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

0x

UC

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C

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E.CC-)

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

po!ymorphonuclear neutrophils. Rev Infect Dis 2:505, 1980

3. Babior BM, Kipnes RS, Curnutte JT: Biological defense

mechanisms. The production by leukocytes of superoxide, a potential

bactericidal agent. J Clin Invest 52:74!, 1973

4. Root RK, Metcalf J, Oshino N, Chance B: H202 release from

human granulocytes during phagocytosis.I. Documentation, quanti-

tation, and some regulating factors. J Clin Invest 55:945, 1975

5. Haber F, Weiss J: The catalytic decomposition of hydrogen

peroxide by iron salts. Proc R Soc Lond (A) 147:332, 1934

6. Koppenol WH, Butler J, van Leeuwen JW: The Haber-Weiss

cycle. Photochem Photobiol 28:655, !978

7. Cohen G: The generation of hydroxyl radicals in biologic

systems: Toxicological aspects. Photochem Photobiol 28:669, 1978

8. Kellogg EW III, Fridovich I: Superoxide, hydrogen peroxide,

and singlet oxygen in lipid peroxidation by a xanthine oxidase

system. J Biol Chem 250:8812, 1975

9. McCord JM, Day ED Jr: Superoxide-dependent production of

hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett

86:139, 1978

10. Rosen H, Klebanoff SJ: Bactericidal activity of a superoxide

anion-generating system. J Exp Med 149:26, 1979

I 1 . Salin ML, McCord JM: Free radicals and inflammation.

Protection of phagocytosing leukocytes by superoxide dismutase. J

Clin Invest 56:1319, 1975

I 2. Tsan M-F: Phorbol myristate acetate induced neutrophil

autotoxicity. J Cell Physiol 105:327, 1980

I 3. Van Asbeck BS, Verbrugh HA, van Oost BA, Marx JJM,

Imhof WH, Verhoef J: Listeria monocytogenes meningitis and

decreased phagocytosis associated with iron overload. Br Med J

284:542, 1982

14. Halliwell B: Free radicals, oxygen toxicity and aging, in Sohal

RS (ed): Age Pigments. Amsterdam, E!sevier/North Holland Bio-

medical, 1981, pp 1-62

I 5. rosen H, Klebanoff Si: Chemiluminescence and superoxide

production by myeloperoxidase-deficient leukocytes. J Clin Invest

58:50, 1976

16. Johnston RB Jr. Keele BB Jr. Misra HP, Lehmeyer JE,

Webb LS, Baehner RL, Rajagopalan KV: The role of superoxide

anion generation in phagocytic bactericidal activity. J Clin Invest

55:1357, 1975

17. Buettner GR, Oberley LW, Chan Leuthauser SWH: The

effect of iron on the distribution of superoxide and hydroxyl radicals

as seen by spin trapping and on the superoxide dismutase assay.

Photochem Photobiol 28:693, 1978

1 8. Ambruso DR. Johnston RB ir: Lactoferrin enhances

hydroxyl radical production by human neutrophils, neutrophil par-

ticulate fractions, and an enzymatic generating system. J Clin Invest

67:352, 198!

