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From the Intestinal Disease Research Program,*
McMaster
University, Hamilton; and the Division of Gastroenterology and
Nutrition,
Research Institute, The Hospital
for Sick Children, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Isc to forskolin] and barrier (transepithelial
electrical resistance) parameters were measured in Ussing chambers.
LPS-activated PBMs from both controls and patients with Crohn’s
disease significantly increased Isc (
300%) and reduced
transepithelial electrical resistance (40%). Epithelial function
was not altered after co-culture with control LPMCs ± LPS.
However, LPMCs from patients with Crohn’s disease
spontaneously secreted tumor necrosis factor-
, and induced
epithelial changes similar to those produced by LPS-activated PBMs.
Co-culture with control Escherichia coli and PBMs
induced comparable changes in epithelial physiology, which were
abrogated by anti-tumor necrosis factor- antibody. We conclude that
LPMCs of patients with Crohn’s disease are spontaneously
activated, possibly by gram-negative luminal bacteria,
and can directly cause significant alterations in epithelial ion
transport and barrier functions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, attention has focused on cells of the
monocyte/macrophage lineage and their inflammatory products. These
phagocytic cells are scattered beneath the intestinal epithelium and
represent a first line of defense against foreign antigens. Previous
studies indicate that enteric inflammation can be associated with
increased heterogeneity and activation of intestinal macrophages: there
is a distinct population of recently recruited monocyte-like
macrophages in actively inflamed IBD lesions.7,8
These
cells are more reactive than resident macrophages, releasing bioactive
products and cytokines such as tumor necrosis factor-
(TNF-).9
Indeed, TNF- has recently become the target
of clinical investigations in studies inhibiting its effects using a
human-murine chimeric monoclonal antibody, Remicade (previously known
as cA2; Centocor Inc., Malvern, PA).10-12
The intestinal epithelium is a dynamic barrier that regulates absorption of nutrients and water and at the same time restricts uptake of microbes and other noxious material from the gut lumen. One of the distinct features of Crohn’s disease is impaired intestinal epithelial function, characterized by increased permeability (altered barrier function) and ion secretion, often resulting in a luminally directed driving force for water movement causing diarrhea. Previously we showed that co-culture of a model epithelium with LPS-activated normal monocytes resulted in epithelial abnormalities reminiscent of those observed in resected tissue from patients with IBD.13
Despite increasing evidence of an association between the commensal
bacterial microflora and intestinal inflammation,14-16
little information is currently available on the direct role of such
bacteria in altering gut epithelial function. We recently described a
co-culture model of epithelial cells and immune cells13
that was adapted for the current studies. The aim of the present study
was twofold: 1) to compare the ability of peripheral blood monocytes
(PBMs) and lamina propria mononuclear cells (LPMCs) from patients with
Crohn’s disease and control patients ± LPS activation to affect
enteric epithelial ion transport and barrier functions; and 2) to
assess the ability of a luminally applied nonpathogenic (
commensal)
strain of bacteria to modulate epithelial function directly and in the
context of monocytes/macrophages in a subepithelial position. Our
objective was to gain further knowledge regarding the role of commensal
microorganisms and the responsiveness of gut-derived immune cells to
bacterial products in affecting epithelial function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was collected from 12 ambulatory patients with mild to moderate Crohn’s disease attending the IBD clinic at McMaster University Medical Center and 14 healthy volunteers. Volunteers were excluded if they showed any signs of illness (eg, common cold, flu, allergy, and so forth) or were currently on medication. Whole blood from each donor was diluted in phosphate-buffered saline (PBS) and subjected to one-step density centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). The interface containing mononuclear cells was collected, washed in PBS, and resuspended in fresh T84 culture medium [1:1 mixture of Dulbecco’s modified Eagle’s medium and F-12 medium supplemented with 1.5% HEPES, 2% penicillin-streptomycin, and 10% fetal calf serum (FCS)] at 106 cells/ml.16 The monocyte/macrophage population was obtained by plastic-plating of peripheral blood mononuclear cells (4 hours at 37°C) and subsequent removal of nonadherent T and B cells. Fresh medium was added to the adherent cells, which were then incubated for 18 hours at 37°C before use in co-culture studies.
