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Division of Clinical Immunology, Mount Sinai Medical Center, New York, NY 10029; and
Department of Medicine, University of California at San Diego, Lo Jolla, CA 92093
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Receptors for
IL-1ß, IL-6, and GM-CSF were readily detectable in all epithelial
cell preparations at levels equal to (GM-CSFR) or lower than those seen
on monocytes. However TNF-R were not detectable on freshly isolated
IECs. Receptor density was greater in surface vs crypt epithelial
cells, but no significant differences were seen between normal and IBD
epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and
IFN-
. Functionally, IL-1ß enhanced proliferation of the IEC cell
line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like
DLD1 and HT29 cells resulted in enhanced expression of ICAM-1.
Furthermore, TNF- treatment enhanced the secretion of IL-8 and
GRO- in HT29 cells, but not in freshly isolated IEC cultures.
The differential binding and function of proinflammatory cytokines on
IEC support the hypothesis that these cytokines may be involved in
normal physiological processes as well as in regulating mucosal immune
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have been
reported to be increased in serum (11), cultures of biopsy tissues
(12, 13, 14), or isolated lamina propria lymphocytes (15, 16, 17, 18, 19) from
inflammatory bowel disease (IBD) patients. There is increasing evidence
that intestinal epithelial cells (IEC) may serve as targets for these
locally produced cytokines. In this scenario, local cytokines may
result in alterations of epithelial cell function or integrity. For
example, the combination of TNF- and IFN- is cytotoxic for the
colonic epithelial cell line, HT29 (20), and IL-1ß up-regulates IL-6
synthesis (a proinflammatory cytokine) by epithelial cells (21).
Alternatively or additionally, cytokines may play a role in the normal
regulatory mechanisms involved in epithelial cell growth and
differentiation ensuring maintenance of homeostasis. An imbalance of
the normal cytokine milieu seen in IBD might lead to the disruption of
epithelial function. There is evidence for selective effects of various
cytokines on IEC growth and differentiation, phenotype, and function
through an autocrine/juxtacrine or paracrine pathway. One hypothetical
model based on these scenarios might be that cytokine-mediated changes
in the epithelial cell integrity might initiate pathologic events in
the gut. The ability of cytokines to function in this manner depends on
the presence of specific receptors expressed on IECs.
Therefore, we analyzed the binding of the proinflammatory cytokines
IL-1ß, IL-6, GM-CSF, and TNF- on both freshly isolated intestinal
epithelial cells and cell lines by flow cytometric analysis. Our
results show that IECs from both IBD and normal control samples express
receptors for GM-CSF, IL-1ß, and IL-6 at comparable levels. TNF-R
were not detected on freshly isolated epithelial cells, although they
were present on colonic adenocarcinoma cell lines. Functionally, IL-1
enhanced while GM-CSF inhibited IEC growth. Furthermore, GM-CSF could
induce or up-regulate ICAM-1 expression preferentially on crypt-like
epithelial cells. Taken together, these results support the potential
for an autocrine or paracrine effect of proinflammatory and
prophagocytic cytokines as potential modulators of epithelial cell
integrity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epithelial cells from resected bowel specimens were isolated by
dispase or EDTA (TNF-R) treatment as previously described (22, 23).
Surgical specimens included those from "normal" controls, i.e.,
patients with adenocarcinoma (10 cm from tumor), colonic inertia, and
familial polyposis, as well as those from patients with active and
inactive UC and CD. In the latter cases, tissues were obtained from
both areas of active inflammation and areas where no histologic
inflammation was noted (where available). Briefly, after vigorous
washing in PBS, the mucosal layer was dissected from the underlying
submucosa. The dissected mucosal tissue was washed several times in
HBSS supplemented with 1% penicillin-streptomycin, amphotericin, and
gentamicin (50 µg/ml), minced into small pieces, and followed by
treatment with the mucolytic agent DTT (1 mM) (Sigma, St. Louis, MO) in
RPMI 1640 medium for 5 min at room temperature. Tissue pieces were
washed in PBS and subjected to dispase (1 mg/ml; Boehringer Mannheim,
Indianapolis, IN) or EDTA (0.75 mM; Sigma) treatment for 30 min at
37°C. During this 30-min incubation, tissue pieces were agitated
every 5 min by vortex mixing, resulting in the liberation of epithelial
cells and intraepithelial lymphocytes into the solution. This cell
suspension was collected and immediately diluted 10-fold in PBS to
minimize any effect of dispase on cytokine receptors (see below). Cells
were spun down and washed twice in PBS. Epithelial cells were further
separated from the contaminating intraepithelial lymphocytes and RBCs
by percoll density gradient centrifugation (23). The cells on top of
the 30% layer contained 95% pure epithelial cells (<1%
CD45+ cells or CD14+ cells) with a viability of
95 to 100%. There was no difference in the viability of normal IECs vs
those obtained from patients with IBD.
