| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Dumont-University of California Los Angeles Transplant Center, Department of Surgery, University of California Los Angeles School of Medicine, Los Angeles, CA 90095;
Institute of Medical Immunology, Humboldt University, Berlin, Germany; and
Institute of Medical Biochemistry, University of Rostock, Rostock, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
remained depressed, whereas that of
IL-4 and IL-10 reciprocally increased selectively in the combined
treatment group, data supported by ELISA studies. In parallel, only
adjunctive treatment triggered intragraft induction of molecules with
anti-oxidant (HO-1) and anti-apoptotic
(Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32)
functions, both in the early and late posttransplant phases. Hence,
systemic infusion of regulatory cells potentiates the effects of local
adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven
up-regulation of protective molecule programs at the graft site, such
as of anti-oxidant HO-1 and/or anti-apoptotic
Bcl-xL and Bag-1, may contribute, at least in part, to the
maintenance of the infectious tolerance pathway in transplant
recipients. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A series of elegant studies from Dr. Herman Waldmann and coworkers have shown that short courses of anti-T cell mAbs can reprogram and then guide the immune system of experimental animals to accept a transplant, or to arrest autoimmunity (15). The most prominent feature was the demonstration that once induced, tolerance could be maintained and perpetuated by what has been termed as infectious T cell-dependent regulatory mechanism (16). Our own studies have confirmed that features of infectious tolerance as described to minor histocompatibility-mismatched skin grafts in thymectomized mice conditioned with nonlytic CD4 and CD8 mAbs (16) may be applied to euthymic-primed rats rendered tolerant to MHC-incompatible vascularized organ allografts by CD4-targeted monotherapy (3, 4, 5). Hence, the tolerant cells in CD4 mAb-treated hosts could disable naive or even alloreactive cells so that they failed to trigger graft rejection (3). Moreover, a donor-specific but organ nonspecific unresponsive state could be transferred by regulatory spleen T cells in a dose-dependent manner into new cohorts of test graft recipients’ cells. The CD4+ T cell subset was instrumental in the induction of such adoptively transferred tolerance. Interestingly, unlike in the original CD4 mAb-treated tolerant hosts, selective up-regulation of IL-4 at the graft site was required for tolerance maintenance in test rat recipients conditioned i.v. with regulatory cells (4). Hence, local cytokine expression patterns in the graft itself and systemic infusion of regulatory cells may both influence allograft outcome in the infectious tolerance pathway.
On the basis of these findings, our present study was designed to test a hypothesis that administration of regulatory cells may potentiate the effects of intragraft adenovirus (Ad)5-mediated IL-4 gene transfer in the infectious tolerance pathway. Indeed, our results demonstrate striking synergistic effects, as evidenced by long-term test cardiac allograft survival after intragraft Ad-IL-4 gene therapy combined with i.v. infusion of a suboptimal dose of spleen cells from tolerant hosts. Only such a combined treatment increased intragraft expression of anti-oxidant heme oxygenase-1 (HO-1) as well as anti-apoptotic (Bcl-xL, Bag-1) molecules, in parallel with preferential induction of IL-4 and IL-10. Hence, our results are the first to document the benefits of systemic infusion of regulatory cells upon local Ad-IL-4 gene therapy in transplant recipients. Perhaps, Th2-driven up-regulation of protective molecules at the graft site may contribute, at least in part, to the effects of adjunctive Ad-IL-4 and regulatory cells in the infectious tolerance pathway.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Inbred male adult rats weighing 200–250 g were used (Harlan Sprague-Dawley, Indianapolis, IN). Lewis (LEW, RT1l) served as recipients of cardiac allografts from (LEW x BN)F1 (LBNF1) hybrids. Brown-Norway (BN; RT1n) rats were used as skin donors, and Wistar-Furth (WF; RT1u) served as heart donors for specificity studies. Full-thickness skin was sutured bilaterally to appropriate defects in the chest of prospective recipients. Hearts were transplanted to the abdominal great vessels by standard microvascular techniques. Graft function was monitored daily by palpation, and rejection was defined as the day of cessation of heartbeat.
