Am J Physiol Gastrointest Liver Physiol 293: G446–G452, 2007.
First published May 17, 2007; doi:10.1152/ajpgi.00097.2007.
PECAM-1 (CD 31) mediates transendothelial leukocyte migration
in experimental colitis
Emile Rijcken, Rudolf B. Mennigen, Sebastian D. Schaefer, Mike G. Laukoetter, Christoph Anthoni,
Hans-Ullrich Spiegel, Matthias Bruewer, Norbert Senninger, and Christian F. Krieglstein
Department of General Surgery, Muenster University Hospital, Germany
Submitted 23 February 2007; accepted in final form 16 May 2007
Rijcken E, Mennigen RB, Schaefer SD, Laukoetter MG,
Anthoni C, Spiegel H-U, Bruewer M, Senninger N, Krieglstein
CF. PECAM-1 (CD 31) mediates transendothelial leukocyte mi-
gration in experimental colitis. Am J Physiol Gastrointest Liver
Physiol 293: G446–G452, 2007. First published May 17, 2007;
doi:10.1152/ajpgi.00097.2007.—Transendothelial migration of circu-
lating leukocytes into the colonic wall is a key step in the development
of the inflammatory infiltrate in inflammatory bowel disease (IBD).
The platelet-endothelial cell adhesion molecule-1 PECAM-1 (CD31)
is expressed in the tight junction area of endothelial cells, where it is
supposed to support the transmigration process. The aim of this study
was to determine the role of PECAM-1 in experimental IBD and to
show whether blockade of PECAM-1 has therapeutic effects. Chronic
colitis was induced in female BALB/c mice by cyclic oral adminis-
tration of dextran sodium sulfate (DSS) 3% (wt/vol). Expression of
PECAM-1 was visualized by immunohistochemistry. In the treatment
group animals received 1 mg/kg anti-PECAM-1 (2H8) ip daily start-
ing on day 26. On day 30 leukocyte adhesion and migration was
measured during N2O-isoflurane anesthesia in the distal colon by
intravital microscopy. Disease activity index (DAI), histology, and
MPO levels were compared with healthy and diseased controls.
PECAM-1 was expressed in colitic mice. Chronic DSS colitis was
characterized by a marked increase in rolling, adherent, and transmi-
grated leukocytes compared with healthy controls. Immunoblockade
of PECAM-1 reduced leukocyte transmigration significantly and also
diminished leukocyte rolling and sticking in an indirect manner. It
also resulted in a significantly diminished DAI and MPO levels, as
well as an amelioration of the histological inflammation score.
PECAM-1 plays an important role in transendothelial leukocyte
migration in DSS colitis. PECAM-1 could be a novel target for
antibody-based treatment in IBD.
platelet-endothelial cell adhesion molecule-1; leukocyte transmigra-
tion; dextran sodium sulfate colitis; intravital microscopy; inflamma-
tory bowel disease
LEUKOCYTE RECRUITMENT FROM the blood circulation into the
tissues is a crucial event in the generation and maintenance of
the inflammatory infiltrate in inflammatory bowel disease
(IBD) as in other inflammatory disorders (19, 25). The initial
mechanisms of leukocyte margination, tethering, rolling, and
firm adhesion to the vessel wall of postcapillary venules have
been extensively studied in recent years, but particularly in
inflammatory bowel disease relatively little is known about
leukocyte migration through the endothelium and the underly-
ing basal membrane into the mucosa and submucosa itself.
There is growing evidence that activated leukocytes drift to the
borders of the endothelial cells and then pass across them in an
Address for reprint requests and other correspondence: E. Rijcken, Dept. of
General Surgery, Muenster Univ. Hospital, Waldeyerstrasse 1, D-48149
Muenster, Germany (e-mail: rijcken@uni-muenster.de).
G446 0193-1857/07 $8.00 Copyright © 2007 the American Physiological Society
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amoeboid fashion. Compared with the intestinal epithelium,
the junctions between endothelial cells in intestinal postcapil-
lary venules are leakier. In vitro, neutrophils accomplish this
process in ⬃90 s.
The process of leukocyte transendothelial migration, also
called diapedesis, involves several families of cell adhesion
molecules, such as VE-cadherin, ICAM-2, CD99, JAM-A,
JAM-B, JAM-C, and platelet-endothelial cell adhesion mole-
cule-1 (PECAM-1, CD31) (19). In contrast to leukocyte rolling
or sticking, leukocyte transmigration involves homophilic in-
teractions between adhesion molecules such as PECAM-1 and
CD99 both on leukocytes and endothelial cells (6). Other
ligands of PECAM-1 are integrin ␣v␤3, ␣6␤1, or CD38.
