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Keywords:

  • allograft rejection;
  • cardiac transplant;
  • dendritic cell;
  • ischemia/reperfusion injury

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Ischemic reperfusion injury (IRI) enhances allograft immunogenicity, worsens transplantation outcome, and is the primary cause of activation of the recipient innate immune response, resulting in subsequent amplification of the alloimmune adaptive response. Here, we aimed at demonstrating that the link between innate injury and alloimmunity occurs predominantly through activation of allograft-derived dendritic cells (ADDC). Perfusion of MCI-186, a free radical scavenger, into donor cardiac allografts prior to transplantation resulted in prolongation of complete MHC-mismatched allograft survival in the absence of immunosuppression (MST of 8 vs. 26 days). This prolongation was associated with a reduction in trafficking of ADDC to recipient lymphoid tissue as well as a reduction in T cell priming. Depleting ADDC with diphtheria toxin (using DTR-GFP-DC mice as donors) 24 h prior to transplant resulted in abrogation of the prolongation observed with MCI-186 treatment, demonstrating that the beneficial effect of MCI-186 is mediated by ADDC. This donor-specific anti-ischemic regimen was also shown to reduce chronic rejection, which represents the primary obstacle to long-term allograft acceptance. These data for the first time establish a basis for donor anti-ischemic strategies, which in the ever-expanding marginal donor pools, can be instituted to promote engraftment.


Abbreviations: 
 

ADDC, allograft-derived dendritic cells

BM12

B6.C-H2-Ab1bm12

BMDC

bone marrow-derived dendritic cells

DAPI

4′,6-diamidino-2-phenylindole

DC

dendritic cells

DLN

draining lymph node

DT

diphtheria toxin

DTR

diphtheria toxin receptor

DTR-GFP-DC

C.FVB-Tg (Itgax-DTR/EGFP) 57Lan/J

Flt3L

fms-related tyrosine kinase 3 ligand

GFP

green fluorescent protein

H&E

hematoxylin and eosin

IEL

internal elastic lumina

IFN

interferon

IL

interleukin

IRI

ischemia reperfusion injury

LN

lymph node

MHC

major histocompatibility complex

MLR

mixed lymphocyte reaction

MST

mean survival time

OCT

optimum cutting temperature

Treg

T regulatory cell

WT

wild-type

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

As an unavoidable consequence of solid organ transplantation, ischemia reperfusion injury (IRI) has been demonstrated to enhance allograft immunogenicity and to thus increase the rate of acute allograft rejection (1,2). IRI to donor organs results in the release of proinflammatory cytokines, upregulation of MHC molecules, and ultimately, greater risk of acute allograft rejection (3–5). Chronic allograft rejection, the predominant impediment to long-term allograft acceptance, is similarly attributed to initial ischemic injury (6). Notably, recent reports have highlighted the importance of attenuating inflammation and innate immunity pathways to improve tolerogenic strategies via costimulatory blockade (7). Nevertheless, mechanisms by which IRI serves as a link between the innate and adaptive arms of immunity are poorly explored. Through direct presentation of alloantigens to recipient T cells, residing dendritic cells in the graft, or allograft-derived DC (ADDC), serve as the primary initiators of the alloimmune response (8), and we have previously shown that induction of ischemia to in vitro-cultured DC enhances their immunogenicity (9). It is likely that the interplay of innate immunity and alloimmunity is primarily mediated through an increase in the immunogenicity of ADDC, caused by the ischemic insult that ADDC inevitably endure over the course of transplantation. The antioxidant MCI-186, a novel free-radical scavenger also known as edaravone, has been shown to offer considerable protection in a multitude of ischemic models (10). Moreover, clinical trials are currently ongoing in which MCI-186 has been found to be efficacious in reducing the burden of ischemic injuries (11). While MCI-186 has been investigated with regard to protection from IR injury, its specific effect on ADDC and the consequences therein have not yet been elucidated. Transplant waiting lists are growing worldwide with an increasing gap between demand for and availability of organs; this shortage has prompted clinicians to expand their criteria to include marginal organs, which are subject to a higher degree of IRI (12). Here, we examine the effects of donor antioxidant therapy using MCI-186 in models of acute and chronic murine cardiac allograft rejection.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Mice

C57BL/6 (H-2b), BALB/c (H-2d), B6.C-H2-Ab1bm12 (BM12) mice aged 6–8 weeks were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). GFP transgenic C.FVB-Tg (Itgax-DTR/EGFP) 57Lan/J (DTR-GFP-DC) mice were purchased from Jackson Laboratory and bred in our animal facility.

