IFN-
downregulates expression of
Na+/H+ exchangers NHE2 and NHE3 in rat
intestine and human Caco-2/bbe cells
Flavio
Rocha,
Mark W.
Musch,
Leonid
Lishanskiy,
Cres
Bookstein,
Kazunori
Sugi,
Yue
Xie, and
Eugene B.
Chang
The Martin Boyer Laboratories, University of Chicago, Chicago,
Illinois 60637
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ABSTRACT |
Diarrhea associated with inflammatory bowel diseases has
traditionally been attributed to stimulated secretion. The purpose of
this study was to determine whether chronic stimulation of intestinal
mucosa by interferon-
(IFN-
) affects expression and function of
the apical membrane Na+/H+ exchangers NHE2 and
NHE3 in rat intestine and Caco-2/bbe (C2) cells. Confluent C2 cells
expressing NHE2 and NHE3 were treated with IFN-
for 2, 24, and
48 h. Adult rats were injected with IFN-
intraperitoneally for
12 and 48 h. NHE2 and NHE3 activities were measured by
unidirectional 22Na influx across C2 cells and in rat
brush-border membrane vesicles. NHE protein and mRNA were assessed by
Western and Northern blotting. IFN-
treatment of C2 monolayers
caused a >50% reduction in NHE2 and NHE3 activities and protein
expression. In rats, region-specific, time- and dose-dependent
reductions of NHE2 and NHE3 activities, protein expression, and mRNA
were observed after exposure to IFN-
. Chronic exposure of intestinal
epithelial cells to IFN-
results in selective downregulation of NHE2
and NHE3 expression and activity, a potential cause of
inflammation-associated diarrhea.
inflammatory bowel disease; inflammation; mucosa; sodium transport; sodium absorption; water and electrolyte transport; diarrhea; malabsorption; transporter; intestinal adaptation
 |
INTRODUCTION |
TWO MAJOR
FUNCTIONS of the intestinal epithelium are to provide a selective
barrier to luminal contents and to transport water, nutrients, and
electrolytes in a vectorial manner (6). In chronic inflammatory bowel diseases (IBDs), aberrations in barrier and transport functions result in malabsorption and diarrhea, with the
latter hypothesized to arise from active anion secretion stimulated by
the actions of numerous immune and inflammatory mediators
(20). This hypothesis is based on numerous experimental
observations that these agents can stimulate active anion secretion and
assumes that the intestinal epithelium of chronically inflamed mucosa is operationally intact. However, recent clinical observations and in
vivo studies do not support this notion and, in fact, suggest that the
chronically inflamed intestinal epithelium may have not only defective
absorption but also impaired secretion and diminished barrier function
(3, 11-13, 23).
Apical membrane Na+/H+ exchange of intestinal
epithelial cells is the major route for electroneutral,
non-nutrient-dependent Na+ absorption (17). In
contrast to the ubiquitously expressed basolateral membrane
Na+/H+ exchanger (NHE1) (5), the
brush-border membrane Na+/H+ exchangers NHE2
and NHE3 (5, 14, 29) appear to be active even under basal
conditions in the intestine, where they serve an important purpose
acting as transport pathways whenever luminal Na+ is
present (17, 18).
In this study, we examined the effect of chronic inflammation as
mediated by interferon-
(IFN-
), a well-characterized cytokine produced by Th1 lymphocytes and present in high quantities in the gut
of patients with IBD that has many effects on intestinal epithelial
cells (1, 4, 9, 16, 31) and on Na+ absorption
by NHE2 and NHE3 in both an in vitro cell culture system and
physiologically relevant brush-border membranes of rat intestine. In
both cases, there was a time- and dose-dependent downregulation of NHE2
and NHE3 activity, protein expression, and mRNA expression. These
findings support the hypothesis of impaired or downregulated intestinal
epithelial function associated with chronic inflammatory states. They
further implicate IFN-
as an important mediator of these effects and
in the pathogenesis of inflammation-associated diarrhea.
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MATERIALS AND METHODS |
Cell culture.
Caco-2/bbe (C2) cells generously provided by Dr. Mark Mooseker (Yale
University, New Haven, CT) were grown as confluent monolayers on
Transwells in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 µg/ml transferrin (GIBCO, Grand
Island, NY), 50 U/ml penicillin, and 50 µg/ml streptomycin in a
humidified atmosphere of air with 5% CO2.
Apical membrane unidirectional 22Na influx as a
measure of NHE activity.
C2 cells were grown on Transwells for 14 days before experiments began
and were treated with DMEM supplemented with glutamine, penicillin,
streptomycin, and transferrin as described in Cell culture; however, the FBS was increased to 30% (vol/vol)
for 4 days to induce apical membrane NHE2 and NHE3 activity. Two days before flux measurements were taken, cells were treated with 3 or 30 ng/ml of IFN-
for 2, 24, and 48 h. The lots of IFN-
used in
the present studies were all 107 U/mg and were obtained
from Endogen (Woburn, MA). Unidirectional apical membrane
Na+ uptake was determined in flux buffer (130 mM choline
chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 15 mM HEPES, pH 7.4, and 20 mM NaCl, with 1 µCi/ml 22NaCl)
for 10 min. Na+ influx was stopped by four washes in cold
buffer (140 mM NaCl, 5 mM KCl, 15 mM HEPES, pH 7.4, and 1 mM
Na3PO4) and was calculated by dividing the
accumulated disintegrations per minute by the specific Na+
activity in the medium. Dimethylamiloride (DMA; 500 µM) and HOE-642 (30 µM) were used to distinguish NHE2 and NHE3 activities; the former
was defined as the HOE-642-sensitive, and the latter as the
HOE-642-insensitive, components of the DMA-inhibitable unidirectional 22Na influx. All fluxes were measured under acid-loaded
conditions as previously described (18). Briefly, cells
were incubated at 37°C for 60 min in acidifying saline (in mM: 50 NH4Cl, 70 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 15 HEPES, pH 7.0). Fluxes in
experimental groups were expressed as a percentage of fluxes in control
wells with each dose and time point having its own control value.
