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Divisions of
* Infectious Diseases and
Nephrology, Departments of Medicine and
Pathology, University of Virginia, Charlottesville, VA 22908; and
Fralin Biotechnology Center, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, or inducible NO
synthase. Infection was a chronic and nonhealing cecitis that
pathologically mirrored human disease. Characterization of the
inflammation by gene chip analysis revealed abundant mast cell
activity. Parasite-specific Ab and cellular proliferative responses
were robust and marked by IL-4 and IL-13 production. Depletion of
CD4+ cells significantly diminished both parasite burden
and inflammation and correlated with decreased IL-4 and IL-13
production and loss of mast cell infiltration. This model reveals
important immune factors that influence susceptibility to infection and
demonstrates for the first time the pathologic contribution of the host
immune response in amebiasis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production, yet such studies
originated from hospitalized patients who required antibiotic therapy
for cure (5). In vitro a protective role for IFN-,
TNF-
, and NO in phagocyte killing of E. histolytica
trophozoites has been demonstrated (6, 7). The paucity of animal models of intestinal infection has detracted from the ability to define the mechanisms of innate and acquired immunity in amebic colitis. The naturally transmissible cyst form of the parasite has never been successfully cultured in vitro; therefore, attempts at experimental intestinal infection have relied on challenge with the invasive trophozoite form. Since Lösch reproduced intestinal amebiasis in dogs with human dysenteric stool in 1875 there have been numerous attempts at an intestinal model using oral or intraintestinal inoculation of trophozoites into outbred, inbred, and immune-deficient animals. Difficulties have arisen in achieving either durable infection beyond 10 days (8, 9) or reproducibility (10). Ghosh and colleagues (11, 12, 13) reported a promising model using the C3H mouse with intracecal inoculation of laboratory strain trophozoites, characterized by systemic Ab and delayed-type hypersensitivity responses to the parasite and infiltrating IgA+ cecal lymphocytes. Yet fundamental questions of whether innate immunity can resist establishment of infection and whether parasite-specific humoral or CMI responses can protect from disease have remained unanswered.
In this work intracecal inoculation of mice with E. histolytica trophozoites has been used to explore the nature and function of the immune response in amebic colitis. BALB/c and C57BL/6 mice were innately resistant to intestinal challenge with E. histolytica. Infection in C3H mice was a chronic cecitis, characterized by massive inflammation and epithelial ulceration. A deleterious acquired CD4+ T cell response exacerbated disease in these animals, as evidenced by parasite burden and intestinal pathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-week-old female C3H/HeJ, C3H/HeOuJ, BALB/c, C57BL/6, as well
as IFN-, IL-12 p40, and inducible NO synthase (iNOS) knockout mice
were purchased from The Jackson Laboratory (Bar Harbor, ME); C3H/HeN
mice were purchased from the National Cancer Institute (Frederick, MD).
Golden Syrian hamsters were purchased from Harlan (Indianapolis, IN).
Animals were maintained under specific-pathogen-free conditions at the
University of Virginia and all protocols were approved by the
Institutional Animal Care and Use Committee.
Parasites and Ags
Trophozoites were cultured in trypsin-yeast-iron (TYI-S-33) medium (14). Laboratory strain HM1:IMSS trophozoites were passaged through hamster liver (15) then grown in the bacterial flora of the xenic strain CDC:0784 supplemented with 0.01% erythromycin (Sigma-Aldrich, St. Louis, MO). Log-phase growth trophozoites were iced and spun (900 x g for 5 min) before intracecal inoculation. E. histolytica lectin was purified as described (16). Soluble amebic Ag (SAA) was obtained from the supernatant of axenic trophozoites washed in sterile HBSS, lysed by freeze-thaw, and spun (10,000 x g for 10 min). Both lectin and SAA were endotoxin free using the Limulus amebocyte lysate assay (<0.1 EU/ml).
