1 Department of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103; and 2 Inotek Corporation, Beverly, Massachusetts 01915
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ABSTRACT |
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Na+/H+ exchangers
(NHEs) are integral transmembrane proteins found in all mammalian
cells. There is substantial evidence indicating that NHEs regulate
inflammatory processes. Because intestinal epithelial cells express a
variety of NHEs, we tested the possibility that NHEs are also involved
in regulation of the epithelial cell inflammatory response. In
addition, since the epithelial inflammatory response is an important
contributor to mucosal inflammation in inflammatory bowel disease
(IBD), we examined the role of NHEs in the modulation of disease
activity in a mouse model of IBD. In human gut epithelial cells, NHE
inhibition using a variety of agents, including amiloride,
5-(N-methyl-N-isobutyl)amiloride, 5-(N-ethyl-N-isopropyl)- amiloride,
harmaline, clonidine, and cimetidine, suppressed interleukin-8 (IL-8)
production. The inhibitory effect of NHE inhibition on IL-8 was
associated with a decrease in IL-8 mRNA accumulation. NHE inhibition
suppressed both activation of the p42/p44 mitogen-activated protein
kinase and nuclear factor-B. Finally, NHE inhibition ameliorated the
course of IBD in dextran sulfate-treated mice. Our data demonstrate
that inhibition of NHEs may be an approach worthy of pursuing for the
treatment of IBD.
cytokines; lipopolysaccharide; mitogen-activated protein kinase; Crohn's disease; ulcerative colitis
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INTRODUCTION |
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THE INTESTINAL
INFLAMMATION during inflammatory bowel disease (IBD) is a result
of a complex interplay of immune and nonimmune cell interactions
(12, 49). Emerging evidence indicates that of all the
classical "nonimmune" cells present in the gut, the epithelial
cells of the mucosa may play a central role in inducing and maintaining
intestinal inflammation. During gut inflammation, intestinal epithelial
cells (IECs) receive their activating signals from basically two
sources: 1) the classical immune cells of the gut via
humoral factors, such as interleukin-1 (IL-1
) and tumor necrosis
factor-
(TNF-
), and 2) direct interactions with
bacteria and bacterial products. These signals activate IECs to produce a wide range of inflammatory mediators, including the chemokine IL-8
(13, 55) and the free radical nitric oxide (28, 42, 54). Central to this inflammatory response is the transcription factor nuclear factor-
B (NF-
B), whose activation is responsible for the transcription of inflammatory genes in IECs during IBD (24). IL-8 is a powerful neutrophil chemoattractant and
activator. Colonic IL-8 levels correlate with the macroscopic grade of
local inflammation in IBD patients, in which large numbers of
neutrophils are found in crypt abscesses (6, 33). In
addition, IL-8 neutralization has been shown (19, 27) to
abolish the neutrophil-activating effect of rectal dialysate or colonic
organ cultures taken from IBD patients. Thus it appears that IL-8 of
epithelial origin plays an important role in the amplification and/or
maintenance of intestinal inflammation in IBD patients.
Na+/H+ exchangers (antiporters, NHE) are a family of ubiquitous plasma membrane transport proteins that catalyze the exchange of extracellular Na+ for intracellular H+ (8, 15). Recent molecular cloning studies (50, 56, 57) have confirmed that NHEs constitute a gene family from which seven mammalian isoforms (NHE1, NHE2, NHE3, NHE4, NHE5, NHE6, and NHE7) have been cloned and sequenced. IECs have been shown (22, 31, 51) to express NHE1, NHE2, NHE3, and NHE4, but the exact function of the various isotypes is unknown. The "housekeeping" NHE1 is present on the basolateral membrane, where it is involved in intracellular pH maintenance and cell volume regulation. The apically expressed NHE3 plays an important role in Na+, bicarbonate, and water reabsorption (7). NHEs are reversibly and selectively inhibited by the diuretic drug amiloride and its analogs as well as by a variety of nonrelated drugs, including cimetidine, clonidine, and harmaline (50, 56).
