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,
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Departments of Microbiology, Immunology, and Medicine, Dartmouth Medical School, Lebanon, NH 03756;
Institut Pasteur, Unite Biologie Moleculaire du Gene, Paris, France;
Departement de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, Nouzilly, France; and
Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, and NO, as has
been reported in other experimental models of human inflammatory bowel
disease. In this study we have investigated the role of
CD4+ T cells from the lamina propria (LP) in the early
inflammatory events after T. gondii infection using
isolated and primary cultured intestinal cells from infected mice and
immortalized mouse mICcl2 intestinal epithelial cells.
Primed LP CD4+ T cells isolated from parasite-infected mice
produce substantial quantities of both IFN- and TNF-. IFN--
and TNF--producing LP CD4+ T cells synergize with
infected mICcl2 and enhance the production of several
inflammatory chemokines including macrophage-inflammatory protein-2,
monocyte chemoattractant protein-1, monocyte chemoattractant protein-3,
macrophage-inflammatory protein-1
, and IFN--inducible
protein-10. Furthermore, primed LP CD4+ T cells cocultured
with infected mICcl2 inhibited replication of the parasite
in the intestinal epithelial cells. Thus, LP CD4+ T cells
can interact with parasite-infected intestinal epithelial cells and
alter the expression of several proinflammatory products that have been
associated with the development of intestinal inflammation. The
interaction between these two components of the gut mucosal compartment
(CD4+ T cells and enterocytes) may play a role in the
immunopathogenesis of this pathogen-driven experimental inflammatory
bowel disease model. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF- (7, 8), IL-2 (9),
IL-4 (10), IL-5 (11), GM-CSF
(12), and IL-10 (13). Moreover, the
overproduction of Th1-type cytokines by LP CD4+ T
cells has been implicated in the development of IBD.
Toxoplasma gondii is an obligate intracellular parasite
acquired by oral ingestion of tissue cysts containing either
bradyzoites or sporozoites from contaminated soil. Significant
differences in resistance to parasite infection have been observed
among various inbred strains of mice (14). BALB/c and
CBA/J mice are resistant to the acute phase of the infection, whereas
C57BL/6 mice develop an acute lethal ileitis 10 days after oral
infection with tissue cysts containing bradyzoites. The epithelium of
the intestine represents a large and critical interface between the
host and invading pathogen. It has been observed that after oral
infection with tissue cysts containing bradyzoites, the intestinal
epithelial cells (IECs; enterocytes) and LP are invaded by parasites
(15). Parasite infection induces a strongly biased Th1
response in the gut that in susceptible C57BL/6 mice leads to a lethal
acute ileitis. We and others have determined that an imbalance between
pro- and down-regulatory inflammatory factors is responsible for the
hyperinflammatory condition (16, 17). Further inducible NO
synthase (iNOS) knockout mice (on C57BL/6 background) or mice treated
with a neutralizing anti-IFN- Ab failed to develop lethal
ileitis despite a detectable parasite burden (18, 19).
Although the acute ileitis is known to result from the overproduction
of a Th1-like immune response, the exact role of LP
CD4+ T cells in this process remains
uncertain.
Protozoan infection of enterocytes can stimulate the production of a wide range of cytokines (20, 21, 22) and chemokines (17, 23, 24). The chemokines lead to paracrine and autocrine activation of surrounding cells in addition to chemoattraction of lymphocytes, neutrophils, monocytes, macrophages, and dendritic cells (25, 26, 27). During inflammation, enterocytes are bathed in a cytokine-rich milieu that can enhance both their innate defense capacity and their ability to interact with and influence local leukocyte populations. In the present study, we have used isolated and primary cultured intestinal cells purified from parasite-infected mice as well as a differentiated mouse enterocyte line (mICcl2) (28) to analyze the role of LP CD4+ T cells and their interplay with enterocytes in this inflammatory process and control of parasite multiplication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female 8- to 10-wk-old inbred C57BL/6 mice and CD4-/- mice (C57BL/6 genetic background) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed under approved conditions of the Animal Research Facility at Dartmouth Medical School (Lebanon, NH). Tachyzoites from the RH strain were used for in vitro studies and 76K strain cysts isolated from the brains of chronically infected mice were used for in vivo studies. Mice were infected per os by intragastric gavage with 30 cysts, a condition that is lethal for C57BL/6 wild-type mice.
