CNS endothelium expression of ADAM-17 and its role in multiple sclerosis pathogenesis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterised by focal lesions of inflammation and demyelination, with subsequent axonal damage. Various factors contribute to the pathogenesis of MS, including the breakdown of the blood-brain barrier (BBB), and increased local expression of inflammatory cytokines, matrix metalloproteinases (MMPs) and adhesion molecules. Upregulation of a distintegrin and metalloproteinase (ADAM)-17 has been previously reported in MS lesional white matter, associated with endothelial cells, astrocytes and macrophages. ADAM-17 is also increased in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) (an animal model of MS), suggesting a role for the enzyme in MS pathogenesis. ADAM-17 is responsible for the proteolytic cleavage of a number of membrane-bound proteins, including tumour necrosis factor (TNF) and the chemokine and adhesion molecule, fractalkine, both of which have been implicated in MS pathogenesis. To determine the functional role of ADAM-17 in MS pathogenesis, an adult human brain endothelial cell line, hCMEC/D3 (a model of the BBB), was studied to determine the relationship between ADAM-17, its inhibitor (tissue inhibitor of metalloproteinase (TIMP)-3), fractalkine, and a number of other proteins shed by the enzyme. This work revealed that under pro-inflammatory cytokine (TNF) treatment, fractalkine expression is dramatically increased at the mRNA and protein level as determined by real-time RT-PCR and immunocytochemistry, respectively, and is shed at high concentrations from the endothelium as determined by ELISA. ADAM-17 expression was not significantly affected at the mRNA, protein, or activity level, suggesting other proteases play a role in fractalkine release, following TNF treatment of brain endothelial cells.To further elucidate the relationship between ADAM-17 activity and fractalkine shedding, small interfering (si) RNA silencing of ADAM-17 was performed in the hCMEC/D3 endothelial cells line. This work revealed that despite knockdown of ADAM-17, fractalkine shedding was not significantly reduced. Again this suggests other mechanisms of regulating ectodomain shedding following TNF treatment are responsible for the release of fractalkine. Fractalkine expression in MS CNS tissue was observed in endothelial cells, astrocytes, macrophages and axons, however this expression was not significantly altered in comparison to control CNS white matter.From the studies undertaken in this thesis, ADAM-17 is not the major sheddase of fractalkine from the CNS endothelium under pathological conditions. Further investigation into the mechanisms involved are required using both in vitro and in vivo methods. The observation of fractalkine shedding following cytokine treatment is another significant finding, as demonstrated here in vitro, which requires further investigation to understand the functional implications this may have in vivo.