sevier.com/locate/parint

Parasitology Internationa

Leukocyte activation by malarial pigment

Nguyen Tien Huy a, Dai Thi Xuan Trang a, Tohru Kariu a,b, Motohiro Sasai a, Katsuya Saida a,

Shigeharu Harada a, Kaeko Kamei a,*

a Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japanb Venture Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Received 7 September 2005; accepted 11 October 2005

Available online 28 November 2005

Abstract

Malarial pigment, a unique hemozoin crystal composed of unit cells of heme dimers, is present in large amounts in circulating monocytes and

neutrophils and can persist unchanged in macrophages for several months. In the present study, we investigated the effect of hemozoin not only on

macrophages, but also on neutrophils. We used h-hematin (BH), a chemically synthetic crystal structurally identical to hemozoin, for these studies.

In vitro, BH up-regulated the expression of tumor necrosis factor-a in whole blood and in isolated peritoneal macrophages, indicating that

hemozoin is able to stimulate monocytes. BH stimulated murine peritoneal neutrophils to express macrophage inflammatory protein-2 (MIP-2), a

homologue of human interleukin-8 that is used as a marker of neutrophil activation. Injecting BH into the peritoneal cavity resulted in a dose-

dependent migration of neutrophils and a high level of myeloperoxidase activity of peritoneal cells. Finally, BH directly induced neutrophil

chemotaxis in vitro. Taken together, these results suggest that the malarial pigment hemozoin can activate leukocytes and may participate in the

pathology of severe malaria.

D 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cellular response; Cytokines/tumor necrosis factor; Monocytes/macrophages; Neutrophils; Malaria

1. Introduction

Malaria is one of the most common diseases in tropical

countries. Each year, there are 300 million new malaria

infections and millions of deaths due to malaria worldwide.

Fast spreading resistance to current quinoline antimalarials has

made malaria a major global problem. Because a vaccine for

malaria is not available, it is necessary to study the molecular,

biochemical, and immunological aspects of malarial parasites

to develop vaccines and new antimalarials.

During development and proliferation in host erythrocytes,

the malarial parasite degrades hemoglobin for use as a major

source of amino acids. This is accompanied by the release of

free heme. Free heme is oxidatively active and toxic to both the

host cell and malarial parasites, and it causes parasite death.

Due to the absence of heme oxygenase, the parasite is unable to

1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.parint.2005.10.003

Abbreviations: BH, h-hematin; HZ, hemozoin; HTA-Br, hexadecyltri-

methylammonium bromide; i.p., intraperitoneally; MPO, myeloperoxidase.

* Corresponding author. Tel.: +81 75 724 7553; fax: +81 75 724 7541.

E-mail address: kame@kit.ac.jp (K. Kamei).

cleave heme into an open-chain tetrapyrrole, which is

necessary for cellular excretion [1].

To protect itself, the malarial parasite detoxifies free heme

via neutralization with histidine-rich protein 2 [2,3], degrada-

tion with reduced glutathione [4], or crystallization into

hemozoin (HZ), a water-insoluble malarial pigment produced

in the food vacuole [2]. As parasites mature and rupture, HZ

is ingested by phagocytes and accumulated in the reticuloen-

dothelial system of the host where it may persist unchanged

in macrophages for several months [5]. Until the 1990s, a

wide belief was that HZ is an inert catabolite of heme that

does not affect the underlying pathophysiological processes.

However, recently, it has been found that during malaria

infection, HZ loading severely impairs the function of

monocytes, including the generation of oxidative burst, the

reinitiation of phagocytosis, and the activation of protein

kinase C [5]. Other studies have also shown that phagocytosis

of opsonized HZ impairs the expression of the major

histocompatibility complex class II antigen, CD54, as well

as CD11c in human monocytes [5]. In contrast, other reports

have demonstrated that HZ induces monocytes/macrophages

l 55 (2006) 75 – 81

www.el

N.T. Huy et al. / Parasitology International 55 (2006) 75–8176

to produce tumor necrosis factor-a (TNF-a) and inflammatory

mediates [6,7], and enhances the immune response of den-

dritic cells and spleen cells [8,9].

Monocytes and neutrophils are always recruited to an area

of infection and inflammation. They are considered to be

important cells because they are capable of phagocytosis and

killing infected red blood cells via mechanisms that depend on

degradative enzymes, reactive oxygen, and nitrogen intermedi-

ates [10]. However, when over-activated, these cells produce

high levels of inflammatory cytokines, leading to severe

malaria [11,12]. Although the effect HZ in monocytes has

been examined, the interaction between HZ and neutrophils has

not been examined. In the current studies, we investigated the

ability of HZ to act as a stimulator of not only monocyte but

also neutrophil.

2. Materials and methods

2.1. Animals

Six-to ten-week-old male ddY mice (Japan SLC Company,

Hamamatsu, Japan), were used in all experiments. These mice

were bred in our own facilities under pathogen-free conditions,

and they were matched for body weight in each experiment. All

mice procedures were reviewed and approved by the animal

care and use committee of the Kyoto Institute of Technology

(Kyoto, Japan).

