Chronic rejection is a major problem in transplantation medicine, largely resistant to therapy, and poorly understood. We have shown previously that basophil-derived IL-4 contributes to fibrosis and vasculopathy in a model of heart transplantation with depletion of CD4+ T cells. However, it is unknown how basophils are activated in the allografts and whether they play a role when cyclosporin A (CsA) immunosuppression is applied. BALB/c donor hearts were heterotopically transplanted into fully MHC-mismatched C57BL/6 recipients and acute rejection was prevented by depletion of CD4+ T cells or treatment with CsA. We found that IL-3 is significantly upregulated in chronically rejecting allografts and is the major activator of basophils in allografts. Using IL-3–deficient mice and depletion of basophils, we show that IL-3 contributes to allograft fibrosis and organ failure in a basophil-dependent manner. Also, in the model of chronic rejection involving CsA, IL-3 and basophils substantially contribute to organ remodeling, despite the almost complete suppression of IL-4 by CsA. In this study, basophil-derived IL-6 that is resistant to suppression by CsA, was largely responsible for allograft fibrosis and limited transplant survival. Our data show that IL-3 induces allograft fibrosis and chronic rejection of heart transplants, and exerts its profibrotic effects by activation of infiltrating basophils. Blockade of IL-3 or basophil-derived cytokines may provide new strategies to prevent or delay the development of chronic allograft rejection.

Fibrosis and vasculopathy are major causes for the chronic failure of cardiac allografts and major hurdles to successful long-term outcomes (13). Registry data show a high incidence of vasculopathy with minimal improvement over the past two decades from 32–29% to 46–40% at 5 and 8 years posttransplant, respectively (4, 5). Using a model of chronic rejection with transient depletion of CD4+ T cells, we have shown previously that basophils and basophil-derived IL-4 markedly contribute to the development of myocardial fibrosis and vasculopathy (6). Although there is a large number of factors that recruit and activate basophils (7), it is currently not known which factors activate basophils within the allografts and whether IL-4 is the only basophil-derived cytokine contributing to fibrogenesis. We and others have shown that IL-3 is a potent activator of basophils, prolongs their survival, and induces the release of large amounts of IL-4 and L-6 (820). IL-3, IL-5 and GM-CSF belong to the family of hematopoietic cytokines with four short α-helices (21). Activated T cells are the main source of IL-3 and increase their IL-3 expression following costimulation with CD28 or in the presence of TLR-activated costimulatory cells like monocytes and B cells (8, 22). Apart from T cells, some B cells (23) and human basophils (24) can also produce IL-3. Hence, we hypothesized that IL-3 could trigger development of cardiac allograft fibrosis by activation of basophils.

We primarily addressed this question in a fully MHC-mismatched model of heart transplantation with transient depletion of CD4+ T cells. Depletion of CD4+ T cells prevents acute rejection and leads to the appearance of typical signs of chronic rejection already 20 days after transplantation (6, 25, 26). We quantified the expression of IL-3 in the allografts and analyzed the impact of IL-3 on the frequency of basophils, on the production of IL-4 and IL-6, and on the development of myocardial fibrosis and luminal vascular occlusion. We found that IL-3 is a major profibrotic cytokine and significantly interferes with long-term transplant survival.

To corroborate our findings in a second model of chronic rejection, we have established a model of allograft fibrosis and vasculopathy using the clinically widely applied calcineurin inhibitor cyclosporin A (CsA) (27). Calcineurin inhibitors are still the most frequently applied drugs for maintenance immunosuppression in heart transplant recipients (5). CsA is known to suppress cytokine expression in T cells and potentially in other cells like basophils and may thus considerably alter the profibrotic network in allograft recipients. Although CsA almost completely suppressed the expression of IL-4 by basophils in vitro and in vivo, basophils and IL-3 still played a predominant role in chronic allograft fibrosis, which was largely dependent on IL-6 expression. Our data indicate that basophils, IL-3, and IL-6 might be valuable targets for preventing chronic allograft remodeling.

Wild-type (wt) C57BL/6 (B6) and BALB/c (Bc) mice and mice deficient for IL-6 on a C57BL/6J background (B6.129S2-IL6tm1Kopf/J, IL-6 knockout [IL-6 ko]) were purchased from The Jackson Laboratory (Bar Harbor, MA). Mice deficient for IL-3 (Cg-IL3tm1Glli, IL-3+/−, and IL-3−/−) on a Bc background and backcrossed for six generations into C57BL/6N were obtained from RIKEN BioResource Center (RBRC No. 02298; Tsukuba, Japan). All mice were bred and housed under specific pathogen-free conditions in the animal facility of the University Hospital Regensburg (Regensburg, Germany). Food and water were provided ad libitum. All experiments were approved and performed in accordance with the institutional guidelines (Az. 2532–2-239).

