Interferon-
downregulates ion transport in murine small
intestine cultured in vitro
Dongjin
Yoo,
Winson
Lo,
Stephen
Goodman,
Wasif
Ali,
Carol
Semrad, and
Michael
Field
Division of Digestive and Liver Diseases, Departments of Medicine
and Physiology and Cellular Biophysics, College of Physicians and
Surgeons, Columbia University, New York, New York 10032
 |
ABSTRACT |
Effects of IFN-
on
mammalian small intestinal ion transport were studied in vitro using
incubated sheets of murine small intestine in Ussing chambers. In
oxygenated standard culture medium containing hydrocortisone and
antibiotics, they maintained their short-circuit current
(Isc) responses to glucose and theophylline for
48 h. Histological examination revealed a 50% diminution of villus
height over 36 h but no change in crypts. Height was better maintained during a 36-h incubation of small intestine from SCID mice,
suggesting a role for B or T lymphocytes in villus atrophy. Exposure of
small intestine to 100 U/ml IFN-
for 36 h decreased basal
Isc by 40% and Isc
responses to glucose and theophylline by ~70%; at 1,000 U/ml for
36 h, IFN-
inhibited these Isc responses by 90%. An inhibitor of inducible NO synthase did not reverse these
effects, suggesting that they are not mediated by NO. Tissue resistance, mucosal K+ content, and epithelial morphology
were not affected. Ouabain-sensitive ATPase activity in homogenates was
inhibited 60% by IFN-
(100 U/ml for 36 h). IFN-
inhibition
of Isc responses to glucose and theophylline
also occurred in SCID mouse small intestine. Thus murine small
intestinal sheets can be maintained viable in vitro for at least
48 h, although villus blunting develops (but less so in SCID mouse
small intestine). Also, prolonged exposure to IFN-
downregulates
Na+-coupled glucose absorption, active Cl
secretion, and Na+-K+-ATPase activity, effects
unlikely to be mediated by enhanced NO.
intestinal absorption; intestinal secretion; sodium transport; glucose transport; chloride transport; intestinal villus architecture; severe combined immunodeficiency disease mouse; organ culture
 |
INTRODUCTION |
TO EFFECTIVELY STUDY
INTERACTIONS between the intestinal immune system and the
intestinal epithelium, an intact epithelial preparation that can
maintain its transport and barrier properties for an extended period of
time is needed. In T84 cells, for example, interferon-
(IFN-
)-induced phenotypic changes require 24-48 h to manifest
themselves (6). Studies of such slow-to-appear cytokine
effects have been accomplished with colon cancer cell lines such as T84
but not heretofore with normal intestine. To accomplish the latter, we
have developed an in vitro method for maintaining murine small
intestine viable and intact for at least 48 h.
We selected IFN-
as the cytokine for initial study because of its
central role in inflammation and because its epithelial effects had
previously been studied in T84 cells (6). T helper-1 (Th1)
lymphocytes, when activated, secrete IFN-
(31), and
Crohn's disease, the panenteric, transmural subtype of inflammatory
bowel disease, is characterized by a Th1 cell cytokine response
(4, 30, 34, 37). A Th1 cell response is also
characteristic of celiac disease (gluten-sensitive enteropathy), in
which there is small intestinal malabsorption associated with villus
atrophy and crypt hypertrophy (20). In colon cancer cell
lines, IFN-
has been shown to upregulate major histocompatibility
complex (MHC) class I and II expressions, enhance neutrophil adhesion, downregulate cAMP-activated Cl
secretion, and attenuate
intestinal barrier function, enhancing the absorption of macromolecules
(6, 42). Whether this so-called "phenotypic switch"
caused by IFN-
in colon cancer cell lines also occurs in normal
mammalian small intestine in primary culture has not heretofore been determined.
In this study, we developed a primary culture system for sheets of
murine small intestine large enough to place in Ussing chambers, and
using this system, we demonstrated that IFN-
downregulates both
Na+-coupled glucose absorption, which arises in villus
cells, and cyclic nucleotide-activated active Cl
secretion, which arises mainly in crypt cells. It also inhibited Na+-K+-ATPase activity, as measured in
homogenates. These ion-transport changes developed without significant
changes in epithelial morphology or K+ content.
 |
METHODS |
Primary organ culture.