2.2. Synthesis of b-hematin (BH)

BH, a chemically synthetic crystal, was used in this study

because it is structurally [13] and biologically [14] identical to

HZ. BH was synthesized from heme using an acetic acid-

treatment described previously [15] with slight modifications.

Heme (Sigma, St. Louis, MO; 163 mg) was dissolved in 10

ml of 0.4 M NaOH. The solution was passed through a 0.2-

Am pore membrane filter to remove insoluble particles. After

adding 140 ml of 1 M acetate buffer (pH 4.5), the solution

was kept overnight at 80 -C. The solution was then

centrifuged for 30 min at 7000 �g, and the supernatant

was discarded. The pellet was suspended in 150 ml of 2.5%

sodium dodecyl sulfate (SDS) buffered with 0.1 M sodium

bicarbonate (pH 9.1) and then sonicated briefly. After shaking

at room temperature for at least 2 h to dissolve unreacted

heme, the remaining insoluble material was recovered by

centrifugation. This step was repeated 10 times followed by

three repeated washes with ethanol and then 5 times with

phosphate-buffered saline (PBS; 20 mM phosphate buffer [pH

7.4] containing 0.9% NaCl). The characteristics of purified

BH were confirmed by infrared spectroscopy as described

previously [16]. The purified of BH was suspended in PBS

for following experiments.

The BH concentration used in this paper was expressed as

micromolar heme after BH was depolymerized into heme.

Briefly, the SDS-insoluble pellet of BH was depolymerized and

dissolved completely by incubation for 2 h at room temperature

in 1 M NaOH–2% SDS. The heme concentration was

calculated from the absorbance at 400 nm with an extinction

coefficient of 105 as described previously [17].

Since monocytes and neutrophils are sensitive to endotoxin

contamination, extreme caution was taken to avoid contami-

nation, such as wearing gloves and using endotoxin-free

solutions. In addition, endotoxin was not detected (<5 pg/ml)

in synthesized BH solution, culture medium, Hanks solution by

a Limulus lysate test (Endospec-ST; Seikagaku, Tokyo, Japan).

2.3. Analysis of TNF-a expression in whole blood

Mouse blood was collected by cardiac puncture under ether

anesthesia, and 0.2-ml aliquots were placed in microcentrifuge

tubes. The blood samples were incubated for 2 h at 37 -C with

various concentrations of BH (7 to 80 AM), lipopolysaccharide

(LPS; 1 Ag/ml) as a positive control, or PBS as a negative

control. At the end of the incubation, total RNA was isolated,

and the expression of TNF-a mRNA was measured as

described below.

2.4. Analysis of TNF-a expression in murine peritoneal

macrophages

Mice were injected intraperitoneally (i.p.) with 1 ml of 4%

thioglycollate broth (Sigma). After four days, peritoneal

macrophages were collected following peritoneal lavage with

7 ml Ca/Mg-free Hanks solution (Sigma). The cells were

isolated by centrifugation for 5 min at 500 �g and then washed

twice with the same solution. Peritoneal macrophages were

suspended at 2�106/ml in RPMI 1640 containing 5% fetal

bovine serum and were allowed to adhere to tissue culture

plates for 1 h. The adherent cells, which consisted of >95%

viable peritoneal macrophages as judged by Trypan blue dye

exclusion, were then used for the analysis of BH-induced TNF-

a expression. Adherent macrophages were treated for 2 h at 37

-C with medium, LPS (1 Ag/ml), or BH (80 AM). Total cellular

RNA was then isolated, and the expression of TNF-a mRNA

was analyzed by semi-quantitative reverse transcription-poly-

merase chain reaction (RT-PCR).

2.5. Isolation of murine neutrophils

To obtain mouse neutrophils, 1 ml of 2% (wt/vol) casein

sodium (Wako Co., Ltd., Osaka, Japan) was injected i.p. into

the 6–10-week-old mice (n =2) as previously described [18].

Four hours after the injection, the mice were sacrificed using

diethyl ether. Seven milliliters of cold Ca/Mg-free Hanks

solution was injected i.p. into the mice, after which the fluids

were removed by aspiration. The cell suspension from the

abdomen was layered onto 5 ml Histopaque 1077 (Sigma).

The mixture was centrifuged at 500 �g for 30 min at room

temperature. The neutrophil pellet was washed once with Ca/

Mg-free Hanks solution, subsequently hemolyzed with

distilled water to remove erythrocytes and then washed twice

with the same solution. The purity and viability of the

isolated neutrophils were both greater than 99% and 90%,

respectively.

N.T. Huy et al. / Parasitology International 55 (2006) 75–81 77

2.6. Leukocyte viability

A dye exclusion assay was performed after a 1- or 4-

h incubation of leukocytes with 80 AM BH. Trypan blue (2%)

was added, and 100 cells were counted in a microscope. Blue-

stained cells were counted as nonviable. The viabilities of

neutrophils and macrophages were found to be similar in the

presence or absence of BH.