Evidence indicates that NHEs are rapidly activated in response to a
variety of inflammatory signals, such as IL-1 (5), TNF-
(52), interferon-
(IFN-
) (41) and
lipopolysaccharide (LPS) (39, 52). Conversely, NHEs have
been shown to regulate the inflammatory functions of "professional"
inflammatory cells, including monocytes (44), macrophages
(35, 41), and neutrophils (45, 53). We
hypothesized that NHEs may regulate the IEC inflammatory response
because various extracellular stimuli present during gut inflammation,
such as cytokines, bacteria, and bacterial products, activate NHEs and
NHEs are involved in the regulation of inflammatory processes. Our
results demonstrate an essential role for NHEs in mediating the IEC
inflammatory response. We show that a functional NHE is required for
both maximal NF-
B activation and IL-8 production in IECs.
Furthermore, we demonstrate that the NHE regulation of intestinal
inflammation is also operational in vivo, because NHE inhibition
dramatically attenuates disease activity in the mouse dextran sulfate
model of IBD.
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MATERIALS AND METHODS |
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In Vitro Studies
Cell lines. The human colon cancer cell lines HT-29 and Caco-2 were obtained from American Type Culture Collection (Manassas, VA). HT-29 cells were grown in modified McCoy's 5A medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Caco-2 cells were grown in DMEM with high glucose containing 10% fetal bovine serum (26).
Drugs and reagents.
Amiloride HCl,
5-(N-methyl-N-isobutyl)amiloride (MIA), and
5-(N-ethyl-N-isopropyl)-amiloride (EIPA) were
obtained from Research Biochemicals (Natick, MA). Cimetidine,
harmaline, and clonidine were purchased from Sigma (St. Louis, MO). All
NHE inhibitors were dissolved in DMSO. Human IL-1 and TNF-
were
obtained from R&D Systems (Minneapolis, MN). The p42/44 pathway
inhibitor PD-98059 was purchased from Calbiochem (San Diego, CA). LPS
(Escherichia coli 055:B5) and pyrrolidinedithiocarbamate
were purchased from Sigma.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
obtained from Fisher Scientific (Pittsburgh, PA).
IL-8 measurement.
To study the effect of NHE inhibitors on IL-8 production, cells
in 96-well plates were treated with IL-1 (20 ng/ml), TNF-
(20 ng/ml), or LPS (10 µg/ml) for 24 h. However, because harmaline was toxic to the cells when the incubation lasted for 24 h, the effect of these agents on IL-8 production was tested 4 h after IL-1
stimulation. Furthermore, the effect of both MIA and EIPA was
tested 4 h after stimulation with IL-1
. Amiloride or its vehicle (0.5% DMSO) was added to the cells at various time points before and after stimulation with cytokines or LPS. Human IL-8 levels
were determined from the cell supernatants using commercially available
ELISA kits (R&D Systems), according to the manufacturer's instructions.
Western blot analysis.
HT-29 cells in six-well plates were pretreated with amiloride
(300 µM) or vehicle (0.5% DMSO), and 30 min later the cells were
stimulated with IL-1 (20 ng/ml) for 15 min. After being washed with
PBS, the cells were lysed by the addition of modified radioimmunoprecipitation buffer [50 mM Tris · HCl, 150 mM
NaCl, 1 mM EDTA, 0.25% Na deoxycholate, 1% Nonidet P-40, 1 µg/ml
pepstatin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride
(PMSF), and 1 mM Na3VO4]. The lysates were
transferred to Eppendorf tubes and centrifuged at 15,000 g,
and the supernatant was recovered. Protein concentrations were
determined using the Bio-Rad protein assay (Hercules, CA). Ten
micrograms of sample were separated on a 8-16% Tris-glycine gel
(Invitrogen) and transferred to a nitrocellulose membrane. The
membranes were probed with anti-phospho-mitogen-activated protein
kinase (MAPK) antibody (p42/p44; Promega, Madison, WI) and subsequently
incubated with a secondary horseradish peroxidase-conjugated donkey anti-rabbit antibody (Boehringer-Mannheim, Indianapolis, IN).
Bands were detected using enhanced chemiluminescence Western blotting
detection reagent (Amersham Life Science, Arlington Heights, IL).