For the purpose of obtaining crypt epithelial cells, minced tissue pieces were incubated with dispase for up to five treatments (5 min for the first treatment, 30 min for the second and third treatments, and then 40 min each for the next two treatments). At each interval, sample tissue (for histologic examination to detect the disappearance of surface epithelium and subsequent crypt cell liberation) and cell suspensions were collected. Epithelial cells were separated by percoll density gradient, and purity of IECs was >95% (as evaluated by staining with anti-CD3, CD14, CD20, CD45 mAbs) and L12 (anti-epithelial cell) Abs. Hematoxylin and eosin staining of histologic sections of the treated tissue samples demonstrated that the surface epithelium was completely removed by the first three dispase treatments with maintenance of the crypt epithelium. After the fifth treatment histologic sections revealed the absence of crypt epithelium. The IEC from the fourth and fifth dispase treatments were used as a source of crypt cells whereas the IEC from the first and second dispase treatments were used as the source of surface epithelium. Purity of the surface or crypt cell populations in the cell suspension from each treatment was determined by morphology (by phase contrast microscopy and histochemical staining), measurement of alkaline phosphatase (as a measure of surface epithelium) (24, 25), and [3H]thymidine uptake incorporation (as a measure of the crypt cell population) for 4, 16, and 24 h. Consistent with the findings in the histologic sections, alkaline phosphatase was detectable only in the lysates from the cells isolated in the first three dispase treatments, suggesting that these fractions contained an enriched population of surface epithelial cells. [3H]Thymidine uptake incorporation was minimal in these cells, whereas the cells liberated in the fourth and fifth dispase treatments were able to proliferate with a 4- to 5-fold increase in thymidine uptake seen at 24 h. These data suggest that the latter fractions are enriched for crypt epithelial cells. No alkaline phosphatase activity was noted in lysates of cells from the fourth and fifth dispase treatments. In some experiments intact crypts were liberated during the treatment, and these could be visualized by conventional light microscopy. Such crypts were teased apart by incubation with 0.15 mol/liter NaCl3 mmol/liter tetraphenyl borate (26, 27) and subjected to analysis.
Isolation of peripheral blood monocytes
Peripheral venous blood was obtained from the same individual from whom the epithelial cells were derived and monocytes were isolated by Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient centrifugation and plastic adherence, as described (28). The nonadherent cells were removed, and the adherent cells were washed with PBS to remove all loosely bound contaminating cells. Adherent monocytes were harvested by a rubber policeman. Purity and viability were assessed by staining with the anti-CD-14 mAb Leu M3 (Becton Dickinson, San Jose, CA) and propridium iodide (29), respectively. In all cases purity was >95% and viability was 100%.
Cell lines
Cell lines (EL-4, L929, DLD1, HT-29, and Caco2) were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The IL-6 responsive plasmacytoma cell line B9, was kindly provided by Dr. Lester May (New York Medical College, Valhalla, NY). Cell lines were either used as positive controls for receptor assays or as sources of intestinal epithelium. Cells were thawed and grown in T75 tissue culture flasks (Nunc, Naperville, IL). DLD1, HT29, and L-929 cell lines were grown in RPMI 1640 medium supplemented with 10% FCS (Life Technologies, Gaithersburg, MD), Caco2 in DMEM with 10% FCS, EL-4 in DMEM with 10% horse serum, and B9 cells in RPMI 1640 with 10% FCS and 20 U/ml rIL-6 (kind gift of Dr. Edward Siden, Mount Sinai Medical Center, NY). All culture media contained 1% penicillin-streptomycin (ICN-Flow. McLean, VA) and L-glutamine (ICN-Flow). Cells from suspension cultures (EL-4, B9) or confluent monolayers (DLD1, HT29, Caco2), dissociated from the plastic surface by treating with a nonenzymatic cell dissociation solution (Sigma), were washed and used for staining. In some experiments, control cell lines or IEC lines were treated with dispase or EDTA to assess the effects of the isolating procedure on cytokine binding. For receptor regulation studies secondary cultures were generated in the presence or absence of modulatory agents for varying time periods. Receptor density was compared between stimulated and unstimulated cells by analysis of mean fluorescence intensity or peak channel (see below).
