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B decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts
1
* Departments of Nephrology and Hypertension,
Vascular Medicine, and
§ Pathology, University Medical Center, Utrecht, The Netherlands; and
German Cancer Research Center, Heidelberg, Germany
1Correspondence: Department of Vascular Medicine, University Medical Center, G02.228, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail T.RABELINK{at}DIGD.AZU.NL
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B-dependent transcription
of genes encoding adhesion molecules and chemokines. Here we
demonstrate that cationic liposomes containing phosphorothioated
oligodeoxynucleotides (ODN) with the B binding site serving as
competitive binding decoy, can prevent TNF-
-induced NFB activity
in endothelial cells in vitro. In an allogenic rat
kidney transplantation model (BN to LEW), we show that perfusing the
renal allograft with this decoy prior to transplantation abolishes
nuclear NFB activity in vivo and inhibits VCAM-1
expression in the donor endothelium (P<0.05). At
24 h postreperfusion, periarterial infiltration of
monocytes/macrophages was significantly reduced in decoy ODN-treated
allografts compared to control allografts (3.7±0.7 vs. 9.2±1.2
macrophages/vessel; P<0.01). At 72 h, there was a
reduction of tubulointerstitial macrophage infiltration in decoy
ODN-treated kidneys compared to controls (75.6±13.9 vs. 120.0±11.2
macrophages/tubulointerstitial area; P<0.05). In
conclusion, perfusion of the renal allograft with NFB decoy ODN
prior to transplantation decreases the initial inflammatory response in
a stringent, nonimmunosuppressed allogenic transplantation model.
Therefore, the NFB decoy approach may be useful to explore the role
of endothelium and macrophages in graft rejection and may be developed
into a graft-specific immunosuppressive strategy allowing reduction of
systemic immunosuppression on organ transplantation.—Vos, I., Govers,
R., Gröne, H.-J., Kleij, L., Schurink, M., de Weger, R.,
Goldschmeding, R., Rabelink, T. J. NFB decoy
oligodeoxynucleotides reduce monocyte infiltration in renal allografts
Key Words: transplantation • adhesion molecules • macrophages • endothelium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
, which occurs secondary to ischemia and
reperfusion, probably also has profound effects on early and late
allograft function (3
4
5)
. Upon infiltration they develop
into macrophages that present donor alloantigens via major
histocompatibility complex II molecules, initiating the early immune
response. In addition, these macrophages secrete cytokines, such as
interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-)
(6)
, that attract and activate T cells, leading to
infiltration of T cells in the graft.
The transendothelial migration of inflammatory cells relies on the
presence of adhesion molecules on the endothelium, such as ICAM-1,
VCAM-1, ELAM-1, and selectins (7
, 8)
and on the presence
of their counter receptors (9
, 10)
. Transcriptional
activation of the genes encoding these adhesion molecules is tightly
regulated by transcription factors, including nuclear factor-B
(NFB) (11
12
13)
. This transcription factor is a DNA
binding protein complex that is usually present in the cytosol as an
inactive complex. IB, an associated protein, renders this complex
inactive by shielding the nuclear localization signal. Upon IB
phosphorylation and its subsequent degradation, the heterodimeric
NFB complex translocates to the nucleus, where it binds to specific
DNA sequences in the promoter region of several genes and up-regulates
their transcription. IB phosphorylation and proteolysis are induced
by inflammatory cytokines, such as TNF- and IL-1, and affected by
the redox status of the cell (11)
. It has been
demonstrated that oxidative stress increases NFB activity (14
, 15)
. Reperfusion and subsequent reoxygenation of an allograft
induce the release of both reactive oxygen species (i.e., oxidative
stress) and TNF- from the endothelium (6)
, resulting in
NFB-mediated transcription of genes encoding inflammatory
chemokines, including monocyte chemoattractant protein-1, cytokines,
and cell adhesion molecules (16
, 17)
.
