Yonago Acta medica 2009;52:37–46

Suppression of Chemokine Gene Expression and Productionin LPS-Stimulated Macrophages by a 130 kDa Glycoproteinfrom Plerocercoids of Spirometra erinaceieuropaei
Haruyo Matsuura, Asumi Tomioku, Satoko Kono, Sayuri Tademoto* and Soji
Fukumoto
Division of Medical Zoology, Department of Microbiology and Immunology, School of Medicine
and *Technical Support Section, Tottori University Faculty of Medicine, Yonago 683-8503 Japan

Previous studies have shown that excretory/secretory (ES) products from plerocercoidsof Spirometra erinaceieuropaei have immunosuppressive activities. We report here thata 130 kDa glycoprotein (ES130) purified from ES products as a suppressive factor of
nitric oxide synthesis in LPS-stimulated RAW 264.7 cells inhibited the gene expressionof 3 chemokines, regulated on activation normal T cell expressed and secreted (CCL5/
RANTES), macrophage inflammatory protein 2 (CXCL2/MIP-2), interferon-inducible
protein 10 kDa (CXCL10/IP-10) in RAW 264.7 cells and mouse peritoneal macrophages
stimulated with LPS for 3 h. These chemokines are important factors for recruitment ofinflammatory leukocytes. RANTES acts on monocytes, basophils, lymphocytes, naturalkiller cells and eosinophils. MIP-2 is a potent chemoattactant for neutrophils, while IP10 binds to Th1 cells. Nearly 80% of MIP-2 gene expression and 50% of IP-10 gene expression in peritoneal macrophages stimulated with LPS for 8 h was suppressed as wellas these chemokine production by the preincubation with 100 ng/mL of ES130 or 5000
ng/mL crude ES products for 24 h. On the other hand the mRNA expression of RANTES in macrophages stimulated with LPS for 8 h or 24 h was not inhibited by ES130 or
crude ES products, while the RANTES chemokine levels in the incubation medium weresignificantly suppressed. These results suggest that ES130 may attenuate inflammation
around the plerocercoids by inhibiting these chemokine production.
Key words: macrophage; chemokine; excretory/secretory products; lipopolysaccharide;
Spirometra erinaceieuropaei

The larval plerocercoids of Spirometra erinaceieuropaei are known to cause “sparganosis” invarious tissues in the human (Kudesia et al., 1998).
When the plerocercoids are taken orally in many
mammals including humans or rodents, the headportion of the larva migrated into the peritoneal
cavity from the intestines. These plerocercoids

may have various bacteria on their surface andthey bring these bacteria into the peritoneal cavity which, in turn, activates the peritoneal macrophages by the bacterial lipopolysaccharide (LPS).
LPS is a ligand of Toll-like receptor 4, and has
been known to be a potent activator of the immune system that induces local inflammation and

Abbreviations: ELISA, enzyme-linked immunosorbent assay; ES, excretory/secretory; FBS, fetal bovine serum; IFN, interferon; IL-1, interleukin-1; ISRE, IFN-stimulated response element; IP-10, IFN-inducible protein 10 kDa; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MIP, macrophage inflammatory protein; RANTES, regulated on activation
normal T cell expressed and secreted; RT, reverse transcriptase; STAT, signal transducer and activator of transcription; TNF,
tumor necrosis factor