For the animal experiments, adult Sprague-Dawley rats (200-255 g)
were injected with 10,000 or 25,000 units (U) of IFN-
IP for 12 and
48 h before death, while controls received saline injections of
equal volume for the same time points. The activity of NHE2 and NHE3 in
the brush-border membranes was measured as previously described
(7, 8). Briefly, ileal and colonic mucosal scrapings were
weighed and added to 30 ml of hypotonic lysis buffer (10 mM Tris, pH
7.4, and 3 mM EDTA, with protease inhibitors as described previously)
and homogenized for 30 s at a speed of 15,000 rpm in an
Ultra-Turrax homogenizer. Samples were taken for enzyme enrichment
studies, and the samples were spun at 2,000 g for 5 min at
4°C to remove nuclei and unbroken cells. The supernatants were
removed and spun at 10,000 g for 10 min at 4°C to remove mitochondria. The supernatants were removed, and 15 mM
CaCl2 was added. Samples were gently stirred in the cold
for 15 min and then spun at 8,000 g for 8 min to remove the
endoplasmic reticulum, Golgi, and basolateral membranes. The
supernatants were spun at 45,000 g for 45 min at 4°C to
obtain brush-border membranes. The membranes were resuspended in a
small volume of intravesicular transport buffer (10 mM MES, pH 6.1, 3 mM EDTA, and 80 mM mannitol) and resuspended using a Teflon pestle
homogenizer. A sample was removed for protein determination and enzyme
enrichment studies. Five microliters of the vesicles were added to
forty-five microliters of extravesicular transport buffer (10 mM HEPES,
pH 7.4, 1 mM Na, with 1 µCi/ml 22Na giving a specific
activity of 2,200 dpm/nmol, and 80 mM mannitol). All brush-border
uptakes were of 10-s duration. Uptake of Na+ into the
vesicles was stopped by the addition of 2 ml of ice-cold 90 mM mannitol
and immediate placement onto a 0.45-µm cellulose filter (HAWP;
Millipore, Milford, MA). The filter was washed once with 4 ml of
ice-cold 90 mM mannitol, and the filter was removed and solubilized in
liquid scintillation fluid. 22Na was determined by liquid
scintillation spectroscopy. For the present experiments, the
22Na uptakes were always performed with both HOE-642 (30 µM) and DMA (500 µM) so that NHE2 and NHE3 activities could be
distinguished. With 1 mM Na, NHE2 is completely inhibited by
the amiloride analog HOE-642 at 30 µM, whereas NHE3 is inhibited
<5% (18). Both exchangers are sensitive to DMA. Fluxes
in experimental rats were expressed as a percentage of the flux in
untreated control rats.
Western blotting.
C2 cells grown on Transwell membranes were harvested by scraping in
PBS, washed with PBS, lysed with hypotonic buffer (10 mM Tris, 5 mM
MgSO4, 5 U/ml RNase, 50 U/ml DNase, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and allowed to sit on ice for 15 min. An aliquot was removed for protein determination, and the remaining cell protein was then solubilized in
Laemmli stop buffer. Proteins were separated by 7.5% SDS-PAGE and
transferred to the polyvinylidene difluoride membranes. After blocking with Tris-buffered saline (TBS)-0.1% Nonidet P-40 (NP-40)-5% milk, blots were incubated with primary antibody diluted in TBS-1% BSA
overnight at 4°C. Blots were incubated with specific polyclonal antisera developed and characterized in our laboratory to NHE2 and NHE3
(5, 18). After three washes with TBS-0.1% NP-40-5% milk
and one wash with TBS-0.1% NP-40-1% milk, blots were incubated with
horseradish peroxidase-conjugated secondary antibody diluted in
TBS-0.1% NP-40-1% milk for 1 h at 25°C. After three washes with TBS-0.1% NP-40-1% milk and one wash with TBS-0.1% NP-40, the
membrane was developed by using an enhanced chemiluminescence system.
For rat intestinal scrapings, NHE2 and NHE3 protein levels were
analyzed by taking an aliquot of the brush-border membranes used for
flux studies. The brush-border proteins were solubilized in Laemmli
stop solution and resolved on a 7.5% SDS-PAGE, and Western blots were
performed as described above.
Northern blotting.
For RNA isolation, intestinal scrapings were homogenized in Trizol. RNA
was then extracted once with acid phenol-chloroform, reprecipitated,
and quantified by absorbance at 260 nm. Twenty micrograms were
size-separated on a denaturing formaldehyde agarose gel, transferred to
a positively charged nylon membrane by capillary action, and RNA-linked
to the membrane by ultraviolet light. Blots were prehybridized and
hybridized in XOTCH solution as previously described (5)
with the use of the cDNA probes for rat NHE2 and NHE3. Glyceraldehyde
phosphate dehydrogenase was used as a constitutive control. Probes were
labeled with [32P]dCTP by random prime labeling. Blots
were hybridized at 55°C overnight and then washed up to a stringency
of 0.5× saline sodium citrate-0.5% SDS at 55°C.
Effect of IFN-
on differentiation.
To determine whether IFN-
had effects on the differentiation of the
C2 cells or the rat intestine, we measured activities of the
brush-border enzymes sucrase and alkaline phosphatase. Additionally,
levels of the microvillus structural protein villin were determined by
Western blotting.