RNA isolation and RT-PCR. Total RNA was isolated from HT-29 and Caco-2 cells using TRIzol reagent (Invitrogen). RNA RT was performed using Moloney murine leukemia virus RT from Perkin-Elmer (50 U/µl; Foster City, CA). RNA (5 µg) was transcribed in a 20 µl reaction containing 10.7 µl of RNA, 2 µl of 10× PCR buffer, 2 µl of 10 mM dNTP mix, 2 µl of 25 mM MgCl2, 2 µl of 100 mM dithiothreitol (DTT), 0.5 µl of RNase inhibitor (Perkin-Elmer, 20 U/µl), 0.5 µl of 50 mM oligo(dT)16, and 0.3 µl of RT. The reaction mix was incubated at 42°C for 15 min for reverse transcription. Thereafter, the RT was inactivated at 99°C for 5 min. RT-generated DNA was amplified using Expand high-fidelity PCR system (Boehringer-Mannheim). The reaction buffer (25 µl) contained 2 µl of cDNA, water, 2.5 µl of PCR buffer, 1.5 µl of 25 mM MgCl2, 1 µl of 10 mM dNTP mix, 0.5 µl of 10 µM oligonucleotide primer (each), and 0.2 µl enzyme. cDNA was amplified using the following primers: IL-8 (36), 5'-ATGACTTCCAAGCTGGCCGTGGCT-3' (sense) and 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3' (antisense); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CGGAGTCAACGGATTTGGTCGTAT-3' (sense) and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3' (anti-sense). The conditions were as follows: an initial denaturation at 94°C for 5 min; 27 and 23 cycles at 94°C for 30 s for IL-8 and GAPDH, respectively; 58°C for 45 s; 72°C for 45 s; and 72°C for 7 min. The expected PCR products were 289 and 306 bp for IL-8 and GAPDH, respectively. The PCR products were resolved on a 1.5% agarose gel and stained with ethidium bromide.
NF-B electrophoretic mobility shift assay and supershift
assay.
IECs were stimulated with IL-1
(20 ng/ml; R&D Systems) for 45 min, and nuclear protein extracts were prepared as described previously
(11, 34). To determine the effect of NHE blockade, we
pretreated cells with amiloride (300 µM), harmaline (3 mM), cimetidine (6 mM), clonidine (3 mM), or vehicle (0.5% DMSO) 30 min
before IL-1
stimulation. All nuclear extraction procedures were
performed on ice with ice-cold reagents. Cells were washed twice with
PBS and harvested by scraping into 1 ml of PBS and pelleted at 6,000 rpm for 5 min. The pellet was resuspended in 1 packed cell volume of
lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM
MgCl2, 0.2% vol/vol Nonidet P-40, 1 mM DTT, and 0.1 mM
PMSF) and incubated for 5 min with occasional vortexing. After
centrifugation at 6,000 rpm, 1 cell pellet volume of extraction buffer
(20 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2,
25% vol/vol glycerol, 1 mM DTT and 0.5 mM PMSF) was added to the
nuclear pellet and incubated on ice for 15 min with occasional
vortexing. Nuclear proteins were isolated by centrifugation at 14,000 g for 15 min. Protein concentrations were determined using
the Bio-Rad protein assay. Nuclear extracts were stored at
70°C
until used for electrophoretic mobility shift assay (EMSA). The
oligonucleotide probe used for the EMSA was purchased from Promega.
Oligonucleotide probes were labeled with [
-32P]ATP
using T4 polynucleotide kinase (Invitrogen) and purified in Bio-Spin
chromatography columns (Bio-Rad). For the EMSA analysis, 10 µg of
nuclear proteins were preincubated with EMSA buffer [12 mM HEPES, pH
7.9, 4 mM Tris · HCl, pH 7.9, 25 mM KCl, 5 mM
MgCl2, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly(dI-dC), 12%
glycerol (vol/vol), and 0.2 mM PMSF] on ice for 10 min before the
radiolabeled oligonucleotide was added for an additional 25 min. The
specificities of the binding reactions were tested by incubating
duplicate samples with 100-fold molar excess of the unlabeled
oligonucleotide probe. Protein-nucleic acid complexes were
resolved using a nondenaturing polyacrylamide gel consisting of 5%
acrylamide (acrylamide/bisacrylamide, 29:1) and run in 0.5× TBE (45 mM
Tris · HCl, 45 mM boric aid, and 1 mM EDTA) for 1 h at a
constant current (30 mA). Gels were transferred to Whatman 3M paper,
dried under vacuum at 80°C for 1 h, and exposed to photographic
film at
70°C with an intensifying screen. For supershift studies,
after addition of the radiolabeled probe, samples were incubated for
1 h with control, p65, or p50 antibody and then loaded on the gel.