Heterotopic transplantation of hearts was performed according to a previously described method (28 ), with the modification that donor hearts were perfused via the abdominal vena cava and the pulmonary artery with cold 0.9% saline containing 500 IU heparin (3 ml each; Ratiopharm, Ulm, Germany). To deplete CD4+ T cells, recipients were treated on day −1, 0, and 7 with i.p. injections of 1 mg of anti-CD4 Ab (Clone: GK1.5; Bio X Cell, West Lebanon, NH). Alternatively, recipients were treated daily from day −1 to day 14 with i.p. injections of 30 mg/kg CsA diluted in olive oil; allografts and recipients were examined at day 15 and day 20. For analysis of allograft survival, assessed by daily palpation, recipients were treated with CsA until the end of allograft function.

Recipient mice were injected i.p. on day −3, −2, and −1 prior to transplantation with 10 μg anti–FcεRI-a Ab (MAR-1; eBioscience, San Diego, CA) (13); purified hamster IgG was used as isotype control Ab. Alternatively, depletion of basophils was performed by i.v. injection of 30 μg of anti-CD200R3 mAb (Ba103) on day −2 and −1; purified rat IgG2b was used as isotype control Ab (BioLegend, San Diego, CA) (6, 18, 29).

Donor hearts were harvested on day 20, and the tissue was embedded in paraffin or cryopreserved in Tissue-Tek compound (Sakura Finetek Germany, Staufen, Germany). Paraffin sections (2–3 μm) were stained with Masson trichrome staining according to the manufacturer’s protocol (Sigma-Aldrich, Munich, Germany). For immunohistochemical staining of basophils, paraffin sections (2−3 μm) were incubated with rat anti-MCPT8 mAb (Clone: TUG8; BioLegend, Fell, Germany) at 4°C overnight. After rinsing with PBS, slides were incubated with secondary biotinylated goat anti-rat Ab (Santa Cruz Biotechnology, Heidelberg, Germany) and with SensiTek-HRP (ScyTek Laboratories, Logan, UT). Positive signals were visualized using a DAB-Kit (3,3′-diamino-benzidine-tetrahydrochlorhydrate; Merck, Darmstadt, Germany). Immunofluorescence staining was performed on 3-μm cryosections. Sections were fixed with ice-cold acetone, blocked with superblock blocking buffer (Thermo Fischer Scientific), and incubated with different primary Abs against collagen-1 (ab21286; Cambridge, U.K.), alpha smooth muscle actin (αSMA; clone C04018; Biocare Medical, Concord, CA), fibronectin-1 (clone AV41490; Sigma-Aldrich), CD4 (clone RM4-5; BD Biosciences, San Jose, CA), or CD8 (clone ab4055; Cambridge, U.K.). For detection, secondary Alexa Fluor 594–labeled F(ab′)2 fragments of goat anti-rabbit IgG (A-11072; Invitrogen, Carlsbad, CA), and goat anti-rat IgG (A-11007; Invitrogen) were used. DNA was labeled with Hoechst 33342 (Invitrogen). Images were taken with an AxioObserver-Z1 microscope (Carl Zeiss, Oberkochen, Germany). For automated analysis, at least 10 high-power fields (HPF) were selected per slide and analyzed with MetaMorph software (Version 4.6; Universal Imaging, Dowingtown, PA). Analysis was performed in a blinded fashion.

RNA was isolated from cardiac grafts on day 20 posttransplantation by homogenization of the tissue in 800 μl TRI reagent (Sigma-Aldrich), according to the manufacturer’s instructions. Total RNA (4 μg) was reverse transcribed using Oligo (dT)20 primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Carslbad, CA). Quantitative real-time PCR was performed using the QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) and the ViiA7 detection system (Life Technologies). Relative gene expression was determined using the 2−∆CT method (normalized to the geometric mean of the expression levels for the housekeeper hprt1, β-microglobulin, and gapdh).

Graft tissue was cut into small pieces, and single-cell suspensions were obtained using cell strainers of 70 μm, followed by cell strainers of 30 μm (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were stained with Abs against CD45 (Clone: 30-F11) and CD11b (clone M1/70), all from eBioscience (San Diego, CA). For intracellular staining of collagen-1, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences, Heidelberg, Germany), washed twice with PBS containing 0.1% saponin, and incubated with biotinylated rabbit anti–mouse collagen type I Ab or biotinylated rabbit IgG as isotype control Ab (Rockland Immunochemicals, Gilbertsville, PA). After washing two times with PBS/0.1% saponin, allophycocyanin-labeled streptavidin (BD Biosciences) was used for detection. Counting beads (Invitrogen) were added to each sample tube. Cells were analyzed with a FACS Canto II flow cytometer (BD Biosciences) using FlowJo (Tree Star, OR) and FACS DIVA software (BD Biosciences).

Basophils were isolated from single-cell suspensions of female B6 bone marrow cells using mouse CD49b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described (30). Basophils (5 × 104 cells/200 μl) were preincubated for 2 h at 37°C with medium or CsA at 0.2 μg/ml or with prednisolone at 20 μg/ml in cell culture medium (RPMI + 10% FCS). Subsequently, IL-3 (10 ng/ml) was added for 20 h at 37°C to activate basophils. IL-6 and IL-4 were measured in the supernatant by ELISA (BioLegend; R&D Systems, Minneapolis, MN).