For the C2 cells, cells were harvested from Transwells 14 days
postplating and, when appropriate, treated with IFN-
. Cells were
scraped from the filter in PBS and lysed in 10 volumes of hypotonic
buffer (10 mM Tris and 3 mM EDTA with protease inhibitors as described
previously). Nuclei, unbroken cells, and mitochondria were removed by
centrifugation (10,000 g for 5 min), and the microsomal membrane fraction was obtained by centrifugation (100,000 g
for 20 min). The amount of protein in this membrane fraction was
measured with the bicinchoninic acid procedure, and alkaline
phosphatase and sucrase activities were immediately measured. Alkaline
phosphatase was measured colorimetrically by using
para-nitrophenol phosphate and measuring absorbance at 490 nm as described by Cox and Griffin (10). Sucrase
activities were measured as described in the microassay procedure of
Messer and Dahlquist (19) by measuring the glucose generated by sucrase activity. Glucose oxidase and
ortho-dianisidine were used to generate a colored end
product, and the absorbance was measured at 450 nm. To determine the
effect of IFN-
on differentiation in the rat intestine, we measured
alkaline phosphatase activities in brush borders of both the ileum and
colon that had been isolated for use in Na+ flux studies.
As another marker of differentiation, the C2 cells as well as the
brush-border membranes from rat ileum and colon were analyzed for
villin by Western blotting. The protocol used was essentially the same
as that used for NHE Western blots, except that a monoclonal anti-villin antibody (Transduction Labs, Lexington, KY) was used.
 |
RESULTS |
Effect of IFN-
on C2 cells.
The time- and dose-dependent effects of IFN-
on apical membrane NHE2
and NHE3 expression and function were studied in human colonic C2 cell
monolayers. NHE2 and NHE3 are the two major NHE isoforms responsible
for non-nutrient-dependent Na+ absorption across the
brush-border membrane of enterocytes. C2 cell monolayers were selected
for these studies because they form tight, polarized monolayers,
display phenotypic properties characteristic of mature absorptive
epithelium, and correctly sort NHE2 and NHE3 to the apical membrane,
where they are found in intestinal enterocytes (5, 14,
29).
As shown on Fig. 1, cell
monolayers treated with 30 ng/ml IFN-
for 2 h exhibited little
change in NHE2 activity (88.6 ± 3.8% compared with controls).
However, as time progressed, IFN-
treatment resulted in a
progressive decrease in NHE2 activity as shown by a reduction to
53.1 ± 6.0% of control values observed after 24 h. A
further decrease of NHE2 function to 44.6 ± 6.4% of control values was observed by 48 h of treatment. At a lower dose of
IFN-
(3 ng/ml), similar, but more slowly developing, effects on NHE2 activity were observed (Fig. 2). NHE2
activity at 24 h was 112 ± 11.3% compared with controls;
however, by 48 h, NHE2 activity decreased to 50.4 ± 8.1% of
control values (Fig. 2).

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Fig. 1.
Apical membrane Na+
uptake in Caco-2/bbe (C2) cells by Na+/H+
exchangers NHE2 and NHE3 after exposure to 30 ng/ml interferon-
(IFN- ). Fluxes are expressed as a percentage of the 22Na
flux in untreated control C2 cells, where 100% represents the
22Na flux in these control cells. C2 monolayers, 14 days
postconfluent, were treated with 30% fetal bovine serum (FBS) for 4 days (days 15-18) and with 30 ng/ml human recombinant
IFN- on day 16. 22Na fluxes were measured at
2, 24, and 48 h posttreatment with IFN- . Unidirectional
22Na flux was measured for 10 min under acid-loaded
conditions, and Na+ flux due to NHE2 was defined as the
HOE-642 (30 µM)-sensitive component of the dimethylamiloride
(DMA)-inhibitable 22Na flux (n = 3), while
Na+ flux due to NHE3 was defined as the HOE-642 (30 µM)-insensitive component of the DMA-inhibitable 22Na
flux (n = 3). The absolute fluxes (in
nmol · Transwell 1 · 10 min 1) for controls for 2, 24, and 48 h treatments
with 30 ng/ml of IFN- were 5.2 ± 0.3, 6.4 ± 1.4, and
6.2 ± 1.7 for NHE2 and 10.2 ± 0.4, 10.3 ± 0.5, and
11.1 ± 0.9 for NHE3, respectively (+P < 0.01, ++P < 0.001).
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Fig. 2.
Apical membrane Na+
uptake in C2 cells by NHE2 and NHE3 after exposure to 3 ng/ml IFN- .
The fluxes are expressed as a percentage of the 22Na flux
in untreated control C2 cells, where 100% represents the
22Na flux in these control cells. C2 monolayers, 14 days
postconfluent, were treated with 30% FBS for 4 days (days
15-18) and with 3 ng/ml human recombinant IFN- on
day 16. 22Na fluxes were measured at 24 and
48 h posttreatment with IFN- , since no effects were noted at
2 h. Unidirectional 22Na flux was measured for 10 min
under acid-loaded conditions, and Na+ flux due to NHE2 was
defined as the HOE-642 (30 µM)-sensitive component of the
DMA-inhibitable 22Na flux (n = 3), while
Na+ flux due to NHE3 was defined as the HOE-642 (30 µM)-insensitive component of the DMA-inhibitable 22Na
flux (n = 3). The absolute fluxes (in
nmol · Transwell 1 · 10 min 1) for controls for 24- and 48-h treatments with 3 ng/ml of IFN- were 4.5 ± 0.7 and 5.2 ± 0.7 for NHE2 and
8.7 ± 0.9 and 7.3 ± 1.1 for NHE3, respectively
(*P < 0.05).
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Time- and dose-dependent effects with IFN-
treatment on apical
membrane NHE3 activity also were observed. As shown in Fig. 1, after
2 h of exposure of monolayers to 30 ng/ml IFN-
, little change
in apical membrane NHE3 activity was observed (97.4 ± 2.6% of
control values). However, by 24 h, NHE3 activity was significantly reduced to 34.1 ± 4.9% of control values with a further decrease to 18.0 ± 2.0% of control values at 48 h. As shown in Fig.