Isolation of LPMCs
Surgical specimens of inflamed small or large intestine were
obtained from nine patients undergoing resection for severe Crohn’s
disease (Table 1)
; four small bowel and
six large bowel resections were used. Tissues supplied for this study
were adjacent to the most severely inflamed or ulcerated region and
were described by the pathologist as moderately inflamed, a decision
made before full histological assessment. Noninflamed control small or
large bowel was obtained from patients undergoing surgery for cancer
(n = 8), and was taken at least 5 cm from the
tumor margin. LPMCs were isolated by a modification of the technique
described by Bull and Bookman.17
Briefly, surgical
specimens were washed extensively in RPMI 1640 (Life Technologies,
Inc., Grand Island, NY) containing 1 mmol/L dithiothreitol (Sigma
Chemical Co., St. Louis, MO) and 5% FCS (Life Technologies, Inc.) and
mucosa dissected free from the muscle layer. Tissue was then cut into
small pieces, washed, and incubated for 20 minutes in RPMI containing
dithiothreitol at 37°C. Epithelial cells were removed by two
30-minute incubations in Hanks’ balanced salt solution (Life
Technologies, Inc.) containing 1 mmol/L ethylenediaminetetraacetic acid
(EDTA; Sigma) and 10% FCS at 37°C. The mucosal samples were
washed and incubated overnight with gentle stirring in RPMI
1640 containing 1 U/ml collagenase (Sigma), 6 U/ml DNase II (Sigma),
and 10% FCS. The tissue digest was then washed and filtered through a
200-µm steel mesh. The cells were washed twice in RPMI 1640
containing 10% FCS and subjected to Percoll density gradient
centrifugation. The interface was carefully removed, washed, and
resuspended in T84 culture medium. For each resection the size of
tissue available was highly variable and thus total cell recovery was
not a priority (most tissues yielded 2.5 x
106
cells per cm2
of
tissue). For comparative purposes with PBMs, LPMCs were resuspended and
at 1 x 106/ml and incubated for 18 hours at
37°C before use in co-culture studies.
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The use of patient blood samples and intestinal resections was approved by the Human Ethics Committee at McMaster University/Chedoke Hospital, Hamilton Health Sciences Corporation.
Epithelial Cells
Human T84 colonic epithelial cells (passage 45 to 60) were seeded
onto tissue culture-treated semipermeable filter supports (0.4-µm
pore size, 1.0-cm2
surface area; Costar
Corporation, Cambridge, MA) at a concentration of
106
cells/ml and grown in T84 culture medium.
Cells were grown at 37°C for 7 days to form polarized monolayers,
displaying transepithelial electrical resistances >750
.cm2
as measured by a voltmeter and associated
chop-stick electrodes (Millicell-RES; Millipore Corp., Bedford, MA). At
the outset of the study, the stock epithelial cells were tested and
found to be negative for mycoplasma contamination.
Bacterial Strain and Growth Conditions
Bacteria of the nonpathogenic, laboratory control strain of
Escherichia coli, HB10118,19
were grown in
nonaerated Trypticase soy broth for 6 hours at 37°C. Bacteria were
pelleted by centrifugation at 2,400 x g for 15 minutes
and then resuspended in sterile antibiotic-free T84 culture medium to a
concentration of 1 x 109
colony forming
units/ml. Viable bacteria were enumerated by serial 10-fold dilutions
plated onto horse-blood agar plates.
Co-Culture Studies
Culture of Immune Cells with Epithelial Cells
Experiments with PBMs: PBMs were activated by addition
of 10 µg/ml LPS (either E. coli O111:B4, O127:B8, or
Salmonella minnesota; Sigma) to the culture medium at the
time of co-culture.20
Our previous study13
showed that maximal changes in epithelial function occurred after 48
hours of co-culture with activated macrophages/monocytes. Thus,
confluent T84 monolayers were co-cultured for 48 hours with normal or
Crohn’s disease PBMs (1.5 x 105
cells/well) with or without LPS placed into the basal compartment of
the culture wells. Controls were time-matched naive T84 monolayers and
monolayers cultured with LPS or nonactivated PBMs only. The latter two
treatments did not significantly impact on T84 ion transport or
permeability and so naive monolayers only were predominantly used as
the standard control condition.