One-way MLR were performed to evaluate alloreactive responses of CD4+ and CD8+ T cells from the allograft recipients. T cells were isolated from the spleens of B6 recipients with a magnetic bead negative isolation kit (Miltenyi Biotec) and stained with 3.3 μM Vybrant CFDA SE Cell (CFSE) tracer reagent (Invitrogen). Allogeneic stimulator cells were prepared from naive Bc mice. Stimulator cells from naive B6 (syngeneic) and naive C3H (third party) mice were used as controls. Stimulator splenocytes were depleted of CD4+ and CD8+ T cells, and CD49b+ (DX5) NK cells using MicroBeads kits against CD4 (L3T4), CD8a (Ly-2), and CD49b (DX5), according to the manufacturer’s protocol (Miltenyi Biotec). A total of 1 × 105 CFSE-labeled recipient T cells were cultured with 5 × 105 stimulator cells (200 μl/well), incubated for 3 d, and stained with Abs against CD4 (clone: RM4-5; eBioscience) and CD8 (clone: 53-6.7; eBioscience). The number of proliferated CD4+ or CD8+ T cells was determined according to their CFSE signal by FACSDiva software. IL-3 and IFN-γ were measured in supernatant of the culture by ELISA (BioLegend).

All data, unless otherwise specified, are shown as the mean ± SEM and were compared using a one-tailed Student t test. The log-rank (Mantel–Cox) test was used to compare the percent allograft survival between the groups. Graph Pad Prism software, version 5.0 was used for analysis.

We first analyzed whether IL-3 is upregulated in allogeneic compared with syngeneic grafts. B6 recipients were depleted of CD4+ T cells and transplanted with Bc donor hearts (Bc→B6, allogeneic) or with B6 donor hearts (B6→B6, syngeneic). On day 20 after transplantation, IL-3 expression was significantly increased in the allografts but not in the syngrafts or in nontransplanted donor hearts (Bc naive) (Fig. 1A). To functionally analyze the contribution of IL-3 to the development of allograft fibrosis, we used heterozygous and homozygous IL-3–deficient mice as recipients. Deficiency of IL-3 markedly diminished the number of basophils within the allografts (Fig. 1B) and the local expression of IL-4 and IL-6 (Fig. 1C, 1D). IL-4 and IL-6 are the two predominant cytokines released by activated basophils (6, 18, 30, 31). The lower number of basophils in IL-3–deficient mice might be explained by antiapoptotic effects of IL-3 on basophils (8, 20) and the IL-3–dependent recruitment of basophils (3236). IL-3–deficient recipients also displayed a reduced expression of TGF-β in the allografts (Fig. 1E), which might be a consequence of the reduced expression of IL-4 and IL-6 because both cytokines are known to induce TGF-β expression and to play an important role in allograft fibrosis development (6, 3741). We found that a complete or partial lack of IL-3 significantly reduced vascular luminal occlusion (Fig. 1F) and interstitial deposition of collagen-1 in the allografts (Fig. 1G). In addition, a complete lack of IL-3 in the recipients significantly prolonged allograft survival from a mean time of 48.9 ± 6.5 and 49.7 ± 5.9 d in wt or heterozygous ko mice, respectively, to 71.1 ± 7.2 d in homozygous IL-3–deficient mice (Fig. 1H). Allograft survival is limited in this model mainly by the progressive development of chronic rejection. Therefore, our data show that IL-3 substantially contributes to the development of chronic allograft rejection in this model.

FIGURE 1.

IL-3 contributes to chronic rejection in a model with depletion of CD4+ T cells. (A) B6 recipients were depleted of CD4+ T cells and transplanted with B6 (B6→B6; syngeneic) or Bc hearts (Bc→B6; allogeneic) on day 0. IL-3 mRNA was quantified in nontransplanted Bc hearts (Bc naive) and in the transplants on day 20 (n = 12 per group; n.d., not detectable). (BH) Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous (Bc→IL-3+/−), and homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients and analyzed on day 20. CD4+ T cells were depleted in the recipients. (B) Quantification and representative stainings of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 6–11 per group). (C–E) Expression of IL-4, IL-6, and TGF-β in the allografts (n = 6–11 per group). (F) Quantification of luminal vascular occlusion by elastin staining (n = 6–11 per group). (G) Quantification of collagen-1 by immunofluorescence in the allografts. Representative stainings of collagen-1 in gray (n = 6–11 per group). (H) Allograft survival assessed by cardiac palpation (Bc→IL-3+/+, n = 12, mean survival 49.7 ± 5.9 d; Bc→IL-3+/−, n = 16, mean survival 48.9 ± 6.5 d; Bc→IL-3−/−, n = 16, mean survival 71.1 ± 7.2 d). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

IL-3 contributes to chronic rejection in a model with depletion of CD4+ T cells. (A) B6 recipients were depleted of CD4+ T cells and transplanted with B6 (B6→B6; syngeneic) or Bc hearts (Bc→B6; allogeneic) on day 0. IL-3 mRNA was quantified in nontransplanted Bc hearts (Bc naive) and in the transplants on day 20 (n = 12 per group; n.d., not detectable). (BH) Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous (Bc→IL-3+/−), and homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients and analyzed on day 20. CD4+ T cells were depleted in the recipients. (B) Quantification and representative stainings of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 6–11 per group). (C–E) Expression of IL-4, IL-6, and TGF-β in the allografts (n = 6–11 per group). (F) Quantification of luminal vascular occlusion by elastin staining (n = 6–11 per group). (G) Quantification of collagen-1 by immunofluorescence in the allografts. Representative stainings of collagen-1 in gray (n = 6–11 per group). (H) Allograft survival assessed by cardiac palpation (Bc→IL-3+/+, n = 12, mean survival 49.7 ± 5.9 d; Bc→IL-3+/−, n = 16, mean survival 48.9 ± 6.5 d; Bc→IL-3−/−, n = 16, mean survival 71.1 ± 7.2 d). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01, ***p < 0.001.

