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Nuffield Departments of
*
Surgery and
Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; and
National Heart and Lung Institute, Imperial College, School of Medicine, Harefield Hospital, Middlesex, United Kingdom.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-12, and it also
increases APC and endothelial expression of adhesion molecules and
costimulatory ligands B7.1 (CD80) and B7.2 (CD86) (3, 4).
Its ligand, CD154, is mainly expressed on activated T cells and
platelets (2). Blockade of the CD40-CD154 pathway, either
alone or in combination with the B7-CD28 pathway, has been shown to
inhibit autoimmune disease (5). The demonstration that
blockade of the CD40-CD154 interaction can lead to long-term allograft
survival in small (6) as well as large animal models
(7) raised hopes that this therapeutic approach would
translate into the clinic. However, several recent reports have clearly shown that anti-CD154 targets predominantly CD4+ T cells and is unable to prevent immune responses mediated by CD8+ T cells such as virus-specific CTLs (8) and allograft rejection (9, 10, 11, 12, 13). Even in the absence of CD8+ T cells, CD154 blockade was ineffective at preventing the formation of transplant arteriosclerosis, suggesting that additional mechanisms contribute to the development of this disease in the absence of a functional CD40-CD154 pathway (13). Therefore, it is essential to explore mechanisms of allograft rejection that are resistant to CD40-CD154 blockade because this might help to elucidate alternative rejection pathways that can lead to graft rejection.
The aim of this study was to investigate and characterize the mechanisms responsible for the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation. To address this question, CD40-knockout (CD40-/-) mice were used as transplant recipients to investigate whether the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation is a general feature of an interrupted CD40-CD154 costimulatory pathway. CD40-/- mice have been shown to be defective in producing IgG Abs (14) and unable to mount effective immune responses to infectious agents such as Leishmania (15). In the context of transplantation, APCs from CD40-/- recipients are unable to fully activate T cells and to mount IgG alloantibody responses.
The abdominal aortic allograft model was used for this study because it allows the precise quantification of vascular lesions (16). Furthermore, we have demonstrated the suitability of the aortic graft to study transplant arteriosclerosis as compared with the vascularized cardiac allograft model (17). The aortic graft has the advantage that it is a nonparenchymal transplant, which therefore contains only a minimal number of donor-derived passenger leukocytes that might be able to interfere in this system by initiating an alloresponse.
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Materials and Methods |
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C57BL/6 (H-2b) mice were originally purchased from Harlan Olac (Bichester, U.K.) and used as recipients. CD40-deficient mice (SV 129, H2b) were generated by homologous recombination in embryonic stem (ES) cells (A. Rahemtulla and H.V. Sorensen, unpublished observations). Briefly, a targeting vector was constructed by inserting a G418 resistance gene (neomycin) into the third exon of an 8-kb CD40 genomic fragment. The targeting vector was transfected into ES cells, and G418 resistant clones were isolated and screened for homologous recombination by Southern blot analysis. The homologously recombined ES cells were injected into 3.5-day-old C57BL/6 blastocysts, and injected blastocysts were transferred into the uteri of mice that had been pseudopregnant for 2.5 days. Germline transmission of the mutation by the chimeric mice resulted in heterozygous mutant mice. The heterozygous mutant mice were interbred to homozygosity for the CD40 mutation. In the phenotypic characterization, CD40-/- mice showed no CD40 expression on lymphocytes, elevated levels of IgM and IgG3, reduced levels of IgG1 and IgG2b, low levels of IgG2b, and undetectable IgE, confirming an impaired Ig class switch. B cells from mutant mice, unlike wild-type B cells, did not respond to stimulation with anti-CD40 Ab. Furthermore, mutant mice did not form germinal centers after stimulation with thymus-dependent Ag (keyhole limpet hemocyanin in CFA), in contrast to wild-type mice (18). These results confirm a phenotype similar to other published CD40-/- strains (14, 19). At the time of experiments, the C57BL/6 CD40-/- mice used in this study were backcrossed for five generations onto the C57BL/6 background, and heterozygous C57BL/6 CD40+/- littermates and normal C57BL/6 mice were used as controls. BALB/c (H2d) mice were used as donors of aortic allografts. Mice were bred and maintained in the Biomedical Services Unit at the John Radcliffe Hospital (Oxford, U.K.). All mice used in this study were between 6 and 12 wk of age at the time of experimental use and were treated in strict accordance with the Home Office Animals (Scientific Procedures) Act of 1986.
