Departments of 1 Molecular and Cellular Physiology and 2 Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, Louisiana 71130; 3 Department of Gastroenterology, University of Barcelona, Barcelona, Spain; 4 School of Physiology and Pharmacology, University of New South Wales, Sydney, Australia; and 5 Department of Surgery, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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The objectives of
this study were to quantify cytokine mRNA levels and endothelial cell
adhesion molecule message and protein expression in healthy wild-type
and interleukin-10-deficient (IL-10/
) mice
that develop spontaneous and chronic colitis. We found that colonic
message levels of IL-1, IL-6, tumor necrosis factor-
, interferon-
, lymphotoxin-
, and transforming growth factor-
were elevated in colitic mice 10- to 35-fold compared with their healthy wild-type controls. In addition, colonic message levels of
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) were found to be increased 10-, 5-, and 23-fold,
respectively, in colitic IL-10
/
mice compared
with their wild-type controls. Immunoradiolabeling as well as
immunohistochemistry revealed large and significant increases in
vascular surface expression of colonic ICAM-1, VCAM-1, and MAdCAM-1 in
the mucosa as well as the submucosa of the colons of colitic mice.
These data are consistent with the hypothesis that deletion of IL-10
results in the sustained production of proinflammatory cytokines,
leading to the upregulation of adhesion molecules and infiltration of
mononuclear and polymorphonuclear leukocytes into the cecal and colonic interstitium.
leukocytes; inflammation; immunology
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INTRODUCTION |
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INFLAMMATORY BOWEL DISEASE (IBD) is a recurrent
inflammation of the small and/or large bowel of unknown etiology.
Increasingly, experimental and clinical data (17, 18, 21, 55) suggest that the induction and pathogenesis of this disease is a multifactorial process involving interactions among genetic, immune, and environmental factors. Indeed, there is substantial experimental evidence to suggest
that chronic gut inflammation may arise from a dysregulated immune
response to components of the normal gut flora (25, 36, 43-45).
This hypothesis is based on several different studies (16, 49-52,
56) demonstrating that targeted deletion of certain genes known to be
important in the immune response induces spontaneous colitis in mice
that is dependent on the presence of normal enteric bacteria. One such
model of IBD is the interleukin-10 deficient (IL-10/
) model of colitis (4, 14, 31, 33,
46-48). IL-10 is a T helper cell 2 (Th2)-type cytokine with a
spectrum of biological activities. It is produced by several different
cell types, including T cells, macrophages, and CD5+ B
cells, and functions to inhibit the synthesis of T helper cell 1 (Th1)-type and/or macrophage-derived cytokines [e.g., tumor necrosis factor-
(TNF-
), IL-12, interferon-
(IF-
)] by
different populations of leukocytes (23, 32). In addition, IL-10
attenuates the formation of reactive oxygen and nitrogen metabolites as
well as inhibiting surface expression of major histocompatibility
complex class II molecules and intercellular adhesion molecule-1
(ICAM-1). Not surprisingly, IL-10
/
mice
develop moderate to severe enterocolitis when raised under conventional
housing conditions (4, 14, 31, 33, 46-48). The colitis develops in
the cecum and proximal colon and is associated with the infiltration of
large numbers of mononuclear leukocytes such as lymphocytes, monocytes,
and plasma cells (4, 14, 31, 33, 46-48). Animals derived and
raised under germ-free conditions fail to develop this distal bowel
inflammation (4, 47, 48).
Recent studies suggest that this model of IBD expresses more of a
Th1-type response, indicating uncontrolled activation of T cells and
macrophages (4, 47, 48). These proinflammatory cytokines are thought to
initiate and/or perpetuate chronic gut inflammation by virtue of their
ability to upregulate the surface expression of different endothelial
cell adhesion molecules (ECAMs), thereby enhancing the adhesion and
infiltration of different populations of leukocytes (13, 20, 24, 27).
Although the IL-10/
model of enterocolitis
has been used by several laboratories to explore certain immunologic
aspects of chronic gut inflammation, no systematic or quantitative
information is available regarding the relationship between cytokine
and ECAM expression in the IL-10
/
model of
colitis. Thus the objectives of this study were to quantify message
levels for a variety of different Th1 and Th2 cytokines and ECAM
message and protein expression in the colons of healthy wild-type and
IL-10
/
mice with distal bowel disease.