Transient transfection and luciferase activity.
For transient transfections, 2-4 × 105 HT-29
cells were seeded per well of a 24-well tissue culture dish 1 day
before transient transfection. Cells were transfected with 3 µl of
Lipofectamine 2000 (Invitrogen) and 5-10 µg/ml of DNA containing
a NF-B luciferase promoter construct (Clontech, San Diego, CA) or
the empty vector, according to the manufacturer's instructions. This
NF-
B luciferase vector contains four tandem copies of the NF-
B
consensus sequence fused to a TATA-like promoter region from the herpes
simplex virus thymidine kinase promoter. After 5 h at 37°C, 10%
fetal bovine serum was added, and cells were allowed to recover at
37°C for 20 h. The cells were pretreated with amiloride or its
vehicle for 30 min and then stimulated with IL-1
for 16 h.
Luciferase activity was measured by the luciferase reporter assay
system (Promega) and normalized relative to micrograms of protein.
Measurement of mitochondrial respiration. Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondria-dependent reduction of MTT to formazan. Cells in 96-well plates were incubated with MTT (0.5 mg/ml) for 60 min at 37°C. The culture medium was removed by aspiration, and the cells were solubilized in DMSO (100 µl). The extent of the reduction of MTT to formazan within cells was quantitated by an optical density measurement at 550 nm using a Spectramax 250 microplate reader (17).
In Vivo Studies
Animals. Male BALB/c mice (6-8 wk of age) purchased from Taconic Farms (Germantown, NY) were used for the colitis studies. Animals were kept on a standard chow pellet diet with tap water ad libitum on a 12:12-h light-dark cycle. Animals were housed at four per cage in a room with controlled lighting (lights on from 800 to 2000) and temperature (maintained at 21-23°C) and used in experiments 3-7 days after arrival. The studies were conducted after approval of the protocols by the local institutional animal care and use committee.
Dextran sulfate-induced colitis.
Colonic inflammation was induced by the administration of dextran
sodium sulfate (DSS) in the drinking water (25, 30, 48).
The animals were exposed to 5% DSS (molecular mass, 40-44 kDa) ad
libitum. Mice were treated via one oral gavage with amiloride (10 mg · kg1 · day
1; gavage
given twice a day with 5 mg/kg amiloride administered each time) or
vehicle (saline) starting on day 1 and continuing throughout
the study. Water and food intake was monitored and did not differ
between the various experimental groups. The parameters recorded were
mortality, body weight, colon length, and myeloperoxidase (MPO) and
malondialdehyde (MDA) levels. In addition, macrophage inflammatory
protein-2 (MIP-2) levels were also measured in colonic homogenates
using ELISA. The body weight of the animals was expressed as the mean
percent decrease over the 10-day experimental period. To measure colon
length, the mice were killed 10 days after the start of DSS
administration. The colon was resected between the ileocecal junction
and the proximal rectum, close to its passage under the pelvisternum.
The dissected colon was placed on a nonabsorbent surface and measured
with a ruler, taking care not to stretch the tissue. The feces was then
removed from the colon using physiological buffer before the biopsies
were taken and flash frozen for biochemical analysis.
Statistical evaluation. Values are expressed as mean ± SE of n observations. Statistical analysis of the data was performed by one-way ANOVA followed by Dunnett's test, as appropriate.
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RESULTS |
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Inhibition of NHEs Suppresses IL-8 Production by IECs
To investigate the role of NHEs in regulating IL-8 production by epithelial cells, we first examined whether the inhibition of Na+/H+ exchange by amiloride influenced IL-8 production in the human epithelial cell line HT-29 stimulated with IL-1
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To examine whether the effect of amiloride was cell-type
specific, we investigated the effect of this agent on IL-1-induced IL-8 production in the Caco-2 human epithelial cell line. Amiloride suppressed IL-8 production by these cells as well (Fig. 1C),
with a similar potency to that observed in HT-29 cells. This
observation suggests that the reduction of IL-8 production by NHE
blockade is not cell-type specific, but rather a general phenomenon. To determine whether the inhibitory effect of amiloride treatment was time
dependent, we administered 300 µM amiloride 30 min before, concurrent
with, or 0.5, 1.5, 3, 4.5, 6, 7.5, or 9 h after stimulation with
IL-1
. As shown in Fig. 2, concomitant
treatment with amiloride resulted in maximal suppression of IL-8
production, which amounted to 57 ± 8.2% (n = 6, P < 0.01). In addition, a significant inhibition of
IL-8 production was also observed up to 4.5 h after IL-1
stimulation. This finding demonstrates that the amiloride-induced
suppression of IL-8 production does not require pretreatment.