Two- to four-month-old mice of CD-1 strain, obtained from Charles River
Laboratories (Wilmington, MA), were gavaged with a mixture of 1.5 mg
metronidazole and 7.5 mg neomycin, fasted, except for water, for
12-18 h, and then killed by cervical dislocation. The small
intestine, from the ligament of Treitz to the ileocecal junction, was
rapidly excised, immersed in oxygenated cold normal saline, cut open
longitudinally with a fine iridotomy scissors, rinsed with normal
saline to remove intestinal contents, and then transferred to cold
bicarbonate-buffered Ringer solution gassed with 95%
O2-5% CO2. Sections 2-cm long were placed,
mucosa up, on a polyester mesh screen (Spectrum, Laguna Hills, CA),
which was positioned over pins set into a circular plastic frame
surrounding a curved end-rectangular opening measuring 1.6 cm in length
and 0.4 cm in width (Fig. 1). The lateral
margins of the intestine were stretched to fit over the pins. This
preparation was then placed in a 6-cm diameter petri dish (Fisher
Scientific, Springfield, NJ) to which ~13 ml of culture medium were
added, creating a level just covering the tissue surface. In some
experiments (those for tissue K+ and
Na+-K+-ATPase measurements), the muscle layers
were stripped off before mounting tissues.

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Fig. 1.
Plexiglas frame for incubating stretched intestinal sheet
in vitro. Three-dimensional (upper) and cross-sectional
views (lower) are shown. Arrowheads indicate grooves
permitting fluid exchange. Outside dimensions: height, 6.5 mm;
diameter, 26 mm. Opening for tissue (indicated by vertical arrow):
length, 16 mm; width, 4.0 mm; depth, 3.0 mm. Recess on underside of
frame created to minimize dead space between well-stirred medium and
serosal surface of tissue.
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The culture medium consisted of bicarbonate-buffered Ringer solution to
which the following were added: 0.5 µg/ml
FeSO4 · 7H2O, 0.08 µg/ml
MnSO4 · H2O, 0.15 µg/ml
ZnSO4 · 7H2O, 0.25 µg/ml CuSO4, MEM vitamins and amino acids (Life Technologies,
Grand Island, NY), insulin-transferin-selenium (Life Technologies), 20 mM fructose, 5 mM L-glutamine (Life Technologies), 0.8 µg/ml hydrocortisone, 300 µg/ml ascorbate, 0.2 µg/ml vitamin
B12, 15 µg/ml glutathione, 15 µg/ml phenol red, 10%
fetal bovine serum (Life technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Murine recombinant IFN-
(Genzyme, Cambridge, MA) was added to some cultures. The loaded petri
dishes were placed in the bases of loosened, 120-ml containers
(Allegiance, Montgomery, NY), which were set on a slowly rotating
platform and gassed with a mixture of 95% O2-5%
CO2 for 30 min. The containers were then screwed shut, and
their interphases were sealed with oil. Then the containers were placed
on a slowly rocking shaker in an incubator at 37°C for varied time
intervals. Unless otherwise specified, materials were purchased from
Sigma Chemical (St. Louis, MO).
Electrical measurements.
The murine intestinal explants were clamped into Ussing chambers with
tissue openings of the same size as specified above for the frames used
for tissue incubation. Short-circuit currents, potential differences,
and tissue resistances were determined with automatic voltage clamps,
which corrected for fluid resistances.
Morphological studies.
Tissue explants, taken either directly from incubation or from Ussing
chambers, were placed in a gelatin capsule (Polysciences, Warrington,
PA) containing OCT (Sakura Finetek, Torrance, CA), gradually frozen in
a dry ice-acetone mixture, and then stored at
70°C until further
processing. Sections (5 µm) were sliced by cryostat at
10 to
20°C, placed on slides, and stained with hematoxylin and eosin.
Measurements of villus height and width, crypt depth, and number of
villi and crypts per 100-fold magnified high-power field were made on
photographs of these images. Measurements were made on a minimum of
three villi per image, and results from multiple images were averaged.
Because longitudinally cut crypts were less frequent, 2-4 crypt
lengths were measured for each mouse. Small intestines from three mice
were used for histological evaluation. Intestinal sheets from each of
the mice were first tested in Ussing chambers and shown to respond
normally to IFN-
, glucose, and theophylline.
Tissue K+ measurements.
For K+ determinations, exposed sections of muscle-stripped
intestinal explants were cut out and dried at ~80°C overnight,
weighed, and extracted in equal volumes of glacial acetic acid and 3 M TCA. The extracts were sonicated until clear, diluted with two volumes
of distilled water, and centrifuged. K+ concentrations in
the supernates were then determined by atomic absorption spectroscopy.
As a control for this method of intracellular K+ assay,
K+ concentration was determined in mucosae incubated at
37°C for 3 or 5 h with 10 mM ouabain and 15 µg/ml amphotericin
B and compared with concentrations in mucosae incubated for the same
time periods without addition of ouabain and amphotericin.