Abs and injection protocols
Abs to CD8 (YTS 169) (20), CD11b (M1/70) (21), and IL-4 (11B11) (22) were grown from hybridomas obtained from American Type Culture Collection (Manassas, VA). Biotinylated Abs to CD4, CD8, and CD40 were purchased from BD PharMingen (San Diego, CA), and those to IgM, IgG1, IgG2a, and IgG3 were purchased from Serotec (Oxford, U.K.). HRP-labeled anti-mouse IgG Abs were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
For depletion of CD8+ T cells, C57BL/6 CD40-/- or C57BL/6 CD40-/- recipients were injected i.p. with a mAb to CD8 (YTS 169; 250 µg) 7 days, 3 days, and 1 day before transplantation and on day 14 after transplantation. This protocol for CD8 depletion has been shown to maintain complete depletion of the CD8+ T cell subset (<1%; data not shown) throughout the posttransplant course of 30 days (23). For neutralization of IL-4, either the IL-4 mAb (11B11) or control rat IgG (Sigma, St. Louis, MO) was injected i.p. on days 2, 4, 6, 10, 14, and 21 after transplantation (24). In one additional group, a mAb to CD154 (MR1) was injected i.p. (500 µg on days 0, 2, and 4). This treatment protocol for anti-CD154 blockade has been shown to prevent acute allograft rejection and to prolong cardiac allograft survival (6).
Abdominal aortic transplantation
The procedure was performed using a modified technique initially described by Koulack et al. (16). Briefly, the donor thoracic aorta was isolated and resected and transferred to the recipient animal. A proximal end-to-end anastomosis was performed by using an 11–0 monofilament nylon suture. Then the aortic graft was repositioned, and the anastomosis continued with single interrupted sutures.
Histology and analysis of aortic grafts
Aortic grafts were removed under anesthesia on day 30. Grafts were perfused with saline and were flash-frozen in OCT medium (Tissue-Tek; Sakura Finetek, Leiden, The Netherlands) in liquid nitrogen, and 7-µm cryostat sections were prepared. Sections were stained with H&E or azoeosin for the detection of eosinophils. Eosinophils infiltrating in the intima, medium, and adventitia were counted manually by two investigators (S.M.E. and O.W.) who were blinded to the experimental conditions; counting was done at a magnification of x200 in each of 100 fields defined by a graticule on day 30 following transplantation. For morphometric analysis, five sections from each graft harvested on day 30 were stained with Miller’s elastin/van Gieson and analyzed by two independent examiners (S.M.E. and O.W.) at an original magnification of x100 using a conventional light microscope. A digitized image of each section was captured, and the areas within the lumen and the internal and external elastic lamina were circumscribed manually and measured as previously described (25). From these measurements, a quotient for the thickness of the intima (Qint) was calculated. Qint indicates the relative thickness (percentage) of the intima (Qint = intima/(lumen + intima) x 100). Therefore, a Qint value of 0% indicates no intimal thickening, and a Qint value of 100% indicates a total occlusion of the lumen. All image analyses were conducted on a color display monitor using Lucia Image Analysis software (Nikon, Kingston, U.K.).