Allograft model
LEW rats were sensitized with BN skin grafts (day -7), followed 1 wk later by transplantation of LBNF1 hearts (day 0). These cardiac allografts are rejected inevitably in an accelerated manner in <36 h (acute rejection occurs in 7–8 days) (3, 4, 5). Animals were treated with CD4 nondepleting (RIB-5/2) mAb (17) (5 mg/rat i.v. at day -7 (day of skin graft), -4, -1, 0 (day of heart graft) and on days 1, 4, 7, 10, 14, and 21 thereafter). This regimen induces permanent (>100 days) cardiac allograft acceptance with concomitant features of infectious tolerance (3, 4, 5).
Adoptive transfer studies
Spleens were harvested from long-term (>100 days) CD4 mAb-treated allograft recipients. Erythrocyte-free splenocytes were collected after treatment with RBC lysing buffer (Sigma, St. Louis, MO) and then administered i.v. into lightly total body gamma-irradiated (450 rad) syngeneic secondary rats. These were then challenged 24 h later with donor-specific (LBNF1) or third-party (WF) test cardiac allografts. Such an adoptive transfer of regulatory cells may impose donor-specific but organ nonspecific tolerance upon secondary hosts; gamma irradiation (450 rad) alone does not significantly affect cardiac allograft survival in this model (3).
Ad vectors
The Ad vector encoding for rat IL-4 (Ad-IL-4) was constructed, as described (18). Briefly, the cDNA for IL-4 was subcloned into pACCMV, and the resulting pACCMV-IL-4 was cotransfected with pJM17 vector. Homologous recombination resulted in a replication-defective Ad-IL-4. The titration of the virus was analyzed by plaque assay. Virus stocks of 2.5 x 1010 pfu/ml were stored at -80°C. As a reporter construct for gene transfer studies, recombinant Ad encoding for E. coli ß-galactosidase (Ad-ßGal) was used (19).
Ad-mediated transfection of donor hearts
Before in situ perfusion of the donor heart, the left superior
vena cava and the distal aorta were ligated to retain the perfusate.
The aorta was then held in position by suture to facilitate perfusion
using a syringe with a 30-gauge needle. The heart was perfused with 100
µl Ringer’s lactate solution containing Ad-IL-4 or Ad-ßGal
[2.5 x 109 pfu]. After perfusion, hearts
were placed in iced Ringer’s solution for 
1 h before
transplantation.
Experimental design
Our present studies were designed to evaluate the effects of in vivo interactions between systemically infused suboptimal regimen of regulatory spleen cells from CD4 mAb-treated tolerant rat recipients and local intragraft Ad-IL-4 gene transfer. We have previously shown that adoptive transfer of regulatory spleen cells alone (50 x 106) or a 1:1 mixture of regulatory (50 x 106) and naive (50 x 106) spleen cells may confer infectious tolerance from CD4 mAb-treated hosts as evidenced by long-term (>100 days) cardiac allograft survival in test rat recipients (3, 4, 5). In the present study, we investigated the influence of three distinct treatment regimens upon allograft outcome in the infectious tolerance pathway. In the first, LEW rats were infused i.v. with a mixture of tolerant (5 x 106) plus normal (45 x 106) spleen cells (1:10 ratio) 1 day before transplantation of LBNF1 hearts. In the second, LBNF1 hearts were transfected with Ad-IL-4 or Ad-ßGal gene and then transplanted into test LEW rats with no adjunctive cell transfer. And finally, in the third regimen, LEW rats received a 1:10 mixture of tolerant plus normal cells (5 x 106 + 45 x 106), followed 1 day later by transplantation of LBNF1 hearts transfected with Ad-IL-4 or Ad-ßGal. All animals were followed for allograft survival. Separate groups of rats were killed at 8 and >100 days, and test cardiac grafts were harvested for future analyses.