Histological analysis
Samples of ileum (day 7 postinfection) were removed from each mouse (n = 3 per group), fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with H&E.
Purification of LP CD4+ T lymphocytes and IECs
Isolation of LP CD4+ T cells was modified as previously described (29). For all experiments, LP CD4+ T lymphocytes were isolated and pooled from at least eight mice per condition. Briefly, the small intestines including the ileum and jejunum were removed, washed with PBS, and placed in ice-cold Ca2+, Mg2+-free RPMI 1640 medium. Peyer’s patches and fat were removed. Intestines were opened longitudinally, cut into small fragments (0.3 cm long), and incubated in RPMI 1640 supplemented with 25 mM EDTA, 100 IU/ml penicillin-streptomycin, 1% fungizone, and 50 IU/ml gentamicin (Sigma-Aldrich, St. Louis, MO). The suspension was stirred for 20 min at room temperature to remove epithelial cells and intraepithelial lymphocytes. This washing procedure was repeated five times. Intestinal fragments were then washed in 10% FCS-supplemented RPMI 1640 (RPMI-10% FCS) and incubated for an additional 2 h at 37°C in RPMI-10% FCS containing 125 IU/ml collagenase VIII (Sigma-Aldrich). Digested intestinal suspensions containing LP mononuclear cells and remaining epithelial cells were filtered and resuspended in RPMI-10% FCS medium supplemented with antibiotics. Finally, LP mononuclear cells were isolated by centrifugation on a Ficoll layer (density (d) = 1.077; Sigma-Aldrich, St. Louis, MO). The subpopulation of CD4+ T cells was purified by positive selection using anti-CD4-coated magnetic beads (L3T4 microBeads) using MidiMACS columns (Miltenyi Biotec, Auburn, CA). The purity of the magnetic-sorted CD4+ cell population was >90%, whereas the remaining 10% of cells corresponded to contaminating enterocytes.
Freshly purified IECs were isolated as described elsewhere
(30). Pooled IECs were isolated from at least three mice
per condition. Small intestines were flushed with
Ca2+- and Mg2+-free HBSS
(Cellgro, Herndon, VA) containing 2% glucose, 25 ng/ml amphotericin B,
and 100 IU/ml penicillin-streptomycin. Intestines were opened
longitudinally, cut into small fragments, and incubated for 45 min in
HBSS supplemented with 300 IU/ml collagenase XI (Sigma-Aldrich), 0.1
mg/ml dispase I (Boehringer Mannheim, Indianapolis, IN), 2% BSA, and
0.2 mg/ml soybean trypsin inhibitor. Digested cells were centrifuged in
DMEM with 2% sorbitol at 120 x g for 3 min.
Supernatants contained essentially monodispersed IECs and contaminating
lymphocytes (CD4+ and CD19+
LP lymphocytes and CD8+ intraepithelial
lymphocytes). Contaminant cells were depleted by magnetic cell sorting
in a positive selection using a mixture of Abs tagged with magnetic
beads (Miltenyi Biotec), including anti-CD8 (Ly-2), -CD4 (L3T4)
(GK1.5), -CD19 (1D3), and -CD11c (KB-90) Abs. IECs were negatively
sorted using MidiMACS columns. The purity of sorted IECs was >90%, as
identified by positive anti-cytokeratin labeling. The remaining
contaminating cells (
10%) included CD8+ and
dead cells.
