Intestinal Disease Research Program, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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We examined the
ability of monocytes (M) activated by bacterial products to alter
epithelial physiology. Confluent monolayers of the T84 colonic
epithelial cell line were grown on filter supports and then cocultured
in the presence of human M
with or without the activating agents
bacterial lipopolysaccharide and the bacterial tripeptide
formyl-methionyl-leucyl-phenylalanine. After 24 or 48 h, monolayers
were mounted in Ussing chambers where parameters of epithelial function
were measured. Exposure to activated M
resulted in a significant
increase (P < 0.05) in baseline
short-circuit current (250% after 48 h) that was associated with
enhanced secretion of Cl
.
In addition, epithelial permeability was significantly increased as
shown by reduced transepithelial resistance and increased flux of
51Cr-EDTA. Activated M
produced
substantial amounts (~3 ng/ml at 48 h) of tumor necrosis factor-
(TNF-
). TNF-
was identified as a key mediator acting via an
autocrine mechanism to induce epithelial pathophysiology. Our data show
that M
, when activated by common bacterial components, are potent
effector cells capable of initiating significant changes in the
transport and barrier properties of a model epithelium.
epithelium; ion transport; permeability
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INTRODUCTION |
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MUCOSAL SURFACES, particularly the intestine, are exposed to a wide variety of commensal and potentially pathogenic bacteria. Several lines of evidence now point to a role for bacteria and/or their products in the pathogenesis of mucosal inflammatory disorders (11, 41), especially in the context of aberrant immune function (10, 18). Inflammatory bowel diseases (IBD) are often characterized by altered epithelial physiology, typically increased permeability and electrolyte secretion that can create a luminally directed driving force for water movement resulting in diarrhea. Epithelial pathophysiology may be caused by activated immune cells, since many studies have provided unequivocal evidence that the transport and barrier functions of the epithelial lining of mucosal surfaces are regulated by cells such as lymphocytes, mast cells, and neutrophils (29, 30).
Although many studies have confirmed the concept of
immunomodulation of epithelial physiology, few studies
have considered the ability of cells of the monocyte/macrophage (M)
lineage to directly affect epithelial function. However, these cells
are among the first immune cells to react on initial exposure to
antigens and infective organisms. For instance, monocytes exposed to
lipopolysaccharide (LPS) and the bacterial tripeptide
formyl-methionyl-leucyl-phenylalanine (FMLP) respond with the
production of mediators and the synthesis of cytokines/growth factors
such as the interleukins (IL), IL-1, IL-6, tumor necrosis factor-
(TNF-
), and transforming growth factor-
(1, 8, 16, 27, 44). These
factors can directly, or indirectly, affect epithelial function,
altering transport and barrier characteristics (24, 25, 38, 49). In
addition, processes from tissue macrophages occur close to the basement membrane of the overlying epithelium, and this spatial association may
facilitate bidirectional communication between the two cell types.
Finally, monocyte chemoattractant peptide-1 (MCP-1) has been
immunocytochemically demonstrated in the surface epithelium of human
colonic biopsies, and its expression is enhanced in tissues from
patients with Crohn's disease or ulcerative colitis (35). These
findings indicate a clear potential for M
to regulate epithelial physiology.
Resident intestinal macrophages do not normally express the LPS receptor CD14 (22). However, it was recently reported that macrophages in resected intestinal segments from patients with IBD express a unique phenotype with unusually high levels of CD14 (12, 13), presumably because of rapid recruitment of monocytes from the circulation to the gut (4, 39). Similarly, it has been shown that monocytes are recruited to the airways during an inflammatory response, and these newly recruited cells are more active in tissue damage than resident macrophages (6).
Integrating these themes, this study examined the specific hypothesis
that human M activated by the bacterial products LPS and FMLP can
influence epithelial electrolyte transport and barrier functions. Here,
we used the human colonic T84 cell line as a model epithelium (9) and a
coculture approach analogous to that used to define the ability of
lymphocytes and polymorphonuclear cells (PMN) to regulate epithelial
physiology (19, 24). Our data show that purified M
(in the absence
of other classes of immune cells) significantly increased luminally
directed Cl
secretion and
disrupted epithelial barrier function. These changes in epithelial
function were inhibited by inclusion in the coculture system of a
neutralizing antibody against TNF-
, implicating this cytokine as a
critical mediator in gut pathophysiology. Further studies indicated an
important autocrine mechanism of action for TNF-
on M
. Thus M
have been identified as being capable of directly modulating epithelial
function. We speculate that given appropriate environmental conditions,
activation of M
could be a precipitating event in the onset of
pathophysiology leading to chronic secretory or inflammatory disease in
the intestine.
