Institute for Neurobiology, Biological Faculty, University of Amsterdam, 1098 SM Amsterdam, The Netherlands
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ABSTRACT |
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Chronic gastrointestinal
diseases such as ulcerative colitis and Crohn's disease are
characterized by severe diarrhea. Mucosal biopsies of these patients
show enhanced levels of cytokines, secreted by infiltrated inflammatory
cells. In this study, we investigated the effect of the cytokine tumor
necrosis factor- (TNF-
) on ion secretion in human intestinal
epithelial cells. The conventional microelectrode technique in the cell
line HT29cl.19A was used, which allows for simultaneous measurements of
transepithelial potential difference and intracellular potential
difference across the apical membrane. Preincubation (2-78 h) with
10 ng/ml TNF-
did not change basal secretory activity. However, the
secretory response to the muscarinic receptor agonist carbachol was
strongly increased after exposure to TNF-
. Application of the
protein kinase C (PKC) inhibitor GF 109203X (bisindolylmaleimide I)
inhibited the response to carbachol as well as the TNF-
-potentiated
response, indicating that PKC mediates the effect of carbachol in this
cell line. Propranolol, a substance that inhibits the phospholipase D
(PLD) pathway, strongly reduced the response to muscarinic stimulation and its potentiation by TNF-
. The results indicate that activation of PLD is involved in ion secretion induced by muscarinic receptor activation and that TNF-
can potentiate this pathway.
phospholipase D; protein kinase C; intestinal epithelia; carbachol; cytokine; tumor necrosis factor-
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INTRODUCTION |
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CHLORIDE SECRETION ACROSS intestinal epithelium plays a
key role in regulating water secretion into intestinal lumen. The Cl secretion is under close
regulation by hormonal, neural, and paracrine mediators. An increased
Cl
secretion can result in
severe diarrhea, due to excessive water transport from blood to lumen.
Inflammatory bowel diseases (IBD) like ulcerative colitis and Crohn's disease are characterized by diarrhea. The underlying pathophysiological mechanisms for the diarrhea remain unknown. IBD patients show an increase in cytokines in the intestinal wall, which are secreted by infiltrated inflammatory cells (8, 23).
Antibodies against tumor necrosis factor- (TNF-
), one of the
elevated cytokines, have been applied in animal models of experimental colitis. These studies suggested a role for antibodies against TNF-
in the treatment of IBD (37). In a multicenter, double-blind, placebo-controlled trial, a single infusion of a monoclonal antibody (cA2) against this cytokine appeared to be an effective treatment in
patients with Crohn's disease (30). This indicates the importance of
TNF-
in the disease. Several studies have shown that cytokines are
able to alter ion transport and barrier properties of intestinal epithelium (19, 21), which could contribute to the diarrhea.
TNF- may mediate activation of ion secretion in intestinal
epithelium, but data on this matter are quite scarce. In human distal
colon, TNF-
was shown to increase ion secretion via an increased
release of prostaglandins by subepithelial cells (27). A similar study
performed in porcine ileum also showed an indirect effect of TNF-
on
ion secretion (15).
Several epithelial cells are known to express TNF- receptors, and,
especially when the receptor density is increased by
interferon-
(IFN-
), they respond to TNF-
directly by
modulating the permeability of the tight junctions (24).
However, direct effects of TNF-
on ion secretion are not widely
studied. This study aims to gain more insight into the possible
direct effect of TNF-
on human intestinal epithelium. With
intracellular electrophysiological techniques, the effect of human
recombinant TNF-
on the basal as well as the secretagogue-induced
ion secretion in human colonic epithelial cells HT29cl.19A was determined.
The data show that TNF- is able to potentiate the secretion induced
by the secretagogue carbachol, an activator of the
Ca2+/protein kinase C
(PKC)-mediated pathway, but not the secretion induced by forskolin, an
activator of the cAMP pathway. The results indicate that
carbachol-induced secretion involves activation of the phospholipase D
(PLD) pathway and that the potentiation by TNF-
occurred via that
pathway. As far as we know, this is the first study in intestinal
epithelial cells that shows a direct effect of TNF-
on
carbachol-induced ion secretion and the involvement of PLD in the
intestinal secretory response.
