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Critical Role for IL-4 in the Development of
Transplant Arteriosclerosis in the Absence of
CD40-CD154 Costimulation
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of June 17, 2017.
Stephan M. Ensminger, Bernd M. Spriewald, Henrik V.
Sorensen, Oliver Witzke, Emily G. Flashman, Andrew
Bushell, Peter J. Morris, Marlene L. Rose, Amin Rahemtulla
and Kathryn J. Wood
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Copyright © 2001 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2001; 167:532-541; ;
doi: 10.4049/jimmunol.167.1.532
http://www.jimmunol.org/content/167/1/532
Critical Role for IL-4 in the Development of Transplant
Arteriosclerosis in the Absence of CD40-CD154 Costimulation1
Stephan M. Ensminger,2* Bernd M. Spriewald,2* Henrik V. Sorensen,† Oliver Witzke,*
Emily G. Flashman,* Andrew Bushell,* Peter J. Morris,* Marlene L. Rose,‡ Amin Rahemtulla,†
and Kathryn J. Wood3*
S
uccessful T cell activation, a crucial event in allograft
rejection, requires recognition of an allo-MHC-peptide
complex as well as the engagement of appropriate costimulatory molecules and their ligands (1). Signals through the
CD40-CD154 costimulatory pathway play a critical role in the
primary activation of T cells (2). CD40 is a member of the TNFR
family of molecules and is expressed by a wide range of cells
including professional APCs, such as dendritic cells (DCs),4 macrophages, B cells, and epithelial and endothelial cells (2). Its ligation leads to APC secretion of TNF-␣ 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
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.
Received for publication October 23, 2000. Accepted for publication April 24, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from The Wellcome Trust, the British Heart
Foundation, and the National Kidney Research Fund. S.M.E. is supported by the
ADUMED-Stiftung. B.M.S. (DFG, Sp-588/1-1) and O.W. (DFG, Wi-1663/1-1) are
supported by the Deutsche Forschungsgemeinschaft.
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: [email protected]
4
Abbreviations used in this paper: DC, dendritic cell; ES, embryonic stem.
Copyright © 2001 by The American Association of Immunologists
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 antiCD154 targets predominantly CD4⫹ T cells and is unable to prevent immune responses mediated by CD8⫹ T cells such as virusspecific CTLs (8) and allograft rejection (9 –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.
0022-1767/01/$02.00
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Blockade of the CD40-CD154 pathway can inhibit CD4ⴙ T cell activation but is unable to prevent immune responses mediated
by CD8ⴙ T cells. However, even in the absence of CD8ⴙ T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent
the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence
of the CD40 pathway. C57BL/6 CD40ⴚ/ⴚ (H2b) recipients were transplanted with MHC-mismatched BALB/c (H2d) aortas.
Transplant arteriosclerosis was evident in both CD40ⴚ/ⴚ and CD40ⴙ/ⴚ mice (intimal proliferation was 59 ⴞ 5% for CD40ⴚ/ⴚ mice
vs 58 ⴞ 4% for CD40ⴙ/ⴚ mice) in the presence or absence of CD8ⴙ T cells (intimal proliferation was 46 ⴞ 7% for CD40ⴚ/ⴚ
anti-CD8-treated mice vs 50 ⴞ 10% for CD40ⴙ/ⴚ anti-CD8-treated mice), confirming that CD8ⴙ T cells are not essential effector
cells for the development of this disease. In CD40ⴚ/ⴚ recipients depleted of CD8ⴙ T cells, the number of eosinophils infiltrating
the graft was markedly increased (109 ⴞ 24 eosinophils/grid for CD40ⴚ/ⴚ anti-CD8-treated mice vs 28 ⴞ 7 for CD40ⴙ/ⴚ antiCD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the
hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40ⴚ/ⴚ recipients were treated with
anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 ⴞ 5 eosinophils/grid for CD40ⴚ/ⴚ
anti-CD8ⴙ, anti-IL-4-treated mice vs 109 ⴞ 24 for CD40ⴚ/ⴚ anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production,
and the level of intimal proliferation (18 ⴞ 5% for CD40ⴚ/ⴚ anti-CD8ⴙ-, anti-IL-4-treated mice vs 46 ⴞ 7% for CD40ⴚ/ⴚ
anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important
mechanism for CD8ⴙ T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation. The Journal
of Immunology, 2001, 167: 532–541.