2, lower doses of IFN-
(3 ng/ml) had no effect on NHE3 activity at
24 h (96.6 ± 1.8% compared with control), but, by 48 h, a decrease in NHE3 activity to 39.7 ± 2.4% of control values
was observed.
To determine whether the IFN-
effects on apical membrane NHE
activity were secondary to altered protein expression, we performed Western blot analysis of NHE2 and NHE3 with isoform-specific polyclonal antibody. As shown by the representative immunoblots in Figs. 3 and 4,
the effects of both low (3 ng/ml)- and high (30 ng/ml)-dose IFN-
treatment of C2 cells for periods of 24, 48, and 72 h can be
observed. At 24 h, there is no change in the expression of NHE2 or
NHE3 at either dose of IFN-
. By 48 h, treatment of C2 monolayers with 30 ng/ml IFN-
caused a significant decrease in both
NHE2 and NHE3 protein expression, whereas 3 ng/ml IFN-
had little
effect. At 72 h, both doses of IFN-
showed dramatic and nearly
equivalent decreases in NHE2 and NHE3 protein expression.

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Fig. 3.
Western blot analysis of NHE2 protein in C2 cells after
exposure to IFN- . C2 monolayers, 14 days postconfluent and grown
under conditions of 30% FBS for 4 days (days 15-18),
were treated with 3 and 30 ng/ml IFN- for 24 h (day
17), 48 h (day 18), and 72 h (day
19). Results in bar graph are percentages of untreated control C2
cell densitometric units (*P < 0.05, +P < 0.01). Blot shown is representative of 3 separate
experiments.
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Fig. 4.
Western blot analysis of NHE3 protein in C2 cells after
exposure to IFN- . C2 monolayers, 14 days postconfluent and grown
under conditions of 30% FBS for 4 days (days 15-18),
were treated with 3 and 30 ng/ml IFN- for 24 h (day
17), 48 h (day 18), and 72 h (day
19). Results in bar graph are means ± SE and are percentages
of untreated control C2 cell densitometric units (*P < 0.05, +P < 0.01). Blot shown is representative of 3 separate experiments.
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In vivo effects of IFN-
on rat intestinal apical membrane NHE
activity and expression.
To determine whether the in vivo effects of IFN-
reflected changes
observed in vitro with C2 cells, we measured the regional activity and
expression of rat intestinal NHE2 and NHE3 in control and
IFN-
-treated rats. As shown in Fig.
5, NHE2 activity of brush-border membrane vesicle decreased to 83.5 ± 24.7% of control values in the jejunum in response to treatment with 25,000 U of IFN-
IP for
12 h, although this change was not statistically significant. However, a significant inhibition of NHE2 activity to 34.2 ± 6.6% of control values was observed in the jejunum of IFN-
-treated rats by 48 h. IFN-
had a similar effect on ileal and colonic NHE2 activity: there was little effect at 12 h, but at 48 h
there was a dramatic decrease in NHE2 activity. This effect was dose dependent as well, since treatment with 10,000 U of IFN-
at 48 h produced a smaller effect compared with 25,000 U of IFN-
at the
same time point. After 48 h of treatment, for instance, this dose
of IFN-
reduced jejunal, ileal, and colonic NHE2 activities to
56.5 ± 11.5%, 62.0 ± 11.5%, and 53.2 ± 10.2% of
control values, respectively (Fig. 6).

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Fig. 5.
Apical membrane Na+ uptake in rat intestine
by NHE2 and NHE3 after exposure to 25,000 U of IFN- at different
time points. The fluxes are expressed as percentages of the
22Na flux in untreated control animals, where 100%
represents the 22Na flux in these control animals. Adult
male Sprague-Dawley rats were injected intraperitoneally with 25,000 U
of murine recombinant IFN- . Animals were killed at 12 and 48 h
after treatment with IFN- . 22Na fluxes were measured in
brush-border membrane vesicles from the rat jejunum, ileum, and colon.
Unidirectional 22Na flux was measured for 10 min under
acid-loaded conditions, and Na+ flux due to NHE2 was
defined as the HOE-642 (30 µM)-sensitive component of the
DMA-inhibitable 22Na flux, while 22Na flux due
to NHE3 was defined as the HOE-642 (30 µM)-insensitive component of
the DMA-inhibitable 22Na flux (n = 4). The
absolute fluxes (in nmol · mg
protein 1 · 10 s 1) in controls for
the 12-h treatment group in rat jejunum, ileum, and colon were
68.5 ± 4.1, 66.3 ± 4.6, and 138.9 ± 0.9, respectively, for NHE2 and 75.2 ± 2.6, 66.5 ± 7.4, and
42.8 ± 12.1, respectively, for NHE3. The absolute fluxes (in
nmol · mg protein 1 · 10 s 1)
in controls for the 48-h treatment group in rat jejunum, ileum, and
colon were 92.4 ± 18.2, 83.2 ± 18.0, and 137.8 ± 20.2, respectively, for NHE2 and 100.9 ± 6.1, 90.6 ± 4.3, and 86.6 ± 8.1, respectively, for NHE3 (*P < 0.05, +P < 0.01, ++P < 0.001). HRS
IFN- , hours of IFN- treatment.
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Fig. 6.