The mICcl2 cell line
The mICcl2 cells are transimmortalized mouse IECs (H2b) derived from the small intestine of a transgenic mouse carrying the SV40 T and t Ags placed under the control of the L-pyruvate kinase promoter (28). These cells have maintained the main features of crypt intestinal cells (28). The mICcl2 cells develop in culture as a confluent monolayer of cuboid cells with short apical microvilli separated by tight junctions. Cells were grown in 25-cm2 flasks (Falcon; BD Labware, Franklin Lakes, NJ) in a modified defined medium (MDM; DMEM/HAM’s F12 (1/1, v/v; Life Technologies, Grand Island, NY) supplemented with 2% FCS, 5 µg/ml insulin, 10 ng/ml epidermal growth factor, 5 µg/ml human transferin, 50 nM dexamethasone, 30 nM sodium selenate, 1 nM triiodothyronine, 100 IU/ml penicillin-streptomycin, 1% fungizone, 50 IU/ml gentamicin, and 1% nonessential amino acids) and 20 mM HEPES (Sigma-Aldrich) at 37°C in 5% CO2/air atmosphere. The MDM was used within 2 wk of preparation to ensure that the activity of growth factors was maintained. The mICcl2 cells (fortieth to fiftieth passage) were plated on either collagen I-coated culture plates (six wells/condition; Falcon) at a density of 1.5 x 106 cells/well or collagen I-coated permeable polycarbonate filter (3-µm pore size; Falcon) at a density of 5 x 105 cells/filter for the coculture experiments.
The mICcl2 cells were seeded on the lower side of
permeable filters and incubated upside down for 10 days in MDM. When
mICcl2 enterocytes were confluent, they were
infected with tachyzoites at a 1:2 (cell:tachyzoites) ratio for 2
h. Afterward, the filter was carefully rinsed with complete medium to
remove the unpenetrated parasites and was replaced in its normal
orientation and filled with 1 ml of 1 x 106
LP CD4+ T cells from infected (primed) or naive
(unprimed) mice. The integrity of the infected monolayer was carefully
checked by microscopic observation before each experiment. As a
control, the reproducibility of the infection was monitored by laser
confocal microscopic analysis of the monolayer stained with a T.
gondii-specific polyclonal Ab (data not shown). In preliminary
experiments, we have determined that the infectivity ratio was
consistent at 50% and, moreover, that the nonpenetrated tachyzoites
were efficiently removed by the washing procedures. All experiments
using coculture on Transwell filters were performed on six separate
wells for each condition tested. Cocultures of primed or unprimed LP
CD4+ T cells with tachyzoite-infected
mICcl2 were maintained for 4 h. In some
cases, primed LP CD4+ T cells were first
incubated with 100 ng/ml anti-IFN- (BD PharMingen, San Diego,
CA) and/or anti-TNF- (R&D Systems, Minneapolis, MN) Abs. Primed
LP CD4+ T cells incubated with irrelevant Abs
(rat IgG1 and normal goat IgG irrelevant isotype-specific Ab, from BD
PharMingen and R&D Systems, respectively) were used as controls.
In all cases, the mRNA chemokine expression was analyzed in
mICcl2 cells 6 h after infection.
Cytokine assays
Mouse rIFN- produced in Chinese hamster ovary cells was
purchased from Life Technologies and was used at 1000 IU/ml when added
to cell supernatants or between 50 and 1000 IU/ml for dose effect
experiments. Mouse rTNF- from baculovirus-infected cells and mouse
IL-1 from Escherichia coli were purchased from BD
PharMingen and were used at 10 (or between 10 and 30 ng/ml for dose
effect experiments) and 5 ng/ml, respectively, when added into
enterocyte culture supernatants.
IFN- and TNF- production were assayed for both primed and
unprimed LP CD4+ T cells. The LP
CD4+ subpopulation was incubated in flat-bottom
24-well plates at 1 x 106 cells/ml, 37°C,
and 5% CO2, without additional stimulation.
Supernatants from triplicate cultures were collected after 48 h
and were evaluated for IFN- and TNF- production by specific ELISA
Duoset Systems (Genzyme, Cambridge, MA).