In vitro models of crypt and surface epithelial cells
In vitro models of fully differentiated or undifferentiated human colonic epithelial cell lines were developed by using glucose free or high glucose containing conditioned medium, respectively, as previously described by Wice et al. (30). Briefly, HT29 (obtained from Dr. Burton M. Wice, Washington University School of Medicine, St, Louis, MO) and DLD1 cells (from ATCC) were cultured in glucose-free DMEM with 10% dialyzed FCS (Summit Biotechnology, Fort Collins, CO) supplemented with 2.5 mM inosine (Sigma) (for differentiated surface-like cells designated HT29ino or DLD1ino in the text) or 25 mM glucose (for de-differentiated crypt-like cells designated HT29glu or DLD1glu in the text). Cells were grown for at least four passages and maintained in a postconfluent state for at least 10 days with daily replacement of fresh medium before their use in the experiments. Although the features of full differentiation (in inosine cells) or de-differentiation (in glucose cells) of these cells have been characterized previously by Wice et al. (30, 31) under these conditions, we reconfirmed the differentiation states of these cells by demonstrating lower levels of alkaline phosphatase and sucrase isomaltase (markers of a mature enterocyte phenotype) in the "glucose-conditioned" cells compared with the higher levels of these enzymes in the "inosine-conditioned" cells (data not shown).
Cytokines and chemicals
Human recombinant IL-1ß was obtained from Regeneron (Boston,
MA), IL-6 was a generous gift from Dr. Edward Siden, human GM-CSF was
purchased from R&D Systems (Minneapolis, MN), TNF- from Cetus
(Emeryville, CA), IFN- from Genentech (South San Francisco, CA), and
Escherichia coli LPS and phorbol 12-myristate
13-acetate (PMA) from Sigma.
Identification of cytokine binding
Cytokine receptors were identified using fluorochrome-labeled
cytokines (Fluorokine; R&D Systems) and analyzed by flow cytometry. We
utilized phycoerythrin (PE) or biotin (followed by avidin-FITC)
conjugated cytokines as the signal-to-noise ratio was greater than that
seen with fluorescein. Briefly, purified epithelial cells were washed
twice in RDF-1 buffer (wash buffer for fluorokine staining from R&D
Systems) and resuspended at 4 x 106 cells/ml. A total
of 25 µl of cell suspension (105 cells) was incubated
with 10 µl (chosen by performing serial dilution analysis with
control cells) of either PE-conjugated IL-1ß, IL-6, GM-CSF, TNF-,
or streptavidin for 1 h on ice. In the cases where staining was
performed with biotinylated cytokines, 5 µl (chosen from serial
dilution studies in normal control cells) of biotin-conjugated
cytokines or soybean trypsin inhibitor (as a negative control) was
added to cells for 1 h on ice followed by incubation with 5 µl
avidin-FITC for another hour. After this incubation, cells were washed
twice with 2 ml of RDF-1 buffer. Cells were resuspended in 200 µl of
PBS and analyzed by an Epics Profile-I flow cytometer (Coulter,
Hialeah, FL). At least 3000 cells were counted per analysis. In
preliminary studies, specificity of these conjugated cytokines was
determined by the inhibition of specific staining on receptor bearing
control cells by unlabeled cytokine. Cells were first incubated with
unlabeled cytokine at 100-fold excess molar concentration (or the
fluorescent cytokines were neutralized by incubation with specific mAbs
against the cytokines for 10 min before their incubation with cells)
and then incubated with labeled cytokine for another hour on ice
following the protocol described above. To assess purity of the cell
preparations, in each set of experiments parallel immunofluorescence
staining was performed as described previously (21) with Abs against
HLA-class I (W6/32), CD14 (Leu-M3), CD20, CD45 (HLe-1), and an
epithelial cell specific mAb L12 (32) followed by FITC-conjugated
F(ab')2 goat anti-mouse IgG. An irrelevant mouse IgG1
mAb served as a negative control. This allowed us to gate on the L12
positive epithelial cells, thus avoiding any false positive cytokine
binding from contaminating nonepithelial cells. In another series of
experiments, dual labeling analyses were performed. Cells were first
stained with L-12/goat anti-mouse FITC as described above and then
incubated with PE-conjugated cytokines to discriminate between
epithelial cells and other contaminating cells in our IBD epithelial
cell preparations. In each assay, positive control cells for IL-6R
(B9), IL-1R (EL-4), GM-CSFR (monocytes), and TNFR- (L929) binding
were included to compare receptor density and reliability of the
technique.
Measurement of IL-8 and GRO-
Freshly isolated IEC and HT29 colonic adenocarcinoma cell lines
were cultured in the presence or absence of TNF- (33) for 48 h,
and culture supernatants were assayed for IL-8 and GRO- production
by ELISA as described previously (34). Briefly, a polyclonal goat
anti-human (GAH) IL-8 or a monoclonal GAH GRO- (R&D Systems) Ab
were used as capturing Abs with a polyclonal rabbit anti-human IL-8
and monoclonal mouse anti-human GRO- as the detecting Ab. The
IL-8 and GRO- ELISAs were sensitive to 20 and 50 pg/ml,
respectively.