Apical membrane Na+ uptake in rat intestine
by NHE2 and NHE3 after 48-h exposure to different doses of IFN- . The
fluxes are expressed as percentages of the 22Na flux in
untreated control animals, where 100% represents the 22Na
flux in these control animals. Adult male Sprague-Dawley rats were
injected intraperitoneally with either 10,000 or 25,000 U of murine
recombinant IFN- . Animals were killed at 48 h
posttreatment with IFN- , and 22Na fluxes were measured
in brush-border membrane vesicles from the rat jejunum, ileum, and
colon. Unidirectional 22Na flux was measured under
acid-loaded conditions, and Na+ flux due to NHE2 was
defined as the HOE-642 (30 µM)-sensitive component of the
DMA-inhibitable 22Na flux, while Na+ flux due
to NHE3 was defined as the HOE-642 (30 µM)-insensitive
component of the DMA-inhibitable 22Na flux
(n = 4). The absolute fluxes (in nmol · mg
protein 1 · 10 s 1) in controls
for 10,000 U of IFN- for the 48-h treatment group in jejunum,
ileum, and colon were 105.8 ± 3.9, 101.6 ± 2.9, and
122.2 ± 22.0, respectively, for NHE2 and 110.4 ± 8.1, 106.3 ± 7.7, and 108.7 ± 8.6, respectively, for NHE3.
The absolute fluxes (in nmol · mg
protein 1 · 10 s 1) in controls for
25,000 U of IFN- for the 48-h treatment group in jejunum, ileum, and
colon were 92.4 ± 18.2, 83.2 ± 18.0, and 137.8 ± 20.2, respectively, for NHE2 and 100.9 ± 6.1, 90.6 ± 4.3, and 86.6 ± 8.1, respectively, for NHE3 (+P < 0.01, ++P < 0.001).
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As shown in Figs. 5 and 6, IFN-
treatment of rats also caused
a significant decrease in intestinal brush-border membrane NHE3
activity. Jejunal and ileal NHE3 activity were reduced to a
statistically significant level of 74.9 ± 4.9% and
74.1 ± 10.8%, respectively, after 12 h of IFN-
(25,000 U) treatment. However, by 48 h, NHE3 activity of jejunal
and ileal brush-border membranes was further reduced to 34.1 ± 6.5% and 35.3 + 5.5%, respectively. On the other hand, colonic
NHE3 activity was profoundly decreased by IFN-
treatment to
48.1 ± 11.3% at 12 h with no further decrease after 48 h (51.8 ± 5.8%) (Fig. 5) Similar to NHE2, a dose-dependent response to IFN-
treatment was observed (Fig. 6). With 10,000 U of
IFN-
, NHE3 activity was significantly reduced to 49.1 ± 8.6%
of control values in the jejunum and 51.5% ± 7.8% of control values
in the ileum, a lesser inhibition than was observed with 25,000 units
of IFN-
. However, colonic mucosal NHE3 activity was reduced to
53.2% ± 11.4% of control levels, a response that approximated the
changes observed induced by the higher dose of 25,000 U of IFN-
.
Western blot analyses were also performed to assess changes in ileal
and colonic NHE2 and NHE3 protein expression. As shown in Fig.
7, 25,000 U of IFN-
significantly decreased NHE2 protein expression in both ileal and
colonic mucosa after 48 h of treatment (32.6 ± 7.7% and
35.6 ± 9.6% of control densitometric units, respectively, corresponding to changes observed in the functional activity
measurements). NHE3 protein expression in the rat intestine was
affected in a similar fashion by exposure to 25,000 U of IFN-
for
48 h, decreasing to 36.3 ± 9.5% and 27.1 ± 10.6% of
control densitometric units in the ileum and colon, respectively (Fig.
8).

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Fig. 7.
Western blot analysis of NHE2 protein in rat intestine
after exposure to IFN- . Adult male Sprague-Dawley rats were injected
intraperitoneally with 25,000 U of murine recombinant IFN- and
killed at 48 h. Ileal and colonic segments were harvested for
protein content of NHE2. Results are percentages of densitometric units
in untreated rats (+P < 0.01). Blot shown is
representative of 3 separate experiments. C, control.
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Fig. 8.
Western blot analysis of NHE3 protein in rat intestine
after exposure to IFN- . Adult male Sprague-Dawley rats were injected
intraperitoneally with 25,000 U of murine recombinant IFN- and
killed at 48 h. Ileal and colonic segments were harvested for
protein content of NHE3. Results are percentages of densitometric units
in untreated rats (+P < 0.01). Blot shown is
representative of 3 separate experiments.
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Northern blot analyses were also performed to assess whether there were
changes in mucosal NHE2 and NHE3 mRNA abundance, as shown in Figs.
9 and 10.
NHE3 mRNA was significantly reduced after 48-h treatment with 25,000 U
of IFN-
in both the ileum and the colon (28.4 ± 2.7% and
44.1 ± 14.9% of control densitometric units, respectively),
consistent with changes observed in protein expression and functional
activity. In contrast, changes in NHE2 mRNA abundance in ileum and
colon induced after treatment for 48 h with 25,000 U of IFN-
(19.1 ± 4.6% and 65.9 ± 7.8%, respectively) did not correlate in magnitude to the changes observed in protein expression and functional activity. These data may indicate differences in IFN-
regulation of NHE2 expression in different segments of bowel.

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Fig. 9.
Northern blot analysis of NHE2 protein in rat intestine
after exposure to IFN- normalized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Adult male Sprague-Dawley rats were injected
intraperitoneally with 25,000 U of murine recombinant IFN- and
killed at 48 h. Ileal and colonic segments were harvested for mRNA
content of NHE2. Results are percentages of densitometric units in
untreated rats (*P < 0.05, ++P < 0.001). Blots shown are representative of 3 separate experiments.
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Fig. 10.
Northern blot analysis of NHE3 protein in rat intestine
after exposure to IFN- normalized to GAPDH. Adult male
Sprague-Dawley rats were injected intraperitoneally with 25,000 U of
murine recombinant IFN- and killed at 48 h. Ileal and colonic
segments were harvested for mRNA content of NHE3. Results are
percentages of densitometric units in untreated rats
(+P < 0.01, ++P < 0.001). Blots shown
are representative of 3 separate experiments.
|
|
Effect of IFN-
on differentiation.
