Am J Physiol Gastrointest Liver Physiol

278: G734–G743, 2000.

Cytokine and endothelial cell adhesion molecule

expression in interleukin-10-deficient mice
SHIGEYUKI KAWACHI,1 STEPHEN JENNINGS,2 JULIAN PANES,3 ADAM COCKRELL,1
F. STEPHEN LAROUX,1 LAURA GRAY,1 MICHAEL PERRY,4 HENRY VAN DER HEYDE,2
EDWARD BALISH,5 D. NEIL GRANGER,1 ROBERT A. SPECIAN,1
AND MATTHEW B. GRISHAM1
Departments of 1Molecular and Cellular Physiology and 2Microbiology and Immunology, Louisiana
State University Medical Center, Shreveport, Louisiana 71130; 3Department of Gastroenterology,
University of Barcelona, Barcelona, Spain; 4School of Physiology and Pharmacology,
University of New South Wales, Sydney, Australia; and 5Department of Surgery,
University of Wisconsin, Madison, Wisconsin 53706

Kawachi, Shigeyuki, Stephen Jennings, Julian Panes,
Adam Cockrell, F. Stephen Laroux, Laura Gray, Michael
Perry, Henry van der Heyde, Edward Balish, D. Neil
Granger, Robert A. Specian, and Matthew B. Grisham.
Cytokine and endothelial cell adhesion molecule expression
in interleukin-10-deficient mice. Am J Physiol Gastrointest
Liver Physiol 278: G734–G743, 2000.—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-102/2) mice
that develop spontaneous and chronic colitis. We found that
colonic message levels of IL-1, IL-6, tumor necrosis factor-a,
interferon-g, lymphotoxin-b, and transforming growth fac-
tor-b 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-102/2 mice compared with their wild-type controls. Immu-
noradiolabeling 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 cyto-
kines, leading to the upregulation of adhesion molecules and
infiltration of mononuclear and polymorphonuclear leuko-
cytes into the cecal and colonic interstitium.
leukocytes; inflammation; immunology
INFLAMMATORY BOWEL DISEASE (IBD) is a recurrent in-
flammation of the small and/or large bowel of unknown
etiology. Increasingly, experimental and clinical data
(17, 18, 21, 55) suggest that the induction and pathogen-
esis of this disease is a multifactorial process involving
interactions among genetic, immune, and environmen-
tal factors. Indeed, there is substantial experimental
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G734 0193-1857/00 $5.00 Copyright r 2000 the American Physiological Society
evidence to suggest that chronic gut inflammation may
arise from a dysregulated immune response to compo-
nents 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-102/2) 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 CD51 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-a (TNF-a), IL-12, inter-
feron-g (IF-g)] 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 histocompatibil-
ity complex class II molecules and intercellular adhe-
sion molecule-1 (ICAM-1). Not surprisingly, IL-102/2
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 inflam-
mation (4, 47, 48).
Recent studies suggest that this model of IBD ex-
presses more of a Th1-type response, indicating uncon-
trolled activation of T cells and macrophages (4, 47, 48).
These proinflammatory cytokines are thought to ini-
tiate and/or perpetuate chronic gut inflammation by
virtue of their ability to upregulate the surface expres-
sion of different endothelial cell adhesion molecules
(ECAMs), thereby enhancing the adhesion and infiltra-
tion of different populations of leukocytes (13, 20, 24,
27). Although the IL-102/2 model of enterocolitis has
been used by several laboratories to explore certain
immunologic aspects of chronic gut inflammation, no
http://www.ajpgi.org
 CYTOKINE AND ADHESION MOLECULES IN COLITIS
systematic or quantitative information is available
regarding the relationship between cytokine and ECAM
expression in the IL-102/2 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-102/2 mice with distal bowel
disease.
MATERIALS AND METHODS
Animals
IL-102/2 mice were generated by gene-targeting deletion in
embryonic stem cells as described by Kuhn et al. (31). The
mice used in this study were bred onto a 129/SvEv back-
ground. When raised in specific pathogen-free conditions,
IL-102/2 mice develop spontaneous colitis at 8–12 wk of age.
Wild-type mice (129/SvEv) were used as controls at 8–12 wk
