Haptoglobin enhances cardiac transplant rejection

Abstract

Rationale: Early graft inflammation enhances both acute and chronic rejection of heart transplants, but it is unclear how this inflammation is initiated.

Objective: To identify specific inflammatory modulators and determine their underlying molecular mechanisms after cardiac transplantation.

Methods and results: We used a murine heterotopic cardiac transplant model to identify inflammatory modulators of early graft inflammation. Unbiased mass spectrometric analysis of cardiac tissue before and ≤72 hours after transplantation revealed that 22 proteins including haptoglobin, a known antioxidant, are significantly upregulated in our grafts. Through the use of haptoglobin-deficient mice, we show that 80% of haptoglobin-deficient recipients treated with perioperative administration of the costimulatory blocking agent CTLA4 immunoglobulin exhibited >100-day survival of full major histocompatibility complex mismatched allografts, whereas all similarly treated wild-type recipients rejected their transplants by 21 days after transplantation. We found that haptoglobin modifies the intra-allograft inflammatory milieu by enhancing levels of the inflammatory cytokine interleukin-6 and the chemokine MIP-2 (macrophage inflammatory protein 2) but impair levels of the immunosuppressive cytokine interleukin-10. Haptoglobin also enhances dendritic cell graft recruitment and augments antidonor T-cell responses. Moreover, we confirmed that the protein is present in human cardiac allograft specimens undergoing acute graft rejection.

Conclusions: Our findings provide new insights into the mechanisms of inflammation after cardiac transplantation and suggest that, in contrast to its prior reported antioxidant function in vascular inflammation, haptoglobin is an enhancer of inflammation after cardiac transplantation. Haptoglobin may also be a key component in other sterile inflammatory conditions.

Keywords: immunology; inflammation; rejection; transplantation.

Figure 1. Haptoglobin levels increase within cardiac transplants

A: WT C57BL/6 hearts transplanted into WT C57BL/6 recipients. Haptoglobin levels were measured in the cardiac transplant and in sera. Hearts were exposed to 4h cold storage prior to transplantation. B: As per A; intra-graft haptoglobin levels measured in A compared to levels in native heart of the recipient at 24h after transplantation. * p<0.0001 (Mann-Whitney) C: Intra-graft levels of haptoglobin in syngeneic (i.e., donor and recipient both = C57BL/6) cardiac transplants and cardiac allografts (i.e., donor = C57BL/6 and recipient = BALB/c) during the 1^st week after transplantation. At least 4 transplants/time point. D: Haptoglobin levels in the liver from non-transplanted mice and mice that received either syngeneic or allogeneic cardiac allografts, day +3 post transplantation. N = 4–6 mice/group. *p<0.01(Mann-Whitney)

Figure 2. Haptoglobin affects the tempo of cardiac allograft rejection in mice

A: WT BALB/c cardiac transplants implanted into WT C57BL/6 recipients or C57BL/6 Hp^−/− recipients. Rejection time was monitored. P = 0.003 between groups (Log Rank). B: Recipients were treated with peri-operative CTLA4 Ig (200μg day 0, 2, 4 post transplantation). P = 0.002 between groups (Log Rank). C: Histological assessment of WT BALB/c allografts at day +21 post transplantation in either WT C57BL/6 or C57BL/6 Hp^−/− recipients treated with CTLA4 Ig per Figure 2B. Allografts transplanted into WT recipients are diffusely swollen and necrotic (markers as N), with large areas of hemorrhage (H), and thrombosis of myocardial blood vessels (arrow). Hp^−/− recipients exhibited diminished hemorrhage within the allograft and patent myocardial vessels (arrow) with retention of myocardiocyte nuclei and sarcoplasm (arrow heads). M = mineralization. Higher power images from the areas marked (*) appear in lower panels. Upper panel scale bars = 1000 μm, lower panel scale bars = 100 μm. D: WT BALB/c cardiac transplants were implanted into WT C57BL/6 recipients treated with peri-operative CTLA4 Ig (200μg day 0, 2, 4 post transplantation). At day +21 post transplantation allografts were obtained and H and E (top panel) and immune histochemistry (bottom panel) for haptoglobin was performed. Haptoglobin positive cells (brown) include scattered cardiomyocytes (arrows), macrophages (arrow heads) and scattered perivascular cells (*). Scale bar = 50 μm, BV = blood vessel. Isotype control staining was negative, data not shown.