Histology
Cardiac allografts were sliced into pieces and preserved in 10% neutral-buffered formalin. Tissue samples were embedded in paraffin, cut into 5-µm sections, and then assessed by routine staining with hematoxylin and eosin for myocyte damage and graft infiltrating cells.
5-Bromo-4-chloro-indolyl-ß-D-galactopyranoside (X-Gal) staining
Cardiac allografts transfected with Ad-ßGal were harvested at 2 and 7 days, embedded in OCT freezing compound, and snap-frozen in liquid nitrogen. Cryosections of 8 µm in thickness were fixed with 1.25% glutaraldehyde at 4°C and then stained with X-Gal overnight at 37°C. Cells expressing ßGal cleave X-Gal to yield a blue chromophore in the cytoplasm. Representative areas were scored for percentage of infected (blue-stained) cells with a light microscope at x10 and x40 magnification.
RNA extraction/reverse transcription
Frozen cardiac allograft samples were homogenized, total RNA was extracted, and RNA concentration was determined by spectrophotometer. A total of 5 µg of RNA was reverse-transcribed using oligo(dT) primers and superscript reverse transcriptase according to the manufacturer’s instructions (Life Technologies, Grand Island, NY).
PCR primers/competitive template RT-PCR
Oligonucleotide primer pairs for the 5' and 3' rat cytokine
regions were selected based on published sequences (20).
To compare the relative level of each cytokine in different samples,
competitors for IL-2, IFN-, IL-4, IL-10, and ß-actin were
constructed, and the competitive template RT-PCR amplification was
performed, as described (4, 20). According to the varying
contents of specific cDNA and varying amplification efficiencies, the
samples were subjected to different cycle numbers at the annealing
temperature that was optimized empirically for each primer pair: 40,
60°C (IL-2), 45, 60°C (IL-4), 40, 55°C (IL-10), 35, 60°C
(IFN-), and 32, 63°C (ß-actin), respectively. PCR products were
analyzed in ethidium bromide-stained 2% agarose gel and photographed
with Polaroid film (Cambridge, MA) under UV light. The PCR results were
scanned, and the density of wild-type (WT) cDNA and gene-specific
competitor bands were analyzed using Kodak Digital Science 1D Analysis
Software (Version 2.0; Kodak, Rochester, NY). All samples were
normalized against the respective ß-actin WT cDNA/CT DNA ratio.
IL-4 ELISA
Sample supernatants were obtained from homogenized cardiac allografts with PBSTDS buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, and 1% Triton X-100, pH 7.2). Serum samples were collected from recipients’ peripheral blood. Rat IL-4 was quantitated by using PharMingen technical protocols (San Diego, CA). Briefly, flat-bottom 96-well microtiter plates (Costar, Corning, NY) were coated with 50 µl/well of diluted polyclonal rabbit anti-IL-4 Ab (BioSource International, Camarillo, CA) overnight at 4°C. Nonspecific binding sites were blocked with Blocking Buffer (10% FBS, 10% newborn calf serum ,or 1% BSA in PBS) and incubated for 90 min at room temperature (RT). Plates were rinsed with PBS/Tween, and then diluted standard and sample supernatant (100 µl) in triplicate were added, followed by incubation for 4 h at RT. Plates were washed, followed by the addition of 100 µl/well biotinylated rabbit anti-IL-4 Ab (2 µg/ml in Blocking Buffer), and incubation for 1 h at RT. After washing, streptavidin-peroxidase conjugate (PharMingen) was added, and the plates were incubated for 30 min at RT. Plates were washed again, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate solution (PharMingen) was added. The plates were incubated at RT for color development, and the reaction was terminated with 50 µl of stopping solution (20% SDS, 50% dimethylformamide). Plates were read at 405 nm in an ELISA reader. The linear region of IL-4 standard curves were obtained in a series of eight 2-fold dilutions of IL-4 standard, from 2000 pg/ml to 15 pg/ml.