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MATERIALS AND METHODS |
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Cell culture.
HT29cl.19A cells were cultured as described previously (2). Briefly,
the human intestinal epithelial cell line HT29cl.19A, passages
12-28,
was grown in DMEM supplemented with 10% fetal bovine serum, 8 mg/l
ampicillin, and 10 mg/l streptomycin. Cells were seeded in
25-cm2 flasks at 37°C in 5%
CO2-95%
O2 and passaged weekly. For
electrophysiological experiments, cells were subcultured on Falcon
filters (25 mm in diameter), and medium was replaced every other day.
Confluency was reached 7 days after seeding, and the cells were used
between 13 and 26 days after seeding. TNF- incubations were
performed in culture media for the indicated time.
Electrophysiological experiments.
The filter was cut from the ring, divided into four pieces, and rinsed
with mannitol-Ringer. One piece was mounted in a small horizontal
Ussing chamber, leaving an oblong area of 0.35 cm2. The apical and basolateral
compartments were continuously perfused with mannitol-Ringer buffer at
a temperature of 37°C and gassed with 5%
CO2-95%
O2. The composition of the Ringer
solution was (in mM) 117.5 NaCl, 5.7 KCl, 25.0 NaHCO3, 1.2 NaH2PO4,
2.5 CaCl2, 1.2 MgSO4, and 28 mannitol. To
increase the driving force for Cl efflux, we used a
low-Cl
(0.1 mM) solution
containing (in mM) 0.1 NaCl, 117.2 sodium gluconate, 5.7 potassium
gluconate, 2.5 CaSO4, 1.2 MgSO4, 1.2 NaH2PO4,
25 NaHCO3, and 28 mannitol. This
solution was applied to the apical side of the monolayer 15 min before
addition of carbachol. Therefore, the electrophysiological response to
carbachol was not affected by junction potentials.
Histology.
Filters containing cells were exposed to 10 ng/ml TNF- for 24 h,
after which they were cut into small pieces. The filters were fixed in
2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h
at room temperature and rinsed three times for 20 min each in 0.1 M
sodium cacodylate buffer (pH 7.4). The filters were rinsed overnight
(4°C) in 0.1 M sodium cacodylate buffer (pH 7.4) and then processed
for routine electron microscopy.
Chemicals.
TNF-, GF 109203X (bisindolylmaleimide I),
4-
-phorbol-12,13-dibutyrate (PDBu), U-73122, and U-73143 were
obtained from Calbiochem. Cycloheximide, propranolol, and forskolin
were purchased from Sigma, and carbachol was from Brunschwig. TNF-
,
propranolol, and carbachol were dissolved in water. Forskolin and
cycloheximide were dissolved in ethanol (maximal concentration in the
Ussing chamber was 0.1%). PDBu and GF 109203X were dissolved in DMSO. U-73122 and U-73143 were dissolved in chloroform. The maximal concentration of the latter two solvents was 0.01%. Maximal
concentrations of carrier solvents were without electrophysiological
effect on the cells.
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RESULTS |
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TNF- potentiates carbachol-induced secretion.
The electrophysiological response to muscarinic receptor activation
with carbachol and its relation to
Cl
secretion in HT29cl.19A
cells have been previously reported (4). A typical electrical response
to 100 µM carbachol is presented in Fig.
1. The mean
values of the electrical parameters and their changes induced by
carbachol are shown in Table 1. For the
present description, the response was divided into two phases, which
are marked by the two thin lines in Fig. 1. The data in Table 1 were taken at the time points indicated by these lines.
Phase 1 was a fast depolarization of
the Va together
with an increase in
fRa while the
Vt decreased.