Na+-K+-ATPase
measurement.
Na+-K+-ATPase activity was measured as
ouabain-inhibitable phosphate production from ATP by a modification of
prior methods (1, 2, 32). Briefly, mucosa stripped of
muscle was homogenized at 650 rpm with 20 vertical strokes for 2 min.
The homogenization medium contained 250 mM sucrose, 30 mM histidine,
and 1 mM EGTA (pH 7.2). Tissues were washed with normal saline and then
twice with each of two EDTA-containing Tris-buffered solutions (pH
7.2), the first 5 mM EDTA in 50 mM Tris and the second 1 mM EDTA in 50 mM Tris. The homogenates were then incubated for 1 h at 4°C with
SDS (4.8 nmol/mg protein), following which they were centrifuged at 750 g for 3 min to remove cellular debris. Aliquots (120 µl) were then added to 1.1 ml of prewarmed reaction medium (130 mM NaCl, 20 mM KCl, 5 mM MgCl2, 3 mM NaN3, and 30 mM Tris,
pH 7.2) and incubated with or without 10 mM ouabain for 30 min. ATP
(final concn, 3 mM) was then added, and the incubation continued for an
additional 15 min. The assay temperature was 37°C. Free phosphate was
measured in a spectrophotometer at 700 nm. Enzyme activities were
expressed as micromoles of phosphate liberated per milligram of protein
per hour.
Statistical analysis.
Isc differences, crypt depths on histological
sections, and enzyme activities were analyzed by Student's
t-test, tissue K+ concentrations by ANOVA, and
villus height and width by the Kruskal-Wallis test for significance.
All results are presented as means ± SE.
 |
RESULTS |
Effects of incubation time on ion transport and resistance.
Isc responses to both glucose and theophylline
were not significantly affected by incubation time up to 36 h
compared with responses in freshly removed tissues not incubated in
vitro (Fig. 2). At 48 h, the glucose
response was still unchanged from the time 0 response but
the theophylline response had declined by ~40% [P < 0.02; at 36 h, the mean theophylline response had declined by
28% but this change is not statistically significant
(P > 0.2)]. Thus sheets of murine small intestine can
be incubated in vitro for an appreciable number of hours without loss
of either sodium-coupled glucose absorption or theophylline-stimulated
active Cl
secretion. In a single experiment,
Isc responses were tested after 5 days of
incubation in vitro: both glucose and theophylline responses were in
excess of 175 µA/cm2.

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Fig. 2.
Effect of duration of incubation on short-circuit current
(Isc) responses to glucose and theophylline.
Bars are means ± SE for 4-6 mice. Results at each time point
are compared with results for unincubated (t = 0) tissues
from the same mice. Glucose (10 mmol/l) was added on the luminal side
and theophylline (5 mmol/l) to the serosal side. * P < 0.05, significantly different from t = 0; other
values are not significantly different.
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Tissue resistance declined with incubation time. In six mice,
resistance declined by 38% over 36 h (from 27.1 ± 2.46
· cm2 at t = 0 to 16.9 ± 1.62
· cm2 at t = 36 h;
P < 0.03). Because measurements were made on
unstripped tissue, it is uncertain whether this decline in resistance
arose from epithelial changes, smooth muscle changes, or both.
Effects of incubation time on histology and
K+ content.
Histological sections made from both fresh (unincubated) intestine and
intestine incubated in vitro for 36 h are shown in Fig.
3. Measurements of mean villus heights
and widths at both times are shown in Table
1. An ~50% decrease in villus height developed over the 36-h incubation period, whereas villus width did not
change. Examination of the crypt region indicates no significant change
in crypt histology or diminution in crypt number over the 36-h
incubation period. The number of crypts divided by the number of villi
was 2.29 ± 0.21 at t = 0 and 2.08 ± 0.13 at
t = 36 h. Examination of crypt depth in a limited
number of crypts cut longitudinally indicated no change over 36 h
[at t = 0, 49.3 ± 1.21 µm (n = 9); control at t = 36 h, 53.0 ± 3.98 µm
(n = 9); IFN-
treated at 36 h, 46.4 ± 2.44 µm (n = 8); no statistically significant
differences].

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Fig. 3.
Effects of in vitro incubation for 36 h in the
presence and absence of interferon- (IFN- ) on mucosal histology
of small intestine of CD-1 mice. Representative photomicrographs of
hematoxylin and eosin-stained sections (×100). Top: freshly
removed, unincubated intestine. Middle: intestine incubated
for 36 h without IFN- . Bottom: intestine incubated
for 36 h with IFN- (100 U/ml).
