Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114-2696
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ABSTRACT |
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Intestinal
epithelial cells may be actively involved in the immunoregulatory
pathways leading to intestinal inflammation. The aim of this study was
to assess expression by intestinal epithelial cells of cytokines with
potential involvement in the development of intestinal inflammation in
interleukin (IL)-2-deficient [(/
)] mice.
Wild-type mice, mice heterozygous for the disrupted IL-2 gene, and
IL-2(
/
) mice were studied at 6, 16, and 24 wk of age. The
mRNA levels of transforming growth factor-
1 (TGF-
1), tumor necrosis factor-
(TNF-
), IL-1
, IL-6, IL-15, KC,
JE, and CD14 in colonic and small intestinal epithelial
cells were assessed by Northern blot analysis. CD14 was also measured
by Western blotting and reverse transcriptase polymerase chain reaction
(RT-PCR). TGF-
1 mRNA was constitutively expressed in both colonic
and small intestinal epithelial cells with increased expression in the
colonic epithelium of colitic mice. CD14 was detected only in colonic epithelial cells, and mRNA levels increased severalfold in
IL-2(
/
) mice with colitis. Northern analysis demonstrated
increased levels of TGF-
1 and CD14 mRNA in colonic epithelial cells
of IL-2(
/
) mice before the development of signs of
colitis. CD14 mRNA and protein expression in the epithelial cells of
colitic mice were confirmed by RT-PCR and Western blot analysis of
isolated cells. In addition, IL-2(
/
) mice also expressed
increased levels of IL-15 mRNA in small intestinal and colonic
epithelial cells compared with heterozygous control mice. TNF-
,
IL-1
, IL-6, KC, and JE mRNAs were only detectable in colonic
epithelial cells of mice after the onset of colitis. Enhanced
expression of TGF-
1, IL-15, and CD14 by colonic epithelial cells may
play a role in the subsequent development of colitis in
IL-2(
/
) mice.
lipopolysaccharide; CD14; interleukin-15; transforming growth
factor-
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INTRODUCTION |
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MICE DEFICIENT [(/
)] in
interleukin (IL)-2 develop normally during the first 3-4 wk of age
but subsequently develop anemia leading to ~50% mortality between 4 and 9 wk after birth. Surviving mice develop an inflammatory bowel
disease with histological alterations resembling human ulcerative
colitis (26). The absence of intestinal inflammation in germ-free
IL-2(
/
) mice suggests that luminal bacteria are involved
in the development of inflammation (26). Lipopolysaccharide (LPS), a
component of the outer membrane of all gram-negative bacteria, is
present in large amounts in the lumen of the colon. This common
bacterial product is a potent stimulus for the production of a variety
of inflammatory mediators, such as IL-1 (5), IL-6, IL-8, and tumor
necrosis factor-
(TNF-
; see Ref. 15), as well as lipid mediators
derived from arachidonic acid, and toxic oxygen radicals (19). CD14,
the membrane receptor for complexes of LPS and LPS-binding protein on
myeloid cells (33), has recently been shown to be expressed by
nonintestinal epithelial cells (10). LPS is able to induce CD14 mRNA in
most tissues, including epithelial cells in the respiratory,
gastrointestinal, urinary, and reproductive tracts and the liver (10).
Increased numbers of macrophages expressing CD14 have been found in
inflammatory bowel disease mucosa, which may give rise to increased
production of proinflammatory mediators (12).
Recent studies have shown the importance of transforming growth
factor-1 (TGF-
1) in maintaining homeostasis in intestinal epithelial cell populations. TGF-
1 inhibits intestinal epithelial cell proliferation, stimulates cell differentiation, and promotes epithelial restitution after mucosal injury (7). Although TGF-
1 increases in association with inflammatory activity, evidence for a
protective role for TGF-
1 in intestinal inflammation has also been
obtained (29).
Development of colitis in IL-2(/
) mice suggests that IL-2
plays a role in mucosal immunoregulatory pathways. These mice exhibit
normal thymic development and thymocyte and peripheral T cell subset
composition, suggesting that other cytokines may compensate for the
absence of IL-2. IL-15 is a recently identified cytokine that is
produced by nonlymphoid cells, including intestinal epithelial cells
(11, 23), and utilizes the IL-2 receptor
and common
c subunits
for signal transduction. As a result, IL-15 exerts functional effects
on T and B cells similar to those of IL-2 (11).