Intracecal inoculation
We anesthetized mice with ketamine/xylazine, shaved their
abdomens, incised the skin and peritoneum, exteriorized the cecum, and
injected 150 µl of trophozoite pellet (
1 x
107 trophozoites) into the proximal, middle, and
apical cecum. Sham-challenged mice were injected with 150 µl of
trophozoite culture supernatant. Ceca were blotted, the peritoneum was
sutured, and the skin was stapled. Mice were kept on 37°C warming
blankets throughout. Survival was 
90% in all strains.
Pathology of murine amebic colitis and immunohistochemistry
Mice were sacrificed, the cecum was fixed in 10% buffered formalin phosphate or Hollande’s fixative and then cut into four to six equal cross-sections and paraffin embedded, and 4-µm slides were stained with H&E or periodic acid-Schiff. Cecal thickness was measured at two or more sites with an ocular micrometer at x40 magnification. For c-kit immunohistochemistry rabbit polyclonal Ab to human kit (cross-reacts with mouse c-kit; Research Diagnostics, Flanders, NJ) was used at a 1/250 dilution followed by avidin-biotin peroxidase, diaminobenzidene, and hematoxylin. PBS was used as negative control and revealed no background staining.
Pathology was scored blindly for each section as follows and averaged for each mouse. Numbers of ameba were scored 0–5 (0, none; 1, rare and difficult to locate; 2, occasional, up to 10% of the lumen occupied by ameba; 3, moderate, up to 25% of lumen occupied; 4, heavy, up to 50% of lumen occupied; 5, virtually complete occupation of the lumen by ameba). Extent of ulceration was measured by estimating the percentage of the lumenal circumference that was ulcerated (0–100%). Degree of inflammation was scored 0–5 (0, normal; 1, mucosal hyperplasia, mild to moderate increase of lymphocytes in mucosa and submucosa with no neutrophil infiltration; 2, increased chronic inflammation, with spotty infiltration of neutrophils, not involving the entire thickness of the mucosa; 3, increased chronic inflammation with marked increase in neutrophil infiltration, involving full thickness of mucosa; 4, marked neutrophil infiltration of mucosa and submucosa, with tissue architecture intact; 5, complete destruction of cecal architecture by inflammation).
Fecal Ag detection
The E. histolytica II stool ELISA kit (TechLab, Blacksburg, VA) was used according to the manufacturer’s instructions. Three fecal pellets per mouse were assayed within 1 h of collection and background was subtracted from sample OD450.
Serum and fecal Ab determinations
Serum was obtained from orbital plexus blood and fecal samples were prepared from four stool pellets per mouse vortexed into a suspension with protease inhibitors (Complete; Roche, Mannheim, Germany) and spun at 900 x g and then 10,000 x g. Ab levels were assayed in duplicate by ELISA (17) using lectin-coated plates (3.5 µg/ml) and HRP-conjugated anti-mouse IgG, IgG1, IgG2a, IgA, and IgE secondary Abs (Southern Biotechnology Associates, Birmingham, AL). Background ELISA OD from the serum of naive mice was subtracted from sample ELISA OD.
Mesenteric lymph node (MLN) proliferation assays and cytokine determinations
Single-cell suspensions of MLNs, spleens, and PBMCs were
prepared as described (18). MLN cells (1 x
105) were cultured in round-bottom 96-well plates
(Costar, Corning, NY) in Dulbecco’s complete medium supplemented with
10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml
gentamicin, and 5 x 10-5 2-ME for 72
h at 5% CO2, with or without 0.1 µg/ml lectin,
100 µg/ml SAA, or 0.1 µg plate-bound anti-murine CD3
(145-2C11), then pulsed for 12 h with 10 µCi
[3H]thymidine, and proliferation was counted
using a Trilux scintillation counter (Wallac, Turku, Finland).
Ag-specific proliferation was determined after subtracting background
proliferation in medium alone. Supernatants were pooled at
72 h and analyzed in duplicate for IL-4, IL-13, and IFN-
production using ELISA sets (R&D Systems, Minneapolis, MN) per the
manufacturer’s instructions.
