1 Department of Medicine II, We studied the expression of interleukin-1
(IL-1) receptors and the effect of IL-1
interleukin-1; interleukin-1 receptor antagonist; hydrogen
production; adenosine 3',5'-cyclic monophosphate; D-myo-inositol
1,4,5-trisphosphate; cytosolic calcium; fluorescence microscopy; fura
2-acetoxymethyl ester
THE PROINFLAMMATORY cytokine interleukin-1 Effects of IL-1 In fact, IL-1 Therefore, it was the aim of the present study to directly investigate
mechanisms mediating the inhibitory effect of IL-1 Materials.
All reagents were of analytic grade and were purchased from the
indicated suppliers. Carbachol, nitro blue tetrazolium, and ionomycin
were obtained from Sigma (Munich, Germany). Quickszint 2000 scintillation cocktail and Biolute S tissue solubilizer were from
Zinsser Analytic (Frankfurt, Germany), and guanidium isothiocyanate was
from GIBCO (Berlin, Germany). BSA, DMEM containing HEPES and glutamine,
IBMX, HEPES, and histamine were obtained from Serva (Heidelberg,
Germany). 1,4-Dithiothreitol, Pronase E, trypan blue, Na2-EDTA, and all buffer
constituents were from Merck (Darmstadt, Germany). Fura 2-AM was
obtained from Molecular Probes (Eugene, OR), and recombinant human
IL-1 Media.
Medium A contained 0.5 mM
NaH2PO4,
1.0 mM
Na2HPO4,
20 mM NaHCO3, 70 mM NaCl, 5 mM
KCl, 11 mM glucose, 50 mM HEPES buffer, 1 mM
Na2-EDTA (pH 7.8), and BSA (10 mg/ml). Medium B had a similar composition; EDTA was substituted with 1.0 mM
CaCl2 and 1.5 mM MgCl2. Medium
C consisted of 140 mM NaCl, 1.2 mM
MgSO4, 1.0 mM CaCl2, 15 mM HEPES (pH 7.4), 11 mM
D-glucose, 0.1% BSA, and 0.5 mM
dithiothreitol.
Gastric mucosal cell isolation.
Gastric epithelial cells were isolated as described previously (29)
with slight modifications. Five female nonfasted Sprague-Dawley rats
(200 g, Charles River, Sulzfeld, Germany) per experiment were killed by
cervical dislocation. After excision, the stomachs were rinsed with
0.9% saline and everted to form sacs with the mucosa facing outward
and the serosa facing inward. Three milliliters of Pronase E solution
(1.3 mg/ml) were injected into the sac lumina. The stomachs were
incubated at 37°C for 35 min in oxygenated
medium A. Superficial mucosal cells were
discarded after incubating the stomachs for 10 min in oxygenated
medium
B. After another 30 min in oxygenated
medium
A, cells for elutriation were
harvested by 8 min of stirring in
medium
B and collected by centrifugation at
800 rpm for 5 min (IEC 6000 B centrifuge, Nunc, Bedfordshire, United
Kingdom) (fraction
0). Subsequently, the stomachs were placed again in medium
A for another 20 min, followed by two
more cell collection steps in medium
B (for 8 min). Collected
cells (4.5 ± 0.7 × 108
cells/preparation) were resuspended in
medium
C.
Parietal cell enrichment.
The crude cell suspension was separated according to increasing cell
diameters by counterflow elutriation (rotor JE-6B, equipped with
standard chamber; centrifuge J2-21M/E; Beckman Instruments, Glenrothes, United Kingdom) as previously described (44). Briefly, the
crude suspension of freshly isolated cells
(fraction
0) was loaded into the rotor at a
flow rate of 16 ml/min and a rotor velocity of 2,300 rpm. Erythrocytes,
cell fragments, and cells with diameters <14 µm were removed by
washing at 40 ml/min and 2,000 rpm. Thereafter, a cell fraction was
collected at 62 ml and 2,000 rpm. This fraction consisted of 82%
parietal cells (nitro blue tetrazolium staining).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
on the function of highly
enriched (>97%) rat parietal cells. RT-PCR of parietal cell
poly(A)+ RNA with primers specific
for the rat IL-1 receptor revealed a single 547-kb PCR product highly
homologous to the published sequence of the IL-1 receptor. Northern
blot analysis of poly(A)+ RNA of
rat parietal cells and brain revealed a single RNA species of 5.7 kb.
