Suppression of chemokine by a parasite

RANTES

Discussion* * *Parasite survival may depend on the ability of theparasite to modulate the host immune responseby the release of immunomodulatory moleculesthat protect the organism (Ramaswamy et al.,
1995; Goodridge et al., 2001, 2005). We purified
an immunosuppressive factor (ES130) from ES
products of plerocercoids in the present study andfound that ES130 inhibits the gene expression ofIL-1 and TNF- as well as plerocercoid-immunosuppressive factor reported by Kina et al. (2005).
Besides, ES130 suppressed the gene expressionof 3 chemokines, MIP-2, IP-10 and RANTES, in
LPS-stimulated macrophages.
In monocytes or macrophage cell lines, LPS
has been reported to activate p42/44 (ERK) mitogen-activated protein kinase (MAPK) and JNK aswell as the p38 MAPK cascade (Hambleton et al.,
1996; Carter et al., 1999). Transcription of c-Fos
is upregulated by MAPKs, and members of theFos family dimerise with Jun to form the AP-1
transcription factor, which upregulates transcription of a diverse range of genes in macrophages.
MIP-2 plays an important role in the recrutment of neutrophils. The MIP-2 promoter is transcriptionally activated in a macrophage cell line
RAW 264.7 by LPS. By deletion analysis of theMIP-2 promoter region, Kim et al. (2003) showedthat NF- B and AP-1 binding sites are essential
for LPS-induced MIP-2 gene expression. TheSp-1 binding element is an important determinantof MIP-2 promoter activity, and NF- B, c-Jun andSp-1 can functionally cooperate to elicit maximal
activation of the promoter (Lee et al., 2005).
The p38 MAPK intracellular signaling
pathway plays a central role in regulating a widerange of inflammatory responses in many different cells. In vitro exposure to a novel p38 MAPKinhibitor (M39) blocked MIP-2 release from LPSstimulated murine and human neutrophils andmacrophages, and eliminated migration of murine
neutrophils toward the chemokines MIP-2 and KC(Nick et al., 2000). We previously showed that

* * *MIP-2* * ** * *IP-10* * *ES 130
Crude ES
LPS–––––+

100 200 ––– 5000+++(8 h)* * *–––––+

100 200 ––– 5000+++(24 h)Fig. 5. ES130 suppressed the chemokine production significantly in the incubation medium of LPS-stimulated macrophages. Peritoneal macrophages were left untreated ortreated with ES130 (100 or 200 ng/mL) or crude ES (5000ng/mL) for 24 h, and then stimulated with LPS (100 ng/mL) for 8 h or 24 h. The incubation medium was obtained, and the levels of 3 chemokines, RANTES, MIP2 and IP-10 were quantified by enzyme-linked immunosorbent assay. Data are expressed as the mean ± SE (n =3). *P < 0.005. ES, excretory/secretory; IP-10, interferoninducible protein 10 kDa; MIP, macrophage inflammatory
protein; RANTES, regulated on activation normal T cell
expressed and secreted; RT, reverse transcriptase.

RANTES in the medium of macrophages
stimulated with LPS for 8 h increased nearly 5
times in the medium after LPS stimulation for 24h. Although the mRNA expression of RANTESin macrophages stimulated with LPS for 8 h or24 h was not suppressed significantly by ES130
or crude ES products (Fig.4), the same amountof ES130 and crude ES products suppressed theRANTES chemokine levels in the medium ofthese macrophages significantly (Fig. 5).
43

H. Matsuura et al.

regulation by ES130 is as yet unknown, it is supposed that ES130 plays a role in inhibiting thetranslation, as noiciceptin does.
In LPS-stimulated macrophages, the IP-10
expression is mediated by the intermediate production of IFN- in MyD88-independent pathway(Kawai et al., 2001; Toshchakov et al., 2002).
IFN- activates IFN stimulated gene factor 3, a
trimeric complex composed of signal transducer
and activator of transcription (STAT) 1, STAT2
and IFN regulatory factor-9/p48 (Darnell et al.,1994). Consistent with this, LPS-induced expression of IP-10 is severely impaired in macrophages from STAT1-deficient or IFN- / R–/–mice (Ohmori and Hamilton, 2001). LPS-induced
transcriptional activation of the IP-10 gene in amacrophage cell line is mediated by regulatory
sequences found in the region between –243 and–105 including an ISRE and 2 B sites, and thecooperative interaction between ISRE and B elements is essential for the IP-10 gene expression(Ohmori and Hamilton, 2001). As the IFN- gene
expression in LPS-stimulated macrophages is suppressed by crude ES products (data not shown),and this suppression is supposed to be one of themechanisms in inhibiting the IP-10 gene expression by ES130.
The present results suggest that ES130 attenuates inflammation around the plerocercoids
through suppression of the chemokine gene expressions and chemokine production. Its mechanism needs to be further investigated.

