular killing in four testsperformed in duplicate.
716 VAN ASBECK ET AL.
Percent Phagocytosis�
PMN Pretreatment 2 mm 6 mm 1 2 mm
Number ofof Tests p Value
47±3 79±3 84±2 59
30±4 58±6 69±5 10 <O.0O1�
37±4 69±3 79±4 5 NS
47±4 79±2 88±2 10 <O.0O5�
34±3 58±5 70±5 4 NS
46 ± 10 75 ± 5 87 ± 2 4 <O.O5�
None
Medium alone
Deferoxamine ( 1 �.tM)
Deferoxamine ( 1 mM)
Deferoxamine, iron-saturated ( 1 mM)
Medium alone (4 #{176}C)
PMN (5 x 1O�/2 ml RPMI 1640) were preincubated for 20 hr at 37#{176}C(5% CO2 in air) � at 4#{176}C,and pH 7.4, with medium alone or medium
containing deferoxamine or iron-saturated deferoxamine.
tNonPreincubated � washed pretreated cells were incubated in GHBSS with opsonized S. aureus (ratio 1 : 10) at 37 #{176}C,and phagocytosis wasassessed for the indicated times, expressed as mean ± SEM of the percentage of the total number of bacteria taken up by the PMN from the medium in
the indicated number of tests.
�Significance of differences between values of nonpreincubated and preincubated PMN in medium alone.
§Significance of differences between PMN preincubated in medium alone at 37 #{176}Cand deferoxamine, and between PMN in medium alone at 37#{176}C
and 4#{176}C,as determined by analysis of variance.
NS. No significance of differences between values of preincubated PMN in medium alone.
deferoxamine was increased by approximately 10%
compared with PMN preincubated without deferox-amine. This enhancement of the phagocytosis by PMN
increased to approximately 20% when the overnight
incubation of the PMN was performed in the presenceof 1 mM deferoxamine (p < 0.005). It is also evident
from Table 1 that incubation of control PMN at 37#{176}C
for 20 hr lowered their activity compared with nonin-
cubated PMN (j.’ < 0.001) (data on the nonincubated
cells were obtained in a separate study comprising 59
donors). PMN incubated for 20 hr at 37#{176}Cwith 1 mM
deferoxamine showed a phagocytic activity similar to
that of the nonincubated PMN. After incubation of S.
aureus with PMN for 1 2 mm, the control PMN had
taken up 69% ± 5% (mean ± SEM), whereas the
deferoxamine-treated and nonpreincubated PMN had
taken up 88% ± 2% and 84% ± 2%, respectively. When
deferoxamine was saturated with iron, the enhancing
effect of the chelator was completely abolished, which
indicates that the iron-chelating property of deferox-
amine was important for the effect on the PMN. From
these results, we concluded that incubation of PMN at
37#{176}Cfor 20 hr had a deleterious effect on the phago-
cytic capacity of the cells, as was shown by a dimin-
ished uptake of opsonized staphylococci by these prein-
cubated cells, and that unsaturated deferoxamine pre-
vented this depression of the phagocytic activity. A
similar effect on the PMN function was observed when
PMN were incubated without deferoxamine for 20 hr
at 4#{176}Cinstead of 37#{176}C.The uptake of staphylococci by
PMN preincubated for 20 hr at 4#{176}Cwas also signifi-
cantly increased relative to the uptake of S. aureus by
control PMN preincubated for 20 hr at 37#{176}C(p <
0.05).
The effect of deferoxamine on the bactericidal activ-
ity of PMN was measured as the number of viable
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18
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4
4
DEFEROXAMINE ENHANCES FUNCTION OF PMN 717
Effect ofDeferoxamine on the Chemiluminescence
and Superoxide Production by PMN
It has been generally accepted that toxic oxygen
species play a role in the bactericidal activity of
phagocytic cells.’6’29 Measurement of PMN-induced
luminol luminescence provides information about the
activity of the oxygen metabolism of PMN.’5’30’3’ An
additional and more specific quantification of PMN
metabolic activity is provided by assessment of the
capacity to reduce ferricytochrome c,’6 which is based
on the reaction with O2 32 To study the effect of
deferoxamine on the metabolic activity of PMN, the
leukocytes were incubated for 20 hr at 37#{176}Cwith and
without deferoxamine. After being washed, these cells
were stimulated with opsonized staphylococci and the
chemiluminescence and 02 production were mea-
sured. As can be seen in Figs. 2 and 3, the metabolic
activity of deferoxamine-treated PMN was increased.
