Parasite Immunology, 2005, 27, 127 137

The innate immune response to Entamoeba histolytica

lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4

ORIGINAL
E.
histolytica
ARTICLE
LPPG signals
Blackwell
Publishing,
Ltd. through TLR2 and TLR4

C. MALDONADO-BERNAL,1 C. J. KIRSCHNING,2 Y. ROSENSTEIN,3 L. M. ROCHA,4 N. RIOS-SARABIA,5

M. ESPINOSA-CANTELLANO,6 I. BECKER,7 I. ESTRADA,8 R. M. SALAZAR-GONZLEZ,5 C. LPEZ-MACAS,5
H. WAGNER,2 J. SNCHEZ9 & A. ISIBASI5
1

Infectious Disease Medical Research Unit, Hospital de Pediatra, Centro Mdico Nacional Siglo XXI, IMSS, Mexico City, Mxico, 2Institute
of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Germany, 3Institute of Biotechnology, Universidad
Nacional Autnoma de Mxico, Morelos, Mxico, 4Department of Immunochemistry and Biology, Hospital Infantil de Mxico Federico
Gmez Secretaria de Salud, Mexico City, Mexico, 5Immunochemistry Medical Research Unit, Hospital de Especialidades, Centro Mdico
Nacional Siglo XXI, IMSS, Mexico City, Mxico, 6Department of Experimental Pathology, Cinvestav, Mexico City, Mxico, 7Department
of Experimental Medicine, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, Mexico City, Mxico, 8National School of
Biological Sciences, Instituto Politcnico Nacional, Mexico City, Mxico and 9Faculty of Medicine, Universidad Autnoma del Estado de
Morelos, Morelos, Mxico

SUMMARY
Entamoeba histolytica is a human pathogen that may invade
the intestinal mucosa, causing amoebic colitis or hepatic abscesses
when the trophozoites travel through the portal circulation to
the liver. Lipopeptidophosphoglycan (LPPG) is a molecular
pattern of E. histolytica recognized by the human immune
system. Here we report that LPPG is exposed on the cell
surface of E. histolytica trophozoites, and is recognized
by the host through toll-like receptor (TLR) 2 and TLR4.
Correspondingly, human embryonic kidney (HEK)-293 cells were
rendered LPPG responsive through overexpression of TLR2
or TLR4/MD2. Moreover, co-expression of CD14 enhanced
LPPG signal transmission through TLR2 and TLR4. The
interaction of LPPG with TLR2 and TLR4 resulted in
activation of NF-B and release of interleukin (IL)-10, IL12p40, tumour necrosis factor (TNF)-, and IL-8 from human
monocytes. Consistent with these findings, responsiveness of mouse
macrophages lacking TLR 2 expression (TLR2/) or functional
TLR4 (TLR4d/d) to E. histolytica LPPG challenge was impaired while double deficient macrophages were unresponsive.
In contrast to wild-type control and TLR2/ animals succumbing
to lethal shock syndrome, TLR4d/d mice were resistant to systemic LPPG challenge-induced pathology.

Correspondence: Dr Armando Isibasi, Immunochemistry Medical

Research Unit, Hospital de Especialidades, Centro Mdico
Nacional Siglo XXI, IMSS, Mexico City, Mxico. Avenue,
Cuauhtemoc 330, Col. Doctores. P.O. Box A-047, C.P. 06703
(e-mail: isibasi@prodigy.net.mx).
Received: 27 January 2005
Accepted for publication: Accepted 06 April 2005
2005 Blackwell Publishing Ltd

Keywords Entamoeba histolytica, innate immunity, LPPG,

nuclear factor (NF)-B, pathogen associated molecular
pattern (PAMP), toll-like receptor (TLR)
Abbreviations: TLRs, toll-like receptors; PAMPs, pathogenassociated molecular patterns; LPPG, lipopeptidophosphoglycan; p3CSK4, tripalmitoyl-cysteine-peptide

INTRODUCTION
Amoebiasis is the second most abundant cause of death from
parasitic disease worldwide. Entamoeba histolytica is the causative protozoan parasite, secreting proteases that dissolve host
tissues by killing host cells upon contact. By engulfing red
blood cells, E. histolytica trophozoites invade the intestinal
mucosa causing amoebic colitis. In some cases, amoebas
breach the mucosal barrier and travel through the liver
portal circulation, where they cause abscesses (1). During
invasive amoebiasis, as a result of their penetration through
the colonic epithelium, E. histolytica trophozoites are initially
exposed to the hosts innate immune cells, yet they succeed
in escaping their attack. Although neutrophils and macrophages surround the amoebic lesion in the liver, indicating
recognition of the parasite, these cells are unable to limit
the infection and the patient with amoebic liver abscess
will have a fatal outcome unless promptly treated. Several
mechanisms have been proposed to explain the evasion, first
of the innate, and later of the adaptive immune response,
allowing for the establishment of amoebic infection in
virtually any tissue of the host, but most frequently in the
liver. These include lysis through potent parasitic proteases,
phagocytosis of immune cells, and capping of surface