the ES products from the plerocercoids reduce thephosphorylation of MAPK, particularly extracellular signal-regulated kinase 1/2 (ERK1/2) and
p38 MAPK (Dirgahayu et al., 2002, 2004), which
may attribute to the suppression of MIP-2 mRNA
expression. Besides MAPKs it has been emphasized the importance of NF- B for the inductionof MIP-2 in LPS-activated monocyte-macrophagelineage. However, we found that the ES products
do not affect LPS-induced nuclear translocationof NF- B (Dirgahayu et al., 2004).
Shin et al. (1994) showed the LPS-response
elements in RANTES chemokine gene. Promoter
regions of RANTES contain an LPS-response
element, which is composed of 2 sequences, one
corresponding to an AP-1 half site and another resembling a portion of interferon (IFN)-stimulated
response element (ISRE). There are a number ofproteins capable of binding to AP-1 sites. They
belong to the Jun-Fos family, ATF-CREB family,and NF-E2. Monomeres of these proteins havenegligible affinity toward the AP-1 site, in contrast
to their homo- or heterodimers. The results fromelectrophoretic mobility shift assay demonstrarated the presence of c-Jun and CREB in the protein-LER complex (Shin et al., 1994). However, theISRE-like motif in RANTES does not have ISRE
activity.
Compared to MIP-2 and IP-10, the mRNA
expression of RANTES in macrophages stimulated with LPS for 8 h or 24 h was not inhibitedby ES130 or crude ES products, while the sameamount of ES130 and crude ES products suppressed the RANTES chemokine levels in themedium of macrophages significantly stimulatedwith LPS for the same amount of time. The production of RANTES chemokine protein can besuppressed by nociceptin in both primary CD14+
human monocyte and monocyte-like cell lines.
However, nociceptin does not appear to regulate
the expression of the chemokine at the level oftranscription, as RANTES mRNA levels following nociceptin treatment of monocytes were essentially normal (Kaminsky and Rogers, 2008).
Although the mechanism of RANTES chemokine

References
1 Alonso S, Minty A, Bourlet Y, Buckingham M.
Comparison of three actin-coding sequences in themouse: evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol Evol
1986;23:11–22.
2 Baker MS, Chen X, Rotramel A, Nelson J, Kaufman
DB. Proinflammatory cytokines induce NF- Bdependent/NO-independent chemokine gene expression in MIN6 cells. J Surg Res 2003;110:295–303.
3 Ben-Baruch A, Michiel DF, Oppenheim JJ. Signalsand receptors involved in recruitment of inflammatory cells. J Biol Chem 1995;270:11703–11706.

44

Suppression of chemokine by a parasite

4 Carter AB, Monick MM, Hunninghake GW. BothErk and p38 kinases are necessary for cytokine
gene transcription. Am J Respir Cell Mol Biol
1999;20:751–758.
5 Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response toIFNs and other extracellular signaling proteins. Science 1994;264:1415–1421.
6 Dirgahayu P, Fukumoto S, Miura K, Hirai K. Excretory/secretory products from plerocercoids ofSpirometra erinaceieuropaei suppress the TNFgene expression by reducing phosphorylation ofERK1/2 and p38 MAPK in macrophages. Int J Parasitol 2002;32:1155–1162.
7 Dirgahayu P, Fukumoto S, Tademoto S, Kina Y, Hirai K. Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1 gene expression in murine macrophages.
Int J Parasitol 2004;34:577–584.
8 Fukumoto S, Dirgahayu P, Nunomura K, Matsuura
H, Hirai K. Suppresion of LPS-induced cyclooxygenase-2 (cox-2) gene expression in mouse macrophages by excretory/secretory products of Spirometraerinaceieuropaei plerocercois. Yonago Acta Med
2006;49:39–47.
9 Fukumoto S, Hirai K, Tanihata T, Ohmori Y, Stuehr
DJ, Hamilton TA. Excretory/secretory products
from plerocercoids of Spirometra erinacei reduce
iNOS and chemokine mRNA levels in peritoneal
macrophages stimulated with cytokines and/or LPS.
Parasite Immunol 1997;19:325–332.
10 Goodridge HS, Stepek G, Harnett W, Harnett MM.
Signalling mechanisms underlying subversion of theimmune response by the filarial nematode secreted
product ES-62. Immunology 2005;115:296–304.
11 Goodridge HS, Wilson EH, Harnett W, Campbell
CC, Harnett MM, Liew FY. Modulation of macrophage cytokine production by ES-62, a secreted
product of the filarial nematode Acanthocheilonema
viteae. J Immunol 2001;167:940–945.
12 Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001;13:
85–94.
13 Hambleton J, Weinstein SL, Lem L, DeFranco AL.
Activation of c-Jun N-terminal kinase in bacterial
lipopolysaccharide-stimulated macrophages. Proc
Natl Acad Sci USA 1996;93:2774–2778
14 Heeger P, Wolf G, Meyers C, Sun MJ, O’Farrell SC,Krensky AM, et al. Isolation and characterization ofcDNA from renal tubular epithelium encoding murine Rantes. Kidney Int 1992;41:220–225.
15 Hsi ED, Remick DG. Monocytes are the major producers of interleukin-1 in an ex vivo model of local cytokine production. J Interferon Cytokine Res
1995;15:89–94.
16 Kaminsky DE, Rogers TJ. Suppression of CCL2/