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The data described above suggest that IL-3 exerts its profibrotic effects by activation of basophils. To show this directly, we depleted basophils with the Ab MAR-1 in wt (IL-3+/+), IL-3+/−, and IL-3−/− recipients. MAR-1 is known to efficiently and selectively deplete basophils in cardiac allografts (6, 13); injection of hamster IgG was used as control. Depletion of basophils in wt mice markedly reduced the expression of IL-4, IL-6, and TGF-β in the allografts, whereas depletion of basophils in IL-3+/− and IL-3−/− recipients had no additional impact on the expression of these cytokines (Fig. 2A, 2B, Supplemental Fig. 1A). Measurement of collagen-1 mRNA by RT-PCR, quantification of collagen-1 and fibronectin by immunofluorescence (Fig. 2C–E), and analysis of overall fibrosis (blue) by Masson trichrome staining (Supplemental Fig. 1B) revealed that depletion of basophils had strong antifibrotic effects in wt mice but was largely without additional effects in IL-3+/− and IL-3−/− mice. We also determined the number of collagen-1+ CD45 mesenchymal fibroblasts and collagen-1+ CD45+ hematopoietic fibrocytes in the allografts. Again, depletion of basophils only reduced these parameters in wt but not in IL-3–deficient mice (Supplemental Fig. 1C, 1D). These data suggest that IL-3 requires the presence of basophils to exert its profibrotic effects in allografts.

FIGURE 2.

IL-3 induces fibrosis by activation of infiltrating basophils in the chronic rejection model with depletion of CD4+ T cells. Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous Bc→ (IL-3+/−), or homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients and analyzed on day 20. Basophils were depleted before transplantation by injection of the Ab MAR-1 (+MAR-1). Injection of hamster IgG served as control (+Ham. IgG). CD4+ T cells were depleted in all recipients. (AC) Expression of IL-4, IL-6, and collagen-1 within the allografts (n = 4–8 per group). (D and E) Quantification of collagen-1 (gray) and fibronectin (gray) in the allografts by immunofluorescence (n = 4–8 per group). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01. ns, not significant.

FIGURE 2.

IL-3 induces fibrosis by activation of infiltrating basophils in the chronic rejection model with depletion of CD4+ T cells. Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous Bc→ (IL-3+/−), or homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients and analyzed on day 20. Basophils were depleted before transplantation by injection of the Ab MAR-1 (+MAR-1). Injection of hamster IgG served as control (+Ham. IgG). CD4+ T cells were depleted in all recipients. (AC) Expression of IL-4, IL-6, and collagen-1 within the allografts (n = 4–8 per group). (D and E) Quantification of collagen-1 (gray) and fibronectin (gray) in the allografts by immunofluorescence (n = 4–8 per group). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01. ns, not significant.