Western blot analysis
Protein was extracted from heart tissue samples with PBSTDS buffer. Proteins (30 µg/sample) in SDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, 1% SDS) were subjected to 12% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The gel was then stained with Coomassie blue to document protein loading. The membrane was blocked with 3% dry milk and 0.1% Tween 20 (United States Biochemical, Cleveland, OH) in PBS. Polyclonal rabbit anti-rat HO-1 Ab was kindly provided by Dr. R. Buelow (SangStat, Fremont, CA). Polyclonal Abs against rat anti-apoptotic Bcl-xL and Bag-1, as well as pro-apoptotic CPP-32, were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The membranes were incubated with Abs, and then developed according to the Amersham enhanced chemiluminesence protocol. Relative quantities of HO-1, Bcl-xL, Bag-1, and CPP-32 proteins were determined by densitometry (Kodak Digital Science 1-D Analysis Software).
Statistical analysis
Statistical comparisons between groups were analyzed by Student’s t test. The differences were considered significant at p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
First, we confirmed our previous findings (3, 4, 5) by
demonstrating that 1) LBNF1 cardiac allografts
survived >100 days following treatment of LEW rats with a nondepleting
CD4 (RIB-5/2) mAb (n = 12); 2) adoptive transfer of
spleen cells from tolerant RIB-5/2 mAb-treated hosts (50 x
106) prolonged survival of test
LBNF1 cardiac allografts to >100 days in lightly
gamma-irradiated (450 rad) secondary rat recipients (Fig. 1
); and 3) cardiac allografts were
rejected within 12 days in gamma-irradiated otherwise untreated rats
(n = 4). Interestingly, as shown in Fig. 1, infusion of
a suboptimal mixture of tolerant and normal spleen cells in a 1:10
ratio failed to significantly affect test cardiac allograft survival
(mean survival time (MST) ± SD = 12.2 ± 3.5 days).
Similarly, LBNF1 hearts transfected with Ad-IL-4
alone were rejected within 3 wk (16.8 ± 5.8 days) following
transplantation into gamma-irradiated and otherwise untreated LEW rats.
However, combination of adoptively transferred tolerant and normal
cells (at 1:10 ratio) plus Ad-IL-4 gene transfer extended test cardiac
allograft survival to about 2 mo, with some surviving >100 days
(MST ± SD = >60 ± 38 days, p <
0.05). This effect was 1) Ad-IL-4 specific, because test cardiac
allografts were rejected in <14 days in animals undergoing combined
local transfer of Ad vector expressing nonimmunoregulatory molecule
(ßGal) plus suboptimal dose of regulatory cells (n =
3; not shown); and 2) donor specific, because Ad-IL-4-transfected third
party (WF) hearts were rejected promptly (10.0 ± 1.7 days) in
rats infused with spleen cells from tolerant CD4 mAb-treated syngeneic
recipients of LBNF1 grafts (Fig. 1). Similarly,
WF hearts transfected with Ad-IL-4 were rejected within 12 days
(n = 3) in gamma-irradiated otherwise untreated LEW
rats (not shown). Thus, systemic infusion of the suboptimal dose of
regulatory cells from tolerant hosts plus local intragraft Ad-IL-4 gene
transfer exerted synergistic therapeutic effects in the infectious
tolerance pathway by significantly extending the survival of
donor-specific test cardiac allografts.
|
Test cardiac allografts were harvested at day 8 for
histopathological evaluation. Allografts from animals treated with
suboptimal dose of regulatory cells showed dense areas of mononuclear
cell (MNC) infiltration, myocyte necrosis, severe interstitial edema,
and lymphocytic vasculitis (Fig. 2A). Cardiac allografts
transfected with Ad-IL-4 were characterized by diffuse MNC
infiltration, moderate to severe interstitial edema, and vasculitis
(Fig. 2B). In contrast, combined systemic treatment with
regulatory cells and local Ad-IL-4 gene transfer largely preserved
cardiac architecture and arteries and markedly depressed MNC infiltrate
(Fig. 2C).
|
To analyze putative mechanisms of in vivo synergy between
regulatory cells and intragraft Ad-IL-4 gene transfer, we analyzed
cardiac allografts histologically for X-Gal staining. By day 7, the
mean percentage of ßGal+ cells in cardiac
allografts was 1–5% after Ad-ßGal gene transfer alone (Fig. 3A); it increased to 10–15%
after adjunctive infusion of regulatory cells (Fig. 3B).