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MATERIALS AND METHODS |
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Animals
IL-10Colonic RNA Isolation and Quantification of mRNA by RNase Protection Assay
Colons were removed, cleaned of intestinal contents, and weighed, and total length was determined. The tissue was then divided into proximal and distal colon. Total RNA was isolated from the tissues using TRIzol reagent (GIBCO BRL), according to the manufacturer's instructions. RNase protection assay was performed using [ECAM Expression In Vivo
Monoclonal antibodies used for experiments. The dual radiolabeled monoclonal antibody (MAb) technique was used to quantify ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1 expression in different vascular beds (12, 19, 27). YN-1, a rat IgG2b directed against mouse ICAM-1 (Bayer, West Haven, CT); 3C4 (MIC2/4), a rat IgG2a directed against mouse ICAM-2 (Pharmingen); MK1.9.1, a rat IgG1 targeted against mouse VCAM-1 (Bayer), and MECA-367, a rat IgG2a targeted against mouse MAdCAM-1 (Pharmingen) were used as the MAbs. P-23, a murine IgG1 against human P-selectin (Pharmacia-Upjohn, Kalamazoo, MI), was used as the control nonbinding MAb.
The binding MAbs directed against ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1 were radiolabeled with 125I (Dupont NEN, Boston, MA), whereas the nonbinding MAb (P-23) was labeled with 131I. Radioiodination of the MAbs were performed by the iodogen method as previously described (12, 19, 27).Animal procedures.
At ~12 wk of age, both wild-type and IL-10/
mice were anesthetized with ketamine hydrochloride (150 mg/kg im) and
xylazine (7.5 mg/kg im), and the right jugular vein and carotid artery were cannulated with polyethylene tubing.
Calculation of ECAM expression.
A 14800 Wizard 3 gamma counter (Wallac, Turku, Finland), with automatic
correction for background activity and spillover, was used to count
125I and 131I activities in each organ and in a
100-µl plasma sample. The injected activity in each experiment was
calculated by counting a 4-µl sample of mixture containing the
radiolabeled MAbs. The amount of radioactivity remaining in the tube
used to mix the MAbs and the syringe used to inject the mixture was
subtracted from the total calculated injected activity. The accumulated
activity of each MAb in an organ was expressed as the percentage of the injected dose (%ID) per gram of tissue. ECAM expression was calculated by subtracting the nonspecific tissue accumulation of MAb from the
accumulation of binding MAb as follows: VCAM-1 expression = (%ID/g for
125I) (%ID/g for 131I) × (%ID
for 125I in plasma)/(%ID for 131I in plasma).
This formula corrects the tissue accumulation of nonbinding MAb for the
relative plasma levels of both binding and nonbinding MAbs to estimate
the nonspecific tissue accumulation of MAb. This value, expressed as
%ID/g, was converted to µg MAb/g tissue by multiplying the above
value by the total injected binding MAb (in µg), divided by 100.
Immunohistochemical Assessment of ECAM Expression
Separate groups of animals (n = 3 for each group) were anesthetized with ketamine hydrochloride (150 mg/kg im) and xylazine (7.5 mg/kg im), and the right jugular vein and carotid artery were cannulated with polyethylene tubing. For assessing the immunolocalization of the four ECAMs, the same total amount of MAb against the different ECAMs was used as in the dual radiolabeled MAb technique described above. However, the MAbs were not radiolabeled; 50 µg of ICAM-1 MAb, 70 µg of ICAM-2 MAb, 30 µg of VCAM-1 MAb, and 10 µg of MAdCAM-1 MAb were infused in each experiment. Five minutes after MAb injection, the vasculature was completely flushed with bicarbonate-buffered saline, and tissues were excised exactly as described for the radiolabeled MAb experiments.The colons were divided into proximal and distal portions, and the contents were removed by gentle flushing with Zamboni's fixative using a syringe and a 30-gauge needle. The samples were then placed in Zamboni's fixative at 4°C overnight. The following day, the samples were placed in 80% ETOH to remove picric acid from the solution, cleared in DMSO, and washed three times in PBS. The samples were then stored in PBS containing 30% sucrose and 0.01% sodium azide at 4°C overnight. Samples were placed in OCT embedding medium, and the blocks were frozen with isopentane chilled in liquid nitrogen. Frozen samples were sectioned in a cryostat at 10-µm thickness and collected on poly-L-lysine-coated slides. The slides were then dried over P2O5 in a vacuum desiccator for 30 min.