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CsA is frequently used to prevent graft rejection in patients despite its association with organ fibrosis (27). The profibrotic effects of CsA have been partially explained by upregulation of TGF-β and the supportive effects of CsA on fibrocyte development (27, 4244). CsA is also known to interfere with the expression of cytokines, especially IL-2 and IL-4 in T cells. Whether or not CsA interferes with the expression of cytokines in murine basophils is not known. If CsA prevented the expression of IL-4 and IL-6 in basophils, this could diminish the profibrotic effects of basophils and IL-3. To analyze the impact of CsA on basophils, we purified basophils from the bone marrow of B6 mice, preincubated the basophils with CsA, and activated them for 24 h with IL-3. Measurement of IL-4 and IL-6 in the supernatant revealed that CsA almost completely suppressed the IL-3–induced release of IL-4 but had little impact on IL-6, whereas prednisolone significantly suppressed IL-6 and had no impact on IL-4 (Fig. 3A). To better understand the profibrotic effects of basophils and IL-3, we considered it important to establish a model of chronic allograft rejection involving the immunosuppressant CsA. B6 recipients were daily treated with 30 mg/kg CsA from day −1 to day 14, transplanted on day 0 with allogeneic (Bc→B6) or syngeneic (B6→B6) hearts, and analyzed on day 20. Although expression of IL-6 was significantly upregulated in the allografts compared with the syngrafts, no expression of IL-4 was detectable in either of the grafts (Fig. 3B). This result is consistent with our in vitro data showing that treatment with CsA inhibits expression of IL-4 but not IL-6 (Fig. 3A). Despite the complete suppression of IL-4, clear signs of chronic rejection consisting of a pronounced vasculopathy (luminal occlusion of vessels) and collagen-1 deposition (Fig. 3C) were detectable in the allografts. In addition, mRNA levels for TGF-β, collagen-1, and fibronectin were significantly increased in the allogeneic versus syngeneic transplants (Supplemental Fig. 2A–C). Allogeneic transplants also contained significantly higher numbers of CD8+ and CD4+ T cells than syngeneic grafts (Supplemental Fig. 2D). These data show that withdrawal of CsA results in the rapid development of allograft remodeling with preservation of IL-6 expression but marked suppression of IL-4. To further investigate how fibrosis and alloimmunity develops after withdrawal of CsA on day 14, we performed time kinetic experiments and analyzed the allografts on day 15 and 20. Although there was fibrosis and infiltration of basophils already on day 15, we found a marked increase of both parameters on day 20 (Fig. 3D). Similarly, expression of collagen-1 and TGF-β significantly increased from day 15 to day 20 (Fig. 3E). The appearance of fibrosis correlated with an increase in alloantigen-induced T cell proliferation and the release of IL-3 and IFN-γ in mixed-lymphocyte reactions (Fig. 3F, Supplemental Fig. 3A). Interestingly, not only CD4+ T cells but also CD8+ T cells produced IL-3 on day 20. Little evidence of an alloantigen-induced immune response was detectable in naive mice before transplantation (day −1). To test whether the proliferation of T cells and the release of IL-3 and IFN-γ were alloantigen specific, we stimulated CD4+ and CD8+ T cells obtained from B6 recipients on day 28 after transplantation, with allogeneic Bc cells, syngeneic B6 cells, or third party C3H cells for 3 d. The IL-3 release from CD8+ T cells was clearly alloantigen specific, as a strong release was observed with Bc stimulation and no relevant release with C3H stimulation. INF-γ release from CD8+ T cells, proliferation of CD8+ T cells, and IL-3 release from CD4+ T cells were highest with Bc stimulation but showed a substantial background with C3H stimulation. Stimulation with syngeneic B6 cells failed to induce cell proliferation or cytokine release. Thus, alloantigen specificity is most impressively observed for the IL-3 release from CD8+ T cells (Supplemental Fig. 3B).

FIGURE 3.

Development of fibrosis and vasculopathy in a heart transplantation model with CsA. (A) Basophils purified from B6 mice were preincubated for 2 h with CsA (0.2 μg/ml), prednisolone (Pred, 20 μg/ml), or PBS (−) and then activated for 20 h at 37°C with IL-3. Release of IL-4 and IL-6 into the supernatant of cultured basophils (triplicates). Significance in relation to the PBS control. (BC) B6 recipients were transplanted with hearts from Bc mice (Bc→B6; allogeneic) or B6 mice (B6→B6; syngeneic) on day 0 and treated from day −1 to day 14 with CsA. Grafts were analyzed on day 20. (B) Expression of IL-4 and IL-6 in the grafts (n = 5–10 per group). (C) Quantification of vasculopathy (luminal occlusion) by elastin staining (black) and of collagen-1 deposition (light gray) in the grafts (n = 5–10 per group). (DF) B6 recipients were transplanted with hearts from Bc mice on day 0, treated from day −1 to day 14 with CsA, and analyzed on day 15 or 20. (D) Quantification of myocardial fibrosis by Masson trichrome staining (fibrotic areas in light gray). Quantification of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 8 per group). (E) Expression of collagen-1 and TGF-β within the allografts (n = 8 per group). (F) CD4+ and CD8+ T cells were isolated from the spleens of B6 recipients before (day −1) or at day 15 or 20 after transplantation. Alloantigen-specific proliferation and release of IL-3 was measured in a 3 d MLR with Bc cells as alloantigen-specific stimulators (n = 8 per group). Significance in relation to day 0. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001.

FIGURE 3.

Development of fibrosis and vasculopathy in a heart transplantation model with CsA. (A) Basophils purified from B6 mice were preincubated for 2 h with CsA (0.2 μg/ml), prednisolone (Pred, 20 μg/ml), or PBS (−) and then activated for 20 h at 37°C with IL-3. Release of IL-4 and IL-6 into the supernatant of cultured basophils (triplicates). Significance in relation to the PBS control. (BC) B6 recipients were transplanted with hearts from Bc mice (Bc→B6; allogeneic) or B6 mice (B6→B6; syngeneic) on day 0 and treated from day −1 to day 14 with CsA. Grafts were analyzed on day 20. (B) Expression of IL-4 and IL-6 in the grafts (n = 5–10 per group). (C) Quantification of vasculopathy (luminal occlusion) by elastin staining (black) and of collagen-1 deposition (light gray) in the grafts (n = 5–10 per group). (DF) B6 recipients were transplanted with hearts from Bc mice on day 0, treated from day −1 to day 14 with CsA, and analyzed on day 15 or 20. (D) Quantification of myocardial fibrosis by Masson trichrome staining (fibrotic areas in light gray). Quantification of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 8 per group). (E) Expression of collagen-1 and TGF-β within the allografts (n = 8 per group). (F) CD4+ and CD8+ T cells were isolated from the spleens of B6 recipients before (day −1) or at day 15 or 20 after transplantation. Alloantigen-specific proliferation and release of IL-3 was measured in a 3 d MLR with Bc cells as alloantigen-specific stimulators (n = 8 per group). Significance in relation to day 0. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001.