Cytochemistry of parietal cell IL-1 receptor was performed with
biotinylated recombinant human IL-1
, visualized by avidin-coupled fluorescein. Corresponding to the high degree of parietal cell enrichment, 95% of the cells stained positive. Basal
H+ production
([14C]aminopyrine
accumulation) was not changed by IL-1
(0.25-100 pg/ml) nor was
the response to histamine or carbachol when added simultaneously with
the cytokine. However, when parietal cells were preincubated with
IL-1
(0.5-5 pg/ml) for 10 min before the addition of histamine
or carbachol, the response to these secretagogues was reduced by 35 and
67%, respectively. Inhibition by IL-1
was fully reversed by the
human recombinant IL-1 receptor antagonist. Preincubation of parietal
cells with IL-1
failed to alter histamine-stimulated cAMP production
but markedly inhibited carbachol-induced formation of
D-myo-inositol
1,4,5-trisphosphate. In fura 2-loaded, purified parietal cells, 10 min
preincubation with IL-1
dramatically reduced the initial transient
peak elevation of intracellular
Ca2+ concentration in response to
carbachol. We conclude that rat parietal cells express IL-1 receptors
mediating inhibition of H+
production. The antisecretory effect of IL-1
may contribute to
hypoacidity secondary to acute Helicobacter
pylori infection or during chronic colonization by
H.
pylori preferring the fundic mucosa.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(IL-1
)
is a polypeptide product of monocytes and activated macrophages
released at numerous sites of inflammation. In the gastric mucosa,
infection with Helicobacter pylori is
followed by an intensive infiltration of inflammatory cells releasing
substantial amounts of cytokines, among them IL-1
. Intact
H. pylori as well as soluble
H. pylori surface proteins induce the
production of IL-1 from human monocytes (22). This multifunctional
cytokine not only serves as an important mediator of the inflammatory
response of the host by inducing IL-2 secretion and IL-2 receptor
expression of helper T cells but also modulates the biological function
of different gastric epithelial cell types. In cultured G cells from
the rat antrum the cytokine stimulated gastrin release (55). In resting
rat fundic enterochromaffin-like (ECL) cell cultures, IL-1
moderately stimulated the release of histamine, whereas 20 min
preincubation of these cells with the cytokine markedly inhibited
gastrin-stimulated histamine secretion (31). Moreover,
after 24 h of incubation of rat ECL cell cultures with IL-1
, the
rate of apoptotic cells was more than doubled, an effect partially
mediated by the inducible form of nitric oxide (NO) synthase and by NO
formation (30). Thus, by modulating the physiological function of
gastric epithelial cells, IL-1
potentially contributes to
hypergastrinemia and decreased gastric mucosal content of histamine,
conditions frequently observed in H. pylori gastritis.
on parietal cells have been investigated in less
detail, although such experiments have been proposed earlier based on
animal studies in vivo (32). IL-1
inhibited gastric acid secretion
not only via central nervous mechanisms, interacting at hypothalamic
and medullar structures, but also, after intravenous administration,
via peripheral mechanisms (19, 35, 36, 37, 52). On the basis of these
data, it has been speculated that IL-1
might interact with parietal
cell receptors to inhibit gastric acid secretion (32).
, a 27% sequence-homologous peptide almost
functionally equivalent to IL-1
, inhibited histamine- and
carbachol-stimulated H+ production
of isolated, highly enriched canine parietal cells, an effect not
mediated by endogenous PG formation (28). These data yielded functional
evidence that parietal cells express the IL-1 receptor (IL-1R). In
general, binding to this receptor is determined by the carboxy terminus
of IL-1, which is highly homologous between IL-1
and IL-1
. This
homology is probably the reason for the nearly identical biological
activities of both cytokines (7, 23) and provided a rationale for a
recent study (1) in rabbit parietal cells. In these cells (1), IL-1
was more effective in inhibiting
H+ production stimulated by
carbachol and gastrin than by histamine. This inhibitory effect of
IL-1
was not mediated by changes in radioligand binding to histamine
H2 or
gastrin/CCKB receptors. Rather,
IL-1
appeared to interact with postreceptor mechanisms. These,
however, were not studied directly but rather by measuring the effects
of IL-1
on H+ production in
response to specific postreceptor agonists or inhibitors (1).
on parietal cell
function. Using highly enriched rat parietal cells, we identified IL-1R
by molecular biological and immunocytochemical techniques. Moreover, we
confirmed in this cell type the inhibitory effect that IL-1
, by
interacting with these receptors, exerts on
H+ production. Finally, we found
direct evidence that IL-1
modulates parietal cell function by
interfering with
Ca2+/phospholipid- rather than
cAMP-dependent signaling pathways that mediate the key secretagogue
effects of carbachol and histamine, respectively (50).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
[10 pg = 1.87 World Health Organization (WHO) units] and
recombinant human IL-1R (rhIL-1R) antagonist were from R & D Systems
(Abingdon, United Kingdom). Cell-Tak was from Becton
Dickinson Labware (Bedford, MA).
-subunit of
H+-K+-ATPase
(kindly donated by J. L. Cabero, Gothenburg, Sweden) in seven cell
preparations. Viability of enriched parietal cells was determined by
trypan blue exclusion and exceeded 95%.