PECAM-1 is also involved in leukocyte migration through the
second barrier, the subendothelial basal membrane (6, 19, 31).
PECAM-1 is a 130-kDa glycoprotein that consists of 6 C2
immunoglobulin domains, a transmembrane region, and a
small cytoplasmatic domain (25). Confocal microscopy studies
demonstrated that PECAM-1 is densely expressed on the apical
surface and the borders of endothelial cells (3, 19). Addition-
ally, PECAM-1 is constitutively expressed on platelets, neu-
trophils, lymphocytes, natural killer cells, and monocytes. As a
response to proinflammatory cytokines such as TNF-␣, IL-1, or
IFN-␥ or to endotoxins, PECAM-1 expression is not upregu-
lated transcriptionally (8), but PECAM-1 is redistributed away
from the intercellular junction of endothelial cells, indicating
that spatial allocation of PECAM-1 has a regulatory role in the
processes mediated by PECAM-1 (28). PECAM-1 is suggested
to guide leukocytes through the endothelium via enhanced
expression of PECAM-1 at endothelial cell junctions during
the transmigration process through recycling of PECAM-1-rich
membrane invaginations below the plasma membrane at lateral
junctions of endothelial cells (18). Ligation to PECAM-1 is
also responsible for activation of ␤1-, ␤2-, and ␤3-integrins on
monocytes, neutrophils, and natural killer cells (3, 30). Fur-
thermore, homophilic interaction of PECAM-1 upregulates
␣6␤1 integrins on transmigrated neutrophils in vivo (6). In
summary, these data clearly demonstrate that PECAM-1 is criti-
cally involved in the transendothelial migration process. Various
in vivo experiments could confirm the crucial role of PECAM-1
in different inflammatory conditions. Blockade of PECAM-1 by
means of neutralizing antibodies ameliorated neutrophil extrava-
sation in experimental peritonitis (4, 5), endotoxin-induced liver
injury (5, 22), and ischemia-reperfusion injury (23, 32).
Despite extensive studies on leukocyte transmigration in
other systems, the role of PECAM-1 in intestinal inflammation
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 ROLE OF PECAM-1 IN EXPERIMENTAL COLITIS
has not been elucidated so far. However, PECAM-1 has been
detected in colon tissue samples of patients with active Crohn’s
disease or ulcerative colitis (2, 29, 33), suggesting that
PECAM-1 interactions are also important in leukocyte trans-
migration in human IBD.
In this study we examined the role of the PECAM-1 in a
well-established animal model of murine colitis. The dextran
sodium sulfate (DSS) model of colitis was used to determine
whether 1) PECAM-1 is expressed in colonic inflammation, 2)
leukocyte transmigration in the distal colon can be inhibited by
antibody blockade of PECAM-1, 3) PECAM-1 is involved in
leukocyte rolling and firm adhesion, and 4) therapeutic block-
ade of PECAM-1 leads to a reduction of inflammatory activity
in established disease. We were able to demonstrate that
PECAM-1 mediates transendothelial migration in submucosal
colonic venules in DSS colitis. PECAM-1 expression in DSS
colitis could be visualized by immunochemistry in submucosal
venules in the distal colon. Furthermore, we showed for the
first time that monoclonal antibody blockade of PECAM-1 has
therapeutic effects in this model of intestinal inflammation.
MATERIALS AND METHODS
Induction of inflammation. The experimental protocol was ap-
proved by the Animal Care Committee of the Regional Administra-
tion, Muenster, Germany. Inbred female BALB/c mice (Charles
River, 20 –22 g) were housed in pairs in standard laboratory cages
with standard laboratory chow and drinking water ad libitum. Chronic
colitis was induced by cyclic oral administration of DSS (24), mo-
lecular weight 40,000 (ICN Biomedicals) 3% (wt/vol) solved in
Millipore water (Millipore, Schwalbach, Germany) for 5 days inter-
rupted by 5 days of Millipore water alone. A highly reproducible
chronic colitis is established after completion of a total of three cycles.
It is characterized by mucosal ulceration, crypt destruction, and
infiltrating neutrophils and lymphocytes (24). Starting from the first
day of induction, the disease activity index (DAI) was recorded daily.
This well-established score is based on clinical symptoms and spe-
cifically designed to evaluate the severity of DSS colitis. It includes
weight loss, stool consistency, and the appearance of blood in stools
(21, 26).
