(Received for publication, September 22, 1995; and in revised form, November 11, 1995)
From the
Using metabolic labeling techniques in human intestinal
epithelial cell lines in tissue culture and in situ hybridization techniques in normal and inflamed (Crohn's)
intestine, recent studies have shown that there is synthesis of acute
phase proteins in enterocytes. Moreover, these studies have shown that
acute phase protein biosynthesis in enterocytes is regulated by
inflammatory cytokines in a manner characteristic of the physiologic
acute phase response. In the course of these studies it was noticed
that one inflammatory cytokine, interleukin-6 (IL-6), mediated
selective down-regulation of the enterocyte-specific,
differentiation-dependent integral membrane protein sucrase-isomaltase
(SI) in the Caco2 intestinal epithelial cell line. In the current study
we examined the effect of several other inflammatory cytokines
interleukin-1 (IL-1), tumor necrosis factor
(TNF
), and
interferon
(IFN
) on synthesis of SI in Caco2 cells, examined
the possibility that inflammatory cytokines affect the synthesis of
other enterocyte integral membrane proteins using lactase as a
prototype, and examined the possibility that SI gene expression was
down-regulated in villous enterocytes in vivo during the local
inflammatory response of Crohn's disease. The results show that
IL-6 and IFN
each mediate a decrease and TNF
mediates an
increase in synthesis of SI in Caco2 cells. The magnitude of
down-regulation by IL-6 and IFN
is significantly greater than the
up-regulation by TNF
. IL-1
has no effect on synthesis of SI.
Synthesis of lactase is not affected by any of the cytokines. There is
a marked specific decrease in SI gene expression in villous enterocytes
in acutely inflamed Crohn's ileum as compared to adjacent
uninflamed ileum and normal ileum. Taken together, these data show that
inflammatory cytokines have specific and selective effects on the
expression of the brush border hydrolase SI in tissue culture and in vivo and provide evidence for a previously unrecognized
mechanism for disaccharidase deficiency in intestinal inflammation.
The host response to injury, the acute phase response, is
characterized by a coordinate series of physiological changes,
including marked changes in plasma concentrations of secretory
glycoproteins termed ``acute phase reactants'' (reviewed in (1, 2, 3) ). In recent years several
extracellular soluble factors thought to be generated at the site of
injury and able to circulate via the bloodstream to remote sites have
been identified and implicated in coordination of the acute phase
response. The most well characterized of these cytokines include IL-6, ()IL-1
, IL-1
, TNF-
, and IFN-
. These
cytokines have been shown to be capable of mediating in tissue culture
and in vivo many of the changes in hepatic gene expression
that characterize the acute phase response.
Although the acute phase
reactants are predominantly derived from liver, there is now evidence
that acute phase reactants are produced in extrahepatic tissues and
cell
types(4, 5, 6, 7, 8, 9) .
Moreover, there is evidence that expression of these gene products in
extrahepatic sites is regulated by cytokines in a manner characteristic
of the physiologic acute phase response. For instance, we have found
that positive acute phase reactant 1-antitrypsin (
1-AT) is
expressed in human intestinal epithelial cell lines Caco2 and T84 (10) and, moreover, that IL-6 mediates an increase in synthesis
of
1-AT in these cell lines (11) . Caco2 and T84 cells
were found to synthesize many other positive and negative acute phase
reactants including
1-antichymotrypsin, serum amyloid A (SAA),
fibrinogen, ceruloplasmin, complement proteins C3, C4, and factor B,
-fetoprotein, and transferrin(12) . Expression of all
these acute phase reactants was regulated by cytokines IL-6 and
IL-1
in exactly the same way that they are regulated in liver
cells. In fact, effects of IL-1 and IL-6, which were found to be
additive, synergistic, and antagonistic in liver cells in previous
studies, were also observed in the intestinal epithelial cell lines.
Finally, for at least one of these proteins,
1-AT, in situ hybridization analysis of normal and inflamed human intestine has
shown that the expression and regulation of the acute phase gene in the
cell lines accurately recapitulates expression and regulation of this
gene in vivo(13) .