Determination of the cytokine effect on epithelial cell growth
DLD1 cell lines were seeded (5 x 105 cells per well) in 6-well plates. Cytokines were added at the time of adherence to the culture well. Cells were removed at different time points (24 and 48 h) by treatment with cell dissociation solution and counted in a Neiubauer chamber (Hausser Scientific, Horsham, PA). Three wells were used for each data point and counting was performed by three investigators independently. The results from direct cell counts were validated in a parallel series of [3H]thymidine uptake assays. IEC cell lines were grown in the presence or absence of varying concentrations of cytokines in 96-well (5000 cells/well) flat-bottom tissue culture plates in a humidified 5% CO2 incubator. After 48 h of culture, 1 µCi [3H]thymidine was added, and after 6 h of further incubation the culture medium was aspirated and thymidine incorporation was measured in a 1450 Microbeta Plus counter (Wallac, Gaithersburg, MD).
Detection of cytokine-induced ICAM-1 on in vitro models of surface and crypt epithelial cells
Conditionally differentiated (inosine medium) or de-differentiated (glucose medium) cell lines (DLD1 and HT29) were grown in T25 tissue culture flasks (Falcon, Franklin Lakes, NJ) as described above. Each type of cell line or freshly isolated crypt and surface IEC were cultured either with recombinant human GM-CSF (20 ng/ml) or medium alone for 24 h at 37°C. The cell lines were dissociated by treatment with nonenzymatic cell dissociation solution (freshly isolated IEC do not adhere to the plastic surface and did not require this cell dissociation procedure after stimulation), washed three times in PBS/1% BSA/0.02% sodium azide, and processed for immunofluorescence staining using an anti-ICAM-1 mAb (Becton Dickinson) or an irrelevant mouse IgG1 control mAb.
Flow cytometric analysis
Stained cell suspensions were analyzed on a Epics Profile flow cytometer (Coulter) as previously described (35), gating on viable cells. Mean channel fluorescence (MCF), which correlates with fluorescence intensity, was determined from the peak of positively stained cells and is recorded on a log scale.
Stimulation of epithelial cells
Freshly isolated epithelial cells or cell lines were cultured in
the presence or absence of IL-1ß (100 ng/ml and 1 µg/ml), IFN-
(250 U/ml), LPS (1 µg/ml), GM-CSF (20 ng/ml), or PMA (10 ng/ml) for
24 h in culture medium in a 5% CO2 humidified 37°C
incubator. These concentrations were optimal for induction of specific
receptors in conventional APCs. Cells were washed and checked for
viability, and receptor expression was analyzed by the method described
above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Freshly isolated epithelial cells and appropriate positive control
cells were stained with either PE-conjugated IL-1ß, IL-6, GM-CSF,
TNF-, or streptavidin to detect the constitutive expression of
receptors for these cytokines. The flow cytometric analysis for one
representative experiment is shown in Figure 1
. A peak shift in MCF demonstrates
the presence and density of receptors compared with the streptavidin-PE
negative control. In each case binding of PE-labeled cytokines was
specific since the binding could be inhibited by the corresponding
unlabeled human recombinant cytokine.
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A, upper). The specificity
of IL-1ß PE binding to IL-1ßR was demonstrated by inhibition of
binding of the labeled cytokine to EL-4 cells in a blocking assay using
100 fold excess unlabeled rIL-1ß (Fig. 1A,
middle) but not with IL-6 or GM-CSF (data not shown).
Blocking experiments were also performed in a series of experiments
with freshly isolated IECs (data not shown) with similar results.
Dispase treatment of EL4 cells had no effect on IL-1ßR expression
(Fig. 1A, lower).
As seen in Figure 1B, IL-6R was also expressed on IECs
(upper) but at a lower density compared with IL-1ßR.
Because we previously demonstrated that IL-6 is secreted by IEC (21),
the lower density of IL-6R may relate to autocrine blockade of IL-6R by
IEC-secreted IL-6. Similar to IL-1ßR, IL-6-PE binding was specific
(Fig. 1B, middle) and dispase treatment had no
effect on IL-6R expression (Fig. 1B, lower) on
the IL-6-dependent B9 cell line.
We next examined expression of receptors for GM-CSF, which enhances
phagocytosis in macrophages (36). Figure 1C,
upper, demonstrates that GM-CSFRs are expressed on
epithelial cells at high density. Since, in human, GM-CSF and IL-3 have
been reported to share a common ß2 subunit, we performed blocking
experiments with cold IL-3 as well. Binding of GM-CSF PE was partially
inhibited by both cold GM-CSF (Fig. 1C, middle)
and IL3 (data not shown). However, the ability of GM-CSF but not IL-3
to regulate surface Ags on IECs (37, 38) confirms that the staining
relates to GM-CSFR and not IL-3R. Dispase had no effect on GM-CSFR
expression (Fig. 1C, lower).