In addition to their long-recognized role in absorptive function,
recent evidence suggests that intestinal epithelial cells are closely
integrated into mucosal immune responses. Intestinal epithelial cells
are able to present antigen through major histocompatibility complex class II expression (16) and secrete
proinflammatory cytokines such as TNF-, IL-1
, IL-8 (8), IL-6
(28), and monocyte-chemoattractant protein-1 (MCP-1; see Ref. 22).
The present study demonstrates that altered gene expression in
intestinal epithelial cells may contribute to the development of
intestinal inflammation in IL-2(/
) mice. Markedly
increased levels of CD14 mRNA and TGF-
mRNA preceded epithelial
expression of mRNA of the proinflammatory cytokines TNF-
, IL-1
,
IL-6, KC, and JE during the development of colitis in
IL-2(
/
) mice. Most importantly, the expression of CD14
and TGF-
1 mRNAs increased before histological signs of mucosal
inflammation. In addition, IL-15 gene expression by small intestinal
and colonic epithelial cells of IL-2(
/
) mice was
constitutively increased. In the small and large intestine, IL-15 may
compensate in part for the loss of IL-2 in IL-2(
/
) mice.
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MATERIALS AND METHODS |
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Animals
IL-2(Peritoneal macrophages. Activated mouse peritoneal macrophages were used as positive controls for the expression of CD14 (20). These were obtained by injecting 6-wk-old C57BL/6 mice intraperitoneally with 3 ml of 3% fluid thioglycollate. Four days later, peritoneal exudate macrophage cells were extracted for use.
Intestinal Epithelial Cells
Animals were killed by cervical dislocation, and the intestines were removed. The total small intestine and colon were separated and flushed with 4°C calcium- and magnesium-free phosphate-buffered saline (CMF). Intestinal epithelial cells were obtained using a modification of previously described methods (3). Briefly, the small intestine or colon was everted over an animal feeding needle (Popper, New Hyde Park, NY) and filled with 4°C CMF, and both ends were closed with surgical silk. The everted intestinal segments were incubated in 50 ml freshly made 3 mM EDTA in CMF, pH 7.4, at 37°C and gently shaken every 5 min. After 20 min, the epithelial cells were centrifuged, washed in CMF, and centrifuged again. Cytospin preparations of epithelial cell populations showed <5% of contaminating nonepithelial cells, using CD45 as leukocyte common antigen marker, CD3 (rat anti-mouse and hamster anti-mouse) as T cell marker, CD45R/B220 as B cell marker, and IM290 as intraepithelial lymphocyte marker. Viability of epithelial cells was >95%, as assessed by 0.1% trypan blue exclusion.Northern Blot Analysis
Immediately after isolation, epithelial cells were lysed and homogenized in 4 M guanidium isothiocyanate and stored atSelection of single primary epithelial
cells. A previously described method was used to
isolate pure primary murine intestinal epithelial cells (24). After
EDTA preparation, the extracted cells were resuspended in sodium
tetraphenylborate. Briefly, the crypts were dissociated into single
epithelial cells to allow selection of individual cells with a
micropipette under microscopic inspection. Fifty cells were resuspended
in RPMI media and frozen at 80°C in Trizol reagent
(GIBCO-BRL, Gaithersburg, MD). RNA was isolated after addition of 50 µg of carrier rRNA from Escherichia coli W (Sigma).
Reverse transcription and PCR amplification. Total cellular RNA was isolated from Trizol according to the manufacturer's suggested protocol (GIBCO-BRL). Reverse transcription was performed by using 200 units of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL), 20 units of RNasin (Promega, Madison, WI), 1 µm dGTP, 1 µm dATP, 1 µm dTTP, 1 µm dCTP, and 1 µg of hexanucleotide random primer (Boehringer Mannheim, Indianapolis, IN) in 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, and 10 mM dithiothreitol for 1 h at 37°C.