To determine whether the effect of IFN-
was specific, we measured
activities of the brush-border enzymes sucrase and alkaline phosphatase. Additionally, levels of the microvillus protein villin were analyzed by Western blots. IFN-
did not affect the activities of sucrase or alkaline phosphatase, or the expression of villin, in
either the C2 cells or the rat ileum or colon (Fig.
11). It should be noted that the C2
cells used for these studies were plated onto collagen-coated
Transwells at confluence. This promotes a rapid differentiation, and
thus, over the 4-day course of treatment with media with 30% serum, no
changes in sucrase, alkaline phosphatase, or villin were generally
observed.

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|
Fig. 11.
Effect of IFN- on differentiation markers in C2 cells and rat
intestine. Activities of sucrase and alkaline phosphatase (APase) and
levels of villin were determined with and without IFN- in C2 cells
or in rat intestine. Data are means ± SE for 3 separate
determinations. Blots shown are representative of 4 separate
determinations. BBM, brush-border membrane.
|
|
 |
DISCUSSION |
IBDs are characterized by intestinal mucosal destruction and
functional impairment caused by the chronic effects of immune and
inflammatory mediators, with the clinical consequences often being the
development of severe diarrhea and malabsorption. For many years, the
etiology of inflammation-associated chronic diarrhea was attributed to
stimulated anion secretion, a notion based on experimental observations
demonstrating the acute prosecretory effects of various immune and
inflammatory mediators on normal intestinal tissues (2, 6,
20). However, these studies failed to take into account the fact
that IBDs are chronic diseases and that the effects of these agents
might have different consequences over time. Sandle et al.
(23), for instance, demonstrated that in colonic mucosa
from IBD patients, Na+ absorptive capacity was diminished.
The Cl
exchanger protein DRA is also decreased in areas
of intestinal inflammation (30). In animal models of
chronic enteritis, Na+-dependent glucose absorption
(26), anion exchange, and Na+/Cl
transport were impaired (27), albeit potential causative
agents for these effects were not identified. Furthermore, Sundaram and West (27) reported decreased intestinal mucosal
Cl
/HCO
exchange, but no change in NHE activity, in a model of coccidiococcus-induced enteritis. Finally, studies of trinitrobenzene sulfonic acid-induced chronic colitis showed
that while basal electrolyte transport was normal, the mucosa was less
responsive to secretagogues (3).
In this study, we have shown that IFN-
that is present at high
levels in IBD tissues can significantly downregulate intestinal epithelial NHE2 and NHE3 protein and mRNA expression in C2 cells and in
rat intestinal brush-border membranes and that the effects are both
time and dose dependent. Although we believe that these changes may be
due to IFN-
-induced decreases in NHE gene transcription, the
possibility that posttranscriptional mechanisms have a role in these
effects cannot be ruled out. This may be particularly true for NHE2
where disproportionate decreases in protein and mRNA expression in
certain regions of the bowel were observed. This IFN-
effect appears
to be relatively specific, since concomitant changes in the expression
of the epithelium-specific protein, villin, and the specific activities
of brush-border hydrolases were not observed. Additionally, IFN-
induces the expression of class II myosin heavy chain, suggesting a
phenotypic switch rather than a downregulation of all functions
(1, 9, 16, 22, 31). Furthermore, IFN-
at the doses used
in this study has no effects on mucosal histology in vivo (data not
shown) and has no effect on sucrase, alkaline phosphatase, or villin in
either the C2 cells or rat intestine.
Considerable differences in NHE2 and NHE3 functional regulation have
been noted, both acutely and chronically. With respect to the latter,
NHE3 appears to be highly sensitive to a variety of systemic and
luminal stimuli (7, 8, 21, 28), whereas NHE2 expression
appears to be relatively stable under physiological situations. The
finding that IFN-
affects the expression of both NHE2 and NHE3 was
therefore surprising but could play a major role in causing many of the
mucosal transport alterations associated with chronic inflammation.
IFN-
is known to chronically downregulate active anion secretion and
barrier function in intestinal epithelial and T84 cells (1, 4, 9,
16, 25, 31). Although one group has reported a decrease in
cystic fibrosis transmembrane conductance regulator (CFTR) expression
and no change in Na+-K+-ATPase activity
(4), others have observed the opposite (9, 16), i.e., downregulated Na+-K+-ATPase
activity is decreased and CFTR expression is unchanged. IFN-
-stimulated decreases in active anion secretion were thus attributed to a diminished Na+ gradient and internalization
of apical membrane CFTR. Diminished anion secretion, however, would not
explain the development of inflammation-associated diarrhea. The
importance of this study therefore lies in its examination of the
chronic effects of IFN-
on apical membrane NHEs, the major routes
for non-nutrient-dependent, electroneutral Na+ absorption.
The contributions of NHE2 and NHE3 to intestinal Na+
absorption have been determined in only a limited number of studies. Recent studies in our laboratory (21) using in vitro
measurements of Na+ absorption in Ussing chambers have
demonstrated that 25% of the Na+ absorption is mediated by
NHE2 and 45% by NHE3. In in vivo studies of intestinal perfusions in
canine small intestine, Maher et al. (17) determined that
NHE3 mediates basal Na+ absorption, while NHE2 plays little
role. The differences may relate to differing species,
techniques, or segments of the intestine. It should be emphasized that
apical Na+/H+ exchange is an important route
for Na+ absorption, and the downregulation by IFN-
would
be anticipated to diminish the Na+ absorbed.