Interestingly, receptors for TNF- were not detected in any
epithelial cell preparation (Fig. 1D, upper),
although TNF-R were clearly demonstrated in the colonic
adenocarcinoma cell lines (see Table I below). In this case, TNF-R
expression in control cell lines was reduced by dispase
treatment; therefore, EDTA was utilized to isolate IECs in this series
of experiments. EDTA had minimal effects on TNF-R expression. The
variability in the staining of the L929 cells related to a reduced
background in this experiment.
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Since IECs possess some of the functional properties ascribed to
APCs, it was of interest to compare cytokine receptor expression on
IECs with that of monocytes. Therefore we performed studies using
paired samples of epithelial cells (n = 5) and isolated
monocytes (n = 5) from the same individuals. As can be
seen in Figure 2, with the
exception of GM-CSF, the density of receptor expression in IECs for all
cytokines, was lower than that of monocytes. As described earlier (Fig. 1), no TNF-R was detected in any of the epithelial cell
preparations, whereas TNF-R were present in the monocytes. GM-CSFR
expression was the highest of any of the cytokine receptors tested and
was comparable to monocytes. Cytokine receptor expression and density
on IEC was similar regardless of the site from which the IEC were
isolated (e.g. ileum vs colon, right colon vs left colon). The
variability seen in receptor expression between the different
individuals likely relates to individual variation rather than
differences in isolation technique.
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Given the above data, it is clear that epithelial cells might be
targets for proinflammatory cytokines released by the epithelial cells
themselves as well as by other mucosal cells during the process of
inflammation. Receptor-cytokine interactions could alter the phenotype
of the epithelial cells resulting in abnormal cytokine receptor
expression, alteration in function, growth, or induction of specific
costimulatory signals for T cell activation. To determine whether such
a phenomenon may be responsible for the epithelial cell dysfunction
seen in IBD (39), we studied the presence and density of receptor
expression for the four cytokines in epithelial cells isolated from
inflamed (UC, n = 11; CD, n = 10) and
uninflamed (UC, n = 2; CD, n = 8)
tissues from patients with IBD and compared them with normal control
epithelial cells (n = 16). As shown in Table I, receptor expression on
epithelial cells derived from inflamed tissues was not significantly
different from normal control IECs, although there was a trend toward
an increase in GM-CSFR in uninflamed UC and CD. However, when we
directly compared the cytokine receptor expression and density on
epithelial cells derived from inflamed and uninflamed areas in the
bowel of the same IBD patient, we found that cytokine receptor
expression did not correlate with inflammation in either UC or CD (data
not shown).
Cell lines differ from freshly isolated epithelial cells in cytokine receptor expression
To develop a model for assessing the regulation of cytokine
receptor expression in IECs, we examined receptor expression in the
epithelial cell lines DLD1, CaCO2, and HT29. Receptors for IL-1ß,
IL-6, and GM-CSF were expressed on these cell lines at levels greater
than those seen in freshly isolated IECs (Table II). Furthermore, unlike freshly
isolated IECs, TNF-R were detected on all three cell lines.
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-R in the cell lines but not in freshly isolated
IECs raised concerns about the possibility that TNF-R on the
epithelial surface might be blocked by the binding of endogenous
TNF-, resulting in negative staining by the PE-conjugated cytokine.
Such a possibility was rendered less likely by the finding of an
absence of TNF- protein or mRNA in isolated IEC (21). To ensure that
our isolation procedure was not responsible for stripping off TNF-R,
we subjected macrophage and control cell lines to the same isolation
procedure. As discussed earlier, dispase treatment did not change
receptor density on either macrophages or the cell lines for any
cytokine except for TNF-R. However we were still unable to detect
TNF-R on IEC when the IEC were isolated using EDTA (which had no
inhibitory effect on TNFR expression and in fact had a modest
stimulatory effect). These data suggest that the absence of
TNF-R in freshly isolated epithelial cells is not due to the
isolation procedure but that tumor bearing epithelial cell lines differ
from fresh IECs in their cytokine receptor expression.