Parietal cell culture. Parietal cell culture was performed as described previously (4). Enriched parietal cells were washed three times by low-speed centrifugation in serum-free culture medium (1:1 Ham's F-12 medium/DMEM with HEPES and L-glutamine, without bicarbonate) supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml), hydrocortisone (4 ng/ml), epidermal growth factor (EGF) (25 ng/ml), gentamicin (10 mg/100 ml), and cell culture grade BSA (2 mg/ml). For removal of contaminating fibroblasts, cells were preattached in flasks coated with 10% calf serum in PBS (30 min at 37°C). After preattachment, 5 × 104 parietal cells were cultured on coverslips precoated with Cell-Tak (3.5 µg/cm2). After 48 h in primary culture, the purity of parietal cells was 98-100% as determined by staining with nitro blue tetrazolium and the mouse monoclonal anti-H+,K+-ATPase antibody 4A6. Cell viability (trypan blue exclusion) exceeded 95%.
Poly(A)+ RNA preparation. Total cellular RNA was prepared by disrupting acutely isolated, highly enriched rat parietal cells with guanidium isothiocyanate and cesium chloride. Poly(A)+ RNA was purified by oligo(dT) cellulose, according to standard procedures (33). The RNA yield was quantified by determining the optical density at 260 nm with a Beckmann DU 640 spectrophotometer.
RT-PCR analysis of rat IL-1R. After a DNase I (GIBCO BRL Life Technologies, Gaithersburg, MD) digestion, parietal cell poly(A)+ RNA (100 ng) was reverse transcribed into the corresponding single-strand cDNA with oligo(dT)15 primers, using a first-strand cDNA synthesis kit (AMV, Boehringer Mannheim). PCR was performed in Taq+ DNA polymerase buffer (final concn, 1.5 mM MgCl2) and 1 unit Taq+ DNA polymerase (GIBCO BRL), using the Prime Zyme PCR kit (Biometra, Göttingen, Germany) and oligonucleotide primers under the following conditions: hot start, 94°C for 5 min and 85°C for 5 min; initial step (1 cycle), 62°C for 1 min and 72°C for 3 min; repeating steps (30 cycles), 94°C for 1 min, 62°C for 1 min, and 72°C for 3 min; and extension step (1 cycle), 94°C for 1 min, 62°C for 1 min, and 72°C for 7 min. The sense primer for subunit I of the rat IL-1R was 5'-CTT GCC GCA CGT CCT ACA CAT ACC-3', and antisense primer sequence was 5'-CGG GGA AGA AAA TCA GAG CAG GAG-3'. The calculated size of the PCR product was 547 bp. The sequence of the PCR product was verified by a commercial institute (MediGene, Martinsried, Germany).
Northern blot.
To produce a cDNA probe specific for type I of the rat IL-1R, a 546-bp
fragment of the receptor was amplified by PCR, directly cloned into
pPCR 2.1 (Invitrogen, Leek, The Netherlands), and transformed into
competent bacteria (DH5). Plasmid DNA was extracted from bacteria
(using Quiagen columns, according to manufacturer's suggested
protocol). The IL-1 insert was removed from the plasmid by
EcoR I digestion, separated by agarose
gel electrophoresis (0.8% agarose), and purified with glass beads
(Geneclean II, Dianova, Hamburg, Germany).
32P labeling of the cDNA was
performed by random priming (12). The cDNA probe corresponded to bp
956-1502 of the mRNA of rat type I IL-1R [GenBank accession
no. M95578; base count according to Hart et al. (17)].
Immunolabeling.
Highly enriched parietal cells were grown in primary culture (48 h) on
Cell-Tak-coated glass slides. Biotinylated rhIL-1 (2.5 µg/ml, 30 µl/slide; R & D Systems) was applied to the cells (1 h at 4°C).
This specific ligand is designed to quantitatively determine the
percentage of cells bearing the cytokine receptor within a given
population. For visualization, avidin-coupled fluorescein (10 µl/slide; R & D Systems) was applied in the dark (30 min at 4°C).
After washing with buffer, cells were immediately visualized under a
fluorescence microscope (Axiovert 100 TV, Zeiss, Jena, Germany;
magnification, ×40; excitation wavelength, 480 nm). For negative
control, biotinylated rhIL-1
was incubated with an 800-fold excess
of polyclonal goat IgG anti-human IL-1
antibody (R & D Systems) (15 min at 20°C) before being applied to the cultured cells.
[14C]aminopyrine
accumulation.