Immunohistochemistry
The 7-µm sections were air-dried and then fixed in acetone for 10 min. Endogenous peroxidase activity was blocked with 2% hydrogen peroxide and 0.1% sodium azide in cold TBS. Endogenous biotin was blocked with an avidin solution mixed in 1% BSA in PBS for 15 min and followed by a biotin solution mixed in 1% BSA in PBS for 15 min (Vector Laboratories, Burlingame, CA). The mAbs directly conjugated to biotin were applied to each section and incubated for 60 min. Binding was detected by an avidin-biotin-peroxidase complex, and staining was visualized using diaminobenzidine (Vector Laboratories). Sections were then counterstained. The positive cells in the intima, medium, and adventitia were counted manually by two investigators (S.M.E. and O.W.) who were blinded to the experimental conditions; counting was done at a magnification of x200 in each of 100 fields defined by a graticule on day 30 following transplantation. Evaluation was performed regardless of the thickness of the intima, medium, and adventitia.
Alloantibody detection in the serum
The isotype of circulating alloantibodies specific for the dominant MHC class I molecule H2-Dd present on the BALB/c aortic graft was determined by FACS analysis as described previously (26). L cells transfected with the Dd gene were used as target cells. Serum was incubated with the target cells, then a second-stage biotin-labeled anti-mouse isotype-specific Ab, IgM, IgG1, IgG2a, and IgG3 (Serotec), was added. In a third step, target cells were incubated with streptavidin-PE. The samples were acquired on a BD Biosciences (San Jose, CA) FACSort using CellQuest software (BD Biosciences) for analysis. The amount of alloantibody was determined by determining the mean fluorescence intensity of each sample.
Competitive RT-PCR
Aortic grafts were removed 14 days after transplantation, flushed with sterile saline, and snap-frozen in liquid nitrogen. RNA isolation, cDNA synthesis, and PCR were performed as previously described (27). The multiple competitive construct was kindly provided by S. Reiner and S. Miller (Northwestern University, Chicago, IL) (28). Amplification and construction of a competitor for eotaxin and CCR3 was performed using the following oligonucleotide sequences: eotaxin forward, 5'-CTC CAC AGC GCT TCT ATT-3'; eotaxin reverse, 5'-CCA GGT GCT TTG TGG CAT3'; CCR3 forward, 5'-ATG GCA TTC AAC ACA GAT GAA ATC AAG3'; and CCR3 reverse, 5'-GGA TAG CGA GGA CTG CAG GAA AAC3'. All reactions were performed in triplicate, and the mean was used for further calculations. To account for minor variations in the hypoxanthine phosphoribosyltransferase level in the experimental samples, the final result is given as the ratio of gene of interest/competitor hypoxanthinephosphoribosyltransferase/competitor (in femtograms) of the amount of competitor used for the amplification of the respective gene of interest. The analysis was performed on day 14, as we have previously shown that, in the aortic allograft model, the cellular infiltrate as well as the intragraft cytokine expression is maximum at this time point, facilitating the analysis (29).
Southern blot analysis
Genomic DNA (10 µg) was digested with HindII and subjected to agarose gel electrophoresis. DNA was then transferred onto nylon blotting membranes, and filters were hybridized with radiolabeled probes overnight. Filters were then washed in 0.1x SSC 0.1% SDS at 65°C for several hours before autoradiography.
Statistical analysis
Results are given as the mean per group ± SD. The data were analyzed using a paired two-tailed Student’s t test. A value of p < 0.05 was considered significant.