37

H. Matsuura et al.

septic shock (Rietschel and Brade, 1992; Guha
and Mackman, 2001). It causes multiple effects on
macrophages, including the secretion of chemokines for recruitment of inflammatory leukocytes
and the inflammatory mediators such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)(Hsi and Remick, 1995).
The chemokines are divided into four families on the basis of the arrangement of the first 2
of 4 conserved cysteins (Ben-Baruch et al., 1995;
Rottman, 1999). Many of the CXC chemokines
are potent chemoattactants for neutrophils, but not
monocytes, while CC chemokines act on monocytes, basophils, lymphocytes, natural killer cells
and eosinophils (Wang et al., 1998; Rottman,1999). In the mouse, the CXC family includes
macrophage inflammatory protein 2 (CXCL2/MIP2), interferon-inducible protein 10 kDa (CXCL10/
IP-10) and KC (CXCL1/Gro- ). MIP-2 and KC
bind to CXCR1 and /or CXCR2 expressed on neutrophils, while IP-10 binds to CXCR3 expressedon Th1 cells. The CC family includes JE/monocyte chemoattactant protein (CCL2/MCP-1) andregulated on activation normal T cell expressedand secreted (CCL5/RANTES). RANTES is a ligand for CCR1/3/5 expressed on monocytes, macrophages, T cells, dendritic cells and eosinophils(Wang et al., 1998; Rottman, 1999).
The experimentally infected plerocercoids
do not induce the strong inflammatory responses
around the parasites and can survive for long periods in the tissues of mice, hence we hypothesizedthat the larval plerocercoids of S. erinaceieuropaei secreted an immunosuppressive factor(s).
We previously showed that the excretory/secretory(ES) products from the plerocercoids suppress the
LPS-induced expressions of chemokines KC and
JE (Fukumoto et al., 1997), TNF- (Miura et al.,
2001; Dirgahayu et al., 2002), and IL-1 (Dirgahayu et al., 2004) in murine macrophages.
In the present study, we purified an immunosuppressive factor (ES130) of a 130 kDa glycoprotein from ES products of plerocercoids and examined the effect on the chemokine gene expression.

Materials and Methods
Preparation of ES products from plerocercoids of Spirometra erinaceieuropaei
Plerocercoids of Spirometra erinaceieuropaei werecollected from 2 species of snakes (Elaphe quadrivirgata and Rhabdophis tigrinus) in the southernpart of Ehime Prefecture, Japan and stored for over
6 to 10 months in the subcutaneous tissue of golden hamsters, which were housed and maintained
according to the guidelines for proper treatmentof animals at the Research Center for Bioscienceand Technology, Tottori University, Japan. ES
products were obtained as described previously(Miura et al., 2001). To obtain ES products, 25
plerocercoids aseptically removed from hamsters
were incubated for 24 h in 25 mL of Dulbecco’s
modified Eagle’s medium (DMEM: Invitrogen,
Carlsbad, CA) in a 10 cm dish. The medium wascentrifuged at 10,000 g for 30 min at 4°C toremove insoluble debris, then dialyzed against
25 mM Tris-HCl (pH 7.4) and concentrated with
Amicon Ultra-15 Centrifugal Filter Units (Nihon
Millipore, Tokyo, Japan), and subsequently sterilized with a 0.22 µm filter (Nihon Millipore). The
ES products after sterilization are referred to as
crude ES products hereafter. The protein concentration was measured using a Bio-Rad protein
assay kit (Bio-Rad, Hercules, CA), and adjusted to
500 µg/mL for culture or 250 µg/mL for purification.
Purification of an immunosuppressive factor from ES products
The crude ES products were loaded onto a new
MonoQ HR5/5 anion-exchange column (Amersham Bio Sciences, Piscataway, NJ) equilibrated
in 25 mM Tris-HCl (pH 7.4), and the column waswashed with 10 column volumes of same buffer.
The bound materials were eluted with a step gradient consisting of 10 column volumes each of 25
mM Tris-HCl (pH 7.4) containing 0.1, 0.2, 0.3, 0.4,
38

Suppression of chemokine by a parasite

ter intraperitoneal injection of 2 mL thioglycolate(Sigma-Ardrich). The macrophages in DMEM
containing penicillin G (Banyu Pharmaceutical,
Tokyo), streptomycin (Meiji Seika, Tokyo) and
10% heat-inactivated fetal bovine serum (FBS;
Invitrogen,) were plated in 60 mm tissue culture
dish (Greiner, Bayern, Germany). Then the cells
were incubated at 37˚C in an atmosphere of 5%
CO2 and were left for more than 14 h before eachexperiment.
The culture medium was replaced with afresh DMEM including penicillin G, streptomycin
and 10% FBS, and either with crude ES products(0.5–5.0 µg/mL) or purified ES products (10–200
ng/mL). The cells were incubated for 24 h, andwere stimulated with 100 ng/mL LPS. After 3 h,
8 h or 24 h of incubation, the cells were collected
for isolation of RNA, and the supernatant fluids ofthese dishes were collected for the measurementof chemokines by enzyme-linked immunosorbent
assay (ELISA).

