314

INFLAMMATION AND INFLAMMATORY BOWEL DISEASE

Tumour necrosis factor α potentiates ion secretion

induced by histamine in a human intestinal epithelial cell
line and in mouse colon: involvement of the
phospholipase D pathway
J C J Oprins, C van der Burg, H P Meijer, T Munnik, J A Groot
.............................................................................................................................

Gut 2002;50:314–321

Background and aims: Patients suffering from inflammatory bowel disease show increased levels of
the mast cell products histamine and tumour necrosis factor α (TNF-α). Treating these patients with anti-
See end of article for bodies against TNF-α diminishes the symptoms of diarrhoea. In this study, the effect of TNF-α on ion
authors’ affiliations secretion induced by the mast cell mediator histamine in HT29cl.19A cells and mouse distal colon was
....................... investigated and the possible second messengers involved were studied.
Correspondence to:
Methods: Electrophysiology of filter grown HT29cl.19A cells and isolated mouse distal colon was used
J C J Oprins, to monitor the secretory response to histamine with and without prior exposure to TNF-α for 3–24 hours.
Swammerdam Institute for Phospholipase D (PLD) activity and phosphatidic acid levels were analysed by 32Pi labelling of
Life Sciences, University of HT29cl.19A cells.
Amsterdam, PO Box
94084, 1090 GB
Results: In both experimental systems TNF-α was found to potentiate ion secretion induced by
Amsterdam, the histamine. Phospholipid analysis of HT29cl.19A cells revealed that histamine activates the PLD
Netherlands; pathway. Furthermore, TNF-α pretreated cells were found to have decreased phosphatidic acid levels,
[email protected] the intermediate product of the PLD pathway, which indicates upregulation of the enzyme phosphatidic
Accepted for publication acid phosphatase.
15 May 2001 Conclusions: The mast cell products TNF-α and histamine synergistically stimulate ion secretion in
....................... intestinal epithelium via upregulation of the PLD pathway.

atients suffering from inflammatory bowel diseases (IBD) tion could be a result of increased protein kinase C (PKC)

P such as ulcerative colitis or Crohn’s disease experience

severe diarrhoea. The underlying mechanism of the diar-
rhoea remains uncertain. Patients show increased release of
activity as a consequence of increased 1,2-diacylglycerol
(DAG) levels due to upregulation of the phospholipase D
(PLD) pathway.13 This proposition was corroborated by analy-
mast cell products such as histamine and also tumour necro- sis of membrane phospholipids.14 To our knowledge, this was
sis factor α (TNF-α).1 2 3 A number of studies have supported a the first time a potentiating effect of TNF-α on epithelial ion
predominant role for the cytokine TNF-α in the pathogenesis secretion had been shown.
of IBD. In bowel mucosa of IBD patients, mRNA levels of Based on the similarities between activation of muscarinic
TNF-α are increased4 as well as concentrations of the protein receptors and histamine receptors, our aim was to study if
in blood and stool.5 6 Moreover, TNF-α secretion from TNF-α could also potentiate the effect of the mast cell media-
infiltrated inflammatory cells is enhanced in biopsies from tor histamine. Because it is important to validate the results
these patients.3 Antibodies against this cytokine appear to be obtained in cell lines in native tissue, we also studied the effect
successful in the therapy of patients with Crohn’s disease of TNF-α and histamine on ion secretion in mouse isolated
resulting in diminished symptoms of the disease.7 These data distal colon.
imply that TNF-α plays an important role in the pathogenesis The present data showed that TNF-α potentiates ion secre-
of diarrhoea in these patients. tion induced by histamine in HT29cl.19A cells via de novo
Chloride secretion across the intestinal epithelium plays an protein synthesis and most probably by upregulating the PLD
important role in water secretion into the lumen of the intes- pathway, thereby generating enhanced DAG levels and subse-
tine. Increased chloride secretion can result in severe quent PKC activation. Furthermore, we demonstrated that
diarrhoea due to excessive water secretion into the lumen. In TNF-α also augments the secretory response to histamine in
several models it was shown that histamine increased chloride mouse distal colon indicating the generality of the observed
secretion. Histamine is thought to stimulate ion secretion in effect in the human cell line.
colonic epithelium via increased prostaglandin release or via The present results suggest a synergy between the mast cell
the augmenting effects of endogenously released products histamine and TNF-α in stimulating ion secretion in
neurotransmitter.8–11 In the intestinal epithelial cell line T84,
histamine increases chloride secretion via increased intra-
cellular calcium levels due to activation of the phospholipase C .............................................................
pathway by the histamine receptor.12 This pathway resembles
that of muscarinic receptor activation and in HT29cl.19A cells Abbreviations: TNF-α, tumour necrosis factor α; PKC, protein kinase C;
intracellular electrophysiological effects of histamine resem- DAG, 1,2-diacylglycerol; PLD, phospholipase D; PA, phosphatidic acid;
PAP, PA phosphatase; PBut, phosphatidylbutanol; IBD, inflammatory
ble those of carbachol (see below). bowel disease; Vt, transepithelial potential; Va, intracellular potential; Isc,
Recently, we showed that TNF-α potentiated carbachol short circuit current; Rt, transepithelial resistance; fRa, fractional resistance;
induced chloride secretion in HT29cl.19A cells in a protein PIP2, phosphatidylinositol 4,5-bisphospate; TTX, tetrodotoxin; IFN-γ,
synthesis dependent manner. We deduced that the potentia- interferon γ; IP3, inositol 1,4,5-trisphosphate.