Experiments with LPMCs: Use of LPMCs has the logistical problem of ensuring that a suitable T84 monolayer is available when surgical specimens are provided, the latter being at the discretion of the surgeon. We, and others, have shown that recombinant cytokines and conditioned medium from activated immune cells evokes changes in epithelial ion transport and permeability that are very similar to those observed in co-culture studies.21-23 Thus, in some experiments LPMC-conditioned medium was made (culture 106/ml LPMCs with 10 µg/ml LPS for 24 hours, then collect cell-free conditioned medium) and stored at -70°C. Subsequently confluent monolayers were treated with 50% conditioned medium (diluted 1:1 in fresh culture medium), added into the basal compartment of the co-culture well, and epithelial function examined 48 hours later. Studies using LPMCs mirrored those with PBMs, with confluent monolayers being co-cultured with LPMCs (106/ml) ± LPS for 48 hours. Of the 18 surgical specimens used, eight experiments with conditioned medium and 10 experiments with freshly isolated LPMCs were conducted.
Addition of Nonpathogenic E. coli
Confluent T84 monolayers were co-cultured for 48 hours with PBMs in the basal compartment, and 105 E. coli strain HB101 added to the apical compartment (luminal side) of the transwells. Controls were naive T84 monolayers, those exposed to HB101 alone, or co-cultured with nonactivated PBMs.
Ussing Chamber Experiments
Epithelial Ion Transport
After culture with immune cells or conditioned medium, T84 monolayers on filter supports were mounted into Ussing chambers, as previously described.13 Briefly, monolayers were bathed in oxygenated Krebs buffer (pH 7.35, 37°C), containing 115 mmol/L NaCl, 8 mmol/L KCl, 1.2 mmol/L MgCl2, 1.25 mmol/L CaCl2, 2.0 mmol/L KH2PO4, 25 mmol/L NaHCO3. The buffer bathing the serosal tissue surface contained 10 mmol/L of glucose as an energy source and this was osmotically balanced by inclusion of 10 mmol/L of mannitol in the buffer in the luminal side of the Ussing chamber. The epithelial spontaneous potential difference (in mV) was maintained at zero volts by the continuous injection of an external current by an automated voltage clamp (World Precision Instruments Inc., Sarasota, FL). This short-circuit current (Isc, in µA/cm2) reflects net active ion transport across the preparation. Baseline Isc was recorded after a 15-minute equilibration period. Stimulated ion secretion was measured by adding the adenylate cyclase-activating agent, forskolin (10-5 mol/L; Sigma), to the serosal side of the T84 monolayers and recording the maximum increase in Isc.
Epithelial Barrier Function
Electrical resistance is a measure of the barrier function of the
epithelium to passive ion movement. At intervals during each
experiment, potential difference across the monolayer was clamped at
1.0 mV (differential pulse method, 1 pulse/30 seconds). The resulting
change in current was measured and the transepithelial electrical
resistance (TER, in .cm2) was calculated
according to Ohm’s law. However, TER indicates the epithelial barrier
to the flux of ions only and this need not be paralleled by increased
transepithelial passage of larger molecules. Therefore, as an
indication of epithelial permeability to larger molecules, in
experiments conducted with normal PBMs (±E. coli) the
mucosal-to-serosal movement of the inert probe,
51Cr-EDTA (362.3 Daltons) was measured by adding
6.5 µCi/ml (Radiopharmacy, McMaster-Chedoke Hospital, Hamilton,
Ontario, Canada) to the mucosal buffer. An equal concentration of
nonradioactive Cr-EDTA was added to the serosal buffer to maintain the
osmotic balance. After a 30-minute equilibration period 2 x
30-minute fluxes were performed.23
TNF-
Basal or stimulated production of TNF- by PBMs and LPMCs was
measured at 24 hours by enzyme-linked immunosorbent assay with a
sensitivity of 7.4 pg/ml (TNF--FLEXIA; BioSource International,
Camarillo, CA).
