O24
INDUCTION OF MONOCYTE AND MACROPHAGE APOPTOSIS AMELIORATES
MICROVASCULAR INJURY IN MURINE RENAL ALLOGRAFT REJECTION
Adair, A, Qu, F, Ferenbach, D, Vass, D, Kipari, T, Clay, S, Marson, L, Hughes, J
University of Edinburgh.
INTRODUCTION AND AIMS: Evidence supports a role for macrophages (M) in promoting
allograft rejection, but the mechanisms of injury are unknown. Our previous study
(Transplantation 2007) of human transplant nephrectomies exhibiting interstitial fibrosis and
tubular atrophy demonstrated significant loss of peritubular capillaries (PTC) associated with
M infiltration and expression of inducible nitric oxide synthase (iNOS). Furthermore, our in
vitro studies of cytokine activated co-cultures of bone-marrow derived M and endothelial cells
indicated that M-derived nitric oxide (NO) induced significant endothelial cell apoptosis
(Renal Association 2005). Using a murine model of acute renal allograft rejection and
conditional transgenic M ablation mice (CD11b-DTR) we sought a role for M and NO in the
microvasculature injury injury associated with acute rejection.
METHODS: The CD11b-DTR mice used in this study are transgenic for the human diphtheria
toxin receptor (DTR) under the control of the CD11b promoter such that the administration of
minute quantities of diphtheria toxin (DT) ablates circulating monocytes and intrarenal M.
Balb/c kidneys were transplanted into CD11b-DTR transgenic mice (CD11b-DTR allograft,
n=10) or FVB/N non-transgenic control mice (FVB/N allograft, n=8). Diphtheria toxin (DT)
was administered at days 3 and 5. Renal tissue was harvested at day 7 and stained with F4/80
(M), iNOS, B220 (B cells), CD3 (T cells), CD11c (dendritic cells, DCs) and CD31
(endothelial cells). FVB/N isografts (FVB/N kidneyFVB/N recipient) were controls for the
surgical procedure. Staining was quantified by computer image analysis or direct counting
RESULTS: DT treatment effectively depleted circulating monocytes and infiltrating M in
murine renal allografts at day 7 (10.5 vs 3.80.8 %F4/80+ area, CD11b-DTR allograft vs
FVB/N allograft, p<0.05) without no effect upon B or T cell infiltration. DT treatment also
resulted in reduced numbers of intrarenal CD11c+ DCs (7.21.5 vs 152.1 %CD11c+ area,
CD11b-DTR allograft vs FVB/N allograft, p<0.05) and this may result from markedly reduced
monocyte recruitment or increased DC emigration following the ingestion of apoptotic M.
Acute rejection was associated with significant reduction in the number of PTC (657 vs
13010 CD31+ PTC/100 tubules; FVB/N allograft vs FVB/N isograft, p<0.05) and this was
prevented by monocyte/M ablation (657 vs 1198 CD31+ PTC/100 tubules, FVB/N allograft
vs CD11b-DTR allograft, p<0.05). Double labeling for F4/80 and iNOS demonstrated iNOS+
M and M ablation resulted in a significant reduction in the number of interstitial iNOS+ cells
(50.3 vs 2.50.3 iNOS+ cells/25 point graticle, FVB/N allograft vs CD11b-DTR allograft).
CONCLUSION: These data indicate that the induction of widespread monocyte and M
apoptosis ameliorates acute rejection-related microvasculature rarefaction with an unexpected
reduction in DC infiltration of the allograft. The associated reduction in the number of
interstitial iNOS+ positive cells following M ablation implicates NO generation as a possible
mechanism of endothelial cell injury.