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MATERIALS AND METHODS |
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Cell Culture
Epithelial cells.
T84 cells (passage 45-65) were seeded onto tissue culture-treated
semipermeable filter supports (0.4-µm pore size,
1.0-cm2 surface area; Costar,
Cambridge, MA) at a concentration of
106 cells/ml and grown in culture
media consisting of equal volumes of DMEM and F-12 medium, supplemented
with 1.5% (vol/vol) HEPES, 2% (vol/vol) penicillin-streptomycin, and
10% newborn calf serum (all from GIBCO Laboratories, Grand Island, NY)
(24). After culture for 7 days, confluent T84 monolayers consistently
displayed electrical resistances 1,000
· cm2.
Immune cells.
Human peripheral blood mononuclear cells (PBM) from healthy volunteers
(male and female, ages 23-45 yr) were isolated by one-step density
centrifugation of whole blood over Ficoll-Hypaque (Pharmacia Biotech,
Uppsala, Sweden) and resuspended in fresh media at
106 cells/ml. The M population
was obtained by plastic plating of PBM (4 h at 37°C) and subsequent
removal of nonadherent T and B cells. Fresh media were added to the
adherent cells, which were then incubated for 18 h at 37°C before
use in coculture studies. Assessment of T cells and monocytes in the
adherent cell population was carried out by two-color flow cytometry
analysis [fluorescence-activated cell sorter (FACS)] after staining
the cells with FITC-conjugated anti-CD3 (OKT3, Coulter Immunology,
Hialeah, FL) and phycoerythrin-conjugated anti-CD14
(Caltag Laboratories, San Francisco, CA), respectively. Analysis was
performed using FACScan (Becton Dickinson, Mississauga, Ontario,
Canada) followed by data analysis using PC-Lysys II computer software
(Becton Dickinson).
Immune Cell Activation
MCoculture Studies
Confluent T84 monolayers were cocultured for 24 or 48 h with LPS/FMLP-activated MUssing Chamber Studies
Epithelial ion transport.
After coculture, T84 monolayers were mounted in Ussing chambers as
previously described (24). Epithelial monolayers were bathed in
oxygenated Krebs buffer (37°C) containing 10 mM glucose as an
energy source in the serosal buffer, which was osmotically balanced by
10 mM mannitol in the mucosal buffer. The epithelial spontaneous
potential difference was maintained at 0 V by the continuous injection
of an external current by an automated voltage clamp (World Precision
Instruments, 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-min equilibration period. Stimulated ion secretion was
measured by addition of the cholinergic agonist carbachol
(104 M) or the adenylate
cyclase-activating agent forskolin
(10
5 M) (both from Sigma
Chemical) to the serosal side of the T84 monolayers and recording the
maximum increase in
Isc (24).
Epithelial permeability.
Electrical resistance is a measure of the barrier property 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 s), and the resulting
change in current was used to calculate the transepithelial ion
resistance (R, in
· cm2)
according to Ohm's law (33). As an indication of epithelial permeability to larger molecules, the M
S movement of the inert probe 51Cr-EDTA (362.3 Da,
diameter 1.15 nm) was measured.
51Cr-EDTA (Radiopharmacy,
McMaster-Chedoke Hospital, Hamilton, Ontario, Canada) was added to the
mucosal buffer at a final concentration of 6.5 µCi/ml. Nonradioactive
Cr-EDTA was added to the serosal buffer to maintain the osmotic
balance. Fluxes were determined using 30-min flux periods (14).
Cell Viability
T84 monolayer viability was assessed by measuring release of lactate dehydrogenase (LDH) (20). After coculture, T84 monolayers were removed and rinsed three times in fresh PBS. Epithelial monolayers were lysed by immersing each filter in 0.1% (vol/vol) Triton X-100 (Sigma Chemical)/PBS for 30 min at room temperature followed by vigorous manual pipetting. The lysate was centrifuged at 500 rpm for 5 min, and the supernatant was analyzed for LDH activity using an automated multiple point rate test (Kodak, Rochester, NY).Studies to Determine the Role of TNF-
The role of TNF- in the M
modulation of epithelial 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 CM) (Centocor). An irrelevant isotype-matched antibody
(anti-hepatoma IgG1, AF20; Centocor) was used as control. Additional
studies examined the effect of cA2 in CM added to T84 cells or to M
.