The role of TNF- in E. coli-PBM modulation of epithelial
ion transport and barrier functions was assessed by inclusion of a
neutralizing antibody to TNF-, cA2 (1 µg/ml, >100-fold excess of
the TNF- measured in the conditioned medium; Centocor Inc., Malvern,
PA). An irrelevant isotype matched antibody (anti-hepatoma IgG1, AF20;
Centocor) was used as a control.
Data Presentation and Analyses
Results are presented as mean ± SEM. Because of variability
in absolute values between different batches of T84 cells, data were
normalized to control values in each experiment (expressed as
percentage of control). The range of control responses is given in the
figure legends; n values represent the number of experiments
(different blood donors or tissue samples) in which two to four
monolayers were examined per condition. Data were analyzed using
one-way analysis of variance followed by Newman-Keuls comparison.
Student’s paired t-test was used to compare TNF-
production with or without LPS treatment. Statistically significant
differences were accepted at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epithelial Ion Transport
Control T84 epithelial monolayers displayed a baseline Isc of
2.2 ± 0.4 µA/cm2
and a Isc of 65
± 9 µA/cm2
in response to forskolin
(n = 14). These indices of epithelial ion
transport were not significantly altered after 48 hours of co-culture
with nonactivated PBMs or LPS alone. However, co-culture with
LPS-activated PBMs significantly (P < 0.05)
elevated T84-baseline Isc to 251 ± 16% of control values
(5.4 ± 0.5 µA/cm2; Figure 1A
). Similarly, no change in T84-baseline
Isc was observed after co-culture with nonactivated Crohn’s disease
PBMs, whereas LPS addition did evoke a significant
(P < 0.05) increase in baseline Isc to 296
± 58% of control values (6.1 ± 0.8
µA/cm2; n = 12). Also, T84
monolayers co-cultured with LPS-activated PBMs from patients with
Crohn’s disease displayed reduced Isc to forskolin (76 ± 6%
of control values, P < 0.05), which was comparable to
the changes induced by LPS-activated normal PBMs (69 ± 6% of
control values) (Figure 1B)
.
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Control T84 monolayers displayed a TER of 1,784 ± 179
.cm2
(n = 14).
Co-culture with nonactivated PBMs produced a small drop in TER (to
78 ± 6% and 75 ± 10% of control values; normal PBMs and
Crohn’s disease PBMs, respectively; Figure 1C
). This change in
epithelial barrier function was enhanced after LPS activation of PBMs.
After 48 hours of co-culture with activated PBMs, T84 TER was
significantly (P < 0.05) reduced to 37 ±
5% and 49 ± 7% of control values (normal PBMs and Crohn’s
disease PBMs, respectively; n = 12).
TNF- Production by PBMs
Nonactivated normal PBMs produced no detectable TNF-. LPS
activation of PBMs caused a significant increase in TNF- secretion
after 24 hours (range, 346 to 4,595 pg/ml; mean, 2,332 pg/ml;
n = 10). Similar to normal PBMs, PBMs isolated from
Crohn’s disease patients’ blood did not secrete TNF- under basal
conditions, but did demonstrate TNF- production when activated by
LPS (range, 590–3,304 pg/ml; mean, 1,921 pg/ml; n = 6)
(Figure 2A)
. This increase in TNF-
production was not statistically significantly different from that
observed in LPS-stimulated PBMs from normal volunteers.
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Results obtained with LPMCs ± LPS and LPMC-conditioned mediums were not statistically significantly different (eg, T84 TER was reduced to 27 ± 11% and 29 ± 5% of control values, respectively) and so the data are considered together.
Epithelial Ion Transport
The baseline Isc of T84 monolayers was unchanged after co-culture
with LPMCs isolated from normal mucosa (Figure 3A)
and remained comparable to control
levels after adding LPS to the co-culture (86 ± 6% and 102
± 16% of control values, normal LPMCs and normal LPMCs plus LPS,
respectively). In contrast, 48 hours of co-culture with LPMCs isolated
from Crohn’s disease mucosa resulted in an elevated T84-baseline Isc
(198 ± 40% of control values, P < 0.05), and
LPS addition produced no further increase in the baseline Isc (188
± 21% of control values; n = 8 to 10).