One mechanism of action for IFN-
may be downregulation of
transcription of NHE2 and NHE3 genes. The mRNA decreased for both the
exchangers, which would occur if transcription had decreased or if the
mRNA was being degraded at a quicker rate for the same rate of
transcription. IFN-
may be a potent regulator of the activity of a
variety of transcription factors. IFN-
may directly act through
second messenger pathways to regulate activity of certain transcription
factors. However, other factors may play a role in the transcriptional
effects of IFN-
. Our laboratory has recently shown that chronic
IFN-
stimulation of human intestinal C2 cells causes a dose- and
time-dependent downregulation of activity and expression of several
transport- and barrier-related proteins including the
Na+-K+-ATPase (25). Further
examination of this phenomenon suggests that the concomitant increase
in intracellular sodium was responsible for the downregulatory effects,
since treatment with ouabain, a specific inhibitor of the
Na+-K+-ATPase, and Na+ ionophores
reproduced the IFN-
effect, while measures that lowered Na+ blunted it. Increases in Na+ have been
implicated as an activating factor for several transcription factors
such as p38 and c-Jun NH2-terminal kinase
(15). Inhibition of Na+/H+
exchange leading to increased Na+ also has been shown to
activate stress protein kinases in U-937 cells (24).
Therefore, it is possible that the mechanism of IFN-
downregulation
of NHE expression and function is mediated by this cell signaling pathway.
What purpose would diminished intestinal epithelial transport and
barrier serve in a state of chronic inflammation? We speculate three
potentially mutually inclusive possibilities. The first possibility
involves the initiation of watery diarrhea expulsing pathogens and
noxious agents from the intestinal lumen and delivering them to the
lumen antimicrobial agents and antibodies. The second possibility is
that these changes are part of a phenotypic shift by enterocytes
preparing them for a critical role in wound healing and host defense.
Finally, we speculate that enterocytes specifically downregulate
non-life essential functions to reduce metabolic requirements or to
shift them to processes necessary for phenotypic shift. The maintenance
of active electrolyte and nutrient transport and a normal
Na+ gradient represents a major metabolic demand on most
cells, particularly under conditions of stress. Therefore, their
downregulation during conditions of sustained inflammation can conserve
sufficient energy for survival.
In summary, this study implicates IFN-
as an important cytokine that
causes selective downregulation of intestinal Na+
absorption, in part by decreasing expression and activity of the apical
membrane Na+ transporters NHE2 and NHE3. This effect may
underlie the development of mucosal dysfunction and diarrhea often
found to be associated with chronic inflammatory diseases of the bowel.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-47722 (E. B. Chang) and
DK-38510 (E. B. Chang), National Institutes of Health Digestive Disease Research Core Center Grant DK-42086, National Cancer Institute Grant CA-14599 (Cancer Research Center, University of Chicago), and the
Gastrointestinal Research Foundation of Chicago.
 |
FOOTNOTES |
F. Rocha and M. W. Musch contributed equally to this work.
Address for reprint requests and other correspondence: E. B. Chang, The Martin Boyer Laboratories, Dept. of Medicine, MC 6084, The
Univ. of Chicago, 5841 South Maryland Ave., Chicago, IL 60637 (E-mail:
echang{at}medicine.bsd.uchicago.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.
Received 1 September 2000; accepted in final form 4 December 2000.
 |
REFERENCES |
1.
Adams, RB,
Planchon SM,
and
Roche JK.
IFN-gamma modulation of epithelial barrier function. Time course, reversibility, and site of cytokine binding.
J Immunol
150:
2356-2363,
1993[Abstract/Free Full Text].
2.
Archampong, EQ,
Harris J,
and
Clark CG.
The absorption and secretion of water and electrolytes across the healthy and the diseased human colonic mucosa measured in vitro.
Gut
13:
880-886,
1972[ISI][Medline].
3.
Bell, CJ,
Gall D,
and
Wallace JL.
Disruption of colonic electrolyte transport in experimental colitis.
Am J Physiol Gastrointest Liver Physiol
268:
G622-G630,
1995[Abstract/Free Full Text].
4.
Besancon, F,
Przewlocki G,
Baro I,
Hongre AS,
Escande D,
and
Edelman A.
Interferon gamma downregulates CFTR gene expression in epithelial cells.
Am J Physiol Cell Physiol
267:
C1398-C1404,
1994[Abstract/Free Full Text].
5.
Bookstein, C,
DePaoli AM,
Xie Y,
Niu P,
Musch MW,
Rao MC,
and
Chang EB.
Na/H exchangers NHE1 and NHE3, of rat intestine.
J Clin Invest
93:
106-113,
1994[ISI][Medline].
6.
Chang, EB,
and
Rao MC.
Physiology of the Gastrointestinal Tract, edited by Johnson LR,
Alpers DH,
Christensen J,
Jacobson ED,
and Walsh JH.. New York: Raven, 1994, p. 2027-2082.
7.
Cho, JH,
Musch MW,
DePaoli AM,
Bookstein CM,
Xie Y,
Burant CF,
Rao MC,
and
Chang EB.
Glucocorticoids regulate Na+-H+ exchange expression and activity in region- and tissue specific manner.
Am J Physiol Cell Physiol
267:
C796-C803,
1994[Abstract/Free Full Text].
8.
Cho, JH,
Musch MW,
Bookstein CM,
McSwine RL,
Rabenau K,
and
Chang EB.
Aldosterone stimulates intestinal Na+ absorption in rats by increasing NHE3 expression of the proximal colon.
Am J Physiol Cell Physiol
274:
C586-C594,
1998[Abstract/Free Full Text].
9.
Colgan, SP,
Parkos CA,
Matthews JB,
D'Andrea L,
Awtrey CS,
Lichtman AH,
Delp-Archer C,
and
Madara JL.
Interferon-gamma induces a cell surface phenotype switch on T84 intestinal epithelial cells.
Am J Physiol Cell Physiol
267:
C402-C410,
1994[Abstract/Free Full Text].
10.
Cox, RP,
and
Griffin MJ.
Alkaline phosphatase.