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receptors (2). However, an alternative explanation could be

modulation of immune effector cells.
Toll-like receptors (TLRs) play an essential role in microbial
recognition by mediation of anti-microbial gene activation,
as well as regulation of the adaptive immune response (35).
Signalling via TLRs leads to the activation of genes encoding
inflammatory cytokines, chemokines, and co-stimulatory
molecules, whose participation in the initial immune response
determines the outcome of an infection. Moreover, recognition
of specific pathogen associated molecular patterns (PAMPs)
through TLRs triggers DC maturation, augments T cellmediated adaptive immunity, activates endogenous CD25+
CD4+ regulatory T cells (TR) and ultimately modulates the
immune response (3). Several TLRs like TLR2 and TLR4
may cooperate to recognize combinations of PAMPs specific
to certain pathogens (6).
Few studies have examined the role of TLRs in immunity
to parasites. Recently, lysophosphatidylserine from S. mansoni
(7), glycosylphosphatidylinositol (GPI) anchors (8), and
Tc-52 from T. cruzi (9), lipopeptidoglycan from L. major (10)
and Gal-lectin of E. histolytica (11) have been shown to
signal through TLR2. Interestingly, TLR2 triggering by
these diverse PAMPs leads to different immune scenarios
such as human dendritic cell maturation (9), initiation of
IL-12, TNF- and NO production in vivo (8), as well as
activation of NK cells (10).
Entamoeba histolytica lipopeptidophosphoglycan (LPPG)
and lipophosphoglycan () are highly immunogenic
molecules directly exposed to the hosts immune system (12
17). Recently LPPG and LPG have been grouped together
and designated GPI-anchored proteophosphoglycans (PPGs)
(18). Entamoeba histolytica LPPG was first described by Isibasi
et al. (19) and has been widely used in different studies regarding the immunogenic properties of the parasite (1214,20).
LPPG is a polysaccharide-type molecule that contains 7585%
carbohydrates, 8% proteins, 25% lipids and 1% phosphates
(14,19). It has proven to be a highly immunogenic molecule,
since specific antibodies against LPPG have been detected
in patients diagnosed with amoebic liver abscess (13).
Additionally, anti-LPPG IgA antibodies have been found
in human colostrum of mothers infected with Entamoeba
histolytica (12). The immunochemical characterization of the
polysaccharide portion of LPPG has revealed differences
between virulent and non-virulent clones of Entamoeba
histolytica strain HM1:IMSS trophozoites (14). LPPG is
structurally similar to LPG of Leishmania, which is a PAMP
with potential immuno-stimulatory capacity (10).
Here we investigated the ability of E. histolytica LPPG to
be recognized and to induce an innate immune response
through TLRs. Our preliminary experiments indicated that
E. histolytica LPPG down-regulates TLR2 expression and
induces IL-10 release (21). In this paper we extended these

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observations and evaluated the potential of E. histolytica

LPPG to influence the transcriptional control of the TLR2
receptor as well as the synthesis of tumour necrosis factor
(TNF)-, IL-12 and IL-8 by human monocytes. In addition,
by using human embryonic kidney (HEK)-293 cells separately
overexpressing TLR1 to TLR10, we found that the interaction of E. histolytica LPPG with TLR2 and TLR4 resulted
in NF-B activation. Moreover, we show that response to
LPPG of macrophages isolated from TLR2/ and functional
TLR4-deficient (TLR4d /d) mice was impaired to a significant
degree and that TLR4d /d mice were resistant to an LPPG
challenge capable of inducing lethal shock in wild-type mice
and TLR2/ mice. Taken together, our results indicate that
E. histolytica LPPG initiates an innate immune response by
interacting with TLR2 and TLR4 and that, through NF-B
activation, it can regulate an early proinflammatory response
followed by an anti-inflammatory response LPPG.

MATERIALS AND METHODS

Reagents and antibodies
The following reagents were from Sigma (St. Louis, MO):
E. coli (strains 0111:B4 and 055:B5) LPS, RPMI 1640 medium,
polymyxin B, PMA, penicillinstreptomycin and anti-rabbit
IgG mAb. DMEM was from Gibco-BRL (Rockville, MD).
Anti-CD14 monoclonal antibody (mAb) was obtained from
PharMingen International (San Diego, CA). Anti-TLR2
polyclonal antibody (pAb; N-17) was from Santa Cruz
Biotechnology (USA). Monoclonal antibody IQ-Mn01, and
polyclonal antibody IQ-P01, directed against LPPG, was
generated by immunizing Balb/c mice with membrane antigens
from trophozoites of E. histolytica strain HM-1:IMSS, as
described previously (22) or by immunizing New Zealand
rabbits with LPPG from trophozoites of E. histolytica
HM-1:IMSS, respectively. FITC-conjugated F (ab)2 fragment
of affinity-isolated rabbit anti-human IgD from DAKO
(Denmark). Cell isolation kits were from Miltenyi Biotec
(Germany) and the endotoxin detection kit was purchased
from Bio-Whittaker, Inc. (Walkersville, MD). The phosphothioated sulfhydryl-modified oligodeoxynucleotides (ODN;
TriLink Biotechnologies, La Jolla, CA) used in this study
consisted of 20 bases and contained a CpG motif (1668: 5S-TCCATGACGTTCCTGATGCT-3).

