Yonago Acta medica 2006;49:39–47

Suppression of LPS-induced Cyclooxygenase-2 (cox-2)Gene Expression in Mouse Macrophages by
Excretory/Secretory Products ofSpirometra erinaceieuropaei Plerocercoids
Soji Fukumoto, Paramasari Dirgahayu*, Kozue Nunomura, Haruyo Matsuura andKazumitsu Hirai
Division of Molecular Medical Zoology, Department of Microbiology and Pathology, School ofMedicine, Tottori University Faculty of Medicine, Yonago 683-8503 JapanThe escape mechanism of parasites from inﬂammatory processes has been thought to be
one of the most important tools for their survival in infected tissues. On the other hand,inducible cyclooxygenase-2 (cox-2) has been shown to play a major role in the process ofinﬂammation. We investigated the effect of excretory/secretory (ES) products from theplerocercoids of Spirometra erinaceieuropaei on cox-2 gene expression in a murine macrophage cell line (RAW 264.7). Preincubation of the macrophages with the ES products
inhibited LPS-induced cox-2 mRNA expression by 75% as well as its protein production.
Dibutyryl cAMP enhances LPS-induced cox-2 mRNA expression. The ES products
also inhibited this enhanced expression by 46%. Inhibition of p38 mitogen-activatedprotein kinase (MAPK) with SB203580 or that of extracellular signal-regulated protein
kinase with PD98059 reduced LPS-induced cox-2 mRNA expression by 75% or 52%,
respectively, along with its protein production. This evidence suggests that both MAPK
pathways are crucial for full induction of cox-2 gene expression. One experiment using atranscriptional inhibitor, actinomycin-D, showed that the ES products destabilized LPSinduced cox-2 mRNA in RAW 264.7 macrophages. These results show that ES products from the plerocercoids of Spirometra erinaceieuropaei suppress LPS-induced cox-2
mRNA expression in murine RAW 264.7 macrophages, and may attenuate inﬂammation
around the plerocercoids of S. erinaceieuropaei.
Key words: Spirometra erinaceieuropaei; lipopolysaccharide; macrophage; cyclooxygenase-2;
mitogen-activated protein kinase

Cyclooxygenase (cox) catalyzes the synthesis ofprostaglandins (PGs) from arachidonic acid. Two
isozymes, cox-1 and cox-2, have been identified,
and they are encoded by separate genes. The
cox-1 isozyme is constitutively present in most tissues and seems to catalyze the synthesis of PGs

for normal physiological functions (O’Neill andFord-Hutchinson, 1993). In contrast, cox-2 is notor is scarcely detectable in most tissues under basal conditions, but is dramatically induced by bacterial lipopolysaccharide (LPS), cytokines, growth
factors or carcinogens (Kujubu et al., 1991).

* Present address: Department of Parasitology, Faculty of Medicine, Sebelas Maret University, Surakarta, Indonesia
Abbreviations: cox, cyclooxygenase; dbcAMP, dibutyryl cAMP; DMEM, Dulbecco’s modiﬁed Eagle’s medium; ERK,
extracellular signal-regulated kinase; ES, excretory/secretory; gapdh, glyceraldehyde-3-phosphate dehydrogenase;
MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; PG, prostaglandin

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S. Fukumoto et al.

activated protein kinase (MAPK), particularly
extracellular signal-regulated kinase 1/2 (ERK1/2)
and p38 MAPK (Dirgahayu et al., 2002; Dirgahayu et al., 2004).
Studies using MAPK inhibitors, SB203580
and PD98059 revealed that MAPK is involved in
LPS-induced cox-2 transcription in RAW 264.7
macrophages (Chanmugam et al., 1995; Hwanget al., 1997). SB203580 inhibits the p38 MAPKpathway (Ajizian et al., 1999), and inhibits thepromoter activities of cox-2 (Ho et al., 2004),
while PD98059 is a specific inhibitor for theERK1/2 pathway (Alessi et al., 1995). In addition
to the transcriptional effects of MAPK, AgC10,a mucin from Trypanosoma cruzi destabilizedcox-2 mRNA by inhibiting the activation of p38
MAPK (Alcaide and Fresno, 2004).
Besides MAPKs, the importance of NF-κB
for the induction of cox-2 in LPS-activated monocyte-macrophage lineage has been emphasized (D’Acquisto et al., 1998). However, we found that ES
products do not affect the LPS-induced nuclear
translocation of NF-κB (Dirgahayu et al., 2002).
Since ES products have the ability to suppress the phosphorylation of ERK1/2 and p38
MAPK in LPS-activated macrophages, we hypothesized that ES products could suppress LPSinduced cox-2 gene expression in macrophages.
We have shown in this paper that ES products
suppress LPS-induced cox-2 mRNA expression
and cox-2 protein in RAW 264.7 cells, and ES
products destabilize cox-2 mRNA. This is theﬁrst report demonstrating that ES products fromthe parasitic helminth reduce cox-2 mRNA andcox-2 protein in macrophages.