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, the Isc evoked by forskolin was unaltered by
co-culture with normal LPMCs (94 ± 8% of control) even in the
presence of LPS (98 ± 10% of control). However, there was a
significantly diminished response to forskolin after co-culture with
LPMCs from Crohn’s disease mucosa, and inclusion of LPS resulted in
no additional effects on T84 responses (70 ± 8% and
72 ± 8% of control values; Crohn’s disease LPMCs and Crohn’s
disease LPMCs plus LPS, respectively; n = 8 to 10). Epithelial Barrier Function
T84 monolayers co-cultured with normal LPMCs, in the absence or
presence of LPS, displayed TERs that were not different from control
monolayers (Figure 3C)
. In contrast, co-culture with LPMCs from
Crohn’s disease mucosa, led to a decrease in TER (34 ± 9% of
control values, P < 0.005) with no further reduction
when LPS was added (28 ± 5% of control values; n
= 8 to 10).
TNF- Production by LPMCs
The spontaneous secretion of TNF- by LPMCs isolated from normal
mucosa was below the detection limit (<7.4 pg/ml) for all samples
(n = 6) and stimulation by LPS had no effect on
TNF- secretion. In contrast, the spontaneous secretion of TNF- by
LPMCs obtained from Crohn’s disease tissue was markedly elevated
(range, 29 to 236 pg/ml; mean, 168 pg/ml; n = 8)
compared to normal LPMCs and was further stimulated by LPS by
approximately twofold (range, 76 to 590 pg/ml; mean, 354 pg/ml; Figure 2B
).
Comparison of LPS-Activated PBMs and Nonactivated LPMCs from Crohn’s Disease
Striking similarities were found between the T84 functional
changes induced by Crohn’s disease LPMCs in the absence of LPS and
those induced by LPS-activated PBMs (Table 2)
. These data suggest that macrophages
in the mucosa of patients with Crohn’s disease are spontaneously
activated, possibly by bacteria or their products, and directly
influence intestinal epithelial physiology in a manner similar to
LPS-activated circulating PBMs. Therefore, we used normal PBMs to
examine if a nonpathogenic control strain of E. coli
(HB101), added to the luminal aspect (physiological side) of T84 cell
monolayers, could induce changes in epithelial function.
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Epithelial Ion Transport
The baseline Isc of T84 monolayers was not significantly altered
by co-culture with luminally applied E. coli only (ie,
123 ± 8% of control values; Figure 4A
). However, co-culture with luminal
bacteria in the presence of basal PBMs resulted in a significantly
(P < 0.05) elevated T84-baseline Isc (217
± 39% of control values). Also, the Isc evoked by forskolin
remained unchanged after co-culture with E. coli alone
(93 ± 11% of control values) but was significantly
(P < 0.05) diminished in the presence of basal
PBMs (61 ± 7% of control values) (Figure 4B)
.
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Co-culture of the T84 monolayers with E. coli alone had
no effect on the TER. However, TER was reduced (54 ± 7% of
control values; P < 0.05) after 48 hours of co-culture
in the presence of PBMs (Figure 5A)
.
Similarly, after 48 hours co-culture with E. coli plus PBMs
(but not bacteria alone), the serosal-to-mucosal flux of
51Cr-EDTA across T84 monolayers was significantly
(P < 0.05) elevated compared with control
monolayers (n = 6 to 8; Figure 5B
). This
increase in epithelial permeability is in agreement with our previous
study, in which normal PBMs activated by bacterial products induced an
increase in the flux of 51Cr-EDTA.13
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Production and Effect of Anti-TNF- Treatment
PBMs stimulated indirectly by co-culture with the nonpathogenic
E. coli secreted substantial amounts of TNF- after 24
hours (range, 984 to 2,907 pg/ml; mean, 2,240 pg/ml; n
= 5). Addition of anti-TNF- antibody at the start of the co-culture
completely prevented the increase in T84-baseline Isc (113 ± 19%
of control values) and restored the diminished Isc response to
forskolin (to 98 ± 12% of control values) (Figure 4)
. In
addition, anti-TNF- ameliorated both the changes in the TER and the
increased flux of 51Cr-EDTA caused by E.