Oligonucleotide primers were designed by using OLIGO 4.0 software and synthesized on a PCR-mate DNA synthesizer (Applied Biosystems). Primers were intron spanning to allow for differentiation of mRNA from genomic DNA (Table 1). PCR was performed using 1× PCR buffer, 2 mM MgCl2, all four dNTPs (each at 50 µM), each 5' and 3' primer at 1 µM, and 2.5 units AmpliTaq DNA polymerase LD (Perkin-Elmer) in a total volume of 100 µl. Five microliters of the reverse-transcribed RNA from the single cell isolations was run in each reaction in the thermal reactor (Perkin-Elmer). Forty-five cycles were run for 95°C for 1 min, 62°C for 1 min, and 72°C for 1.5 min. PCR products from peritoneal macrophages were cloned into pCR2.1 vector (Invitrogen, San Diego, CA) and sequenced by using the Sequenase 2.0 kit (United States Biochemical, Cleveland, OH). In addition, negative control PCRs were performed with water to rule out amplification contamination products.
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Immunoblot analysis. Intestinal epithelial cell isolates and peritoneal macrophages were suspended in lysis buffer (1% Nonidet P-40, 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 µg/ml aprotinin, 100 µM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin). After 30 min on ice, cell lysates were cleared by centrifugation at 12,000 g for 20 min. The protein concentration in each sample was quantified by the Bradford method (Bio-Rad).
Protein preparations were denatured for 5 min at 95°C in Laemmli buffer and separated by 10% SDS-gel electrophoresis. After transfer onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), the blots were saturated in 5% milk proteins in CMF-0.05% Tween 20. Subsequent washings were in CMF-0.05% Tween 20. The primary antibody was rat anti-mouse CD14 monoclonal antibody (Pharmingen, San Diego, CA) diluted 1:250 in CMF-1% bovine serum albumin. The bound antibodies were detected by peroxidase-labeled sheep anti-rat immunoglobulin (Ig; Amersham International, Buckinghamshire, UK). After incubation with Renaissance chemiluminescence reagents (NEN), the immunoblots were exposed to autoradiography film (Kodak, Rochester, NY).
Histology
At 6, 16, and 24 wk of age, mouse tissues were fixed in 4% paraformaldehyde, treated with graded alcohols and xylenes, and embedded in paraffin, and sections were cut at 4 mm. Light microscopy examination of tissues from IL-2(Immunohistochemical Detection of TGF-1
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RESULTS |
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CD14 Gene Expression by Intestinal Epithelial Cells
Using Northern blot analysis, we found CD14 mRNA to be constitutively expressed by colonic epithelial cells in all mice. As demonstrated in Fig. 1, colonic epithelial cells of colitic IL-2(
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The epithelial derivation of CD14 was confirmed using isolated primary
epithelial cells. As shown in Fig. 2, the
absence of leukocyte contamination in preparations of isolated primary
colonic intestinal epithelial cells was demonstrated by the absence of CD45 (leukocyte common antigen) transcript. The ability to detect even
minimal contamination by this technique was confirmed by the
demonstration of transcripts for CD45 in a preparation of isolated
epithelial cells to which two leukocytes were added (Fig. 2,
lane 7). After confirmation of the
purity of isolated primary intestinal epithelial cell preparations,
epithelial cells were tested for the presence of CD14 and GAPDH. As
shown in Fig. 2, primary epithelial cells from IL-2(/
)
mice contained transcripts for CD14. In contrast, the epithelial cell
isolates from control mice did not demonstrate CD14 mRNA.
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The presence of CD14 protein in the colonic epithelial cells of colitic
mice was demonstrated by Western analysis. As shown in Fig.
3, colonic epithelial cells from
IL-2(/
) mice expressed CD14 protein, whereas CD14 was
undetectable in epithelial cells from IL-2(+/+) mice. CD14 could not be
detected by Western blotting in small intestinal epithelial cell
preparations from either wild-type or IL-2(
/
) mice (data
not shown).
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TGF-1 Gene and Protein Expression by Intestinal
Epithelial Cells
|
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Using immunohistochemical analysis, we localized active TGF-1 in the
supranuclear cytoplasm of both colonic (Fig.