Thus, the infusion of regulatory cells from tolerant hosts increased
the expression of the reporter gene at the graft site.
|
Because of our previous findings on the role of Th2-type cytokines
in the infectious tolerance pathway (3, 4, 5), we then used
competitive template RT-PCR to analyze intragraft Th1 and Th2 cytokine
gene programs in our transplantation models. As shown in Fig. 4, by day 8, the expression of both IL-2
and IFN- remained consistently increased after infusion of cells or
Ad-IL-4 gene transfer alone, as compared with combined treatment
regimen (p < 0.05). In contrast, the level of
transcripts coding for IL-4 and IL-10 remained significantly
(p < 0.01) elevated after i.v. infusion of
regulatory cells plus local Ad-IL-4 gene transfer both in the early
(day 8) and late (day 114) posttransplant period, as compared with
individual treatment regimens. Thus, adjunctive infusion of regulatory
cells from tolerant hosts and local Ad-IL-4 gene transfer induced
Th2-type deviation in well-functioning test cardiac allografts in the
infectious tolerance pathway.
|
To validate RT-PCR-based data, we then performed serial ELISAs to
assess IL-4 levels in groups of engrafted hosts. As shown in Fig. 5, intragraft expression of IL-4 protein
(ng/ml) at day 8 in the Ad-IL-4 gene therapy group was significantly
higher (p < 0.05), as compared with
corresponding graft samples following regulatory cell treatment
(mean ± SD = 0.025 ± 0.007 and 0.14 ± 0.03,
respectively). The combined treatment regimen further increased IL-4
levels (p < 0.05) both in the early (day 8,
0.043 ± 0.003) and late (day 114, 0.24 ± 0.04;
p < 0.005 as compared with day 8 samples)
posttransplant periods. Unlike in the graft itself, little if any IL-4
could be detected in the serum (<0.03 ng/ml) regardless of the
treatment regimen (data not shown). Hence, combined regulatory cells
and Ad-IL-4 gene transfer synergistically enhanced intragraft but not
systemic IL-4 protein levels.
|
Finally, because of the emerging role of "protective" genes in
long-term allograft maintenance in transplant recipients (21, 22), we used Western blots to analyze the expression of
anti-oxidant (HO-1), anti-apoptotic (Bcl-xL,
Bag-1), and pro-apoptotic (CPP-32) gene products in our model. The
protein was extracted from cardiac allografts harvested at day 8 or
>100 in separate groups of rats that received suboptimal dose of
regulatory cells and/or Ad-IL-4 gene transfer. The relative intragraft
expression levels of individual protective molecules was determined by
densitometry and expressed in absorbance units (AU). As shown in Fig. 6, the expression of anti-oxidant
HO-1 and anti-apoptotic Bcl-xL and Bag-1 was strongly
up-regulated after combined (regulatory cells plus Ad-IL-4 gene
transfer) therapy both in the early and late phases after
transplantation (1.7–2.0 AU, 1.8–2.2 AU, and 1.6–2.1 AU,
respectively), as compared with Ad-IL-4 gene transfer (0.5 AU, 0.6 AU,
and 0.1 AU, respectively) or infusion of regulatory cells (0.1 AU, 0.3
AU, and 0.7 AU, respectively) alone. In contrast, the expression of
pro-apoptotic CPP32 was decreased after combined regulatory cells plus
Ad-IL-4 gene transfer (0.2–0.3 AU), as compared with Ad-IL-4 (1.8 AU)
or cells (1.9 AU) alone groups. Hence, test allograft survival after
adjunctive treatment with regulatory cells and Ad-IL-4 correlated with
the selective induction of molecules with anti-oxidant (HO-1) and
anti-apoptotic (Bcl-xL, Bag-1) functions.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
That IL-4 might play a role in the acquisition of infectious tolerance
has been shown by the ability of neutralizing IL-4 mAb to partially
antagonize the induction of donor-specific unresponsiveness in an
adoptive transfer system (23). Moreover, in our own
ongoing studies, spleen cells from tolerant animals tend to produce a
higher level of IL-4 (but not IL-10 or TGF-ß) upon in vitro
stimulation with donor cells or Con A than those from naive animals (Y.