coli infection in the presence of PBMs (Figure 5)
. Use of an
irrelevant control antibody did not prevent the changes in epithelial
responsiveness to forskolin or TER, that were reduced to 66% and 52%
of control values, respectively (n = 4 to 6
monolayers from two experiments).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we show that PBMs from normal patients and those from patients
with Crohn’s disease were not spontaneously active with respect to
TNF- production and, apart from a slight decrease in TER, did not
alter epithelial physiology. LPS-induced activation, however, resulted
in comparable changes in T84 cell function after co-culture with PBMs
from both groups—increased baseline Isc, reduced responses to
forskolin and reduced TER were observed. Similarly, and in accordance
with Bouma and colleagues,26
there was no significant
difference in the intrinsic capacity of normal PBMs and Crohn’s
disease PBMs to produce TNF- after stimulation with LPS. We have
reported that the increase in T84-baseline Isc elicited by co-culture
with activated monocytes/macrophages is because of active
Cl- secretion13
and this is mostly
likely the cause of the increased Isc observed here. Also, the
magnitude of the reduction in the Isc response to forskolin suggests
that it is not solely a consequence of the concomitant increase in
baseline Isc, and likely reflects a specific perturbation in ion
transport signaling. Oprins and colleagues27
recently
reported that TNF- potentiates the Isc response elicited in the
HT-29 epithelial cell line in response to cholinergic stimulation.
These data complement our findings, demonstrating that TNF- can
affect enteric epithelial ion transport, however our data indicate that
TNF- (in the context of other mediators) reduces the response to
forskolin, indicating distinct effects of this cytokine on
Ca2+- and cAMP-driven Cl-
transport.
We also document, for the first time, that LPMCs from Crohn’s disease
differ from those of normal patients in their ability to directly
influence enteric epithelial physiology. LPMCs isolated from normal
intestinal mucosa induced neither secretory (baseline Isc, Isc to
forskolin) nor permeability (TER) changes in the T84 monolayers after a
48-hour co-culture period. These cells did not respond directly to LPS
(using TNF- production as an indication of activity) nor did they
induce epithelial physiological changes on LPS exposure. In contrast,
LPMCs isolated from affected Crohn’s disease mucosa displayed
spontaneous TNF- production and evoked significant changes in
epithelial function, as evidenced by a marked increase in T84-baseline
Isc, diminution of the response to forskolin and impairment of barrier
function (ie, reduced TER) after co-culture.
The fact that normal LPMCs were incapable of activation by LPS could be explained by the lack of CD14+ macrophages.8 This limitation of the macrophage response is important in avoiding an inflammatory reaction to every day exposure of the epithelium to LPS from commensal bacteria in the normal intestine. In the inflamed intestine, however, the continuing influx of blood monocytes to the mucosa results in the presence of a population of macrophages that is strongly CD14+ and, consequently, sensitive to LPS. This sensitivity to LPS exposure becomes particularly important when associated with the damaged epithelial cells in active IBD or in the presence of a primary abnormality in epithelial tight junctions,28 when large quantities of LPS may leak across the intestinal epithelium and gain access to the underlying mucosa. Exposure to LPS could activate newly recruited macrophages in Crohn’s disease mucosa, thus causing changes in epithelial function (ie, ion secretion and permeability) analogous to those observed in this in vitro model system.