Demonstration of a lack of functional response to TNF- by
freshly isolated IEC
Although our results of TNF- binding (receptor expression) by
malignant epithelial cell lines have been consistent with previously
described functional effects of TNF- on these cell lines, the lack
of TNF- binding on freshly isolated IEC raises the concern as to
whether the nonbinding of TNF- on isolated IEC is an artifact of
either endogenous TNF- production or the isolation procedure. To
address this issue we compared the functional response of the HT29 cell
line and freshly isolated IEC to TNF- stimulation by examining
chemokine (IL-8 and GRO-) production by these cells. As can be seen
in Table III, consistent with
previously reported results, TNF- stimulation of HT29 cells resulted
increased production of both IL-8 and GRO- in a dose-dependent
fashion, whereas the freshly isolated IECs from two different
individuals showed no response to TNF- stimulation even at the
higher concentration of 10 ng/ml. Furthermore, it appeared less likely
that endogenous TNF- was present since baseline IL-8 and GRO- was
comparable to the HT29 cell line. These data support the lack of
TNF-R expression on freshly isolated IEC.
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The differences in cytokine receptor expression between the
different epithelial cell lines raised the possibility that cytokine
receptor expression may vary depending upon the differentiation
state of the cells. Previous studies have shown that the response to
various hormones and physiological modulators differs in crypt and
surface epithelial cells. Therefore, to determine whether the two cell
types (crypt and surface IEC) display any difference in the cytokine
receptor expression, we isolated surface and crypt epithelial cells
from 4 individual specimens. As can be seen in Figure 3, the surface epithelial cells
expressed cytokine receptors (IL-1ß and IL-6) at a higher density
than crypt epithelium. These findings were reconfirmed in an in vitro
cell line model system of crypt and surface epithelial cells (see
Materials and Methods). HT29 and DLD1 cell lines were
conditionally differentiated (HT29ino and DLD1ino cells, comparable to
surface/villus IEC) or de-differentiated (HT29glu and DLD1glu,
comparable to crypt cells) by using glucose free or high glucose
containing medium (see Materials and Methods). After growth
under such conditions, experiments were performed to detect cytokine
receptor expression on these cells. Consistent with our results with
freshly isolated crypt and surface epithelial cells, IL-1ß and IL-6R
expression was greater in the differentiated (HT29ino and DLD1ino)
cells than the de-differentiated (HT29glu and DLD1glu) crypt-like cells
(data not shown). As seen with freshly isolated IEC, the GM-CSFR
density was similar in both differentiated and dedifferentiated cell
lines. The finding of cytokine receptor heterogeneity in the crypt vs
surface epithelial cells may relate to specific cytokine requirements
for the modulation of growth, differentiation, function, or apoptosis
of epithelial cells as they migrate from the crypt to the surface.
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Several laboratories have recently shown that inflammatory
cytokine production is elevated in IBD mucosa and that these cytokines
can modulate expression of various cytokine receptors. Therefore we
assessed the regulation of cytokine receptor expression in IECs. Based
on the receptor profile of the epithelial cell lines, we chose DLD1
cells for these studies since it most closely mimicked normal IECs. A
total of 106 DLD1 cells/well were cultured in the presence
or absence of LPS (1 µg/ml), IFN- (250 U/ml), or PMA (10 ng/ml)
for 24 h in 6-well tissue culture plates (Falcon), and receptor
expression and density were analyzed by flow cytometry and expressed as
fold increase over baseline MCF. As shown in Figure 4 (average of five experiments),
IL-1ßR expression was enhanced by all of the stimuli used, although
this did not achieve statistical significance (Fig. 4A).
Unlike monocytes (40), IL-6Rs on IEC (Fig. 4B) were
up-regulated by LPS and unchanged with PMA, suggesting a different
regulatory pathway for IL-6R expression in both conventional and
mucosal APCs. This lack of induction of IL-6R cannot relate to the
production of (unlabeled) IL-6 in the cell line as we have previously
seen that DLD1 cells do not secrete this cytokine (A. Panja and L.
Mayer, unpublished data). GM-CSFR expression was unaffected by PMA,
LPS, or IFN- (Fig. 4C). Finally, TNF-R was neither
altered in DLD1 cells nor induced in freshly isolated epithelial cells
(data not shown) by any of these stimuli.
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IL-1ß, IL-6, and GM-CSF differentially regulate epithelial cell growth
The effect of cytokines on epithelial cell growth was studied by
culturing DLD1 cells in the continuous presence of IL-1ß (10–100
ng/ml), IL-6 (60 u/ml), GM-CSF (20 ng/ml), or medium only. Cell growth
at various time points was quantitated by direct cell counts. As can be
seen in Figure 5, IL-1ß
enhanced the cell growth by 3- to 4-fold, whereas GM-CSF and to a
lesser extent IL-6 inhibited growth. In a second series of experiments
cultures were grown in 96-well flat-bottom plates (5000 cells/well) for
24 h in 1% FCS with 0.5% BSA. Cells were then treated with
various concentrations of cytokines in the presence of 2% FCS for
72 h, and cell proliferation was assessed by a thymidine
incorporation assay. Again, a stimulatory effect of IL-1ß and an
inhibitory effect of GM-CSF was seen in a dose-dependent fashion (data
not shown). There was minimal or no effect of IL-6 in this system.