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Results |
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Transplant arteriosclerosis was equally pronounced in BALB/c
(H2d) aortic allografts transplanted into either
CD40-/- or heterozygous littermates
(CD40+/-), and the degree of intimal
proliferation was not significantly different from that observed in
wild-type C57BL/6 recipients (intimal proliferation was 59 ± 5%
for CD40-/- mice vs 58 ± 4% for
CD40+/- mice vs 62 ± 11% for
CD40+/+ mice; n = 5) (Fig. 1
A, panels A and
B, and B). Syngeneic grafts did not show any
signs of transplant arteriosclerosis at any time point analyzed. To
investigate whether donor wild-type APCs play a role in this model,
CD40-/- recipients were treated with an
anti-CD154 mAb (MR1) at the time of transplantation, which showed
no reduction in intimal proliferation (intimal proliferation was
56 ± 7% for CD40-/- MR1-treated mice vs
59 ± 5% for CD40-/- mice;
n = 5). Furthermore, in an additional group, aortic
transplants were performed in the reverse order using
CD40-/- and CD40+/- mice
as donors of the aortic grafts implanted into BALB/c recipients. Again,
there was no detectable difference in intimal proliferation between
these groups (intimal proliferation was 52 ± 8% for C57BL/6
CD40-/- mice and 57 ± 10% for C57BL/6
CD40+/- mice; n = 5),
demonstrating that donor APCs do not play a role in this model. Recent
data (9, 12, 13) suggest that anti-CD154 treatment is
unable to prevent CD8+ T cell-mediated allograft
rejection effectively and that CD40-CD154-independent
CD8+ T cells might be responsible for the
development of transplant arteriosclerosis. Therefore, we next depleted
CD8 T cells in CD40-/- and heterozygous
littermates. However, treatment of CD40-/-
recipients with an anti-CD8 mAb did not result in a significant
reduction of intimal proliferation as compared with anti-CD8
depleted heterozygous littermates (intimal proliferation was 46 ±
7% in CD40-/- anti-CD8-treated mice vs
50 ± 10% for CD40+/- anti-CD8-treated
mice; n = 5) (Fig. 1A, panels C
and D, and B) or with untreated
CD40-/- recipients (intimal proliferation was
59 ± 5 for CD40-/- mice vs 46 ± 7%
for CD40-/- anti-CD8-treated mice;
n = 5) (Fig. 1A, panels B and
D, and B). This suggested that additional
pathways can trigger the development of transplant arteriosclerosis in
the absence of the CD40-CD154 pathway and CD8+ T
cells.
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Although an equivalent degree of intimal proliferation within the
aortic graft was observed in CD40-/- recipients
and heterozygous littermates, infiltration of the grafts transplanted
into CD40-/- recipients by T cells and
macrophages (CD11b+) was significantly reduced
(Fig. 2A, panels B,
D, and F, and B, panels
A–C) compared with the number of T cells and macrophages
infiltrating grafts transplanted into heterozygous littermates (Fig. 2A, panels A, C, and E, and
B, panels A–C). As expected, no
CD40+ cells could be detected in any of the
grafts recovered from CD40-/- recipients (Fig. 2B, panel D), whereas CD40+
cells were present in aortic grafts from heterozygous littermates (Fig. 2B, panel D).
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B). Interestingly, depletion
of CD8+ T cells resulted in a further dramatic
increase in the eosinophil infiltrate in grafts recovered from
CD40-/- recipients and to a lesser degree in
heterozygous littermates (109 ± 24 eosinophils/grid for
CD40-/- anti-CD8-treated mice vs 28 ±
7 for CD40+/- anti-CD8-treated mice;
p < 0.01; n = 5) (Fig. 3, A
and B).
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and IL-12 mRNA was
significantly reduced
When compared with untreated heterozygous littermates,
CD40-/- mice exhibited significantly decreased
intragraft mRNA expression of the inflammatory cytokines IFN-
(-89%), IL-10 (-129%), and IL-12 (-377%), but markedly increased
IL-4 (+181%) and TGF-
(+207%) production (Fig. 4). Depletion of
CD8+ T cells did not result in a further
significant reduction in IFN-, IL-10, and IL-12 mRNA expression.
However, in the absence of CD8+ T cells, IL-4 and
TGF- mRNA expression was even more elevated in
CD40-/- recipients and heterozygous littermates
(Fig. 4). mRNA for IFN-, IL-4, IL-10, IL12, inducible NO synthase
was undetectable in syngeneic controls, and TGF- mRNA levels were
<500 fg (data not shown).
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To investigate whether alloantibodies played a role in the
development of transplant arteriosclerosis seen in
CD40-/- recipients, circulating alloantibodies
specific for MHC class I molecule H2-Dd were
measured on days 14 and 30 after transplantation (Fig. 5). Heterozygous littermates produced
high levels of IgM and IgG3 alloantibodies (Fig. 5, A and
D) on day 14, and IgG1 and IgG2a alloantibodies became the
dominant subclasses on day 30 (Fig. 5, B and C).