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Effects of TNF-α on ion secretion induced by histamine 315

intestinal epithelium, and this may contribute to symptoms in Histamine

patients with IBD. Control
TNF-α

MATERIAL AND METHODS Vt

Cell culture
HT29cl.19A cells were cultured as described previously.15
Briefly, the human intestinal epithelial cell line HT29cl.19A, 1 mV
passages 17–29, was grown in Dulbecco’s modified Eagle’s
medium, 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
the experiments, cells were subcultured on Falcon filters (25
mm Ø), and medium was replaced every other day. Confluency
was reached seven days after seeding, and filters with cells
were used between 13 and 19 days post seeding. TNF-α incu- Va
bations were performed in culture media during the indicated 10 mV
times.

Electrophysiological experiments with HT29cl.19A cells

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 con-
tinuously perfused with mannitol-Ringer buffer at a tempera-
ture of 37°C and gassed with 5% CO2–95% O2. The composition fRa
0.25
of Ringer’s solution was (in mM): NaCl 117.5, KCl 5.7,
NaHCO3 25.0, NaH2PO4 1.2, CaCl2 2.5, MgSO4 1.2, and manni-
tol 28. Agar bridges were placed in apical and basolateral
compartments and were connected to Ag-AgCl electrodes for
monitoring the transepithelial potential difference (Vt). An
extra Ag-AgCl electrode was placed in the apical bath, serving
as a common ground. Apical membrane potential (Va) was
measured by impalement with a glass microelectrode, pulled
from capillaries (1 mm od; Clark Electromedical, Reading, Rt
50 Ω × cm2
UK) with a Flaming Brown P-87 micropipette puller. The
microelectrode was filled with 0.5 M KCl solution. Tip
resistance was 100–200 MΩ and tip potential 2–5 mV. Current
electrodes (Ag-AgCl) were placed in the walls of both
compartments; these were used to apply bipolar current
pulses from a floating current source of 10 and 50 µA at 30
second intervals to calculate transepithelial resistance (Rt)
and fractional resistance fRa (fRa=Ra/(Ra+Rb)). The equivalent
short circuit current (Isc) was calculated from Vt and Rt.
The potentials were measured differentially with M-4A
electrometer probes (W-P-Instruments, New Haven, USA). Isc

Potential differences were continuously recorded on a

multipen recorder and on a computer using custom made
software. 25 µA/cm2
Measurements were corrected for the offset of the
electrodes and for the resistances of the fluid and filter with-
out cells.

Phospholipid analysis in HT29cl.19A cells

Cells were labelled for 24 hours with 1.85×106–3.7×106 Bq 32Pi
at the mucosal side and were washed with Ringer’s solution to
1 minute
remove unincorporated 32Pi. Cells were stimulated for 30 min-
utes with 100 µM histamine in the presence of 0.05% Figure 1 Changes in the electrophysiological parameters of
1-butanol (v/v). The concentration of 1-butanol and the time HT29cl.19A cells after addition of 100 µM histamine in the absence
of incubation were chosen based on results described in a pre- or presence of 10 ng/ml tumour necrosis factor α (TNF-α). Two
vious study.14 Reactions were stopped with 0.1 N HCl. Cells are tracings (control and TNF-α treated groups) represent experiments in
frozen for 30 minutes (−20°C) and scraped off the filters, and which intracellular recordings were obtained. Two thin lines divide
the response into phases 1 and 2, as described in the results. Vt,
lipids were extracted as described previously.16
transepithelial potential; Va, intracellular potential; fRa, fractional
Extracted lipids were separated on heat activated TLC plates apical resistance (fRa= Ra/(Ra+ Rb)); Rt, transepithelial resistance; Isc,
using an ethyl acetate solvent system (ethyl acetate/iso-octane/ short circuit current. (For statistics see table 1.)
formic acid/water (13:2:3:10, by vol)).17 The TLC plate was
exposed to a photographic film overnight and levels of 32P Tissue preparation
labelled phosphatidic acid (PA) and phosphatidylbutanol Female Balb/C mice were used. They had access to food and
(PBut) were quantified by phosphoimaging (Storm, Molecu- water ad libitum. The animals were killed by cervical disloca-
lar Dynamics). tion. The intestine was cut proximal from the caecum and

