Background: Antibody-mediated rejection is one of the major causes of acute and chronic rejection. This is mediated by endothelium microvascular inflammation and leukocyte migration to the graft. However, the role of donor specific HLA class I antibodies in inducing allograft rejection in the absence of complement is not fully understood. In this project, the mechanisms by which HLA class I antibodies induce endothelial cell-leukocyte interactions were examined. Methods: Human microvascular endothelial cells (HMEC-1) were stimulated with HLA class I antibody either mouse monoclonal (W6/32) or from sensitized kidney patients. The activation of cell signaling pathways was examined using Western blotting. The expression of adhesion molecules and chemokines were determined using flow cytometry and q-PCR, respectively. Monocyte adhesion and migration was examined using chemotaxis and flow based adhesion assays. Results: HMEC-1 cells stimulated with W6/32 antibody showed phosphorylation of a transcription factor CREB by a mechanism dependent on the protein kinase A pathway. W6/32 also induced significant expression of the adhesion molecules VCAM-1 and ICAM-1 (P<0.001) in a mechanism dependent on the PI3K/Akt pathway. Additionally, stimulated cells showed significant up-regulation in IL-6, CXCL8, CXCL1, CCL5 and CXCL10. The expression of CXCL8 was significantly reduced by knocking down CREB (p<0.001). Conditioned media from W6/32-treated cells was able to induce significant THP-1 monocyte migration compared to control (p<0.001). Furthermore, monocytes flowing at 0.5 dyne/cm2 significantly adhered to HMEC-1 cells-treated with F(ab)2-fragments of W6/32 (p<0.001). HLA class I alloantibodies from patients induced phosphorylation of CREB and a significant upregulation of VCAM-1, ICAM-1 and CXCL8. Monoclonal human HLA-B58 antibody was also able to induce CREB phosphorylation. Conclusion: Exposure of microvascular endothelial cells to HLA class I antibodies induce an activation of endothelial cell signaling that are responsible for upregulation of adhesion molecules and chemokines. These mediators enhance the interaction between donor endothelium and recipient leukocytes during cellular rejection. Strategies that block endothelium-leukocyte interaction might reduce the incidence of allograft rejection and improve allograft survival.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:639784 |
| Date | January 2014 |
| Creators | Naemi, Fatmah Mohammed A. |
| Publisher | University of Newcastle upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10443/2539 |
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