Cell-surface staining of LPPG

Trophozoites of pathogenic E. histolytica strain HM-1:IMSS
were cultured axenically at 37C in TYI-S-33 medium, and
harvested during the logarithmic phase of growth. E. histolytica
trophozoites (25 105) were incubated with 10, 50, 150,
or 250 g/mL of an anti-LPPG pAb (IQ-P01) followed by
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Volume 27, Number 4, April 2005

FITC-labelled anti-rabbit mAb. Trophozoites incubated with

rabbit preimmune serum, with an irrelevant Ab [F (ab)2 rabbit
anti-human IgD] and only using the secondary Ab, were
used as negative controls. FACS analysis was performed with
a FACScan flow cytometer (Becton Dickinson); data were
analysed using the CellQuest software (BD Biosciences).
Additionally, E. histolytica trophozoites were harvested from
72-h cultures, placed on coverslips and allowed to adhere for
1 h at 37C. Cells were washed with PBS and fixed with
4% paraformaldehyde/01% glutaraldehyde for 1 h. Reactive
sites were blocked with 5% of goat serum for 1 h at 37C.
Monoclonal anti-E. histolytica LPPG antibody was added
and were incubated overnight at 37C. Samples were washed
and incubated for 90 min with an anti-mouse antibody coupled
to FITC subsequently. After mounting with Vectashield, samples
were analysed with a Zeiss 430 LSM confocal microscope.

LPPG isolation and identification

LPPG was isolated from trophozoites through a phenol extraction procedure as described by Westphal and Jann and
modified by Isibasi et al. (19). Briefly, washed trophozoites
were resuspended in distilled injectable water and disrupted
by five cycles of freezethawing at 20C and 37C, respectively.
The homogenate was ultracentrifuged at 100 000 g for 2 h.
The pellet was extracted twice with an equal volume of 90%
phenol at 68C for 30 min with constant mixing, and the
aqueous phase was recovered after centrifugation at 17 000 g
for 30 min, dialysed extensively against distilled water at 4C,
before ultracentrifugation at 100 000 g for 3 h. The pellet
containing LPPG was collected and strict quality controls
were carried out including analysis of the carbohydrate content by the method described by Dubois et al. (23), protein
content was quantified using the Lowry colorimetric protein
assay and LPS contamination was ruled out with the Limulus
test. A single batch of LPPG was used for all the experiments.
The LPPG sample used for the present study contained
344 mg /mL of carbohydrate, 412 mg /mL of protein, and
0020 endotoxin units (EU) per 1 g of carbohydrate, as
determined by the Limulus amoebocyte assay.
LPPG samples were separated by SDSPAGE on a 10% gel
under non-reducing conditions. Gels were stained using the
method of Tsai and Frasch for carbohydrate identification
(24). Samples were transferred to nitrocellulose, and Western
blotted with an anti-LPPG mAb IQ-Mn01, using goat antimouse horseradish-peroxidase-conjugated secondary antibodies
and 4-chloro-1-naphthol as a substrate (Sigma, St. Louis, MO).

E. histolytica LPPG signals through TLR2 and TLR4

with 09% saline solution, and peripheral blood mononuclear

cells (PBMC) were collected by density gradient centrifugation
using Lymphoprep (Nycomed, Oslo, Norway). Isolated PBMCs
were diluted in phosphate-buffered saline (PBS) containing
2 m EDTA, and monocytes were enriched by depleting
T cells, NK cells, B cells, dendritic cells, and basophils (Miltenyi
Biotec, Germany). Approximately 98% of the recovered cells
were monocytes, as determined by anti-CD14 FACS analysis.
Following purification, 1 106 monocytes were transferred
to 12-cell culture plates and maintained in RPMI 1640 tissueculture medium with 2% heat-inactivated FBS, at 37C under
a 5% CO2 atmosphere for 24 h prior to stimulation.

Stimulation of cells
Human monocytes (1 106) were stimulated with 010 g
of E. histolytica LPPG or E. coli 0111:B4 LPS for 2, 4, 6, or
24 h. For specific experiments, polymyxin B (5 g/mL) was
mixed with LPPG or LPS prior to usage for stimulation. In
order to block TLR2, cells were incubated with an anti-TLR2
polyclonal antibody (pAb, 2 g /mL at 37C) (N17; Santa
Cruz Biotechnology) 1 h prior to addition of the stimulant.

and
Quantification of human IL-10, IL-12p40, TNF-
and IL-6
IL-8, as well as murine TNF-
Cell-free culture supernatants were harvested and the concentrations of hIL-10, hIL-12p40, TNF- and hIL-8 were measured
(BD PharMingen, San Diego, CA). The concentration of each
sample was calculated by regression analysis using the mean
absorbance (average of duplicate readings of each sample was
used). The detection limit of this assay was 15 pg /mL. Additionally, hTNF- levels were determined by measuring the cytokine
cytotoxic effects of cell supernatants on TNF- sensitive murine
L929 fibroblasts, as previously described (25). Briefly, L929
cells (1 106) in 50 L DMEM supplemented with 10% FCS
were plated in 96-well plates for 2 h at 37C in the presence
of actinomycin D (1 g/mL; Sigma, St. Louis, MO). Fifty
L of the supernatant was added to each well and, after 18 h
incubation, cells were stained with 50 L of 01% neutral
red (Sigma, St. Louis, MO). Cultures were incubated for 2 h
at 37C under 5% CO2. The dye was extracted with 005
Na2HPO4 in 100 L v/v methanolwater, and the absorbance was measured at 570 nm. Recombinant human TNF (Sigma, St. Louis, MO) was used as reference standard
(average of triplicate readings of each sample was determined).