In contrast, no IgG and only low levels of IgM alloantibodies were
detectable in CD40-/- recipients (Fig. 5, A–D), suggesting that alloantibodies did not contribute to
the development of transplant arteriosclerosis in this model.
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Abundant intragraft IL-4 mRNA expression has been reported to play
an important role in tissue eosinophilia (30, 31) and was
an important feature, together with the eosinophil infiltrate, of
aortic allografts transplanted into CD40-/-
recipients. Therefore, we wanted to investigate the effects of
neutralizing the functional activity of IL-4 on the development of
transplant arteriosclerosis. For this purpose,
CD8+ T cell-depleted mice, which showed the
highest intragraft IL-4 mRNA expression (Fig. 4
E), received
either the neutralizing IL-4 mAb (11B11) or control rat IgG (Sigma).
When mice were treated with anti-IL-4, the development of
transplant arteriosclerosis was significantly inhibited (intimal
proliferation was 18 ± 5% for CD40-/-
anti-CD8+-,
anti-IL-4-treated mice vs 46 ± 7% for
CD40-/- anti-CD8-treated mice) (Fig. 1A, panel F, and B), and the
eosinophil infiltrate was abolished. This effect was not seen in grafts
harvested from mice treated with control rat IgG (data not shown).
Treatment with the anti-IL-4 mAb had only a minor effect in
CD8+ T cell-depleted heterozygous littermates
(intimal proliferation was 39 ± 7% for
CD40+/- anti-CD8+-,
anti-IL-4-treated mice vs 50 ± 10% for
CD40+/- anti-CD8-treated mice) (Fig. 1A, panel E, and B).
Intragraft eotaxin and CCR3 mRNA expression correlated with the eosinophil infiltrate and was significantly reduced after anti-IL-4 mAb treatment in CD8+ T cell-depleted CD40-/- recipients
To determine whether the increased infiltration of the grafts by
eosinophils in CD40-/- recipients paralleled
the production of eotaxin, a strong chemoattractant for eosinophils and
expression of the CCR3 chemokine receptor at the graft site, intragraft
eotaxin and CCR3 mRNA production was measured. Eotaxin mRNA expression
was at the highest level in CD40-/- recipients
after CD8+ T cell depletion (Fig. 6A). After treatment with the
anti-IL-4 mAb, eotaxin mRNA expression in the grafts was
significantly reduced (5-fold; p < 0.01), a finding
that correlated with the eosinophil infiltrate of the graft (Figs. 3B and 6A). The pattern of expression of CCR3
mRNA was similar to that observed for eotaxin and was also
significantly reduced after anti-IL-4 treatment (3-fold;
p < 0.05) (Fig. 6B). These findings were
not observed in the CD8+ T cell-depleted
heterozygous littermates, which showed high eotaxin and CCR3 expression
without an eosinophil infiltrate. This most likely reflects the overall
more pronounced cellular infiltration of these grafts by T cells and
macrophages and the increased proliferation of smooth muscle cells
(Fig. 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This was somewhat surprising, because three of
the major effector mechanisms implicated in the development of this
disease were either reduced or impaired in
CD40-/- mice. First, leukocyte infiltration by
CD4+, CD8+, and
CD11b+ cells was significantly reduced in grafts
recovered from CD40-/- recipients (Fig. 2).
Second, being defective in their Ag presentation,
CD40-/- mice were unable to mount an effective
alloantibody response after transplantation (14) (Fig. 4).
Third, IL-12 mRNA production, a strong stimulator of IFN-
production, was reduced in the absence of the CD40 pathway (Fig. 5).
Previous studies have shown that the absence of IFN- markedly
reduced intimal proliferation in several models of allotransplantation
(32, 33) and that IFN- can induce arteriosclerotic
changes by directly acting on vascular smooth muscle cells
(34).