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Using the above described model of CsA withdrawal, we investigated whether basophils have a relevant role in fibrogenesis. B6 recipients were depleted of basophils with the Ab Ba103 (6, 29) on day −2 and −1. Significantly reduced numbers of MCPT8+ basophils in the allografts were still visible on day 20 after transplantation (Fig. 4A). Depletion of basophils led to significantly lower expression of IL-6 and TGF-β within the allografts, whereas in accordance with the results described above, IL-4 was almost undetectable (Fig. 4B, 4C). In addition, signs of chronic allograft rejection as illustrated by expression of collagen-1 and fibronectin mRNA, luminal vascular occlusion, myocardial deposition of collagen-1, and development of αSMA+ myofibroblasts were clearly reduced following depletion of basophils (Fig. 4D–H). Overall, these results indicate that even in a transplant model using conventional immunosuppression, basophils have a pronounced profibrotic effect and are an important source of IL-6.

FIGURE 4.

Basophils trigger allograft fibrosis and vasculopathy in the heart transplantation model with CsA. wt B6 recipients were transplanted with Bc hearts (Bc→B6) on day 0 and treated from day −1 to day 14 with CsA. Basophils were depleted before transplantation with the Ab Ba103 (+Ba103). Injection of rat IgG2b isotype Ab served as control (+rat IgG2b). Transplants were analyzed on day 20. (A) Injection of Ba103 reduced the number of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 3–7 per group). (BE) Expression of IL-4, IL-6, TGF-β, collagen-1, and fibronectin in the allografts. (FH) Quantification of vasculopathy (luminal occlusion), interstitial deposition of collagen-1, and αSMA following depletion of basophils. Representative stainings for elastin, collagen-1 (gray) and αSMA (gray) on the right. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Basophils trigger allograft fibrosis and vasculopathy in the heart transplantation model with CsA. wt B6 recipients were transplanted with Bc hearts (Bc→B6) on day 0 and treated from day −1 to day 14 with CsA. Basophils were depleted before transplantation with the Ab Ba103 (+Ba103). Injection of rat IgG2b isotype Ab served as control (+rat IgG2b). Transplants were analyzed on day 20. (A) Injection of Ba103 reduced the number of MCPT8+ basophils (black spots as indicated by the arrows) per HPF in the allografts (n = 3–7 per group). (BE) Expression of IL-4, IL-6, TGF-β, collagen-1, and fibronectin in the allografts. (FH) Quantification of vasculopathy (luminal occlusion), interstitial deposition of collagen-1, and αSMA following depletion of basophils. Representative stainings for elastin, collagen-1 (gray) and αSMA (gray) on the right. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01, ***p < 0.001.

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To analyze the role of IL-3 in this model, wt (IL-3+/+), heterozygous (IL-3+/−), and homozygous (IL-3−/−) IL-3–deficient recipients were transplanted with Bc donor hearts. Partial or complete deficiency of IL-3 resulted in significantly reduced numbers of basophils in the allografts and a clearly reduced expression of IL-6 and collagen-1 on day 20 after transplantation (Fig. 5A–C). Most importantly, IL-3–deficient recipients showed significantly less vasculopathy (luminal occlusion) (Fig. 5D), less interstitial deposition of collagen-1 and fibronectin (Fig. 5E, 5F), and less expression of αSMA as an indicator of myofibroblast development (Fig. 5G). These data indicate that IL-3 and basophils have a critical role in the rapid development of fibrosis and vasculopathy after the withdrawal of CsA in mice and suggest that IL-6 may be an important mediator of allograft remodeling in this setting.

FIGURE 5.

IL-3 contributes allograft fibrosis and vasculopathy in the heart transplantation model with CsA. Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous (Bc→IL-3+/−), and homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients on day 0. Recipients were treated from day −1 to day 14 with CsA and analyzed on day 20. (A) Quantification of MCPT8+ basophils in black spots indicated by the arrows and per HPF within the allografts with representative stainings on the right (n = 6–11 per group). (B and C) Expression of IL-6 and collagen-1 in the allografts (n = 6–11 per group). (DG) Quantification of vasculopathy (luminal occlusion), collagen-1, fibronectin, and αSMA (n = 6–11 per group) in the allografts. Representative stainings for elastin, collagen-1 (gray), fibronectin (gray), and αSMA (gray) are depicted for each group. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01.

FIGURE 5.

IL-3 contributes allograft fibrosis and vasculopathy in the heart transplantation model with CsA. Bc hearts were transplanted into wt (Bc→IL-3+/+), heterozygous (Bc→IL-3+/−), and homozygous (Bc→IL-3−/−) IL-3–deficient B6 recipients on day 0. Recipients were treated from day −1 to day 14 with CsA and analyzed on day 20. (A) Quantification of MCPT8+ basophils in black spots indicated by the arrows and per HPF within the allografts with representative stainings on the right (n = 6–11 per group). (B and C) Expression of IL-6 and collagen-1 in the allografts (n = 6–11 per group). (DG) Quantification of vasculopathy (luminal occlusion), collagen-1, fibronectin, and αSMA (n = 6–11 per group) in the allografts. Representative stainings for elastin, collagen-1 (gray), fibronectin (gray), and αSMA (gray) are depicted for each group. Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, **p < 0.01.