Statistics
Results are presented as means ± SE. 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); n values represent the number of experiments (different blood donors) in which two to four monolayers were examined for each condition. Data were analyzed using one-way ANOVA followed by Newman-Keuls comparison. Student's t-test was used where appropriate for individual comparisons. Statistically significant differences were accepted at P < 0.05. ![]() |
RESULTS |
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Immune Cells
FACS analysis showed that >95% of the adherent immune cell population expressed CD14 and were the appropriate size for monocytes (n = 6). Less than 5% of cells expressed CD3, indicating that virtually no T cells were present. MEpithelial Physiology After Coculture With Monocytes
T84 monolayers cultured with LPS/FMLP in the absence of MEpithelial Ion Transport
Baseline Isc of T84 monolayers was unaltered after 24-48 h of coculture with nonactivated M
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The Isc increase
evoked by forskolin (88.0 ± 9.3 µA/cm2) was unaltered by
coculture with nonactivated M (24-48 h) or by exposure to
activated M
for 24 h. However, after 48 h of culture with activated
M
, there was a significantly diminished response (Fig. 2). In contrast, the stimulated
Isc evoked by
carbachol was unaffected by coculture (24 or 48 h) with M
or
activated M
(119 ± 24 and 95 ± 21% of control values,
respectively).
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Epithelial Permeability
Coculture with nonactivated M
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The degree of the epithelial barrier defect was further assessed by
determination of the flux of the radiolabeled probe
51Cr-EDTA. After 48 h of coculture
with activated M (but not nonactivated M
), the M
S
movement of 51Cr-EDTA across the
T84 monolayers was significantly increased compared with control
monolayers (3.12 ± 1.49 vs. 0.76 ± 0.66 nmol · h
1 · cm
2,
respectively) (Fig. 4).
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Epithelial Viability
After 48 h, there was no significant difference in LDH released from T84 epithelial cells cultured in media only or cocultured with activated MEpithelial Physiology After Culture With CM
The altered epithelial ion transport properties (elevation of baseline Isc and reduced responsiveness to forskolin) were also observed after 48-h culture with CM from activated M
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Role of TNF-
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However, exposure of naive T84 monolayers to 3 or 6 ng/ml of
recombinant TNF- (amount measured in CM from activated M
after 24 and 48 h, respectively) for 48 h had no significant effect on baseline
secretory properties and transepithelial resistance of the epithelial
cells (data not shown). In addition, cA2 in CM from activated M
added to T84 cells did not prove beneficial in correcting the altered
epithelial ion secretion (baseline
Isc of 276 ± 66 vs. 263 ± 57% of control values) or barrier properties (resistance of 49 ± 5.6 vs. 48 ± 10% of control values) for CM vs. CM + cA2, respectively. This suggested an indirect effect of
TNF-
, perhaps via autocrine action on M
themselves. To examine this possiblity, T84 monolayers were exposed to the CM that was prepared in the presence of cA2 antibody. Under these conditions, the
increased baseline
Isc was
completely returned to control levels, and the reduced T84 resistance
was significantly corrected (Table 2). This hypothesis was confirmed,
since T84 monolayers cocultured with M
activated with human
recombinant TNF-
for 48 h consistently showed elevated baseline
Isc, diminished
responses to forskolin, and lowered epithelial resistance that were
significantly different from control values (Fig.
6). These changes in epithelial function
were very similar to those observed in coculture with LPS/FMLP-activated M
.
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DISCUSSION |
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We have used an in vitro coculture model system to demonstrate that
M activated with common bacterial products, in the absence of other
immune cell types, can alter epithelial ion transport and permeability.
Our results showed that after 48 h of coculture, activated M
stimulated epithelial Cl
secretion and increased permeability leading to impaired epithelial barrier function. We also demonstrated that M
-derived TNF-
was a
key factor in mediating these abnormalities as addition of a neutralizing antibody against TNF-
in the coculture system inhibited the epithelial defects.