B dependent reporter gene assay

Transfections and NF-
Cell purification
Peripheral blood obtained from healthy volunteers was mixed
with 10 U/mL heparin or acid citrate dextrose, diluted 1 : 2
2005 Blackwell Publishing Ltd, Parasite Immunology, 27, 127 137

Human embryonic kidney (HEK) 293 cells were plated in

96-well dishes (3 104 cells/well) and transfected on the
following day by calcium phosphate precipitation (26) with

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expression plasmids for specific TLRs (TLR1 to TLR10), as

well as CD14, endothelial cell-leucocyte adhesion molecule
(ELAM-1) promoter-luciferase reporter, Rous sarcoma virus
(RSV) promoter--galactosidase reporter and MD2 (kind gift
from Kensuke Miyake, University of Tokyo, Japan). Cells were
treated with PAMPs for 18 h, lysed and luciferase activities
were measured using reagents from Promega Corp. (Madison,
WI). Transfection efficiency was normalized by relation of
luciferase activity to RSV--galactosidase activity. Values in
all diagrams represent mean values SD for one representative experiment in which each transfection was performed
in duplicate. Each experiment was repeated at least twice.

Mice and preparation of peritoneal macrophages

C3H/HeN mice were purchased from The Jackson Laboratory
(Bar Harbor, ME, USA). TLR2/ mice were provided by
Tularik Inc. Functional TLR4 deficient C3H/HeJ (TLR4d /d)
and double deficient mice (TLR2//TLR4d /d) were generated
as described (27). All mice were bred under specific pathogenfree conditions. Female mice (6 8 weeks old) were intraperitoneally injected with 2 mL of 4% thioglycollate medium
(Sigma, St. Louis, MO). Five days later, peritoneal exudate
cells were isolated by washing the peritoneal cavity with
ice-cold phosphate buffered saline (PBS), plated on 96-well
tissue culture-plates (12 105 cells/well) and cultured overnight in RPMI 1640 medium supplemented with 10% FBS.
Cells were then stimulated with 10 g /mL of LPPG or 1 ng/
mL CpG DNA (1668) for 18 h as indicated.

Viability of wild-type, TLR2 / , TLR4d/d and TLR2 / /

TLR4d/d mice upon LPPG challenge
Wild-type mice, TLR2/, TLR4d/d and TLR2//TLRT4d/d
were injected intraperitoneally with LPPG (1 mg) and galactosamine (20 mg) (Sigma) and viability was monitored
for 7 days. Three to four mice were used for each genotype
(experimental group).

Statistics
All experiment were carried out in duplicate and repeated
at least two times; results were expressed as the mean SD.
Comparisons between test were done by Students t-test with
statistical significance considered to be P < 005.

RESULTS
Entamoeba histolytica LPPG is a cell surface molecule
Purity of LPPG extracted from E. histolytica trophozoites
by the phenolwater method was assessed by electrophoretic

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mobility assay, silver-staining, Western blotting and Limulus

amoebocyte assay. LPPG migrated as polydisperse bands
between 10 and 107 kDa MW markers (Figure 1a). The antiLPPG mAb IQ-Mn01 specifically recognized various LPPG
variants distinguishable through their different sizes, whereas
it did not cross-react with two independent LPS samples
obtained from E. coli serotypes 0111:B4 and 055:B5 used
as negative controls (Figure 1b). Staining of E. histolytica
trophozoites with the LPPG-specific polyclonal antibody
(IQ-P01) for flow cytometry and monoclonal antibody
(IQ-Mn01) for fluorescence microscopy, showed that LPPG
is localized on the surface of E. histolytica trophozoites
(Figure 1c,d). These data indicated that the purified LPPG
preparation that was used throughout the experiments, was
specifically recognized by monoclonal and polyclonal antiLPPG antibodies and was endotoxin-free, as determined by
the Limulus amoebocyte assay.

and IL-8 production

LPPG induces IL-12, TNF-
Incubation of human monocytes with increasing amounts
of LPPG resulted in a consistent down-regulation of TLR2
mRNA (data not shown and Ref. 21), suggesting that downstream events such as expression of molecules involved in proand anti-inflammatory reactions might be affected as well.
We reported previously that IL-10 production was induced
in response to E. histolytica LPPG (21). To further characterize
the effects of LPPG, we estimated the production of IL12 and TNF- by human peripheral blood monocytes in
response to LPPG or LPS. Following 24 h of incubation
with LPPG (110 000 ng /well), considerable amounts of
IL-12p40 were detected in the supernatants of monocytes.
Maximum levels were reached at 24 h (6972 pg/mL, corresponding to a three-fold increase); and no IL-12 p40 was
detectable after 2, 4, or 6 h (Figure 2a). In contrast, a dosedependent production of TNF- was detected in response
to LPPG as early as 6 h after the onset of the experiment
(Figure 2c). As for LPPG, LPS-induced production of IL12p40 was maximal 24 h after stimulation start (1200 pg/
mL; Figure 2b) while TNF- release was detectable as early
as 6 h after stimulation (Figure 2d). When evaluating the
ability of LPPG (10 g/mL) to induce the production of
TNF- and IL-8 by monocytes and macrophages, we found
that macrophages produced high levels of TNF- and IL-8,
while monocytes produced 45 times lower amounts of both
cytokines. TNF- production by monocytes in response to
LPS was 810 fold higher than that produced upon LPPG
challenge (Figure 2e,f). Overall, the doses of LPPG required
to induce a detectable response were considerably higher
(10 g /mL) than those of LPS (1 ng/mL).
To eliminate the possibility that stimulation was due to
contaminant LPS in the LPPG sample, the effect of polymyxin
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E. histolytica LPPG signals through TLR2 and TLR4