2.7. Analysis of MIP-2 expression in peritoneal neutrophils

Isolated neutrophils were suspended at 5�106/ml in RPMI

1640 containing 5% fetal bovine serum and then were

incubated for 2 h at 37 -C with medium, LPS (1 Ag/ml), or

BH (80 AM). Total cellular RNA was then isolated, and the

expression of murine macrophage inflammatory protein 2

(MIP-2) was assessed by RT-PCR.

2.8. Reverse transcription and semi-quantitative PCR

Total cellular RNA was prepared using the Trizol reagent

according to the manufacturer’s instructions (Life Technolo-

gies, Tokyo, Japan). Each RNA sample was quantified by

spectrophotometry. The A260 /A280 values of the RNA samples

were all greater than 1.7.

Using a Superscript one-step RT-PCR kit (Life Technologies,

Tokyo, Japan), we subjected 1 Ag of total RNA to the following

sequence: 30min at 50 -C; 5min at 94 -C; repeated cycles of 30 sat 94 -C, 30 s at 55 -C for TNF-a (60 -C for MIP-2 and h-actin),and 40 s at 72 -C; and 5 min at 72 -C for the final extension. The

primers for the housekeeping gene h-actin and for TNF-a and

MIP-2 were purchased from Qiagen (Osaka, Japan) and the

nucleotide sequences were as follows: for h-actin (347-bp

amplified fragment), 5V-TGG AAT CCT GTG GCATCC ATG

AAA-3V (sense) and 5V-TAA AAC GCA GCT CAG TAA CAG

TCC G-3V (antisense) [19]; for TNF-a (228-bp amplified

fragment), 5V-ATG AGC ACA GAA AGC ATG ATC-3V (sense)and 5V-GTC TGG GCC ATAGAA C-3V (antisense) [19];and forMIP-2 (302-bp amplified fragment), 5V-ATG GCC CCT CCC

ACCTGCCG-3V (sense) and 5V-TCAGTTAGCCTTGCCTTT

GT-3V (antisense) [20]. To minimize the problems associated

with DNA contamination, the primer pairs spanned at least one

intron in the corresponding genomic DNA. In addition, negative

controls were performed by omitting the RT step or the cDNA

template from PCR amplification, while positive RNA controls

were performed previously to confirm specificity of primer pairs

(data not shown). The amplified products were detected by 2%

agarose gel electrophoresis followed by ethidium bromide

staining. Gel photos were scanned and the intensity of signals

was quantified using the NIH Image 1.62 program (Research

Services Branch, National Institutes of Health). The levels of h-actin were used for normalization.

2.9. Measurement of MIP-2 production

Isolated neutrophils (105 cells) were suspended in 200 Al ofRPMI 1640 containing 5% fetal bovine serum and were

incubated for 5 h at 37 -C with medium, LPS (1 Ag/ml), or BH

(80 AM). Supernatants were harvested, and MIP-2 release was

assessed using a MIP-2 enzyme-linked immunosorbent assay

(ELISA) kit (R&D Systems, Osaka, Japan) according to the

manufacturer’s instructions.

2.10. Effect of BH on neutrophil migration into the peritoneal

cavity

PBS (100 Al) with or without 80 AM BH was injected i.p.

into healthy mice. For positive controls, the same volume of

2% casein was injected. After 4 h, animals were killed by

diethyl ether, and 6 ml of PBS containing 1% bovine serum

albumin (Sigma) was injected into the peritoneal cavities, after

which cells were recovered by collecting the peritoneal fluid

(approximately 4 ml). A 20-Al aliquot of the recovered fluid

was diluted 20-fold with Turk’s solution (0.01% crystal violet

in 3% acetic acid), and the total number of cells was counted

with a hemocytometer. For differential counting of neutrophils

and mononuclear cells, the collected cells were applied to a

glass slide and stained with Giemsa. Two hundred cells were

counted with an optical microscope using a 100� immersion

objective.

Alternatively, the level of neutrophil influx after 4 h was

evaluated in peritoneal cells using the myeloperoxidase (MPO)

activity, which is a marker for the presence and activity of

neutrophils in inflammatory sites [21]. Results are expressed as

meansTSEM of the number of cells per peritoneal cavity from

nine different animals.

2.11. MPO assay for peritoneal cells

MPO activity of peritoneal exudates cells was measured as

previously described [22] with a slight modification. Briefly,

suspensions of collected peritoneal cells (0.5 ml) were

centrifuged for 5 min at 500 �g. The resulting cell pellets

were solubilized in 1.0 ml of ice-cold 0.5% hexadecyltri-

methylammonium bromide (HTA-Br; Sigma) in 50 mM

phosphate buffer (pH 6.0). Aliquots (50 Al) of the supernatant

fractions from HTA-Br solubilized cell extracts were assayed

in 96-well plates for MPO activity in 150 Al of reaction

buffer (50 mM phosphate [pH 6.0], 0.25 mM HTA-Br, 300

AM hydrogen peroxide, and 1.5 mM o-dianisidine [Sigma]).