of age and were obtained from Taconic Labs (Germantown,
NY). Development of colitis was assessed as previously
described by Berg et al. (4), using serum amyloid A protein
levels as an indicator of ongoing inflammation.
Colonic 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 protec-
tion assay was performed using [a-32P]UTP-labeled MCK2b
and MCK3b RiboQuant probe sets (Pharmingen) for the
different Th1 and Th2 cytokines. ICAM-1, vascular cell
adhesion molecule-1 (VCAM-1), and mucosal addressin cell
adhesion molecule-1 (MAdCAM-1) message levels were quan-
tified using a custom probe set obtained from Pharmingen
(San Diego, CA). Probe synthesis, hybridization, RNase treat-
ment, gel preparation, and electrophoresis were all per-
formed according to the manufacturer’s instructions. Autora-
diographs of the resulting protected species were processed
into digital images analyzed and quantified using Image-
Quant software (Molecular Dynamics). The relative levels of
mRNA for the two different cytokines and ECAMs were
determined from the ratio of the density obtained for each
cytokine to the density obtained for the mRNA of the house-
keeping gene glyceraldehyde-3-phosphate dehydrogenase.
Multiple exposures of each reaction were generated to ensure
accuracy of the comparison of the two signals under different
signal density conditions.
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 tar-
geted 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
G735
NEN, Boston, MA), whereas the nonbinding MAb (P-23) was
labeled with 131I. Radioiodination of the MAbs were per-
formed by the iodogen method as previously described (12,
19, 27).
Animal procedures. At ,12 wk of age, both wild-type and
IL-102/2 mice were anesthetized with ketamine hydrochlo-
ride (150 mg/kg im) and xylazine (7.5 mg/kg im), and the right
jugular vein and carotid artery were cannulated with polyeth-
ylene tubing.
For assessing the ICAM-1 expression, a mixture of 10 µg of
125I anti-ICAM-1 MAb and 40 µg of unlabeled anti-ICAM-1
MAb was given with an amount of 131I-labeled P-23 necessary
to ensure a total 131I-injected activity of 400,000–600,000 cpm
through the jugular vein cannula (total vol 200 µl). In the
ICAM-2 experiments, a mixture of 10 µg of 125I-labeled
anti-ICAM-2 MAb and 60 µg of unlabeled anti-ICAM-2 MAb
was given with an appropriate amount of 131I P-23 (400,000–
600,000 cpm). In the VCAM-1 experiments, a mixture of 10
µg of 125I-labeled anti-VCAM-1 MAb and 20 µg of unlabeled
anti-VCAM-1 MAb was administered with an appropriate
amount of 131I-labeled P-23 through the jugular vein. In the
MAdCAM-1 experiments, only 10 µg of 125I-labeled anti-
MAdCAM-1 MAb were administrated with an appropriate
amount of 131I-labeled P-23 through the jugular vein. These
dosages were selected on the basis of pilot studies demonstrat-
ing optimum activity and receptor saturation in the tissues
examined under constitutive and stimulated conditions.
A blood sample was taken 5 min after MAb injection.
Bicarbonate-buffered saline was isovolumically exchanged by
infusion of buffer through the jugular vein and simultaneous
withdrawal of blood and buffer from the artery. After the
vascular system was completely flushed with bicarbonate-
buffered saline, the small intestine, cecum, large intestine,
and a variety of other tissues were harvested and weighed.
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 calcu-
lated 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 expres-
sion 5 (%ID/g for 125I) 2 (%ID/g for 131I) 3 (%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 5 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,
G736 CYTOKINE AND ADHESION MOLECULES IN COLITIS
and 10 µg of MAdCAM-1 MAb were infused in each experi-
ment. 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 embed-
ding 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-102/2
mice were recovered from animals at necropsy and fixed in
Zamboni’s fixative. The tissues were rinsed in 0.1 M phos-
phate buffer and dehydrated with 95% ethanol. The tissues
were then embedded in glycol methacrylate (JB-4; Poly-
sciences, Warrington, PA). Sections (1–2 µm) were cut on