Antibodies and reagents. 2H8, a hamster-anti-mouse IgG mono-
clonal antibody vs. PECAM-1, and LO-DNP-1, an unspecific control
antibody, were purchased from Serotec, Oxford, UK. The antibodies
were dissolved in PBS before injection.
For immunohistochemistry a rat monoclonal antibody against
mouse PECAM-1 (MEC 13.3) was obtained from BD Pharmingen
(San Diego, CA). Secondary reagents were biotinylated goat anti-rat
IgG (H⫹L) (VECTOR Laboratories, Burlingame, CA) and avidiny-
lated Alexa Fluor 488 conjugate, Molecular Probes (Invitrogen,
Karlsruhe, Germany).
IVM. In isoflurane/N2O inhalation anesthesia, spontaneous breath-
ing animals were placed in a supine position on a heating pad.
Polyethylene catheters (Portex, Lythe, UK) were inserted into the
right jugular vein for intravenous administration of contrast dyes and
in the left carotid artery for monitoring of arterial blood pressure and
heart rate throughout the experiment (Servomed, Hellige, Germany).
Blood samples (0.1 ml in EDTA) were taken before intravital micros-
copy (IVM) to evaluate an effect of the antibodies applied on leuko-
cyte counts by using a hematology cytometer (Coulter counter, Beck-
man Coulter, Fullerton, CA). The intestine was kept moist and warm
by continuous superfusion with normal saline warmed to 37°C.
Leukocytes were stained in vivo by repeated intravenous injections of
acridine orange (Sigma Aldrich, Steinheim, Germany) (20 ␮g/ml)
during IVM. In epiluminescence technique leukocyte-endothelial cell
interaction was visualized after 15 min of equilibration in five adja-
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G447
cent submucosal venules of the distal colon. Final magnification ⫻760
was achieved by ⫻16/0.5-mm water immersion objective (Plan-
Neofluar, Zeiss) and a video camera using 0.5 zoom (FK 6990-IQ,
Pieper). Microscopic images were recorded for off-line quantitative
assessment of microcirculatory parameters by a computer-assisted
analysis system (analySIS, Soft Imaging System, Muenster, Ger-
many). In each vessel the mean value of five venular diameters (D)
was calculated. Central line leukocyte velocity (V) and leukocyte
rolling velocity (Vroller) were determined by calculating the mean
velocity of five single frame-to-frame tracked fluorescent free flowing
or rolling cells, respectively. Flow rate (Ft) was calculated by the
formula Ft ⫽ [␲ ⫻ (D2/4) ⫻ V ⫻ t]/109 where t ⫽ time. Leukocytes
were defined as firm adherent (sticker) when attached to the vessel
wall for at least 30 s and as rolling (roller) when moving with a
velocity less than 2/5 of that of leukocytes at the centerline of the
observed microvessel. Rolling and firm adherent cells were counted
over a period of 30 s in a 100-␮m section of the vessel and given as
numbers per 0.01 mm2 endothelial surface (26). Vroller is a marker for
the intensity of selectin interactions enabling rolling (17). For deter-
mination of leukocyte extravasation mucosal IVM was performed by
opening the colon by a longitudinal incision along the antimesenteric
border using microcautery (12, 36). In earlier studies we were able
to show that the microcirculation in the studied area was not
disturbed by surgery. After cleaning of feces and mucus the
mucosal microcirculation was visualized in the mucosa in epilu-
minescence technique. The amount of extravasated leukocytes was
assessed by counting the fluorescent cells outside the microvessels
surrounding the crypts and averaged over five areas of 100 ⫻ 100
␮m at the center of the opened bowel, given as numbers per square
millimeter.
Tissue analysis. For histological studies the entire colon was
excised. The colon was opened longitudinally and rinsed with saline.
Each colon was divided in four parts, representing cecum with
appendix, proximal, middle, and distal colon. Hematoxylin and eosin
staining was performed on formalin-fixed, paraffin-embedded sec-
tions. From each region of the colon, four transverse slides were
made, resulting in 16 slides for evaluation of colitis for each animal as
described before (26). Histological inflammation was scored in a
blinded fashion by a score introduced by Dieleman et al. (9), consid-
ering the degree of inflammation, the vertical extent of inflammation,
and the crypt damage score, related to the percentage of involvement
in each single slide (1, 9).