The Journal of Immunology
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.
Materials and Methods
Animals
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
␮g) 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 ␮g 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-␮m
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 ⫻200 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 ⫻100 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) ⫻ 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-␮m 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 ⫻200 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).
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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.
533
534
Southern blot analysis
Genomic DNA (10 ␮g) 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 0.1⫻ 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.
Results
Presence or absence of CD40 expression on recipient APCs did
not affect the development of transplant arteriosclerosis
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. 1A, 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
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FIGURE 1. A, Histopathological evaluation of the
morphology of fully allogeneic BALB/c aortic grafts recovered from either untreated heterozygous littermates
(C57BL/6 CD40⫹/⫺) (panel A), CD8⫹ T cell-depleted
heterozygous littermates (panel C), CD8⫹ T cell-depleted and anti-IL-4 (11B11)-treated heterozygous littermates (panel E), untreated C57BL/6 CD40⫺/⫺ recipients (panel B), CD8⫹ T cell-depleted C57BL/6
CD40⫺/⫺ recipients (panel D), and anti-IL-4 (11B11)treated and CD8⫹ T cell-depleted C57BL/6 CD40⫺/⫺
recipients (panel F). Snap-frozen sections were stained
with Miller’s elastin/van Gieson stain. Magnification,
⫻100. The data shown are representative of grafts from
five independent experiments. B, Morphometric analysis
of the degree of intimal thickening in fully allogeneic
BALB/c aortic allografts implanted in either C57BL/6
CD40⫹/⫺ or C57BL/6 CD40⫹/⫺ recipients, harvested
on day 30 after transplantation. For morphometric measurements, Miller’s elastin/van Gieson-stained sections
were used. Areas within the lumen and the internal and
external elastic lamina were circumscribed manually
and measured. From these measurements, a quotient for
the thickness of the intima (Qint) was calculated. Qint
indicates the relative thickness (percentage) of the intima. Five measurements from different areas of each
aortic graft were obtained for this analysis from five
grafts in each group.
IL-4 AND TRANSPLANT ARTERIOSCLEROSIS
The Journal of Immunology
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.
CD40⫺/⫺ recipients showed reduced graft infiltration by CD4⫹,
CD8⫹, and CD11b⫹ cells, but also showed significantly
increased eosinophil infiltration
CD40⫺/⫺ recipients showed elevated intragraft IL-4 mRNA
expression, whereas expression of IFN-␥ 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).
⫺/⫺
CD40
recipients could not mount alloantibody responses
against the MHC class I molecule Dd
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.
Neutralization of IL-4 in CD40⫺/⫺ recipients dramatically
reduced intimal proliferation and infiltration of the graft by
eosinophils in the absence of CD8⫹ T cells
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. 4E), 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⫹-, antiIL-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 antiIL-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).
Discussion
The first significant finding from this study was that the levels of
transplant arteriosclerosis seen in grafts recovered from CD40⫺/⫺
recipients and heterozygous littermates were similar (Fig. 1). 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
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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).
The number of eosinophils infiltrating the graft was significantly
increased in CD40⫺/⫺ recipients as compared with heterozygous
littermates (52 ⫾ 21 eosinophils/grid for CD40⫺/⫺ mice vs 8 ⫾ 5
for CD40⫹/⫺ mice; p ⬍ 0.05; n ⫽ 5) (Fig. 3B). 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).