FACScan analysis
Purity of IECs and LP CD4+ T cells was
evaluated with anti-pan cytokeratin-26 FITC-conjugated
(Sigma-Aldrich) and anti-CD4 (H129.19) PE-conjugated (BD
PharMingen) Abs, respectively. FACScan analysis was also performed to
phenotype the LP CD4+ T cells with PE-conjugated
Abs to CD25 (3C7), CD95 (Jo2), and CD95 ligand (Kay-10) or
biotinylated Abs to CD44 (IM-7), CD45RB (16A), and CD69 (H1.2F3) (BD
PharMingen). Cells (106 per well) were first
incubated with 100 µl of 1% Fc-block (CD32/CD16) (2.4G2) in 96-well
plates for 20 min at 4°C to prevent nonspecific Ab staining. After
washing, cells were incubated with a variety of PE or biotinylated Abs
for 2 h at 4°C. Cells were then washed and incubated with
streptavidin-PE (for biotinylated Abs) or fixed with 2%
paraformaldehyde in PBS. Fluorescence acquisition was done on FACS (BD
PharMingen) the next day. Data analysis was done using CellQuest
software (BD Biosciences). Twelve mice per condition were used
for the LP CD4+ T cell phenotyping. Results were
the means of three separate experiments and were expressed as
percentages of cells expressing the molecule.
mRNA extraction and RNase protection assay
Total mRNA from freshly isolated LP CD4+ T
cells, IECs, and cultured mICcl2 cells was
extracted using the TRIzol reagent according to the manufacturer’s
instructions (Life Technologies). Chemokine, chemokine receptor, and
iNOS mRNA expression in cells were detected using the
RiboQuant multiProbe RNase Protection Assay System kit (BD
PharMingen). Briefly, probes labeled with 32P
(custom probes and the mCK-5 MultiProbe template set from BD
PharMingen, no. 556146) used for specific mRNA detection were as
follows: for chemokines (mCK-5), macrophage-inflammatory protein
(MIP)-1 and -1, MIP-2, lymphotactin (Ltn), monocyte
chemoattractant protein (MCP)-1/JE, MCP-3, IFN--inducible
protein (IP)-10/CRG-2), C-10, eotaxin, RANTES, T cell activation
gene-3, L32, GAPDH, or (custom probe) iNOS. For the chemokine receptors
(custom probe), CCR1, CCR5, CCR2, CCR7, CXCR3, and CCR6 were assessed.
RNA samples were digested by RNase treatment for 45 min at 30°C. The
RNase digests were extracted carefully. Samples were loaded into a 5%
acrylamide gel. The value of each hybridized probe was normalized to
that of GAPDH, included as internal standard (arbitrarily set to 1).
For quantification, autoradiographs were scanned and band densities
were analyzed by National Institutes of Health Image 1.61/ppc software
(National Institutes of Health, Bethesda, MD) using a Macintosh
computer (Apple Computer, Cupertino, CA). Results were expressed as the
percentage of the intensity of the band analyzed relative to the
intensity of the housekeeping (GAPDH/L32) RNA.
T. gondii intracellular replication
Confluent mICcl2 cells were seeded (1
x 104 cells/well in 200 µl of culture medium)
in collagen I-precoated 96-well plates and grown for 10 days at 37°C
in a 5% CO2 atmosphere. Thereafter, various
concentrations of IFN-, TNF-, or 2 x
104 of unprimed or primed LP
CD4+ T cells were added, and 2 h later
T. gondii tachyzoites (5 x 104
tachyzoites/well) were added. Plates were then incubated for 2 h
at 37°C, and 0.5 µCi 5,6-[3H]uracil (New
England Nuclear, Boston, MA) was added to each well. After 16 h,
nonincorporated [3H]uracil, extracellular
tachyzoites, and unprimed or primed LP CD4+ T
cells were removed by careful washing with PBS. Cells were then lysed
with 0.1% SDS (Sigma-Aldrich) and nucleic acids were precipitated with
3 M TCA. Cell lysates were transferred to glass fiber filters using an
automated cell harvester and the radioactivity was counted. Infected
mICcl2 were also cultured on the bottom side of a
system filter and incubated without contact with primed or unprimed LP
CD4+ T cells placed on the top side of the
filter. Monolayer integrity was assessed by microscopic observation,
and parasite multiplication was appreciated by
[3H]Uracil incorporation as
described.