A segment of the distal colon in some healthy as well as some
colitic animals was snap frozen in liquid nitrogen for immunohisto-
chemical studies. Fluorescence staining of PECAM-1 was performed
by the avidin-biotin technique in cryostat sections. Additional nucleus
staining was achieved by using DAPI (Sigma Aldrich, Steinheim,
Germany). Before antibody application, unspecific protein binding
was blocked by incubation with BSA (Sigma Aldrich). Negative
controls were prepared by omission of the specific antibody. The
slides were given random numbers for blinding before examina-
tion.
Myeloperoxidase (MPO) is contained in cytoplasmatic granules of
neutrophils and monocytes (13), and MPO activity correlates with the
presence of neutrophils in intestinal inflammation (15). Tissue MPO
activity was measured in units per gram by the o-dianisidine assay. In
brief, samples of the distal colon were snap frozen in liquid nitrogen
and stored at ⫺80°C until thawed for MPO activity determination.
The samples were homogenized and sonicated in hexadecyltrimeth-
ylammonium bromide 0.5% (Sigma Aldrich) while kept on ice and
finally centrifuged at 12,000 rpm at 4°C. The supernatant was trans-
ferred into 3-ml glass tubes containing 50 mM KPi, pH 6, 20 mM
H2O2, and 30 ␮l of o-dianisidine 20 mg/ml to start the reaction. The
reaction was stopped after 10 min by adding sodium azide 2%
(Fischer Scientific, Fair Lawn, NJ). The change in absorbance was
read at 460 nm in a spectrophotometer (Biochrom Libra S11, Cam-
bridge, UK). MPO activity was expressed as the amount of enzyme
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G448 ROLE OF PECAM-1 IN EXPERIMENTAL COLITIS
necessary to evoke a change in absorbance of 1.0 per minute per wet
weight gram of tissue.
Experimental protocol. In the first experiment, the effect of mono-
clonal antibodies against PECAM-1 (2H8) on leukocyte adhesion and
transmigration and their effect on intestinal inflammation were stud-
ied. After completing the third DSS cycle, 2H8 (1 mg/kg body wt)
was administered in the treatment group by daily intraperitoneal
injection starting on day 25 over a period of 5 days. The dosage was
based on previous studies in ischemia-reperfusion injury (32). DAI
scores were recorded daily. On day 30 leukocyte-endothelial interac-
tions in colonic submucosal venules were studied by use of IVM.
After opening of the distal colon, leukocyte extravasation was visu-
alized in the mucosa. The treatment group was compared with a
healthy control group receiving Millipore water alone and a diseased
control group, which received the antibody-carrier PBS alone (n ⫽ 10
for each group). At the end of the IVM experiments the animals were
killed and colon was prepared for histology, immunohistochemistry,
and MPO measurements.
The second experiment was designed to evaluate a direct effect of
the antibody 2H8 on leukocyte rolling and sticking. IVM was per-
formed in chronic inflamed animals. A submucosal venule in the distal
colon was randomly selected, and leukocyte rolling and firm adhesion
were visualized after in vivo staining by repeated intravenous injec-
tions of acridine orange (Sigma Aldrich). After the baseline leukocyte
adhesion was recorded for 10 min after an equilibration period, the
antibody 2H8 (1 mg/kg body wt) was injected intravenously under
direct IVM vision. All standard parameters of IVM that define
leukocyte-endothelial adhesive interactions were measured in submu-
cosal venules in the distal colon 1, 5, 10, 20, 30, 45, and 60 min after
antibody administration. Control animals received 1 mg/kg body wt of
the unspecific antibody LO-DNP-1. Each group included seven ani-
mals (n ⫽ 7).
Statistics. Results are given as mean values ⫾ SE. Statistical
evaluations were performed by using the Kruskal-Wallis ranking test
for unpaired samples supplemented by Dunn’s method. Mann-Whit-
ney-Wilcoxon signed-rank test was applied for nonparametric paired
samples. P values of ⬍0.05 were considered significant.
Fig. 1. Representative fluorescence-immu-
nohistochemical stains of platelet-endothelial
cell adhesion molecule-1 (PECAM-1) in sub-
mucosal venules (arrowheads) in the distal
colon in healthy controls (A) and dextran
sodium sulfate (DSS)-induced colitis (B). Nu-
clei are counterstained with DAPI. Small ar-
rows indicate epithelial cells in colonic
crypts. PECAM-1 is constitutively expressed
on endothelial cells. During DSS colitis there
is no marked upregulation of PECAM-1 in
colonic submucosal venules.
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RESULTS
Expression of PECAM-1 in DSS colitis. Immunofluores-
cence was used to evaluate PECAM-1 expression in the distal
colon during chronic DSS colitis. Cryosections were prepared
from the distal colon of healthy and chronically inflamed mice.
