Neutrophils are efficient phagocytic cells that form the body’s first line defence against entry of foreign infectious microorganisms, but also contribute to tissue damage and non-infectious, chronic inflammation. Neutrophils contain a variety of separate classes of proteinase containing granules: primary (azurophilic) granules [containing serine proteinases elastase (NE), proteinase 3 (Pr3), and cathepsin G (Cat G)], secondary granules [containing metalloproteinase 8 (MMP-8)] and tertiary granules [containing metalloproteinase (MMP-9)]. These proteases are important molecules in immune and inflammatory processes. Sustained inflammation is associated with accumulation of these proteinases and is assumed to contribute to normal parenchymal damage and pathology. Regulation of proteolysis induced by these proteases is crucial to avoid self-induced damage. In the first part of my PhD thesis, I sought to examine the surface expression of Pr3 and CD177 (a surface receptor of Pr3) on neutrophils, in presence of physiological inhibitors of proteases (alpha-1-antitrypsin; AAT). I have demonstrated that membrane-bound Pr3 (mPr3) is still detectable on the surface of neutrophils in the presence of purified inhibitors and autologous serum as a source of physiological inhibitors. The interaction between CD177 and Pr3 was also examined by expressing CD177 cDNA on the surface of non-neutrophil cells (CHO cells), as was the ability of purified AAT and serum to interfere with these interactions. AAT was able to remove Pr3 from the surface of CD177-CHO cells, and similar results were observed for AAT removal of Pr3 binding to CD177-expressing neutrophils. In the second part of this thesis, I examined the change in neutrophil proteinases (Pr3, MMP-8 and -9) expression following neutrophil transmigration. In vitro transmigrated neutrophils showed no significant change in mPr3 expression compared to un-migrated neutrophils and both CD177-positive and CD177-negative subsets were able to migrate across HUVEC cells. In addition, intracellular Pr3 and MMP-8 also showed no change after in vitro transmigration. For comparison I also examined the CD177 and proteinase expression in salivary neutrophils (in vivo low inflammation transmigration) relative to matched volunteer blood neutrophils. In contrast to the in vitro data, I found only CD177-positive (with Pr3 bound to the surface) neutrophils present in the saliva of healthy individuals. I also found that levels of MMP-8 and MMP-9 were completely depleted in salivary neutrophils, relative to the matched levels in blood neutrophils from the same donor. In the third part of this thesis I used confocal microscopy to examine the intracellular distribution of neutrophil proteinases within different granule subsets. It was found that selected neutrophil proteinases co-localised with two different granule markers; showing their location in either azurophilic granules (CD63) or secondary granules (CD66b). However, the relationship with Pr3 revealed some discrepancies compared to previous reports. Some neutrophil proteins were co-localized both before and after neutrophil stimulation; whereas others were co-localisation before but not after stimulation. This suggested that degranulation of subsets of granules had occurred.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642508 |
| Date | January 2015 |
| Creators | Bshaena, Amina |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/71246/ |
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