Accumulation of the weak base
[14C]aminopyrine
served as a quantitative index of parietal cell
H+ production (49). Our
modifications of the original procedure have been described previously
(41, 42). Acutely isolated highly enriched parietal cells (5 × 106/ml) were preincubated for 2 h
at 37°C in DMEM in the absence of test agents. Next, 400 µl of
the cell suspension (3 × 106
cells/ml) were incubated together with
[14C]aminopyrine (0.04 µCi/tube) in flat-bottomed plastic vials in a shaking bath (37°C)
in the absence or presence of IL-1 for various time intervals
(0-60 min). Histamine (3 × 10
6 M) or carbachol
(10
5 M) was added, and the
incubation was continued for another 40 min. Incubation was stopped by
layering 200 µl of the suspension over 1 ml of
medium
C, followed by sedimentation in an
Eppendorf table centrifuge at 15,000 rpm for 10 s. The pellet was
dissolved with 300 µl Biolute S. Four milliliters of scintillation
cocktail were then added, and radioactivity was determined in a liquid scintillation counter (LSD 1801, Beckman Instruments). In only the
experiments with histamine, the phosphodiesterase inhibitor IBMX
(10
4 M) was added for
amplification of the response. At this concentration IBMX did not exert
an effect of its own on
[14C]aminopyrine
accumulation in rat parietal cells (42). In experiments with carbachol,
the incubation medium was supplemented with
CaCl2 to reach a final
concentration of 3 mM Ca2+ to
achieve a more pronounced response to carbachol (38). The ratio of
[14C]aminopyrine taken
up in the parietal cells to that in the undiluted incubation medium was
calculated as described previously (49). [14C]aminopyrine
accumulation in the presence of 0.1 mM dinitrophenol represents
nonspecific incorporation and was subtracted from the test values. Data
were normalized as the percentage of the maximal effect of the
respective stimulus.
cAMP production.
Acutely isolated highly enriched parietal cells (385 µl; 5 × 106/ml) were preincubated for 10 min at 37°C in the absence (control cells) or presence of IL-1
(2.5 pg/ml). Subsequently, the cells were incubated for 3, 6, or 10 min
with IL-1
in combination with IBMX
(10
4 M) and histamine. In
this time frame histamine is known to elicit full stimulation of
parietal cell cAMP production (40). In the present cell system, maximal
stimulation of cAMP production is induced by
10
4 to
10
3 M histamine (40).
Therefore, to induce submaximal stimulation of cAMP formation
corresponding to submaximal stimulation of
[14C]aminopyrine
accumulation, we used 10
5 M
histamine to induce the adenylate cyclase. It is well known that
isolated parietal cells of different species require slightly higher
concentrations of histamine to induce cAMP production compared with
H+ production (44, 49, 50). This
phenomenon potentially reflects intracellular amplification of the
cAMP-induced signal. All test conditions were studied in duplicate
incubations. To terminate the reaction, the samples were shock-frozen
in liquid nitrogen and stored at
70°C until assayed for cAMP
(3). After thawing, samples were extracted in 65% ethanol (final
concn). After centrifugation at 8,000 g for 15 min, aliquots of the extracts
were SpeedVac dried (4 h) and resuspended in the sample buffer of the
commercial kit used for cAMP determination (NEK-033, DuPont, Boston,
MA). Cross-reactivity of the antibody was <0.02% for other
nucleotides (cGMP, GMP, ATP, ADP, AMP). Results
(pmol · 106
cells
1 · 30 min
1) were normalized as
the percentage of the maximal response to the respective stimulus.
D-myo-inositol
1,4,5-trisphosphate production.
Acutely isolated highly enriched parietal cells (5 × 106 cells/ml; 385 µl/sample)
were preincubated for 10 min at 37°C in the absence (control cells)
or presence of IL-1 (2.5 pg/ml). Subsequently, the cells were
incubated with IL-1
in combination with
10
4 M carbachol for 30 s.
In preliminary experiments, this interval had proven optimal for
stimulation of phosphoinositol breakdown (not shown). In the present
cell system, 10
4 M
carbachol yields maximal stimulation of
D-myo-inositol
1,4,5-trisphosphate (IP3)
formation (44), corresponding to maximal stimulation of [14C]aminopyrine
accumulation by 10
5 M
carbachol. All test conditions were studied in quadruplicate incubations. After addition of 400 µl ice-cold TCA (15% in PBS), the
precipitates were placed on ice for 5 min before being sedimented by
centrifugation (2,000 g for 15 min at
4°C). The supernatants (500 µl) were washed three times with
water-saturated diethyl ether that subsequently was evaporated by
incubation for 5 min at 70°C in a shaking bath. From the aqueous
phase 400 µl were adjusted to pH 7.5 by
NaHCO3 and stored at
20°C until being assayed for
IP3
(3H assay system, Amersham Life
Science, Braunschweig, Germany).
Video image analysis of intracellular
Ca2+
concentration.