Clearly, defects in the above-mentioned effector mechanisms did not
lead to a reduction of transplant arteriosclerosis in aortic allografts
transplanted into CD40-/- recipients. We also
confirmed that the development of lesions in this model was not due to
CD40 expression by the graft (Fig. 2B, panel D)
or the presence of alternative ligands for CD154 in
CD40-/- recipients, because administration of
anti-CD154 to CD8+ T cell-depleted
CD40-/- recipients did not have a beneficial
effect, and there was no difference in intimal proliferation in the
reciprocal transplantation setting when CD40-/-
mice were used as donors of the aortic grafts when compared with the
littermate controls. These results are in accordance with a
recent study by Shimizu et al. (35) showing that, in the
mouse cardiac allograft model, host CD154 deficiency induced long-term
graft survival but failed to prevent the development of transplant
arteriosclerosis independently of an additionally administered
CD40 Ab.
Previous work from others and our group has demonstrated that CD154
blockade does not inhibit the activation of CD8+
T cells (5, 9, 10, 12, 13). However, in this study,
CD8+ T cells were not found to play a role in the
development of transplant arteriosclerosis in CD40-deficient
recipients, because CD8+ T cell depletion of
CD40-/- recipients did not result in a
significant reduction of transplant arteriosclerosis compared with
littermate controls (Fig. 1). Thus, CD8+ T cells
are not major contributors to the formation of transplant
arteriosclerosis in the absence of CD40-CD154 costimulation (Fig. 1).
A potential mechanism for the development of transplant
arteriosclerosis in the CD40-/- recipients
could be the strong intragraft IL-4 expression (Fig. 3). CD40-CD154
interactions are important for the regulation of IL-12 production by
DCs and macrophages, and interruption of this pathway may therefore
result in an immune deviation toward the Th2 phenotype in Ag-specific
systems. Accordingly, in our model, the intragraft IL-12 expression was
markedly reduced, whereas IL-4 expression was up-regulated. An immune
deviation toward a Th2 phenotype was also observed in long-term
surviving cardiac allografts following treatment with donor spleen
cells and anti-CD154 Ab (36). Although a Th2 phenotype
has been suggested to be beneficial with respect to graft acceptance,
several reports indicated that it may contribute to chronic rejection
processes and the development of transplant arteriosclerosis (37, 38).
Therefore, to investigate the role of IL-4 in the development of
transplant arteriosclerosis in the absence of CD40-CD154 costimulation,
CD40-/- mice were treated with an anti-IL-4
mAb in the absence of CD8+ T cells. This markedly
reduced the level of transplant arteriosclerosis and also abolished the
eosinophil infiltrate in the graft almost completely, demonstrating
that IL-4 was responsible for the formation of transplant
arteriosclerosis in the absence CD40-CD154 costimulation and
CD8+ T cells. There are several possible
explanations of how IL-4 could mediate the development of transplant
arteriosclerosis in the absence of CD40-CD154 costimulation and
CD8+ T cells. In a recent study, Bagley et al.
(39) reported that IL-4 can enhance the induction of
alloimmune responses of CD4+ T cells. They could
demonstrate that IL-4 was required for the activation and expansion of
alloreactive IL-2-, IL-4-, and IFN-producing CD4+
T cells by enhancing the costimulatory activity of the APCs through
increased expression of B7.1 (CD80) and B7.2 (CD86). However, to which
extent this effect might be functional in APCs deficient of CD40, as in
our system, is currently unclear. Shimizu et al. (35)
found a decreased expression of B7 molecules on graft-infiltrating
macrophage APCs in heart grafts transplanted into CD154-knockout
recipients, although no measurements for IL-4 have been reported. In
contrast, Larsen et al. (40) found no alterations of B7
and IL-4 expression after treatment with anti-CD154 Ab to prevent
cardiac allograft rejection. An effect of IL-4 on Ag-presenting DCs was
also recently reported by King et al. (41), who
investigated the effect of IL-4 on the inhibition of
CD8+ T cell-mediated autoimmune diabetes. In this
model, IL-4 increased the expression of B7.2 on the APCs but decreased
the expression of B7.1, resulting in an increased expansion of
Ag-specific CD8+ T cells while inhibiting their
acquisition of cytolytic function. However, this potentially negative
effect of IL-4 on CD8+ T cell differentiation is
irrelevant in our CD8+ T cell-depleted system.