Previously it was shown that NFB activation could be blocked by
decoy strategy, providing a potential therapeutic approach for the
prevention of myocardial infarction (18
, 19)
. The decoy
used for this purpose comprised a double-stranded oligodeoxynucleotide
(ODN) containing a sequence corresponding to the consensus sequence of
the B binding sites (20
, 21)
.
In the present study we evaluated the use of NFB decoy strategy in
kidney transplantation. We postulated that the decoy approach might be
a useful tool to reduce monocyte infiltration in the renal allograft by
preventing the interaction of NFB with specific promoter sequences
of genes encoding cell adhesion molecules (22)
, thereby
reducing the initial inflammatory response. Therefore, we investigated
in a rat renal allograft model whether NFB decoy treatment of the
isolated donor kidney prior to transplantation affects NFB activity
in the kidney, decreases endothelial NFB-induced expression of
adhesion molecules, and prevents the early influx of monocytes in the
renal allograft. Our findings demonstrate that the NFB decoy
approach is a potent therapy to reduce perivascular and
tubulointerstitial monocyte infiltration at the early stages of the
inflammatory response after allogenic kidney transplantation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B decoy sense strand was 5' AGT TGA GGG GAC TTT
CCC AGGC 3', containing both the specific p50 (GGGAC) and p65 (TTCC)
B binding sites (23)
. As scrambled ODN, a nonsensed
sequence was used: 5' TTG CCG TAC CTG ACT TAG CCGT 3'
(19)
. Cationic liposomes were generated from Tfx-50
Reagent (Promega) and consisted of a mixture of the synthetic cationic
lipid molecule
N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3,-dioleoyloxy-1,4-butanediaminium
iodide and the fusogenic lipid L-dioleoyl phosphatidylethanolamine
(DOPE). Liposomes were used according to the supplier’s instructions.
RF24 cell culture and transduction
Immortalized human umbilical vein endothelial RF24 cells were
cultured as originally described by Fontijn et al. (24)
.
Cells were cultured in RPMI 1640 medium (Life Technologies, Eggenstein,
Germany) containing 20% HSP (normal human serum pool), 50 U/ml
penicillin and 50 µg/ml streptomycin. RF24 cells between the 24th and
30th passage were used for experiments.
DNA–liposome complexes were prepared just before use and added to the
cells in a 2:1 liposome/DNA charge ratio. RF24 monolayers were
pretreated with either liposomes containing 0.5 µM ODN (NFB decoy
ODN or scrambled ODN) or vehicle in serum-free RPMI 1640 medium for 30
min at 37°C. After washing once with phosphate-buffered saline, RF24
cells were stimulated for 30 min at 37°C with 100 U/ml TNF- (Pepro
Tech Inc., Rocky Hill, N.J.) in RPMI 1640 medium, supplemented with
20% HSP.
Animals and transplant transduction
Brown Norway (BN=RT1n) kidneys were
transplanted heterotopically into male Lewis
(LEW=RT1l) recipients, with one native kidney
in situ. Donors (150–200 g) and recipients (300–350 g)
were obtained from Iffa Credo Broekman (Zeist, Netherlands) and Harlan
(Bicester, U.K.), respectively. Maintenance occurred under standard
conditions with water and chow ad libitum. The protocol was
approved by the Utrecht University Committee for study in experimental
animals.
Rats were anesthetized with a mixture of fentanyl citrate and fluanison
(0.55 ml/kg), and midazolam (0.50 ml/kg). After endotracheal
intubation, recipients were mechanically ventilated with
O2/N2O (60/30). Donor nephrectomy and
heterotopic kidney transplantation were performed as described
previously (25)
. The left donor kidney was slowly perfused
in situ with 1 ml of warm (37°C) perfusion solution
(Custodiol-HTK; Dr. F. Köhler Chemie GmbH, Germany). Perfusion
solution contained 14 µM of NFB decoy ODN or scrambled ODN. The
perfused kidney was transplanted immediately into the recipient. After
30 min of warm ischemia the clamps were released, resulting in
reperfusion of the left kidney. Recipients did not receive
immunosuppressive medication and were killed 24 h (n=8
and 6 for decoy and scrambled ODN treatment) or 72 h
(n=7 and 6 for decoy and scrambled ODN treatment)
postreperfusion.