The MPO activities were determined by measuring the

change in absorbance at 450 nm at room temperature over

a 15-min period using a MTP-120 microplate reader (Corona

Electric Co., Ibaragi, Japan). Enzymatic MPO activity was

routinely confirmed by its inhibition (>95%) with sodium

azide (10 mM).

2.12. In vitro chemotaxis assay

In vitro chemotaxis was performed in a 96-well micro-

chemotaxis cell containing a polycarbonate 3-Am pore filter

(Kurabo, Osaka, Japan) and using a photometric assay for the

quantitative measurement of neutrophil migration as described

previously [23]. Briefly, suspensions of neutrophils in RPMI

0

0.1

0.2

0.3

0.4

0 7 21 63 189

BH (µM)

Den

sity

arb

itrar

y un

its

Fig. 2. Dose response of BH-induced expression of TNF-a mRNA in whole

blood. TNF-a mRNA level was expressed as arbitrary density units of ethidium

bromide staining from the RT-PCR for TNF-a mRNA as described in Fig. 1

Results are representative of two independent experiments.

N.T. Huy et al. / Parasitology International 55 (2006) 75–8178

1640 medium (60 Al) were added to the upper compartments in

triplicate and at a final concentration of 1.0�106 cells/ml. One

hundred microliters of various concentration of BH, N-formyl-

Met-Leu-Phe (fMLP; 0.2 AM; Sigma) as a positive control, or

medium alone as a negative control was added to the lower

compartment. After incubation for 60 min at 37 -C in a

humidified incubator in the presence of 5% CO2, the cells in

the lower compartments were collected by centrifugation and

solubilized in 1.0 ml of ice-cold 0.5% HTA-Br in 50 mM

phosphate buffer (pH 6.0). Their MPO activity was then

measured, and the results are expressed as the chemotaxis

index (mean MPO activity in the presence of fMLP or BH

divided by the mean MPO activity in the presence of medium)

TSEM from two separate experiments.

2.13. Statistical analysis

Results are presented as meanTSEM. Student’s t-test (one-

tailed) for unpaired samples was used to compare the means. A

probability value of P <0.05 was considered significant.

Correlations between the MPO activities and the number of

neutrophils that migrated into the peritoneal cavity were

assessed using the Spearman correlation coefficient.

3. Results

3.1. BH up-regulates TNF-a expression in whole blood samples

Inflammation is considered to play an important role in

severe malaria [11]. To test the hypothesis that BH might serve

as a pro-inflammatory molecule, we studied ability of BH to

activate monocytes and neutrophils.

To examine the effect of BH on monocytes, whole blood

was incubated with BH, and TNF-a mRNA levels were

examined by RT-PCR (Fig. 1). The results indicated that 80

AM BH significantly increased the expression of TNF-a

compared to the negative control. TNF-a mRNA levels,

expressed as arbitrary density units of ethidium bromide

staining, were 2.2-fold higher in BH-treated cells than in the

negative control. In addition, the level of TNF-a expression

positively correlated with the BH concentration between 7

and 189 AM (Fig. 2). This stimulatory effect was detected at a

0

0.1

0.2

0.3

0.4

PBS LPS BH

Den

sity

arb

itrar

y un

its

Fig. 1. Analysis of BH-induced gene expression in whole blood by RT-PCR.

The intensity of the signal from the amplified products was quantified and the

TNF-a signal was normalized by the h-actin signal. Results are expressed as

meansTSEM (n =3). LPS and BH significantly increased the level of TNF-a

mRNA compared with the negative control (PBS). *P <0.001 and **P <0.01

vs. negative control.

Den

sity

arb

itrar

y un

its

Medium LPS BH0

0.3

0.6

0.9

1.2

1.5

Fig. 3. Induction of TNF-a expression in murine peritoneal macrophages by

BH. The intensity of the signal from the amplified products was quantified, and

the TNF-a signal was normalized by the h-actin signal. Results are expressed

as meansTSEM (n =3). *P <0.05 vs. negative control.

.

concentration of BH as low as 7 AM and reached an almost 3-

fold induction at 160 AM.

3.2. BH induces TNF-a expression in murine peritoneal

macrophages

To further investigate the ability of BH to activate

monocytes, we evaluated the TNF-a mRNA levels in isolated

peritoneal macrophages by RT-PCR. Incubation of macro-

phages with 80 AM BH resulted in a significant increase

(P <0.05) in TNF-a expression compared with the negative

control (Fig. 3). As a positive control of TNF-a expression,

macrophages were incubated with LPS, which caused the

expected increase in mRNA. These results, together with the

findings in whole blood, demonstrate that BH can activate

monocytes to enhance the expression of TNF-a, a well-

established pro-inflammatory cytokine.