The cox-2 isozyme is responsible for highlevels of PG production, and secreted PGs areinvolved in various physiological events including
progression of inflammation, immunomodulation, and transmission of pain (Vane and Booting, 1998; Kim et al., 2004). The central role ofPGs in inﬂammation was ﬁrmly established after
the discovery that the anti-inflammatory actionof nonsteroidal anti-inﬂammatory drugs, such asacetylsalicylic acid, was mediated by the inhibition of cox. It has been widely accepted that macrophages play an important role in the regulationof inflammation and immune response. In inﬂammatory reactions, macrophages are the main
producers of large quantities of PGE2 (Giroux andDescoteaux, 2000).
Parasite survival may depend on the abilityof the parasite to modulate host immune responseby the release of immunomodulatory moleculesthat protect the organism (Maizels et al., 2001;
Harnett et al., 2004). When larval plerocercoidsof S. erinaceieuropaei are taken orally in many
mammals including humans and rodents, theseplerocercoids invade the peritoneal cavity fromthe intestine, and can survive in the tissues ofhosts. In humans, infection with larval plerocercoids causes “sparganosis” in various tissues(Kudesia et al., 1998). Experimentally infected
plerocercoids do not induce the strong inﬂammatory responses around the parasites and they cansurvive for long periods in the tissues of mice;hence, we hypothesized that larval plerocercoidsof S. erinaceieuropaei secrete an immunosuppressive factor(s). We previously showed that excretory/secretory (ES) products from plerocercoids
suppress LPS-induced expressions of chemokines
KC and JE, the murine homologues of neutrophil chemoattractant growth related oncogene α(CXCL1/GRO-α) and monocyte chemoattractant
protein-1 (CCL2/MCP-1), respectively (Fukumotoet al., 1997), tumor necrosis factor (TNF)-α (Miura et al., 2001; Dirgahayu et al., 2002), and IL-1β(Dirgahayu et al., 2004) in murine macrophages.
We also found that these suppressions are causedby the reduction of phosphorylation of mitogen-Materials and Methods
Plerocercoids of Spirometra erinaceieuropaei and their ES products
Plerocercoids of Spirometra erinaceieuropaei
were collected from two species of snakes (Elaphequadrivirgata and Rhabdophis tigrinus) in Ehime40

Parasite inhibits cox-2 gene expression2001), and similar activation pathways such as
NF-κB and MAPK are involved in LPS-induced
cox-2 and TNF-α gene expressions.

Prefecture, Japan and stored for 6 to 10 months
in the subcutaneous tissues of golden hamsters,
which were housed and maintained according to
the guidelines for proper treatment of animals at
the Research Center for Bioscience and Technology, Tottori University, Japan. ES products wereobtained as described previously (Miura et al.,2001). To obtain ES products, 25 plerocercoids
aseptically removed from hamsters were incubated for 24 h in serum-free 25 mL Dulbecco’s modiﬁed Eagle’s medium (DMEM) in a 10 cm dish,and the incubation medium was dialyzed against
distilled water and lyophilized. The protein concentration of this sample dissolved in DMEM
was assayed using a protein assay kit (Bio-Rad,Melville, NY) and adjusted to 50 µg/mL. The average amount of total protein secreted from eachplerocercoid for 24 h was approximately 30 µg in
the present experiment.