Close modal

To interrogate the role of IL-6 in the development of graft fibrosis in CsA-treated recipients, B6 wt (IL-6 wt) and B6 IL-6 ko (IL-6 ko) mice were transplanted with Bc wt donor hearts on day 0. Recipients were treated with CsA from day 1 to day 14. Analysis on day 20 revealed that the allografts of IL-6–deficient recipients expressed significantly less TGF-β and collagen-1 (Fig. 6A, 6B), developed significantly less fibrosis (less deposition of collagen-1 and fibronectin), and contained less αSMA+ activated myofibroblasts (Fig. 6C–E, Supplemental Fig. 4A). In addition, mRNA transcripts of αSMA, fibronectin, arginase 1 (Arg-1), and IL-11 (Supplemental Fig. 4B–E), as well as the number of collagen-1+ CD45 mesenchymal fibroblasts and collagen-1+ CD45+ hematopoietic fibrocytes in the allografts (Supplemental Fig. 4F, 4G), were significantly reduced in IL-6-deficient recipients. These data indicate that the profibrotic network consisting of alternatively activated Arg-1+ macrophages (AAM) (45), myofibroblasts, and profibrotic cytokines like TGF-β and IL-11 (46) is, to a major degree, dependent on the expression of IL-6.

FIGURE 6.

IL-6 triggers chronic rejection of allografts in the heart transplantation model with CsA. Bc hearts were transplanted into wt (Bc→IL-6 wt) and IL-6 ko (Bc →IL-6 ko) B6 recipients on day 0. Recipients were treated from day −1 to day 14 with CsA and analyzed on day 20 (n = 6 per group). (A and B) Expression of TGF-β and collagen-1 in the allografts. (CE) Measurement of collagen-1, fibronectin, and αSMA by immunofluorescence (gray) in the allografts with representative stainings on the right. (F) Allograft survival: recipients were treated daily from day −1 until the end of allograft function with CsA (Bc→IL-6 wt, n = 12, mean survival 15.9 ± 1.8 d; Bc→IL-6 ko, n = 7, mean survival 35.4 ± 4.4 d). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, ***p < 0.001.

FIGURE 6.

IL-6 triggers chronic rejection of allografts in the heart transplantation model with CsA. Bc hearts were transplanted into wt (Bc→IL-6 wt) and IL-6 ko (Bc →IL-6 ko) B6 recipients on day 0. Recipients were treated from day −1 to day 14 with CsA and analyzed on day 20 (n = 6 per group). (A and B) Expression of TGF-β and collagen-1 in the allografts. (CE) Measurement of collagen-1, fibronectin, and αSMA by immunofluorescence (gray) in the allografts with representative stainings on the right. (F) Allograft survival: recipients were treated daily from day −1 until the end of allograft function with CsA (Bc→IL-6 wt, n = 12, mean survival 15.9 ± 1.8 d; Bc→IL-6 ko, n = 7, mean survival 35.4 ± 4.4 d). Data are mean ± SEM. Scale bar, 50 μm. *p ≤ 0.05, ***p < 0.001.

Close modal

To investigate whether the diminished development of fibrosis and vasculopathy in IL-6–deficient recipients translates into a prolonged graft survival, we performed long-term experiments. Dosing of CsA was continued beyond day 14 to avoid acute rejections. As shown in Fig. 6F, allograft survival was significantly prolonged from a mean of 15.9 ± 1.8 d in wt mice to a mean of 35.4 ± 4.4 d in IL-6–deficient mice. In summary, our data show that a sequence of events involving IL-3, basophils and basophil-derived IL-6 plays a major role in the development of chronic allograft rejection in CsA-treated recipients.

Although great progress has been achieved in the prevention of acute allograft rejection, chronic rejection is still largely resistant to therapy and mechanistically not well understood. Chronic rejection of cardiac allografts is characterized by vasculopathy and interstitial (myocardial) fibrosis (13). We have used a model of chronic rejection of heart transplants that is based on the depletion of CD4+ T cells to prevent acute rejections (6, 25). In addition, we have established a second model of chronic transplant rejection in which mice are initially treated with CsA to prevent immediate acute rejections. Withdrawal of CsA after 14 d resulted in the appearance of clear signs of chronic rejection (interstitial fibrosis and vasculopathy) within ∼1 wk. Insufficient levels of CsA or other immunosuppressive drugs can occur in patients (e.g., because of noncompliance or increased metabolism). The model involving CsA appears clinically more relevant and takes into account that CsA interferes with the expression of cytokines, especially the profibrotic cytokine IL-4. Although many studies associate the use of CsA with the occurrence of solid organ fibrosis, the underlying mechanisms are still unclear (15, 3436).