RNA protection analysis and Affymetrix gene chip analysis
Total RNA was isolated from rinsed cecal tissue and MLN with the Qiagen RNAEasy kit (Qiagen, Valencia, CA). RNase protection analysis was performed using the mck-1b template (BD PharMingen, San Diego, CA) per the manufacturer’s instructions. Affymetrix gene chip analysis (Affymetrix, Santa Clara, CA) was performed per the manufacturer’s instructions using murine MgU74Av2, MgU74Bv2, and MgU74Cv2 arrays. The in vitro transcription reaction product was purified and analyzed by gel electrophoresis to confirm the size range. Results were analyzed using D-chip analysis software (19), which reported average difference of hybridization intensity between perfect match and mismatch and fold change between the three infected vs three sham-challenged cecal samples.
In vivo CD4 cell depletion and flow cytometry
One milligram of rat IgG2a anti-murine CD4 (GK1.5) or
purified rat IgG (catalog no. I8015; Sigma-Aldrich) was injected i.p.
on days -1, +2, +5, and +8 relative to intracecal challenge. Flow
cytometry was performed using FITC-labeled Abs to murine CD4 (GK1.5),
CD8 (53-6.7) (both from BD PharMingen), and Ig H and L chains
(Rockland, Gilbertsville, PA), and to rat Ig 
(RG7) as previously
described (18). Staining with anti-rat Ig indicated
no masking of CD4 by persistent GK1.5 Ab.
Statistics
Group means were compared by the Student t test or the alternate Welch test and infection rates were compared by Fisher’s exact test. All p values are two-tailed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intracecal injection of trophozoites into C3H/HeJ mice led to
chronic cecal infection in 60% (112 of 186) of mice as determined by
histopathology (Fig. 1
A).
Infection did not spontaneously clear and was documented to persist
beyond 18 mo postchallenge. Infected ceca were thickened and contracted
on gross inspection. Histopathologic changes were evident as early as 4
days after challenge, including crypt hyperplasia, epithelial
ulceration, and submucosal infiltration. At such early time points
viable ameba were usually seen only at areas of epithelial ulceration,
yet by 3 wk postinoculation ameba had extended into the lumen.
Inflammation was severe and contained plasma cells, neutrophils, and
mast cells. By 10 wk inflammation obscured the entire mucosa and
morphologically resembled human colitis (Fig. 1B). Disease
was limited to the cecum (the most common segment affected in humans;
Ref. 20), which the gastrointestinal tract was able to
bypass, perhaps explaining why infected mice did not become ill or lose
weight. The model mirrored human infection in important ways: the
morphology of the inflammatory infiltrate, the burden of trophozoites
within the mucosa without submucosal invasion or liver abscess
formation (these are the exception in human amebic colitis; Ref.
21), and the absence of parasite cyst development with
invasive infection.
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C). Fecal Ag excretion was thus measured after the first
week for the purposes of detecting and quantifying successful
infection, and the sensitivity of the test was 58% (152 of 263) for a
single measurement and 90% (43 of 38) for three measurements. Cecal
pathology was entirely normal at all time points in
challenged/uninfected mice as demonstrated by cecal thickness (Fig. 1D). Susceptibility to intestinal infection with E. histolytica is parasite and mouse strain dependent
The infection rate in C3H/HeJ mice was higher for
trophozoites cultured in bacterial flora before intracecal challenge
(15 of 39 vs 8 of 48, p = 0.03) and ultimately 60%
(112 of 186) with a trophozoite strain that was in vivo passaged
through hamster liver abscess. Using these same trophozoites we found
that C3H/HeOuJ and C3H/HeN strains had similar infection rates and
disease severity (Table I). We
quantified disease severity in infected mice by fecal Ag excretion and
scoring of histopathology. Cross-sections of infected ceca were
examined histopathologically for numbers of ameba, extent of
ulceration, and severity of inflammation as detailed in Materials
and Methods. The comparable infection rates and disease severities
by histopathology of the C3H/HeOuJ strain ruled out the defective LPS
signaling of the C3H/HeJ (22) as the explanation for its
susceptibility. C57BL/6 mice and BALB/c mice were relatively resistant
to initial infection; however, when it occurred (in 4 of 27 C57BL/6
mice) fecal Ag and disease severity was again similar to that of
C3H/HeJ (Table I). To understand whether innate resistance in the
C57BL/6 or BALB/c strains was due to more robust innate production of
IL-12, IFN-, or NO we tested susceptibility in genetically deficient
mice but found that resistance persisted. Thus, there were mouse
strain-dependent differences in the initial susceptibility to infection
but not in the development of amebic colitis once successful infection
occurred.