Flow cytometric analysis

Cells were run on a FACSCalibur™ (Becton Dickinson, San Jose, CA, USA), and data were analyzed using FlowJo software version 6.3.2 (Treestar, Ashland, OR, USA). Annexin V and 7-AAD staining was used to assess cell death and apoptosis according to the manufacturer's instructions (BD Biosciences), and staining with anti-mouse CD4, CD25, (BD Biosciences,) and Foxp3 (eBioscience, San Diego, CA, USA) was performed to assess Treg frequency. Cells were stained using anti-mouse CD40, CD80, CD86, I-Ab (class II) (BD Biosciences), TLR2, TLR4 and CCR7 (eBioscience) antibodies to assess DC immunogenicity.

In vitrogeneration of bone marrow-derived DC (BMDC) and H2O2 treatment of DC

BMDC were generated as previously described (9). For H2O2 treatment, BMDC were plated in 24-well plates for 60 min at 37°C with 5, 50 or 500 μm H2O2 in the presence or absence of 10 μm MCI-186, and cells were washed and then subjected to flow cytometric analysis or used for MLR studies, in which 1 × 105 DC were incubated for 72 h with 1 × 105 CD4+ BALB/c cells, followed by pulsing with 1 μCi tritiated thymidine (Perkin Elmer, Waltham, MA, USA). In some experiments, H2O2-treated or control cells were plated and incubated for 24 h in medium, followed by surface staining and flow cytometric analysis.

Isolation of heart DC

Hearts of naïve C57BL/6 mice previously implanted with Flt3L hybridoma cells as previously described (13) were recovered, incubated for 60 min with 1 μg/mL collagenase D (Roche, Indianapolis, IN, USA) and homogenized through a 70 μm cell strainer. Cells were subjected to Percoll density gradient centrifugation, the leukocyte layer was isolated and DC were extracted using CD11c microbeads (Miltenyi Biotec, Auburn, CA). DC were incubated with H2O2 as above and washed, and 1 × 104 DC were incubated for 72 h with 1 × 105 BALB/c CD4+ cells followed by pulsing with tritiated thymidine.

Diphtheria toxin (DT) treatment

DTR-GFP-DC mice express the simian DT receptor (DTR) proximal to the CD11c promoter, so that administration of DT to these mice selectively depletes the DC population (13). DTR-GFP-DC and WT mice were injected with 250 ng DT (Sigma Aldrich, St. Louis, MO, USA), i.e. 24 h before transplantation, after which they were used as donors of heterotopic cardiac transplants.

Heterotopic cardiac transplantation

MCI-186 (courtesy of Drs. Niimi and Shirasugi, Mitsubishi Tanabe Pharma Corporation, Osaka, Japan) treatment was performed by perfusing donor hearts directly prior to transplant with 0.25 mL of 1 mg/mL MCI-186 injected into the ascending aorta in a retrograde fashion, and successful perfusion was visually confirmed. Injection of phosphate-buffered saline using the same method served as a control. Cardiac grafts were transplanted intra-abdominally using microsurgical techniques as described by Corry et al. (13). Rejection was determined as complete cessation of cardiac contractility and was confirmed by direct visualization.

ELISPOT

An ELISPOT assay was performed according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA) in order to assess production of murine IFN-γ, IL-4, IL-6 and IL-10 as previously described (13).