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In subsequent experiments, we examined the effect of selective
inhibition of NHEs by the amiloride analogs MIA and EIPA on IL-1-induced IL-8 production by HT-29 cells. Figure
3 demonstrates that both MIA and EIPA
suppressed IL-8 production. MIA suppressed IL-8 production with an
IC50 of ~10 nM (Fig. 3A). MIA was most efficacious at 100 nM; however, at higher concentrations, MIA became
less effective. EIPA had a biphasic effect (Fig. 3B). The first phase of the EIPA suppression of IL-1
-stimulated IL-8
production reached its maximum at 300 nM, followed by an elevation of
IL-8 levels that reached its peak at 3 µM EIPA. At higher EIPA
concentrations, the curve showed another downward turn. However, IL-8
levels at 50 µM EIPA were still higher than those at 300 nM. The
inhibitory effect of MIA or EIPA was not due to a decrease in cell
viability, because neither of these agents decreased mitochondrial
respiration at the concentrations tested (data not shown).
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To further corroborate a role for NHEs in the regulation of IL-8
production, we next determined the effect of a series of NHE inhibitors
that are structurally unrelated to amiloride on IL-1-induced IL-8
production by HT-29 cells. The NHE inhibitors cimetidine, clonidine,
and harmaline decreased IL-1
-induced IL-8 production by HT-29 cells
(Fig. 3, C, D, and E). The effect of all three nonamiloride NHE inhibitors was concentration dependent (Fig.
3, C, D, and E). Under the conditions
studied, none of these agents decreased cell viability at the
concentrations tested (data not shown). These data confirm that NHE
inhibition attenuates IL-8 production by HT-29 cells.
Finally, we tested whether amiloride inhibited the production of IL-8
induced by LPS or TNF-. Similar to our findings with IL-1
-induced
IL-8 production, amiloride pretreatment of HT-29 cells attenuated LPS-
or TNF-
-induced IL-8 production (Fig.
4). In addition to suppressing cytokine-
or LPS-induced IL-8 production, amiloride also decreased basal,
unstimulated IL-8 production (data not shown).
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Amiloride Suppresses IL-1-Induced IL-8 mRNA Accumulation in IECs
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NHE Blockade Inhibits IL-1-Induced NF-
B Activation and
Extracellular Signal-Regulated Kinase Phosphorylation
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Next, we investigated whether the decrease in NF-B activation
corresponded with a decrease in NF-
B-dependent gene transcription. To this end, HT-29 cells were transiently transfected with a
NF-
B-luciferase reporter construct. The transfectants were
pretreated with amiloride or its vehicle for 30 min and then stimulated
with IL-1
for 16 h. The effect of amiloride on
NF-
B-dependent gene transcription was assessed using the luciferase
assay. Similar to its effect on NF-
B DNA binding, amiloride
suppressed IL-1
-stimulated NF-
B-dependent gene transcription
(Fig. 7A). This effect was
specific, because amiloride did not decrease luciferase activity when
the cells were transfected with an enhancerless empty vector (data not
shown). To further support the notion that the reduction of NF-
B
activation by NHE inhibition is an important mechanism whereby NHE
inhibition reduces IL-8 production, we conducted further
pharmacological experiments using the NF-
B inhibitor
pyrrolidinedithiocarbamate (59). Figure
7B shows that pyrrolidinedithiocarbamate decreased, in a
concentration-dependent manner, the release of IL-8 by
IL-1
-stimulated HT-29 cells, corroborating the idea that NF-
B is
a pivotal inducer of IL-8 production in this system.
Pyrrolidinedithiocarbamate was not toxic to the cells as measured using
the MTT assay (data not shown).