Histology
Animals were anesthetized with inactin. Kidneys were harvested,
weighed, and processed for immunohistochemistry. For monitoring
in vivo transduction, rabbit-anti-FITC antibody (Dako A/S,
Denmark) was used in combination with the TSA Direct kit (DuPont/NEN
Life Science products, Boston, Mass.). Sections were analyzed by
fluorescence microscopy.
For evaluation of NFB-mediated protein expression, monoclonal
antibodies for ICAM-1-, VCAM-1-, and CD8-antigen (Serotec/Carnon,
Germany) were used in combination with alkaline phosphatase
anti-alkaline phosphatase detection (Dako, Germany). Controls, omitting
first or second antibody were negative. VCAM-1 expression was evaluated
semiquantitatively and scored from 0 to 2, with 0 indicating no
staining, 1 moderate, and 2 profound intensity of staining in
endothelium.
For evaluation of monocyte infiltration, kidney sections were stained with monoclonal ED1 antibody (Serotec/Carnon, Germany). The number of ED1+ monocytes/macrophages in the perivascular area (i.e., vasa vasorum and adventitial area) was determined for all arteries per whole kidney section with a minimum of 20 arteries. In grafts harvested 3 days after transplantation, the number of ED1+ cells in the allograft tubulointerstitium was also determined. ED1+ cells were counted in 10 high-power fields (40x objective) of tubulointerstitium and expressed as amount of ED1+ cells per tubulointerstitial area.
Apoptosis was evaluated with the TUNEL method using dig11'UTP to incubate cryosections. Apoptotic nuclei were scored semiquantitatively in 10 high power fields from 0 to 2, where 0 indicates no apoptosis, 1 indicates few spread apoptotic nuclei, and 2 demonstrates areas of apoptosis.
Preparation of nuclear protein extract
Nuclear protein extracts from RF24 cells were prepared as
described by Andrews et al. (26)
with some modifications.
Cells were lysed in buffer containing 10 mM HEPES pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, and
0.1% Nonidet P-40. After centrifugating 5 min 14.000 rpm at 4°C,
nuclear proteins were extracted by suspending the nuclei in 10 µl of
low salt buffer (20 mM HEPES pH 7.9, 1.5 mM
MgCl2, 20 mM KCl, 0.2 mM EDTA, 25% glycerol, 0.2
mM PMSF, 0.5 mM DTT) and subsequently adding dropwise 40 µl of high
salt buffer (20 mM HEPES pH 7.9, 1.5 mM MgCl2,
400 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT).
Samples were centrifuged for 15 min at 14.000 rpm at 4°C. Protein
concentration of nuclear protein extract (supernatant) was determined
by Bradford assay.
Nuclei from kidney tissue were isolated essentially according to the
method of Blobel et al. (27)
and resuspended in hypotonic
buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM
KCl, 0.2 mM PMSF, 0.5 mM DTT). Preparation of nuclear protein extract
was performed as described for the RF24 cells.
Electromobility shift assay (EMSA)
ODN were end-labeled with [
-32P]ATP
(Amersham, Little Chalfont, U.K.) using T4 polynucleotide kinase
(Pharmacia Biotech, Brussels, Belgium). The oligo was purified using a
Sephadex G-50 spin column. Nuclear proteins (10 µg) were incubated
with 0.1 pmol 32P-labeled ODN in 10 µl binding
buffer [20 mM HEPES pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.1
µg poly (dI:dC)] in the absence or presence of 2 µg anti-p50 or
anti-p65 NFB antibody (Santa Cruz, Santa Cruz, Calif.).
Electrophoresis was performed on a 4% polyacrylamide gel with a 0.5x
TBE running buffer (45 mM Tris, 45 mM boric acid, and 1 mM EDTA).
