Neutrophil extravasation into tissues is an essential process required for the inflammatory response. Upon receiving an inflammatory cue, neutrophils begin accumulating on the luminal surface of the endothelium. Neutrophil recruitment is initiated by selectin-mediated tethering and rolling of neutrophils along the endothelial monolayer, followed by integrin-mediated firm adhesion. Adherent neutrophils then traverse the endothelium in a process known as transendothelial migration. The events mediating the rolling and adhesion steps are well characterised, but research into the molecular mechanisms regulating transendothelial migration is an area of intense focus. A previous study conducted in our laboratory found that the activation of endothelial extracellular signal-regulated kinase (ERK) 1/2 was required for neutrophil transmigration. Furthermore, it was found that endothelial ERK was activated in response to a soluble protein produced by fMLP- or IL-8-stimulated neutrophils. In the present study, the soluble ERK-activating neutrophil protein was identified as annexin A1, which was selected as a possible candidate following mass spectrometry analysis of proteins secreted from activated neutrophils. Annexin A1 antibodies (Abs) were found to block endothelial ERK activation induced by conditioned medium harvested from stimulated neutrophils. Annexin A1 Abs were additionally able to inhibit neutrophil transmigration across human umbilical vein endothelial cell (HUVEC) monolayers in an in vitro transmigration assay. Following the purification of recombinant annexin A1, it was demonstrated that it could activate endothelial ERK in a similar manner to neutrophil conditioned medium. Upon further investigation, ERK activation was found to be induced by a truncated form of annexin A1 present in the protein preparation rather than the full length protein. Calpain I, a calcium dependent protease that is activated upon neutrophil stimulation and is known to cleave annexin A1 within the N-terminal domain, was shown to process full length inactive recombinant annexin A1 into an unidentified product that could activate endothelial ERK. A calpain I inhibitor was also found to prevent stimulated neutrophils from secreting an ERK-activating protein, thus further suggesting a role for calpain I in this process. As full length annexin A1 has been reported to signal through the formyl peptide receptor (FPR) family, a pan-FPR antagonist was incubated with endothelial cells and was found to inhibit ERK activation induced by neutrophil conditioned medium, indicating that pro-inflammatory annexin A1 is also a FPR ligand. Endothelial projections termed “transmigratory cups” form around neutrophils during extravasation, of which ICAM-1 is a major component. Using an assay that examined transmigratory cups during neutrophil transmigration, it was found that annexin A1 Abs could inhibit neutrophil adhesion and transmigration through HUVEC monolayers by interfering with transmigratory cup formation around neutrophils, as shown by monitoring ICAM-1 during the process. Quantification of transmigrating neutrophils highlighted that the majority of neutrophils were emigrating via a transcellular pathway, which is in opposition to many in vitro studies where paracellular transmigration predominates. The results generated from this study identified a novel pro-inflammatory role for annexin A1 in neutrophil transendothelial migration. Preliminary experiments suggested that the pro-inflammatory annexin A1 responsible for endothelial ERK activation was a truncated form. Calpain I appears to be a likely candidate responsible for the generation of this uncharacterised, truncated annexin A1 product, however further experiments are required to confirm this hypothesis. Pro-inflammatory annexin A1 represents a new target for the treatment of inflammatory disorders. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374554 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009
| Identifer | oai:union.ndltd.org:ADTP/269183 |
| Date | January 2009 |
| Creators | Williams, Samantha Louise |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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