Another possibility is that IL-4 could mediate the development of
transplant arteriosclerosis via a direct effect on the vasculature, as
suggested by a study of acute vascular proliferative disease in a
carotid artery injury model (42). In this system,
up-regulation of signal transducer and activator of transcription
protein 6 following vessel injury in vascular smooth muscle cells
correlated with the increased expression of the IL-4/IL-13 receptor
-chain and the platelet-derived growth factor- receptor. Because
signal transducer and activator of transcription protein 6 is involved
in the induction of transcription of the IL-4 and IL-4R -chain, this
finding may indicate a possible autocrine loop in vascular smooth
muscle cells after arterial injury involving IL-4 (42). A
direct effect of IL-4 on the vasculature has also been demonstrated in
a preclinical evaluation of recombinant human IL-4. The administration
of recombinant human IL-4 to cynomolgus monkeys resulted in
dose-depending toxic effects and the varying occurrence of chronic
arteritis, which was frequently associated with medial smooth muscle
cell proliferation and an infiltration of eosinophils
(43).
In our study, the high intragraft IL-4 expression in
CD8+ T cell-depleted
CD40-/- recipients was also associated with a
strong eosinophil infiltrate, which was almost abolished entirely by
treatment with an anti-IL-4 mAb. This effect of anti-IL-4
treatment has been described in several previous reports that showed
that tissue eosinophilia was dependent on the presence of IL-4
(30, 44) or IL-5 (45). Eosinophils have also
been detected during allograft rejection in both experimental models
and clinical transplantation (46, 47). The vascular
lesions seen in CD8+ T cell-depleted
CD40-/- recipients are consistent with the
ability of activated eosinophils to induce fibrotic lesions in several
chronic inflammatory diseases (48, 49), probably by
secreting a large number of cytotoxic mediators such as eosinophil
cationic proteins and activated oxygen radicals (48).
Eosinophils are also a major source of TGF- (50), a
cytokine that has been implicated in the development of transplant
arteriosclerosis because it is able to modulate cellular
differentiation, cell proliferation, and extracellular matrix formation
(51, 52, 53, 54). In the CD40-/-
recipients, the levels of intragraft TGF- expression correlated well
with the eosinophil infiltrate and the amount of transplant
arteriosclerosis (Fig. 4). Consequently, treatment with anti-IL-4,
which abolished the eosinophil infiltrate, resulted in a significant
reduction of the intragraft TGF- production and intimal
proliferation. Although these findings do not prove a causative role
for eosinophils, they suggest that eosinophils may contribute to the
IL-4-mediated transplant arteriosclerosis.
One mechanism by which IL-4 could contribute to tissue eosinophilia
would be to induce the production of eotaxin within the graft. Eotaxin
has been shown to induce chemotaxis and migration of eosinophils in
vivo (55, 56) via the CCR3 receptor expressed by
eosinophils (57). Besides almost completely preventing
eosinophil infiltration, neutralization of IL-4 also reduced the
expression of eotaxin and CCR3 mRNA in grafts in
CD8+ T cell-depleted
CD40-/- recipients (Fig. 6). More importantly,
transplant arteriosclerosis of aortic grafts implanted in these
recipients was almost abolished. Eotaxin not only plays an important
role in attracting eosinophils to inflammatory sites, but is also
involved in the development of tissue damage by activating the
proinflammatory effector functions of eosinophils (58);
these data, together with the morphological findings, suggest a
functional role of the infiltrating eosinophils in the development of
transplant arteriosclerosis.