3.3. BH induces MIP-2 expression and production in murine

peritoneal neutrophils

Another type of cell from the immune system that contributes

to the pathology of malaria is the neutrophil [12]. Neutrophils are

the first cells attracted to a site of infection or inflammation.

Interleukin (IL)-8, one of the most important chemoattractants for

neutrophils, has been used as an indicator of neutrophil activation

[24]. In this experiment, we used the expression of MIP-2, a

mouse homologue of human IL-8, as a marker of murine

MP

O a

ctiv

ity(A

bsor

banc

e/15

min

)

0

0.4

0.8

1.2

1.6

2

PBS Casein

BH (µM)

0.5 5 50 500

Fig. 5. BH dose-dependently stimulates neutrophil migration into the peritoneal

cavity of mice. The MPO activity of cells that migrated into the peritoneal

cavity are expressed as the meanTSEM (n =9) of the increase in absorbance at

450 nm over a 15-min period.

N.T. Huy et al. / Parasitology International 55 (2006) 75–81 79

neutrophil activation. Incubation of murine neutrophils with LPS

as positive control significantly increased MIP-2 expression

(P<0.05). When incubated with 80 AMBH, the neutrophils also

significantly up-regulated the expression of MIP-2 (P<0.05),

suggesting that BH activates neutrophils (Fig. 4A).

To confirm the up-regulation of MIP-2 expression in

neutrophils by BH, the MIP-2 released by the isolated

peritoneal neutrophils was measured using an ELISA kit.

LPS and BH induced 34- and 27-fold increases in released

MIP-2 protein, respectively (Fig. 4B).

3.4. BH induces neutrophil migration to the peritoneal cavity

The activation of neutrophils by BH was further explored by

studying neutrophil migration in vivo. Mice were injected i.p.

with BH, and, after 4 h, cells were recovered from the

peritoneal fluid and counted. BH significantly induced neutro-

phil influx but did not cause the migration of mononuclear cells

(data not shown) into the peritoneal cavity. This induction of

neutrophil migration was dose-dependent. The stimulatory

effect of BH was evident at 5 AM, and the number of migrating

neutrophils reached a maximum (4-fold) at 50 AM.

As suggested previously [21], we used the neutrophil enzyme

MPO as a marker for neutrophils. First, we confirmed that this

assay can be used by comparing the relationship between the

MPO activities of collected peritoneal cells and the number of

neutrophils that migrated into the peritoneal cavity using all of

the samples, including mice injected with BH, PBS (negative

control), or casein (positive control). For induction by BH, the

Spearman rank coefficient (r) was 0.86 (n =54; P <0.001),

0

0.5

1

1.5

2

Medium LPS BH

Den

sity

arb

itrar

y un

its

Medium LPS BH

400

500

300

200

100

0

A

B

MIP

-2 p

rodu

ctio

n (p

g/m

l)

Fig. 4. Induction of MIP-2 expression in murine peritoneal neutrophils by BH.

(A) The intensity of the signal from the amplified products are expressed as

meansTSEM (n =3). MIP-2 expression in positive control (LPS) or BH-treated

cells was significantly higher than that in the negative control (medium).

*P <0.05 vs. negative control. (B) MIP-2 released in the cell culture

supernatant are expressed as the meanTSEM for three experiments. *P <0.01

and **P <0.05 vs. negative control.

indicating a good positive correlation between the MPO activity

and the number of neutrophils that migrated into the peritoneal

cavity over a wide range. There was no change in the Spearman

rank coefficient when the negative and positive controls were

excluded from the analysis (r =0.81 [n =36]; P <0.001),

suggesting that accumulation of neutrophils by BH leads to an

increase in the level of MPO in peritoneal cells. These results

further suggest that MPO is a good indicator for evaluating the

number of neutrophils that migrated into the peritoneal cavity.

Given this good correlation, we used the MPO activity to

follow the effect of different stimuli on neutrophil accumulation

in the peritoneal cavity. The data in Fig. 5 represent the average

changes in absorbance over 15 min for nine mice in each group.

The average MPO activity of the collected peritoneal cells

injected with 5 AM BH was 2.5-fold higher than the average of

the negative control (PBS). The MPO level reached maximum

at 50 AM BH. This level was almost as high as for the positive

control (casein), indicating that BH causes a significant

induction of neutrophil influx into the peritoneal cavity.

3.5. BH induces neutrophil chemotaxis in vitro

To directly demonstrate the ability of BH to elicit neutrophil

migration, we used an in vitro assay of chemotaxis performed

BH

0

20

40

60

80

Medium fMLP 5 µM 50 µM

Che

mot

axis

Inde

x

Fig. 6. Induction of neutrophil chemotaxis by BH in vitro. The chemotaxis

index indicates the mean of MPO activity in the presence of stimulators divided

by the mean of MPO activity in the presence of medium. The results represent

the meansTSEM (n =6). *P <0.0001 compared to the negative control.