Figure 1 LPPG expression by E. histolytica

trophozoite, gel mobility, carbohydrate
profiles, and surface localization. (a) Silver
stained polyacrylamide gel after
electrophoresis of isolated LPPG from
trophozoites of E. histolytica HMI:IMSS
shown as polydispersed bands (lane 2) or
LPS from E. coli 0111:B4 or 055:B5 (lane 1
and lane 3). (b) Immunoblot probed with
anti-LPPG mAb (IQ-Mn01). (c) LPPG
detection by flow cytometry in E. histolytica
trophozoites HM1:IMSS incubated with
an anti-LPPG pAb, and a FITC-labelled
anti-rabbit secondary mAb. Unstained
trophozoites (line 1), trophozoites incubated
with rabbit preimmune serum and
secondary mAb (line 2), with anti-LPPG
pAb and irrelevant mAb as secondary Ab
[FITC-F (ab)2 rabbit anti-human IgD]
(3) and irrelevant mAb and the secondary
Ab (4) were used as negative controls. LPPG
staining with 10 g/mL, 50 g /mL, 150 g /
mL, or 250 g/mL anti-LPPG pAb (IQP01) (lines 58). (d) Surface localization of
LPPG on fixed E. histolytica trophozoites
incubated with a monoclonal
anti-E. histolytica LPPG antibody and
a secondary anti-mouse antibody coupled
to FITC. Samples were analysed with
a confocal microscope.

B pre-treatment was assessed. The addition of polymyxin B

(5 g /mL) did not inhibit the production of TNF- by
monocytes in response to LPPG, yet it substantially reduced
the effect of E. coli LPS (Figure 3a,b). These results indicate
that the production of cytokines is due to intrinsic LPPG
activity and not to contaminant LPS, since the co-incubation
with polymyxin B did not alter the stimulatory effect of LPPG,
but only inhibited the stimulatory effect of LPS, as shown in
Figure 3a and 3b, respectively.

B through TLR2 and TLR4

LPPG activates NF-
TLR2-mediated signals have been shown to promote the
synthesis of pro-inflammatory cytokines. Since we had
previously shown that LPPG induced the synthesis of IL-10
(21), we assessed the ability of TLR2 to mediate recognition of LPPG and to induce the synthesis of anti- and
pro-inflammatory cytokines, such as IL-10 and TNF- in
monocytes preincubated with a TLR2-specific Ab prior to
stimulation with 10 g /mL LPPG or 100 ng /mL LPS for
12 h. Preincubation of cells with anti-TLR2 resulted in a
partial decrease of IL-10 and TNF- synthesis in response
to LPPG (Figure 3c,d). When cells were stimulated with
2005 Blackwell Publishing Ltd, Parasite Immunology, 27, 127137

LPS under the same experimental conditions, IL-10 production was significantly impaired, while TNF- production
was as only partially diminished. Thus, IL-10 and TNF-
production in response to LPPG is partially dependent on
TLR2-specific signal transduction.
The NF-B family of transcription factors controls the
expression of numerous cytokine gene, including those of
IL-10 and TNF-. Addition of 10 g /mL of LPPG, resulted
in NF-B translocation to the nucleus 2 h after stimulation
(data not shown). To determine if other TLRs were involved
in the LPPG-dependent NF-B recruitment, we transiently
transfected HEK-293 cells with expression-plasmids for
each of TLR1 to TLR10 and tested the ability of these cells
to respond to LPPG by application of an NF-B-dependent
ELAM-1 promoter luciferase reporter gene assay. Activation of the reporter gene in response to LPPG was detected
only in cells expressing TLR2 or TLR4 (Figure 4a,b). TLR2dependent NF-B activation increased in a LPPG dosedependent manner. Interestingly, TLR4-dependent NF-B
activation reached maximal levels at 1 g /mL of LPPG, while
TLR-2-dependent NF-B activation required higher doses.
Negative controls using TLR2 and TLR4 transfected cells,
which were not stimulated by LPPG, showed no response.

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Parasite Immunology

Figure 2 Activation of human monocytes

with LPPG from E. histolytica HM1:IMSS
induced IL-12p40, TNF- and IL-8
production. Cells (1 106) were stimulated
for 2, 4, 6, or 24 h with increasing amounts
of LPPG from E. histolytica HM-1:IMSS or
LPS from E. coli 0111:B4, as indicated.
IL-12p40 (a and b) concentrations were
measured in cell-free culture supernatants
by ELISA. TNF- release from cells
stimulated for 6 or 24 h, with varying
amounts (10 ng to 10 g) of LPPG or
LPS were measured by bioassay (c and d).
Each point represents the mean of three
determinations. Human monocytes and
macrophages were stimulated with either
LPPG from E. histolytica HM-1:IMSS
(10 g /mL) or LPS from E. coli 0111:B4
(100 ng /mL), for 6 h. TNF- and IL-8 levels
in cell culture supernatants of human
monocytes and macrophages were measured
by ELISA (e and f ). Each point represents
the mean of two determinations. One
representative experiment of two is shown.