The segment of the distal colon corresponded to the segment
that was used for IVM. Immunohistochemically, marked
PECAM-1 expression was detected in submucosal venules in
healthy animals (Fig. 1A). Also, strong staining of PECAM-1
was found in submucosal venules in animals with DSS-induced
colitis (Fig. 1B). This indicated that PECAM-1 expression in
colitic mice was not upregulated compared with healthy con-
trols but rather expressed constitutively in marked levels inde-
pendent from the inflammatory stimulus.
IVM. Chronic DSS colitis was characterized by a marked
increase in rolling leukocytes (116.4 ⫾ 19.8 vs. 51.1 ⫾ 8.6 per
0.01 mm2/30 s, P ⬍ 0.05) and adherent leukocytes (28.0 ⫾ 3.6
vs. 4.3 ⫾ 1.2 per 0.01 mm2/30 s, P ⬍ 0.05) in submucosal
venules of the distal colon compared with healthy controls.
Accordingly, leukocyte rolling velocity was markedly de-
creased (23.9 ⫾ 2.3 vs. 48.8 ⫾ 4.7 ␮m/s, P ⬍ 0.05). The
number of transmigrated leukocytes was significantly in-
creased in chronic DSS colitis compared with healthy control
animals (404 ⫾ 40.8 vs. 38.8 ⫾ 8.4 per mm2/30 s, P ⬍ 0.05)
(Fig. 2). In the first set of experiments, treatment with anti
PECAM-1 antibodies resulted in a significant reduction of
leukocyte transmigration by ⬃84% in the distal colon (65.8 ⫾
6.8 mm2/30 s, P ⬍ 0.05). After 5 days of treatment, also the
number of rolling (59.3 ⫾ 14.2 vs. 116.4 ⫾ 19.8 per 0.01
mm2/30 s, P ⬍ 0.05) and firm adherent leukocytes (5.6 ⫾ 0.6
vs. 28.0 ⫾ 3.6 per 0.01 mm2/30 s, P ⬍ 0.05) was significantly
reduced compared with diseased controls. However, rolling
velocity was not decreased in 2H8-treated animals compared
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 ROLE OF PECAM-1 IN EXPERIMENTAL COLITIS G449
Table 2. Peripheral blood counts after intraperitoneal anti-
PECAM-1 (2H8) antibody administration
Healthy DSS ⫹ Anti-PECAM-1
Controls DSS Colitis (2H8)

Leukocytes, 1,000/␮l 5.2⫾0.9 3.1⫾0.6 4.9⫾0.8

Hb, g/dl 13.2⫾1.8 10.8⫾1.3 14.9⫾0.3
Hct, % 32.6⫾2.9 30.3⫾3.8 37.6⫾2.8
Thrombocytes, 1,000/␮l 288⫾28 408⫾14 352⫾14
Values are means ⫾ SE. All differences are not significant.

observed in long-term-treated animals was caused by an im-

Fig. 2. Epiluminescence mucosal intravital microscopy. Crypts are clearly
recognizable as pale gray rings surrounded by dark-colored intramucosal
microvessels. The extravasated leukocytes, stained with acridine orange, are
the bright white spots. In chronic DSS colitis there is a marked increase of
leukocyte extravasation compared with healthy control animals. This was
significantly blunted after treatment with an antibody vs. PECAM-1.
with diseased animals (27.9 ⫾ 3.0 ␮m/s, P ⫽ not significant).
The microcirculatory parameters central line leukocyte veloc-
ity, vessel diameter, blood flow, or shear rate were not different
in healthy controls, DSS colitis, or 2H8-treated colitic animals
(Table 1), showing stable microcirculatory conditions in all
IVM experiments. There were no complications related to
intraperitoneal administration of the antibodies. Peripheral leu-
kocyte counts were not affected by 2H8 (Table 2). Hemoglobin
levels in chronic colitic animals tended to be lower than in
healthy mice as a sign of fecal blood loss, but this did not reach
statistical significance.
In the second set of experiments, we intended to study
whether the reduction of leukocyte rolling and sticking as
mediate blockade of PECAM-1, implicating a direct involve-
ment of PECAM-1 in leukocyte rolling and firm adherence in
the distal colon. Therefore we administered 2H8 intravenously
in chronically diseased animals under direct IVM vision in
submucosal venules. Neither 2H8 nor the unspecific control
antibody LO-DNP significantly reduced leukocyte rolling, roll-
ing velocity, or leukocyte firm adherence during constant
observation over a period of 60 min (data not shown).
Effects of blockade of PECAM-1 on intestinal inflammation.