glass knives, stained with hematoxylin and eosin, dried on a
warming plate, and mounted in Permount. Samples were
evaluated, and photographs were taken with a SenSys digital
camera and processed with Metamorph.
Statistical Analysis
All values are presented as means 6 SE. Data were
analyzed using standard statistical analyses, i.e., one-way
ANOVA and a paired and unpaired Student’s t-test. Statisti-
cal significance was set at P , 0.05.
RESULTS
Histopathology
Of the IL-102/2 mice, 100% developed moderate to
severe inflammation with marked thickening of the
intestinal wall, cecum, and proximal colon at 3 mo of
age. Corresponding to this spontaneous colitis was a
dramatic increase in serum amyloid A protein levels (73
vs. 2,381 µg/ml for wild-type vs. IL-102/2 mice; P ,
0.01). The colon in IL-102/2 mice was significantly
longer than that of wild-type mice (9.23 6 0.18 vs.
7.02 6 0.22 cm), and the weight of colon in IL-102/2
mice was significantly heavier than that of wild-type
mice (0.57 6 0.03 vs. 0.20 6 0.04 g). Histopathology
revealed moderate to severe inflammation of the colon.
The colonic mucosa exhibited epithelial hyperplasia
with infiltration of chronic inflammatory cells such as
lymphocytes, plasma cells, and macrophages but also
contained small-to-moderate numbers of neutrophils.
This infiltrate was present in both the mucosa and the
submucosa. Transmural inflammation, multiple small
superficial erosions of the mucosa covered by inflamma-
tory exudate, mucus, and cellular debris, and crypt
abscesses were present as well.
Cytokine mRNA Expression
The pattern of cytokine mRNA expression in wild-
type and colitic IL-102/2 mice revealed enhanced expres-
sion of IL-1a, IL-1b, interleukin-1 receptor antagonist,
and IL-18 as well as IL-6, IFN-g, TNF-a, and lympho-
toxin (LT)-b compared with their healthy controls (Fig.
1). LT-a was present at low levels in colitic colons yet
significantly above the levels detected in the healthy
wild-type mice (Fig. 1). IL-10 mRNA was totally absent
in colitic IL-102/2 mice, as expected; however, TGF-b1
mRNA levels were increased approximately threefold
(Fig. 1). Colitic IL-102/2 mice also expressed low but
detectable levels of mRNA for the IL-12 p35 and p40
subunits (Fig. 1).
ECAM Expression
Colonic message levels of ICAM-1, VCAM-1, and
MAdCAM-1 in colitic IL-102/2 mice were found to be
significantly increased by 10-, 5-, and 23-fold, respec-
tively, compared with their wild-type controls (Fig. 2).
Corresponding to these large and significant increases
in ECAM mRNA was enhanced surface expression of
colonic ICAM-1, VCAM-1, and MAdCAM-1 (Fig. 3).
Colonic VCAM-1 and MAdCAM-1 expression were en-
hanced eight- and fourfold, respectively, whereas
ICAM-1 surface expression was increased by 50% (Fig.
3). Immunolocalization of each ECAM using the same
method described for the radiolabeled MAb technique
revealed enhanced vascular staining of ICAM-1,
VCAM-1, and MAdCAM-1 in colitic mice compared
with their wild-type controls (Fig. 4). Constitutive expres-
sion of VCAM-1 in healthy wild-type mice was below
the sensitivity of the immunolocalization method; how-
ever, we were able to observe enhanced vascular expres-
sion of VCAM-1 in the IL-102/2 mice (Fig. 4). We
observed low levels of constitutive expression of ICAM-1
that increased in submucosal and mucosal vessels in
the IL-102/2 mice (Fig. 4). Enhanced vascular expres-
sion of MAdCAM-1 was also observed in colitic mice;
however, the staining pattern for MAdCAM-1 was quite
different from those of the other ECAMs. We observed
staining of large mucosal and submucosal vessels that
appeared approximately two to three times larger than
those vessels from wild-type animals with constitutive
expression of MAdCAM-1 (Fig. 4). Furthermore, the
staining appeared to be junctional in nature. Because
ICAM-2 is constitutively expressed and not upregu-
lated in response to a variety of proinflammatory
mediators and/or cytokines (40), we quantified this
ECAM to assess vascular surface area in wild-type vs.
colitic mice. We found, using either the dual radiolabel
or immunohistochemical techniques, that ICAM-2 ex-
 CYTOKINE AND ADHESION MOLECULES IN COLITIS G737