Figure 3 Activation of human monocytes

by LPPG from E. histolytica HM1:IMSS
TLR2-dependent. LPPG activation of
human monocytes is unaffected by
polymyxin B. TNF- release from cells
stimulated with LPPG (10 g /mL) (a) or
LPS (100 ng /mL) (b) in the presence
of polymyxin B (5 g/mL) is shown.
Data represent mean values of three
determinations. One representative
experiment of three is shown. Human
monocytes were treated for 12 h with
10 g /mL LPPG or 100 ng /mL LPS with
or without 2 g /mL anti-TLR2 pAb.
Release of IL-10 (c) and TNF- (d) was
assessed. Data represent mean values of
two determinations. One representative
experiment of two is shown.

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E. histolytica LPPG signals through TLR2 and TLR4

Figure 4 LPPG from E. histolytica HM-1:IMSS activates TLR2 and TLR4. HEK-293 cells were transfected with TLR2/CD14 (a) or TLR4/
D2/CD14 expression plasmids (b) and ELAM-1 luciferase reporter plasmid. After 24 h, cells were stimulated with 01000 g /mL of LPPG,
1 g /mL of P3CSK4 or 1 g /mL of LPS (a, b, c) for 16 h. Luciferase activities were determined and related to empty vector control. All
diagrams are shown as mean ED for one representative experiment in which each transfection was performed in duplicate. Each experiment
was repeated at least twice in all cases. For control experiments HEK-293 cells were transfected with TLR1 TLR10/CD14/MD2 expression
plasmids and ELAM-1 luciferase reporter plasmid. After 24 h, cells were stimulated with LPPG (10 g /mL) and with specific stimulants
(TLR1, TLR4, TLR6 and TLR10: 1 g/mL LPS; TLR2: 1 g /mL Pam3Cys; TLR3: Poly IC; TLR5: flagellin of Listeria monocytogenes;
TLR7 and TLR8: Ibiquimod 848; TLR9: CpG 1668) for 16 h. Luciferase activities were determined and related to empty vector control (d).

On the other hand, positive controls using TLR2 and TLR4

transfected cells stimulated with specific positive stimulants
(P3Cys and LPS, respectively) showed an intense response
(Figure 4c). Cells transfected with TLR1, TLR3, or TLR5
to TLR10 did not respond to LPPG stimulation. Even though
similar expressions of the different TLRs were observed in
unstimulated cells, the stimulation with LPPG only led to
an increase in activity in cells transfected with TLR2 and
TLR4. The activation of the cells transfected with the other
TLRs was only achieved using specific ligands for each TLR
(Figure 4d). However, in the absence of good positive control
data for the other TLRs, a role for other TLRs cannot be ruled
out. Altogether these data strongly suggest that recognition
of LPPG through TLR2 and TLR4 initiates a signalling
cascade leading to cell activation.

Serum dependence of LPPG signalling through TLR2 but

not through TLR4
To evaluate the impact of serum components in the relative
participation of TLR2 and TLR4 in LPPG recognition,
we transfected HEK-293 cells with TLR2/CD14 or TLR4/
MD2/CD14 and tested their ability to respond to LPPG or
to a specific stimulus (P3CSK4 TLR2; LPS TLR4) in the
NF-B-dependent reporter gene assay applied in the presence
2005 Blackwell Publishing Ltd, Parasite Immunology, 27, 127137

or absence of serum. Luciferase activity in cells expressing

TLR2/CD14 was stronger when activated with LPPG in the
presence of serum as compared to their activation under
serum-free conditions (Figure 5a), whereas the response to
P3CSK4 was independent of serum. In contrast, when cells
were transfected with TLR4/CD14/MD2, no enhancement
in the LPPG-induced response was observed in the presence
of serum as compared to serum-free conditions (Figure 5b).
To assess the role of CD14 in TLR2 or TLR4 dependent cell
activation upon LPPG challenge, we measured the response
of HEK-293 cells transiently transfected with or without
CD14. When CD14 was co-expressed with TLR2 or TLR4/
MD2, the activity of the reporter gene in response to LPPG
was increased by approximately six-fold for TLR2 (Figure 5c)
and four-fold for TLR4 (Figure 5d) as compared to cells
expressing TLR2 or TLR4/MD2 alone. These data suggest
that CD14 potentiates the ability of TLR2 and TLR4 to mediate
cell activation upon E. histolyticas LPPG challenge.

TLR2/ and TLR4d/d macrophages are impaired in

and IL-6 release in response to LPPG and LPPG
TNF-
response in vivo
We tested whether macrophages isolated from TLR2/ and
TLR4d/d mice would respond to LPPG. Peritoneal macrophages

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C. Maldonado-Bernal et al.