All mice fed with DSS survived the colitis-induction protocol
and were killed on day 30 after the IVM experiments. Mice
receiving DSS developed symptoms of colitis after 3– 4 days,
when weight loss and diarrhea became apparent. Bloody stools
were frequent after 5– 6 days of DSS 3%. All colitic animals
exhibited a similar DAI course until day 26, when treatment
with 2H8 was initiated (Fig. 3). On day 30, treatment with
anti-PECAM-1 significantly reduced DAI compared with DSS
controls (2.8 ⫾ 0.3 vs. 5 ⫾ 0.4 points, P ⬍ 0.05). This effect
could already be observed after 2 days of treatment (Fig. 3). In
chronic DSS colitis MPO levels in the distal colon were
significantly increased compared with healthy controls (46.1 ⫾
4.2 vs. 12.9 ⫾ 1.3 U/g, P ⬍ 0.05). Accordingly, MPO levels
were significantly decreased in 2H8-treated animals (25.8 ⫾
4.7 U/g, P ⬍ 0.05), although still being significantly higher as

Table 1. Microcirculatory and hemodynamic parameters in

the distal colon
Healthy DSS ⫹ Anti-
Controls DSS Colitis PECAM-1 (2H8)

Central line velocity, ␮m/s 591.7⫾47.5 843.5⫾119.5 932.0⫾142.8

Vessel diameter, ␮m 79.0⫾8.4 74.8⫾8.0 88.4⫾7.1 Fig. 3. Disease activity index (DAI) scores of all mice during the last 10 days
Bloodflow, ␮l/min 21.4⫾3.7 30.6⫾8.2 44.4⫾9.7 of the experiment. Treatment by daily intraperitoneal injections of anti-
Shear rate, s⫺1 70.5⫾11.1 94.8⫾12.1 87.6⫾12.2 PECAM-1 was initiated on day 26. There was a significant reduction of clinical
inflammation as early as 2 days after the beginning of the therapy. However,
Values are means ⫾ SE. PECAM-1, platelet-endothelial cell adhesion DAI of 2H8-treated animals did not reach the values of the healthy control
molecule-1. All differences are not significant. group (values given as means ⫾ SE).

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G450 ROLE OF PECAM-1 IN EXPERIMENTAL COLITIS
Fig. 4. Myeloperoxidase (MPO) activity in the distal colon as a marker for
leukocyte infiltration. MPO levels were significantly decreased after treatment
with anti-PECAM-1. Values are means ⫾ SE.
in healthy control animals (Fig. 4). Upon histological exami-
nation, healthy animals exhibited a normal microscopic anat-
omy of the colon, with infrequent areas of mild inflammation,
leading to a slightly elevated histological inflammation score.
After three cycles of DSS, colonic tissue showed a discontin-
uous chronic inflammation, including a mixed inflammatory
infiltrate containing mononuclear cells and neutrophils and a
loss of epithelium and crypt shortening, as described previ-
ously. In chronic DSS colitis there was an increase of the
severity of histological inflammation toward the distal colon
(Fig. 5). Administration of 2H8 blunted histological alterations
significantly in all parts of the colon. In the distal colon
blockade of PECAM-1 lead to significant decrease of the
inflammation score from 23.9 ⫾ 1.2 points (DSS colitis) to
15.4 ⫾ 1.5 points (treatment group) (P ⬍ 0.05).
DISCUSSION
The present study provides new insights in the mechanisms
involved in leukocyte transmigration in intestinal inflamma-
tion. We examined the role of PECAM-1 in the recruitment of
leukocytes in experimental inflammatory bowel disease. By
immunoblockade of PECAM-1 using the highly specific anti-
PECAM-1 monoclonal antibody 2H8, leukocyte transmigra-
tion was reduced by ⬃80%, and furthermore the inflammation-
associated tissue injury was significantly ameliorated. This
implicates that the generation of the inflammatory infiltrate in
DSS colitis is largely dependent on the presence of PECAM-1
on leukocytes and endothelial cells.