Because the Rt
was not affected, the decrease of
Vt must be caused
by a small but significant decrease of the Isc. On the basis
of previous experiments (4), phase
1 is attributed to a sharp increase of
the intracellular Ca2+ activity,
primarily from the inositol 1,4,5-trisphosphate
(IP3)-sensitive intracellular
pool, which activates Cl
conductances located in the apical membrane and, more prominently, in
the basolateral membrane. Phase
2 was characterized by repolarization leading to a hyperpolarization of the apical membrane, concomitant with
an increase in Vt
and a return of the
fRa and
Rt to control values. The increase of the
Isc during
phase
2 of the carbachol response is
ascribed to the opening of basolateral
Ca2+-sensitive
K+ channels (4), which increase
the driving force for Cl
efflux through apical Cl
channels. Previous work in HT29cl.19A cells suggests that these channels are different from the
Ca2+-sensitive
Cl
channels and that PKC
is involved in their activation (4, 35).
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Dose-response curve of TNF-.
To define the dose dependence of the action of TNF-
, filters were
incubated for 24 h with different concentrations of TNF-
varying
between 1 and 100 ng/ml. Figure 2 shows the
effects of the different concentrations of TNF-
on the
carbachol-induced Isc. The optimal
potentiation takes place at a concentration of 10 ng/ml TNF-
.
Therefore, this concentration was used in the following experiments.
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Time dependence of TNF- action.
To define the time dependence of the potentiating effect of TNF-
,
cells were incubated with 10 ng/ml TNF-
for different times, varying
between 30 min and 78 h. Figure 3 shows the
relation between the carbachol-induced
Isc and the time
of exposure to TNF-
. The maximal change in
Isc is presented
as the percentage of the carbachol response of monolayers not exposed
to TNF-
(= 100%). Until 2 h of incubation, TNF-
did not show a
significant potentiation. However, incubation for 2.5 h with TNF-
enhanced the carbachol response significantly (206 ± 7%), and,
with increasing incubation times, the potentiating effect of TNF-
on
the carbachol response became larger
(r = 0.70). After 78 h of incubation,
the response was increased to >900%. We decided to use a shorter
exposure time for TNF-
in some of the experiments, since this
permitted more stable intracellular measurements.
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Histology.
Figure 4,
A and
B, shows typical electron micrographs
(×3,100) of HT29cl.19A monolayers with (Fig.
4B) or without (Fig.
4A) exposure to 10 ng/ml TNF- for
48 h. No differences are seen compared with time-matched control
monolayers. Propidium iodide staining combined with Hoechst 33258 was
used to check for apoptosis. Even after 48 h of exposure to TNF-
, no
indication was found for TNF-
-induced apoptosis (not shown).
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TNF- action is dependent on protein synthesis.
We examined whether the action of TNF-
was dependent on protein
synthesis using the protein synthesis inhibitor cycloheximide. We
incubated the cells with 10 µg/ml cycloheximide (bilaterally) for 1 h
before TNF-
exposure (41). The cells were then exposed to TNF-
in
the presence of the inhibitor for 4 h, and the maximal change in
Isc induced by
100 µM carbachol was measured. Figure 5 shows the effects of
cycloheximide on the carbachol response with or without exposure to
TNF-
. The increase in
Isc induced by
carbachol in control monolayers was not affected by incubation with
cycloheximide (7.1 ± 1.2 µA/cm2 with cycloheximide vs.
5.8 ± 1.5 µA/cm2 for
control, n = 4, P > 0.1). However, in the presence
of cycloheximide, the potentiating effect of TNF-
was completely
abolished (31.8 ± 11.2 µA/cm2 for TNF-
vs. 6.5 ± 1.1 µA/cm2 for cycloheximide + TNF-
, n = 4, P < 0.05). This, together with the
time lag, suggests that TNF-
potentiates the carbachol response via
a mechanism that requires protein synthesis.
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PKC is involved in electrical response to carbachol.