Reverse transcription-PCR analysis
Total RNA was prepared using an ISOGEN Kit(Nippon Gene, Tokyo, Japan) according to themanufacturer’s protocol. Total RNA (1 µg) was
reverse-transcribed, and PCR reactions were performed on a Perkin Elmer DNA Thermal Cycler(Takara Bio, Kusatsu, Japan). Primers used wereas follows: cox-2: 517 bp, 5'-CTT CCT GAT CAA
AAG AAG TGC TG-3' (sense) and 5'-ACT TGAGTT TGA AGT GGT AAC CGC-3' (antisense);and β -actin: 349 bp, 5'-TGG AAT CCT GTG
GCA TCC ATG AA-3' (sense) and 5'-TAA AAC
GCA GCT CAG TAA CAG TCC-3' (antisense).
The PCR cycles consisted of denaturation at 94˚Cfor 30 s, annealing at 60˚C for 1 min and extension at 72˚C for 1 min. The number of cycles used
was 29 for semi-quantitative analysis of cox-2
or 25 for β -actin. A 15-µL aliquot of each PCR
product was separated by electrophoresis on a
1.5% agarose gel, stained with ethidium bromide
and photographed. For quantification, photographs showing PCR products were scanned andanalyzed using the software Densitograph version
4.0 (ATTO, Tokyo, Japan). The amount of cox-2
mRNA was normalized to that of β -actin.

Culture of macrophages
A murine macrophage cell line RAW 264.7(RCB0535) was purchased from RIKEN Cell
Bank (Wako, Japan). The cells were cultured in
DMEM supplemented with 5% heat-inactivatedfetal bovine serum (FBS) (Invitrogen, Carlsbad,CA) containing 100 U/mL penicillin G (Banyu
Pharmaceutical, Tokyo, Japan) and 100 µg/mL
streptomycin (Meiji Seika, Tokyo, Japan), plated
in tissue culture dishes (Greiner Bio-One, Tokyo,Japan) and ﬁnally incubated at 37˚C in an atmosphere of 5% CO2 in a humidiﬁed incubator. Inthe present study, 5 × 106 cells were preincubatedin a 10 cm culture dish for 3 h or 6 h in the presence or absence of ES products (5 µg/mL). Thecells were washed with PBS and stimulated withLPS (100 ng/mL) in DMEM supplemented with
5% FBS. After 3 h of incubation with LPS, large
amounts of cox-2 mRNA were expressed in atime-course experiment. Then, the cells wereincubated with LPS for 3 h in the present experiments. A concentration of 5 µg/mL ES products
was used, because this concentration had beeneffective in TNF-α gene expression (Miura et al.,Preparation of total RNA and Northernblot analysis
Northern hybridization analysis was performed
as previously described (Fukumoto et al., 1997).
Brieﬂy, 10 µg of total RNA was separated on an
1% agarose gel containing 2.2 M formaldehyde,
and subsequently blotted onto a MagnaGraph
Nylon transfer membrane (Micron Separation,Westboro, MA) and then cross-linked to the membrane using an UV cross-linker (Funa-UV-Linker
Fs 1500, Funakoshi, Tokyo, Japan). After prehybridization, the blots were hybridized with cDNA
plasmid probes radiolabelled by [α-32 P]dCTP
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S. Fukumoto et al.

for 30 min. Insoluble material was removed bycentrifugation at 4˚C, 12,500 rpm for 10 min andprotein concentration in the clear supernatant wasdetermined using a protein assay kit (Bio-Rad).
The solubilized proteins (80 µg) were boiled in
SDS-loading buffer, then separated on a 10%
SDS-polyacrylamide gel with a running buffer
containing 25 mM Tris, 192 mM glycine, pH 8.3,
and 0.1% SDS. After electrophoresis, the separating gel was soaked in transfer buffer (25 mMTris-HCl, 0.2 M glycine, 20% (v/v) methanol, pH8.5) for 15 min and electrophoretically transferred
to Hybond-ECL nitrocellulose (Amersham Biosciences, Piscataway, NJ) using a semidry blotting
apparatus (Novablot, Amersham Biosciences),and then blocked overnight in Tris-buffered saline/tween-20 (0.05%) supplemented with 3%
non-fat milk. The membranes were washed,incubated overnight with phospho-specific antibody recognizing cox-2 (1:300) detected with
anti-rabbit horseradish peroxidase-conjugatedantibodies (1:3,000) and subsequently visualized
by an enhanced chemiluminescence reagent (Amersham Biosciences). The gel was stained with(MP Biomedicals, Irvine, CA) using a Takara
BcaBEST Labelling Kit (Takara Bio), and thenautoradiography was performed. The murine-517
bp of the cox-2 cDNA fragment was prepared by
RT-PCR, followed by conﬁrmation with sequencing. A plasmid encoding the glyceraldehyde-3phosphate dehydrogenase (gapdh) cDNA, used asa control, was kindly provided by Dr. David Stern(Columbia University). Relative cox-2 mRNA
expressions were analyzed using a Molecular Imager, Model GS-535 (Bio-Rad) with reference tothe expression of gapdh mRNA.
Preparation of cell lysate and Western blot
After the treatment, cell lysates were prepared
as previously described (Dirgahayu et al., 2002).
Brieﬂy, the adherent cells were disrupted in cold
lysis buffer (20 mM Tris, pH 7.5, 120 mM EDTA,
10% glycerol, 1% Triton X-100, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl ﬂuoride, 5 µg/mL aprotinin, 5 µg/mL pepstatin and5 µg/mL leupeptin), homogenized for 5 s using
a microtube homogenizer, and then kept on ice��Fig. 1. Suppression of LPS-induced cox-2 mRNA expression in RAW 264.7 macrophages by ES products. RAW
264.7 cells were left untreated or treated with ES products (5 µg/mL) for 3 h or 6 h, and the medium was replaced
prior to stimulation with LPS (100 ng/mL) for 3 h.
A: Total RNA was obtained from the cells, and the expression of cox-2 and gapdh mRNA was assessed by Northern blot analysis.
B: The cox-2 mRNA level was quantiﬁed and normalized to gapdh. The relative cox-2 mRNA levels are presented as percentages of the LPS-induced control in the absence of ES products.
C: The cox-2 protein in each cell lysate was analyzed by Western blot analysis. The same volume of each celllysate was resolved by SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue to show that total
protein was equally loaded. One representative of 2 independent experiments is shown.