Z. and J. W. K.-W., unpublished data). Direct evidence for an IL-4
requirement in the induction of regulatory mechanism was demonstrated
in a murine cardiac transplant model in which CD2- plus CD3-targeted
therapy failed to induce tolerance in IL-4-deficient recipients, and
infusion of IL-4 mAb abrogated tolerance in normal hosts
(24). Although neutralizing IL-4 at the time of
donor-specific blood transfusion plus CD4 mAb-induced tolerance had no
effect on allograft outcome in primary recipients, neutralizing IL-4 in
adoptively transferred donors prevented long-term engraftment in the
majority of secondary hosts. Moreover, although in our recent study,
treatment with IL-4 mAb did not prevent tolerance induction in primary
hosts, this tolerant state was not "infectious," as further
transfers of splenocytes failed to prevent rejection in new cohorts of
engrafted rats (25). Our present results show that
selective induction of IL-4 after Ad-based gene therapy alone failed to
significantly affect cardiac allograft survival in two different
(LBNF1
LEW and WFLEW) rat strain
combinations. This may have resulted from limitations of the system in
which the actual Ad-ßGal transfection rate in rat myocytes was very
low (1–5% at day 7), consistent with our previous report
(18). Interestingly, we now show that an adjunctive
suboptimal dose of regulatory cells markedly increased transgene
expression by recombinant Ad in cardiac allografts. Consistent with our
present RT-PCR data, this may have resulted from depression of an
intense proinflammatory Th1-type response that otherwise down-regulates
the transgene expression-driven CMV promoter (26).
Interestingly, mouse cardiac isografts transfected with ßGal were
spared from inflammatory response, despite the presence of an immune
response to the vector (27). We have recently reported
that treatment with RIB-5/2, a mAb used in our present tolerogenic
regimen, prolonged lung-directed Ad-mediated gene expression
(28). As our present RT-PCR assay has not been constructed
to differentiate between Ad- and endogenously derived IL-4, enhanced
intragraft expression of this Th2 cytokine at both mRNA (RT-PCR) and
protein (ELISA) levels in the combined treatment group could come from
Ad vector, host lymphocytes, and/or adoptively transferred lymphocytes.
Hence, our results support the idea that ex vivo-generated regulatory
cells might be considered as an adjunctive measure to enhance the
efficacy of Ad-mediated cytokine gene therapy in transplant
recipients.
It is becoming increasingly appreciated that transplantation tolerance exists at multiple levels (29), and its maintenance may require utilization of several immune mechanisms, including deletion, anergy, and the emergence of regulatory T cells. These mechanisms may not only overlap, but their individual contributions may vary depending on MHC barrier, the type of organ graft, therapeutic strategy applied, and species being studied. Our present findings complement recent reports that provide compelling evidence for the role of regulatory T cells in host unresponsiveness, especially in the infectious tolerance (30). We have previously shown that at least 50 x 106 spleen cells were required to confer operational tolerance from tolerant hosts to new cohorts of test rat recipients (3, 4). By choosing the suboptimal regimen, we have now elected to use 1/10 of the effective dose (5 x 106), and to keep with the total cellular load these regulatory cells were mixed with 45 x 106 of naive splenocytes. Such a mixture alone produced marginal prolongation of test graft survival after adoptive transfer, consistent with our previous dose-response trials (3, 4). However, if combined with Ad-IL-4 gene therapy, a significant prolongation of donor-type (LBNF1) but not third party (WF) test graft survival was observed. Hence, our results demonstrate that 1) Ad-IL-4 gene transfer potentiates the operational activity of regulatory cells, and 2) regulatory cells maintain MHC specificity of the infectious tolerance pathway. CD4+ T cells are instrumental in that pathway, as their selective depletion in the original tolerant animals or after transfer into new cohorts of test recipients prevents induction of tolerance (3, 31, 32). In all animal models tested to date, the maintenance of regulatory T cells was shown to depend on the presence of donor Ag (30). However, unlike in mice (16), the induction of regulatory T cells in rats (5) or pigs (33) was thymus dependent, suggesting that both central and peripheral immune mechanisms are required for the acquisition of infectious tolerance.