N.T. Huy et al. / Parasitology International 55 (2006) 75–8180

in 96-well plates. As illustrated in Fig. 6, BH significantly

induced the migration of neutrophils in vitro compared with

random migration in the presence of RPMI medium. The

activity in the presence of 50 AM BH was higher than with 0.2

AM fMLP, an established neutrophil chemoattractant that we

used as a positive control.

4. Discussion

It is estimated that 200 Amol of HZ is released into the

circulation of a P. falciparum infected patient [14]. Since brain

capillaries are filled with infected erythrocytes in cerebral

malaria, the level of HZ could locally reach 100 AM in brain

capillaries [25].

The severity of malaria correlates with the level of TNF-a

[11], neutrophil activation [12], and the level of malarial HZ

loaded in circulating monocytes and neutrophils [26]. TNF-a is

a major mediator of the acute inflammatory response, including

the induction of cellular functions in leukocytes. This cytokine

is important in reducing parasitemia at early production [27].

However, the over-expression of TNF-a leads to severe disease

[11]. Excess TNF-a production is also important in the

activation of endothelial cell expression of ligands for adhesion

molecules, which results in the subsequent sequestration of

infected erythrocytes, leukocytes, and platelets and leads to

microvascular obstruction [28].

In malarial infections, high levels of TNF-a production are

thought to arise from activated macrophages at the time of

schizont rupture [29]. This is thought to be due to the

stimulation of TNF-a production by infected red blood cells,

some parasite molecules such as glycosylphosphatidylinositol

[30], and specific anti-malaria IgE-containing immune com-

plexes [31]. In this study, we found that HZ induces a high

level of TNF-a expression not only in whole blood but also in

macrophage cultures.

The whole blood assay that we used to study BH-induced

TNF-a expression has multiple advantages: first, in this

system, cell populations and serum factors can interact with

each other and with BH; second, the cells are not activated

during isolation procedures [32]; and third, only a small sample

size is required, which is important because it is difficult to

collect large amounts of blood from a mouse. Our findings in

the whole blood system and in macrophage cultures, coupled

with previous reports [6], indicate that malarial HZ may be a

novel TNF-a inducer.

Most studies have focused on the interaction of HZ and

monocytes and macrophages, and little is known about the

interaction of HZ and neutrophils. Neutrophils are the first cell

type to be recruited to sites of infection and inflammation.

They are considered to be terminally differentiated effector

cells, capable of phagocytosis as well as the killing of infected

red blood cells via mechanisms involving degradative

enzymes, reactive oxygen, and nitrogen intermediates [10].

Despite these beneficial effects of neutrophils, neutrophil

hyperactivity is well known to cause multiple organ failure in

circulatory shock and to be related to the severity of malaria

[12]. Indeed, depletion of neutrophils prevents the development

of cerebral malaria in experimental mouse model. The

hyperactivity of neutrophils in Plasmodium-infected indivi-

duals appears to lead to an increased production of inflamma-

tory cytokines [12], the release of lysosomal enzymes, and the

amplification of reactive oxygen species production. The

consequence may be an enhancement of vascular damage

[33], which may contribute to severe malaria.

Very little work has been done to determine the conse-

quences of the interaction of parasite molecules with intact

neutrophils. Neutrophils might be stimulated either directly by

the parasites or by cytokines or other mediators produced

during the malarial attack. We have studied the ability of BH to

activate neutrophils by measuring MIP-2 expression and

production and the in vivo and vitro migration of neutrophils.

The increase in MIP-2 expression suggests that BH activates

neutrophils because MIP-2 is homologue of human IL-8 [34],

which is produced by activated neutrophils [35], and is a

chemokine that can attract neutrophils to inflammatory sites.

Furthermore, high production of MIP-2 correlates with the

severity and outcome of malaria [36].

MPO activity has been widely used as another marker of

neutrophil activation, for example in bronchial fluid and the

pleural and peritoneal cavities [21]. Our demonstration of MPO

activity in the peritoneal cavity following i.p. injection of BH is

a good indicator of neutrophil influx. The data show a good

positive correlation between the MPO activity and the number

of neutrophils that migrated into the peritoneal cavity over a

wide range. This indicates that the accumulation of neutrophils

by BH leads to an increase in the MPO level in peritoneal cells.

These results suggest that MPO activity is a good measure for

evaluating the number of neutrophils that have migrated into

the peritoneal cavity.

Increased neutrophil influx into the lung is related to the

severity of malaria in primates [37]. Our findings suggest

that HZ, which is structurally identical to BH, may be a

good candidate for inducing the high level of neutrophil

migration into an inflammatory site. This probably is

mediated by the release of MIP-2, a neutrophil chemotactic

cytokine, which may result in further neutrophil recruitment.