TNF- was not detected in the supernatants of normal LPMCs under
basal or LPS-stimulated conditions. In contrast, LPMCs obtained from
affected Crohn’s disease mucosa spontaneously produced TNF- and
were further stimulated by LPS to produce greater amounts of this
cytokine. Infiltrating monocyte-like macrophages are an important
source of TNF- in Crohn’s disease.9
Currently, there
is much interest in the role of TNF- in Crohn’s disease. Several
studies have shown increased TNF- protein and mRNA levels in
biopsies obtained from patients with Crohn’s
disease.25,29,30
The increased level of TNF- may lead
to enhanced activation of multiple mucosal cells, including
macrophages, lymphocytes, and epithelial cells, and thereby contribute
to mucosal damage in the intestine. In our experiments, a key role for
TNF- in the pathogenesis of the epithelial abnormalities was
found—both ion transport and barrier defects were prevented by
inclusion of an anti-TNF- antibody in the co-culture.13
The importance of TNF- in the pathophysiology of Crohn’s disease is
further supported by recent clinical trials demonstrating the efficacy
of anti-TNF- antibody in treatment of Crohn’s
disease.10-12
There is considerable evidence from both animal models and clinical
investigations supporting a pivotal role for bacteria in the initiation
or exacerbation of the intestinal inflammation. For instance, bacteria,
or their products, have been detected in inflamed mucosa of patients
with Crohn’s disease,15
antibiotic
treatment31
or diversion of the fecal
stream14
can reduce disease severity in some patients with
Crohn’s disease, and in the majority of the spontaneous models of
murine colitis the inflammation does not occur when the animals are
housed under germ-free conditions.16,32-34
However,
little data are available on the role of commensal bacteria in altering
epithelial function. Because LPMCs from patients with Crohn’s disease
evoke epithelial abnormalities virtually identical to those elicited by
LPS-activated PBMs (Table 2)
, we used PBMs (a convenient and readily
available source of monocytes) to explore the affect of a nonpathogenic
strain of E. coli (HB101) on epithelial physiology.
Our investigations revealed that a nonpathogenic strain of bacteria
could indeed, via activation of monocytes/macrophages, induce
irregularities in enteric epithelial function that are characteristic
of those observed in tissue resections from patients with IBD. We
showed a marked increase in T84-baseline Isc, reduced responsiveness to
forskolin and a simultaneous disruption of barrier function (ie,
reduced TER and increased 51Cr-EDTA flux) after
co-culture with luminal bacteria, but only in the presence of PBMs.
These results indicate that nonpathogenic commensal bacteria, in the
presence of CD14+ macrophages, have the potential
to alter intestinal epithelial secretory and barrier functions via
immune activation. In vivo, similar events could result in a
cascade of immune events, the consequences of which may be tissue
damage leading to chronic inflammation. In this context, Duchmann and
colleagues1,5
have shown that LMPCs isolated from patients
with IBD, or from mice with a chemically induced colitis, proliferate
when exposed to autologous intestinal bacteria. Collectively these data
support the concept that the tolerance toward autologous intestinal
flora in healthy individuals is lost during inflammation associated
with IBD. It is also noteworthy that the altered epithelial functions
observed in our in vitro tripartite model were negated by
use of an anti-TNF- antibody, adding further support for the current
hypothesis that an inappropriate immune response to bacteria
contributes to the pathophysiology of IBD.
In summary, this study extends our previous findings,13,20
illustrating the ability of PBMs from patients with Crohn’s disease
exposed directly to LPS, or indirectly (ie, separated by an epithelial
layer) to nonpathogenic bacteria to affect the transport and barrier
properties of a model gut epithelium. Moreover, we have identified that
co-culture with LPMCs from patients with Crohn’s disease, unlike those
from controls, resulted in increased epithelial permeability and
altered ion transport in the absence of LPS stimulation and that
inclusion of LPS did not accentuate the changes in epithelial function.
We conclude that LPMCs from patients with Crohn’s disease are
spontaneously altered compared to controls, and that their response to
bacteria/bacterial products may lead to epithelial dysfunction through
a TNF--dependent mechanism. Finally, while one must always bear in
bind the inherent cellular complexity of the gut, we present an easily
manipulated tripartite model that can be used to dissect signaling
pathways between enterocytes, immune cells, and microflora in the
regulation of gut physiological and pathophysiological reactions.
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Footnotes |
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Supported by grants from the Crohn’s and Colitis Foundation of Canada, the Hospital for Sick Children Foundation, and the Medical Research Council of Canada.