Statistical analysis
Results are expressed as the mean ± SD. Statistical differences between groups were analyzed using the Student t test. A value of p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The susceptibility of C57BL/6 to oral T. gondii
infection is associated with an exaggerated expression of inflammatory
cytokine mRNAs in the small intestine (19) that leads to a
lethal intestinal hyperimmune response. CD4+ T
cells are an important component in regulating the gut injury process.
Histological analysis of C57BL/6 small intestines from day 7
postinfection revealed the typical lesions of ileitis, including
reduced length and increased thickness of the villi and leukocytic
infiltration into the LP (Fig. 1
A). Focal loss of superficial
epithelial cells lining the tip of villi, indicating the earliest stage
of necrosis, was also observed. In contrast, the small intestine from
CD4-/- mice at day 7 postinfection displayed an
intact epithelium with mild leukocytic infiltration (Fig. 1B) compared with the intestine of naive C57BL/6 wild-type
mice (Fig. 1C). The involvement of
CD4+ T cells located in the LP was specifically
assessed in this intestinal hyperimmune response. The number of
magnetic-sorted intestinal LP CD4+ T cells
isolated from mice at day 7 postinfection was three times greater than
the number of LP CD4+ T cells isolated from
naive mice (9 ± 0.89 x 105 LP
CD4+ T cells/mouse vs 3 ± 0.45 x
105). Both unprimed and primed LP
CD4+ T cells expressed high, but not
significantly different, levels of CD69 (73% ± 5), CD44 (75% ± 5),
CD95 (50% ± 4), and CD45RBhigh/low
(85% ± 3). In contrast, the expression of CD25 (9% for unprimed
LP CD4+ T cells vs 23% for primed cells) and
CD95 ligand (18% for unprimed LP CD4+ T cells vs
36% for primed cells) were significantly higher for the primed LP
CD4+ T cells compared with unprimed cells. Sorted
unprimed and primed LP CD4+ T cells were assayed
for the production of IFN- and TNF-. As shown in Fig. 2, the level of IFN- and TNF-
production was significantly higher (p < 0.01)
for primed CD4+ LP T cells compared with that of
unprimed CD4+ LP T cells. Proinflammatory
chemokine expression by the LP CD4+ T cells was
also assessed (Fig. 3). Primed LP
CD4+ T cells exhibited a significant increase in
the expression of several CC chemokine mRNAs including RANTES, MCP-1,
MCP-3, and the CXC chemokine IP-10. Compared with unprimed LP
CD4+ T cells, no significant difference in the
level of mRNA expression was found for MIP-2, C-10, MIP-1,
and Ltn.
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We have observed that oral infection with T. gondii
enhances the expression of inflammatory chemokines in the gut (data not
shown). To investigate whether these chemokines were produced by
enterocytes, freshly isolated IECs were isolated. Intestines were
harvested from naive mice, then tachyzoites or medium were added ex
vivo directly into the lumen (n = 3 mice/condition),
and freshly isolated IECs were purified from a pool of ileum sections
(1 cm) after a 6-h incubation period. IECs expressed a high level of
MIP-2 and MCP-1 mRNA and a lesser but significant amount of IP-10 mRNA
(Fig. 4). MIP-2, IP-10, and MCP-1 mRNA
expression were, respectively, 1.8-, 2.2-, and 4-fold higher in IECs
isolated from the ex vivo infected intestines than in those from
noninfected intestines. A very similar pattern of chemokine expression
was observed when cultured mICcl2 cells were
infected with T. gondii (Fig. 4). We have developed an in
vitro cell culture system to better appreciate the physiologic
interaction between LP CD4+ T cells and
enterocytes. Because the infected mICcl2 cells
and the freshly isolated IECs from infected mice exhibited similar
chemokine-induced profiles, the mICcl2 cell line
