Croft, Daniel Richard; (1999) Expression and regulation of VCAM-1 and CD44 by cultured fibroblast-like synoviocytes. Doctoral thesis (Ph.D.), University College London.

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Abstract

The fibroblast-like synoviocyte (FLS) has been implicated in the destructive process associated with rheumatoid arthritis. In this thesis I demonstrate the expression and regulation of several adhesion molecules expressed on cultured FLSs obtained from inflamed synovium. Unlike fibroblasts from other areas of the body, FLSs constitutively express vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 on RA FLSs is constitutively expressed at high levels during the first 2 weeks of culture. At later time points (4 weeks in culture), VCAM-1 expression declined to low basal levels. A number of strategies were employed to determine the exogenous factors that determine the initially high levels of VCAM-1 in FLSs. Of the extracellular matrix components examined only collagen type I enhanced VCAM-1 expression but this had only limited success. The pro-inflammatory cytokines, IL-1β (10 ng/ml) and TNF-α (10 ng/ml), were also tested and found to induce only transient increases in cell surface VCAM-1 expression. However, the chronic administration of IL-4 or IL-13 in combination with TNF-α resulted in elevated levels of VCAM-1 with prolonged expression Prolonged VCAM-1 expression was found to result in part from the capacity of IL-4 and IL-13 to stabilize VCAM-1 mRNA transcripts. CD44 splice variants, isoforms of the CD44 receptor, that are implicated in the progression of a number of human tumours were also expressed by FLSs isolated from inflamed synovium. Immunohistochemistry, flow cytometry and RT-PCR analysis of CD44 splice variant expression revealed differential expression of a number of variant isoforms. Splice variant expression, at both the mRNA and cell surface protein level, was observed in a large percentage of RA FLSs, it is variable in those from OA synovium, and is absent in cells isolated from non-inflamed joints. However, RA FLSs showed a greater intensity of staining for variants v3, v5 and v7/8. VCAM-1-positive FLSs also demonstrated complex splice variant mRNA transcripts, comprising v3, v6, v7, v8, v9 and v10 in a variety of splicing combinations. These results indicate that the nature of CD44-splice variant expression is closely linked to the inflammatory state of the synovial joint. Moreover, expression of the CD44v7/8 epitope is associated with an increased cellular proliferation rate and could thus be functionally implicated in the hyperplasia observed in RA synovium.

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