Fig. 1. Relative message levels of different colonic cyto-

kines in wild-type and interleukin-10-deficient (IL-
102/2) mice normalized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). Three representative wild-
type mice (filled bars) and three colitic IL-102/2 mice
(open bars) were used for these studies. Relative in-
creases in cytokine mRNA were determined from ratio
of density obtained for each cytokine to density deter-
mined for housekeeping gene GAPDH. Specific proto-
cols are described in MATERIALS AND METHODS. Data are
expressed as mean values. LT-a or -b, lymphotoxin-a or
-b; TNF-a, tumor necrosis factor-a; IL-1Ra, interleu-
kin-1 receptor antagonist; IFN-g, interferon-g; TGF,
transforming growth factor; MIF, macrophage migra-
tory inhibitory factor.

Fig. 2. Colonic message levels of inter-

cellular adhesion molecule-1 (ICAM-1),
vascular cell adhesion molecule-1
(VCAM-1), and mucosal addressin cell
adhesion molecule-1 (MAdCAM-1) in
wild-type and IL-102/2 mice. Specific
methods for RNA isolation and RNase
protection assay are described in MATE-
RIALS AND METHODS. L32, ribosomal
structural protein. B: filled bars, wild-
type mice; open bars, IL-102/2 mice.
G738 CYTOKINE AND ADHESION MOLECULES IN COLITIS
Fig. 3. Endothelial cell adhesion molecule (ECAM) ex-
pression in colons from wild-type (wt) and IL-102/2
mice. Vascular surface expression of the different ECAMs
was quantified using the dual radiolabeled monoclonal
antibody (MAb) technique described in MATERIALS AND
METHODS. ECAMs were quantified in mice at 3 mo of
age. A: ICAM-1. B: VCAM-1. C: MAdCAM-1. D: ICAM-2.
* P , 0.05 vs. wild type.
pression was similar in wild-type and IL-102/2 mice
(Figs. 3 and 4). In addition to the colon, we observed
significant increases in expression of ICAM-1, VCAM-1,
and MAdCAM-1 in the cecum of colitic IL-102/2 mice,
whereas only VCAM-1 was enhanced in the small
bowel of the colitic animals (Figs. 5 and 6).
DISCUSSION
The distal bowel is continuously exposed to numer-
ous environmental antigens and bacterial products,
resulting in a constant threat of a potentially danger-
ous 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 pro-
vided 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-b regulate expression of potentially damaging
cytokines, such as IFN-g and TNF-a (17, 43, 48). One of
the best-studied models of spontaneous and chronic
colitis is the IL-102/2 model bred to the 129 strain
background (4, 47, 48). All of these mice develop colitis
when housed under conventional conditions, due princi-
pally 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 manifesta-
tions of this uncontrolled activation of T cells within the
cecal and colonic interstitium is the sustained produc-
tion of proinflammatory mediators such as the Th1 and
macrophage-derived cytokines. In the present study,
we found large and significant increases in IL-1a,
IL-1b, IL-6, IFN-g, and TNF-a mRNA in colons of
IL-102/2 mice compared with their healthy controls
(Fig. 1). Each of these cytokines was strongly associ-
ated with ongoing colitis. These findings are consistent
with previous reports (2, 7, 14, 38, 39, 44) implicating
IFN-g and TNF-a as major mediators in the pathophysi-
ology of chronic of colitis.
However, the patterns of other cytokine species were
unexpected. In the IL-102/2 colitis model, it was ex-
pected 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 regula-
tory CD41 T cells, designated Tr-1 (25). Interestingly,
it has been reported that IL-10 is an important growth
and differentiation factor for Tr-1 CD41 T cells, which
exert their regulatory function principally through the
production of the inhibitory cytokine TGF-b (25). How-
ever, the level of mRNA for TGF-b1 was significantly
elevated, yet profound disease was still present. These
results suggest that TGF-b 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-b 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-a and
LT-b mRNA species. In healthy wild-type mice, low but
discernible levels of both mRNA species are present.
However, in colitic mice, LT-b mRNA was very high,
whereas LT-a 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-a and two chains of LT-b (1). The absence of the LT-a
mRNA species may indicate that, during chronic inflam-
mation, functional LT is not secreted by activated CD41
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-b receptor (LT-bR) is blocked by a soluble
 CYTOKINE AND ADHESION MOLECULES IN COLITIS G739

Fig. 4. Immunolocalization of different ECAMs in colons obtained from wild-type (wt) and IL-102/2 mice. At 3 mo of
age, unlabeled rat anti-mouse MAb specific for the different ECAMs was injected (iv), and tissues were processed as
described in MATERIALS AND METHODS. Frozen sections were stained with donkey anti-rat secondary antibody
conjugated to Cy3 fluorochrome. Note vascular immunolocalization of each MAb, indicating little or no staining of
extravascular cells and tissue (magnification, 3100).