We examined the involvement of PKC in the response to carbachol with
and without exposure to TNF- by using an inhibitor of PKC, GF
109203X, that is specific for PKC but can also inhibit PKA at high
concentrations (32). We tested whether GF 109203X could inhibit PKC
specifically, but not PKA, in HT29cl.19A cells. Figure
6 shows the effects of bilateral
application of consecutive 0.1 and 1 µM GF 109203X on the changes in
Va induced by PKA
and PKC activation. Forskolin stimulates PKA-activated
Cl
channels, via direct
activation of adenylyl cyclase. Application of 1 µM PDBu results in
activation of PKC (6). Addition of forskolin or PDBu, although with
different time constants, resulted in a depolarization of
Va, due to the
increase in apical Cl
conductance (3). Application of GF 109203X did not affect the
forskolin-induced depolarization. However, 1 µM GF 109203X reduced
the depolarization of
Va induced by
PDBu. Because the full effect of GF 109203X required ~20 min, in the
following experiments preincubation was performed for 30 min. As shown
in Table 2, 1 µM GF 109203X did not show
an effect on the basal electrical parameters. The increase of
Isc induced by
forskolin was not different with or without 30 min of preincubation
with the inhibitor (43 ± 5 and 47 ± 11 µA/cm2, respectively;
n = 9 and 4, respectively). However, the
Isc induced by
application of 1 µM PDBu in the presence of the inhibitor was significantly reduced by 64% from 12 ± 2 to 5 ± 1 µA/cm2
(n = 6, P < 0.05). Thus 30 min of exposure
to 1 µM GF 109203X specifically inhibits PKC in HT29cl.19A. GF
109203X was therefore used to test whether PKC was involved in the
response to carbachol and its potentiation by TNF-
. The cells were
preincubated for 30 min with 1 µM GF 109203X, after which the effects
of carbachol and of exposure to TNF-
were measured. After TNF-
exposure, one part of the filter was used to measure the potentiated
carbachol response. The second part of the filter was preincubated for
30 min with 1 µM GF 109203X, after which the effect of carbachol was
measured. Figure 7 shows the relative
decrease of the carbachol-induced increase in
Isc after
preincubation with GF 109203X with or without exposure to TNF-
. In
the presence of the inhibitor, the maximal change in
Isc after
carbachol alone was significantly reduced by 71 ± 14%.
After exposure to TNF-
, the potentiated carbachol response was
inhibited by 64 ± 6%. We conclude that PKC activation is involved
in the normal carbachol-induced secretion as well as in the
TNF-
-potentiated response in this cell line.
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TNF- does not affect PDBu- and forskolin-induced
secretion.
One mechanism by which TNF-
could potentiate the effect of carbachol
is a direct upregulation of PKC. To investigate this possibility, we
preincubated the cells with TNF-
for between 6 and 24 h and measured
the Isc induced
by the PKC activator PDBu. The change in
Isc induced by 1 µM PDBu alone was 9.9 ± 1.2 µA/cm2
(n = 5), and the change induced by
PDBu after TNF-
was 9.3 ± 0.7 µA/cm2
(n = 4). In HT29cl.19A,
the secretory response activated by PDBu is mediated by the same
Cl
channels as are the
PKA-activated channels, namely the cystic fibrosis transmembrane
conductance regulator (5). To test whether TNF-
had an effect on the
PKA-mediated activation of the apical Cl
channels, we applied
forskolin. The
Isc induced by 10 µM forskolin alone was 43 ± 5 µA/cm2
(n = 9). After exposure to TNF-
for
48 h, the Isc
induced by forskolin was 51 ± 3 µA/cm2
(n = 4), which is not significantly different.
PLD activation is involved in carbachol-mediated secretory response.
The foregoing results indicate that the potentiation by TNF- must be
sought upstream from PKC. One possibility is that the effect is based
on a stronger activation of PKC by an increased production of
diacylglycerol (DAG). DAG can be produced by several reactions: first,
the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by
phosphatidylinositol-specific phospholipase C (PI-PLC; this pathway has been demonstrated for muscarinic and histaminic receptor activation in intestinal epithelial cells; Ref. 13); second,
hydrolysis of phosphatidylcholine by phosphatidylcholine-specific PLC
(PC-PLC); or third, hydrolysis of phospholipids to phosphatidic acid
(PA) by PLD and further to DAG by phosphatidate phosphohydrolase (PAP;
reviewed in Ref. 11). However, as far as we know, the two last
mechanisms are not documented in intestinal epithelial cells. To test
the possible involvement of the PI-PLC pathway, we attempted to block
PLC with U-73122, a putative
PLC-PIP2-specific antagonist (39).