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To further evaluate the physiological role of the cytokine
receptors on IEC, we examined whether treatment of IECs with GM-CSF
(since GM-CSF production by IECs is increased in active inflammation
(71) and GM-CSFR are present in both crypt and surface IEC) could
regulate ICAM-1 expression. This integrin has been shown to be of
importance in Ag-specific T cell responses and in lymphocyte homing to
endothelium with subsequent migration of lymphocytes into inflammatory
sites (41). As shown in Figure 6,
de-differentiated crypt-like cell lines (DLD1glu and HT29glu) lack
constitutive ICAM-1 expression. Treatment with GM-CSF induced ICAM-1
expression in both of these cell lines (significant level of expression
in DLD1glu and a very low level of expression in HT29glu). However, the
differentiated surface-like (DLD1ino and HT29ino) cell lines
constitutively expressed ICAM-1 (low in HT29ino and high in DLD1ino)
and this was not further enhanced by GM-CSF treatment in either HT29ino
or DLD1ino cells. Similar results were seen in experiments
(n = 2) with freshly isolated crypt epithelial cells
although the response in these cells was weaker than in the cell lines
and there was no constitutive expression in either cell type (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility that IECs could serve as a target for these
proinflammatory cytokines is supported by similar studies with
endothelial cells in vascular processes. Several groups have
demonstrated that IL-1 induces PGE2 secretion by
fibroblasts, smooth muscle, and endothelial cells (42), which may have
a role in promoting local cytoprotection or in some cases tissue
destruction. In addition, IL-1, as well as other cytokines, has been
shown to be able to induce the expression of adhesion molecules (ICAM,
ELAM-1) on endothelial cells (43), which promotes cell-cell
interactions and contributes to the inflammatory response.
Proinflammatory cytokine receptor expression by various epithelial
cells has also been reported. IL-1R expression has been documented in
the rat intestinal epithelial cell line, IEC-18 (44, 45), whereas
Krueger and coworkers (46) have reported mitogenic effect of GM-CSF on
basal keratinocytes, suggesting the presence of GM-CSFRs on these
cells. This latter observation is of interest since GM-CSF is a potent
stimulator of phagocytosis and class II Ag expression in APCs
(macrophages) (34). Specific examples of IECs serving as targets for
such cytokines include studies by Shanahan and coworkers (20), who
demonstrated that the combination of IFN- and TNF- is cytotoxic
for the colonic epithelial cell line HT-29; Madara et al. (47), who
demonstrated that IFN- alters the permeability of tight junctions in
T84 cells allowing for neutrophil transmigration; Chang et al. (48),
who reported that IL-1 induces chloride secretion and short circuit
current by chicken enterocytes when added to the serosal side of
chicken intestine mounted in an Ussing chamber (although these effects
may have been mediated through stromal cells); and Molmenti et al.
(49), who documented acute phase response regulation by IL-1 and IL-6
binding on Caco2 cells. Such data support the potential for autocrine
and paracrine effects of cytokines produced by epithelial cells and/or
lamina propria mononuclear cells. Given the potential implications of
such possibilities, we studied the expression and density of IL-1ß,
IL-6, TNF-, and GM-CSF receptors on epithelial cells from normal and
IBD tissues. These cytokines were chosen because they have significant
effects on the APC function of monocytes and have been reported to be
increased in the mucosa of patients with IBD. The findings reported
here demonstrate that receptors for IL-1ß, IL-6, and GM-CSF were
comparably expressed in both normal and IBD epithelial cells. However,
considerable differences did exist in receptor density within
differentiated and undifferentiated epithelial cells. TNF-R were not
detected in any of the freshly isolated epithelial cell preparations.
In contrast, conventional APCs from the same individuals as well as
malignant epithelial cell lines expressed receptors for all four
cytokines including receptors for TNF-. Thus freshly isolated
epithelial cells differ from professional APCs in their inability to
express receptors for TNF-. This latter finding is of importance as
TNF has been shown to be an important factor in regulating cytokine
production by macrophages and as a potentially cytotoxic agent for IEC.
The absence of TNF-R on IEC suggests that these cells may not be
targets for killing by TNF- in diseases such as IBD where TNF-
levels are high.