6) and small intestinal epithelial cells
(data not shown). In the colon, TGF-
1 protein was detected in
colonocytes along the crypts. Expression of active TGF-
1 in
intestinal epithelial cells was increased in IL-2(
/
) mice
before the onset of inflammation (Fig.
6B) and during active colitis (Fig.
6C) compared with in wild-type mice (Fig.
6A). Staining for active TGF-
1
was specific, as demonstrated by the absence of staining when the
primary antibody was replaced with normal rabbit serum (Fig.
6D). In the terminal ileum,
immunoreactivity for TGF-
1 was present in the enterocytes of the
middle and upper crypts and along the villi. The expression of TGF-
1
in small intestinal epithelial cells did not change during the
development of colitis in IL-2(
/
) mice (data not shown).
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IL-15 Gene Expression by Intestinal Epithelial Cells
IL-15 mRNA expression from colonic and small intestinal epithelial cells from IL-2(
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Expression of Proinflammatory Cytokines and Chemokines by Mouse Intestinal Epithelial Cells
Finally, the expression of the proinflammatory cytokines TNF-
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DISCUSSION |
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Animal models of colitis offer the opportunity to gain new insight into
the mucosal immune system in inflammatory bowel disease. Several new
rodent models support the importance of the bacterial flora in causing
intestinal inflammation (9). IL-2(/
) mice that do not
succumb to an early anemia develop progressive colonic inflammation by
6-15 wk of age, resulting in death by 10-35 wk of age (26). A
role for the enteric bacterial flora in intestinal inflammation is
supported by the absence of disease in IL-2(
/
) mice bred
in a germ-free environment (26). Another model of colitis, the
HLA-B27/
2m transgenic rat, also
fails to develop intestinal inflammation when raised in germ-free
conditions (31). IL-10(
/
) mice develop enteritis when
reared under conventional conditions but have attenuated disease when
raised in a specific pathogen-free environment (14). Antibiotic
treatment of CD45RBhi
CD4+ T cell reconstituted severe
combined immunodeficient mice diminishes intestinal inflammation (18).
Collectively, these animal models show that, in addition to an
immunologic defect, nonpathogenic bacteria are required for the
development of inflammatory bowel disease.
At present, the exact mechanism by which bacterial components
contribute to the pathogenesis of inflammatory bowel disease is
unknown. Binding of LPS in the presence of LPS binding protein to the
LPS receptor CD14 results in protein tyrosine phosphorylation and the
initiation of multiple cellular events, resulting in the production of
proinflammatory cytokines (5, 15). The results of Northern studies
demonstrated that mouse colonic epithelial cells constitutively express
CD14 mRNA. However, wild-type control mice demonstrated low levels of
CD14 mRNA compared with IL-2(/
) mice. This could be
caused by downregulation of CD14 mRNA expression in wild-type mice,
upregulation of CD14 gene transcription in IL-2(
/
) mice,
or contamination by leukocytes.
To fully determine if the CD14 mRNA detected was from colonic
intestinal epithelial cells, individual cells were isolated to allow
for a pure population of epithelial cells. These were found to be free
of mRNA for the leukocyte common antigen CD45. This excluded possible
contamination by any cells belonging to leukocyte lineage, including
macrophages, 90% of which express CD45 (13). Using single cell reverse
transcriptase-PCR, we found colonic epithelial cells from colitic
IL-2(/
) mice to express CD14 mRNA, whereas age-matched
wild-type mice did not.
To assess whether CD14 protein was produced, immunoblotting was
performed. Colonic epithelial cells from IL-2(/
) mice
expressed CD14, whereas IL-2(+/+) mice lacked CD14. Thus the CD14 mRNA
found in the colonic epithelial cells of colitic IL-2(
/
)
mice is translated to protein.
CD14 expression has been shown to be upregulated in
other systems in response to LPS (10). Tolerance to LPS seen in
wild-type mice might be lost in IL-2(/
) mice, resulting
in the activation of a proinflammatory cascade. Of note, no expression
of CD14 mRNA or protein was found in the small intestinal epithelial
cells in any of the studied mice populations. This is consistent with the inference that CD14 expression by colonic intestinal epithelial cells may be driven directly or indirectly by the higher concentrations of bacteria in the large intestine. This may influence the development of colitis in IL-2(
/
) mice, since they express the
receptor for LPS on their epithelial cells and do not develop disease
when raised germ free.