Statistics
Student’s t test was used to compare monocyte
infiltration in decoy ODN- vs. scrambled ODN-treated transplants.
VCAM-1 staining was analyzed for significance using Mann-Whitney rank
sum test. P < 0.05 was considered significant. Data are
presented as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B decoy treatment of endothelial cells in vitroB inactivation in
regard to proinflammatory responses of the endothelium on
transplantation, we analyzed the effects of NFB decoy strategy
in vivo as well as in vitro. For in
vitro experiments, we used the human umbilical vein endothelial
cell line RF24 (24)
. EMSAs showed that TNF--induced
activation of transcription factor NFB in RF24 cells is inhibited by
incubation of the cells with liposome-entrapped NFB decoy ODN
(Fig. 1
), which is in accordance with the data of Tomita et al.
(28)
. Control incubations with excess nonlabeled NFB
oligo, nonspecific oligos, and antibodies directed against the p50 and
p65 subunits of NFB demonstrated that the TNF--induced shift was
specific for NFB (lanes 4–7). Treatment of cells with scrambled ODN
did not prevent the induced NFB shift. Moreover, EMSA of untreated
RF24 cells with excess unlabeled scrambled ODN confirmed that these ODN
do not bind NFB (lane 4). FACS analysis showed that the transduction
efficiency of FITC-labeled decoy and scrambled ODN was comparable (data
not shown). After 30 min of transduction, 95% of the cells contained
FITC label, which disappeared after 20 h.
|
Transduction of endothelial cells with NFB decoy in
vivo
Since our results demonstrated that the NFB decoy is a potent
tool to inhibit nuclear NFB activity in cultured endothelial cells,
we examined in a rat renal allograft model whether the NFB decoy
approach could be applied as a strategy to reduce NFB-mediated
expression of adhesion molecules on endothelial cells of the allograft,
resulting in a reduced infiltration of monocytes. Therefore, allografts
were perfused with liposome-loaded NFB decoy ODN prior to
transplantation. Liposome-mediated ODN transduction was monitored by
means of the FITC label, which was conjugated to the ODN. Fluorescence
microscopy demonstrated that after 30 min of transduction, ODN were
mainly present in peritubular capillaries, whereas macrophages and
mesangial cells as well as the contralateral kidney were negative
(Fig. 2
). Most of the peritubular vessels were positive, suggesting that
in vivo transfection was high. At 24 h after
transplantation, only a limited amount of peritubular vessels were
positive for FITC label, whereas at 72 h the FITC label of the ODN
could no longer be detected (data not shown).
|
To examine the effect of NFB decoy transduction on NFB activity
in the endothelium of renal allografts, nuclear extracts of these
tissues were subjected to NFB EMSA (Fig. 3
). Scrambled ODN-treated tissue showed a shift that was specific for
NFB and could be supershifted with antibodies directed against the
p50 and p65 subunits of NFB, whereas decoy ODN-treated kidneys
showed no specific NFB shift. Thus, perfusion of the renal allograft
with decoy ODN reduced activation of NFB.
|
NFB decoy reduces endothelial expression of VCAM-1 in
vivo
Since NFB is known to induce the expression of adhesion
molecules, we evaluated the expression of adhesion molecules VCAM-1 and
ICAM-1 in decoy- and scrambled ODN-treated renal transplants at 24 h after transplantation. Kidney grafts treated with decoy ODN showed a
significantly reduced VCAM-1 expression on the endothelium of both
arteries and venules compared with scrambled ODN-treated allografts
(P<0.05, Fig. 4
). Moreover, decoy treatment increased the number of arteries in which
VCAM-1 staining could not be detected. Venules of both decoy- and
scrambled ODN-perfused kidneys were positive for VCAM-1, though decoy
ODN prevented the profound expression of VCAM-1 seen in scrambled
ODN-treated grafts. ICAM-1 was detected on glomerular endothelial
cells, as well as on the endothelium of large intrarenal vessels and on
infiltrating cells, after both decoy and scrambled ODN treatment (data
not shown). Endothelial ICAM-1 staining of decoy- and scrambled
ODN-treated kidneys was comparable.
