Supplementary Materials
Figure S1: Adhesion of post-migrated PMNs to epithelial monolayers
promotes IEC wound healing without adding activating effects on PMNs (a)
Surface expression of CD11b, as an index of PMN activation was examined in
pre- and post-migrated PMNs, as well as in PMNs that remained adherent to
apical epithelial membrane after competing TEM using flow cytometry. A flow
diagram representative of 3 independent experiments shows that while TEM
results in significant PMN activation, no additive, activating effect was observed
in adherent PMNs post-TEM. (b) PMN chemotaxis or TEM in the physiological
basolateral to apical direction (1x106 cells/well) was induced by a transepithelial
gradient of fMLF. Following 3 hours of transmigration, the number of PMNs that
completed migration was quantified. N=3 independent experiments, with three
technical replicates for each condition. (c) Confluent, unstimulated (no ICAM-1
expression) T84 IEC monolayers were scratch wounded, and the healing
process was monitored over 48 h. Addition of either anti-ICAM-1 cross-linking
Abs or post-migrated PMNs to IEC monolayers had no significant effect on
wound healing. N=3 independent experiments, with three technical replicates for
each condition.
Figure S2: Expression and subsequent ligation of ICAM-1 leads to
activation of Akt and β-catenin signaling. CHO-K1 cells, which lack
endogenous expression of ICAM-1 but can activate Akt and β-catenin signaling,
were induced to stably express ICAM-1 (CHO-ICAM-1). (a) A flow diagram
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representative of 3 independent experiments shows expression of ICAM-1 in the
induced CHO-ICAM-1 cells. (b) ICAM-1 expression (green, right panel) was also
confirmed by immunofluorescence staining. (c) Activity (phosphorylation) of Akt
and β-catenin following ICAM-1 expression and following addition of cross-linking
Abs was examined using Western blot analysis. Blots representative of 3
independent experiments demonstrate that overexpression of ICAM-1 in CHO-K1
cells was sufficient to induce increases in Akt and β-catenin phosphorylation.
These increases were further potentiated following Ab-mediated ICAM-1 crosslinking. N=3 independent experiments.
Figure S3: ICAM-1 expression is induced at wound-edge epithelium.
Superficial wounds were introduced to mice colons in-vivo using colonoscopicbiopsy-based technique. Punch-biopsied, colonic-mucosal wounds were
analyzed for ICAM-1 expression using Western blot (a) and immunofluorescence
(b) analyses. Induction of ICAM-1 in wounded mucosa by wound-edge epithelial
cells was observed. Images are representative of 3 independent experiments.
The scale bar represents 200µm. (c) To confirm the generation of Ab complexes,
non-boiled, non-reduced, and non-complexed versus complexed IgGs were run
on an 8% acrylamide gel and stained with Coomassie. Line order as follows: 1)
marker; 2) non-complexed IgG (anti-ICAM-1 primary Abs); 3) complexed antiICAM-1 Abs; and 4) complexed IgG2a control Abs. The generation of higherorder complexes can be seen in lines 3 and 4. Some of the large aggregates
remained in the stacking gel and were not able to run on the resolving gel.
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Figure S4: Specific, engagement of ICAM-1 promotes IEC wound closure.
(a) Surface expression of ICAM-1 in control and scratch wounded IECs was
examined by flow cytometry. A flow diagram representative of 3 independent
experiments demonstrates constitutive expression of ICAM-1 on Coco-2 IECs,
and that wounding does not alter its expression. (b) Scratch-wounded,
unstimulated Caco-2 IEC monolayers were treated with anti-ICAM-1 or antiCD55 cross-linking Abs immediately following wounding. Wound healing was
monitored over 48 h. As can be seen, cross-linking of ICAM-1 but not CD55
significantly enhanced wound closure. N=3 independent experiments, with three
technical replicates for each condition. *p<0.05, **p<0.01. (c) Scratch wounded
Caco-2 IECs, immediately following wounding were incubated with post-migrated
PMNs (2.5x105 cells/well) in the presence or absence of CD11b and CD11a
inhibitory Abs (20µg/ml). Consistent with Ab-ligation of ICAM-1, PMN adhesion to
ICAM-1 enhanced IEC wound closure. PMN effects were CD11b dependent and
were prevented in the presence of anti-CD11b but not CD11a inhibitory Abs. N=3
independent experiments, with three technical replicates for each condition. *
p<0.05, ** p<0.01.
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