www.gutjnl.com
316 Oprins, van der Burg, Meijer, et al

Table 1 Electrophysiological parameters of HT29cl.19A cells before and after application of histamine
Vt (mV) Rt (Ω×cm2) Va (mV) fRa Isc (µA/cm2)

Control
Baseline 1.7 (0.7) 195 (10) −62 (6) 0.67 (0.02) 9.5 (5)
Histamine phase 1 1.6 (0.7) 195 (10) −58 (3) 0.73 (0.05) 8.9 (5)
Histamine phase 2 2.5 (0.8) 190 (11) −66 (7) 0.65 (0.02) 14 (6)
∆Histamine 0.8 (0.1) −5 (2) −4 (0.4) −0.02 (0.01) 5 (1)
TNF-α
Baseline 2.2 (0.7) 173 (12) −57 (2) 0.70 (0.06) 13 (4)
Histamine phase 1 2.1 (0.7) 173 (12) −54 (3) 0.73 (0.05) 12 (4)
Histamine phase 2 8.4 (1)** 154 (11)** −38 (2)** 0.47 (0.07)** 58 (11)**
∆Histamine 6.2 (0.8)** −20 (5)** 19 (3)** −0.24 (0.05)** 45 (10)**

TNF-α, tumour necrosis factor α; Vt, transepithelial potential; Va, intracellular potential; Isc, short circuit current; Rt, transepithelial resistance; fRa, fractional
resistance.
Effects of addition of 100 µM histamine to the serosal side of the cells. ∆Histamine represents the difference between phase 2 and baseline.
n=5 for control experiments; n=7 for TNF-α exposed monolayers except for Va and fRa (n=5).
Mean incubation time for TNFα (10 ng/ml) was 6–24 hours.
Statistical significance was evaluated using the Students t test: **p<0.01 compared with control histamine.