During the course of these
studies of the acute phase response in the Caco2 cell
line(12) , we found that IL-6 had a marked and selective
down-regulating effect on the expression of sucrose-isomaltase (SI). SI
is not an acute phase reactant, but it is an enterocyte-specific,
differentiation-dependent integral membrane protein. It is expressed in
the adult small intestine where, as a brush border hydrolase, it is
involved in the final steps of digestion of sucrose, starch and
glycogen. Its expression is limited to the villous
enterocyte(14, 15, 16) . In fact, together
with lactase and intestinal alkaline phosphatase it is often used as a
marker of terminal differentiation of enterocytes. SI is synthesized as
a co-translationally glycosylated high mannose precursor with a
molecular mass of 210 kDa(17) . This precursor is also
subject to O-glycosylation in the Golgi and is finally
targeted to the apical membrane. After SI has reached the apical
membrane as a single-chain glycoprotein, it undergoes post-insertional
cleavage by intraluminal pancreatic trypsin into the sucrase (molecular
mass
130 kDa) and isomaltase (molecular mass
145 kDa)
subunits(18, 19) .
In this study we examined the possibility that inflammatory cytokines other than IL-6 mediate changes in expression of the SI gene in the model enterocyte cell line Caco2, whether inflammatory cytokines regulate expression of other brush border hydrolases in Caco2 cells, and whether SI gene expression is regulated in a local site of inflammation, acutely inflamed Crohn's ileum.
Figure 1:
Effect of inflammatory cytokines on
synthesis of SI, factor B (Bf), C3, and C4 in Caco2 cells.
Separate monolayers of Caco2 cells were incubated at 37 °C for 24 h
in control serum-free medium alone (lane 1) or medium
supplemented with 50 ng/ml IL-6 (lane 2), 25 ng/ml IL-1 (lane 3), 50 ng/ml IL-6 and 25 ng/ml IL-1
(lane
4), or 1,000 units/ml IFN
(lane 5). Cells were then
pulse-labeled at 37 °C for 30 min with
[
S]methionine, 250 µCi/ml, Dulbecco's
modified Eagle's medium lacking methionine. Cells were then
homogenized and cell lysates prepared for sequential
immunoprecipitation with antibodies to SI, factor B, C3, and C4 as
indicated at the top of each panel. Immunoprecipitates were analyzed by
SDS-PAGE/fluorography as described under ``Experimental
Procedures.'' Molecular mass markers are shown at right of each panel.
Next
we examined the possibility that regulation of SI biosynthesis by
inflammatory cytokines was concentration-dependent. In Fig. 2,
the results show that IL-6 and IFN-, indeed, mediate
concentration-dependent decreases in SI biosynthesis (Fig. 2, upper panels). An effect was evident at concentrations of IL-6
as low as 10 ng/ml and of IFN
as low as 200 units/ml. These
concentration-dependent effects were specific as shown by sequential
immunoprecipitation for C3. There is a concentration-dependent increase
in C3 mediated by IFN
(Fig. 2, lower panels). IL-6
has minimal effects on synthesis of C3.
Figure 2:
Effect of several different concentrations
of IL-6 and IFN on synthesis of SI (top panels) and C3 (bottom panels) in Caco2 cells. The protocol was identical to
the protocol described in the legend for Fig. 1. Lane
1, control serum-free medium; lane 2, 10 ng/ml IL-6; lane 3, 50 ng/ml IL-6; lane 4, 100 ng/ml IL-6; lane 5, 200 units/ml IFN
; lane 6, 1000 units/ml
IFN
; lane 7, 5000 units/ml IFN
. In this particular
experiment, there was a greater decrease in SI after treatment with 200
units/ml IFN
than after 100 units/ml or 5000 units/ml IFN
.
This is probably due to loading because there was a lesser decrease in
SI after treatment with 200 units/ml IFN
than after 1000 or 5000
units/ml in three other experiments (data not
shown).
We also examined the effect
of TNF on synthesis of SI (Fig. 3, left panel). In
contrast to IL-6 and IFN
, TNF
mediated a modest
concentration-dependent increase in SI synthesis in Caco2 cells. The
effect was evident at concentrations of TNF
as low as 10 ng/ml.
The magnitude of this increase was less than that mediated by TNF
on synthesis of C3 (Fig. 3, right panel) and less than
the magnitude of the decrease in SI biosynthesis mediated by IL-6 and
IFN
. Quantitative analysis by densitometric scanning of five
separate experiments shows that the optimal effect of IL-6 on SI
biosynthesis resulted in a decrease to 37.4 ± 19.0% of control,
of IFN
resulted in a decrease to 51.3 ± 22.1% of control,
and of TNF
resulted in an increase to 170.6 ± 21.0% of
control.