0.5 M NaCl. These samples were determined fortheir inhibitory effect on the nitrite production in
RAW 264.7 macrophages (RCB00535) that were
stimulated with LPS (Escherichia coli serotype
005:B5) (Difco Laboratories, Detroit, MI). The
strongest inhibition was obtained in the fraction
eluted with Tris-HCl containing 0.3 M NaCl. For
further purification, the fractions eluted with 0.3
M NaCl were applied on Ricinus communis agglutinin (RCA120)- and wheat germ agglutinin(WGA)-agarose column (Seikagaku, Tokyo)
sequentially according to the manufacture’s instructions. To measure the protein concentration,
the purified proteins and bovine serum albumin(Sigma Aldrich, St. Louis, MI), as a standard protein, were applied to 5% to 20% linear gradientpolyacrylamide gel (SPG-R520L; ATTO, Osaka,
Japan) for SDS-PAGE. After that, the polyacrylamide gel was stained with SYPRO Ruby gel
staining (Invitrogen), and the concentration of thepurified proteins was quantified by densitometry(ATTO Densitograph 4.0; ATTO).
Preparation and culture of macrophage

RNA isolation and semi-quantitative reverse
transcription-PCR

RAW 264.7 cells were cultured according to
Dirgahayu et al. (2004). Male C57/BL6 mice
(8-week-old) were purchased from Japan SLC(Shizuoka, Japan). These mice were housed andmaintained at the Research Center for Bioscienceand Technology, Tottori University. Peritoneal
macrophages were harvested with 10 mL of icecold phosphate-buffered saline on the 3rd day af-

Total RNA was prepared using an ISOGEN Kit(Nippon Gene, Tokyo) according to the manufacture’s protocol. Total RNA (1 µg) was reversetranscribed into cDNA using M-MLV Reverse
Transcriptase (Promega, Tokyo) and random
primers. The cDNA was subjected to PCR amplification with Taq DNA polymerase (Gene Taq)
(Nippon Gene). The primers for IL-1 and TNF-

Table 1. Chemokine primers and cycle number of PCR
Gene
CCL5/RANTESCXCL2/MIP-2
CXCL10/IP-10-actinPrimer5'-CCT CAC CAT CAT CCT CAC TGC A-3'
5'-CAT CCC CAA GCT GGC TAG GAC T-3'
5'-AGT TTG CCT TGA CCC TGA AGC C-3'5'-TGG GTG GGA TGT AGC TAG TTC C-3'5'-CCT ATC CTG CCC ACG TGT TGA G-3'5'-CGC ACC TCC ACA TAG CTT ACA G-3'5'-TGG AAT CCT GTG GCA TCC ATG AAA-3'
5'-TAA ACC GCA GCT CAG TAA CAG TCC-3'(forward)(reverse)(forward)(reverse)(forward)(reverse)(forward)(reverse)

Product size (bp)

Cycle

334

34

466

28

431

23

349

22

IP-10, interferon-inducible protein 10 kDa; MIP, macrophage inflammatory protein; RANTES, regulated on activation
normal T cell expressed and secreted.

39

H. Matsuura et al.

1
(kDa)

2

3

4

ELISA analysis(kDa)

The culture medium of 4.0 106 macrophages in
60 mm dishes was harvested after incubation with
LPS for 8 h or 24 h. The collected supernatants
were stored at –80˚C before measurement. The
concentrations of RANTES, MIP-2 and IP-10
were assayed using ELISA kit (R & D Systems,
Minneapolis, MN), following the manufacturer’s
protocol.
Statistical analysis was performed by non-repeated measures analysis of variance. If there wassignificant difference between the positive control
and treatment groups, Dunnett’s test was subsequently performed. Data were analyzed with statistical package software SPSS 12.0 for Windows.