proximal from the rectum. The colon was rinsed with inosine- In the corresponding text, p values are given and in the
Ringer to remove any faeces and placed over a thin plastic rod. legends the test is indicated.
The distal colon was taken 2 cm distal from the caecum and
cut along the mesenteric border across a length of 1 cm. The
excised segments were mounted on a sledge18 and placed in a
RESULTS
modified Ussing chamber kept at a temperature of 37°C. The
Electrophysiology in HT29.cl19A cells
exposed area was 0.2 cm2 and the volume of the serosal and
mucosal bath was 2 ml each. The composition of the Ringer’s TNF-α potentiates ion secretion induced by histamine in
solution was (in mM): NaCl 117.5, KCl 5.7, NaHCO3 25.0, HT29cl.19A cells
NaH2PO4 1.2, CaCl2 2.5, MgSO4 1.2, inosine 5, β-OH-butyrate A typical electrical response to 100 µM histamine in
0.5, glutamine 2.5+50 mg/l azlocillin (modified from Grotjo- HT29cl.19A cells is shown in fig 1. The response is divided into
hann and colleagues19). Ringer’s solution in the two compart- two phases: the first from the time of histamine application to
ments was refreshed every 30 minutes to avoid bacterial over- the first vertical thin line (fig 1) and the second as the period
flow. The solution was circulated by a gas lift using 5% thereafter. In table 1, the electrophysiological parameters for
CO2/95% O2. the control responses are summarised. They were taken at the
two thin vertical lines indicated in fig 1. Application of 100 µM
Electrophysiological measurements in distal mouse histamine to the serosal side of the cells resulted in fast depo-
colon larisation of the apical membrane potential (Va) together with
Agar bridges were placed in apical and basolateral compart- an increase in fRa during phase 1 without a significant change
ments and were connected to Ag-AgCl electrodes for monitor- in Isc or Vt. In phase 2, depolarisation of Va was followed by
ing Vt. Platina electrodes were placed in serosal and mucosal repolarisation leading to hyperpolarisation, concomitantly
chambers and current injections of +10 µA and −10 µA were with an increase in Vt, decrease in Rt, and increase in Isc. fRa
given to measure Rt of the epithelia. From Vt and Rt the short returned to basal values. To our knowledge, this response has
circuit current Isc was calculated. not been described previously in detail. Muscarinic receptor
Potential difference was continuously recorded on a activation by carbachol triggers the same intracellular
notebook using custom made software. Measurements were response (although usually larger) and thus based on earlier
corrected for the offset of the electrodes and for the resistance studies with carbachol23 we explain the changes in the param-
of the fluid. eters as follows. The first phase—depolarisation without a
change in Vt—is attributed to opening of the chloride channels
Materials located in both membranes of the cells which are activated by
TNF-α and (San Diego, California, USA) GF109203X (bisin- increased intracellular calcium levels, primarily from the
dolylmaleimide I) were from Calbiochem. Propranolol, hista- inositol 1,4,5-trisphosphate (IP3) sensitive intracellular pool.
mine, cycloheximide, azlocillin, tetrodotoxin (TTX), β-OH- The following hyperpolarisation of Va and increase in Vt and Isc
butyrate, indomethacin, and amiloride were purchased from is ascribed to increased calcium dependent potassium
Sigma (St Louis, Missouri, USA), and 1-butanol from conductance located in the basolateral membrane. This
Brocades (Delft, the Netherlands). Silica 60 TLC plates and increases the driving force for chloride efflux through the api-
reagents for lipid extraction were from Merck (Darmstadt, cal membrane through chloride channels, which are believed
Germany). [32P] Orthophosphate (32Pi; carrier free) was from to be activated by PKC-α.24
Amersham International (Uppsala, Sweden). All drugs were Incubation for 24 hours with 10 ng/ml TNF-α (bilaterally)
dissolved in water except for indomethacin and cycloheximide did not change basal electrophysiological parameters. The
which were dissolved in ethanol with a maximal concentra- parameters, representing seven experiments, are presented in
tion of 0.1% ethanol in the Ussing chamber. Concentrations of table 1. In fig 1 a typical electrophysiological response to his-
GF109203X, TNF-α, cycloheximide, and 1-butanol were tamine after exposure to TNF-α is shown. After exposure of
chosen based on earlier studies with this cell line.13 cells to TNF-α, the first phase depolarisation was not affected.
Concentrations were taken from the literature for histamine, However, instead of repolarisation in the second phase, a pro-
propranolol, indomethacin, and amiloride.20–22 longed depolarisation of Va occurred in TNF-α exposed cells.
The decrease in fRa was significantly larger. The change in Vt
Statistics was increased and the change in Rt was also significantly
Data are presented as means (SEM). Statistical significance larger. The histamine induced increase in Isc was ninefold
was evaluated using a Student’s t test or a non-parametric test. larger after exposure to TNF-α. The effects of exposure to

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Effects of TNF-α on ion secretion induced by histamine 317

60
* Histamine PIP2
PI-PLC
IP3
50

∆Isc (µA/cm2) Ca2+

40

30
P-choline DAG PKC
20
PC-PLC
PC

DGK
PAP
PLD
10

0
α Choline
PA
Hist Cyclo TNF- Cyclo +

+ hist + hist TNF- α + hist

Figure 2 Effect of cycloheximide on maximal change in short Figure 4 Schematic representation of the signalling mechanisms
circuit current (Isc) induced by histamine (hist) with or without involved in histaminic receptor activation. PC, phosphatidylcholine;
exposure to tumour necrosis factor α (TNF-α). Cells were incubated PIP2, phosphatidylinositol 4,5-bisphosphate; PI-PLC,
with 10 µg/ml cycloheximide (cyclo) bilaterally for one hour before phosphatidylinositol phospholipase C; PLD, phospholipase D; DAG,
TNF-α exposure (4–5 hours). Data are presented as means (SEM) 1,2-diacylglycerol; PA, phosphatidic acid; IP3, inositol
from 3–7 monolayers. Statistical significance was evaluated using 1,4,5-trisphosphate; DGK, DAG kinase; PAP, phosphatidic acid
the non-parametric Mann-Whitney test: *p<0.05. phosphatase.

* 60 *
60

*
50 50

∆Isc (µA/cm2)
∆Isc (µA/cm2)

40 40

30 30

20 20

* *
10 10

0 0
Hist GF + Hist + GF + hist Hist Prop + Hist + Prop + hist

hist TNF- α + TNF- α hist TNF- α + TNF- α

Figure 3 Effect of GF109203X on the maximal change in short Figure 5 Effect of propranolol on the maximal change in short
circuit current (Isc) induced by histamine with or without exposure to circuit current (Isc) induced by histamine with or without exposure to
tumour necrosis factor α (TNF-α). Cells were incubated with 1 µM tumour necrosis factor α (TNF-α). Cells were incubated with 100 µM
GF109203X (GF) 30 minutes prior to addition of histamine (hist). propranolol (prop) 10 minutes prior to addition of histamine (hist).
Data are presented as means (SEM) from 5–6 monolayers. Cells Data are presented as means (SEM) from 3–4 monolayers. Cells
were exposed to TNF-α for 5–24 hours. Statistical significance was were exposed to TNF-α for 5–24 hours. Statistical significance of the
evaluated using the Student’s t test: *p<0.05. differences in change in Isc was evaluated using the non-parametric
Mann-Whitney test: *p<0.05.