In inflammatory bowel disease, TGF-1 can play dual roles. TGF-
1
has proinflammatory activities by modulation of isotype switching among
B lymphocytes, enhanced cytokine expression by T lymphocytes and
macrophages (32), and chemotactic activity for neutrophils and
monocytes (21). TGF-
also has anti-inflammatory properties,
including suppression of lymphocyte proliferation, induction of IL-1
receptor antagonist (32), and enhancement of the process of restitution
after mucosal injury (7). In the present study, we show
that colonic and small intestinal epithelial cells in mice
constitutively express TGF-
1 mRNA. In noncolitic IL-2(
/
) mice, the levels of TGF-
1 mRNA in colonic and
small intestinal epithelial cells are higher than wild-type and
heterozygous control mice, which could result in proinflammatory
activity in IL-2(
/
) mice. In colitic mice, TGF-
1 mRNA,
minimal in small intestinal epithelial cells, is expressed in high
amounts by colonic epithelial cells, suggesting that it could play a
role in migration of the colonic epithelial cells into a wounded
surface. The increased levels of TGF-
1 mRNA correlated with an
increased amount of TGF-
1 protein in colonic epithelial cells in
IL-2(
/
) mice. These observations are in accordance with
recent studies in which increased contents of TGF-
1 mRNA were found
in the colonic mucosa of patients with inflammatory bowel disease (1,
17).
IL-15 appears to be abundantly and constitutively expressed in a
variety of tissues and shares many biological properties with IL-2. The
present data suggest a regulatory pathway between IL-2 and IL-15
expressions. Increased expression of IL-15 mRNA by epithelial cells may
compensate for the lack of IL-2 in IL-2(/
) mice. Further
studies are needed to determine whether increased IL-15 mRNA expression
contributes directly to the initiation of colitis in
IL-2(
/
) mice.
The expression of proinflammatory cytokines TNF-, IL-1
, IL-6, KC,
and JE by intestinal epithelial cells was only upregulated after the
onset of inflammation. Previously, the production of these
proinflammatory cytokines has been largely attributed to secretion by
leukocytes. This study demonstrates that a similar repertoire of
proinflammatory cytokines and chemokines can be expressed by colonic
epithelial cells of colitic IL-2(
/
) mice. The cytokine
pattern during colitis in the IL-2(
/
) mice resembled the
expression of their human homologs in the intestinal mucosa of patients
with ulcerative colitis and Crohn's disease (8, 22, 25). Increased
expression of TNF-
, IL-1
, IL-6, and the chemokine macrophage
inhibitory protein-1 in mouse colon tissue has been demonstrated in a
mouse model after induction of colitis by acetic acid (6). In this
model, the expression of proinflammatory cytokines was closely
correlated with the histological signs of inflammation. These cytokines
may therefore be a marker for "nonspecific" inflammation and
tissue repair. In contrast, increased gene expression of CD14,
TGF-
1, and IL-15 in IL-2(
/
) mice is present before signs of colitis and therefore may play a role in the initiation of
colitis in these mice. These findings implicate intestinal epithelial
cells in the initiation and subsequent development of colitis in
IL-2(
/
) mice.
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ACKNOWLEDGEMENTS |
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We thank Dr. Stephen Simpson, Center for the Study of Inflammatory Bowel Disease (CSIBD) Genetic Animal Model Core for providing interleukin-2-deficient mice and Dr. Emiko Mizoguchi, CSIBD Morphology Core, for performing analysis of cytospin preparations.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41557, DK-07191, and DK-43351 (to D. K. Podolsky) and DK-51003, a research grant from the Crohn's and Colitis Foundation of America (to H.-C. Reinecker), the Dutch Organization for Scientific Research, a Fulbright Scholarship, and Tramedico (M. A. C. Meijssen).
Address for reprint requests: D. K. Podolsky, Gastrointestinal Unit, GRJ-719, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114-2696.
Received 3 September 1996; accepted in final form 19 November 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Babyatsky, M. W.,
G. Rossiter,
and
D. K. Podolsky.
Expression of transforming growth factors and
in colonic mucosa in inflammatory bowel disease.