Purified parietal cells were cultured on sterile coverslips (diameter,
42 mm; 5 × 104
cells/coverslip) precoated with Cell-Tak (3.5 µg/cm2). After 48 h of
culture, cells were washed twice in culture medium without EGF and
incubated for one more hour. The permeable, hydrolyzable ester fura
2-AM was added at a final concentration of 1 µM, and the cells were
incubated for another 15 min at 37°C, before being washed three
times in medium
C. The coverslips with fura 2-loaded parietal cells were then transferred to a perfusion chamber (vol, 2 ml;
30°C) (POC System, Zeiss, Oberkochen, Germany). The chamber was
continuously perfused (2 ml/min
medium
C) without or with the indicated
test agents (carbachol with or without IL-1). After each exposure to
test agents, cells were washed with
Ca2+-free
medium
C for 5 min, followed by 5 min with
Ca2+-containing
medium
C to allow for restoration of cellular
Ca2+ stores. During exposure to
test agents, cells were observed under an Axiovert 100 TV microscope
(Zeiss) in the epifluorescence mode using a dichroic filter (395 nm).
Excitation by alternating wavelengths (334 nm, 380 nm) was performed by
a motorized filter wheel. The image field was monitored by an Attofluor
charge-coupled device camera (Zeiss). Image pairs were
captured (<1 s apart) under the control of Attofluor/Ratio Vision
software (Atto Instruments, Rockville, MD) and stored as digitized 256 gray level images. Free cytosolic
Ca2+ concentration
([Ca2+]i)
was determined according to the equation
[Ca2+]i = Kd(R
Rmin/Rmax
R)[Dlow/Dhigh],
where Kd is the
dissociation constant for fura 2-AM (224 nM) and R the measured ratio.
Rmin is the limiting value of R
when all the indicator is in the
Ca2+-free form [low
standard, 2 mM EGTA (pH = 7.5)],
Rmax is the limiting value of R
when all the indicator is saturated with
Ca2+ (high standard, 10 µM
ionomycin).
Dlow/Dhigh
is the ratio of measured fluorescence intensity when all the indicator
is Ca2+ free, to the intensity
when all the indicator is Ca2+
bound (both measurements performed at 380 nm). This
equation was used as described previously (16, 57) to relate the ratio of chosen 334/380 pixel pairs to
[Ca2+].
Statistics.
Data were expressed as means ± SE from four to seven independent
experiments, each performed in quadruplicate
([14C]aminopyrine
accumulation) or duplicate (cAMP and
IP3 formation). On the basis of
the absolute values, ANOVA for multiple determinations was performed.
Student's t-test for unpaired data
was performed as a post hoc test for calculation of statistically
significant differences between individual treatments.
P 0.05 was considered significant.
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RESULTS |
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Parietal cell IL-1R: PCR, Northern blot analysis, and immunolabeling. RT-PCR was performed with single-strand cDNA derived from reverse transcription of rat parietal cell poly(A)+ RNA using primers specific for the known sequence of type I of the rat IL-1R. The reaction yielded a single primer product at the expected size of 547 bp (n = 5 independent experiments; Fig. 1). The product was cut out, separated, and sequenced and revealed complete homology with the known receptor sequence.
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Effect of IL-1 on parietal cell
[14C]aminopyrine accumulation.
In the presence of the phosphodiesterase inhibitor IBMX
(10
4 M), basal
[14C]aminopyrine
accumulation in acutely isolated, highly enriched rat parietal cells
was stimulated 11.7-fold by 3 × 10
6 M histamine. This
submaximal concentration elicited 57 ± 7% of the maximal
response to 10
4 M
histamine, an effect quite similar to the stimulation by
10
5 M carbachol (see Fig.
5A). IL-1
(1.0-1,000.0
pg/ml) had no effect on basal H+
production (Table 1). When
IL-1
was added to the cells simultaneously with 3 × 10
6 M histamine, the
cytokine failed to affect the response (Table 2). However, when the cells
were preincubated for 10 min in the presence of IL-1
, the cytokine
moderately inhibited the stimulation by 3 × 10
6 M histamine; maximal
inhibition by 35% was observed with 2.5 pg/ml IL-1
(P < 0.05) (Fig.
4A).
Preincubation with 2.5 pg/ml IL-1
for 20 and 30 min did not result
in more effective inhibition (Table 2). When IL-1
(0.5-5.0
pg/ml) was added 10 min before the maximally effective concentration of
histamine (10
4 M), the
cytokine failed to induce significant inhibition (Table 3).
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Effect of IL-1 on parietal cell cAMP and
IP3 production.
cAMP production of acutely isolated highly enriched rat parietal cells
was studied in the presence of the phosphodiesterase inhibitor IBMX
(10
4 M). The submaximally
effective concentration of
10
5 M histamine was used
for stimulation. In control cells preincubated in the absence of
IL-1
, histamine (10
5 M)
stimulated cAMP production 5.4-fold above the basal rate already after
3 min. This response increased to a sevenfold stimulation after 6 and
10 min of exposure to histamine. Cells of the identical preparations
were preincubated uniformly in the presence of 2.5 pg/ml IL-1
for 10 min. Thereafter, cells were stimulated by
10
5 M histamine for 3, 6, and 10 min, respectively. Compared with control cells, there was only a
trend toward a small inhibitory effect of IL-1
at 3 min of exposure
to histamine (P > 0.05). At 6 and 10 min of exposure to histamine, preincubation with the cytokine did not
affect the response to the agonist (Fig.