TNF-α on the response to histamine are similar to its potenti-

ating effect on the response to carbachol where we argued that GF109203X decreased the TNF-α potentiated histamine
this must be due to increased apical chloride conductance.13 response from 37.3 (9.7) to 13.7 (3.6) µA/cm2 (n=6; p<0.05)
but the potentiating effect of TNF-α was still present. These
Cycloheximide prevents the potentiating effect of TNF-α results indicate that PKC activation is involved in the secretory
To examine whether the action of TNF-α was dependent on response to histamine as well as in the TNF-α potentiated
protein synthesis, we incubated cells with the protein synthe- response.
sis inhibitor cycloheximide one hour prior to TNF-α applica-
tion and measured Isc induced by histamine. Addition of 10 Involvement of PLD in the histamine response
µg/ml cycloheximide did not affect basal parameters (not In our previous study13 we argued that exposure of cells to
shown). Figure 2 shows that Isc induced by histamine was not TNF-α would make them more susceptible to PKC activation
significantly affected after application of cycloheximide over by muscarinic receptor related intracellular messengers—for
one hour (5.0 (1) µA/cm2 for controls v 3.5 (0.4) µA/cm2 for example, via increased DAG production. DAG can be generated
cycloheximide; n=3). However, in the presence of cyclohex- via different pathways. The phosphatidylinositol-PLC pathway
imide the potentiating effect of TNF-α on histamine-induced has been identified as necessary for histamine receptor activa-
Isc was completely inhibited (45 (9.6) µA/cm2 for TNF-α v 3.7 tion in intestinal epithelial cells and hydrolyses the membrane
(0.7) µA/cm2 for cycloheximide+TNF-α; p<0.001; n=4), as lipid phosphatidylinositol 4,5-bisphospate (PIP2) into DAG
shown in fig 2. These results show that the effect of TNF-α is and IP3.25 IP3 is related to increased intracellular calcium which
dependent on de novo protein synthesis. determines the first phase of the electrophysiological re-
sponse. Because this phase was unchanged, we excluded acti-
Involvement of PKC in the histamine response vation of this pathway by TNF-α. Furthermore, DAG can be
To investigate the role of PKC in the secretory response to his- generated by hydrolysis of structural phospholipids, such as
tamine and in the potentiating effect of TNF-α, cells were pre- phosphatidylcholine by PLD via the intermediate product PA.
incubated with 1 µM GF109203X on both sides of the cells PA is subsequently hydrolysed by phosphatidic acid phos-
over 30 minutes. We demonstrated previously that at this con- phatase (PAP) to DAG. A schematic presentation of the second
centration GF109203X can be used as an inhibitor of PKC.13 In messenger pathways involved in histaminic receptor activa-
fig 3 it is shown that incubating cells with GF109203X prior to tion is presented in fig 4.
addition of histamine decreased histamine induced Isc from To investigate the role of the PLD/PAP pathway in the hista-
5.6 (0.3) to 3.1 (0.3) µA/cm2 (n=5; p<0.005), suggesting a role mine response, we used propranolol to inhibit PAP.21 In a pre-
for PKC in histaminic receptor activation. Pre-exposure with vious study we have shown that in these cells levels of PA

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318 Oprins, van der Burg, Meijer, et al

130 A 150 A *
120 * *
*

32P-PA (% of control)
32P-PA (% of control) 110 100
100
90
80 50
80
40
20 0
Control TNF-α Hist Hist +
Control Histamine TNF-α

120 B 150 B
* * *

32P-Pbut (% of control)
110
32P-Pbut (% of control)