Prolonged allograft survival after adjunctive Ad-IL-4 and regulatory
cell treatment in this study was accompanied by overexpression of
protective molecules at the graft site, including anti-oxidant HO-1
and anti-apoptotic Bcl-xL and Bag-1 but depression of
pro-apoptotic CPP-32. HO-1 expression can be detected by
immunohistology studies by all rat cardiac cells, with endothelial and
smooth muscle cells expressing far better than myocytes; MNC infiltrate
expresses both Bcl-xL and HO-1 variably (W. W.
Hancock, J. W. Kupiec-Weglinski, unpublished data). Up-regulation
of these genes blocks activation of the transcription factor NF-
B in
endothelial cell cultures, and thereby suppresses induction of
proinflammatory genes in Ag-activated endothelium (21, 34). We have recently shown striking cytoprotective effects of
HO-1 overexpression in rat liver models of ischemia/reperfusion injury
(35), consistent with other reports documenting the role
of HO-1 overexpression in xenograft "accommodation"
(36), prolonged allografts survival (37), or
prevention of transplant arteriosclerosis and interstitial fibrosis
characteristic for chronic rejection (38). Moreover, in a
parallel Fas ligand gene therapy study in rat renal allografts
(39), we have recently detected down-regulation of Bag-1,
a prototype of a novel type of Bcl-2-binding anti-cell death gene,
which has been implicated in T cell activation-induced apoptosis
(40). Hence, as combined Ad-IL-4 gene transfer and
infusion of regulatory cells were required to achieve therapeutic
effect, both graft and host factors might play a role in the induction
of protective genes in the infectious tolerance pathway.
Putative mechanisms that may trigger intragraft expression of protective genes are ill defined. Interestingly, as in xenograft "accommodation" (21), the induction of protective molecules in the infectious tolerance pathway correlated with preferential infiltration of cardiac allografts by Th2-like IL-4- and IL-10-producing cells. Indeed, the Th2 cytokine program has been shown to trigger the expression of protective genes in MNC (41) and endothelial cells (39). This is consistent with our findings on the role of Th2-like cells and local secretion of Th2-type cytokines in animals rendered tolerant by transfer of regulatory cells (3, 4, 25). We favor the notion that the expression of protective molecules is Th2-type dependent, because treatment of rats with neutralizing IL-4 mAb depressed intragraft expression of HO-1 and Bcl-xL and prevented the acquisition of tolerance in cohorts of adoptively transferred rats (J. W. Kupiec-Weglinski, unpublished data). Perhaps future studies in knockout mice will determine whether the host Th2 environment might promote graft survival by influencing natural mechanisms of protection.
In conclusion, our present study demonstrates that systemic infusion of regulatory cells from tolerant hosts potentiates the effects of local Ad-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway. This pathway is important, and defining the underlying mechanisms may lead to new insights into the complexities of establishing immune tolerance to solid organ transplants. Clearly, tolerogenic strategies that trigger generation and then support the maintenance of regulatory cells in rodent transplantation models warrant more attention in future primate and human studies.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Institute of Medical Immunology, Humboldt University, Berlin, Germany.