Histological analysis

Cardiac allografts were removed from recipients to evaluate acute and chronic rejection and fixed in 10% buffered formalin. Ventricular short-axis sections were cut and stained with hematoxylin and eosin (H&E). The International Society for Heart and Lung Transplantation nomenclature system was used to score acute rejection of cardiac allografts as previously described (13). For the assessment of chronic rejection, coronary arteries were digitally photographed, and blindly stored in an image analysis system (NIH image, version 1.62). Luminal occlusion was calculated as percent intimal thickening as follows: the area encompassed by internal elastic lamina (IEL) and luminal area (square pixels) was carefully analyzed, and the cross-sectional area luminal stenosis was calculated as previously described: luminal occlusion (%) = (IEL area-luminal area)/IEL area × 100 (14). The number of CD3- and F4/80-positive cells was examined per high power field, and sections were scored on a scale from 0 to 4, as follows: score 0: no positive cells; score 1: 1–10 positive cells; score 2: 11–30 positive cells; score 3: 31–100 positive cells and score 4: more than 100 positive cells (15). Spleen samples recovered from experimental animals were embedded in OCT and snap frozen. 4–8 mm thick cryosections were stained for DAPI, and ADDC were characterized as GFP-positive cells with DAPI-stained nucleoli and are represented as cell counts per section. Four to six random high power fields of spleen sections were examined (16).

Immunohistology

Cardiac allografts, spleen and paraaortic lymph nodes (LNs) were embedded in OCT, frozen and immunohistological studies were performed as previously described (13). Fibronectin staining was performed using a rabbit polyclonal antibody (Abcam, Cambridge, MA, USA) at a 1:150 dilution, and the Verhoeff procedure was used to perform elastin staining.

Statistics

Kaplan–Meier survival graphs were constructed and log rank comparison of the groups was used to calculate p values for the survival comparisons between the groups. Student's t-test was used for comparison of means between experimental groups examined in our in vitro assays. Differences were reported to be significant with p ≤ 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Oxidative stress increases DC immunogenicity

To address the effect of oxidative stress on DC activation, we treated C57BL/6 BMDC with 5, 50 or 500 μm H2O2 for 60 min as described previously (17,18). Flow cytometric analysis of apoptosis and cell death as assessed by 7-AAD and Annexin V staining showed no significant decrease in cell viability (<4%) following H2O2 treatment (Figure 1A). DC were also analyzed for their expression of CCR7, CD40, CD80, CD86, class II, TLR2 and TLR4 to determine their maturation status and immunogenic profile. While no substantial increase was found in the expression of these molecules, CCR7 was found to be slightly increased upon treatment with 500 μM H2O2 (5.5 ± 0.22% and 6.51 ± 0.22% for control and 500 μM H2O2-treated, respectively; p = 0.03, data not shown). In order to determine whether upregulation of surface expression occurs at a later time point, cells were incubated for 24 h in medium following treatment and were then subjected to flow cytometric analysis. At 24 h, H2O2 treatment was not associated with any significant increase in cell death (Figure 1B). As shown in Figures 1B and C, CCR7, CD80, CD86, class II, and TLR4 were found to be significantly increased in response to 500 μM H2O2 treatment, demonstrating that free radical injury results in increased DC immunogenicity (n = 3, p < 0.05). Treated DC were also evaluated for their ability to stimulate allogeneic T cell proliferation, and H2O2-treated BMDC were found to stimulate BALB/c T cells more potently than untreated DC as measured by tritium uptake, an effect which was inhibited with prior MCI-186 treatment (Figure 1D, n = 3, p < 0.0001 for all doses of H2O2 alone). While isolation of DC from naïve hearts is problematic due to the low number of constitutive heart tissue DC, we and others have recently shown that implantation of Flt3L hybridoma cells (which produce Flt3L) increases the DC population in the heart as well as in lymphoid tissues, and DC obtained do not exhibit any phenotypical or functional alterations (13,19). To demonstrate the effect of ischemic insult in the specific context of heart tissue DC, isolated naïve C57BL/6 heart DC of mice pretreated with the Flt3L hybridoma were treated with H2O2 as above and were cocultured with allogeneic T cells. Indeed, H2O2-treated heart DC exhibited greater immunogenicity in comparison to control heart DC, as evidenced by augmented proliferation of allogeneic T cells in response to coculture with H2O2-treated DC (Figure 1E, n = 4, p < 0.0001).