MCP-1 and CCL5/RANTES expression by nociceptin in human monocytes. J Neuroimmune Pharmacol 2008;3:75–82.
17 Kawai T, Takeuchi O, Fujita T, Inoue J, Muhlradt
PF, Sato S, et al. Lipopolysaccharide stimulates theMyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expressionof a subset of lipopolysaccharide-inducible genes. JImmunol 2001;167:5887–5894.
18 Kim DS, Han JH, Kwon HJ. NF- B and c-Jundependent regulation of macrophage inflammatory
protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells. Mol Immunol 2003;40:633–643.
19 Kina Y, Fukumoto S, Miura K, Tademoto S, Nunomura K, Dirgahayu P, et al. A glycoprotein fromSpirometra erinaceieuropaei plerocercoids suppresses osteoclastogenesis and proinflammatory cytokine
gene expression. Int J Parasitol 2005;35:1399–1406.
20 Kudesia S, Indira DB, Sarala D, Vani S, Yasha TC,Jayakumar PN, et al. Sparganosis of brain and spinal
cord: unusual tapeworm infestation (report of twocases). Clin Neurol Neurosurg 1998;100:148–152.
21 Lee KW, Lee Y, Kwon HJ, Kim DS. Sp1-associatedactivation of macrophage inflammatory protein-2
promoter by CpG-oligodeoxynucleotide and lipopolysaccharide. Cell Mol Life Sci 2005;62:188–198.
22 Miura K, Fukumoto S, Dirgahayu P, Hirai K. Excretory/secretory products from plerocercoids ofSpirometra erinaceieuropaei suppress gene expressions and production of tumor necrosis factorin murine macrophages stimulated with lipopolysaccharide or lipoteichoic acid. Int J Parasitol
2001;31:39–47.
23 Nick JA, Young SK, Brown KK, Avdi NJ, Arndt PG,
Suratt BT, et al. Role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation. J Immunol 2000;164:2151–2159.
24 Ohmori Y, Hamilton TA. Requirement for STAT1in LPS-induced gene expression in macrophages. J
Leukoc Biol 2001;69:598–604.
25 Ramaswamy K, Salafsky B, Potluri S, He YX, LiJW, Shibuya T. Secretion of an anti-inflammatory,immunomodulatory factor by schistosomulae ofSchistosoma mansoni. J Inflamm 1995;46:13–22.
26 Rietschel ET, Brade H. Bacterial endotoxins. Sci
Am 1992;267:54–61.
27 Rottman JB. Key role of chemokines and chemokine
receptors in inflammation, immunity, neoplasia, andinfectious disease. Vet Pathol 1999;36:357–367.
28 Shin HS, Drysdale BE, Shin ML, Noble PW, Fisher
SN, Paznekas WA. Definition of a lipopolysaccharide-responsive element in the 5'-flanking regions ofMuRantes and crg-2. Mol Cell Biol 1994;14:2914–2925.

45

H. Matsuura et al.

29 Su YH, Yan XT, Oakes JE, Lausch RN. Protective antibody therapy is associated with reduced
chemokine transcripts in Herpes simplex virus type1 corneal infection. J Virol 1996;70:1277–1281.
30 Toshchakov V, Jones BW, Perera PY, Thomas K,
Cody MJ, Zhang S, et al. TLR4, but not TLR2, mediates IFN- -induced STAT1 / -dependent gene expression in macrophages. Nat Immunol 2002;3:392–398.

31 Wang JM, Su S, Gong W, Oppenheim JJ. Chemokines, receptors, and their role in cardiovascular pathology. Int J Clin Lab Res 1998;28:83–90.
Received December 19, 2008; accepted January 8, 2009
Corresponding author: Soji Fukumoto, MD, PhD46