appeared to be a suitable and convenient mouse IEC model for coculture
experiments. The chemokine profile expressed by infected
mICcl2 was examined in a permeable membrane
culture dish after their incubation with primed or unprimed LP
CD4+ T cells. In the presence of primed LP
CD4+ T cells, the overall chemokine mRNA
expression by infected mICcl2 cells was
substantially increased compared with unprimed LP
CD4+ T cells cocultured under the same conditions
(Fig. 5A). Coculture of primed
LP CD4+T cells with infected
mICcl2 enterocytes led to the up-regulation of a
largenumber of proinflammatory chemokines (including C-10,
MIP-2, MCP-3, MIP-1, MCP-1, IP-10, MIP-1, and RANTES) compared
with that observed with unprimed LP CD4+ T cells
(Fig. 5B). LP CD4+ T cells were found
to express a large array of mRNA chemokine receptors (Fig. 5C), including CCR1 (MCP-3, RANTES, MIP-1R), CCR2
(MCP-1R), and CCR5 (MIP-1 and , RANTES, MCP-2R). CCR1, CCR2, and
CCR5 mRNA production, respectively, were enhanced by 2.3-, 3.4-, and
2.5-fold in primed LP CD4+ T cells compared with
unprimed LP CD4+ T cells. Addition of a
neutralizing anti-IFN- Ab reduced the mRNA expression of
MIP-1 but did not alter the mRNA expression of the chemokines MCP-2,
MCP-1, and IP-10 in mICcl2 cells cocultured with
primed LP CD4+ T cells (Fig. 6). The neutralizing anti-TNF- Ab
led to a significant decrease in the expression of MIP-1, MCP-1, and
IP-10 chemokines but did not alter expression of MCP-2 and other
proinflammatory chemokine mRNAs (data not shown). When both
anti-IFN- and anti-TNF- neutralizing Abs were added to
the coculture, a more pronounced decrease of the effect of the primed
LP CD4+ T cells was observed (Fig. 6). In this
case, the mRNA expression of all four chemokines tested were
synergistically down-regulated, especially MIP-1 (Fig. 6). However,
the addition of anti-TNF- and anti-IFN- neutralizing Abs
to the coculture system never completely reversed the effects exerted
by the primed LP CD4+ T cells on the
enterocytes.
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To better investigate the role of enterocytes on the
proinflammatory response to T. gondii infection, TNF- and
IFN- were added to confluent mICcl2 cells. As
already described (17), parasite infection has been found
to up-regulate MIP-2, MCP-1, and IP-10 mRNA expression by the
mICcl2 cells. IFN- by itself increased MCP-1
and IP-10 (Fig. 7). Parasite infection
performed in the presence of exogenous IFN- increased the production
of MIP-2 and MIP-1 compared with the uninfected condition. A
synergistic effect of parasite infection plus IFN- was observed on
the MIP-1 mRNA expression. IFN- and/or the parasite have the same
effect on MCP-1 mRNA production. It is of note that IFN-
down-regulated the production of MIP-2, as recently reported
(31). TNF- up-regulated the expression of a large array
of chemokine mRNA, but in the present study parasite infection it
failed to further enhance this response (data not shown). TNF- and
IFN-, when added together, promoted the maximum effect, in
particular for MIP-2 and IP-10. Parasite infection or IL-1 addition
did not further this response (data not shown). The MCP-3 chemokine
mRNA expression was also evaluated in this experiment and followed the
same variation of expression as MIP-1 for all the conditions
tested.
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To investigate the influence of cytokine production on parasite
survival, parasite replication was measured in
mICcl2 cells when cocultured with primed or
unprimed LP CD4+ T cells. The
mICcl2 cells were in direct contact with primed
or unprimed LP CD4+ T cells for 1 h,
followed by the addition of freshly prepared tachyzoites to the
mICcl2 cells. Eighteen hours later, the cultures
were rinsed and mICcl2 cells were harvested.