6). Thus preincubation of the cells with
IL-1
at the identical concentration and for the identical time
interval yielding inhibition of histamine-stimulated [14C]aminopyrine
accumulation failed to significantly reduce cAMP production in response
to histamine.
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|
Effect of IL-1 on
[Ca2+]i
in single parietal cells.
Carbachol-induced changes in
[Ca2+]i
were studied in 48 h primary cultures of highly enriched rat parietal
cells, loaded with fura 2-AM. In control cells, the chamber was
perfused continuously with medium
C containing
10
4 M carbachol. Single
cells were chosen for recording of
[Ca2+]i
(8-10 single cell recordings per cell preparation,
n = 8 independent cell preparations).
At an overview of 10-20 adherent cells per observation field,
carbachol elicited a fluorescence signal in 93 ± 9% of the cells.
Carbachol elicited a biphasic response, an initial transient followed
by a lasting plateau phase. The initial peak response was observed
within 40 s on stimulation with carbachol, and the plateau phase
decayed to the baseline after carbachol and
Ca2+ were removed from the
perfusion medium (Fig.
8A).
After recovery of the cells in
Ca2+-containing medium,
10
4 M carbachol was added
to the perfusion medium for a second period of stimulation yielding the
identical biphasic response of
[Ca2+]i,
i.e., initial peak transient and sustained phase as observed after the
initial stimulation of the identical cells (Fig.
8A). Thus, under the
conditions used, parietal cells responded reproducibly and
identically to two consecutive challenges with carbachol.
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DISCUSSION |
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The present study demonstrates that rat parietal cells express
functionally relevant IL-1R mediating impairment of
H+ production. The cytokine
inhibited carbachol-stimulated parietal cell function more effectively
than the response to histamine. The cellular mechanisms mediating the
inhibitory effect of IL-1 involved impairment of the
IP3-dependent increase in cellular [Ca2+]i
rather than a decrease of the production of cAMP.
Characterization of the parietal cell IL-1R revealed similarities with
IL-1R expressed by rat fundic ECL cells (31). The parietal cell IL-1R
is type I (11) as evidenced by the RT-PCR and Northern blot analysis in
this study. Type I IL-1R is an 80-kDa transmembrane protein mediating
the biological effects of IL-1. On the other hand, the type II
receptor is a 68-kDa membrane protein without an apparent signaling
function. Rather, the type II receptor appears to act as a decoy target
for IL-1, inhibiting IL-1 activities by preventing the binding of this
cytokine to type I receptors. We performed a database search using the
TBLASTN algorithm. This search revealed no sequence
homology allowing for cross hybridization of our IL-1R type I-specific
probe with type II mRNA. However, the potential expression of type II
IL-1R by rat parietal cells was not examined in the present study. Our
Northern blot analysis of parietal cell RNA with a cDNA probe specific
for the rat IL-1R yielded a signal of a size quite similar to that
initially determined in mouse T cells (~5.0 kb; Ref. 46).
The parietal cell IL-1R is of functional relevance and mediates
inhibition of H+ production.
Reversal of the inhibitory response by the IL-1R antagonist supported
the view that inhibition by IL-1 is a specific, receptor-mediated
effect. The IL-1R antagonist is a naturally occurring protein inducible
in many cell types and constitutively expressed in intestinal
epithelial cells. The IL-1R antagonist lacks a site binding a receptor
accessory protein necessary for inducing biological effects mediated
via type I IL-1R. Thus, on binding to these receptors, the IL-1R
antagonist exerts no agonist activities but rather inhibits the binding
of IL-1 and the subsequent biological effects. In our rat parietal cell
system a 100-fold excess of rhIL-1R antagonist over the maximally
effective concentration of IL-1
was sufficient to completely prevent
the inhibitory response to the cytokine. This ratio is compatible with
the view that the parietal cell IL-1R mediating inhibition of
H+ production is type I, since the
affinity of the antagonist to type II receptors is 1,000-fold lower
than that of IL-1 (9, 14).
The potency at which IL-1 acted on our rat parietal cell system was
similar to that by which the cytokine modulated the growth of the
murine helper T cell line D10.G4.1 (51). Moreover, the IL-1
concentrations effective at rat parietal cell IL-1R closely resembled
those at which the cytokine modulated hormone secretion from cultured
rat pituitary cells (2) and impaired the function of cultured rat
pancreatic islets (10, 34, 47), insulinoma INS-1 cells (20), and fundic
ECL cells (30, 31). On the other hand, 1,000-fold higher concentrations
of IL-1
were required to stimulate gastrin release from rabbit
antral G cells (55) or to inhibit rabbit parietal cell
H+ production (1). Finally,
IL-1
acted 200,000-fold more potently on our rat parietal cells than
IL-1
did on canine parietal cells (28). This suggests that, at
parietal cell IL-1R, IL-1
may be the functionally more important
agonist compared with IL-1
.