Haber-Weiss reaction, and that this can be interpreted

in terms of the chelation of iron. Hitherto, chelation of

iron has only been discussed in relation to iron over-

loading. The results of our study and those of others

suggest that chelators with a high affinity for iron,

such as deferoxamine, might be useful for the protec-

tion of biologic structures against the deleterious effect

19. Halliwell B: Superoxide-dependent formation of hydroxyl

radicals in the presence of iron chelates. FEBS Lett 92:321 , 1978

20. Gutteridge JMC, Richmond R, Halliwel! B: Inhibition of the

iron-catalysed formation of hydroxyl radicals from superoxide and

of lipid peroxidation by desferrioxamine. Biochem J 184:469, 1979

21 . Keberle H: The biochemistry of desferrioxamine and its

relation to iron metabolism. Ann NY Acad Sci 1 19:758, 1964

22. B#{246}yumA: Isolation of mononuclear cells and granulocytes

from human blood. Scand J Clin Invest (Suppl) 97:77, 1968

23. Verbrugh HA, Peters R, Peterson PK, Verhoef J: Phagocyto-

sis and killing of staphylococci by human polymorphonuclear and

mononuclear leukocytes. J Clin Pathol 31:539, 1978

24. Aisen P: Some physicochemical aspects of iron metabolism,

in: Iron Metabolism. Ciba Foundation Symposium. Amsterdam,

Elsevier/Exerpta Medica/North Holland, 1977, p 1

25. Spiro TG, Allerton SE, Renner J, Terzis A, Bils R, Saltman

P: The hydrolytic polymerization of iron (III). J Am Chem Soc

88:2713, 1966

26. Dwyer FP: Enzyme-metal ion activation and catalytic phe-

nomena with metal complexes, in Dwyer FP, Mellor DP (eds):

Chelating Agents and Metal Chelates. New York, Academic, 1964,

p335

27. Mills EL, Rholl KS, Quie PG: X-linked inheritance in

females with chronic granulomatous disease. J Clin Invest 66:332,

1980

28. Dixon WJ, Massey Fi: Introduction to Statistical Analysis.

New York, McGraw-Hill, 1969, p 150

29. Klebanoff Si: Oxygen metabolism and the toxic properties of

phagocytes. Ann Intern Med 93:480, 1980

30. Cheson BD, Christensen RL, Sperling R, Kohler BE, Babior

BM: The origin of the chemiluminescence of phagocytosing granulo-

cytes. i Clin Invest 58:789, 1976

3 1 . Allen RC, Loose LD: Phagocytic activation of a luminol-

dependent chemiluminescence in rabbit alveolar and peritoneal

macrophages. Biochem Biophys Res Commun 69:245, 1976

32. McCord JM, Fridovich E: Superoxide dismutase. An

enzymic function for erythrocuprein (hemocuprein). J Biol Chem

244: 6049, 1969

33. Chandra RK: Reduced bactericidal capacity of polymorphs

in iron deficiency. Arch Dis Child 48:864, 1973

34. Chandra RK, Saraya AK: Impaired immunocompetence

associated with iron deficiency. J Pediatr 86:899, 1975

35. MacDougall LG, Anderson R, McNab GM, Katz J: Immune

response in iron-deficient children. Impaired cellular defense mecha-

nisms with altered humoral components. J Pediatr 86:833, 1975

36. Srikantia SG, Prasad iS, Bhaskaram C, Krishnamachari

KAVR: Anaemia and immuno response. Lancet I :1307, 1976

37. Prasad iS: Leukocyte function in iron-deficiency anemia. Am

i Clin Nutr 32:550, 1979

38. Gladstone GP, Walton E: The effect of iron and haematin on

the killing of staphylococci by rabbit polymorphs. Br J Exp Pathol

52:452, 1971

For personal use only.on April 12, 2019. by guest www.bloodjournal.orgFrom

720 VAN ASBECK ET AL.

39. Kaplan SS, Quie PQ, Basford RE: Effect of iron on leukocyte

function: lnactivation of H202 by iron. Infect Immun 12:303, 1975

40. Ward PA, Goldschmidt P. Greene ND: Suppressive effects of

metal salts on leukocyte and fibroblastic function. J Reticuloendo-

thelSoc 18:313, 1975

41. Fong K-L, McCay PB, Poyer JL, Keele BB, Misra H:

Evidence that peroxidation of lysosomal membranes is initiated by

hydroxyl free radicals produced during flavin enzyme activity. i Biol

Chem 218:7792, 1973

42. Noguchi T, Nakano M: Effect of ferrous ions on microsomal

phospholipid peroxidation and related light emission. Biochim Bio-

phys Acta 369:446, 1974

43. Gutteridge JMC, Rowley DA, Halliwell B: Superoxide-

dependent formation of hydroxyl radicals in the presence of iron

salts. Detection of “free” iron in biological systems by using bleomy-

cm-dependent degradation of DNA. Biochem J 199:263, 1981

44. Heys AD, Dormandy TL: Lipid peroxidation in iron-

overloaded spleens. Clin Sci 60:295, 1981

45. Floyd RA: DNA-ferrous iron catalyzed hydroxyl free radical

formation from hydrogen peroxide. Biochem Biophys Res Commun

99:1209, 1981

46. Blake DR. Hall ND, Bacon PA, Dieppe PA, Halliwell B,

Gutteridge JMC: The importance of iron in rheumatoid disease.