Tachyzoite proliferation was estimated by using the
[3H]uracil method. The levels of uracil
incorporated into mICcl2 cells cocultured with
primed LP CD4+ T cells were 50% lower
(p < 0.01) than those measured in control
cells either infected alone or cocultured with unprimed LP
CD4+ T cells (Fig. 8A). These data strongly
suggest that LP CD4+ T cells control the parasite
replication and/or invasion into enterocytes. Because a cytotoxic
effect of LP CD4+ lymphocytes cannot be ruled out
at the conditions used, LP CD4+ T cells isolated
at day 7 after infection were cocultured on the upper part of Transwell
filters for 16 h with parasite-infected enterocytes
(mICcl2) grown on the bottom of the well. The
membrane of the Transwell prevents direct contact between the effector
and target enterocytes. The results from microscopic analysis
demonstrated that the integrity of the epithelial layer cocultured with
either primed or unprimed LP CD4+ T cells was
unaffected. Under these conditions, a similar effect on parasite
replication was observed: a 50% reduction of uracil incorporation by
the parasite, when the infected enterocytes were cocultured with primed
LP CD4+ T cells (70 ± 6.2 x
102 cpm/filter; n = 3) compared
with those cocultured with either medium (182 ± 12.6 x
102 cpm/filter; n = 3) or
unprimed LP CD4+ T cells (165 ± 2.9 x
102 cpm/filter; n = 3). We also
found that mICcl2 cells pretreated with IFN-
and TNF- were more resistant to T. gondii infection (Fig. 8B). Incubation of mICcl2 in direct or
indirect contact with LP CD4+ T cells or with
rIFN- and rTNF- did not alter the integrity of the monolayer, as
determined by direct microscopy and dye exclusion assay. IFN-
induced a dose-dependent decrease in uracil incorporated into
parasite-infected enterocytes, whereas the effect of TNF- did not
appear to be dose dependent at the concentrations usedin this
experiment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF- in the interaction between LP
CD4+ T cells and enterocytes in response to the
infection. After T. gondii infection, LP
CD4+ T cells that have been isolated ex vivo are
activated. This activation leads to an increased production of Th1-type
cytokines as well as enhancing the mRNA expression of specific
chemokines that have previously been shown to be important in the
immunopathogenesis of IBD (32, 33). The control of the
enhanced immunological interaction between gut enterocytes and T cells
appears to be regulated by a specific population of
CD4+ T cells that expresses the surface marker
CD45RBhigh (34). Thus, the
cross-talk between these two components of the gut-associated lymphoid
compartment may be a potentially important mechanism of the immune
intestinal mucosal regulation.
Chemokines are pleiotropic and are elicited early in the intestinal
inflammatory process (35, 36). Our data indicate that
Ag-primed LP CD4+ T cells express high levels of
RANTES, MCP-3, and IP-10 that are regulated by IFN-. IP-10 is
critical for effector T cell trafficking and host survival during
T. gondii infection (37). Thus, IP-10 may be
attractive for other T cell subsets in the infected bowel. This
overproduction of chemokines can also account for the migration and
trafficking of monocytes, lymphocytes, polymorphonuclear cells,
macrophages, and dendritic cells to the site of inflammation that is
under the control of some of these CC chemokines (38). In
other infectious models, such as Trypanosoma cruzi and
RANTES (39) as well as MCP-1 and MIP-1
(40), are responsible for the control of inflammation in
the tissue of infected animals. Moreover, the production of epithelial
cell-derived MCP-3 is enhanced in IBD (41). Besides
attraction, chemokine receptor ligation may participate in
CD4+ activation (42). Mice deficient
in MIP-1 and CCR5 (receptor for RANTES, MIP-1) or CCR2
(receptor for MCP-1) exhibit reduced production of IFN- upon
activation. Furthermore, chemokine receptors may interact with TCR and
costimulatory T cell signaling pathways (43), which will
in turn alter the production of Th1-type cytokines.
Immune cytokines, in particular IFN- and TNF-, are important
modulators of inflammation and chemokine production. Our data suggest
that Ag-primed LP CD4+ T cells secrete IFN-
and TNF-, which enhance enterocyte expression of several important
chemokines. In previous studies, we demonstrated that infection by
T. gondii in vitro up-regulated chemokine expression by the
enterocyte cell line mICcl2 (17).