100
100
90 50
80
80
40 0
20 Control TNF-α Hist Hist +
Control Histamine TNF-α
Figure 6 Effect of histamine on levels of 32P-phosphatidic acid (PA)
(A) and 32P-phosphatidylbutanol (PBut) (B) in HT29cl.19A cells. Cells Figure 7 Effect of tumour necrosis factor α (TNF-α) on basal and
were stimulated for 30 minutes with 100 µM histamine in the histamine induced levels of 32P-phosphatidic acid (PA) (A) and
presence of 0.05% 1-butanol. Data are shown as percentage of 32
P-phosphatidylbutanol (PBut) (B). Cells were exposed to 10 ng/ml
control monolayers (100%). The basal 32P-PA level was 2.32 (0.28)% TNF-α over the 24 hour labelling period. Cells were stimulated with
of total labelled phospholipids and basal 32P-PBut was 0.38 (0.06)% 100 µM histamine (hist). Application of histamine resulted in
of total labelled phospholipids. About 20% of the total label was significantly increased levels of 32P-PA and 32P-PBut, as indicated in
incorporated into phosphatidylcholine. The other labelled fig 5. Data are shown as percentage change from control
phospholipids cannot be distinguished on this system. Data are experiments. Data are presented as means (SEM) of five monolayers.
presented as means (SEM) of five monolayers. Statistical significance Statistical significance was evaluated using the Student’s t test:
was evaluated using the Student’s t test: *p<0.05. *p<0.05.

increase twofold after treatment with propranolol, which con- shows that levels of 32P-PA decreased after 24 hours of
firms the inhibitory action of the substance in these cells.14 exposure to 10 ng/ml TNF-α to 91 (3)% compared with cells
Cells were incubated with 100 µM propranolol on the apical without TNF-α (100%; p<0.005; n=5). Also, the increase in
side of the cells for 10 minutes prior to histamine application. 32
P-PA level by histamine was decreased to 88 (6)% (n=5;
The change in Isc induced by histamine is shown in fig 5. In the p<0.01) with respect to histamine alone. Exposure to TNF-α
presence of propranolol the increase in Isc by histamine was did not affect the level of 32P-PBut (101 (4)% compared with
reduced from 5.7 (0.6) to 1.6 (0.6) µA/cm2 (n=3; p<0.05). control 100%; n=5) (fig 7B). However, the presence of TNF-α
After exposure to TNF-α, Isc induced by histamine was reduced significantly decreased 32P-PBut levels to 81 (7)% compared
by propranolol from 43.7 (7.7) to 3.3 (1.3) µA/cm2 (n=4; with cells exposed to histamine alone (100%; n=5; p<0.005).
p<0.05). Thus the potentiating effect of TNF-α was com- These results suggest that histaminic receptor activation
pletely abolished. These results suggest that the PAP/PLD results in activation of the PAP/PLD pathway and that TNF-α
pathway is involved in the histamine response in these cells upregulates expression or activity of PAP, resulting in
and that TNF-α possibly exerts its potentiating effect on decreased PA levels. Reduction in the stable 32P-PBut levels
histamine induced secretion via upregulation of the PAP/PLD seems anomalous. A possible explanation will be given in the
pathway. discussion.

Phospholipid analysis Electrophysiology of distal colon of the mouse

PBut and PA formation by histamine TNF-α potentiates the histamine induced secretory
Previous results suggested that the PAP/PLD pathway is response in mouse distal colon
involved in histaminic receptor activation and that TNF-α It is important to know whether the previous results can be
exerts its potentiating effect via this pathway. To investigate extrapolated to native tissues. Therefore, the effect of TNF-α
this we measured PLD activity. The enzyme PLD has the on ion secretion induced by histamine in mouse distal colon
unique ability to transfer the phosphatidyl group of its was studied. We chose the mouse colon as the cell line we used
substrate to a primary alcohol such as 1-butanol instead of represents colonic epithelia and because mouse recombinant
water, forming phosphatidylbutanol (Pbut).26 Unlike PA, this TNF-α was available.
is a stable product and therefore the relative amount of PBut One piece of distal colon was removed from each mouse and
formed is a measure of PLD activation. After labelling the cells mounted in an Ussing chamber, as described in the materials
with 32Pi, cells were stimulated with histamine for 30 minutes and methods section. To diminish Isc, which may be induced by
in the presence of 0.05 % 1-butanol. Incubation time and con- subepithelial and neuronal factors in distal colon, we used the
centration were chosen based on results described in a previ- following agents. Amiloride (100 µM) was added immediately
ous study.14 Incubation with 100 µM histamine resulted in an after mounting to block mucosal Na+ influx. On the serosal
increase in PA and PBut levels to 114 (3)% and 111 (3)%, side, TTX 0.3 µM was added to block neuronal activity and
respectively (n=5; p<0.05) compared with cells not exposed indomethacin 1 µM to block ion secretion induced by release
to histamine (100%) (fig 6A and 6B, respectively). Figure 7A of prostaglandins. Figure 8A and B show two typical

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Effects of TNF-α on ion secretion induced by histamine 319

120

110

∆I

Isc ( A/cm )
100

2
sc

90

µ
80
Control

α
70
TNF-
60

50
8 10 12 14 16 18

Time (minutes)