image

Figure 1. Oxidative stress and upregulation of DC immunogenicity. (A) 60 min H202 treatment has no effect on DC death or apoptosis, as assessed by Annexin V and 7-AAD staining (n = 3–5, p = ns). (B) Cell viability and surface expression of DC markers indicative of enhanced immunogenicity were examined following 500 μM H2O2 treatment and incubation in medium for 24 h. No difference in cell viability between treated and control groups was observed, while DC expression of CCR7, CD80, CD86, class II and TLR4 was found to be significantly enhanced. (C) CCR7, CD80, CD86, class II and TLR4 expression increased significantly in response to H2O2 treatment (*p < 0.05, n = 3). (D) In an MLR, H202-treated BMDC stimulated allogeneic T cells to a greater degree than untreated DC in a dose-dependent manner, an effect inhibited by pretreatment with 10 μm MCI-186 (n = 3, *p < 0.0001). (E) 5 μm H202 treatment was also shown to augment the immunogenicity of isolated heart DC (n = 3, p < 0.0001), in which 1 × 104 C57BL/6 heart DC were incubated with 1 × 105 BALB/c CD4+ cells.

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Donor MCI-186 therapy prolongs cardiac allograft survival

To examine the effect of MCI-186 antioxidant therapy on the alloimmune response in vivo, we performed fully mismatched cardiac allograft transplants using hearts from DTR-GFP-DC mice (on a C57BL/6 [H-2b] background) into BALB/c (H-2d) recipients. Using the DTR-GFP-DC mouse model also enabled us to deplete ADDC by administration of DT, as transfer of a primate diphtheria toxin receptor (DTR) proximal to the CD11c promoter selectively confers DT sensitivity to murine DC. In some experiments, naïve DTR-GFP-DC mice were administered an injection of 250 ng DT intraperitoneally, and 24 h following administration, naïve hearts were removed and examined for the percentage of GFP+ cells to confirm CD11c+ depletion. As compared with untreated hearts, administration of DT was shown to deplete GFP+ ADDC (Figure 2A). Prior to heterotopic cardiac transplantation, donor hearts were perfused with MCI-186 or normal phosphate-buffered saline as a control. As shown in Figure 2B, compared with the control DTR-GFP-DC donor group, MCI-186-treated cardiac allografts enjoyed significantly prolonged survival (MST of 8 vs. 26 days, 6–7 mice/group, p = 0.0005). To assess allograft infiltrate and cardiac muscle integrity, we also performed histological examination of cardiac allografts at day 7 posttransplantation. Compared with control cardiac allografts, donor treatment with MCI-186 was associated with marked muscle preservation and reduced infiltration of CD3+ T cells and macrophages (Figure 2C) and was concomitant with reduced rejection, CD3 and F4/80 scores (rejection score: 3.76 ± 0.14 vs. 1.96 ± 0.14, p < 0.01; CD3 score: 3.25 ± 0.25 vs. 1.5 ± 0.29, p = 0.004; F4/80 score: 1.5 ± 0.29 vs. 0.25 ± 0.25, p = 0.017, for control and MCI-186-treated, respectively) (Figures 2C and D).

image

Figure 2. Donor MCI-186 therapy prolongs cardiac allograft survival of fully mismatched cardiac allografts. (A) 250 ng of DT was injected i.v. into DTR-GFP-DC mice. Twenty-four hours postinjection, hearts were removed and examined for the presence of GFP+ DC (green). Compared to untreated mice (left), DT was shown to deplete heart DC. DAPI was used to reveal cell nuclei (blue). (B) Cardiac allografts of treated donors were perfused with 1 mg/mL MCI-186 solution, while control cardiac allografts were perfused with phosphate-buffered saline. Allograft survival was significantly prolonged in the MCI-186-treated group compared to the control group (MST of 26 and 8 days, respectively, n = 6–7 mice/group, p = 0.0005). Compared to the ADDC-depleted control group (+DT), allograft survival was prolonged by administration of MCI-186 (MCI-186+DT) to the donor (MST of 8.5 and 12.5 days, respectively, n = 4 mice/group, p = 0.0169). (C) Histological examination at 7 days posttransplantation revealed that, compared to control allografts (left), donor MCI-186 therapy results in protection of cardiac allografts, as demonstrated by preserved muscle and fewer infiltrating CD3+ cells and macrophages (right). (D) Quantitative analysis of acute rejection and CD3 or F4/80 infiltration at day 7 was also performed as described in the methods section, and rejection score (3.76 ± 0.14 vs. 1.96 ± 0.14, p < 0.01), CD3 score (3.25 ± 0.25 vs. 1.5 ± 0.29, p = 0.004) and F4/80 score (1.5 ± 0.29 vs. 0.25 ± 0.25, p = 0.017) was significantly reduced in response to MCI-186 treatment. (Data are means ± SEM and are shown as control and MCI-186-treated, respectively.)