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As an additional measure of epithelial viability, we determined mucosal
K+ content (µmol/mg dry wt), comparing values at
t = 0 and t = 36 h. We used
muscle-stripped intestine for these determinations to avoid
contamination from the K+ content of smooth muscle. We
first tested the suitability of the stripped preparation by measuring
its Isc responses after 36 h incubation:
means and ranges of Isc responses for stripped tissues from three mice were 346 µA/cm2 (range
135-527) for glucose and 246 µA/cm2 (range
173-381) for theophylline. Clearly, vigorous
Isc responses can be elicited from stripped
intestine after prolonged incubation in vitro. The
K+ contents of stripped tissues, both those incubated for
36 h and those tested without preincubation, are compared in Table
2. The two values do not differ
significantly.
Effects of IFN-
on ion transport and resistance.
Prolonged incubation of tissues with IFN-
diminished both baseline
Isc and Isc responses to
glucose and theophylline. Baseline Isc, measured
~15 min after mounting tissues in vitro and just before mucosal-side
addition of glucose, was decreased by IFN-
(100 U/ml) by ~40%
(Table 3). The time course of the effect
of IFN-
on Isc responses to glucose
and theophylline is shown in Fig. 4. At
100 U/ml IFN-
, the magnitude of inhibition progressed from 24 to
36 h and, at least with respect to the glucose response, from 36 to 48 h. At 1,000 U/ml, inhibitions of Isc
responses were greater than at 100 U/ml. Inhibition of
Isc responses at concentrations of IFN-
from
25 to 1,000 U/ml are compared in Table 4.
No significant differences in percentage inhibition from 25 to 500 U/ml
were found, although, as also noted in Fig. 4, inhibition was greater at 1,000 U/ml. The present results for time course and dose dependency are similar to those obtained by Colgan et al. (6) using
T84 cell monolayers.

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Fig. 4.
Effect of IFN- on Isc responses to
glucose and theophylline. Values for Isc in
the presence of IFN- are expressed as % of values in its absence.
Bars are means ± SE for 24, 36, and 48 h incubation and for
100 and 1,000 U/ml IFN- . Each bar represents results for 4-6
mice. See Fig. 2 legend for further details.
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In contrast to the results of Colgan et al. (6), however,
tissue resistance was unaffected by IFN-
: in tissues from six mice
incubated for 36 h in either the presence or absence of 100 U/ml
IFN-
, resistances in control and IFN-
-treated tissues were identical (16.9 ± 1.62 vs. 16.8 ± 1.53
· cm2). It should be noted, however, that the
baseline resistance of the T84 cell monolayers was in the 600-800
· cm2 range. Because of the much greater
leakiness of murine small intestine, a change in cellular or
paracellular resistance properties brought on by IFN-
may have been
missed. During prolonged incubation in vitro, there may be significant
cell desquamation. This would produce holes of several micrometers in
diameter and would likely outweigh the effect of any change in tight
junction permeability.
The inhibitory effect of IFN-
on Isc
responses to glucose and theophylline could represent, as previously
suggested for T84 cells (6), a shift in phenotype:
downregulation of ion and nutrient transport function and upregulation
of other functions more related to the immune system and inflammation.
Alternatively, it could represent a loss of epithelial viability. To
exclude the latter, the effects of IFN-
on tissue morphology and
K+ content were evaluated. Morphological characteristics
are shown in Fig. 3 and Table 1 and K+ content in Table 2.
It can be seen that incubation for 36 h in the presence of IFN-
(100 U/ml) did not significantly affect either. Thus tissue viability
does not appear to have been compromised by prolonged incubation with
IFN-
.
Absence of a role for nitric oxide in ion transport inhibition by
IFN-
.
In murine proximal tubule epithelial cells, IFN-
, in combination
with lipopolysaccharide, inhibits Na+-K+-ATPase
activity by a nitric oxide (NO)-mediated mechanism [i.e., the NO
synthase (NOS) inhibitor
N
-nitro-L-arginine prevented this
inhibition] (18). To evaluate whether the ion-transport
effects of IFN-
were secondary to upregulation of NOS by IFN-
, we
added the inducible NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME) to incubations with IFN-
. Results are shown in
Table 5. L-NAME did not even
slightly reduce the effect of IFN-
. Although there is some arginine
in the incubation medium (0.36 mM), the concentration of
L-NAME was 14-fold greater and L-NAME is
reported to have an at least fivefold greater affinity for NOS than
does arginine (19).
Effect of IFN-
on
Na+-K+-ATPase
activity.