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Parasite inhibits cox-2 gene expression

Coomassie Brilliant Blue to demonstrate that anequal amount of proteins was loaded in each lane.

Results
Suppression of LPS-induced cox-2 mRNAexpression by ES products
No appreciable band was observed in controls
without LPS stimulation. Firstly, the effect of ES
products on cox-2 mRNA expression and protein production were investigated. RAW 264.7
cells were either left untreated or preincubatedwith ES products (5 µg/mL) for 3 h or 6 h. The
cells were stimulated with 100 ng/mL LPS for 3h. Total RNA or total protein was subjected to
Northern blot or Western blot, respectively. The
result shows that ES products strongly suppressed
the levels of LPS-induced cox-2 mRNA (Figs. 1Aand B) and cox-2 protein (Fig. 1C) to a similar degree. The level of constitutively expressed gapdh
mRNA was unaltered by exposure to ES products.
Since preincubation for 3 h or 6 h showed no signiﬁcant differences on cox-2 mRNA expression orprotein production, a 6 h pretreatment was usedfor the following experiments.

Fig. 2. The suppressive effect of ES products on cox-2mRNA expression induced by dbcAMP and LPS. RAW
264.7 cells were left untreated or preincubated with ES
products for 6 h prior to medium change. The cells werethen either left untreated or treated with dbcAMP (0.1mM) for 15 min and were then stimulated with LPS (100ng/mL) for 3 h. Total RNA was obtained from the cells
and cox-2 and gapdh mRNA expression was assessed by
Northern blot analysis (A). The cox-2 mRNA level wasquantified and was normalized to gapdh. The relativecox-2 mRNA levels are presented as percentages of theLPS-induced control in the absence of ES products anddbcAMP (100%) (B). The ﬁgure is representative of 3
independent experiments.

Suppressive effect of ES products on cox-2mRNA induced by dbcAMP and LPS

preciable band was detected in the untreated control (Lane 1), nor in cells treated with dbcAMPor ES products without LPS-induction (data notshown).

The plerocercoids release PGE2 (Fukushima etal., 1993; Gao et al., 1998), which enhances theinduction of LPS-induced cox-2 mRNA by an adenyl cyclase/cAMP-dependent pathway (Hinz etal., 2000). We tested the possibility of ES products on LPS-induced cox-2 mRNA expression in
the presence of dibutyryl cAMP (dbcAMP). As
seen in Fig. 2, LPS-induced cox-2 mRNA expression (Lane 2) was further enhanced by treatment
with dbcAMP 1.7 times (Lane 4). Pretreatment ofmacrophages with ES products prior to stimulation with LPS (Lane 3) or with LPS and dbcAMP(Lane 5) strongly suppressed cox-2 mRNA expression by 75% and 46%, respectively. No ap-Involvement of MAPK in LPS-induced cox-2mRNA
We examined the effects of MAPK inhibitors
for the ERK1/2 pathway (PD98059) and for p38
MAPK (SB203580) in LPS-induced cox-2 mRNA
expression. The macrophages were pretreated for
1 h with SB203580 (10 µM), PD98059 (50 µM)
or both, and these cells were then stimulated with
LPS for 3 h. The total RNA or protein was sub43