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Critical Role for IL-4 in the Development of Transplant Arteriosclerosis in the Absence of CD40-CD154 Costimulation This information is current as 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 References Subscription Permissions Email Alerts This article cites 57 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/167/1/532.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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). Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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- Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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). Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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. 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Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017 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 (51–54). 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. 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