The concept of neutralizing adhesion molecules to control
intestinal inflammation has been successfully applied both in
experimental and, in part, in clinical studies. However,
PECAM-1 has not recognized much attention in this regard. In
contrast to other adhesion molecules such as ICAM-1 or
P-selectin that show elevated levels in tissue and plasma
samples of patients with active IBD, PECAM-1 seems not to
be upregulated on endothelial cells (33). Quantification of
basal expression of PECAM-1 in the vascular beds of the
murine distal colon under unstimulated conditions was dem-
onstrated by the dual radiolabeled monoclonal antibody tech-
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nique (34). By means of immunohistochemistry we could
demonstrate that there was no enhanced expression of
PECAM-1 in submucosal venules of the distal colon of in-
flamed animals compared with healthy controls. Because of its
marked constitutive expression on endothelial cells, PECAM-1
has been used as an immunohistochemical marker for endo-
thelium by many groups (34). Nonetheless it becomes apparent
that upregulation is not required to play a significant role in the
process of leukocyte recruitment in an inflammatory setting.
In our model, PECAM-1 was mainly involved in leukocyte
transmigration across the endothelial layer and basal mem-
brane as the amount of leukocytes in the crypts was signifi-
cantly reduced after treatment with 2H8. After 5 days of
treatment with 2H8 also leukocyte rolling and firm adhesion
appeared to be significantly diminished. We interpreted this
observation as an indirect effect due to the general downregu-
lation of inflammatory activity. To test this hypothesis we
performed additional IVM experiments in which 2H8 was
injected intravenously under direct intravital microscopic ob-
servation of submucosal venules in colitic animals. Although
there was a large number of adherent leukocytes present in the
vessel, in this experiment no significant alteration in leukocyte-
endothelial cell-to-cell adhesive interactions was observed.
This indicates that PECAM-1 is not participating in the early
stages of leukocyte recruitment such as rolling or firm adhe-
sion. This is consistent with the observation that anti-PECAM-1
antibodies or soluble recombinant PECAM-1 administered to
leukocytes or endothelial cells could specifically inhibit trans-
endothelial migration in vitro while leukocytes remain firm
adherent to the apical surface of the endothelial cells (20),
demonstrating that PECAM-1 does not influence leukocyte
firm adhesion.
Platelets have been shown to mediate leukocyte adhesive
interactions with endothelial cells in many physiological and
pathophysiological conditions. This process has been demon-
strated to be largely regulated by CD40-CD40 ligand interac-
tions in the inflamed colon (35). Since platelets express high
levels of PECAM-1, too, it might be suggested that 2H8 may
also interfere with PECAM-1 homophilic interactions in plate-
Fig. 5. Histological score: in chronic DSS colitis inflammation increases
toward the distal colon. Five-day treatment with 2H8 reduced histological
inflammation in all parts of the colon significantly compared with diseased
controls. Values are means ⫾ SE.
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 ROLE OF PECAM-1 IN EXPERIMENTAL COLITIS
lets as well as in platelet leukocyte interactions and thereby
limit leukocyte extravasation. This might be another explana-
tion for the observed reduction of leukocyte rolling and firm
adhesion in this study. However, a direct involvement of CD40
in the extravasation process has not been demonstrated yet (35).
In similarity to other adhesion molecules such as CD40,
there is increasing evidence that PECAM-1 has important
signaling properties, too. For example, CD31 is an important
amplifier of ␤1-integrin (CD29)-mediated T cell adhesion to
endothelial cells (30). ␤1-Integrin as a compound of VLA-4 is
a major ligand for VCAM-1, which is involved in the firm
adhesion of eosinophils, lymphocytes, monocytes, and natural
killer cells to cytokine-activated endothelium. CD31 also acti-
vates ␤2-integrins (CD18) on the surface of monocytes, neu-
trophils, and natural killer cells (3). ␤2-Integrin is a compound
of LFA-1 and Mac-1, both serving as a ligand for ICAM-1,
which also plays an important role for leukocyte firm adhesion
(27). Thus an inhibition of integrin activation must also be
considered to play a role in the observed reduction of rolling
and firm adherence of leukocytes as seen in the 5-day treatment
experiments as well as in the general reduction of inflammatory
activity.
Therapeutic blockade of PECAM-1 did not entirely cure
intestinal inflammation since DAI, histological score, and
MPO levels were still significantly increased compared with
healthy controls. Also, different in vivo studies using antibody
blockade of PECAM-1 in various inflammatory settings only
achieved a significant reduction but not a complete blockade of
leukocyte transmigration (4, 5, 23, 32). Possible explanations
are 1) the compensation of PECAM-1 function by other adhe-
sion molecules involved in leukocyte transmigration such as
CD99 or ICAM-2 (10, 19), 2) an increased rate of migration of
leukocytes, or 3) nonsaturating doses of 2H8. Studies using
PECAM-1-deficient mice showed that these CD31 knockout
mice had no impaired leukocyte migration in the models of
IL-1␤- or thioglycolate-induced peritonitis and showed a nor-
mal cutaneous hypersensitivity response (10). These results
suggest that there are CD31-independent pathways, although
they might be quantitatively less important.