The efficacy with which in the present study IL-1 inhibited rat
parietal cell H+ production was
quite similar to that with which the cytokine reduced histamine release
from rat fundic ECL cells (31) and H+ production by canine (IL-1
;
Ref. 28) and rabbit parietal cells (IL-1
; Ref. 1). Moreover, the
present study demonstrates that, in inhibiting submaximally stimulated
rat parietal cell function, IL-1
is as effective as
PGE2 and somatostatin, which are
established endogenous inhibitors of
H+ production (40, 43, 45).
However, in inhibiting fully stimulated H+ production in response to
10
4 M histamine
plus IBMX, we found IL-1
to be less effective than PGE2 and somatostatin. In
accordance with recent experiments in rabbit parietal cells (1), we
observed that IL-1
inhibits rat parietal cell
H+ production in response to
carbachol more effectively than in response to histamine. In canine
parietal cells IL-1
was more effective in inhibiting the response to
histamine alone than the potentiating interaction between histamine and
IBMX (28). It has to remain open whether this holds also for isolated
rat parietal cells, which, in the absence of IBMX, fail to respond to
histamine (42).
Inhibition of parietal cells by IL-1 depended on preincubation of
the cells with the cytokine 10 min before addition of the stimulus.
Apparently, IL-1
alters the signaling machinery of rat parietal
cells in a manner that reduces the efficacy of subsequent, not of
simultaneous, stimulation. In our parietal cell system, induction of
this process required 10 min. Preincubation with IL-1
for longer
time intervals resulted in less effective inhibition. The complexity of
signaling mechanisms at which IL-1
may interfere to inhibit
H+ production is still unresolved.
However, our data shed some light on the effects of IL-1
on
essential signaling pathways of parietal cells.
Key stimuli of the parietal cell function are histamine and ACh. They
interact with H2 and
M3 receptors governing
cAMP/protein kinase A- and phosphatidyl
inositol/Ca2+-dependent signaling
pathways, respectively (50). In this study, our finding that IL-1
inhibited rat parietal cell H+
production in response to carbachol more effectively than that stimulated by histamine suggested that the cytokine interferes with
Ca2+/phospholipid- rather than
with cAMP-dependent signaling pathways. In fact, we observed that in
rat parietal cells IL-1
had no significant effect on
histamine-stimulated cAMP production. In contrast, we found that the
cytokine substantially inhibited carbachol-induced formation of
IP3. In parietal cells, this
second messenger mediates the M3
receptor-dependent peak increase in
[Ca2+]i.
Accordingly, our analysis of single parietal cells by fluorescence microscopy revealed a substantial reduction of this peak. Thus we
conclude that interfering with the
IP3-dependent peak increase in
[Ca2+]i
is an essential mechanism by which IL-1
inhibits rat parietal cell
function.
In contrast to the present study, a recent study (1) in rabbit parietal
cells did not directly investigate IL-1 effects on intracellular
signaling mechanisms but rather drew indirect conclusions from
[14C]aminopyrine
accumulation in response to postreceptor agonists and inhibitors. In
that study (1), inhibition by IL-1
of forskolin- but not of
dibutyryl-cAMP-stimulated H+
production suggested that the cytokine interfered at the level of cAMP
production rather than at the protein kinase A or subsequent signaling
steps. The inhibitory IL-1
effect on histamine- or forskolin-induced
[14C]aminopyrine
accumulation was reversed by pertussis toxin and herbimycin, suggesting
that the cytokine interfered at G proteins and tyrosine kinases
governing H2 receptor-dependent
cAMP production (1). This interpretation is at variance with our
present finding that IL-1
had no effect on histamine-stimulated cAMP
production. However, inhibition by IL-1
of rabbit parietal cell
H+ production in response to the
Ca2+ ionophore A-23187 (1) fits
our conclusion that the cytokine interferes with
Ca2+/phospholipid-dependent
signaling. We propose that in parietal cells interference with this
pathway is the key effect by which IL-1
inhibits primarily
H+ production in response to
carbachol. By interfering with the IP3-dependent peak increase in
[Ca2+]i,
IL-1
might impair the potentiating cross talk between
Ca2+/phospholipid- and
cAMP-dependent transduction. Therefore, IL-1
may also inhibit, to a
minor degree, cAMP-mediated H+
production in response to histamine.