3 Current address: Institute of Medical Biochemistry, University of Rostock, Rostock, Germany.
4 Address correspondence and reprint requests to Dr. Jerzy W. Kupiec-Weglinski, Dumont-University of California Los Angeles Transplant Center, Room 77-120 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095.
5 Abbreviations used in this paper: Ad, adenovirus; Ad-IL-4, adenovirus coding for IL-4 gene; ßGal, ß-galactosidase; HO-1, heme oxygenase-1; MNC, mononuclear cells; MST, mean survival time; RT, room temperature; X-Gal, 5-bromo-4-chloro-indolyl-ß-D-galactopyranoside; LEW, Lewis rat; BN, Brown-Norway rat; LBNF1, (LEW x BN)F1 hybrid rat; WF, Wistar-Furth rat; WT, wild type; AU, absorbance unit.
Received for publication December 3, 1999. Accepted for publication March 15, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in long-surviving mouse heart allografts after brief CD4-monoclonal antibody therapy. Transplantation 59:559.[Medline]
and tumor necrosis factor-
inhibit transgene expression. Hum. Gene Ther. 8:2019.[Medline]
B. J. Clin. Invest. 103:543.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. Vassalli, M.-E. Roehrich, P. Vogt, G. B. Pedrazzini, F. Siclari, T. Moccetti, and L. K. von Segesser Modalities and future prospects of gene therapy in heart transplantation Eur. J. Cardiothorac. Surg., June 1, 2009; 35(6): 1036 - 1044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P Cobbold T cell tolerance induced by therapeutic antibodies Phil Trans R Soc B, September 29, 2005; 360(1461): 1695 - 1705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Pace, C. Pioli, and G. Doria IL-4 Modulation of CD4+CD25+ T Regulatory Cell-Mediated Suppression J. Immunol., June 15, 2005; 174(12): 7645 - 7653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Zenclussen, K. Gerlof, M. L. Zenclussen, A. Sollwedel, A. Z. Bertoja, T. Ritter, K. Kotsch, J. Leber, and H.-D. Volk Abnormal T-Cell Reactivity against Paternal Antigens in Spontaneous Abortion: Adoptive Transfer of Pregnancy-Induced CD4+CD25+ T Regulatory Cells Prevents Fetal Rejection in a Murine Abortion Model Am. J. Pathol., March 1, 2005; 166(3): 811 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Vassalli, S. Fleury, J. Li, J.-J. Goy, L. Kappenberger, and L. K. von Segesser Gene transfer of cytoprotective and immunomodulatory molecules for prevention of cardiac allograft rejection Eur. J. Cardiothorac. Surg., November 1, 2003; 24(5): 794 - 806. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Hammer, G. Schroder, K. Risch, A. Flugel, H.-D. Volk, M. Lehmann, and T. Ritter Antigen-Dependent Transgene Expression in Kidney Transplantation: A Novel Approach Using Gene-Engineered T Lymphocytes J. Am. Soc. Nephrol., February 1, 2002; 13(2): 511 - 518. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhai, X.-D. Shen, M. Lehmann, R. Busuttil, H.-D. Volk, and J. W. Kupiec-Weglinski T Cell Subsets and In Vitro Immune Regulation in ""Infectious"" Transplantation Tolerance J. Immunol., November 1, 2001; 167(9): 4814 - 4820. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, Spleen cells from tolerant hosts alone
(50 x 106) (MST = >100 days;
n = 5); 
, 1:10 mixture of spleen cells from
tolerant hosts (5 x 106) and normal naive rats
(45 x 106) (MST = 12.2 days;
n = 4); 
, intragraft Ad-IL-4 gene transfer alone
(MST = 16.8 days; n = 4); 
, 1:10 mixture of
tolerant and normal spleen cells plus intragraft Ad-IL-4 gene transfer
(MST = >60 days; n = 5); •, 1:10 mixture of
tolerant and normal spleen cells plus Ad-IL-4 gene transfer into
third-party WF cardiac allograft (MST = 10.0 days;
n = 3).
) and 114 days (