Arch Biochem Biophys
122:
552-562,
1967[ISI].
11.
Donowitz, M,
Charney AN,
Hynes R,
Formal SB,
and
Collins H.
Significance of abnormal rabbit ileal histology in the pathogenesis of diarrhea.
Infect Immun
26:
380-386,
1979[ISI][Medline].
12.
Edmonds, CJ,
and
Pilcher D.
Electrical potential difference and sodium and potassium fluxes across rectal mucosa in ulcerative colitis.
Gut
14:
784-789,
1973[ISI][Medline].
13.
Hawkey, PC,
McKay JS,
and
Turnberg LA.
Electrolyte transport across colonic mucosa of patients with inflammatory bowel disease.
Gastroenterology
79:
508-511,
1980[ISI][Medline].
14.
Hoogerwerf, WA,
Tsao SC,
Devuyst O,
Levine SA,
Yun CH,
Yip JW,
Cohen ME,
Wilson PD,
Lazenby AJ,
Tse C-M,
and
Donowitz M.
NHE2 and NHE3 are human and rabbit intestinal brush border proteins.
Am J Physiol Gastrointest Liver Physiol
270:
G29-G41,
1996[Abstract/Free Full Text].
15.
Kuroki, D,
Minden A,
Sanchez I,
and
Wattenberg EV.
Regulation of a c-Jun amino-terminal kinase/stress-activated protein kinase cascade by a sodium-dependent signal transduction pathway.
J Biol Chem
273:
23905-23911,
1997.
16.
Madara, JL,
and
Stafford J.
Interferon-gamma directly affects barrier function of cultured intestinal epithelial monolayers.
J Clin Invest
83:
724-727,
1989[ISI][Medline].
17.
Maher, MM,
Gontarek JD,
Bess RS,
Donowitz M,
and
Yeo CJ.
The Na/H exchange isoform NHE3 regulates basal canine ileal Na absorption in vivo.
Gastroenterology
112:
174-183,
1997[ISI][Medline].
18.
McSwine, RL,
Musch MW,
Bookstein C,
Xie Y,
Rao MC,
and
Chang EB.
Regulation of apical membrane Na/H exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe.
Am J Physiol Cell Physiol
275:
C693-C701,
1998[Abstract].
19.
Messer, M,
and
Dahlquist A.
A one-step ultra-micro method for the assay of intestinal disaccharidases.
Anal Biochem
14:
376-392,
1966[ISI][Medline].
20.
Musch, MW,
and
Chang EB.
Inflammatory Bowel Disease: From Bench to Bedside, edited by Targan SR,
and Shanahan F.. Baltimore, MD: Lippincott Williams and Wilkins, 1994, p. 239-254.
21.
Musch, MW,
Bookstein C,
Yue X,
Sellin JH,
and
Chang EB.
SCFA increase intestinal Na absorption by induction of NH3 in rat colon and human intestinal C2/bbe cells.
Am J Physiol Gastrointest Liver Physiol
280:
G687-G693,
2001[Abstract/Free Full Text].
22.
Ruemmele, FM,
Dionne S,
Levy E,
and
Seidman G.
Effects of interferon gamma on growth, apoptosis, and MHC class II expression of immature rat intestinal crypt (IEC-6) cells.
J Cell Physiol
176:
120-126,
1998[ISI][Medline].
23.
Sandle, GI,
Higgs N,
Crowe P,
Marsh MN,
Venkatesh S,
and
Peters TJ.
Cellular basis for defective electrolyte transport in inflamed human colon.
Gastroenterology
99:
97-105,
1990[ISI][Medline].
24.
Shrode, LD,
Rubie EA,
Woodgett JR,
and
Grinstein S.
Cytosolic alkalinization increases stress activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and p38 mitogen-activated protein kinase by a calcium-independent mechanism.
J Biol Chem
272:
13653-13659,
1997[Abstract/Free Full Text].
25.
Sugi, K,
Musch MW,
Field M,
and
Chang EB.
Initiation and cellular basis of downregulated intestinal epithelial transport and barrier function by interferon-gamma (Abstract).
Gastroenterology
116:
A827,
1999[ISI].
26.
Sundaram, U,
Weisel S,
Rajendren VM,
and
West AB.
Mechanism of inhibition of Na-glucose cotransport in the chronically inflamed rabbit ileum.
Am J Physiol Gastrointest Liver Physiol
273:
G913-G919,
1997[Abstract/Free Full Text].
27.
Sundaram, U,
and
West AB.
Effect of chronic inflammation on electrolyte transport in rabbit ileal villus and crypt cells.
Am J Physiol Gastrointest Liver Physiol
272:
G732-G741,
1997[Abstract/Free Full Text].
28.
Turnamian, SG,
and
Binder HJ.
Aldosterone and glucocorticoid receptor-specific agonists regulate ion transport in rat proximal colon.
J Clin Invest
84:
1924-1929,
1989[ISI][Medline].
29.
Wormmeester, L,
Sanchez de Medina F,
Kokke F,
Tse CM,
Khurana S,
Bowser J,
Cohen ME,
and
Donowitz MM.
Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush border Na/H exchange.
Am J Physiol Cell Physiol
274:
C1261-C1272,
1998[Abstract/Free Full Text].
30.
Yang, H,
Jean W,
Furth EE,
Wen X,
Katz JP,
Salon RK,
Silber DG,
Analis TM,
Schweinfest CW,
and
Wu GD.
Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea.
Am J Physiol Gastrointest Liver Physiol
275:
G1445-G1453,
1998[Abstract/Free Full Text].
31.
Yoakim, A,
and
Ahdieh M.
Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin.
Am J Physiol Gastrointest Liver Physiol
276:
G1279-G1288,
1999[Abstract/Free Full Text].
Am J Physiol Cell Physiol 280(5):C1224-C1232
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