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Activation of extracellular signal-regulated kinase (ERK) 1/2 is
an important step in the cascade of cellular events leading to IL-8
production in IECs (18, 23). Because amiloride has been
demonstrated (3) to regulate MAPK activation, we evaluated whether the amiloride suppression of IL-8 production could be explained
by an effect on this pathway. To this end, we pretreated HT-29 cells
with amiloride or vehicle, and 30 min later the cells were stimulated
with IL-1. Whole cell extracts were prepared 30 min after the
IL-1
challenge, and subsequently the activation of ERK1/2 was
determined by Western blotting using an antibody against the active,
phosphorylated form of ERK1/2. As shown in Fig.
8A, IL-1
increased the
activation of ERK1/2 compared with IL-1
-untreated controls, and
pretreatment of the cells with amiloride blunted this increase.
Finally, to further ascertain that a decrease in ERK1/2 activation
could contribute to the inhibitory effect of NHE blockade on IL-8
production, we tested whether ERK1/2 blockade by pharmacological means
prevented IL-8 production by IL-1
-induced HT-29 cells. Figure
8B demonstrates that exposure of HT-29 cells to a selective
inhibitor of the ERK1/2 pathway (PD-98059) suppresses the production of
IL-8. PD-98059 did not cause any toxicity as determined using the MTT
assay (data not shown).
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Taken together, these observations suggest that the decreased activation of ERK1/2 may represent an important mechanism by which NHE inhibition attenuates IL-8 production.
Amiloride Attenuates Course of Colitis in DSS-Treated Mice
Administration of DSS in the drinking water induced marked colonic inflammation, as evidenced by significant weight loss (Table 1), colonic shortening (Table 1), and histological injury, as well as increased MIP-2, MPO, and MDA levels in colonic samples (Fig. 9). Treatment with amiloride (10 mg/kg) induced significant protection against these alterations (Table 1 and Fig. 9).
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DISCUSSION |
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The activation of NHEs by inflammatory stimuli crucially
contributes to inflammatory gene expression in professional
inflammatory cells, such as monocytes (39) and macrophages
(35, 41). The increased activity of NHEs promotes
several macrophage functions, including TNF- and IL-8 production
(38, 39, 44), prostaglandin release (10),
upregulation of Ia expression (41), and Fc
receptor
expression (4). There is also evidence that NHE activation is involved in promoting neutrophil migration (45) as well
as MPO activity and release (53). We (35)
recently demonstrated that NHE inhibition by a variety of amiloride
analogs suppressed IL-12, MIP-1
, and MIP-2 production by
LPS-stimulated macrophages. Furthermore, amiloride decreases IL-8
and IL-6 production in respiratory epithelium infected with
respiratory syncytial virus (32). The results presented in
the current study demonstrate, for the first time, that NHEs are
involved in the regulation of the intestinal epithelial inflammatory
response. Although amiloride is a relatively selective inhibitor of
NHEs, it has effects on a number of other systems, including epithelial
Na+ channels and the Na+/Ca+
exchanger (21, 40, 46). However, the fact that the
selective NHE inhibitors MIA (IC50, ~10 nM) and EIPA
(IC50, ~300 nM) mimicked the inhibitory effect of
amiloride on IL-8 production suggests that the primary target of
amiloride in decreasing IL-8 production is the NHE. This idea was
further confirmed by using the structurally different NHE inhibitors
clonidine, harmaline, and cimetidine, because all three inhibitors
suppressed IL-1
-induced IL-8 production. It is important to note
that we (34) recently found that lithium induced both
NF-
B activation and IL-8 production in human IECs. We
(34) demonstrated that lithium, which is an agent known to stimulate NHEs, induced the intestinal epithelial inflammatory response. This lithium-induced intestinal cell inflammatory response is
suppressed by the same NHE inhibitors as the IL-1
-induced response.
The potency of these inhibitors in reducing lithium-induced IL-8
production is similar to the potency of these inhibitors in decreasing
IL-1
-induced IL-8 production. Thus these data with lithium further
highlight the importance of NHEs in the regulation of the IEC
inflammatory response.
