Macrophage mediated endothelial injury and proliferation in renal transplant rejection.
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Date
2008Author
Adair, Anya
Metadata
Abstract
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.