Gruenewald, J.K.G.; (2008) Mechanisms involved in leukocyte transendothelial migration: studies of the role of hydrogen peroxide and the ecto-5'-nucleotidase, CD73. Doctoral thesis , University of London.

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Abstract

During inflammation, leukocytes are recruited from blood vessels into the site of injury or infection. A key event in this process is transendothelial migration (TEM), a multi-step process involving various adhesion molecules on both leukocytes and endothelial cells. Oxidative stress has been linked to inflammation in the development of various diseases. To investigate the effect of oxidative stress on TEM, leukocytes were treated with hydrogen peroxide (H2O2). A defect in TEM was observed across human umbilical vein endothelial cells (HUVECs), but not across a porous membrane, suggesting that H2O2 affects the interaction between leukocytes and endothelial cells. H2O2-treated leukocytes exhibited higher motility and adhesion on HUVECs, whereas their adhesion on substrates such as VCAM-1 and ICAM-1 was unaltered. The number of leukocytes exhibiting protrusions on HUVECs was decreased after H2O2 treatment, which is likely due to the increase in nitric oxide (NO) that was detected. Incubation of THP-1 cells with a NO donor led to a decrease in TEM and reduction of protrusions. The effects of H2O2 on several signalling molecules implicated in cell migration were investigated and changes in the activity of the RhoGTPases RhoA, Rac1 and Cdc42 were observed. CD73 is a 5'-ectonucleotidase expressed on endothelial cells and has been postulated to be involved in TEM. The inhibition of endothelial CD73 activity after leukocyte binding was reduced when H2O2-treated leukocytes were bound. Immunofluorescence staining showed local accumulation of CD73 around transmigrating leukocytes. RNAi knock-down of endothelial CD73 led to an inflammatory phenotype with elongated cells, increased surface levels of ICAM-1, VCAM-1, E- and P-selectin and increased permeability. Together these findings suggest that ROS have multiple effects on leukocytes leading to a defect in TEM, one of which is reduced CD73 inhibition after endothelial binding.

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