500
A Control
500
B TNF- α
400 400
Isc ( A/cm )

Isc ( A/cm )
2

2
300 300

Histamine Histamine
µ

µ
200 200

100 100

0 0
0 50 100 150 200 0 50 100 150 200

Time (minutes) Time (minutes)

Figure 8 Changes in short circuit current (Isc) across mouse distal colon over a three hour period of measurements for two typical experiments
of control (A) and tumour necrosis factor α (TNF-α) exposed tissues (B). At t=0 tissues were mounted and transepithelial potential and
transepithelial resistance were continuously recorded. Every 30 minutes Ringer’s solution was replaced which shows as a drop in Isc during the
recording. Histamine was added at the time point indicated by the arrow. The frames are enlarged in the inset. Two typical recordings of Isc
from mouse distal colon of control and TNF-α exposed tissues are presented in the inset. The change in current with respect to baseline levels is
indicated by ∆Isc. The recordings represent four experiments for each group. The corresponding electrophysiological data are presented in table
2. Statistical significance was evaluated using the Student’s t test.

recordings, each representing four experiments for control These results confirm that TNF-α also potentiates hist-
and TNF-α exposed epithelia (50 ng/ml; three hours), respec- amine induced ion secretion in mouse native tissue.
tively. Every 30 minutes Ringer’s solution containing antibiot-
ics was replaced to remove possible accumulated bacteria in
the chambers. After changing Ringer’s solution, Isc recovers, DISCUSSION
which can be seen in figure 8A and 8B. In the first hour, Isc In the present study we have shown that exposure to 10 ng/ml
decreases rapidly after which it remains at a steady level. No TNF-α potentiated ion secretion induced by histamine in the
differences in transepithelial potential or resistance were colonic epithelial cell line HT29cl.19A. The effect of TNF-α was
observed between control epithelia and epithelia exposed for completely blocked by cycloheximide, which indicates that de
three hours to 50 ng/ml TNF-α. In table 2 changes in electro- novo synthesis of a protein is involved in the action of TNF-α.
physiological parameters on histamine application are pre- TNF-α did not affect basal electrophysiological parameters.
sented. In the inset in fig 8, enlargement of the frames This, together with our previous finding that TNF-α potenti-
indicated are presented. Addition of histamine resulted in an ates ion secretion induced by carbachol,13 suggests that TNF-α
increase in Vt, preceded by a small decrease in Vt. Rt slightly exerts its effect downstream of the histamine receptor. The
decreased after addition of histamine (−4 (1) Ω/cm2) and Isc intracellular recordings show that after exposure to TNF-α
increased to 21 (5) µA/cm2 with respect to baseline values. activation of chloride conductance, located in the apical mem-
After exposure to TNF-α, the change in Vt was increased com- brane, is enhanced. It is unlikely that this chloride conduct-
pared with control epithelia (n=4; p<0.01) and the change in ance is activated by increased calcium levels as the first phase
Rt was larger than that in control experiments (n=4; p<0.01). depolarisation is not affected by TNF-α. Moreover, earlier
Isc induced by histamine was 2.6-fold larger than in control results showed that the response to the calcium ionophore
epithelia (n=4; p<0.01). ionomycin was not affected by TNF-α exposure.13 Apical chlo-
ride conductance can also be increased by PKC activation.27
The present results indicate that PKC activation is involved in
Table 2 Electrophysiological parameters of mouse the histamine response in these cells and in TNF-α
distal colon before and after application of 100 µM potentiated ion secretion. Because even in the presence of
histamine GF109203X (which only partially inhibits PKC28) TNF-α exerts
Vt (mV) Rt (Ω×cm2) Isc (µA/cm2) its potentiating effect and because we found previously that
TNF-α does not affect 4β-phorbol-12,13-dibutyrate induced
Control
Baseline 2.4 (0.6) 35 (5) 67 (8)
ion secretion,13 we hypothesise that TNF-α stimulates the syn-
Histamine 2.8 (0.7) 31 (4) 88 (13) thesis of a protein involved in the second messenger pathway
∆Histamine 0.4 (0.1) −4 (1) 21 (5) between histaminic receptor activation and PKC activation.
TNF-α The diminished electrophysiological response to histamine
Baseline 3.6 (0.6) 51 (11) 77 (12) after propranolol exposure implicates PLD/PAP activation in
Histamine 4.8 (1.0) 38 (7) 130 (16)**
∆Histamine 1.1 (0.2)** −13 (5)** 54 (7)**
histaminic receptor activation in these cells. The marked sup-
pression (over 70%) suggests that PLD activation plays a major
TNF-α, tumour necrosis factor α; Vt, transepithelial potential; Isc, short role in ion secretion induced by histamine. Furthermore,
circuit current; Rt, transepithelial resistance. blocking PAP with propranolol completely prevented the
Effects of addition of 100 µM histamine to the serosal side of the
tissues with or without exposure to 50 ng/ml TNF-α over three hours
potentiating effect of TNF-α, implying that TNF-α acts via this
(n=4 for the two groups). pathway. Measurement of levels of PA and the stable PLD
Statistical significance was evaluated using the Student’s t test: product PBut established that application of histamine leads
**p<0.01 compared with control histamine.
to activation of PLD in these cells. Activation of the PAP/PLD
pathway by histamine has been described previously. In