Recent studies have revealed that there is a cytokine-specific
effect for PECAM-1-mediated leukocyte transmigration (7, 23,
31). Leukocyte migration through venular walls provoked by
injection of IL-1␤ was found to be PECAM-1 dependent (31),
whereas PECAM-1 was not required for leukocyte transmigra-
tion induced by TNF-␣ (7, 23, 31). PECAM-1 seems to be
crucial for induction of integrin ␣6␤1 on the surface of neutro-
phils (6). This PECAM-1-mediated activation is unnecessary
following TNF-␣ stimulation, because TNF-␣ in contrast to
IL-1␤ is able to directly activate neutrophils (3, 7, 23). Taken
together, PECAM-1 is suggested to be more involved in
IL-1␤-triggered inflammation than in TNF-␣-mediated inflam-
mation. Chronic DSS colitis is a much more complex state of
inflammation than direct stimulation of endothelium by addi-
tion of purified cytokines and it is consensus that it is not
dominated by a certain cytokine. Egger et al. (11) reported a
progressive upregulation of Th1 cytokines such as IL-12,
IFN-␥, IL-1, and TNF-␣ during the induction of chronic DSS
colitis, whereas in that study the expression of Th2 cytokines
(e.g., IL-4) was only modest. On the other hand, Dieleman et
al. (9) found that DSS colitis is mediated by both Th1 and Th2
cells, since high mucosal levels of IL-4 and IFN-␥ were
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G451
detected especially in the chronic phase of DSS colitis. DSS
colitis can be ameliorated by both TNF-␣- and IFN-␥-neutral-
izing antibodies, indicating that these cytokines are both im-
portant for maintenance of inflammation in DSS colitis. In
contrast, antibody-blockade of IL-1 failed to reduce DSS
colitis, referring to a subordinated role of IL-1 in this model
(14). Interestingly, in our study blockade of PECAM-1 reduced
leukocyte transmigration and inflammatory activity in DSS
colitis, which cannot be attributed only to IL-1-mediated and
TNF-␣-independent mechanisms. Therefore pathways other
than IL-1␤ might be important for leukocyte transmigration in
colitis, too. Furthermore, in other models of inflammation
driven by both TNF-␣ and IL-1␤, for example murine endo-
toxemia, antibody blockade of PECAM-1 was shown to reduce
MPO levels in the tissue as an index of leukocyte transmigra-
tion (22). In chronic DSS colitis as in active human IBD
integrins on leukocytes and endothelial cells are already acti-
vated by many proinflammatory stimuli. The reduced activa-
tion of ␤1 and ␤2 integrins might be important in our model,
but also direct inhibition of PECAM-1-mediated transmigra-
tion should be considered.
By blocking the step of leukocyte transmigration, the repet-
itive influx of inflammatory cells into the bowel wall is inter-
rupted, giving opportunity for tissue regeneration. Further-
more, the engagement of ␣1␤1 integrins on the surface of
monocytes and leukocytes has been shown to play a major role
in binding to extracellular matrix structures such as collagen 1
and thereby stabilizing the inflammatory infiltrate (16). This
might lead to a reduced shedding of proinflammatory cytokines
and a reduced cytokine gradient guiding the inflammatory cells
into the tissue, resulting in decreased leukocyte activation in
the microcirculation.
In conclusion, the present study shows that PECAM-1 plays
a major role in this experimental model of chronic colitis and
should therefore be considered as a valuable target for future
anti-PECAM-1-based strategies in human IBD, too. The fact
that we could successfully treat established chronic DSS colitis
by anti-PECAM-1 and not work in a prophylactic manner as in
the acute DSS colitis model further supports our recommen-
dation. However, whether therapeutic blockade of PECAM-1
alone will have clinical implications in human IBD remains
speculative. So far many adhesion molecules have been tested
for treatment of IBD in clinical studies without achieving a
final breakthrough. This might be due to the diversity of
overlapping functions of single adhesion molecules. Therefore
a combined therapeutic blockade with several monoclonal
antibodies, each directed at a different level of the leukocyte
recruitment cascade, for example PSGL-1 for leukocyte rolling
and PECAM-1 for leukocyte transmigration, might be a more
promising strategy than the blockade of a single adhesion
molecule alone.
GRANTS
This work was supported by a grant of the Deutsche Morbus Crohn/Colitis
ulcerosa Vereinigung DCCV e.V. (Crohn and Colitis Foundation of Germany).
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