In conclusion, we have shown that IL-4 plays a pivotal role in the development of transplant arteriosclerosis in CD40-/- recipients in the absence of CD8+ T cells. Moreover, IL-4 was shown to be responsible for the strong intragraft eosinophil infiltration and eotaxin mRNA expression of aortic grafts implanted in CD40-/- recipients. This study also shows that IL-4-mediated transplant arteriosclerosis is an obstacle that has to be overcome if CD40-CD154 costimulatory blockade is to be developed into a successful strategy in clinical transplantation.
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Acknowledgments |
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Footnotes |
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2 S.M.E. and B.M.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Kathryn J. Wood, Nuffield Department of Surgery, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, Oxford, U.K. E-mail address: kathryn.wood{at}nds.ox.ac.uk
4 Abbreviations used in this paper: DC, dendritic cell; ES, embryonic stem.
Received for publication October 23, 2000. Accepted for publication April 24, 2001.
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  M. Isobe, H. Kosuge, and J.-i. Suzuki T Cell Costimulation in the Development of Cardiac Allograft Vasculopathy: Potential Targets for Therapeutic Interventions Arterioscler Thromb Vasc Biol, July 1, 2006; 26(7): 1447 - 1456. [Abstract] [Full Text] [PDF]
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 S. Abele, M. Weyand, M. Wollin, N. E. Hiemann, F. Harig, T. Fischlein, and S. M. Ensminger Clopidogrel reduces the development of transplant arteriosclerosis J. Thorac. Cardiovasc. Surg., May 1, 2006; 131(5): 1161 - 1166. [Abstract] [Full Text] [PDF]
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 M. Matsui, O. Moriya, T. Yoshimoto, and T. Akatsuka T-bet Is Required for Protection against Vaccinia Virus Infection J. Virol., October 15, 2005; 79(20): 12798 - 12806. [Abstract] [Full Text] [PDF]
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 M. Terme, E. Tomasello, K. Maruyama, F. Crepineau, N. Chaput, C. Flament, J.-P. Marolleau, E. Angevin, E. F. Wagner, B. Salomon, et al. IL-4 Confers NK Stimulatory Capacity to Murine Dendritic Cells: A Signaling Pathway Involving KARAP/DAP12-Triggering Receptor Expressed on Myeloid Cell 2 Molecules J. Immunol., May 15, 2004; 172(10): 5957 - 5966. [Abstract] [Full Text] [PDF]
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C. Guillonneau, C. Louvet, K. Renaudin, J.-M. Heslan, M. Heslan, L. Tesson, C. Vignes, C. Guillot, Y. Choi, L. A. Turka, et al. The Role of TNF-Related Activation-Induced Cytokine-Receptor Activating NF-{kappa}B Interaction in Acute Allograft Rejection and CD40L-Independent Chronic Allograft Rejection J. Immunol., February 1, 2004; 172(3): 1619 - 1629. [Abstract] [Full Text] [PDF]
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 U. Schonbeck, G. K. Sukhova, N. Gerdes, and P. Libby TH2 Predominant Immune Responses Prevail in Human Abdominal Aortic Aneurysm Am. J. Pathol., August 1, 2002; 161(2): 499 - 506. [Abstract] [Full Text] [PDF]
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 W. Palinski and S. Tsimikas Immunomodulatory Effects of Statins: Mechanisms and Potential Impact on Arteriosclerosis J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1673 - 1681. [Abstract] [Full Text] [PDF]
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C. Guillot, C. Guillonneau, P. Mathieu, C. A. Gerdes, S. Menoret, C. Braudeau, L. Tesson, K. Renaudin, M. G. Castro, P. R. Lowenstein, et al. Prolonged Blockade of CD40-CD40 Ligand Interactions by Gene Transfer of CD40Ig Results in Long-Term Heart Allograft Survival and Donor-Specific Hyporesponsiveness, But Does Not Prevent Chronic Rejection J. Immunol., February 15, 2002; 168(4): 1600 - 1609. [Abstract] [Full Text] [PDF]
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