|
Monocyte influx in renal allografts is inhibited by NFB decoy
We also examined whether the reduction in NFB activity
and adhesion molecule expression affected monocyte influx in the
allograft. Infiltration pattern and amount of monocytes/macrophages
were evaluated for the entire kidney cross section using anti-ED1
antibody (Fig. 5
). Scrambled ODN-treated grafts showed a dense infiltrate in the
adventia and periarterial area and a minor infiltrate in the
tubulointerstitium. Perivascular infiltrate consisted mainly of
ED1+ macrophages, whereas
CD8+ cells were rarely seen (not shown). The
amount of CD4+ cells in the infiltrate was not
determined. In contrast, decoy ODN-treated kidneys showed a minor
influx of ED1+ cells and no influx of
CD8+ cells in adventititia and surrounding area.
Quantification of the number of ED+ cells in the
perivascular area of all arteries per cross section clearly
demonstrated that decoy ODN treatment increased the amount of arteries,
with few or no macrophages in its perivascular region. (Fig. 5C
). The mean number of macrophages per vessel was also
significantly decreased in decoy-treated allografts (3.7±0.7 vs.
9.2±1.2 macrophages/vessel; P<0.01; Fig. 5D
).
This decrease was not due to transduction of circulating cells caused
by systemic overspill of the decoy, since infusion of
liposome-entrapped decoy ODN directly into the vena cava inferior did
not reduce renal monocyte infiltration (data not shown).
|
Three days after ODN perfusion, the density of infiltrating cells,
including macrophages and CD8+ cells, was
increased and infiltration extended from the perivascular to the
tubulointerstitial region (Fig. 6
). The distribution of perivascular ED1+
infiltrates was comparable for decoy ODN and scrambled ODN treatment.
Mean number of infiltrating monocytes in adventitia and adjacent area
tended to be lower in decoy ODN-treated grafts, but this difference
appeared not to be significant (Fig. 6C
). The pattern of
tubulointerstitial infiltrate three days after transplantation was
affected by decoy ODN treatment. In scrambled ODN-treated grafts the
infiltration pattern was cortical diffuse, whereas in decoy ODN-treated
grafts infiltration was more focal and restricted to the adventitia and
adjacent area. In addition, the mean number of infiltrated macrophages
in the tubulointerstitium was significantly reduced by the decoy
treatment (75.6±13.9 vs. 120.0±11.2 macrophages/tubulointerstitial
area; P<0.05; Fig. 6D
).
|
Both at 24 h and 3 days after transplantation, no apoptosis could be observed in glomeruli, peritubular endothelium, and infiltrating cells. There was some apoptosis in tubular epithelium, which did not differ between decoy and scrambled ODN-treated allografts; 24 h after transplantation: 0.46 ± 0.08 (decoy) vs. 0.32 ± 0.10 (scrambled); 3 days after transplantation: 0.20 ± 0.2 (decoy) vs. 0.33 ± 0.12 (scrambled).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B decoy ODNs
inhibit the activation of NFB in cultured endothelial cells
(in vitro) as well as in renal allografts (in
vivo). In an experimental renal transplantation model, we show
that a single 30 min exposure of the renal allograft to double-stranded
NFB decoy ODN prior to transplantation results in a decreased
expression of adhesion molecules. In addition, these ODN reduce the
initial perivascular infiltration of monocytes and the subsequent
development of tubulointerstitial infiltration in nonimmunosuppressed
recipients.
Our in vitro studies demonstrated that a 30 min transduction
of endothelial cells with liposome-entrapped NFB decoy ODN results
in a near 100% transduction efficiency and in a complete inhibition of
NFB activation. At 20 h post-transduction, most of the
FITC-labeled ODN had disappeared. In vivo studies showed
that transduction was mainly limited to the peritubular vasculature of
the renal allograft. Also, the amount of FITC label was strongly
reduced at 24 h after transplantation. Nevertheless, NFB
activity was still reduced. Apparently, the FITC label has a short
half-life whereas the phosphorothioate-modified ODNs are more stable.
