TITLE

CHARACTERISATION OF MUCOSAL ADDRESSIN CELL ADHESION MOLECULE (MADCAM-1) IN INFLAMMATORY BOWEL DISEASE

AUTHOR(S)
Ala, A.; Stubbs, M.; Coward, S.; Brown, D.; Hodgson, H.J.
PUB. DATE
April 2003
SOURCE
Gut;Apr2003 Supplement 1, Vol. 52, pA39
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Introduction: MAdCAM-1 is an endothelial cell adhesion glycoprorein that regulates α4β7 lymphocyte trafficking. It is upregulated in active inflammatory bowel disease (IBD). No satisfactory in vitro models of MAdCAM-1 expression have been described. In vitro, brain endothelium (bEnd3) is reported to express MAdCAM-1 in response to cytokines. In this study, we used a mouse lymph node endothelial cell line (SVEC4-10), which expresses functional levels of MAdCAM-1 in response to TNF-α and is a more realistic model of high endothelial venules. Methods: Cells were exposed to TNF-α (20 ng/L) for 0-72 h and incubated with anti-MAdCam-1 antibody, followed by FITC anti-Rat IgG. For quantification of fluoresence, 5 fields were selected for each time point and imaged using a Nikon Coolpix 990 digital camera. Using imaging software the total amount of fluorescence was measured for each field. Mean values were calculated for each time point. Results: Unstimulated SVEC cells exhibited no staining. After TNF-α there was marked increase in MAdCAM-1 immunostaining especially at cell junctions. Surface expression of MAdCAM-1 increased to 48 h and remained elevated at 72 h. Quantitation of fluorescence revealed that MAdCAM-1 expression was increased by 74% (9 h), 187% (23 h), 443% (48 h) and 505% (72 h). The elevation of MAdCAM-1 at 24 h was confirmed using microwell plate immunofluoresence of cultured SVEC cells. TNF-α treated cells expressed a 326% increase in MAdCAM-1 compared with unstimulated cells. MAdCAM-1 expression in normal and inflamed tissues of the gut was also characterised using immunostaining and RT PCR, showing upregulation in active IBD. Conclusion: Our studies demonstrate that MAdCAM-1 is upregulated by TNF-α on SVEC 4-10 cells and in inflamed gut tissues. This will provide the basis For investigating the expression and modulation of MAdCAM-1 both in vitro and in human disease.
ACCESSION #
9747531

 

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