200
116
94.5

130

66
45

31

21.5
14.5
6.5

Fig. 1. An immunosuppressive factor, ES130, purified with
ion-exchange chromatography and lectin chromatography
was analyzed by SDS-PAGE with SYPRO Ruby gel staining. Lane 1, molecular marker; lane 2, crude ES products;
lane 3, the fraction eluted from the MonoQ column with
Tris-HCl containing 0.3 M NaCl; lane 4, the fraction eluted
from the RCA120- and WGA-agarose columns sequentially.
ES, excretory/secretory; RCA, Ricinus communis agglutinin;
WGA, wheat germ agglutinin.

Results
Purification of an immunosuppressive factor (ES130)
A suppressive effect on NO synthesis in LPS-stimulated RAW 264.7 cells was found in 0.3 M NaCl
fraction of crude ES products by an anion exchange
chromatograpy. Then we purified a glycoprotein of
130 kDa, which suppressed NO synthesis in RAW
264.7 cells, using 2 lectin columns, RCA-agaroseand WGA-agarose-columns sequencially (Fig. 1).

were used according to Kina et al. (2005), andthose for cyclooxygenase-2 (COX-2) were basedon Fukumoto et al. (2006). The primers that weused for RANTES (Heeger et al., 1992), MIP-2(Su et al., 1996), IP-10 (Baker et al., 2003) and-actin (Alonso et al., 1986) are listed in Table 1.
The PCR cycles consisted of denaturation at 94°C
for 30 s, annealing at 60°C for 1 min and extension at 72°C for 1min. The number of cycles forsemi-quantitative reverse transcriptase-PCR (RTPCR) of each gene was also listed in Table 1. The
PCR products were separated by electrophoresis
on 1.5 % agarose gels, and stained with ethidium
bromide and photographed under UV light with adigital camera. For quantification, electrophoretic
gels were analyzed using the software Image J
version 1.33 (National Institutes of Health Bethesda, MD). The amount of mRNAs was normalized
to that of -actin.

Suppression of chemokine mRNA expression in RAW 264.7 cells by ES130
Preincubation with 100 ng/mL of ES130 for 24 h
suppressed the mRNA expression of 3 proinflammatory genes, IL-1 , TNF- and COX-2, and 3
chemokine genes, MIP-2, RANTES and IP-10 aswell as 5 µg/mL of crude ES in RAW 264.7 cells
stimulated with LPS for 3 h (Fig. 2).
Suppression of chemokine mRNA expression in peritoneal macrophages by ES130
No appreciable expression of chemokines wasobserved in control macrophages without LPS
40

Suppression of chemokine by a parasite

TNF-

COX-2

RANTES

MIP-2

IP-10

Relative expression

Relative expression

IL-1

—

ES 130 Crude ES——

ES 130 Crude ES

ES 130 Crude ES

Fig. 2. ES130 inhibited the mRNA expression of proinflammatory genes including 3 chemokines in LPS-stimulated RAW
264.7 macrophages. RAW 264.7 cells were left untreated or incubated with ES130 (100 ng/mL) or crude ES products (5 µg/mL) for 24 h, then stimulated with LPS (100 ng/mL) for 3 h. Total RNA was obtained and the mRNA expression of IL-1 ,TNF- , COX-2, MIP-2, RANTES, IP-10 and -actin was analyzed by semi-quantitative RT-PCR. The mRNA levels of
proinflammtory genes were quantified and were normalized to -actin. Relative mRNA levels of these genes were presented
as percentages of the LPS-induced control in the absence of ES130 or crude ES products. ES, excretory/secretory; IL-1, interleukin-1; IP-10, interferon-inducible protein 10 kDa; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein;
RANTES, regulated on activation normal T cell expressed and secreted; RT, reverse transcriptase.