There appears to exist no uniform signal-response transduction
secondary to IL-1R activation. Although IL-1 reduced cAMP production in
a pertussis toxin-sensitive manner in rat pancreatic islet cell
cultures (47), the cytokine had no effect on cAMP formation in Jurkat
(6) and U-373 MG human astrocytoma cells (4) and increased cAMP
production and protein kinase A activity in human FS-4 fibroblasts
(58). Breakdown of membrane inositol phosphates and
IP3 formation were not affected by
IL-1 in the human leukemia-derived HSB-2 subclone (26), Jurkat (6), and
U-373 MG human astrocytoma cells (4), while the cytokine induced
IP3 formation in mouse peritoneal
macrophages (56) and renal mesangial cells (8). IL-1 did not induce
Ca2+ influx in the human
leukemia-derived HSB-2 subclone (26) and Jurkat cells (6). In this cell
type IL-1 increased the synthesis of phosphatidylserine, a necessary
cofactor for the activation of protein kinase C (6). However, the
cytokine lacked an effect on protein kinase C in human FS-4 fibroblasts
(58) and the human leukemia-derived HSB-2 subclone (26).
PGE2 formation was induced by
IL-1
in renal mesangial cells (8). On the other hand, indomethacin failed to prevent inhibition by IL-1
of
H+ production in canine parietal
cells (28), arguing against endogenous PGs as mediators of IL-1 effects
on this cell type. IL-1
induced NO formation in renal mesangial
cells (8) and rat fundic ECL cell cultures (31). Moreover, in the
latter cell type (31), in Jurkat cells (6), and cultured rat pancreatic
islets (15), IL-1
induced cGMP formation (31). cGMP and NO have been
implicated previously in the inhibition of rat parietal cell function:
an NO donor induced cGMP production and inhibited
H+ production in isolated rat
parietal cells (3). However, in contrast to these findings, we have
found (39) no evidence of cGMP production and specific cGMP-dependent
signaling systems in the present rat parietal cell system.
Moreover, a cGMP analog had no effect on
[14C]aminopyrine
accumulation in response to histamine or carbachol but rather acted to
stimulate basal H+ production by
cross-activation of the cAMP-dependent protein kinase (39). These
findings clearly argue against a role of cGMP as mediator of the
antisecretory IL-1
effect observed in the present study.
The inhibitory effect of IL-1 on isolated parietal cells is in line
with previous in vivo studies (32). Thus, in addition to interacting
with central nervous sites, peripheral IL-1
acting at parietal cell
receptors may be a paracrine antisecretory agent. Our data indicate
that the cytokine is more effective in inhibiting the parietal cell
response to cholinergic neural stimulation than that to the
physiologically more important humoral secretagogue histamine. Release
of the latter, however, is efficiently impaired by a direct effect of
IL-1
on rat fundic ECL cells (31). Thus, by interacting with
specific receptors on both ECL and parietal cells, peripheral IL-1
has the potential to reduce histamine-dependent gastric acid secretion
as well as the parietal cell response to cholinergic stimulation. The
antisecretory effect of peripheral IL-1
may be of physiological
relevance. This hypothesis is not disproved by the observation that in
pylorus-ligated rats basal gastric acid secretion was unaffected by an
IL-1R antagonist (35), because in this study the antagonist was applied
intracerebroventricularly, compromising firm conclusions as to the role
of peripheral IL-1
. On the other hand, a pathophysiological
relevance of the antisecretory effect of peripheral IL-1
is
supported by the general observation that the cytokine is not produced
constitutively but rather in response to inflammation, endotoxins, burn
injury, or sepsis (18, 27, 53). It may be speculated that under these
conditions the antisecretory effect of IL-1
helps to prevent
acid-induced lesions of the gastric mucosa. In line with that,
increased gastric mucosal expression of IL-1
has been observed
during the healing phase of experimental ulcers (54). The direct
inhibitory effect of IL-1
on parietal (present study) and ECL cells
(31), potentially together with effects of other inflammatory mediators
and bacterial factors, may contribute to hypochlorhydria during acute
infection with H. pylori (13, 24, 25,
48) when major parietal cell damage is morphologically absent.
Furthermore, compared with preferential H. pylori infection of the gastric antrum mucosa, patients
with intense H. pylori
colonization of the gastric body are relatively protected against
duodenal ulcers (21), which are usually associated with hypersecretion
of gastric acid. It may be speculated that the high local release of
antisecretory IL-1
in close vicinity to the parietal cells
contributes to this relative protection.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank A. Fütterer, Department of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany for technical support.
![]() |
FOOTNOTES |
---|
This study was supported by grants to W. Schepp from Deutsche Forschungsgemeinschaft (Sche 229/7-2) and from Else-Kröner-Fresenius Foundation, Bad Homburg, Germany.
A portion of this study was presented at the Annual Meeting of the American Gastroenterological Association in Washington, DC, May 1997, and has been published previously in abstract form (Gastroenterology 112: A1185, 1997).
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: W. Schepp, Dept. of Medicine II, Bogenhausen Academic Teaching Hospital, Englschalkinger Strasse 77, D-81925 Munich, Germany.
Received 4 May 1998; accepted in final form 21 July 1998.
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