In the CD4+ T cell depletion model, we have shown previously that basophil-derived IL-4 is critical for the development of chronic rejection (6). However, it remained completely unclear which factors activate basophils and whether the contribution of basophils to fibrosis is restricted to this particular model. Among the many factors known to activate basophils, IL-3 appeared as promising candidate because it is expressed primarily by T cells. We have shown that following transplantation, IL-3 is not only expressed by CD4+ T cells but also by CD8+ T cells. IL-3 induces the release of large amounts of IL-4 and IL-6 from basophils and markedly prolongs their survival (816, 1820). Little is known about the potential profibrotic properties of IL-3. In a model of lupus nephritis in MRL-lpr mice, we have previously observed reduced renal fibrosis in anti–IL-3–treated animals (45), and in a recently published model of myocarditis, deletion or inhibition of IL-3 reduced development of myocardial fibrosis (47). However, the pathways of IL-3–induced fibrosis were not investigated.

We now show that IL-3 expression is markedly upregulated in chronically rejecting allografts. Deficiency of IL-3 reduced the number of basophils and the overall expression of IL-4 and IL-6 in the allografts. A similar reduction of IL-4 and IL-6 expression was observed when basophils were depleted with a mAb. Depletion of basophils in IL-3–deficient mice did not further reduce the expression of IL-4 and IL-6, indicating that IL-3–activated basophils are the main source of IL-4 and IL-6 in allografts. The lower number of basophils in IL-3–deficient mice may be explained by the fact that IL-3 blocks apoptosis (8, 13, 20) and induces migration (3236) of basophils. Single interventions like ko of IL-3 or depletion of basophils reduced expression of collagen-1, fibronectin, myocardial fibrosis, and vasculopathy, whereas the combination of both interventions did not further enhance these beneficial effects, suggesting that basophils are the predominant cells mediating the profibrotic effects of IL-3.

We considered it important to confirm the role of IL-3 and basophils in a model involving immunosuppression with CsA, because CsA could potentially interfere with the expression of IL-4 in basophils, which was considered previously as the main profibrotic factor of basophils (6). Indeed, CsA almost completely suppressed the expression of IL-4 by basophils in vitro and in vivo. Despite this lack of IL-4, recipients developed clear signs of allograft fibrosis that markedly increased from day 15 to 20 after transplantation. The appearance of fibrosis correlated with the development of an alloantigen-induced immune response and the release of IL-3 by both CD4+ and CD8+ T cells. Stimulation with allogeneic, syngeneic, and third party cells revealed that alloantigen specificity was most pronounced for the release of IL-3 from CD8+ T cells. This suggests that IL-3 and basophils could still contribute to allograft fibrosis in the CsA model. We showed this experimentally by depletion of basophils and the use of IL-3–deficient recipients. To find out how IL-3–activated basophils could induce fibrosis when IL-4 is suppressed by CsA, we studied the expression of IL-6, the second most abundantly expressed cytokine of basophils. We showed that CsA is unable to suppress the release of IL-6 from basophils in vitro or in vivo and that basophils are the main source of IL-6 in the allografts because depletion of basophils reduced the expression of IL-6 by more than 50%. Using IL-6–deficient recipients we finally showed that IL-6 plays an important role in development of fibrosis and chronic allograft rejection in the CsA model. Fewer basophils and a lack of IL-3 and IL-6 led to a reduction in profibrotic factors like TGF-β (29), IL-11 (38), and arginase 1 (48, 49), suggesting that all these factors are interconnected in a profibrotic network. TGF-β and other profibrotic factors were recently shown to stimulate the release of IL-11 (46), which belongs to the IL-6 family of cytokines using the same gp130 receptor subunit for signal transduction (50). The decreased expression of arginase in IL-6–deficient mice points toward a reduced alternative activation of macrophages, which are known to support fibroblast proliferation and collagen production (45). By flow cytometry we were able to detect collagen production by CD45 and CD45+ cells. CD45 collagen-producing cells predominate and correspond most likely to mesenchymal fibroblasts, whereas the CD45+ collagen-producing cells, sometimes called fibrocytes, are known to be of hematopoietic origin and are thought to be monocyte derived (6, 43, 5155). The presence of fibrocytes in fibrotic organs has frequently been described and recently been shown by cell type specific deletion of col1a1 to directly contribute to production of collagen-1 in models of renal fibrosis (56). Whether or not CD45+ collagen-producing cells significantly contribute to allograft fibrosis needs to be addressed in further studies.

Chronic allograft rejection is the main barrier to long-term transplant survival. Deletion of IL-3 and IL-6 not only inhibited allograft fibrosis and vasculopathy, it also significantly prolonged the survival of the transplanted hearts. In previous studies, we and others readily detected basophils in biopsies of patients with chronic rejection of heart and kidney transplants (6, 57), suggesting that similar pathways could be active in human allografts. Our study supports the hypothesis that missing or insufficient immunosuppression with CsA induces allograft fibrosis by mechanisms involving IL-3, basophils, and IL-6. Blockade of IL-3 or IL-6 may offer new strategies to prevent or delay the development of chronic allograft rejection in patients.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft.

The online version of this article contains supplemental material.

Abbreviations used in this article:

B6

C57BL/6

Bc

BALB/c

CsA

cyclosporin A

HPF

high-power field

IL-6 ko

IL-6 knockout

αSMA

alpha smooth muscle actin

wt

wild-type.

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The authors have no financial conflicts of interest.

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