INF-
has been shown in T84 cells to downregulate several epithelial
transport proteins including Na+-K+- ATPase
(6, 37a). Because the Na pump is involved in both Na-coupled glucose
transport and theophylline-stimulated Cl
secretion, we
determined the effect of 36 h incubation with IFN-
on
Na+-K+-ATPase activity. Enzyme activity was
assayed in homogenates of muscle-stripped small intestine as
ouabain-sensitive phosphate production from ATP as described in
METHODS. Results are shown in Table
6. Although mean ouabain-sensitive ATPase
activity was 38% less in 36-h incubated control mucosa than in
unincubated mucosa, this difference was not statistically significant
(P > 0.3). Incubation with IFN-
for 36 h
inhibited ouabain-sensitive ATPase activity by ~60%, which compares
fairly closely to the percentage inhibitions of
Isc responses to both glucose and theophylline (~70%).
Effects of IFN-
in SCID mice.
Because the intestinal mucosa contains an appreciable number of
leukocytes and other mesenchymal cells, it remains uncertain whether
the effects of the added IFN-
were exerted directly on enterocytes
or only indirectly via another cytokine released from mesenchymal
cells. Activated T cells, for example, release a number of cytokines
that directly or indirectly may affect ion transport [in addition to
IFN-
, tumor necrosis factor-
, interleukin-2 (IL-2), IL-4, IL-5,
IL-10, and IL-13; Ref. 31]. To provide a partial answer
to this question, we tested the effect of IFN-
on the small
intestine of severe combined immunodeficiency disease (SCID) mice since
they are devoid of mature T and B lymphocytes (25). In
this preparation, IFN-
(100 U/ml for 36 h) inhibited Isc responses to glucose and theophylline by
~60% (Fig. 5), suggesting that the
observed in vitro effects of IFN-
were not mediated by mucosal T or
B lymphocytes. It is also of interest, as shown in Table 3, that the
basal Isc of SCID mouse small intestine is lower
than that of CD-1 mouse small intestine and is unaffected by IFN-
.

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Fig. 5.
Effect of IFN- on Isc responses to
glucose and theophylline in sheets of small intestine from severe
combined immunodeficiency disease (SCID) mice. Results are means ± SE for tissues from 5 mice. * P < 0.001, significantly different from control.
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Figure 6 shows the morphology of SCID
mouse intestine incubated for 36 h with or without IFN-
. It can
be seen that IFN-
did not significantly alter intestinal morphology
in SCID mice. Figure 6 does reveal one surprising finding: After a 36-h
incubation, villus height in the SCID mouse intestine shown is clearly
greater then in normal intestine. This result is borne out by the
histology of two additional incubated SCID mouse small intestinal
preparations. We also examined the histology of two unincubated
(time 0) SCID mouse small intestinal mucosae. Villus height
appeared to be well within the range of values for CD-1 mouse small
intestine at time 0. We did not attempt to quantitate villus
height in SCID mouse small intestine because the n was small
and there is significant variability in height along the length of
intestine from proximal jejunum to distal ileum. Thus the greater
villus height in 36-h incubated SCID mouse small intestine appears to
be due to less villus blunting and not to a greater villus height at
time 0. Villus height in normal (CD-1 mouse) small intestine
appears, therefore, to diminish during prolonged incubation in vitro
due to one or more factors absent from SCID mouse intestine. The
factors that come to mind are mucosal lymphocytes and enteric
microorganisms (SCID mice are raised and maintained in a relatively
germ-free environment).

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Fig. 6.
Effect of IFN- on mucosal morphology of small
intestine of SCID mouse. Representative photomicrographs of hematoxylin
and eosin-stained sections (×100). Top: intestine incubated
in vitro for 36 h without IFN- . Bottom: intestine
incubated in vitro for 36 h with IFN- . The sections shown are
representative of intestine from 3 SCID mice. Examination of the
histology of unincubated small intestine from 2 SCID mice indicates
that villus height is not greater than in CD-1 mouse intestine.
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DISCUSSION |
Primary culture of mammalian small intestine.
We have developed a culture system for intact sheets of murine small
intestine that maintains viability for at least 48 h. The exposed
area of these epithelial sheets (1.6 cm × 0.4 cm), together with the
surrounding pinned area, was large enough to mount in Ussing chambers
specially designed for elongated tissue sections. Cultured tissues
maintained their Isc responsiveness to glucose
(reflecting Na+-coupled glucose absorption, a villus cell
function) and theophylline (reflecting active Cl
secretion, largely a crypt cell function). Both muscle-stripped and
full-thickness intestine could be maintained in this way. Despite
maintaining these absorptive and secretory functions at their
nonincubation rates, cultured intestine underwent morphological change,
villi being 50% shorter at 36 h than at time 0 without evident changes in crypts. Furthermore, electrical resistance decreased
by ~40% over 36 h of incubation in vitro; this may have been
due to shedding of epithelial cells during incubation.