Monocytes/macrophages play a central role in immune and inflammatory
events in the intestinal mucosa. Resident macrophages are located close
to the basal membrane of the intestinal epithelium and represent the
first line of defense by immune cells. Additional monocytes may be
attracted to the intestinal mucosa during inflammation by locally
produced MCP-1 (35). Recent studies have demonstrated the appearance of
monocyte subpopulations with a different phenotype in IBD mucosa (22,
39) that unlike resident macrophages in normal intestine express high
levels of the LPS receptor CD14 (4, 12, 39). The newly recruited cells
are more easily activated, resulting in the production of excessive
amounts of potent inflammatory mediators (2, 39). Isolated mononuclear cells from the colonic mucosa of IBD patients have an increased ability
to undergo respiratory burst and to stimulate immunoglobulin secretion
and an enhanced antigen-presenting activity (5). Furthermore, it has
been recently reported that the
CD14+ subset of macrophages from
IBD mucosa have a different cytokine profile compared with the resident
macrophages and are primed for the production of TNF-, IL-1, and
IL-6, all of which can directly or indirectly affect epithelial
function (40). Despite these data, the ability of M
to directly
regulate epithelial physiology has not been examined. Additionally, LPS
is present in large quantities in the intestinal lumen, and when
exposed to LPS, M
synthesize a plethora of proinflammatory mediators (8, 16, 27, 36) that are capable of inducing local tissue damage
through their interactions with T cells, leukocytes, and endothelial
cells (28). The bacterial tripeptide FMLP (43) induces monocyte
chemotaxis and adherence as well as the production of oxygen radicals
and proinflammatory eicosanoids (26). Because LPS and FMLP are usually
present simultaneously in the gut lumen, we chose to add both agents
directly to M
(mimicking events after their uptake from the lumen)
and then determine the consequence of this M
activation on
epithelial ion transport and permeability.
Our study clearly shows that M activated by LPS/FMLP cause
significant changes in epithelial ion transport and barrier functions. Coculture of T84 cells with activated M
elicited a significant increase in epithelial ion secretion as shown by the elevation of T84
baseline Isc. The
elevated baseline secretion was most pronounced 48 h after coculture
and was associated with increased Cl
secretion. However, the
Isc increase was
less than would be expected based on
Cl
secretion alone,
suggesting that there is another transport event that is decreasing the
measured Isc that
is not accounted for. From the data presented in Table 1, it appears
that altered Na+ is not
responsible. These observations complement findings documenting that
LPS-stimulated alveolar macrophages altered ion transport in isolated
rat lung epithelial cells (6). In comparison with our findings, T84
monolayers cocultured with PMN also display an increase in baseline
Isc (19).
Activated M increased epithelial ionic permeability as illustrated
by a significant reduction in transepithelial resistance of the
monolayer after 48 h of coculture. Concomitant with the reduced
resistance was an increased transepithelial flux of the inert probe
51Cr-EDTA, which is suggestive of
increased paracellular permeability (34, 42). These changes in
epithelial permeability were evoked by remarkably few cells
(2.5-5 × 104
M
). Because epithelial cells were plated at an original density of
106 T84 cells/filter, a
significant increase in epithelial permeability was observed at a ratio
of
40:1 of epithelial cells to M
. Similar changes in epithelial
physiology have been documented at a ratio of 20:1 of epithelial cells
to PMN (15, 19). Thus our data indicate that M
have a potent ability
to alter epithelial ion transport and permeability. It is clear that
M
can be added to the growing list of immune cells (T cells,
neutrophils, and eosinophils; Refs. 19, 23, 24, 37) that regulate
epithelial physiology.
Increased permeability of the epithelial monolayers could potentially
be because of epithelial cell cytotoxicity. However, the effects of
activated M on T84 cells were not simply the result of decreased
epithelial viability, since release of LDH from M
cocultured
epithelial monolayers was not different from control monolayers. A
number of previous studies have shown similar increases in permeability
of T84 epithelial monolayers cocultured with other immune cells (20,
24) or infected with enteropathogenic E. coli (31) without any significant epithelial
cytotoxicity. Moreover, after exposure to activated M
, the
epithelial monolayer was still capable of substantial vectorial ion
secretion as indicated by the elevated baseline
Isc of the
monolayer and significant cAMP- and normal
Ca2+-mediated
Cl
secretion. These events
indicate a functionally intact monolayer.
Cell-free CM from activated M was equally effective in producing the
epithelial abnormalities observed after coculture with activated M
.