Lancet 2:1 142, 1981

47. Cederbaum Al, Qureshi A: Role of catalase and hydroxyl

radicals in the oxidation of methanol by rat liver microsomes.

Biochem Pharmacol 31:329, 1982

48. Aisen P: Current concepts in iron metabolism. Clin Haematol

11:241, 1982

49. Neta P. Dorfman LM: Pulse radiolysis studies. XIII. Rate

constants for the reaction of hydroxyl radicals with aromatic com-

pounds in aqueous solutions. Adv Chem Sci 8 1 :222, 1968

50. Halliwell B: An attempt to demonstrate a reaction between

superoxide and hydrogen peroxide. FEBS Lett 72:8, 1976

51. Tauber Al, Babior BM: Evidence for hydroxyl radical pro-

duction by human neutrophils. J Clin Invest 60:374, 1977

52. Weiss Si, Rustagi PK, LoBuglio AF: Human granulocyte

generation of hydroxyl radical. J Exp Med 147:316, 1978

53. Repine JE, Eaton JW, Anders MW, Hoidal JR. Fox RB:

Generation of hydroxyl radical by enzymes, chemicals, and human

phagocytes in vitro. Detection with the anti-inflammatory agent,

dimethyl sulfoxide. i Clin Invest 64:1642, 1979

54. Rosen H, Klebanoff Si: Hydroxyl radical generation by

polymorphonuclear leukocytes measured by electron spin resonance

spectroscopy. i Clin Invest 64:1725, 1979

55. Bock iL, Lang G: M#{246}ssbauer spectroscopy of iron chelated by

deferoxamine. Biochim Biophys Acta 264:145, 1972

56. Martell AE, Gustafson R, Chaberek 5: Metal chelate com-

pounds in homogeneous aqueous catalysis, in Farkas E (ed):

Advances in Catalysis IX. New York, Academic, 1957, p 319

57. Harris DC, Aisen P: Facilitation of Fe(II) autoxidation by

Fe(III) complexing agents. Biochim Biophys Acta 329:156, 1973

58. Schwarzenbach G, Schwarzenbach K: Hydroxamatkomplexe

I. Die Stabilit#{228}t der Eisen(I11)-Komplexe einfacher Hydroxam-

s#{227}urenund des Ferrioxamins B. Helv Chim Acta 46:1390, 1963

59. Goodwin iF, Whitten CF: Chelation of ferrous sulphate

solutions by desferrioxamine B. Nature 205:281, 1965

60. Rosen H, Klebanoff SJ: Role of iron and ethylenediamine-

tetraacetic acid in the bactericidal activity of a superoxide anion-

generating system. Arch Biochem Biophys 208:512, 1981

61. Fischer Li, Hamburger SA: Inhibition of alloxan action in

isolated pancreatic islets by superoxide dismutase, catalase and a

metal chelator. Diabetes 29:213, 1980

62. Ishibashi F, Howards BV: Alloxan and H202 action on

glucose metabolism in cultured fibroblasts. Generation of oxygen

containing free radicals as a mechanism of alloxan action. i BioI

Chem 256:12,134, 1981

63. Grankvist K, Marklund 5, Sehlin i, T#{228}ljedal I-B: Superoxide

dismutase, catalase and scavengers of hydroxyl radical protect

against the toxic action of alloxan on pancreatic islet cells in vitro.

Biochemi 182:17, 1979

64. Warner RC, Weber I: The cupric and ferric citrate complex-

Cs. J Am Chem Soc 75:5086, 1953

65. Lind MD, Hamor Mi, Hamor TA, Hoard JL: Sterochemis-

try of ethylenediaminetetraacetate complexes. morgan Chem 3:34,

196466. Martell AE: The design and synthesis of chelating agents, in

Martell AE, Anderson WF, Badman DG (eds): Development of Iron

Chelators for Clinical Use. New York, Elsevier/North Holland,

1981, p 67

67. Gutteridge JMC, Paterson 5K, Segal AW, Halliwell B:

Inhibition of lipid peroxidation by the iron-binding protein lactofer-

rin. Biochem i 199:259, 1981

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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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