It is intriguing that villus height diminished by 50% over 36 h
without any reduction in the Isc response to
glucose. This apparent discrepancy can perhaps be explained by the
results of a prior study (9) in which the surface location
of the Na+-coupled glucose transporters was determined in
rat ileum. Using [3H]phloridzin as a label, these
transporters were found bunched at the tips of villi and only minimally
present along the sides of villi. In contrast, in the ileum of rats
rendered diabetic with streptozocin (glucose absorption is enhanced in
experimental diabetes), the glucose transporters were found spread
evenly over the entire villus surface, sides as well as tips. Whether
these observations on rat ileum apply also to mouse small intestine and
jejunum as well as ileum is uncertain. Certainly in vitro changes in
cell proliferation and apoptosis could also influence the proportion of
intestinal epithelial cells engaged in glucose absorption. Whatever the
explanation, our results do demonstrate that there is not a tight
correlation between villus surface area and glucose absorptive
capacity. We are unaware of any evidence that glucose absorption is
diminished in celiac disease, for example.
Primary culture of intact small intestine for periods up to 48 h
has been reported previously (10, 16, 28, 43) for rabbit,
mouse, and human small intestine with good, although not perfect,
preservation of brush-border enzyme activities, cellular functions, and
epithelial structure. As in the present study, some villus blunting has
been noted. Fetal small intestine appears to be easier to maintain in
vitro, survival for multiple days having been accomplished
(26). In all of these studies, the sizes of incubated
tissue sections were quite small, mostly ~1 mm3. To our
knowledge, this is the first published report of maintaining in vitro
for up to 48 h small intestinal sections large enough to mount in
Ussing chambers for measurements of transepithelial electrical
properties (microchambers for biopsies would enable use of smaller
mucosal sections, but these chambers are very difficult to use). We
believe that a key element in the prolonged survival of these tissues
in vitro was the stretch imposed in pinning opened intestine onto
frames. Through stretch, better oxygenation of intervillus spaces and
crypts was likely provided. Stretch has also been shown to increase
proliferation of fetal rat lung epithelial cells (21) and
to activate immediate early genes such as c-fos, c-jun, c-myc, JE, and EGR-1 in cardiac muscle
cells (36). These immediate early genes, if activated in
the intestine by stretch, could act as promoters of enterocyte proliferation.
The reasons for the development of villus atrophy during prolonged in
vitro incubation remain to be established. Of interest is a report by
MacDonald and Spencer (23) showing that human fetal small
intestine in organ culture develops villus atrophy and crypt
hypertrophy when stimulated with T cell activators (PWM or anti-CD3
antibody) and that these changes could be prevented by addition of
cyclosporin A, which prevents T cell activation. Some T cell activation
may have occurred spontaneously in our culture system. In future
experiments, we will explore the effect of cyclosporin A and the role
of Th1 cell cytokines on the villus atrophy that we have observed.
Effects of IFN-
.
Incubation with IFN-
for 24-48 h downregulated short-circuit
responses to both glucose and theophylline and decreased basal Isc, producing these effects without
significantly altering tissue resistance, morphology, or K+
content. The time course and concentration dependence of these effects
were similar to those previously observed for T84 cells (6). The inhibitory effect of IFN-
was seen in SCID as
well as in CD-1 mice, suggesting that its intestinal epithelial effects were not indirectly exerted via a primary effect on T or B lymphocytes.
IFN-
has been shown previously to downregulate ion transport or,
more specifically, active Cl
secretion in the colon
cancer cell lines T84 (3, 6) and HT-29 (3)
and in a cystic fibrosis transmembrane conductance regulator (CFTR)
gene rescued pancreatic cancer cell line derived from a patient with
cystic fibrosis (CF-PAC) (22). Downregulation of the
apical Cl
channel, CFTR, has been demonstrated at
functional, mRNA, and protein levels (3, 6, 22).
Downregulation of the Na+-K+-2Cl
cotransporter and Na+-K+-ATPase has also been
shown (6). The present study, in addition to demonstrating
the previously observed inhibition of active Cl
secretion, extends the transport effects of IFN-
to include Na+-coupled sugar absorption. Thus IFN-
interferes with
transport functions in both villus and crypt cells.