HZ has also been shown to increase the expression of pro-

inflammatory cytokines in mice after i.v. or air pouch

injection of BH [7], and this effect seems to involve an

interaction with neutrophils. Although the role of the HZ in

the outcome of malaria is not well understood, the presence

of HZ in the circulation monocytes and neutrophils is

associated with mortality and morbidity [26]. Understanding

the effect of HZ may allow blocking its signaling pathways,

which could improve the outcome of treatments for malaria.

It has been reported that the Toll-like receptor 9 is

responsible for hemozoin activities in activation of dendritic

and spleen cells [8,9], and it would be the case in HZ-

neutrophil interaction.

Acknowledgments

This work was supported in part by a Grand-in-Aid for

Scientific Research (C) and a JSPS postdoctoral fellowship to

N.T. Huy et al. / Parasitology International 55 (2006) 75–81 81

Huy NT from the Ministry of Education, Science, Sports, and

Culture.

References

[1] Eckman JR, Modler S, Eaton JW, Berger E, Engel RR. Host heme

catabolism in drug-sensitive and drug-resistant malaria. J Lab Clin Med

1977;90:767–70.

[2] Sullivan Jr DJ, Gluzman IY, Goldberg DE. Plasmodium hemozoin

formation mediated by histidine-rich proteins. Science 1996;271:219–22.

[3] Huy NT, Serada S, Trang DT, Takano R, Kondo Y, Kanaori K, et al.

Neutralization of toxic heme by Plasmodium falciparum histidine-rich

protein 2. J Biochem (Tokyo) 2003;133:693–8.

[4] Huy NT, Kamei K, Yamamoto T, Kondo Y, Kanaori K, Takano R, et al.

Clotrimazole binds to heme and enhances heme-dependent hemolysis:

proposed antimalarial mechanism of clotrimazole. J Biol Chem 2002;277:

4152–8.

[5] Schwarzer E, Alessio M, Ulliers D, Arese P. Phagocytosis of the malarial

pigment, hemozoin, impairs expression of major histocompatibility

complex class II antigen, CD54, and CD11c in human monocytes. Infect

Immun 1998;66:1601–6.

[6] Biswas S, Karmarkar MG, Sharma YD. Antibodies detected against

Plasmodium falciparum haemozoin with inhibitory properties to cytokine

production. FEMS Microbiol Lett 2001;194:175–9.

[7] Jaramillo M, Plante I, Ouellet N, Vandal K, Tessier PA, Olivier M.

Hemozoin-inducible proinflammatory events in vivo: potential role in

malaria infection. J Immunol 2004;172:3101–10.

[8] Coban C, Ishii KJ, Sullivan DJ, Kumar N. Purified malaria pigment

(hemozoin) enhances dendritic cell maturation and modulates the

isotype of antibodies induced by a DNA vaccine. Infect Immun 2002;

70:3939–43.

[9] Coban C, Ishii KJ, Kawai T, Hemmi H, Sato S, Uematsu S, et al. Toll-like

receptor 9 mediates innate immune activation by the malaria pigment

hemozoin. J Exp Med 2005;201:19–25.

[10] Kumaratilake LM, Ferrante A, Robinson BS, Jaeger T, Poulos A.

Enhancement of neutrophil-mediated killing of Plasmodium falciparum

asexual blood forms by fatty acids: importance of fatty acid structure.

Infect Immun 1997;65:4152–7.

[11] Day NP, Hien TT, Schollaardt T, Loc PP, Chuong LV, Chau TT, et al. The

prognostic and pathophysiologic role of pro- and antiinflammatory

cytokines in severe malaria. J Infect Dis 1999;180:1288–97.

[12] Chen L, Zhang Z, Sendo F. Neutrophils play a critical role in the

pathogenesis of experimental cerebral malaria. Clin Exp Immunol

2000;120:125–33.

[13] Pagola S, Stephens PW, Bohle DS, Kosar AD, Madsen SK. The structure

of malaria pigment beta-haematin. Nature 2000;404:307–10.

[14] Sherry BA, Alava G, Tracey KJ, Martiney J, Cerami A, Slater AF.

Malaria-specific metabolite hemozoin mediates the release of several

potent endogenous pyrogens (TNF, MIP-1 alpha, and MIP-1 beta) in vitro,

and altered thermoregulation in vivo. J Inflamm 1995;45:85–96.

[15] Slater AF, Swiggard WJ, Orton BR, Flitter WD, Goldberg DE, Cerami A,

et al. An iron-carboxylate bond links the heme units of malaria pigment.

Proc Natl Acad Sci U S A 1991;88:325–9.

[16] Taramelli D, Recalcati S, Basilico N, Olliaro P, Cairo G. Macrophage

preconditioning with synthetic malaria pigment reduces cytokine

production via heme iron-dependent oxidative stress. Lab Invest

2000;80:1781–8.

[17] Sullivan Jr DJ, Gluzman IY, Russell DG, Goldberg DE. On the molecular

mechanism of chloroquine’s antimalarial action. Proc Natl Acad Sci U S A

1996;93:11865–70.