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Given the significant inflammation in murine amebic colitis, yet
the failure of acquired immunity to clear the infection once
established, we sought to characterize this nonhealing immune response
in the C3H/HeJ mouse. We measured Ab against the E.
histolytica adherence lectin, a major virulence factor, by ELISA.
Mice at 5–7 wk duration of infection produced significantly
higher-titer serum antiadherence lectin IgG, IgG1, IgG2a, IgE, and
fecal IgA than challenged/uninfected mice (Fig. 2A). Gross inspection of
infected mice revealed enlarged MLNs, although flow cytometry of MLN
revealed similar percentages of CD4+,
CD8+, and B lymphocytes among infected,
challenged/uninfected, and wild-type mice (mean 41 ± 1.4, 17
± 0.4, 18 ± 1.1; n = 11). However, the MLN cells
from infected mice demonstrated significantly higher proliferation in
response to SAA than those from challenged/uninfected or wild-type mice
(Fig. 2B).
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We analyzed the production of cytokines from the in vitro
stimulated MLN cultures for IL-4, IL-13, and IFN- by ELISA (Fig. 3A). This revealed
significantly higher IL-4 and IL-13 production in response to SAA or
anti-CD3 in infected compared with challenged/uninfected mice.
Significantly higher quantities of IFN- were produced in response to
anti-CD3 in the MLN cultures from infected mice, but
parasite-specific IFN- production was negligible (<0.1 ng/ml).
RNase protection analysis of MLN RNA from infected vs sham-challenged
mice at 10 wk postinoculation confirmed increases in IL-4 with minimal
and perhaps diminished IFN- expression (Fig. 3B).
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B). Increased levels of IL-4
and IL-13 and minimal IFN- mRNA expression were again observed in
infected mice. Thus, the consistent finding in MLN and cecum was the
production of IL-4 and IL-13 in response to E. histolytica
infection; while infected MLN were capable of producing IFN-,
production was not observed in response to parasite Ag or in situ in
the infected cecum. The elevated serum anti-lectin IgG2a (dependent
upon IFN- for its production) might therefore represent Ag-specific
skewing of cytokine production (lectin vs SAA) or dissociation between
splenic and MLN parasite-specific cytokine production, as has been
described in gastrointestinal helminth infections
(23).
To more broadly characterize gene expression during the inflammation of
murine amebic colitis, we performed Affymetrix gene chip hybridization
on cecal RNA from the same three infected and three sham-challenged
mice. Differences in expression by gene chip correlated with visual
differences in expression by RNase protection for 89% (8 of 9) of
genes (the exception was IL-15, where gene chip indicated decreased
expression in infected mice; average difference was 69.3 ± 3.3 vs
164 ± 5.0 for controls (p < 0.0001),
while RNase protection showed increase in MLN and decrease in cecum).
Of the total of 9645 genes or expressed sequence tags probed by
gene chip, mast cell proteases accounted for 4 of the 20 most
overexpressed genes in infected mice vs controls (Fig. 3C).
With 95% confidence, expression of mast cell protease 2, mast cell
protease-like protein, mast cell chymase 2, and mast cell protease 1
were 25-, 23-, 8.4-, and 7.3-fold increased, respectively
(p < 0.0007). There was no evidence for
increased expression of the mast cell-inducing cytokines IL-3 or IL-9
by gene chip (both cytokines scored as absent in five of six mice) or
the mast cell effector cytokine TNF- (scored as absent for six of
six mice by gene chip). Furthermore, there was no evidence for an
increase in the Th3/Tr1 T cell population by TGF-
1 or IL-10
expression levels (both cytokines scored as absent for five of six
mice). Differences in production of these cytokines due to
posttranscriptional regulation or using other detection techniques
remain possible.