Parasite Immunology

Figure 5 Serum dependence of TLR2- but not TLR4-mediated NF-B activation by LPPG and synergistic activation of NF-B by
co-expression of CD14 and TLR2 or TLR4. HEK-293 cells were transfected with TLR2/CD14 expression plasmid (a) or TLR4/MD2/CD14
expression plasmid (b) and ELAM-1 luciferase reporter plasmid. After 24 h cells were left unstimulated (white bar) or stimulated with specific
stimuli (P3CSK4 or LPS) (grey bar) or LPPG (10 g /mL) (black bar) for 16 h either in the presence or absence of 10% FCS before harvest.
All bar diagrams are shown as mean ED for one representative experiment in which each transfection was performed in duplicate. Each
experiment was repeated at least twice in all cases. Luciferase activities were related to that of empty vector control. HEK-293 cells were
transfected with ELAM-1 luciferase reporter plasmid and expression vectors for TLR2 (c), TLR4/MD2 (d) and co-transfected with or without
CD14 as indicated. After 24 h cells were either left untreated (white bar) or stimulated for 16 h with specific stimuli (P 3CSK4 or LPS)
(grey bar) or LPPG (10 g /mL) (black bar) prior to harvest. Luciferase activities were related to empty vector control. All bar diagrams
are shown as mean ED for one representative experiment in which each transfection was performed in duplicate. Each experiment was
repeated at least twice in all cases.

from wild-type mice produced TNF- and IL-6 in response

to LPPG in the presence or absence of IFN- (30 U/mL).
However, the ability of TLR2/ and TLR4d /d macrophages
to produce these inflammatory cytokines in response to LPPG
was significantly impaired under either experimental condition (Figure 6a and data no shown).
Next, wild-type, TLR2/, TLR4d /d and TLR2//TLR4d /d
mice were challenged with LPPG and galactosamine, simulating
a low dose endotoxic shock. Wild-type mice and TLR2/ all
died within 12 h after administration of LPPG, whereas
TLR2//TLR4d /d and TLR4d /d survived the LPPG challenge
(Figure 6b). These data suggest that activation of immune
cells by E. histolytica LPPG causes the release of an inflammatory mediator such as TNF- and IL-6 through TLR2
and TLR4, and that TLR4 drives LPPG-dependent lethal shock.

DISCUSSION
The initial resistance to E. histolytica infection depends on
the innate immunity of the host. Mucins in the colonic

134

epithelium (28) and complement activation have been suggested

as being particularly important for protection (29). However,
the complexity of effects observed during an infection with
E. histolytica suggests that additional components of innate
immunity participate in the hosts response to this parasite.
Because LPPG is a cell surface molecule, it is directly exposed
to the hosts immune system, promoting activation of innate
immunity effector cells.
The balance between pro- and anti-inflammatory cytokines
produced by monocytes and macrophages regulates innate
immune responses. Consistent with a recent report showing
down-regulation of the innate immune response to be harmful
for the host if infected with pathogenic microbes (30), our
results show that following challenge with LPPG for 24 h,
human monocytes released IL-10 and IL-12p40. IL-10 is
an anti-inflammatory and immunosuppressive cytokine
controlling macrophage effector functions. The mechanisms
utilized by IL-10 to reverse inflammation include inhibition
of NF-B activation and TLR synthesis, deactivation of
professional phagocytes, and limitation of ongoing immune
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Volume 27, Number 4, April 2005

Figure 6 Responsiveness and lethality upon challenge of wild-type,

TLR2 /, TLR4d /d, and TLR2//TLR4d /d peritoneal macrophages
and mice with LPPG. (a) Thioglycollate-elicited peritoneal
macrophages from wild-type (C3H/HeN), TLR2/ and TLR4d /d
mice were cultured in presence or absence of IFN- (30 U/mL) for
16 h and subsequent stimulation with CpG DNA (1668) or 10 g /
mL of LPPG for 18 h. Concentrations of IL-6 in the culture
supernatants were measured by ELISA. These data represent
the average of four mice. One representative experiment of two
is shown. (b) Age-matched wild-type, TLR2/, TLR4d /d and
TLR2//TLR4d /d mice were injected i.p. with LPPG (1 mg) and
-galactosamine (20 mg) and survival was monitored for 7 days.
Four sex-matched mice were used in each experimental group
except for TLR2/ for which 3 mice were applied.

responses and prevention of further inflammation (31).

We had previously reported that in the presence of LPPG,
TLR2 mRNA accumulation was inhibited in a dose-dependent
manner (ref 19, and data not shown), thus eventually leading
to a suppression of the immune response which might
contribute to the persistence of E. histolytica in the host.
Whether the LPPG-mediated IL-10 induction initiates this
down-modulation TLR2 expression, promoting a transitory
immunosuppression, allowing survival of amoebic trophozoites
in the liver remains to be demonstrated.
IL-12 is a heterodimer composed of IL-12 p35 and IL-12
p40 subunits bound via disulphide bonds and secreted as
a biologically active IL-12 p70 molecule. IL-12 produced
during the early phase of an infection promotes the differentiation of CD4+ effector T cells to a Th1 phenotype that
supports cell-mediated immunity, cytotoxic T cell generation,
activation of phagocytic cells, and eventually eradication of
2005 Blackwell Publishing Ltd, Parasite Immunology, 27, 127137

