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 the effect of the specific iron chelator. deferoxamine. on the antibacterial function of PMN. PMN were incubated for 20 hr with various concentrations of deferoxamine at 37’C in medium containing 0.54 iM endogenous iron. The cells were then washed, and the phagocytic cell function was assessed. The results were compared with those for control PMN preincubated for 20 hr without deferoxamine, and those of nonincubated PMN. Compared with that of control 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) was used. Bacterial uptake was similarly increased in nonprein- cubated PMN or PMN preincubated for 20 hr at 4#{176}C instead of 3TC. The intracellular killing capacity of both deferox- amine-treated and control PMN exceeded 90%. PMN incu- bated for 20 hr at 37#{176}C with DFO not only phagocytosed more bacteria than control cells, but were also capable of killing the greater number of bacteria ingested. This increased activity of deferoxamine-treated PMN was accompanied by enhanced generation of chemilumines- cence and production of superoxide during phagocytosis of S. aureus. These findings indicate that deferoxamine may enhance the antibacterial activity of PMN by protecting the cells against damage by iron-mediated generation of toxic oxygen 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%. For personal use only. on April 12, 2019. by guest www.bloodjournal.org From
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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%.
For personal use only.on April 12, 2019. by guest www.bloodjournal.orgFrom
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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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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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
BS van Asbeck, JJ Marx, A Struyvenberg, JH van Kats and J Verhoef leukocytesDeferoxamine enhances phagocytic function of human polymorphonuclear
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