As described above, IECs have been shown to express NHE1, NHE2, NHE3, and NHE4; however, the expression of these isotypes varies even between different cell clones (14, 22, 31, 51). The different isotypes have differing sensitivities to inhibition by amiloride and its analogs. At this point, it would be premature to speculate which isoforms mediate the suppressive effect of NHE inhibition on IL-8 production. The high potency of EIPA and MIA is consistent with a possible role for NHE1 or NHE2. However, this picture is complicated by the fact that amiloride was less potent in decreasing IL-8 production than would have been expected. That is, the IC50 of amiloride's suppressive effect was 30 µM, whereas amiloride has been documented to block NHE1 or NHE2 at 1 µM (50, 56). A further complicating factor is that both MIA and EIPA lost their suppressive effects in the micromolar range. We believe that while the inhibitory effect of both MIA and EIPA in the nanomolar range reflects their ability to block NHEs, the reversal of inhibition in the micromolar range may be a nonspecific action. In any case, further studies are needed to characterize the NHE isoform(s) involved in the regulation of epithelial cell inflammatory responses.
Our data documenting that amiloride suppresses IL-8 mRNA
accumulation are in agreement with the results of previous studies, which showed that amiloride suppressed IL-8 mRNA accumulation in human
monocytes (44) or respiratory epithelium
(32). Interestingly, in addition to demonstrating that
amiloride suppresses IL-8 production, both of these previous studies
(32, 44) found that amiloride attenuated the production of
other inflammatory cytokines, including IL-6 and TNF-. This
observation and the fact that the effect of amiloride on cytokine
production is likely to be transcriptional suggest that amiloride
targets an early pathway that is common to the induction of
proinflammatory cytokines. Our results demonstrating that amiloride,
harmaline, cimetidine, and clonidine prevent both NF-
B DNA binding
and NF-
B-dependent transcriptional activity in IECs indicate that
this transcription factor may be the primary mediator of the
anti-inflammatory effects of NHE inhibition. This notion is supported
by the findings of a recent study (16), in which amiloride
blocked NF-
B activation in respiratory epithelial cells.
These findings raise an important question: how is a signal provided by
the membrane protein NHE transmitted to the cytosolic protein NF-B?
One of the most intriguing possibilities is that it is the alteration
of the cytoskeletal organization of actin filaments that links NHEs to
NF-
B and MAPK activation. This possibility is supported by the fact
that NHEs are important regulators of actin filament assembly (9,
47) and that changes in the actin microfilament system are
involved in the activation of the NF-
B system (1, 58).
Clearly, further studies will be necessary to delineate the mechanisms
whereby NHEs couple extracellular inflammatory signals to activation of
the intracellular inflammatory cascade and NF-
B.
We also provide evidence that NHE inhibition by amiloride suppresses
disease activity in the mouse DSS model of IBD. This decrease in IBD
activity was associated with a decrease in MIP-2 production in colonic
samples. MIP-2 or its human analog IL-8 is released from numerous
sources during IBD. These include IECs as well as monocytes/macrophages
(12, 49). Because we have shown that NHE inhibition
suppresses MIP-2/IL-8 production by both epithelial cells (the current
study) and macrophages (35), we propose that NHE
inhibition exerts its beneficial effect, at least in part, by
inhibiting the production of MIP-2/IL-8 by a variety of inflammatory
cell types. Furthermore, we speculate that the mechanism of NHE
promotion of inflammatory processes may have evolved as a positive
feedback signal during inflammatory cell activation, and dysregulation
of NHE activation may contribute to the maintenance of inflammatory
processes during inflammatory/autoimmune diseases. On the other hand,
recent evidence (43) suggests that NHEs and the intestinal
epithelial inflammatory response are interconnected on yet another
level. That is, an interesting study by Rocha et al. (43)
showed that chronic treatment with the inflammatory cytokine IFN-
downregulated the expression of NHEs in IECs. Thus it can be proposed
that the downregulation of NHE expression by inflammatory signals may
serve as a protective mechanism against the NHE-amplified inflammatory
response during chronic inflammatory states.
In summary, our data demonstrate that NHE inhibition has anti-inflammatory effects in the gut, suggesting that the inhibition of Na+/H+ exchange may be a therapeutic approach in IBD.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Haskó, Dept. of Surgery, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Ave., Univ. Heights, Newark, NJ 07103 (E-mail: haskoge{at}umdnj.edu).
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. Section 1734 solely to indicate this fact.
First published March 20, 2002;10.1152/ajpgi.00015.2002
Received 11 January 2002; accepted in final form 15 March 2002.
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