Colon sections were blocked with 10% normal donkey serum and then incubated with a donkey anti-rat secondary antibody against ECAMs conjugated to Cy3 fluorochrome in a humid box for 1 h. The slides were washed in PBS three times for 10 min each time, and the sections around and on the back were dried before mounting. Slides were then mounted in Mowiol-glycerol containing 2.5% 1,4-diazabicyclo[2.2.2]octane (pH 8.5). Sections were also stained with hematoxylin and eosin for standard observation of histology.
Histopathology
Colons from three representative wild-type and IL-10Statistical Analysis
All values are presented as means ± SE. Data were analyzed using standard statistical analyses, i.e., one-way ANOVA and a paired and unpaired Student's t-test. Statistical significance was set at P < 0.05. ![]() |
RESULTS |
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Histopathology
Of the IL-10Cytokine mRNA Expression
The pattern of cytokine mRNA expression in wild-type and colitic IL-10
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ECAM Expression
Colonic message levels of ICAM-1, VCAM-1, and MAdCAM-1 in colitic IL-10
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DISCUSSION |
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The distal bowel is continuously exposed to numerous environmental
antigens and bacterial products, resulting in a constant threat of a
potentially dangerous and debilitating inflammatory response. For this
reason, immune responses of the intestine and colon are under very
strict regulatory control (17, 43, 54). A variety of recently developed
mouse models have provided important insights into the mechanisms that
control the immune response in the intestinal tract and the aberrations
that lead to a disease state. Data obtained from a variety of different
studies using genetically engineered or immune-manipulated models of
colitis suggest a regulatory network in which IL-10 and TGF-
regulate expression of potentially damaging cytokines, such as IFN-
and TNF-
(17, 43, 48). One of the best-studied models of spontaneous and chronic colitis is the IL-10
/
model bred
to the 129 strain background (4, 47, 48). All of these mice develop
colitis when housed under conventional conditions, due principally to
the absence of the major regulatory cytokine IL-10 (4, 47, 48). Studies
with this model, as well as others, suggest that chronic colitis
results from a dysregulated immune response to components of the normal
gut flora (43, 48). One of the earliest manifestations of this
uncontrolled activation of T cells within the cecal and colonic
interstitium is the sustained production of proinflammatory mediators
such as the Th1 and macrophage-derived cytokines. In the present study,
we found large and significant increases in IL-1
, IL-1
, IL-6,
IFN-
, and TNF-
mRNA in colons of IL-10
/
mice compared with their healthy controls (Fig. 1). Each of these cytokines was strongly associated with ongoing colitis. These findings
are consistent with previous reports (2, 7, 14, 38, 39, 44) implicating
IFN-
and TNF-
as major mediators in the pathophysiology of
chronic of colitis.
However, the patterns of other cytokine species were unexpected. In the
IL-10/
colitis model, it was expected that no
IL-10 mRNA would be detected, and this was found to be true. This
finding is also consistent with the concept that IL-10 production is
essential for the development and expression of function of regulatory
CD4+ T cells, designated Tr-1 (25). Interestingly, it
has been reported that IL-10 is an important growth and
differentiation factor for Tr-1 CD4+ T cells, which exert
their regulatory function principally through the production of the
inhibitory cytokine TGF-
(25). However, the level of mRNA for
TGF-
1 was significantly elevated, yet profound disease was still
present. These results suggest that TGF-
alone, in the absence of
IL-10, or in the presence of elevated levels of other cytokines is not
sufficient to limit the development of IBD. The cellular origin of
TGF-
in this model is not known. It is likely that it is derived
from a non-T lymphocyte source within the site of inflammation.