Notably, glomerular endothelium was hardly transduced, although
transient transduction in the initial hours post-transplantation cannot
be excluded. The preference for the peritubular vasculature may well be
due to the fact that cationic liposomes were used. These liposomes have
recently been found to transduce specifically relatively hypoxic
endothelium as can be found in the renal interstitium
(29)
. This phenomenon may also have contributed to the
observed reduction in tubulointerstitial infiltrate. Transduction of
macrophages probably did not contribute significantly to the observed
results, since FITC label could not be retrieved in these cells.
Furthermore, systemic administration of the decoy aiming at
transduction of circulating cells did not effect perivascular
infiltrate formation.
Allografts that were perfused with decoy ODN showed a reduction of
nuclear NFB binding activity and likewise a reduced
immunohistological staining of endothelial adhesion molecule VCAM-1
compared to scrambled ODN-treated kidneys. A clear reduction of ICAM-1
staining could not be detected, probably due to the high constitutive
expression of this adhesion molecule in renal endothelium (7
, 10)
. Since VCAM-1 expression is up-regulated in renal
allografts, it is likely that a reduced NFB-mediated expression of
this adhesion molecule contributes to the reduction in monocyte influx
upon decoy ODN treatment of renal allografts. In contrast, allografts
that were perfused with scrambled ODN did develop a characteristic
perivascular monocyte influx within 24 h after reperfusion, as we
previously described in this Brown Norway x Lewis transplantation model
for saline-treated allografts (25)
. Three days after
transplantation, this monocyte influx had developed into a marked
tubulointerstitial infiltrate. Decoy-treated allografts showed a
reduced tubulointerstitial infiltrate when compared with scrambled ODN
treatment. This reduction was characterized by a reduced presence of
ED1+ macrophages. CD8+ T
cell infiltrate also tended to be reduced in decoy-treated grafts. The
fact that there is a difference in monocyte infiltrate after decoy ODN
perfusion underscores the critical role of the initial NFB-mediated
(pro)inflammatory response in allograft pathology. As NFB has been
shown to regulate transcription of anti-apoptotic genes (30
, 31)
, it is of interest that we were unable to demonstrate
apoptosis in glomerular capillaries, peritubular endothelium, and
infiltrating cells.
Our data are in agreement with and extend previous observations on
ischemia-reperfusion injury of the kidney, where initial monocyte
influx could be reduced by local administration of the soluble form of
p-selectin glycoprotein ligand-1 (32)
or antisense
oligonucleotides against ICAM-1 (33
34
35)
. The current
study extrapolates this concept to allograft transplantation,
supporting the notion that ischemia-reperfusion injury is an important
determinant of inflammation in organ transplantation. In recent years
it has become evident that this initial inflammatory response
conditions the transplanted kidney to both acute rejection as well as
chronic transplant dysfunction. Therefore, our approach to reduce the
initial inflammatory response by NFB decoy may serve as a novel way
to explore organ-specific modulation of the prorejection environment
and the subsequent allograft survival in clinical medicine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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J. Bao, K. Sato, M. Li, Y. Gao, R. Abid, W. Aird, M. Simons, and M. J. Post PR-39 and PR-11 peptides inhibit ischemia-reperfusion injury by blocking proteasome-mediated Ikappa Balpha degradation Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2612 - H2618. [Abstract] [Full Text] [PDF]
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I. H. C. VOS, T. J. RABELINK, B. DORLAND, R. LOOS, B. V. MIDDELAAR, H.-J. GRÖNE, and J. A. JOLES L-Arginine Supplementation Improves Function and Reduces Inflammation in Renal Allografts J. Am. Soc. Nephrol., February 1, 2001; 12(2): 361 - 367. [Abstract] [Full Text]
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