CD4+ T cell depletion paradoxically decreased parasite burden and intestinal inflammation
Because CD4+ T cells are a primary source of
IL-4 and IL-13, we depleted CD4+ T lymphocytes to
test their impact on the course of infection. Mice were administered 1
mg of anti-CD4 mAb (vs control rat IgG) i.p. on days -1, +2, +5,
and +8 relative to inoculation with E. histolytica, which
resulted in >90% CD4+ T cell depletion from
PBMCs, splenocytes, and MLN by FACS analysis during wk 1 and 4
postchallenge (data not shown). Mice were sacrificed at wk 4 and, as
expected, the depletion of CD4+ T cells had no
effect on the infection rate (5 of 17 in CD4-depleted mice vs 8 of 18
control mice, p = 0.5), because this appeared dependent
on innate immune responses. However, fecal Ag was paradoxically
decreased in the CD4-depleted/infected vs control/infected mice
(Fig. 4A). Furthermore, on
histopathology, while CD4-depleted/infected mice still demonstrated
crypt hyperplasia and mild submucosal infiltration, the numbers of
ameba, extent of ulceration, and degree of inflammation were each
significantly decreased compared with control/infected mice (Fig. 4B; representative photomicrographs are shown in Fig. 4, C vs D). Immunohistochemistry on cecal tissue for
the mast cell marker c-kit additionally revealed a
significant decrease in the number of
c-kit+ cells per section in
CD4-depleted/infected mice (171 ± 29 vs 29 ± 16,
p = 0.005) (Fig. 4C). CD4-depleted/infected
mice had comparable numbers of c-kit+ cells
as uninfected and non-CD4-depleted mice (data not shown). Although
surface c-kit expression can be found on other cells such as
precursor T cells and intraepithelial lymphocytes (24),
the c-kit+ cells had mast cell morphology
on adjacent H&E-stained sections. Correlation with cytokine production
was obtained by stimulating MLN with SAA or anti-CD3, and
CD4-depleted/infected mice demonstrated significantly lower IL-4 and
IL-13 production but a similar IFN- pattern compared with
control/infected mice (Fig. 4E). Thus, severity of amebic
colitis correlated with Ag-specific IL-4 and IL-13 production and
submucosal mastocytosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We suspect that the mitigation in disease results from depletion of CD4+ T cells, because production of characteristic T cell cytokines IL-4 and IL-13 was diminished with the depletion (although a role for CD4+ macrophages cannot be ruled out). We thereby envision at least two mechanisms to explain how the CD4 depletion diminishes amebic colitis: 1) parasite-primed CD4+ T cells cause intestinal inflammation through effector cytokine production including IL-4 and/or inducing mastocytosis or 2) the CD4+ depletion acts primarily through decreasing parasite numbers.
This first concept of host CD4+ T cells directly
mediating intestinal inflammation is well established from mouse models
of inflammatory bowel disease (a condition that can resemble amebic
colitis pathologically). These are heterogeneous models of either
spontaneous or induced inflammation, most of which correlate with Th1
cytokine production (29). Yet Th2 models of intestinal
inflammation exist, with a direct role for unbalanced IL-4 production
documented in the pathogenesis of oxazolone-induced, TCR- knockout,
and trinitrobenzene sulfonic acid colitis in the BALB/c mouse
(30, 31, 32). Furthermore, even in Th1 models of colitis,
late-stage disease has been characterized by elevated IL-4 and IL-13
production (33). It is of interest that, similar to this
model of amebic colitis, the Th2 models of inflammatory bowel disease
are generally characterized by their colonic location (as opposed to
small bowel) and by a heavily disrupted epithelium. It is postulated
that the development of Th2 intestinal inflammation depends on a
combination of the gastrointestinal tract location, the specific Ag,
and the mouse strain. As such, the development of amebic colitis may be
a net result of the combination of the cecum and chronic Ag stimulation
with E. histolytica. Some Th2 colitis models are marked by
an anti-inflammatory suppressor TGF- response (31);
thus, given the robust ongoing cell proliferation to parasite Ag and
lack of detectable TGF- and IL-10 mRNA expression in this model, the
role of an insufficient Th3 suppressor response (34) in
the perpetuation of this inflammation seems reasonable.