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ADDC depletion abrogates MCI-186-induced prolongation of cardiac allograft survival

To determine the contribution of ADDC to the prolongation we observed following MCI-186 treatment, we performed cardiac allograft transplantation using hearts from DTR-GFP-DC donors into BALB/c recipients in which DT was previously injected into the donors, i.e. 24 h prior to transplantation. Administration of DT was shown to reduce the effect of MCI-186 treatment alone by almost half (MST of 13 days in the MCI-186+DT group compared to MST of 26 days in MCI-186 treatment alone [Figure 2B], p = 0.0031). Taken together, these data indicate that donor MCI-186 therapy results in prolongation of cardiac allograft survival and that the protective effect is mediated largely by ADDC.

Donor MCI-186 therapy reduces ADDC trafficking to recipient lymphoid tissues

The capacity of ADDC to present alloantigens to T cells and to generate an alloimmune response relies on their ability to traffic efficiently. We thus tested the hypothesis that IR injury increases ADDC immunogenicity in part due to enhanced trafficking by using the DTR-GFP-DC mouse model, in which a GFP protein has been linked to the CD11c promoter. As we have previously shown, this model allows for efficient monitoring of ADDC posttransplantation (13). Transplants were thus performed as above; spleens of recipient mice were recovered at day 3 posttransplantation, and immunohistological sections were examined for the magnitude of trafficked ADDC. Indeed, greater numbers of splenic ADDC were found in the untreated group compared to those in the MCI-186-treated group (11.33 ± 2.2 vs. 5.0 ± 1.12, respectively, p = 0.03) (Figure 3A). These data indicate that donor MCI-186 therapy diminishes trafficking of ADDC to recipient lymphoid tissue.

image

Figure 3. Depletion of ADDC abrogates MCI-186-induced prolongation of cardiac allograft survival. (A) Secondary lymphoid tissues were recovered at day 3 posttransplantation (4 mice/group) and trafficked ADDC were enumerated. Greater numbers of ADDC trafficked to the spleen in the control group (left, 11.33 ± 2.2 ADDC) as compared to the MCI-186-treated group (right, 5.0 ± 1.12 ADDC) (p = 0.03). ADDC were characterized as GFP+ cells containing DAPI-stained nucleoli (blue: DAPI, green: GFP+ DC, magnification of 400x, 4–6 random high power fields counted/section.) (B) Splenocytes from BALB/c recipients of MCI-186-treated or control C57BL/6 cardiac allografts were challenged with irradiated C57BL/6 splenocytes. Recipients transplanted with MCI-186-treated cardiac allografts showed a significant decrease in IFN-γ and IL-6 production compared to recipients transplanted with control cardiac allografts, an increase in IL-10, and no difference in IL-4 production. Data represent mean ± SEM of triplicate wells from 4–6 mice/group (p = 0.0005, p = 0.015 and p = 0.009 for IFN-γ, IL-6 and IL-10, respectively). (C) Surface expression of DC markers was assessed to examine immunogenic status of DC by gating on GFP+CD11c+ cells in the spleen and draining lymph nodes, and no difference in expression was observed. (D) Treg frequency was also examined in the spleen and draining lymph nodes by flow cytometric analysis, and MCI-186 donor treatment was not found to alter Treg percentages.