Both the reactive chemiluminescence of these cells and
the production of 02 were significantly higher than in
the control PMN preincubated for 20 hr at 37#{176}C
without deferoxamine (p < 0.001 and p < 0.01,
respectively). The mean peak chemiluminescence
response of deferoxamine-treated PMN was nearly
twice that found for the control PMN (1,600 cpm x
i03 versus 900 cpm x iO�). In contrast, when
deferoxamine was present in the chemiluminescence
mixture, the mean peak activity of the PMN was 30%
lower than that of control PMN without deferoxamine
in the assay medium, suggesting that deferoxamine
could inhibit the formation of oxygen metabolites
(data not shown).
DISCUSSION
When PMN were preincubated for 20 hr at 37#{176}C
together with the iron chelator deferoxamine, phago-
cytosis of S. aureus was significantly increased com-
pared with control cells preincubated without deferox-
amine. The intracellular killing capacity was not
altered by deferoxamine. This means that the deferox-
amine-treated PMN not only phagocytosed more bac-
teria, but were also capable of killing almost all of the
extra microbes ingested. Furthermore, the metabolic
burst, as measured by the capacity to produce 02 and
chemiluminescence, was significantly enhanced. As
the intracellular killing capacity of the deferoxamine-
treated and control cells was similar, it is possible that
the enhanced chemiluminescence and 02 production
were merely a reflection of the increased number of
bacteria taken up by deferoxamine-treated cells. The
ability of nonpreincubated and deferoxamine-treated
PMN to phagocytose bacteria was similar. Taken
together, these results indicate that, during overnight
incubation of PMN at 37#{176}C,the phagocytic function
of these cells decreases and that this reduction is
prevented by deferoxamine. The abolition of the effect
of deferoxamine by iron in excess supports the hypoth-
esis that the iron-chelating property of deferoxamine is
responsible for the beneficial effect of this compound
on the phagocytic cell.
Although iron appears to be essential for PMN
function, because the microbicidal activity of PMN of
patients with severe iron-deficiency anemia is
impaired,3337 there are also data that indicate that iron
is a potentially toxic agent, affecting the antibacterial
activity of the PMN. Incubation of PMN in the
Incubation time(min)
Fig. 2. Effect of pretreatment with deferoxamine on thegeneration of chemilumineacence by human polymorphonuclearleukocytes (PMN). PMN were incubated for 20 hr at 37#{176}C(5% CO2in air) in RPMI 1640 with or without deferoxamine (1 mM). Theassay mixture contained 5 x 1 0’ washed deteroxamine-treated(#{149})or untreated (0) PMN. 1 .4 nM luminol. and enough GHBSS toobtain a final volume of 2.1 ml (pH 7.4. ambient temperature). Toinitiate the reaction, 5 x 1 O� opsonized Staphylococcus aureuswere added at zero time. Luminescence generated by deferox-
amine-treated PMN in the absence of S. aureus (0). Chemilumi-nescence was measured for 0.1 mm at 1 5-sec intervals over a
12-mm period in a liquid scintillation counter. Data are presentedas the mean of values of counts (cpm x 1O�) within 2-mmintervals in 5 tests.
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60
z
F#{176}:� �E
C
:!e io
‘CU0
>‘
C-, �
A B
Fig. 3. Effect of pretreatment with deferoxamine on thesuperoxide production of human polymorphonuclear leukocytes(PMN). PMN were incubated for 20 hr at 37#{176}C(5% CO2 in air) in
RPMI 1640 with or without deferoxamine (1 mM). Next. 5 x 1Owashed deferoxamine-treated (A) untreated (B) PMN were incu-bated for 30 mm at 37#{176}Ctogether with 2.5 x 10’ opsonizedStaphylococcus aureus and 1 .1 mg ferricytochrome c. in thepresence or absence of 1 10 �ig SOD in GHBSS (final volume 2 ml,pH 7.4). After removal of the PMN and bacteria by centrifugation,the amount of superoxide produced was determined by measuringthe absorbance of the reaction mixture at 550 nm. Data areexpressed as the mean ± SEM of nanomole cytochrome c reducedin 3 tests performed in duplicate.