Accepted for publication November 9, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IL-1ß by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn’s disease. Clin Exp Immunol 1993, 94:174-181[Medline]
for Crohn’s disease. N Engl J Med 1997, 337:1029-1035. Am J Physiol 1998, 275:C932-C939ß-deficient mice fail to develop colitis in the absence of a microbial environment. Am J Pathol 1997, 150:91-97[Abstract]
directly affects barrier function of cultured intestinal epithelial monolayers. J Clin Invest 1989, 83:724-727
and ß in patients with inflammatory bowel disease and healthy controls. Scand J Gastroenterol 1995, 30:1095-1100[Medline]
potentiates the ion secretion induced by muscarinic receptor activation in HT29cl.19A cells. Am J Physiol 2000, 278:C463-C472, interleukin-1ß, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn’s disease. Gut 1996, 39:684-689This article has been cited by other articles:
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  K. Lewis, J. Caldwell, V. Phan, D. Prescott, A. Nazli, A. Wang, J. D. Soderholm, M. H. Perdue, P. M. Sherman, and D. M. McKay Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-{alpha} Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G669 - G678. [Abstract] [Full Text] [PDF]
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 A. Nazli, A. Wang, O. Steen, D. Prescott, J. Lu, M. H. Perdue, J. D. Soderholm, P. M. Sherman, and D. M. McKay Enterocyte Cytoskeleton Changes Are Crucial for Enhanced Translocation of Nonpathogenic Escherichia coli across Metabolically Stressed Gut Epithelia Infect. Immun., January 1, 2006; 74(1): 192 - 201. [Abstract] [Full Text] [PDF]
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 S. Menard, D. Laharie, C. Asensio, T. Vidal-Martinez, C. Candalh, A. Rullier, F. Zerbib, F. Megraud, T. Matysiak-Budnik, and M. Heyman Bifidobacterium breve and Streptococcus thermophilus Secretion Products Enhance T Helper 1 Immune Response and Intestinal Barrier in Mice Experimental Biology and Medicine, November 1, 2005; 230(10): 749 - 756. [Abstract] [Full Text] [PDF]
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 Q S Guo, B Xia, Y Jiang, S A Morre, L Cheng, J Li, J B A Crusius, and A S Pena Polymorphisms of CD14 gene and TLR4 gene are not associated with ulcerative colitis in Chinese patients Postgrad. Med. J., August 1, 2005; 81(958): 526 - 529. [Abstract] [Full Text] [PDF]
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 E. J. Schiffrin, M. E. Yousfi, M. Faure, L. Combaret, A. Donnet, S. Blum, C. Obled, and D. Breuille Milk Casein-Based Diet Containing TGF-{beta} Controls the Inflammatory Reaction in the HLA-B27 Transgenic Rat Model JPEN J Parenter Enteral Nutr, July 1, 2005; 29(4_suppl): S141 - S150. [Abstract] [Full Text] [PDF]
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 J D Soderholm, C Streutker, P-C Yang, C Paterson, P K Singh, D M McKay, P M Sherman, K Croitoru, and M H Perdue Increased epithelial uptake of protein antigens in the ileum of Crohn's disease mediated by tumour necrosis factor {alpha} Gut, December 1, 2004; 53(12): 1817 - 1824. [Abstract] [Full Text] [PDF]
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F Bataille, F Klebl, P Rummele, J Schroeder, S Farkas, P-J Wild, A Furst, F Hofstadter, J Scholmerich, H Herfarth, et al. Morphological characterisation of Crohn's disease fistulae Gut, September 1, 2004; 53(9): 1314 - 1321. [Abstract] [Full Text] [PDF]
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D Berrebi, R Maudinas, J-P Hugot, M Chamaillard, F Chareyre, P De Lagausie, C Yang, P Desreumaux, M Giovannini, J-P Cezard, et al. Card15 gene overexpression in mononuclear and epithelial cells of the inflamed Crohn's disease colon Gut, June 1, 2003; 52(6): 840 - 846. [Abstract] [Full Text]
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S Fujino, A Andoh, S Bamba, A Ogawa, K Hata, Y Araki, T Bamba, and Y Fujiyama Increased expression of interleukin 17 in inflammatory bowel disease Gut, January 1, 2003; 52(1): 65 - 70. [Abstract] [Full Text] [PDF]
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 D. A. van Heel, I. A. Udalova, A. P. De Silva, D. P. McGovern, Y. Kinouchi, J. Hull, N. J. Lench, L. R. Cardon, A. H. Carey, D. P. Jewell, et al. Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF-{kappa}B transcription factors Hum. Mol. Genet., May 16, 2002; 11(11): 1281 - 1289. [Abstract] [Full Text] [PDF]
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