However, addition of this compound (10 µM) did not show inhibitory
effects, and the negative control U-73343 did show the same
quantitative effects on
Va. Thus this
compound could not be used as a tool to investigate the role of PI-PLC.
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T84 cells.
Exposure of T84 cells to 10 ng/ml TNF- for 24 h did not change basal
secretory activity
(Isc was
6.82 ± 1.50 µA/cm2
for control vs. 6.30 ± 1.8 µA/cm2 for TNF-
-exposed
monolayers). Also no change in basal
Rt was found
after exposure to TNF-
(588 ± 88
· cm2 for
control vs. 656 ± 99
· cm2 for
TNF-
). Addition of 100 µM carbachol resulted in an increase in
Isc of 82 ± 25% (n = 3) compared with basal
values. After 24 h of exposure to TNF-
, the change in
Isc was 68 ± 10% (n = 3), which is not
significantly different from that in control monolayers.
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DISCUSSION |
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In the present study, we show that exposure to TNF- (10 ng/ml)
potentiates the Cl
secretion induced by muscarinic receptor activation in human intestinal
epithelial cells (HT29cl.19A). The potentiating effect of TNF-
was
time dependent; a significant increase of the carbachol-induced Isc was observed
after exposure to TNF-
for at least 2.5 h. The rather long lag time
and the fact that blocking protein synthesis by cycloheximide prevented
the potentiating action of TNF-
suggest that de novo synthesis of a
link or activator in the signaling cascade occurs.
There are many potential sites where TNF- could augment the effect
of carbachol.
1) The basal
Cl conductance in the
apical membrane may be increased, so that the carbachol-dependent
increase of K+ conductance in the
basolateral membrane has a larger effect. However, the observation that
the basal electrophysiological parameters did not show increased
secretory activity after prolonged exposure to TNF-
argues against
this possibility.
2) TNF- may increase the number
of Cl
channels in the
apical membrane so that activation induces a larger conductance.
However, activation of the PKA route by forskolin or the PKC route by
PDBu did not show potentiation by TNF-
. Therefore, this possibility is not likely.
3) TNF- may increase the number
of muscarinic receptors. Although we have not tested this possibility
directly, the observation that activation by histamine after exposure
to TNF-
also showed a potentiated secretory response (unpublished
observations) indicates that potentiation is in the pathway between
receptors and the Cl
channel.
4) Because the carbachol effect is
mediated via PKC in HT29cl.19A cells (Ref. 4; presently illustrated by
the effect of GF 109203X), it may be that TNF- upregulates PKC so
that a larger pool of PKC molecules is available. This possibility
should be studied directly. However, because the effect of PDBu was not different in TNF-
-exposed cells, this possibility is less likely. Additionally, the observation that GF 109203X inhibited the PDBu effect
and the potentiated carbachol effect by the same percentage suggests
that the quantitative relation between the blocker and PKC is not
changed by TNF-
. Therefore we hypothesize that the effect of TNF-
is between the receptor and PKC. The absence of an effect of exposure
to TNF-
on the carbachol response in T84 cells may be in line with
this assumption. In these cells, the mechanism leading to increased
Cl
secretion is different
from the mechanism in HT29cl.19A cells. In T84 cells, the
carbachol-induced increase in the
Isc is primarily due to activation of basolateral
K+ channels that leads to an
increased driving force for
Cl
efflux through
conducting apical Cl
channels (34). This view is corroborated by the present intracellular potential measurements. The resting
Va is much lower
than in HT29cl.19A cells, suggesting a much larger
Cl
conductance. The
simultaneous hyperpolarization of the
Va and the
increase of transepithelial potential and
Isc upon
carbachol addition indicate activation of basolateral
K+ channels as the underlying
mechanism for transepithelial current in this cell line. The
refractoriness of the carbachol response to the PKC inhibitor GF
109203X confirms the finding that PKC is not involved in the carbachol
response in these cells (17). Therefore, the absence of an effect of
TNF-
on the electrophysiological effect of carbachol in these cells
is an argument in favor of the hypothesis.