718 VAN ASBECK ET AL.
presence of an excess of iron resulted in impaired
mitochondrial activity,38’39 diminished chemotaxis,�#{176}
and defective phagocytosis.’3 It is becoming increas-
ingly evident that many of the molecular mechanisms
underlying iron-mediated degradation of biologic
structures involve interaction between iron and toxic
oxygen species.41�8 In the present study, it was found
that PMN in the steady state after overnight incuba-
tion at 37#{176}Cin a medium containing traces of ferric
iron had lost about 1 8% of their phagocytic capacity as
measured over a 1 2-mm period. Although on a consid-
erably lower level than phagocytosing PMN, resting
PMN also generate 02, as measured by the reduction
of ferricytochrome c.3”5”6 Ferric iron in the vicinity of
PMN may react with this 02 to yield Fe2�, which in
turn can react with H202, which results in the forma-
tion of the highly toxic .OH.7’9 This oxygen radical is
one of the strongest oxidizing agents known.49 It has
been generally accepted that the formation of . OH by
reaction of 02 and H202 (Haber-Weiss reaction),5
which in itself proceeds sluggishly,5#{176} is catalyzed by
trace metals such as iron.6’9’43 Evidence supporting the
production of . OH by phagocytosing PMN has been
presented,5154 and this radical has also been held
responsible for the premature death of phagocytosing
PMN.” Involvement of oxygen metabolites in the
reduction of the PMN function during overnight incu-
bation, which implies metabolic activity, is supported
by the observation that preincubation of PMN at 4#{176}C
did not result in depressed phagocytic capacity.The peculiarities of the deferoxamine molecule that
render it especially suitable for preventing iron from
exerting its catalytic activity may lie in its hexadentate
structure, comprising three hydroxamic acid moieties
that specifically bind to all six coordination sites of iron
in a 1:1 complex.21’55 Thus, all of the aquo positions,
which are believed to be important in transition metal
catalysis,26’56 are occupied by the donor atoms of the
deferoxamine molecule. Another important feature
may be the ferroxidase activity of Fe3�-complexing
agents,57 such as deferoxamine.24 The chelator only
binds the Fe2� ion weakly,58’59 ifat all,55’59 but neverthe-
less, deferoxamine promotes oxidation of the ferrous
ion,59 thus inhibiting its reaction with H202. Unlike the
neutral effect of the 1 : 1 complex of iron-saturated
deferoxamine on PMN function, incubation of PMN
with iron attached to citrate at a 1 : 1 stoichiometry led
to decreased phagocytic function,’3 which seems to
suggest catalytic activity of the ferric citrate complex
with respect to the generation of toxic oxygen species.
In another study also, iron bound to EDTA in equimo-
lar concentration was catalytically active, as shown by
increased .OH generation by PMN.’8 Recently, it was
shown that Fe(III)-EDTA increased the bactericidal
activity of an enzymic 02 and H202 generating sys-
tem, which was attributed to an increased generation
of .OH,�#{176}whereas this iron effect was eliminated by
deferoxamine and DTPA. In addition, the latter chela-
tor was also found to have a protective effect against
the toxicity of alloxan,61’62 an oxygen free radical
generating agent.62’63 These data support the assump-
tion that the ability of iron to function as a catalyst in
the generation of toxic oxygen species depends, at least
partially, on the nature of the iron-liganding agent.
Citrate only forms three coordination bonds with the
metal.M EDTA, although a hexadentate ligand, is too
small to completely encompass the iron ion, and this
permits the metal to be catalytically active via a
seventh coordination site induced by distortion of the
usual coordination symmetry.65 In contrast, DTPA,
like deferoxamine, can effectively “lock in” the iron
ion (see refs. 66 and 20, respectively), thus sequester-ing the metal in a catalytically inactive form.
When these features are taken into consideration, it
seems possible that inhibition of PMN-induced oxida-
tive stress by prevention of the catalytic activity of iron
in the generation of the highly toxic . OH might well
account for the increased phagocytic cell function of
deferoxamine-treated PMN. This hypothesis is sup-
ported by the recent report on the inhibition of .OH
formation and lipid peroxidation by deferoxamine.20’67
Why deferoxamine was more effective in our study
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enhances the phagocytic cell function of PMN, possi- of activated oxygen species.
bly due to inhibition of . OH production via the
REFERENCES
DEFEROXAMINE ENHANCES FUNCTION OF PMN 719
when it was added in excess of the iron present in the
medium is not clear. One explanation might be that
sufficient iron was left after competition with other
traces of metallic ions, such as Ca2�, Cu2�, or Zn2’,
although the affinity of deferoxamine for the Fe3� ion
is several logs greater.2’
In summary, we have shown that deferoxamine
! . Cohn ZA, Austen FK: Contributions of serum and cellular
factors in host defense reactions. II. Cellular factors in host resis-
tance. N Engl J Med 268:1056, 1963
2. Mills EL, Quie P0: Congenital disorders of the functions of
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3. Babior BM, Kipnes RS, Curnutte JT: Biological defense
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1984 63: 714-720
BS van Asbeck, JJ Marx, A Struyvenberg, JH van Kats and J Verhoef leukocytesDeferoxamine enhances phagocytic function of human polymorphonuclear
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