Figure 3:
Effect of TNF on synthesis of SI (left panel) and C3 (right panel) in Caco2 cells. The
protocol was identical to the protocol described in the legend for Fig. 1. Lane 1, control serum-free medium; lane
2, 10 ng/ml TNF
; lane 3, 50 ng/ml TNF
; lane
4, 100 ng/ml TNF
.
The effects of IL-6, IL-1, IFN
, and TNF
on
SI could not be attributed to general effects on cell number or
metabolic activity because there was no significant effect on DNA
content or on total trichloroacetic acid-precipitable protein
determinations in these experiments (data not shown). Moreover, changes
in cell number or metabolic activity would not explain the increase in
synthesis of some proteins, decrease in others, and no change in still
others on sequential immunoprecipitation. Finally, examination of a
subclone of Caco2 cells expressing high levels of SI under basal
conditions, Caco2/15 cells, gave identical results (data not shown).
Next we examined the effect of cytokines on synthesis of another
enterocyte-specific integral membrane disaccharidase, lactase (Fig. 4). In the same experiments in which selective and
specific changes in synthesis of SI and complement proteins are noted,
there was no significant change in the synthesis of lactase. There was
also no change in the synthesis of another enterocyte integral membrane
protein, alkaline phosphatase (data not shown). At least with respect
to inflammatory cytokines in this model system, there appears to be
discordant regulation of the two disaccharidases and the three
enterocyte membrane proteins. The results of the experiments with
lactase and alkaline phosphatase, which are considered markers of
terminal differentiation in enterocytes and Caco2 cells, make it
unlikely that the down-regulating effect of IL-6 and IFN on SI are
due to a general effect of these cytokines on the state of
differentiation of Caco2 cells.
Figure 4:
Effect of inflammatory cytokines on
biosynthesis of lactase in Caco2 cells. The protocol was identical to
that described in the legend for Fig. 1. Lane 1,
control serum-free medium; lane 2, 5 ng/ml IL-1; lane
3, 25 ng/ml IL-1
; lane 4, 10 ng/ml IL-6; lane
5, 50 ng/ml IL-6; lane 6, 1000 units/ml IFN
; lane 7, 5000 units/ml IFN
; lane 8, 50 ng/ml
TNF
; lane 9, 100 ng/ml TNF
. These results are
representative of three separate experiments. Densitometric scanning of
these three experiments did not reveal any significant changes in
synthesis of lactase.
Figure 5: RNase protection assays for SI (left panel) and SAA (right panel) mRNA levels in normal jejunum, normal ileum, acutely inflamed Crohn's ileum, and adjacent uninflamed Crohn's ileum. The migration of the probe is indicated at the left margin in each panel. RNA from Caco2 cells and IL-6-activated Caco2 cells is shown for comparison in the right panel. For each lane, 10 µg of RNA was loaded. The same preparations of RNA were used for both panels. The migration of the labeled probe is shown at the left margin in each case. The migration of undigested probe is indicated by open arrowheads at the right margin. The specific protected RNA fragment is indicated by a solid arrowhead at the right margin of each panel.
We subjected
cryostat sections from the same tissues to immunostaining for SI to
determine whether the decrease was enterocyte-specific (Fig. 6a). The results show that anti-SI antibody
stains the cell surface of villous enterocytes from normal ileum. There
is a patchy decrease in this staining in uninflamed Crohn's
ileum, but a marked decrease, almost complete absence, of SI
immunostaining in acutely inflamed ileum. Results were identical when
the other normal donor and the other Crohn's donor were examined
(data not shown). When adjacent sections of the same tissue are
subjected to immunostaining with positive acute phase reactant
1-AT (Fig. 6b), there is a patchy increase in
uninflamed Crohn's ileum and a marked increase in acutely
inflamed Crohn's ileum. The staining of enterocytes with
1-AT is cytoplasmic and extends into the crypt as compared to SI,
which is membranous and confined to the villus. These results of
1-AT immunostaining are identical to our previous results in
tissues from 2 other normal individuals and 3 other individuals with
Crohn's disease(13) .
Figure 6:
Immunostaining for SI (a) and
1-AT (b) in normal ileum, acutely inflamed Crohn's
ileum, and adjacent uninflamed Crohn's ileum. For a, the
immunogold silver enhancement staining technique was used. The top
panels show light microscopy, and bottom panels show
epipolarizing microscopy. For b, immunofluorescence was used.
The top panels show polarized microscopy, and the bottom
panels show fluorescence microscopy. Magnification,
125.
