Transcript Regulation of epithelial syndecan

Regulation of epithelial
syndecan-1 expression by
inflammatory cytokines
Richard M. Day, Tracey J. Mitchell, Stella C. Knight,
Alastair Forbes
Cytokine 21 (2003) 224–233
Introduction
Inflammatory bowel disease (IBD)
Inflammatory bowel disease (IBD) refers to the condition that results
when cells involved in inflammation and immune response are called into
the lining of the GI tract.
IBD is characterized by chronic
intestinal inflammation that
results in clinical symptoms
such as diarrhea, bleeding,
abdominal pain, fever, joint
pain, and weight loss.
Dysregulation of epithelial glycosaminoglycans (GAGs) occurs in IBD
Syndecans are a class of heparan sulphate proteoglycans that mediate
both cell adhesion and growth factor binding via GAG side chains.
Syndecan-1
Syndecan-1(CD138)
The best characterized of the four syndeacan core
proteins binding to variety of components of extracellular
matrix, including collagen type1 ,3,5 and fibronectin.
Syndecan-1 may regulate ligand-dependent activation of cell
surface growth factor at two dimernsional surface of
plasma membrane
Fibroblast growth factor(bFGF), hepatocyte growth
factor,platelet-derived growth factor,vascular endothelial
growth factor,and etc.
Mucosal cytokine may regulate epithelial cell
function.
Reduced expression of syndecan-1 observed
in IBD results from the increased presence
of inflammatory cytokines.
Cell culture
Human colorectal carcinoma epithelial cell lines HT29/219
and T84
Cytotoxicity assay
The cytokine effects of TNF-a, IL-1b and IL-6.
Flow cytometry analysis
Epithelial cell surface syndecan-1 expression analysis
Co-culture with peripheral blood mononuclear cells
Co-culture of PBMC and epithelial (HT29) cell were
stimulated with 20 ng/ml PMA and 2mM ionomycin for 24hr.
PMA phorbol 12-mryristate 13-acetate
Detection of soluble syndecan-1
By ELISA
Immunocytochemistry
RNA extraction and amplification by reverse
transcription-polymerase chain reaction
Statistical analysis
Results
Fig. 1
unstimulated
5 ng/ml TNF-a
75 ng/ml TNF-a
IgG1 isotype control
TNF-a
IL-6
IL-1b
TNF-a
B,C,D HT29 cell
E
T84 cell
Cell variability --- ApoAlert cytotoxicity assay
Above 86% for all concentrations of cytokine studied.
Fig. 2A
HT29 cell treated with 5 ng/ml TNF-a for the various time
Fig. 2B
Fig. 3
HT29 cell treated with TNF-a, IL-1b and IL-6 for 24h
Syndecan-1 expression
reduced to 49.7%
Syndecan-1 expression
reduced to 64.3%
Fig. 4
Increase 69.3%
Cytokine stimulation induced
syndecan-1 shedding from the
cell surface
Increase 17.1%
Increase 8.1%
Fig. 5
Fig. 6
Co-culture HT29 cell and PBMC
PBMC endogenous inflammatory cytokine
PMA increase syndecan-1 suppression
Discussion
Epithelial expression of syndecan-1 is down-regulated in the
mucosa of the patients with IBD
Epithelium overlying inflamed mucosa and reparative
epithelium
Wound repair
The expression of
syndecan-1
other model without
inflammation
(up-regulation)
Increased syndecan-1 expression during wound repair
is thought to facilitate growth factor binding and
wound healing.
The reduced expression in IBD maybe related to the
presence of inflammatory cytokines in mucosa.
Does-dependent down-regulate
syndecan-1 mRNA
TNF-a, IL-1b and IL6 stimulate
HT29 cell
Reduced Syndecan-1
expression
T84 cell
Stable expression of
syndecan-1
IL-6 to cause a reduction of syndecan-1
expression in murine B-lymphoid cells
 cell-type specific
Epithelial cell adhesion molecules
Syndecan-1, E-cadherin
Inflammatory cytokine
ex. TNF-a IL-1
dysregulation
Expression of E-cadherin is dependent on syndecan-1 expression
and vice versa.
Cytokine induce loss of cell-adhesion through reduced syndecan-1
and/or E-caderine destablizes the epithelial barrier.
Increase intestinal permeability in IBD
Shedding of syndeacan-1 ectodomain into the culture medium
of HT29 cell monolayers was significantly increase following
the addition of TNF-a .
The increase syndecan-1, shedding following stimulation
correlated with the loss of membrane syndecan-1 expression
Some syndecan-1 detected may also have been released as
the intact proteoglycan from dead cell
Release inflammatory
cytokine
PBMC
Co-culture
HT29 cell
block
Reduced syndecan-1
expression
Increased ICAM-1
expression
HT29 cell
+ TNF-a and IL-1b
The loss of GAG expression in p’t with IBD is associated with
increased density of TNF-a
This study demonstrates dysregulation of epithelial
syndecan-1 expression by inflammatory cytokines and
may have implications for mucosal ulcer healing.
Inflammatory bowel disease refers to the condition that results
when cells involved in inflammation and immune response are
called into the lining of the GI tract. This infiltration thickens the
bowel lining and interferes with absorption and motility (the ability
of the bowel to contract and move food). With abnormal ability to
contract and abnormal ability to absorb, the bowel’s function is
disrupted. Chronic vomiting results if the infiltration is in the
stomach or higher areas of the small intestine. A watery diarrhea
with weight loss results if the infiltration is in the lower small
intestine. A mucous diarrhea with fresh blood (colitis) results if the
infiltration occurs in the large intestine. Of course, the entire tract
from top to bottom may be involved. Many people confuse
Inflammatory Bowel Disease with “Irritable Bowel Syndrome,” a
stress-related diarrhea problem. Treatment for “IBS” is aimed at
stress; it is a completely different condition from “IBD.”
These symptoms can range from mild to
severe, and may gradually and subtly develop
from an initial minor discomfort, or may
present themselves suddenly with acute
intensity.