Epithelial cells are a potentially important source of chemokines known
to regulate tissue inflammation (35, 44, 45). In this
study, we observed that the profile of chemokines (e.g., MIP-2, MCP-1,
and IP-10) expressed by freshly isolated IECs while cultured ex vivo is
similar to that of the mICcl2 enterocyte cell
line. The expression of chemokines by human enterocytes during the
inflammatory response can be regulated by inflammatory cytokines
(46, 47). We found that coculturing
mICcl2 cells with Ag-primed LP
CD4+ cells that produced IFN- and TNF-
enhanced a large array of mRNA chemokine expression by the infected
enterocytes, including MIP-2, MCP-1, and IP-10, but also MIP-1,
MCP-3, and RANTES, which were not up-regulated by the infection alone.
We have also shown that depletion of both IFN- and TNF- is
required to decrease the expression of MIP-2, IP-10, and MCP-1
chemokines by infected enterocytes. Both IFN- and TNF- enhance
MCP-1, MCP-3, and IP-10 mRNA expression in cultured intestinal
mICcl2 cells, but only TNF- induced an
overexpression of MIP-2 and MIP-1. Taken together, the present data
and previous studies from our laboratory (17) strongly
suggest that the LP population of the CD4+ T
cells producing IFN- and TNF- may be associated with the
development of the lethal ileitis induced by oral infection of C57BL/6
mice with T. gondii. Although our observations were
primarily focused on a model of cultured intestinal cells, we also
demonstrate that the response of freshly prepared IECs isolated from
the intestine of susceptible mice is consistent with the established
mICcl2 intestinal cell line. Thus, this ex vivo
model provides sufficient basis to suggest that similar interactions
may occur in vivo during the course of acute inflammation after oral
infection with T. gondii.
Furthermore, we have demonstrated that the intestinal enterocyte
barrier may have an important immunoregulatory capacity. The complex
cross-talk between enterocytes and LP CD4+ T
cells, especially mediated by IFN- and TNF-, may be a critical
pathway during the in vivo induction of the intestinal inflammation. In
turn, the inflammatory process will restrict the parasite’s survival.
Our observations demonstrate that coculture of parasite-infected
enterocytes and Ag-primed LP CD4+ T cells limits
the ability of the parasite to replicate in vitro. This reduction in
parasite replication is not related to any cytotoxic side effect.
Furthermore, the direct contact between the effector and target cells
is not required for the parasiticidal effect. Our data further support
previous studies that show that soluble factors, such as IFN- and
TNF-, can be responsible for this inhibitory effect. The ability of
IFN- to inhibit parasite growth was first documented in human
fibroblast cells by Pfefferkorn and Guyre (48) and was
more recently shown to be involved in the control of parasite
replication in a murine model (49). As reported by others,
the microbiostatic mechanism involved could be iron dependent
(50).
Our data would suggest that the LP CD4+ T cells
have both a host beneficial and host detrimental effect. This
dichotomous response is consistent with our previous observations,
which showed that IFN- is responsible for both the microbicidal
effect as well as the pathologic changes in the intestine of
susceptible C57BL/6 mice after oral infection with T.
gondii. In conclusion, this study demonstrates that a potentially
important interaction between the CD4+ T cell
from the LP and the enterocytes may in part underlie the
immunopathogenesis of pathogen-driven experimental IBD. The importance
of this interaction between T cells and enterocytes in the pathogenesis
of human IBD is novel but remains speculative. However, the potential
association between IBD and exposure of the sensitized host to this
ubiquitous pathogen should not be understated. Characterization and
identification of the intestinal subpopulation of
CD4+ T cells in this unique model may have
important implications for understanding intestinal inflammatory
disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lloyd H. Kasper, Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756. E-mail address: lloyd.kasper{at}dartmouth.edu
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; LP, lamina propria; IEC, intestinal epithelial cell; iNOS, inducible NO synthase; MIP, macrophage-inflammatory protein; MDM, modified defined medium; Ltn, lymphotactin; MCP, monocyte chemoattractant protein; IP, IFN--inducible protein.
Received for publication September 13, 2001. Accepted for publication December 27, 2001.
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