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Title: Macrophage mediated endothelial injury and proliferation in renal transplant rejection
Author: Adair, Anya
ISNI:       0000 0004 2727 9224
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Macrophages (Mφ) have previously been implicated in both acute and chronic renal allograft rejection however the mechanisms remain unclear. In this thesis I set out to explore the effect of the Mφ on the endothelium in the context of renal graft rejection. Initial studies focussed upon human renal allograft tissue from transplant nephrectomies performed because of chronic allograft nephropathy (CAN). Immunostaining was carried out on these tissues (n=29) and control kidney tissue obtained from nephrectomies performed for renal cell carcinoma (n=19). An increased interstitial Mφ infiltrate was found compared to control tissue. Immunostaining for the T cell marker CD3 and the B cell marker CD20 demonstrated that both lymphocyte populations were present in the CAN tissue with almost negligible numbers seen in control tissue. Previous work in the group had demonstrated a reduced number of CD31 positive peritubular capillaries in the tissues used in these studies. In the work undertaken in this thesis, additional analysis was performed to study lymphatic vessels. Immunostaining of control tissue with the lymphatic endothelial cell (LEC) marker podoplanin demonstrated a normal distribution of lymphatic vessels around large interlobular arteries. CAN tissue, however, exhibited an increased lymphatic density with lymphatic vessels evident within the interstitium; a finding verified with two additional LEC markers (LYVE-1 and VEGFR-3). Further investigations examined possible mediators that could be responsible for the reduced microvascular peritubular capillary network and increased lymphatic vessels present in tissues affected by CAN. Previous work had implicated nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) in cardiac allograft rejection. Double immunolabelling for iNOS and the Mφ marker CD68 revealed evidence of Mφ expression of iNOS. No obvious reduction in vascular endothelial growth factor (VEGF)-A was evident although marked expression of VEGF-A was found in CD20 positive B cells within CAN tissue. Occasional interstitial cells expressed the lymphangiogenic growth factor VEGF-C, with double labelling studies indicating occasional CD68 +ve Mø that were positive for VEGF-C. In vitro studies were undertaken to dissect the interaction between Mø and microvascular endothelial cells (MCEC-1) using well established in vitro co-culture techniques. Co-culture of cytokine activated bone marrow derived Mø with MCEC-1 cells (a murine cardiac microvascular endothelial cell line) resulted in increasing levels of MCEC-1 apoptosis and a reduced cell number over a 24-hour time course. Non-activated Mø or cytokines alone were not cytotoxic. Co-cultures were performed in the presence of L-Nimino- ethyl lysine (L-Nil), a specific inhibitor of iNOS (control D-N6- (1-iminoethyl)-lysine (D-Nil)). L-Nil significantly inhibited MCEC-1 apoptosis and preserved cell number implicating a major role for NO in Mø-mediated MCEC-1 death. Importantly, L-Nil treatment did not affect TNFα production by cytokines suggesting that TNFα is not involved in MCEC-1 death in this in vitro experimental system. Experiments were then undertaken involving the depletion of Mø in a murine model of acute renal allograft rejection. Renal transplants were performed between donor Balb/c mice and either FVB/N CD11b-DTR mice transgenic for the diphtheria toxin receptor (DTR) under the CD11b promoter or control non-transgenic FVB/N mice. Diphtheria toxin (DT) was administered on days 3 and 5 to induce Mø depletion and mice sacrificed at day 7. Isograft controls were also performed between FVB/N mice. Murine allografts exhibited marked interstitial F4/80 positive Mø infiltration with expression of iNOS in the allografts. There was significant loss of peritubular capillaries (PTC) in allografts compared to isografts, indicating microvascular injury. DT treated CD11b-DTR mice exhibited 75% reduction in Mø infiltration and this was associated with dramatic microvascular protection. B and T cells were not evident in the isograft but significant accumulation of B and T cells was present in the allograft and not affect by Mø depletion. Interestingly, there was an increase in the number of podoplanin positive lymphatic vessels in the allograft compared to the isograft, which was significantly inhibited following Mø depletion. The final area of study focussed upon attempts to isolate lymphatic endothelial cells in vitro. Two types of vascular cells (HUVECs and HDMECs) were analysed by flow cytometry for LEC markers and immunofluorescence to phenotype the cells. Magnetic bead sorting was then undertaken to isolate discrete populations of endothelial cells expressing LEC markers. The murine studies reinforce the cytotoxic potential of Mø and supports a role for Mø in the deleterious rarefaction of microvascular interstitial vessels with resultant tissue hypoxia and ischaemia. Furthermore, these data support the involvement of Mø in the interstitial lymphangiogenesis that may occur in renal allografts. Furthermore, the study of human allograft tissue indicates that microvascular rarefaction and an increase in intrarenal lymphatic vessels occurs in human disease. Lastly, Mø expression of iNOS and VEGF-C suggests that Mø are involved in key processes that may adversely affect graft outcome.
Supervisor: Hughes, Jeremy. ; Marson, Lorna. Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: macrophage ; renal allograft rejection ; endothelium