The other plausible explanation we can envision, that
CD4+ T cells mount a counterproductive response
against the parasite, is inherently supported by the decrease in ameba
numbers upon CD4+ depletion. Available data
indicate that macrophages and neutrophils kill E.
histolytica trophozoites more efficiently when activated by
IFN- and/or TNF- (35). Thus, a reasonable
explanation for the increased parasite numbers in
CD4+ T cell-depleted mice is through the
down-regulatory activity of IL-4 on protective macrophage function
(36).
Assuming that the primary effect of CD4+ T cell depletion on this model is through increasing parasite numbers, one easily finds support that this parasite can directly cause inflammation. The propensity of E. histolytica to destroy human tissues has been apparent since 1903, when Schaudinn named the parasite "histolytica." The ability of E. histolytica to damage intestinal epithelium, marked by IL-8 and IL-1 release, has been shown in vitro (37) and in vivo using the SCID-HU-INT model (38). Yet the trophozoite’s action on host epithelium is complex, as we have demonstrated previously in this model that in the infected colon intestinal cells undergo apoptosis as well (purportedly an "anti-inflammatory" process) (39).
The association of mast cell activity with deleterious immunity in this model is intriguing. In murine nematode infection, mast cells and their proteases, specifically mucosal mast cell protease-1, enhance parasite clearance (40), possibly through increased vascular and epithelial permeability. Taken with the fact that trophozoites appear to favor areas of epithelial breakdown, one wonders whether to some degree E. histolytica benefits from epithelial cell permeability. Mast cells could also be directly causing inflammation through production of proteases or proinflammatory cytokines, or through IgE-dependent neutrophil and monocyte recruitment (41).
This model also indicates that innate immunity can prevent
establishment of infection in mice, a process that occurs both
mouse-to-mouse within the C3H/HeJ strain and among the C3H, BALB/c, and
C57BL/6 strains. Therefore, the mechanism of this innate protection may
include combinations of genetics, environmental factors such as
different bacterial flora in the intestine, and parasite factors (given
that parasites grown in bacteria led to a higher infection rate). Mast
cells are a critical innate immune component for survival in a cecal
puncture sepsis model through Toll-like receptor-4-dependent processes
(42) and TNF- production (43), but at
present this model does not support these mechanisms in protection
against E. histolytica given the similar susceptibilities
across C3H strains (Toll-like receptor-4 dominant negative or wild
type) and >90% survival in all mice. Indeed, we can only conclude
that the resistance to initial infection with E. histolytica
in the BALB/c and C57BL/56 strains cannot be solely attributed to
robust innate macrophage activity given the persistence of resistance
in IL-12, IFN-, and iNOS knockout animals.
The final issue is how this model relates to the human condition. Foremost, this mouse model of amebic colitis demonstrates compatible pathology with human disease (21). As for observations from humans that CD4+ T cells contribute to disease, early reports of amebic colitis in patients receiving immunosuppression (44) suggest that in some patients CMI responses may be protective. However, the expected increases in invasive amebiasis among HIV-infected patients have not been realized (45) despite an up to 43% incidence of intestinal carriage in such individuals (46). It is an exciting and important possibility that certain individuals are predisposed to either acquiring infection or, based on divergent CD4+ T cell responses to the parasite once infection is acquired, developing inflammation and disease. It is hoped that through much needed field studies on the epidemiology of amebiasis in developing countries combined with future work to define the mechanism of immunopathogenesis in this model we can bring new insight into how to protect humans from this important worldwide disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. William A. Petri, Jr., Division of Infectious Diseases, University of Virginia School of Medicine, 300 Lane Road, P.O. Box 801340, MR4 Building, Room 2115, Charlottesville, VA 22908-1340. E-mail address: wap3g{at}virginia.edu
3 Abbreviations used in this paper: CMI, cell-mediated immune; MLN, mesenteric lymph node; SAA, soluble amebic Ag; iNOS, inducible NO synthase.
Received for publication June 21, 2002. Accepted for publication August 5, 2002.
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References |
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