E. histolytica LPPG signals through TLR2 and TLR4

intracellular pathogens (32). In this study we detected IL-12

p40, which also functions as a subunit of IL-23 (33).
TNF- has been shown to play an important role in the
development of amoebiasis. Its production is related to either
amoebic survival and formation of granulomas, or to amoebic
death through an increase in NO-dependent cytotoxicity
resulting from high-level expression of inducible nitric oxide
synthase (iNOS) (34,35). High levels of TNF- promote
amoebicidal activity, whereas low levels favour the development of amoebic granulomas (35). TNF- stimulates fibroblast growth and granulocyte activation; although it inhibits
migration of neutrophils , it increases their adherence and
tissue-damaging properties. In addition, accumulation of
macrophages and white blood cells, as well as epithelioid
differentiation, have also been attributed to TNF- activity
(36). All these TNF- effects might contribute to the development of liver abscesses. Supporting this hypothesis, a recent
report shows that during the early stages (6 h) of E. histolytica
infection in an experimental amoebic liver abscess model,
TNF- is produced at the inflammatory focus. Although the
number of mononuclear cells increased as the infection progressed, no significant increase in TNF- positive cells was
observed, suggesting that inflammatory cells are not properly
activated to produce large amounts of TNF- (34). This is
consistent with our data showing that TNF- production
by human monocytes in response to LPPG treatment was
detectable 6 h after stimulation, but was no longer detectable
24 h after challenge, indicating transient response.
The fact that cytokine production in response to LPPG was
only partially blocked by an anti-TLR2 antibody suggested
that additional innate immune receptors were involved in
LPPG recognition by human monocytes. This was further
confirmed by data from experiments in which HEK-293
cells were transfected with cDNAs encoding for TLR110.
Specifically, overexpression of TLR2 or TLR4 conferred to
HEK-293 cells the ability to respond via these two receptors
to challenge with LPPG. Furthermore, transfection of TLR2
or TLR4 with CD14 resulted in a synergistic effect on reporter
gene activity, suggesting that in addition to TLR2 and TLR4,
CD14 contributed to generation of intracellular signals that
led to LPPG-dependent NF-B recruitment. Yet a role
for other TLRs cannot be completely ruled out, due to the
absence of good positive data for the other TLRs, since the
stimulatory effect of their specific ligands was low as
compared to the effect obtained with specific ligands for
TLR2 and TLR4.
In this study we found that the interaction of LPPG
with TLR2 and TLR4 resulted in IL-6 and TNF- release.
Consistent with these results, our data obtained with
macrophages of TLR2/ or TLR4d/d mice show that these cells
were severely impaired in terms of IL-6 and TNF- production as compared to macrophages isolated from wild type

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C. Maldonado-Bernal et al.

mice. This in turn could lead to a partial blockade of the

suppressive effect of CD4+CD25+ TR cells, and activation
of a pathogen-specific adaptive immune responses as has
been suggested recently (37).
Induction of pro- and anti-inflammatory cytokines, costimulatory molecules, adhesion molecules and many other
molecules requires specific signalling cascades in order to
activate the transcription of genes (38). Following recognition of specific PAMPs, TLRs activate intracellular signalling cascades leading to activation of NF-B. The NF-B
family of transcription factors plays an essential role in
regulating the transcription of pro- and anti-inflammatory
cytokine genes (39,40). In addition to the NF-B-dependent
reporter gene activity we found in HEK-293 cells expressing
TLR2 or TLR4, we show that the IL-8 gene, which is regulated by NF-B, is induced in monocytes and macrophages
in response to LPPG. IL-8 is a chemokine involved in activation of neutrophil chemotaxis which in turn plays an
important role in E. histolytica physiopathology (41).
Our results from an in vivo experiment with knockout
mice, suggest that endotoxic shock in response to LPPG was
dependent on hyperactivation through TLR4, probably
implicating the lipid moiety of LPPG in this effect, and not
the peptide fraction of the molecule.
Entamoeba histolytica LPPG has been considered as a
virulence factor, since some experiments demonstrated that
an anti-LPPG mAb protected against liver abscess formation (22) and that LPPG can be transferred to enteric cell
layers in vitro and ex vivo (42). The interaction of the LPPG
with enteric cells, probably through TLR4 and NF-B
activation, can explain an inflammatory response mediated
by cytokine secretions that induce high fever, leucocytosis,
tachycardia, etc. The inflammatory response could be quickly
controlled through of the down-regulation of TLR2, with
IL-10 production by compensatory anti-inflammatory process
(21).
Lethality induced through septic shock has been reported
as a complication caused by amoebic liver abscess (ALA) (43)
which could be explained by cellular hyperactivation through
TLR4 induced by LPPG in monocytes/macrophages, resulting in systemic inflammatory response syndrome (SIRS),
acute respiratory insufficiency syndrome (SIRA), multiple
organic failure (MOF) or coagulation intravascular disseminated (CID).
On the other hand, it has been reported that sepsis may
result from amoebic liver abscess. These evidences could be
explained by the observation that E. histolytica suppresses
both the macrophage respiratory burst and antigen presentation by class II major-histocompatibility-complex (MHC)
molecules (2). It could further lead to immunological paralysis, making development of a secondary bacterial infection
likely (44).

136

Parasite Immunology

In summary, our results demonstrate that LPPG from

E. histolytica HM-1:IMSS functions as a immuno-stimulatory
PAMP, inducing NF-B activation and TNF- IL-6 IL-8,
IL-12p40 and IL-10 synthesis, all critical components of an
innate immune response. Moreover, our data demonstrate
that LPPG is a TLR2 and TLR4 agonist and suggest that
within the process of amoebahost interaction, LPPG
participates in the activation of innate immunity, inducing
synthesis and secretion of soluble mediators which regulate
the adaptive immune response (45). This dual effect may
help to understand the broad spectrum of effects observed
during an E. histolytica infection.

ACKNOWLEDGEMENTS
This work was supported by CONACYT, Ref. 31 005-M
and 110562. We thank Antonio Ramrez, Jess Ramos, and
Jos Delgado Domnguez for excellent technical assistance.
We are very grateful to Gustavo Pedraza-Alva and Anglica
Santana for helpful discussions.

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