LT-bR-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 interac-
tions play a critical role in initiating and/or perpetuat-
ing the chronic gut inflammation observed in experimen-
tal as well as human IBD. The microvasculature
occupies a critical position in the inflammatory re-
sponse 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
G740 CYTOKINE AND ADHESION MOLECULES IN COLITIS

Fig. 5. ECAM expression in cecums from wild-type (wt)

and IL-102/2 mice. Vascular surface expression of the
different ECAMs was quantified using the dual radiola-
beled MAb technique described in MATERIALS AND METH-
ODS. ECAMs were quantified in mice at 3 mo of age. A:
ICAM-1. B: VCAM-1. C: MAdCAM-1. D: ICAM-2. * P ,
0.05 vs. wild type.

colonic interstitium and dictate whether an acute and/or
chronic inflammation will ensue.
In the present study, we demonstrate that chronic
enterocolitis in IL-102/2 mice is associated with en-
hanced 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 mem-
bers of the immunoglobulin supergene family (11).
ICAM-1 contains five immunoglobulin-like extracellu-
lar domains of which the first NH2-terminal immuno-
globulin-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-102/2
colitic mice (Figs. 2 and 3). The disparity between the
magnitude of the increase in mRNA vs. surface expres-
sion 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 vascu-
lar 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 oligonucleo-

Fig. 6. ECAM expression in small intestines from wild-

type (wt) and IL-102/2 mice. Vascular surface expres-
sion of the different ECAMs was quantified using the
dual radiolabeled MAb technique described in MATERI-
ALS AND METHODS. ECAMs were quantified in mice at 3
mo of age. A: ICAM-1. B: VCAM-1. C: MAdCAM-1. D:
ICAM-2. * P , 0.05 vs. wild type.
 CYTOKINE AND ADHESION MOLECULES IN COLITIS
tide 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-102/2
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 in-
creases in ICAM-1, VCAM-1, and MAdCAM-1 expres-
sion are due to increases in expression on the vascular
endothelium rather than increases in colonic vascular-
ity.
VCAM-1 is an inducible ECAM, which is known to
mediate the firm adhesion of monocytes and lympho-
cytes to endothelial cells (22). Thus this ECAM may act
to promote adhesion and immigration of chronic inflam-
matory cells into colonic interstitium (22). Proinflamma-
tory cytokines, such as TNF-a and IL-1b, both of which
are upregulated in this model, induce VCAM-1 expres-
sion in various organs via activation of nuclear fac-
tor-kB (24). It is known that lymphocytes, monocytes,
and eosinophils possess a b1 integrin, called very late
activation antigen-4 (a4b1), which binds to VCAM-1
(and MAdCAM-1) (22). The kinetics of VCAM-1 expres-
sion 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 demon-
strate enhanced expression of VCAM-1 in biopsies
obtained from patients with active colitis. These obser-
vations 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 ex-
pression 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 quan-
tify ECAM expression in vivo. Alternatively, the quality
of the antibodies used for the immunolocalization stud-
ies may represent an important determinant for accu-
rate determination of VCAM-1. In fact, our data would
suggest that the dual radiolabeled MAb technique
provides the necessary sensitivity to accurately deter-
mine 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 mol-
G741
ecule that is selectively expressed in mucosal organs
and required for lymphocyte homing to mucosal lym-
phoid tissue (9, 10, 53). Murine endothelial cells can be
induced to express high levels of MAdCAM-1 in re-
sponse to proinflammatory cytokines (8, 9). Recently, it
has been shown (28, 57) that the adhesive interactions
mediated by MAdCAM-1 and its lymphocyte receptor,
a4b7, are involved in leukocyte recruitment to chronic
inflammatory diseases of the bowel. MAbs specific for
b7 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 CD41 T cells
enriched for the CD45RBhigh subpopulation (42). Our
studies indicate that MAdCAM-1 expression in colon is
significantly increased in colitic IL-102/2 mice com-
pared with wild-type mice. Together, these results
suggest that MAdCAM-1 may also play an important
role in initiating and/or maintaining chronic inflamma-
tion of colon in IL-102/2 mice and may be a relevant
therapeutic target for patients with IBD.
In summary, this study represents the first demon-
stration that ICAM-1, VCAM-1, and MAdCAM-1 are
upregulated in the chronically inflamed cecum and
colon of IL-102/2 mice. Furthermore, this study indi-
cates 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 cyto-
kine(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.
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).
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@lsumc.edu).
Received 1 November 1999; accepted in final form 11 January 2000.
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