In T84 cells, IFN-
decreases transmonolayer resistance and increases
permeability to mannitol and inulin (6, 24). This permeability effect has been shown in HT-29 cells to extend to horseradish peroxidase (42). In our study, an effect of
IFN-
on monolayer resistance was not seen, but changes in tight
junctional properties, which appear to be responsible for the drop in
T84 cell monolayer resistance, may not have a measurable impact on overall resistance in small intestine, which has a baseline resistance 40-fold lower. The very high conductance of cultured murine small intestine is likely dominated by cell desquamation, which may leave
holes of several micrometers in diameter.
IFN-
has been shown in T84 cells (6), IEC-6 cells
(35), and biopsies of human colon (14) to
induce MHC class II expression and, in T84 cells, to enhance
2-integrin-dependent neutrophil adhesion
(6). Colgan et al. (6) suggested that IFN-
causes a global phenotypic switch in intestinal epithelial function, enterocytes becoming immune accessory cells. The decrease in
barrier function, by contributing to presentation of luminal antigens to immune cells, may be part of this overall phenotypic switch. To
explore this concept further in murine small intestine, the effects of
IFN-
on luminal uptake of macromolecules and on the prevalence of
proteins involved in the regulation of tight junction permeability,
such as ZO-1 (44), will need to be examined.
In murine small intestine, IFN-
not only decreases the
Isc responses to glucose and theophylline but
also the basal Isc. In large part, this basal
current is due to spontaneous secretion of Cl
and
HCO3
. This has been shown in a study
(12) of jejunal ion transport in cystic fibrosis gene
knockout mice. Small intestines from these mice, which lack a
functional apical membrane anion channel (CFTR), exhibit almost no
basal Isc. The IFN-
-induced decrease in the basal Isc of small intestine from CD-1 mice may
therefore reflect its overall inhibition of anion secretion and is
consistent with its reported downregulation of CFTR in other systems
(3, 6). Ileal HCO3
secretion, like
Cl
secretion, is electrogenic (27) and
probably mediated by CFTR (12). It is interesting to note
that the basal Isc in SCID mice was lower than
in CD-1 mice and was not significantly decreased by pretreatment with
IFN-
. The lower basal or spontaneous secretion in intestine from
SCID mice suggests a role for T or B lymphocytes in the regulation of
basal secretion.
The observed inhibitions of ion transport by IFN-
were not the
result of IFN-
-induced epithelial damage or death since no significant differences in either morphology or epithelial
K+ content between IFN-
-treated (100 U/ml for 36 h)
and control intestine were found. However, both villus height and
epithelial K+ content were, on the average, 15% lower in
IFN-
-treated tissues than in control tissues, suggesting a modest
effect of IFN-
on epithelial structure and viability. Perhaps a
larger number of experiments would confirm this possibility. We have
not yet directly measured apoptosis in our preparation, but IFN-
(29) has been shown to induce apoptosis in several cell
lines/tissues, including HT-29 cells and normal human colon. Other
studies suggest, however, that it does not induce apoptosis by itself
but only in conjunction with other factors (8).
Possible changes in microvillus membrane or apical cytoskeletal
properties could be another cause for the inhibition of transport by
IFN-
. The effects of IFN-
on the appearance of the microvillus membrane on electron microscopy, the density of a few characteristic microvillus membrane proteins, and the organization of actin and other
cytoskeletal proteins in the apical region of the enterocyte remain to
be examined. IFN-
has been shown to disrupt actin organization in
the apical cytoplasm of the T84 cell (44).
Examination of all intestinal ion transport processes downregulated by
IFN-
is beyond the scope of the present study. To gain some insight,
however, we determined the effect of the cytokine on
Na+-K+-ATPase activity, because active
Na+ transport is required for Cl
secretion as
well as for Na+-absorptive processes. We found 60%
inhibition (Table 6) in a 36-h incubation with 100 U/ml of IFN-
. In
comparison, this concentration of IFN-
for this time period
inhibited glucose- and theophylline-stimulated Isc responses by closer to 70%, but this
difference is not significant. It is worth noting that neither glucose
nor theophylline alone or in combination maximally stimulate
Na+ pump activity (the Isc changes
these two agents induce arise from different cells). For example, in
control tissues, the addition of alanine subsequent to glucose causes
an additional large increase in Isc (data not
shown). Because Na+-K+-ATPase activity is not
rate limiting for individual processes in murine small intestine,
IFN-
very likely causes independent downregulation of additional
transport proteins. In colon cancer cell lines, CFTR,
Na+-K+-2Cl
cotransport, NHE2, and
NHE3 have all been shown to be downregulated by IFN-
(3, 6, 33, 37a), but these effects could be secondary to a primary effect of
IFN-
on Na+-K+-ATPase (close to 100%
inhibition); ouabain, for example, downregulates at least some of these
proteins (37a). In the rat, NHE2 and NHE3 have been shown at both
protein and mRNA levels to also be downregulated by IFN-
(33).