B

LPS (3 h)
ES 130
Crude ES

–
–

–
–

10
–

50
–

100
–

200
–

–
500

–
1000 5000

RANTESMIP-2

RANTES
Relative expression

A

IP-10
Relative expression

Fig. 3. Suppression of LPS-induced chemokine mRNA expression inperitoneal macrophages by ES130. Peritoneal macrophages were left
untreated or treated with ES130 (10–200 ng/mL) or crude ES (500–5000
ng/mL) for 24 h, and then stimulated with LPS (100 ng/mL) for 3 h. Total RNA was obtained from the cells, and the expression of RANTES,
MIP-2, IP-10 and -actin mRNA was assessed by semi-quantitative RTPCR (A). The mRNA levels of RANTES, MIP-2 and IP-10 were quantified and were normalized to -actin. The relative mRNA levels of thesechemokines were presented as percentages of the LPS-induced control
in the absence of ES130 or crude ES products (B). The results are representative of independent 2 experiments. ES, excretory/secretory; IP-10,
interferon-inducible protein 10 kDa; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal
T cell expressed and secreted; RT, reverse transcriptase.

MIP-2

IP-10

Relative expression-actinES 130
Crude ES

–
–

–
–

10
–

50
–

100
–

200
–

–
–
–
500 1000 5000

LPS (3 h)h. Crude ES products suppressed 3 chemokine
gene expression in a dose-dependent manner. Ten
to 100 ng/mL of ES130 also suppressed RANTES
and IP-10 in dose-dependent manner, while about

stimulation. Mouse peritoneal macrophages were
either left untreated or preincubated with various
doses of ES130 or crude ES products for 24 h. The
cells were stimulated with 100 ng/mL LPS for 3
41

H. Matsuura et al.

B
LPS
ES 130
Crude ES

8h

–
–

–
–

100
–

200
–

RANTES
Relative expression

A
–
5000

RANTES
MIP-2-actin

24 h

MIP-2

Relative expression

IP-10

RANTES

IP-10

Relative expression

MIP-2
IP-10
-actinES 130
Crude ES

–
–

–
–

100
–

200
–

–
5000

LPS (8 h)Fig. 4. Suppression of LPS-induced chemokine mRNA expression in murine peritoneal macrophages by ES130. Peritoneal
macrophages were left untreated or treated with ES130 (100 or 200 ng/mL) or crude ES (5000 ng/mL) for 24 h, and then stimulated with LPS (100 ng/mL) for 8 h or 24 h. Total RNA was obtained from the cells, and the mRNA expression of RANTES, MIP-2, IP-10 and -actin was assessed by semi-quantitative RT-PCR (A). The mRNA levels of RANTES, MIP-2 and IP10 in macrophages stimulated with LPS for 8 h were quantified and were normalized to -actin. The relative mRNA levelsof these chemokines were presented as percentages of the LPS-induced control in the absence of ES130 or crude ES products(B). The results are representative of 2 independent experiments. ES, excretory/secretory; IP-10, interferon-inducible protein
10 kDa; LPS, lipopolysaccharide; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell
expressed and secreted; RT, reverse transcriptase.

half of the MIP-2 gene expression was inhibited by
10 to 100 ng/mL of ES130 (Fig. 3). However, 200
ng/mL of ES130 did not inhibit the gene expressionof 3 chemokines more than the dose of 100 ng/mL.
Nearly 80% of MIP-2 gene expression and
50% of IP-10 gene expression in peritoneal macrophages stimulated with LPS for 8 h were suppressed by the preincubation with 100 or 200 ng/
mL of ES130 or 5000 ng/mL crude ES products
for 24 h. The suppressive effect on the gene expression of MIP-2 and IP-10 continued in macrophages after stimulation with LPS for 24 h. On
the other hand, the mRNA expression of RANTES in macrophages stimulated with LPS for 8 h
or 24 h was not inhibited by ES130 or crude ES
products (Fig. 4).

Suppressive effect of ES130 on chemokine
production in the medium of macrophages
The effects of ES130 and crude ES products on
3 chemokine leves in the incubation medium of
peritoneal macrophages stimulated with LPS for 8
h and 24 h were examined by ELISA.
MIP-2 concentrations in the medium of LPSstimulated macrophages for 8 h and 24 h weredeeply suppressed by ES130 or crude ES products (Fig. 5) as well as the suppression of MIP-2
mRNA levels (Fig. 4). The IP-10 concentration in
the medium of macrophages stimulated for 8 h or
24 h was also suppressed by ES130 or crude ES
products significantly (Fig. 5).

42