[18] Parker KP, Benjamin WR, Kaffka KL, Kilian PL. Presence of IL-1

receptors on human and murine neutrophils. Relevance to IL-1-mediated

effects in inflammation. J Immunol 1989;142:537–42.

[19] Bang R, Sass G, Kiemer AK, Vollmar AM, Neuhuber WL, Tiegs G.

Neurokinin-1 receptor antagonists CP-96,345 and L-733,060 protect

mice from cytokine-mediated liver injury. J Pharmacol Exp Ther 2003;

305:31–9.

[20] Walzog B, Weinmann P, Jeblonski F, Scharffetter-Kochanek K, Bommert

P, Gaehtgens P. A role for beta(2) integrins (CD11/CD18) in the regulation

of cytokine gene expression of polymorphonuclear neutrophils during the

inflammatory response. FASEB J 1999;13:1855–65.

[21] Nys M, Deby-Dupont G, Habraken Y, Legrand-Poels S, Kohnen S,

Ledoux D, et al. Bronchoalveolar lavage fluids of ventilated patients with

acute lung injury activate NF-kappaB in alveolar epithelial cell line: role

of reactive oxygen/nitrogen species and cytokines. Nitric Oxide 2003;9:

33–43.

[22] Graff G, Gamache DA, Brady MT, Spellman JM, Yanni JM. Improved

myeloperoxidase assay for quantitation of neutrophil influx in a rat model

of endotoxin-induced uveitis. J Pharmacol Toxicol Methods 1998;39:

169–78.

[23] Junger WG, Cardoza TA, Liu FC, Hoyt DB, Goodwin R. Improved rapid

photometric assay for quantitative measurement of PMN migration. J

Immunol Methods 1993;160:73–9.

[24] Frossard JL, Hadengue A, Pastor CM. New serum markers for the

detection of severe acute pancreatitis in humans. Am J Respir Crit Care

Med 2001;164:162–70.

[25] Newton CR, Taylor TE, Whitten RO. Pathophysiology of fatal falciparum

malaria in African children. Am J Trop Med Hyg 1998;58:673–83.

[26] Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ. Intraleucocytic

malaria pigment and prognosis in severe malaria. Trans R Soc Trop Med

Hyg 1995;89:200–4.

[27] Kremsner PG, Winkler S, Brandts C, Wildling E, Jenne L, Graninger W, et

al. Prediction of accelerated cure in Plasmodium falciparum malaria by

the elevated capacity of tumor necrosis factor production. Am J Trop Med

Hyg 1995;53:532–8.

[28] de Souza JB, Riley EM. Cerebral malaria: the contribution of studies in

animal models to our understanding of immunopathogenesis. Microbes

Infect 2002;4:291–300.

[29] Clark IA, Virelizier JL, Carswell EA, Wood PR. Possible importance of

macrophage-derived mediators in acute malaria. Infect Immun 1981;32:

1058–66.

[30] Schofield L, Hackett F. Signal transduction in host cells by a glycosylpho-

sphatidylinositol toxin of malaria parasites. J Exp Med 1993;177:145–53.

[31] Perlmann P, Perlmann H, ElGhazali G, Blomberg MT. IgE and tumor

necrosis factor in malaria infection. Immunol Lett 1999;65:29–33.

[32] De Groote D, Zangerle PF, Gevaert Y, Fassotte MF, Beguin Y, Noizat-

Pirenne F, et al. Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-

6, IL-2, IFN-gamma and GM-CSF) in whole blood: I. Comparison with

isolated PBMC stimulation. Cytokine 1992;4:239–48.

[33] Hemmer CJ, Bierhaus A, von Riedesel J, Gabat S, Liliensiek B, Pitronik P,

et al. Elevated thrombomodulin plasma levels as a result of endothelial

involvement in Plasmodium falciparum malaria. Thromb Haemost 1994;

72:457–64.

[34] Tekamp-Olson P, Gallegos C, Bauer D, McClain J, Sherry B, Fabre M,

et al. Cloning and characterization of cDNAs for murine macrophage

inflammatory protein 2 and its human homologues. J Exp Med 1990;

172:911–9.

[35] Strieter RM, Kasahara K, Allen RM, Standiford TJ, Rolfe MW, Becker

FS, et al. Cytokine-induced neutrophil-derived interleukin-8. Am J Pathol

1992;141:397–407.

[36] Burgmann H, Hollenstein U, Wenisch C, Thalhammer F, Looareesuwan S,

Graninger W. Serum concentrations of MIP-1 alpha and interleukin-8 in

patients suffering from acute Plasmodium falciparum malaria. Clin

Immunol Immunopathol 1995;76:32–6.

[37] Ozwara H, Langermans JA, Maamun J, Farah IO, Yole DS, Mwenda JM,

et al. Experimental infection of the olive baboon (Paplio anubis) with

Plasmodium knowlesi: severe disease accompanied by cerebral involve-

ment. Am J Trop Med Hyg 2003;69:188–94.