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320 Oprins, van der Burg, Meijer, et al

oligodendroglioma cells and pulmonary endothelium, it was hypothesise that hyporesponsiveness may be due to down-
shown that histamine application resulted in PLD regulation of one or more steps in the secretory pathway
activation.29 30 Interestingly, levels of PA are decreased in the because of prolonged activity of PKC. Prolonged activation of
presence of TNF-α. This suggests that TNF-α exerts its effect PKC is known to inhibit basolateral K+ conductance, resulting
on the PAP/PLD pathway by upregulation of expression or in an inability to reveal a secretory response to secretagogues.
activity of the protein PAP, thereby increasing turnover from This suggests that the synergistic effect of TNF-α and
PA to DAG. The increased formation of DAG may activate PKC. histamine on ion secretion is more likely involved in an early
Exposure to TNF-α also results in decreased PBut levels initiating phase of the disease than longer term. Then, the
induced by histamine. PLD activity can be enhanced by PA31 synergistic effect of histamine and TNF-α on activation of PKC
and by PIP2, which is formed by the PA dependent PIP may account for the reduced ion transport in models of IBD.
kinase.32 Thus when PA levels are decreased due to TNF-α- It has been described previously that TNF-α can also act
induced PAP activity, histamine may stimulate PLD to a lesser synergistically with another immunomodulator interferon γ
extent after TNF-α exposure, resulting in less elevated PBut (IFN-γ) in altering epithelial physiology in intestinal epithelial
levels. cells (T84 cells).39 40 This is probably attributed to induction of
Interestingly, in mouse distal colon the histamine response TNF-α receptors by IFN-γ.41 Thus it may be that in the presence
was also potentiated by TNF-α, which indicates the generality of both cytokines the effect of secretagogues is even more
of the observed effect in the cell line. Apparently, TNF-α and potentiated. Recently, it has been shown that TNF-α is a
histamine synergistically stimulate ion secretion in the distal stimulus for mast cells to release histamine42 and because the
colon also. To our knowledge this is the first study in which a release process of TNF-α is regulated differentially from
direct effect of TNF-α on ion transport in intestinal tissue has degranulation,43 it may be that TNF-α activates histamine
been demonstrated. In porcine ileum and human distal colon, release in an autocrine way.
TNF-α was shown to increase basal Isc via increased synthesis In summary, TNF-α potentiates ion secretion induced by
of subepithelial prostaglandins.33 34 We exclude this possibility histamine in HT29cl.19A cells via a protein synthesis depend-
in the present study as we performed our experiments in the
ent mechanism. We hypothesise that TNF-α upregulates
presence of indomethacin, an inhibitor of prostaglandin syn-
expression or the activity of PAP, thereby increasing DAG for-
thesis.
mation and subsequent PKC activation. This augments ion
The effect of histamine on chloride secretion in the colon
secretion induced by histamine. Interestingly, in mouse distal
has been extensively studied in different species. In mouse
colon, TNF-α also potentiated histamine induced ion secre-
caecum, rat colon, guinea pig colon, porcine distal colon, and
tion. It remains to be seen whether the PKC and PAP/PLD
human colon histamine induces a transient increase in
pathway are involved in the secretory response to histamine in
Isc.8–11 20 In the guinea pig, histamine acts via augmenting the
effects of endogenously released neurotransmitters.10 Hard- mouse distal colon and whether this mechanism is involved in
castle and Hardcastle were the first to show that histamine in the pathogenesis of IBD related phenomena.
rat colon induces chloride secretion partly via release of
prostaglandins.9 Also, in mouse caecum histamine causes the .....................
majority of its effect through stimulating arachidonic acid Authors’ affiliations
metabolism.20 The increase in histamine related Isc in our study J C J Oprins, C van der Burg, H P Meijer, T Munnik, J A Groot,
Swammerdam Institute for Life Sciences, University of Amsterdam,
was not due to indirect neuronal stimulation or prostaglandin Amsterdam, the Netherlands
release as we measured the histamine response after blocking
both pathways by TTX and indomethacin, respectively. The
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