Proinflammatory and prophagocytic cytokines have been known to regulate
APC functions as well. However, different APCs display a distinct range
of responses to these cytokines. For example IFN- up-regulates class
II MHC expression on murine macrophages but fails to do so on B cells,
although non-class II Ag presentation is enhanced (50). Similar
differences have been observed with other cytokines (e.g. IL-4) (51, 52). Thus disparate effects of cytokines on different APCs are well
described. Intestinal epithelial cells like conventional APCs express
class II MHC molecules on their cell surface (53, 54) and are capable
of taking up, processing, and presenting Ag to immunocompetent T cells
(55, 56, 57, 58, 59, 60, 61). However, several differences exist as well. These include a
lower density of class II molecules on the IEC surface (62), a limited
cytokine profile (lack of IL-1 production) (15, 21), preferential
stimulation of suppressor T cells in mixed cell culture systems (52),
and a slower rate of Ag processing by IECs (57, 61). These differences
may relate to cytokines in the local environment and their effect on
IEC function. Such correlations have not been addressed in in vivo
systems.
It is well described that cytokines or bacterial products can modulate
cytokine receptor expression (62, 63). This may in turn influence
immune and inflammatory events by promoting or suppressing activation,
surface molecule expression, or costimulatory signals for T cell
activation. The regulatory mechanisms governing cytokine receptor
expression in IECs are not known. Our data suggest that the regulation
of IL-6R is different from that of monocytes, i.e., LPS up-regulates
IL-6R expression in DLD1, a colonic epithelial cell line, whereas it
down-regulates expression in macrophages. This differential regulation
of cytokine receptors at a site where LPS is abundant may reflect a
compensatory mechanism. Still these cytokine receptors may play an
important role in intestinal homeostasis. Reinecker and Podolsky (64)
have recently identified the presence of receptors for T cell-derived
cytokines on IEC describing the expression of the common -chain of
the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors in a rat IEC cell line.
Taken together, one could develop a model where locally produced cytokines by resident T cells, macrophages, myofibroblasts, as well as epithelial cells might regulate the normal growth and differentiation of IEC. It is of interest to note that many of the cytokine knockout mice that develop intestinal inflammation display histologic pictures characterized by marked crypt cell proliferation. Thus alterations in cytokine production could alter normal physiologic processes (i.e., growth and differentiation) and result in epithelial cell dysfunction. Such a scenario may be true in IBD where both T cell-derived as well as proinflammatory cytokines are increased in inflamed tissues. The effects of these cytokines on IEC phenotype and function may account for some of the clinically expressed components of these diseases. Since IBD IEC express similar profiles of cytokine receptors to those seen on normal IEC, the epithelial cell dysfunction seen would relate to the higher concentrations of local cytokine.
Cytokines might be important for the growth, differentiation, and
migration of epithelial cells during maturation from crypt cells as
well. Alteration in any of these events may cause serious pathologic
consequences. Specific roles of cytokines in each of these events
require further exploration, but the identification of candidate
cytokines by virtue of receptor expression is the first step. That
cytokines alter epithelial cell function is well described. IL-4
enhances IEC-6 cell line proliferation (65) whereas the proinflammatory
cytokines IL-1 and GM-CSF either enhance or inhibit DLD1 cell line
growth. IFN-, IL-1, and TNF- have been shown up-regulate ICAM-1
expression on the surface of many cells (66, 67) including intestinal
epithelial cells (68). Although the role of ICAM-1 expression in
intestinal inflammation has been controversial (69, 70), our in vitro
data suggest that an increased GM-CSF concentration in the epithelium
can induce ICAM-1 expression by crypt epithelial cells enhancing the
chances of adhesion between these cells and CD4+ lamina
propria lymphocytes. This finding may represent an important mechanism
for the immune responses seen in IBD.
In conclusion, our data support the concept that IEC can be affected by cytokines in their local environment. Alterations in cytokine concentrations as a consequence of inflammation may therefore have profound effects on epithelial cell growth, phenotype, and function. Selective regulatory roles of proinflammatory cytokines on IEC growth and surface molecule expression may provide evidence for an unrecognized mechanism responsible for inflammation, ulceration, or malignant transformation seen in IBD. Further elucidation of the functional interactions between distinct cytokines would be of importance in the understanding of cytokine mediated regulation of mucosal immune responses.
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Acknowledgments |
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Footnotes |
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2 Present address, correspondence and reprint request to Dr. Asit Panja, Division of Gastroenterology, Hepatology and Nutrition, Winthrop University Hospital, 222 Station Plaza North, Suite 511A, Mineola, Long Island, New York 11501.
3 Abbreviations used in this paper: UC, ulcerative colitis; CD, Crohn’s disease; GM-CSF, granulocyte-macrophage CSF; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; MCF, mean channel fluorescence; PE, phycoerythrin; ECAM-1, endothelial cell adhesion molecule-1; ELAM-1, endothelial leukocyte adhesion molecule-1.
Received for publication October 23, 1997. Accepted for publication May 27, 1998.
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