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-1 b 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 Immunocytochemistry

By ELISA

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-1

b

IL-6 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 Cytokine stimulation induced syndecan-1 shedding from the cell surface

Increase 17.1% Increase 69.3% 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 T84 cell Stable expression of syndecan-1 Reduced Syndecan-1 expression 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

PBMC HT29 cell HT29 cell Release inflammatory cytokine Co-culture block Reduced syndecan-1 expression Increased ICAM-1 expression + TNF a and IL-1 b 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.