Sundaram and co-workers (38-41) have shown in a
rabbit chronic intestinal inflammation model that the activities of a
variety of transporters, including Na+-glucose cotransport,
are altered, usually downward, although constitutive Na+/H
exchange in crypt cells was found to be enhanced. In contrast to most
of these findings (38-41),
Na+-K+-ATPase activity was only slightly
inhibited. The inflammation caused was most likely Th1 in type, meaning
that there was enhanced production of IFN-
, but other cytokines were
also generated, and it is not clear that the several transport changes
documented (38-41) were due to IFN-
alone.
The transport effects of IFN-
described in the present study are
unlikely to be mediated by NO since L-NAME did not prevent the effect of IFN-
on Isc responses to
glucose and theophylline. Whether NO mediates the IFN-
-induced
inhibition of Na+-K+- ATPase activity in
small intestine, as noted in proximal tubule cells (13),
is a separate issue, untested in the present study.
The genes activated by IFN-
that are involved in the downregulation
of transport proteins such as CFTR,
Na+-K+-ATPase, and perhaps also the
Na+-glucose cotransporter remain to be established. In
several cell lines, including HT-29 cells, IFN-
has been shown, via
activation of STAT-1, to induce the cyclin-dependent kinase inhibitor
p21, with a resulting inhibition of cell proliferation
(5). In addition to its effect on cell cycle control, p21
has also been shown in mouse keratinocytes to inhibit terminal
differentiation of these cells (7). In intestine, p21 may
play a similar role because it is highly expressed in postmitotic cells
adjacent to the proliferative compartment but less so at later stages
of differentiation (11). Na+-glucose
cotransport function, which has been shown to localize to the villus
tip region in rabbit ileum (9), is certainly representative of terminally differentiated villus enterocytes. It is
less clear if Cl
-secreting cells, most of which appear to
reside in crypts, can be considered to be terminally differentiated.
Some members of the crypt cell population (e.g., Paneth cells)
are not progenitors of villus cells and may be terminally
differentiated. With light microscopy, evidence of IFN-
-induced
dedifferentiation was not apparent, but this is a relatively crude
measure of cell differentiation. It is of interest, in this regard,
that Kerneis et al. (17) showed that coculture of Peyer's
patch-derived lymphocytes with Caco-2 cells (another colon cancer cell
line) results in the acquisition by the Caco-2 cells of M-cell-like
properties. At least some of this effect was not due to direct contact
between lymphocytes and enterocytes but to a soluble factor released by
the lymphocytes, possibly IFN-
. Induction of p21 synthesis by
IFN-
could play a role in this transformation.
In conclusion, sheets of murine small intestine suitable for mounting
in Ussing chambers can be maintained in vitro for 36-48 h
without decreases in Na+-dependent glucose absorption or
active Cl
secretion. Despite maintenance of glucose
absorption, a 50% diminution of villus height was observed in small
intestine from normal mice (much less so in SCID mouse intestine),
indicating a lack of close correlation between abundance of
Na+-glucose cotransporters and villus surface area and
suggesting a role for T or B lymphocytes in the observed partial villus
atrophy. IFN-
downregulates Na+-dependent glucose
absorption, active anion secretion, and
Na+-K+-ATPase activity without significantly
altering epithelial cell viability as determined by mucosal histology
and K+ content. The transport effects of IFN-
were not
reversed by an inducible NOS inhibitor, suggesting that these effects
were not mediated by NO. The effect of IFN-
on tissue resistance
previously noted in T84 colon cancer cell monolayers could not be
demonstrated in murine small intestine perhaps because of its low
baseline resistance.
 |
ACKNOWLEDGEMENTS |
We thank Professor Joseph Graziano and Vesna Slavkovich for
assaying K+ in our tissue samples.
 |
FOOTNOTES |
This study was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Postdoctoral Training Grant DK-07715 (W. Lo ) and in part by a gift from Dr. and Mrs. Bernard German in memory
of Mrs. German's brother, Daniel V. Kimberg, M.D., former Chairman of
the Department of Medicine at Columbia-Presbyterian Medical Center (W. Lo).
Address for correspondence: M. Field, PS10-508, CPMC, 630 W. 168th St., New York, NY 10032 (E-mail: mf9{at}columbia.edu).
Reprints will not be provided.
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 12 October 1999; accepted in final form 12 June 2000.
 |
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