Figure 3. Recipient haptoglobin alters the intra-graft inflammatory environment after cardiac transplantation and treatment with peri-operative CTLA4 Ig

WT and Hp^−/− recipients were implanted with a WT BALB/c cardiac allograft and were treated with peri-operative CTLA4 Ig (200μg day 0, 2, 4 post transplantation). At day +14 or +21 post transplantation cardiac allografts were obtained and intra-graft IL-6 (A), MIP-2 (B), IL-10 (C) and haptoglobin (D) were measured via ELISA. *p<0.01 (t-test). Arrows in 3D indicate the Hp^−/− groups. Figures represent pooled data from three independent experiments. Error bars = SEM, N = 6–9 mice/time point. E–F: Immune cells (i.e., CD45⁺ cells) were enriched from hearts of recipients at day +21 post transplantation and treatment with CTLA4 Ig and cultured for 12h. IL-6 and MIP-2 were measured in the culture supernatant. Pooled data from 3 independent experiments, n = 2/experiment, *p<0.01 (t-test)

Figure 4. Recipient enhances accumulation of mature DCs after cardiac transplantation and treatment with peri-operative CTLA4 Ig

A: Representative flow cytometric plot showing surface expression of CD80 on CD11c⁺ MHC class II⁺ cells within the graft at day + 7 post transplantation in WT and Hp^−/− mice. Grey shadow shows expression on CD11c⁺ MHC class II⁺ cells in the allograft prior to implantation. B: Median fluorescent intensity (relative units) of CD80 expression on CD11c⁺ MHC class II⁺ cells in the groups shown in A. *p<0.01 (t-test). C–D: Enumeration of DCs and immune cells within the allografts of WT and Hp^−/− recipients treated with CTLA4 Ig at day +7 post transplantation. Pooled data from 2 independent experiments with n =3/experiment, *p<0.01 (t-test). Arrow in 4C indicate pre transplant group.

Figure 5. MyD88 expression within allografts modifies intra-graft inflammation after cardiac transplantation and treatment with CTLA4 Ig

A–C: C57BL/6 WT or MyD88^−/− hearts were transplanted into WT BALB/c recipients that were treated with CTLA4 Ig 200μg day 0, 2, 4 post transplantation. At day +14 post transplantation, allografts were obtained and intra-graft IL-6 (A), MIP-2 (B), and IL-10 (C) were measured via ELISA. *p<0.01 (t-test). Tx = transplanted. D: Immune cells (i.e., CD45⁺ cells) were enriched from non-transplanted WT or MyD88^−/− C57BL/6 hearts and stimulated ex vivo with the indicated dose of haptoglobin. IL-6 was measured in the media via ELISA. ECs and fibroblasts did not produce IL-6 above control levels in response to haptoglobin (data not shown).

Figure 6. Recipient haptoglobin enhances anti-donor T cell responses after cardiac transplantation and perioperative treatment with CTLA4 Ig

A–B. Purified WT or Hp^−/− T cells were stimulated with irradiated donor BALB/c spleen cells in vitro and IFN-γ (A) + IL-2 (B) were measured by ELISPOT. T cells stimulated with syngeneic spleen cells did not induce a response (data not shown). C: As in A–B but cellular proliferation of T cells measured by thymidine incorporation. D–E: Splenic anti-donor IFN-γ (D) IL-2 (E) T cell responses from either WT or Hp^−/− recipients before transplantation or at day +21 after cardiac transplantation and treatment with CTLA4 Ig were measured via ELISPOT. Tx = transplantation, *p<0.01 (t-test). F: As per D–E but CD4⁺CD25⁺FoxP3⁺ cells were enumerated in spleens of mice after relevant staining and flow cytometric analysis. Figures represent pooled data from two experiments. N = 3 mice/experiment. Error bars = SEM

Figure 7. Intra-graft haptoglobin associates with acute allograft rejection in humans

Human heart transplant specimen with no cellular rejection and no staining for Hp (A), and a specimen with evidence of rejection and staining for Hp (black arrow) (B) Blue arrow indicates circumferential staining consistent with endothelial cells. Scale bar = 50 μm