Finally, we subjected cryostat
sections from the same tissues to in situ hybridization
analysis for even more sensitive evaluation of cell-specific SI gene
expression (Fig. 7a). Sections from normal ileum and
from acutely inflamed ileum and adjacent uninflamed ileum from patients
with Crohn's disease were again examined. To make the analysis
strictly comparable, these sections were hybridized together with the
same prep of S-labeled antisense cRNA and sense RNA
probes, washed together, subjected to autoradiography together for the
same length of time, and photographed together. The results show silver
grains within enterocytes lining the villi of normal ileum with
antisense SI cRNA. There were no silver grains in the villous
enterocytes with sense SI probe. Refractile bodies (arrowheads) could be seen over the villous epithelium and
lamina propria with antisense cRNA or sense RNA, or when sections were
hybridized without any probe; thus, these are nonspecific by
definition. There was a patchy decrease in silver grains in uninflamed
Crohn's ileum but a marked and diffuse decrease in silver grains
in the villous enterocytes of acutely inflamed Crohn's ileum. The
difference in density of silver grains between normal ileum and
Crohn's ileum was also confirmed by quantitation of the number of
silver grains per high power field. For this analysis silver grains per
high power field were counted on the base, lower third, middle third,
and upper third of 8 different villi from the normal ileum, on 7
different villi from inflamed ileum, and on 8 different villi from
uninflamed adjacent ileum from 2 normal and 2 Crohn's donors. The
results showed counts of 168 ± 33.6 for normal, 32.0 ±
11.0 for inflamed ileum specimen, and 137.0 ± 37.09 for
uninflamed ileum. The difference between normal and inflamed ileum was
statistically significant (p < 0.001 by ANOVA and by
Tukey's test of multiple comparisons). The difference can also be
seen on multiple villi when the sections are shown at lower
magnification (Fig. 7b). The decrease in
enterocyte-specific SI mRNA in inflamed ileum was specific in that
there was an increase in enterocyte-specific
1-AT mRNA in the same
specimens (data not shown) and in 3 other individuals with
Crohn's disease in our previous studies(13) . Taken
together, the results of ribonuclease protection assays,
immunostaining, and in situ hybridization analysis provide
internally consistent evidence for selective and specific decrease in
SI gene expression in acutely inflamed Crohn's ileum. The results
of immunostaining and in situ hybridization analysis show that
there is a decrease in cell surface SI expression and SI mRNA per
enterocyte in the inflamed Crohn's ileum.
Figure 7:
In situ hybridization for
analysis of cellular expression of SI in normal ileum, acutely inflamed
Crohn's ileum, and adjacent uninflamed Crohn's ileum. a, higher magnification (250); b, lower
magnification (
125). Cryostat sections were hybridized together
with the same prep of
S-labeled antisense SI cRNA probe
and were examined under dark-field microscopy. The lumen is located
toward the top of the panels, and the serosa is located toward
the bottom. Adjacent cryostat sections from the normal ileum
were also hybridized with
S-labeled sense RNA SI probe and
were examined under dark-field microscopy. Open arrows point
to cells in the lamina propria that have refractile properties and are,
therefore, not sites of specific
hybridization.
The results of this study show that inflammatory cytokines
mediate changes in expression of the SI gene in intestinal epithelial
cells. IL-6 and IFN mediate concentration-dependent decreases, and
TNF
mediates a concentration-dependent increase in the
biosynthesis of SI in the human intestinal epithelial cell line Caco2.
On the other hand, IL-1
has no effect on synthesis of SI in Caco2
cells. The effects of each cytokine on SI synthesis are specific and
selective in that they occur in the context of marked changes in
expression of other target genes, particularly acute phase genes. This
includes cytokine-specific and target gene-specific changes. IL-6
mediates a decrease in SI synthesis at the same time as it mediates an
increase in complement proteins factor B, C3, and
1-AT. IL-1
has no effect on synthesis of SI while it mediates an increase in
factor B and C3 synthesis. IL-1
also has no effect on synthesis of
SI when it is used together with IL-6, even though it has an additive
effect on factor B and a synergistic effect on SAA in the same
experiment. IFN
mediates a decrease in SI synthesis at the same
time as it mediates an increase in factor B and C4 synthesis. In
contrast, TNF
mediates an increase in synthesis of SI and C3, but
has little effect on the other genes. The decrease in SI biosynthesis
in Caco2 cells activated by IL-6 or by IFN
could not be attributed
to a general effect on cell number or metabolic activity per cell or to
heterogeneity within the cell line because sequential
immunoprecipitation of the same labeled cell lysates showed an increase
in factor B, C3, and
1-AT in the case of IL-6 and an increase in
C4 in the case of IFN
. Similarly the increase in biosynthesis of
SI in Caco2 cells treated with TNF
could not be attributed to a
general effect or heterogeneity because sequential immunoprecipitation
showed no change in synthesis of
1-antichymotrypsin and
1-AT.