Gastroenterology
110:
975-984,
1996[Medline].
2.
Barcellos-Hoff, M. H.,
R. Derynck,
M. L. Tsang,
and
J. A. Weatherbee.
Transforming growth factor- activation in irradiated murine mammary gland.
J. Clin. Invest.
93:
892-899,
1994[Medline].
3.
Bjerknes, M.,
and
H. Cheng.
Methods for the isolation of intact epithelium from the mouse intestine.
Anat. Rec.
199:
565-574,
1981[Medline].
4.
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald,
and
W. J. Rutters.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
5.
Dentener, M. A.,
V. Bazil,
E. J. U. Von Asmuth,
M. Ceska,
and
W. A. Buurman.
Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor-I, IL-6 and IL-8 release by human monocytes and alveolar macrophages.
J. Immunol.
150:
2885-2891,
1993
6.
Dieleman, L. H.,
C. O. Elson,
G. S. Tennyson,
and
K. W. Beagley.
Kinetics of cytokine expression during healing of acute colitis in mice.
Am. J. Physiol.
271 (Gastointest. Liver Physiol. 34):
G130-G136,
1996
7.
Dignass, A. U.,
and
D. K. Podolsky.
Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor .
Gastroenterology
105:
1323-1332,
1993[Medline].
8.
Eckmann, L.,
H. C. Jung,
C. Schurer-Maly,
A. Panja,
E. Morzycka-Wroblewska,
and
M. F. Kagnoff.
Differential cytokine expression by human intestinal epithelial cell lines: regulated expression of interleukin-8.
Gastroenterology
105:
1689-1697,
1993[Medline].
9.
Elson, C. O.,
R. B. Sartor,
G. S. Tennyson,
and
R. H. Riddell.
Experimental models of inflammatory bowel disease.
Gastroenterology
109:
1344-1367,
1995[Medline].
10.
Fearns, C.,
V. V. Kravchenko,
R. J. Ulevitch,
and
D. J. Loskutoff.
Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide.
J. Exp. Med.
181:
857-866,
1995[Abstract].
11.
Grabstein, K. H.,
J. Eisenman,
K. Shanebeck,
C. Rauch,
S. Srinivasan,
V. Fung,
C. Beers,
J. Richardson,
M. A. Schoenborn,
M. Ahdieh,
L. Johnson,
M. R. Alderson,
J. D. Watson,
D. M. Anderson,
and
J. G. Giri.
Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor.
Science
264:
965-968,
1994[Medline].
12.
Grimm, M. C.,
P. Pavli,
E. Van de Pol,
and
W. Doe.
Evidence for a CD14+ population of monocytes in inflammatory bowel disease mucosaimplications for pathogenesis.
Clin. Exp. Immunol.
100:
291-297,
1995[Medline].
13.
Koller, M.,
M. Willheim,
W. Krugluger,
M. Kurz,
P. Hocker,
O. Forster,
and
G. Boltz-Nitulescu.
Immunophenotyping of human bone marrow-derived macrophages.
Scand. J. Immunol.
43:
626-632,
1996[Medline].
14.
Kühn, R.,
J. Löhler,
D. Rennick,
K. Rajewsky,
and
W. Müller.
Interleukin-10-deficient mice develop chronic enterocolitis.
Cell
75:
263-274,
1993[Medline].
15.
Lord, P. C. W.,
L. M. G. Wilmoth,
S. B. Mizel,
and
C. E. McCall.
Expression of interleukin-1 and
genes by human blood polymorphonuclear leukocytes.
J. Clin. Invest.
87:
1312-1321,
1991[Medline].
16.
Mayer, L.,
D. Eisenhardt,
P. Salomon,
W. Bauer,
R. Plous,
and
L. Piccini.
Expression of class II molecules on intestinal epithelial cells in humans. Differences between normal and inflammatory bowel disease.
Gastroenterology
100:
3-12,
1991[Medline].
17.
McCabe, R. P.,
H. Secrist,
M. Botney,
M. Egan,
and
M. G. Peters.
Cytokine mRNA expression in intestine from normal and inflammatory bowel disease patients.