Another unexpected observation made in the present study was the
divergence in expression of LT- and LT-
mRNA species. In healthy
wild-type mice, low but discernible levels of both mRNA species are
present. However, in colitic mice, LT-
mRNA was very high, whereas
LT-
mRNA was very low to undetectable. This was an unexpected
observation, since the mature, secreted form of the lymphotoxin
molecule is composed of a heterotrimeric complex consisting of one
chain of LT-
and two chains of LT-
(1). The absence of the LT-
mRNA species may indicate that, during chronic inflammation, functional
LT is not secreted by activated CD4+ T cells within the
site of inflammation. However, LT plays a critical role in the
initiation of disease, since IBD does not develop in mice in which the
signaling via the LT-
receptor (LT-
R) is blocked by a soluble
LT-
R-lg fusion molecule (34). A potential consequence
of the uncontrolled production of Th1 and macrophage-derived cytokines
is the transcriptional activation of ECAM expression within the colonic
microvasculature. A number of different experimental and clinical
studies (24) suggest that leukocyte-endothelial cell interactions play
a critical role in initiating and/or perpetuating the chronic gut
inflammation observed in experimental as well as human IBD. The
microvasculature occupies a critical position in the inflammatory
response by virtue of its ability to differentially regulate the
infiltration of specific populations of leukocytes. In this respect,
the postcapillary venules may define the type of inflammatory cell that
may gain access to the colonic interstitium and dictate whether an
acute and/or chronic inflammation will ensue.
In the present study, we demonstrate that chronic enterocolitis in
IL-10/
mice is associated with enhanced
expression of ICAM-1, VCAM-1, and MAdCAM-1 message and
protein in the colon (Figs. 2 and 3). The vascular localization of
these ECAMs was confirmed using immunohistochemistry (Fig. 4).
ICAM-1 and ICAM-2 are ECAMs, which are members of the immunoglobulin
supergene family (11). ICAM-1 contains five immunoglobulin-like extracellular domains of which the first NH2-terminal
immunoglobulin-like domain recognizes CD11a/CD18 and the third
immunoglobulin-like domain recognizes CD11b/CD18 (15). ICAM-1 is
basally expressed on endothelial cells, and its expression is increased
in response to activation of endothelial cells with certain Th1 and/or
macrophage-derived cytokines or bacterial products. Maximal expression
of ICAM-1 is achieved within 4-8 h after activating the
endothelial cells and is associated with maximal levels of leukocyte
adherence. Indeed, we observed a significant increase in both message
levels as well as surface expression of ICAM-1 in the
IL-10/
colitic mice (Figs. 2 and 3). The
disparity between the magnitude of the increase in mRNA vs. surface
expression of ICAM-1 (10-fold vs. 50%, respectively) most probably
reflects the large increase in infiltrating ICAM-1-containing cells
(e.g., lymphocytes, monocytes), whereas the more modest increase in
ICAM-1 surface expression reflects increases in ICAM-1 only on vascular
endothelium (Fig. 6). Several different reports (29, 35, 37, 41) have
demonstrated enhanced staining for ICAM-1 in biopsies obtained from
patients with active ulcerative colitis and Crohn's disease.
Furthermore, recent experimental and clinical studies (3, 58) have
demonstrated that infusion of an antisense oligonucleotide directed
against ICAM-1 message produces clinical improvement in a mouse model of colitis and in steroid-resistant Crohn's disease patients.
ICAM-2 is a truncated form of ICAM-1 containing only two
immunoglobulin-like extracellular domains. Like ICAM-1, ICAM-2 also is
basally expressed on endothelial cells, but its expression is not
increased in response to cytokine exposure (40). We found that the
expression of ICAM-2 was similar between
IL-10/
and wild-type mice, suggesting that
the density of the colonic vascular bed (i.e., vascular surface area)
was similar between colitic and control mice (Figs. 3 and 4). Together,
these data suggest that the observed increases in ICAM-1, VCAM-1, and
MAdCAM-1 expression are due to increases in expression on the vascular endothelium rather than increases in colonic vascularity.