5) The activation of the muscarinic
receptor after exposure to TNF- may lead to increased activation of
PI-PLC, leading to larger amounts of the intracellular messengers
IP3 and the PKC activator DAG.
This possibility should also be studied directly. However, because
IP3 leads to an increase in
Ca2+ and because
Ca2+ in turn leads to the
activation of phase
1 of the carbachol response (4), which
was not different after TNF-
exposure, this possibility seems less
likely. Therefore, another pathway to increase DAG may be involved. The
PI-PLC inhibitor U-73122 was without effect on
phase
1 of the carbachol effect, and
therefore it was not possible to use this inhibitor to show the
involvement of other phospholipase(s) in the generation of DAG.
6) Carbachol, after TNF-
exposure, may lead to increased production of DAG by activation of the
PLD pathway. Although not described for epithelia, TNF-
can increase
the PLD route in a number of cells (7, 9). Direct measurements of DAG
levels and the analyses of the involvement of the PLD pathway have to be performed. However, parallel studies confirmed that, in the presence
of butanol, carbachol increased the synthesis of phosphatidylbutanol, a
specific product of PLD activation. This indicates that muscarinic receptor activation can increase the PLD activity (Oprins et al., unpublished observations). Participation of the PLD pathway in the
carbachol response was also indicated by the electrophysiological effect of propranolol, which is considered an inhibitor of conversion of the PLD product PA to DAG by PAP (11). The inhibitory effect of
propranolol on PAP in these cells was confirmed by the observation that
propranolol increased the
[32P]PA level in
32P-exposed cells, as analyzed
with TLC (Oprins et al., unpublished observations). Other studies have
shown an effect of TNF-
on PC-PLC (28, 36, 38). This route forms DAG
directly without PA as an intermediate and does not require PAP. We
cannot exclude a role for PC-PLC in the (potentiated) carbachol
response. However, the large suppression of the carbachol response by
propranolol suggests that DAG formation by the PLD route plays an
important role in the TNF-
-potentiated carbachol response.
7) According to work with T84 cells,
activation of PI-PLC can generate an inhibitory inositol derivative,
inositol 3,4,5,6-tetrakisphosphate, that may mediate carbachol-induced
inhibition of Cl secretion
(33). The presence of this mechanism in HT29cl.19A cells is unknown,
and the possibility that TNF-
exposure alleviates a similar
inhibition by reducing the synthesis of such a compound remains to be
studied. However, TNF-
was without effect in the T84 cells, and the
inhibitory effect of preexposure to carbachol on the histamine response
in HT29cl.19A cells was not affected by TNF-
(unpublished observations).
8) In human lymphoma cells, TNF-
appeared to increase Na+ channels
(14). If this were the case in HT29cl.19A cells and if the channels
were activated by carbachol, one would not expect to observe an
increased response when the driving force for
Cl
was increased.
Therefore, the most plausible hypothesis for the effect of TNF- in
the potentiation of the effect of muscarinic receptor activation in
HT29cl.19A cells is an upregulation of the PLD route leading to
increased DAG formation and increased PKC-dependent Cl
conductance.
In another clone of HT-29 cells (HT-29/B6), TNF- appeared to have no
direct effect on the secretory status (27). However, as in HT29cl.19A
cells, the effect of carbachol was potentiated (J. D. Schulzke, Freie
Universitat Berlin, personal communication).
The effects of TNF- on ion secretion in human or animal intestine
have not been studied extensively. A difficulty in these studies is
that in the presence of so many other cells one cannot be sure of the
target for the applied TNF-
. For example, in human distal unstripped
colon (27) and in porcine ileum (15), TNF-
increased the
Isc. This effect
could be blocked by indomethacin, indicating the release of
prostaglandins. As far as we know, no experiments have been reported
showing more or less acute effects of TNF-
on responsivity to
secretagogues in isolated intestine. Pathophysiology gives no clear
evidence for effects of TNF-
. TNF-
is increased in inflammatory
intestinal tissue (8, 23), and isolated but unaffected tissue from
inflamed intestine appeared hyporesponsive to secretagogues (1). An
explanation for this finding has not been given. It may be that in some
studies disruption of the tissue or altered morphology plays a role.