Clin. Immunol. Immunopathol.
66:
52-58,
1993[Medline].
18.
Morrissey, P. J.,
and
K. Charrier.
Induction of wasting disease in SCID mice by transfer of normal CD4+/CD45RBhi T cells and regulation of this autoreactivity by CD4+/CD45RBlo T cells.
Res. Immunol.
145:
357-362,
1994[Medline].
19.
Nathan, C. F.
Secretory products of macrophages.
J. Clin. Invest.
79:
319-326,
1987[Medline].
20.
Perera, P. Y.,
S. N. Vogel,
G. R. Detore,
A. Haziot,
and
S. M. Goyert.
CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knock-out mice stimulated with lipopolysaccharide or taxol.
J. Immunol.
158:
4422-4429,
1997[Abstract].
21.
Reibman, J.,
S. Meixler,
T. C. Lee,
L. I. Gold,
B. N. Cronstein,
K. A. Haines,
S. L. Kolasinski,
and
G. Weissmann.
Transforming growth factor , a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways.
Proc. Natl. Acad. Sci. USA
88:
6805-6809,
1991[Abstract].
22.
Reinecker, H. C.,
E. Y. Loh,
D. J. Ringler,
A. Mehta,
J. L. Rombeau,
and
R. P. MacDermott.
Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa.
Gastroenterology
108:
40-50,
1995[Medline].
23.
Reinecker, H. C.,
R. P. MacDermott,
S. Mirau,
A. Dignass,
and
D. K. Podolsky.
Intestinal epithelial cells both express and respond to interleukin 15.
Gastroenterology
111:
1706-1713,
1996[Medline].
24.
Reinecker, H. C.,
and
D. K. Podolsky.
Human intestinal epithelial cells express functional cytokine receptors sharing the common c chain of the interleukin 2 receptor.
Proc. Natl. Acad. Sci. USA
92:
8353-8357,
1995[Abstract].
25.
Reinecker, H. C.,
M. Stellen,
T. Witthoett,
I. Pflüger,
S. Schreiber,
R. P. MacDermott,
and
A. Raedler.
Increased secretion of IL-1, TNF-
, and IL-6 by isolated lamina propria mononuclear cells cultured from biopsies from patients with ulcerative colitis and Crohn's disease.
Clin. Exp. Immunol.
94:
174-181,
1993[Medline].
26.
Sadlack, B.,
H. Merz,
H. Schorle,
A. Schimpl,
A. C. Feller,
and
I. Horak.
Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene.
Cell
75:
253-261,
1993[Medline].
27.
Schorle, H.,
T. Holtschke,
A. Schimpl,
and
I. Horak.
Development and function of T-cells in mice rendered interleukin-2 deficient by gene targeting.
Nature
352:
621-624,
1991[Medline].
28.
Shirato, K.,
L. LeDuy,
S. Y. Yuan,
and
S. Jothy.
Interleukin-6 and its receptor are expressd in human intestinal epithelial cells.
Virchows Arch
58:
303-308,
1990.
29.
Shull, M. M.,
I. Ormsby,
A. B. Kier,
S. Pawlowski,
R. J. Diebold,
M. Yin,
R. Allen,
C. Sidman,
G. Proetzel,
D. Calvin,
N. Annunziata,
and
T. Doetschman.
Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.
Nature
359:
693-699,
1992[Medline].
30.
Suemori, S.,
C. Ciacci,
and
D. K. Podolsky.
Regulation of transforming growth factor expression in rat intestinal epithelial cell lines.
J. Clin. Invest.
87:
2216-2221,
1991[Medline].
31.
Taurog, J. D.,
J. A. Richardson,
and
J. T. Croft.
The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats.
J. Exp. Med.
180:
2359-2364,
1994[Abstract].
32.
Wahl, S. M.,
N. McCartney-Francis,
and
S. E. Mergenhagen.
Inflammatory and immunomodulatory roles of TGF-beta.
Immunol. Today
10:
258-261,
1989[Medline].
33.
Wright, S. D.,
R. A. Ramos,
P. S. Tobias,
R. J. Ulevitch,
and
J. C. Mathison.
CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science
249:
1431-1433,
1990[Medline].