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Donor antioxidant therapy is associated with reduced alloreactivity of recipient lymphocytes

The capacity of recipient lymphocytes to mount a proliferative response upon alloantigen challenge in vitro was assessed using an ELISPOT assay. We restimulated recipient splenocytes with donor antigen at day 5 posttransplantation, and the number of IFN-γ–producing cells was assessed as an index of alloreactivity as described previously (13). Our ELISPOT data show that the frequency of IFN-γ-producing cells was significantly reduced in lymphocytes isolated from recipients transplanted with MCI-186-treated allografts versus control allografts (405 ± 35 vs. 646 ± 55, respectively, p = 0.0005) (Figure 3B), demonstrating a lesser inflammatory response to alloantigen as a result of MCI-186 treatment. We also examined the frequency of the Th2 cytokine IL-4, which showed no significant shift in response to donor MCI-186 treatment, while IL-10 was shown to be enhanced (22 ± 3 vs. 43 ± 8 IL-10-producing cells for control and MCI-186-treated, respectively, p = 0.009, Figure 3B). We have previously demonstrated the central role of IL-6 in ischemic injury of DC (9), and IL-6 production upon alloantigen challenge was indeed diminished following MCI-186 treatment of donor cardiac allografts (90 ± 5 vs. 74 ± 5 IL-6-producing cells for control and MCI-186-treated, respectively, p = 0.015, Figure 3B). No difference was noted in the expression of maturity markers, CCR7, TLR2 or TLR4 on ADDC in spleens or draining lymph nodes of recipients transplanted with control or MCI-186-treated donors as assessed by flow cytometric analysis (Figure 3C), nor was any difference found in the percentage of Tregs in the periphery, examined by enumerating splenic CD4+CD25+Foxp3+ cells (Figure 3D).

Donor MCI-186 therapy attenuates chronic allograft rejection

Due to the impediment that ischemic injury presents to long-term allograft acceptance, we also sought to establish whether our anti-ischemic strategy would reduce the severity of chronic rejection. Chronic rejection was thus evaluated in an MHC II single-mismatch model using BM12 donors and C57BL/6 recipient mice, a transplant combination well established as a model of chronic allograft rejection in which, in the absence of acute rejection, surviving transplants exhibit classical histological features of fibrosis and vasculopathy (14). To test the efficacy of our anti-ischemic treatment, BM12 hearts were perfused with MCI-186 and were then transplanted into C57BL/6 recipients, and cardiac allografts were recovered at 8 weeks posttransplantation. Compared with the control group, cardiac parenchyma was markedly preserved in the MCI-186-treated group, as demostrated by hematoxylin and eosin staining, together with a marked reduction in the number of infiltrating CD3+ and macrophage cells (Figure 4A). Staining for fibronectin and elastin revealed a lesser degree of fibrosis, intimal thickening, and microvascular occlusions in MCI-186-treated hearts as well (Figure 4A), and the degree of cellular infiltration and vasculopathy score (Figure 4B, shown as % luminal stenosis) were both significantly decreased in the MCI-186-treated donors (3.2 ± 0.5 vs. 1.3 ± 0.4 and 81.6 ± 7.5 vs. 32.5 ± 5.5, respectively, p<0.0009). To measure the frequency of cytokine-producing alloreactive T cells in allograft recipients, splenocytes from C57BL/6 recipients were isolated at 8 weeks and were challenged with irradiated naïve BM12 stimulators. Recipient splenocytes showed a significant decrease in the inflammatory cytokines IFN-γ and IL-6 as well as in IL-4 production compared with mice that received control BM12 grafts (IFN-γ-producing cells: 160 ± 6 vs. 359 ± 31; IL-6-producing cells: 90.4 ± 8 vs. 156 ± 29, IL-4-producing cells: 116 ± 8 vs. 189 ± 24, p < 0.02 in all groups) (Figure 4C).

image

Figure 4. Donor MCI-186 treatment attenuates chronic rejection. (A) At 8 weeks posttransplantation, BM12 cardiac allografts transplanted into C57BL/6 recipients were examined for cellular infiltrates, CD3+ cells, Foxp3+ cells and macrophages as well as for the severity of chronic rejection. The degree of cellular infiltration was significantly lower in MCI-186-treated donor hearts as compared to control hearts, and control hearts showed markedly more severe chronic rejection, as shown by increased infiltration and a greater degree of smooth muscle necrosis/inflammation, in addition to intimal thickening. (B) Cellular infiltration and vasculopathy score were significantly increased in control grafts compared to MCI-186-treated grafts. Values shown represent mean ± SEM histological score obtained from three individual mice (p < 0.0009 for rejection score and % luminal stenosis). (C) The frequencies of cytokine-producing T cells were then determined by an ELISPOT assay. C57BL/6 mice that received MCI-186-treated cardiac allografts showed a significant decrease in IFN-γ, IL-6 and IL-4 cytokine production compared with C57BL/6 recipients of control allografts (p < 0.02).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