535
536
IL-4 AND TRANSPLANT ARTERIOSCLEROSIS
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FIGURE 2. A, Immunohistochemical evaluation of snap-frozen sections from BALB/c aortic allografts obtained from untreated C57BL/6 CD40⫹/⫺
(panels A, C, and E) or C57BL/6 CD40⫺/⫺ recipients (panels B, D, and F) on day 30 after transplantation. Staining is shown for CD4 (panels A and B),
CD8 (panels C and D), and CD11b (panels E and F). The adventitia is shown on the right, and the intima and the lumen of the graft are shown on the
left of each individual section. One representative section is shown of five experiments. Original magnification, ⫻200. B, Quantification of the intragraft
cellular infiltrate on day 30 after transplantation. The number of positive cells in the adventitia was counted manually. Counts were obtained per 100 fields
of a grid that covered approximately half of the aortic allograft using an original magnification of ⫻200. Quantification was performed for CD4⫹ (panel
A), CD8⫹ (panel B), CD11b⫹ (panel C), and CD40⫹ cells (panel D) (n ⫽ 5 animals/group; values of p are indicated in the diagram).
The Journal of Immunology
537
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 dif-
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FIGURE 3. A, Histopathological evaluation of the
eosinophil infiltrate in fully allogeneic BALB/c aortic
grafts obtained from either anti-CD8⫹ T cell-depleted
C57BL/6 CD40⫺/⫺ (panels A–C) or C57BL/6 CD40⫹/⫺
recipients (panels D and E) on day 30 after transplantation. Snap-frozen sections were stained with a standard H&E stain. There was a striking difference in eosinophil infiltration between CD40⫺/⫺ recipients (panel
A) and CD40⫹/⫺ recipients (panel D). Magnification,
⫻100. High power magnification of the medial area
(panel B) and the proliferation zone (panel C) of the
same section showed a strong eosinophil infiltrate in the
CD40⫺/⫺ recipient, whereas hardly any could be detected in the medial area (panel E) and proliferation
zone (data not shown) of CD40⫹/⫺ recipients. Magnification, ⫻400. The data shown are representative of
grafts from five independent experiments. B, Quantification of the eosinophil infiltrate. The total number of
eosinophils in the intima, medium, and adventitia was
counted manually using an original magnification of
⫻200. Counts were obtained per 100 fields of a grid that
covered approximately half of the aortic allograft (n ⫽
5 animals/group; values of p are indicated in the
diagram).
ference 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
538
IL-4 AND TRANSPLANT ARTERIOSCLEROSIS
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FIGURE 4. Quantitative RT-PCR analysis of intragraft cytokine production. Fully allogeneic BALB/c aortic allografts were analyzed on day 14 after
transplantation. Analysis was performed for IFN-␥ (A), IL-12 (B), IL-10 (C), inducible NO synthase (iNOS) (D), IL-4 (E), and TGF-␤ (F). Data are shown
as the mean of three animals from the each group (values of p are indicated in the diagram).
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
The Journal of Immunology
539
FIGURE 5. Circulating alloantibody responses against the mismatched murine MHC
class I molecule (H2-Dd) were measured by
FACS analysis on days 14 and 30 after transplantation of aortas from BALB/c donors into
C57BL/6 CD40⫺/⫺ or C57BL/6 CD40⫹/⫺ recipients. Analysis was performed for IgM (A),
IgG1 (B), IgG2a (C), and IgG3 (D) alloantibodies. MFI, mean fluorescence intensity; ⴱ, p
⬍0.05 vs C57BL/6 CD40⫹/⫺; n ⫽ 5
animals/group.
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
FIGURE 6. Quantitative RT-PCR analysis of intragraft chemokine production. Fully allogeneic BALB/c aortic allografts were analyzed on day
14 after transplantation. Analysis was performed for eotaxin (A) and CCR3
(B). Data are shown as the mean of three animals from each group (values
of p are indicated in the diagram).
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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
540
Acknowledgments
We thank Dr. Nick D. Jones for helpful discussions during this study, Dr.
Karen Morrison for her expert help with histology, Helene Beard and
Robin Roberts-Gant for editorial assistance, and the staff of the Biomedical
Services Unit facility at the John Radcliffe Hospital Site for their expert
care of the animals used in this study.
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to be responsible for the strong intragraft eosinophil infiltration
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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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