Alternatively, the hyporesponsivity may be related to downregulation of
one or more steps in the pathway of the secretagogues because the
tissue is still in a secretory state or has been in this state for a prolonged period. A recent abstract claims that TNF-
is not
responsible for the secretory dysfunction caused by inflammation (18).
From cocultures of T84 cells and activated immune cells, there is ample
evidence that products from immune cells can modify the epithelial
response to secretagogues (21, 22, 40). The nature of these products or
the mechanism of action is not known. As shown in the present study,
T84 cells lack the PKC-dependent carbachol route, and it is feasible
that this cell line cannot show the potentiating effect of TNF-.
An interesting question therefore is whether PKC is involved in carbachol secretory responses of small or large intestinal enterocytes. Data concerning this question are scarce, but in rabbit ileum PKC appears to be involved in the response to carbachol (10).
Transepithelial permeability appears to be modified by TNF-
directly. This effect occurred at high concentrations (100 ng/ml) in
Caco-2 BBE cells (20) and in HT-29/B6 cells (26) and at lower
concentrations (10 ng/ml) in HT29cl.19A cells when TNF-
exposure was
performed in combination with IFN-
(24). The cooperative effect of
the cytokines may be due to the expression of TNF-
receptors
triggered by IFN-
(25). In our laboratory, coexposure of the cells
to TNF-
and IFN-
made it totally impossible to obtain
intracellular recordings, and also high concentrations or longer
exposure to TNF-
alone decreased the success rate of impalements
strongly. We have no explanation for this. It appeared not be due to
morphological changes, since electron micrographs of monolayers exposed
to TNF-
for even 48 h were not different from controls. From the
increase of the
Rt induced by
exposure to TNF-
, we can conclude that there is no increased
permeability for ions. The large, transient decrease in
Rt during the
potentiated carbachol response is concomitant with a decrease in
fRa and therefore indicates a transcellular change in conductances. We propose that, after exposure to TNF-
, carbachol may induce an increased activation of PKC, and numerous other studies in various cell types have implicated a role for DAG-stimulated PKC in the effect of TNF-
(reviewed in Ref. 29). Massive stimulation of PKC by phorbol esters
with PDBu caused a slowly increasing paracellular permeability for
macromolecules in HT29cl.19A cells (12). It remains to be studied
whether TNF-
in combination with a secretagogue like carbachol can
induce a similar increase of permeability and, if so, which isotype of
PKC could be involved.
This is the first study that shows a direct potentiation of
receptor-activated ion secretion in intestinal epithelial cells by
TNF-. It remains to be studied whether the results in HT29cl.19A cells can be translated to the living animal. If so, TNF-
could contribute to the diarrhea in patients with IBD, especially when the
cells are primed by other cytokines like IFN-
to express receptors
for TNF-
. The potentiation of secretion induced by muscarinic
receptor activation (and histamine
H1 receptor activation; unpublished observations) suggests that TNF-
upregulates a common intermediate in the transduction pathway and underscores its possible role in mast cell responses. It is conceivable that this intermediate step is also involved in other PKC-dependent secretory mechanisms induced by, for example, bacterial toxins (16). Furthermore, our
results suggest a place for PLD in the secretory mechanism. These
aspects deserve further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. R. de Jonge for providing the T84 cells and Greet
Scholten for the electron micrographs. We thank Dr. Teun Munnik and Dr.
Alan Musgrave for their help with the lipid messenger system and Dr.
J.-D. Schulzke for evaluating the TNF- effect in another clone of
HT-29 cells.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. C. J. Oprins, Institute for Neurobiology, Faculty of Biology, University of Amsterdam, Kruislaan 320, 1098 SM Amsterdam, The Netherlands (E-mail: oprins{at}bio.uva.nl).
Received 19 July 1999; accepted in final form 11 October 1999.
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