Stimulation of M
by LPS and FMLP leads to the production of an array
of proinflammatory cytokines (8, 16, 27), and among these, TNF-
presents itself as a clear candidate for the mediation of the altered
epithelial physiology. Several studies have reported increased TNF-
protein and mRNA levels in biopsies from IBD patients, particularly in
Crohn's disease (17, 32). Significantly increased concentrations of
TNF-
have been reported in stools of children with active chronic
IBD (3). Other studies have also reported a rise in circulating TNF-
and the soluble TNF-
receptor (p55) in patients with active IBD that
were significantly correlated with the clinical and/or
laboratory measures of disease activity (11, 17). Furthermore, in a
recent multicenter, placebo-controlled trial, anti-TNF-
antibody
(cA2 antibody; same antibody used in this study) treatment resulted in
prolonged clinical improvement in some patients with Crohn's disease
(46). Therefore, having demonstrated significant TNF-
production by
M
to our cocktail of LPS/FMLP, we proceeded to examine the role of
TNF-
in M
modulation of epithelial function. Neutralization of
TNF-
in the coculture model by addition of an anti-TNF-
antibody
reduced the T84 baseline Isc response to
control levels and significantly improved the T84 transepithelial
resistance. In addition, anti-TNF-
treatment inhibited the increased
epithelial permeability to
51Cr-EDTA evoked by culture with
activated M
. The correction of the abnormal epithelial function by
anti-TNF-
suggested that TNF-
directly affected T84 physiology,
alone or in concert with other immune mediators, or that the TNF-
effect was one of autocrine activation of M
. A direct effect of
TNF-
on HT-29 cells has been demonstrated (38). However, a number of
studies have not been able to illustrate a direct action of TNF-
on
T84 function after an acute exposure (
72 h; 3-6 ng/ml) (20, 24,
48). In contrast, a recent study has shown a direct effect of TNF-
(100 ng/ml) on transepithelial resistance, but only in the presence of
interferon-
(47). Another study demonstrated that chronic treatment
(4 days) of T84 monolayers with 100 ng/ml recombinant TNF-
caused a
significant increase in inulin movement across the T84 monolayers (21).
In exploring the role of TNF- in our model of epithelial
disfunction, we found that addition of recombinant TNF-
at the concentrations measured in the supernatant from activated M
did not
affect epithelial physiology. Moreover, neutralization of TNF-
in
activated M
CM did not prevent the disrupted secretory responses or
the increased epithelial permeability, whereas inclusion of cA2 at the
time of preparation of the CM resulted in a CM that evoked
significantly less epithelial abnormalities. This suggested that the
TNF-
was autocrinely affecting the M
, and not the epithelium directly. To furthur examine this possibility, recombinant TNF-
was
added to M
-T84 cocultures, and this resulted in elevated baseline
Isc and decreased
transepithelial resistance of T84 monolayers. These results were very
similar to those evoked by LPS/FMLP activation of M
. Taken together,
these results support the hypothesis that TNF-
affected M
in an
autocrine manner (7, 45), causing the release of other as yet
unidentified monocyte-derived mediators, the net result of which was
altered epithelial function. Integrating these findings with previous
studies, we suggest that TNF-
can modulate epithelial function both
directly (studies with HT-29 cells, Ref. 21) and indirectly (our study)
via immune cell activation.
In summary, we have demonstrated that M activated by common
bacterial products can stimulate
Cl
secretion, alter ion
transport responses, and impair the barrier function of the epithelium.
We have also shown that TNF-
is a key factor mediating the
M
-induced pathophysiology, although not affecting T84 cells
directly. It has been suggested that during active IBD, LPS passes
through the mucosal barrier, gaining access to M
, and primes them
such that subsequent contact with luminal bacteria results in excessive
production of potent inflammatory mediators, particularly TNF-
,
resulting in pathology/pathophysiology of intestinal tissue (2).
Modeling this scenario in vitro, we have demonstrated that LPS/FMLP
activation of M
has significant consequences for epithelial ion
transport and permeability functions.
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ACKNOWLEDGEMENTS |
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We thank P. Singh, P. Stetsko and D. Steele-Norwood for expert technical assistance.
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FOOTNOTES |
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This research was funded in part by Centocor, Astra Draco Pharma, the Crohn's and Colitis Foundation of Canada, and the Medical Research Council, Canada.
Address for reprint requests: M. H. Perdue, Intestinal Disease Research Program, HSC-3N5, McMaster University, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
Received 23 November 1997; accepted in final form 10 June 1998.
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