IRI to donor grafts has been shown to activate the innate immune response and to instigate the adaptive alloimmune response, and it is additionally well established that prolonged ischemia can enhance graft immunogenicity and subsequently increase the rate of acute allograft rejection (1). IRI to the donor organ induces an inflammatory milieu in the allograft, which appears to be the initial key event for activation of the innate immune system. In this report, we examined the role of donor antioxidant MCI-186 therapy in prolonging cardiac allograft survival. We first demonstrate that in vitro induction of oxidative stress to DC results in an increase in the expression of maturity markers and CCR7. Ischemic insult of DC resulted in enhanced immunogenicity, as demonstrated by their augmented capacity to induce proliferation of allogeneic T cells compared to untreated DC. In vivo, we show that compared with the untreated WT group, MCI-186-treated donors enjoy significant prolongation of cardiac allograft survival. Notably, depletion of ADDC with DT was not shown to increase cardiac allograft survival. While insertion of the DTR sequence proximal to the CD11c promoter allows for DC depletion, we and others have learned that this DT-induced depletion DT is not complete (13,20). The persistence of residual ADDC, albeit in low numbers, could therefore potentially lead to rejection. To address this issue, we also performed experiments with the aim of obtaining a more robust depletion by administering higher doses of DT to the donors or continuing DT administration in the recipients; however, a high mortality rate associated with the toxicity of this strategy has been a limiting factor in achieving this end. Moreover, depleting ADDC by DT reduced the effect of MCI-186 by almost half, indicating that the protective effect of MCI-186 therapy in the donors is mediated largely by ADDC. Following egress from the bone marrow, DC precursors and progenitors circulate in the blood until they seed tissue paranchyma as immature cells. These immature tissue DC are highly effective in capturing and processing antigens and in responding to various insults; once immature DC encounter local inflammatory mediators, they become activated, undergo a maturation process (21) and release inflammatory cytokines. We therefore postulate that donor heart DC will maintain their immature status and associated tolerogenicity if they are protected from ischemic injuries. In the absence of such protection, ischemic injury will activate heart DC, resulting in DC maturation and production of inflammatory cytokines as well as DC mobilization to secondary lymphoid tissues, after which they become potently immunostimulatory. Thus, depletion of heart DC protected by MCI-186 would diminish DC tolerogenicity and its associated effects on tolerance to allografts. Future studies beyond this brief report, however, are needed to examine the in vivo behavior of tissue DC in chimeric or parabiosis models.

Upon examination of T cell priming, we show that treating donors with MCI-186 resulted in a reduction in the number of IFN-γ- and IL-6-producing cells. IL-6 has been reported to be secreted by ischemic DC, and various strategies have also been applied to reduce IRI through siRNA intervention or donor anti-IL-6 strategies, resulting in organ protection (9,22,23). These data are in accordance with our trafficking study, in which we demonstrate impaired trafficking of ADDC to lymphoid tissue, which is reported to be necessary in the generation of alloimmune responses. It should be noted that the protective effect of MCI-186 could also be exerted via protection from other parenchymal subsets such as endothelial cells; this avenue should be explored in future studies given the complexity of such an investigation. Importantly, our donor anti-ischemic strategy reduced chronic rejection in an MHC II-single mismatched model. Given that chronic rejection remains a primary impediment to long-term allograft acceptance, the results of these studies may have significant clinical implications and may constitute a novel strategy to prevent chronic rejection. The establishment of anti-ischemic strategies specific to donors is a novel approach to promote long-term allograft acceptance by reducing allograft immunogenicity. Donor anti-ischemic treatment may also allow clinicians to reduce both the dose and course of immunosuppressive drugs associated with significant toxicity. While a reduction in immunosuppression is highly advantageous, it has thus far been difficult to attain in clinical transplantation, and our donor anti-ischemic strategy is particularly relevant given the growing usage of ischemic/marginal donors due to the shortage of available organs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References