Richard M. Day, Tracey J. Mitchell, Stella C. Knight, Alastair Forbes

Cytokine 21 (2003) 224–233

Regulation of epithelial syndecan-1 expression by inflammatory cytokines

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.

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.

IBD is characterized by chronic intestinal inflammation that results in clinical symptoms such as diarrhea, bleeding, abdominal pain, fever, joint pain, and weight loss.

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 cultureHuman colorectal carcinoma epithelial cell lines HT29/219 and T84

Cytotoxicity assayThe cytokine effects of TNF, IL-1 and IL-6.

Flow cytometry analysis

Epithelial cell surface syndecan-1 expression analysis

Co-culture with peripheral blood mononuclear cellsCo-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

unstimulated5 ng/ml TNF-a75 ng/ml TNF-aIgG1 isotype control

Fig. 1

TNF-

IL-1

IL-6

B,C,D HT29 cell

E T84 cell

TNF-

Cell variability --- ApoAlert cytotoxicity assay

Above 86% for all concentrations of cytokine studied.

Fig. 2AHT29 cell treated with 5 ng/ml TNF-a for the various time

Fig. 2B

Fig. 3HT29 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 17.1%

Increase 69.3%

Increase 8.1%

Cytokine stimulation induced syndecan-1 shedding from the cell surface

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

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.

other model without inflammation

(up-regulation)Wound repairThe expression of

syndecan-1

HT29 cell

TNF-a, IL-1b and IL6 stimulate

Reduced Syndecan-1 expression

T84 cell

Stable expression of syndecan-1

Does-dependent down-regulate syndecan-1 mRNA

IL-6 to cause a reduction of syndecan-1 expression in murine B-lymphoid cells cell-type specific

Epithelial cell adhesion molecules

dysregulation

Inflammatory cytokine ex. TNF- IL-1

Syndecan-1, E-cadherin

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

PBMC

HT29 cell

Reduced syndecan-1 expression

Co-culture

Increased ICAM-1 expression

HT29 cell + TNF- and IL-1

Release inflammatory cytokine

block

The loss of GAG expression in p’t with IBD is associated with increased density of TNF-

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.