The changes in SI synthesis are also unlikely to reflect a general
effect on the state of differentiation of Caco2 cells because on
sequential immunoprecipitation there were no changes in synthesis of
two other enterocyte integral membrane proteins that are markers of
terminal differentiation in Caco2 cells, lactase and alkaline
phosphatase.
In order to examine the possibility that the effects of
inflammatory cytokines on SI gene expression in enterocytes are
relevant to physiologic and pathophysiologic situations in
vivo, we used RNase protection assays and immunofluorescent and in situ hybridization analyses to determine SI gene expression
in inflamed ileum from patients with Crohn's disease. Previous
studies have shown that there is increased complement proteins in
intestinal loop fluid from patients with Crohn's disease (29) and increased 1-AT gene expression in enterocytes
from acutely inflamed Crohn's ileum(13) . The results of
our studies showed that there is a marked decrease in expression of the
SI gene in villous enterocytes of acutely inflamed Crohn's ileum.
The decrease could not be attributed to generalized damage to the
enterocytes in that there was evidence for substantial increases in SAA
and
1-AT gene expression in the same epithelial cells using
analysis of cell-specific expression by immunostaining and in situ hybridization. Taken together, these results indicate that there
is a specific and selective decrease in SI gene expression in villous
enterocytes in one type of intestinal inflammatory condition and that
the specificity and selectivity of the effect recapitulates the changes
in gene expression elicited in a human intestinal epithelial cell line
in tissue culture in response to IL-6 or IFN
. The data do not
prove that IL-6 and/or IFN-
are responsible for the decrease in SI
gene expression in vivo. Moreover, the data do not address
whether locally- or systemically-derived cytokines are responsible.
However, the fact that there is a gradient in the changes in gene
expression when comparing the acutely inflamed to the adjacent
uninflamed Crohn's ileum is more consistent with an effect of
locally-derived cytokine, or cytokines. Expression of all the cytokines
studied here, IL-6, IL-1
, TNF
, and IFN
, has been
detected in the bowel in inflammatory bowel disease(30) .
Finally, the data do not necessarily imply that TNF
is absent
from, or not acting on, villous enterocytes in acutely inflamed
Crohn's ileum. The net effect on SI gene expression here could
depend on many factors, including the relative endogenous
expression/activity of receptors and soluble receptors/receptor
antagonists.
SI is an integral membrane glycoprotein of enterocytes,
which digests dietary disaccharides. Its expression is confined to the
small intestine in the adult. Its expression is altered by changes in
dietary nutrients and by diabetes mellitus. It is also expressed
aberrantly in colonic polyps, adenomas and adenocarcinomas, probably as
a result of dedifferentiation to a fetal colonocyte program in these
conditions. Several cis-acting DNA binding domains in the upstream
flanking region of the SI gene and trans-acting DNA binding proteins
that regulate its intestine-specific transcription have been identified
in recent studies (26, 31, 32) . However,
cis-acting elements and transcription factors, which could mediate
down-regulation of SI gene expression in response to IL-6 and IFN
or up-regulation of SI gene expression in response to TNF
, have
not been described. There is, however, no a priori reason that
these cytokines directly act on SI gene expression. Cross and Quaroni (24) have shown that epidermal growth factor mediates a marked
decrease in SI gene expression in Caco2 cells, raising the possibility
that the effects of cytokines could be mediated indirectly through
epidermal growth factor, transforming growth factor
, or, for that
matter, through other related growth factors.
The results of this
study do not provide any information about the clinical relevance of
depression in SI gene expression in inflammatory bowel disease. It is
known that lactose malabsorption is as common in inflammatory bowel
disease as it is in the general population(33) , but there is
relatively little information in the literature about sucrose
malabsorption. Nevertheless, the results do raise the possibility that
cytokines can mediate an effect that inhibits SI gene expression in
Crohn's disease, in infectious enteritis, or in perturbations of
the intestinal flora that result in the local release of cytokines such
as IL-6 and IFN.