VCAM-1 is an inducible ECAM, which is known to mediate the firm
adhesion of monocytes and lymphocytes to endothelial cells (22). Thus
this ECAM may act to promote adhesion and immigration of chronic
inflammatory cells into colonic interstitium (22). Proinflammatory cytokines, such as TNF- and IL-1
, both of which are upregulated in this model, induce VCAM-1 expression in various organs via activation of nuclear factor-
B (24). It is known that
lymphocytes, monocytes, and eosinophils possess a
1
integrin, called very late activation antigen-4
(
4
1), which binds to VCAM-1 (and
MAdCAM-1) (22). The kinetics of VCAM-1 expression on vascular endothelial cells closely resemble that seen with ICAM-1. Although a
great deal of interest has been generated regarding the possibility that VCAM-1 may be important in lymphocyte infiltration in IBD, several
investigators (29, 30, 37) have failed to demonstrate enhanced
expression of VCAM-1 in biopsies obtained from patients with active
colitis. These observations are particularly surprising in view of data
obtained in the present study and in three recent reports (5, 6, 26),
which demonstrated that primary cultures of microvascular endothelial
cells isolated from human intestine and colon respond to different
proinflammatory cytokines with enhanced surface expression of VCAM-1.
The reasons for these differences between experimental and human IBD
are not known but may represent the inherent variability known to be
associated with immunohistochemistry compared with the more objective,
radiolabeled MAb method to quantify ECAM expression in vivo.
Alternatively, the quality of the antibodies used for the
immunolocalization studies may represent an important determinant for
accurate determination of VCAM-1. In fact, our data would suggest that
the dual radiolabeled MAb technique provides the necessary sensitivity
to accurately determine low-level constitutive expression of VCAM-1 in
the absence of any immunohistochemical evidence for VCAM-1-positive
vessels in healthy controls (Fig. 4).
One of the more interesting findings in the present study was the
localization of MAdCAM-1 to dilated mucosal and submucosal vessels in
the colon, which appeared to be junctional in nature (Fig. 4).
MAdCAM-1 was first described as an endothelial cell surface
molecule that is selectively expressed in mucosal organs and required
for lymphocyte homing to mucosal lymphoid tissue (9, 10, 53). Murine
endothelial cells can be induced to express high levels of MAdCAM-1 in
response to proinflammatory cytokines (8, 9). Recently, it has been
shown (28, 57) that the adhesive interactions mediated by MAdCAM-1 and
its lymphocyte receptor, 4
7, are involved
in leukocyte recruitment to chronic inflammatory diseases of the bowel.
MAbs specific for
7 and MAdCAM-1 not only blocked
recruitment of lymphocytes to the colitic colon but also reduced the
severity of colonic inflammation in severe combined immunodeficient
mice reconstituted with CD4+ T cells enriched for the
CD45RBhigh subpopulation (42). Our studies
indicate that MAdCAM-1 expression in colon is significantly increased
in colitic IL-10
/
mice compared with
wild-type mice. Together, these results suggest that MAdCAM-1 may also
play an important role in initiating and/or maintaining chronic
inflammation of colon in IL-10
/
mice and may
be a relevant therapeutic target for patients with IBD.
In summary, this study represents the first demonstration that ICAM-1,
VCAM-1, and MAdCAM-1 are upregulated in the chronically inflamed cecum
and colon of IL-10/
mice. Furthermore, this
study indicates that the transcriptional activation of certain Th1 and
macrophage-derived cytokines is associated with the enhanced surface
expression of ECAMs and may mediate the infiltration of immune
modulating and/or potentially injurious leukocytes. The specific
cytokine(s) or ECAM(s) responsible for the initiation and/or
perpetuation of the chronic gut inflammation in this model of colitis
is presently under investigation.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Arthritis Center of Excellence, Feist Foundation of Louisiana State University Medical Center in Shreveport, the Crohn's and Colitis Foundation of America, and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47663 and DK-43785).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. B. Grisham, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Medical Center, P.O. Box 33932, Shreveport, LA, 71130 (E-mail: mgrish{at}lsumc.edu).
Received 1 November 1999; accepted in final form 11 January 2000.
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