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Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts ¹

KENICHIRO YAMASHITA^*,2, JAMES McDAID^*, ROBERT ÖLLINGER^*, TUNG-YU TSUI, PASCAL O. BERBERAT^*, ANNY USHEVA, EVA CSIZMADIA^*, R. NEAL SMITH, MIGUEL P. SOARES^*,¶,3 and FRITZ H. BACH^*,3

^* Department of Surgery and
Department of Medicine, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA;
Department of Surgery, University of Hong Kong Medical Center, Queen Mary Hospital, Hong Kong;
Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA; and
^¶ Instituto Gulbenkian de Ciência, 2781–901 Oeiras, Portugal

²Correspondence: Department of Surgery, Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215, USA. E-mail: kyamashi{at}bidmc.harvard.edu

SPECIFIC AIMS

Bilirubin has been considered a natural waste product of the body. However, several epidemiological studies have shown that individuals with slightly above normal levels of serum bilirubin have less incidence of heart disease than those with low levels, suggesting that bilirubin may have beneficial effects, possibly related to its potent antioxidant properties. We tested our hypothesis that biliverdin, a precursor of bilirubin, has a salutary effect in suppressing alloimmune responses and prolonging cardiac allograft survival.

PRINCIPAL FINDINGS

1. Biliverdin administration induces donor-specific tolerance to cardiac allografts
We first asked whether biliverdin has a beneficial effect in transplantation. Untreated control B6AF1 (H-2^k/d,b) mouse recipients rejected DBA/2 (H-2^d) cardiac allografts in a median of 11.5 days (n=6; Fig. 1 a). In contrast, administration of biliverdin twice (every 12 h) or three (every 8 h) times per day at 50 µmol/kg/dose (n=6 per group) significantly increased graft survival, with 4 of 6 (66.7%) grafts in each group surviving for >200 days (both P<0.001 vs. control, Fig. 1a ). Since mice harboring cardiac allografts over 200 days had not received further treatments after 2 wk post-transplantation, we tested whether they were tolerant to the donor-antigens. The animals were thus challenged with second heart grafts. Strikingly, these animals accepted donor strain DBA/2 hearts for >100 days (n=3) while promptly rejecting third party FVB (H-2^q) grafts within 11 days (n=3; Fig. 1b ).

View larger version (16K): [in this window] [in a new window]   Figure 1. Exogenous biliverdin administration induces donor-specific tolerance to cardiac allografts. a) Kaplan-Meier plotting of the DBA/2 cardiac allograft survival in B6AF1 mouse. Donor and recipient animals received nontreatment (x) or biliverdin (at 50 µmol/kg, i.p.) 2 () or 3 () times a day (n=6 per each group). All treatments were terminated at 2 wk after transplantation. P < 0.001, control vs. biliverdin (both 2 and 3 times/day). b) Kaplan-Meier plotting of the second cardiac allograft survival. Long-term heart graft accepting B6AF1 recipients by biliverdin treatments accepted second heart grafts from theDBA/2 (donor strain; •) but not from FVB (third party; ) mice (n=3 per each group). P < 0.05, DBA/2 vs. FVB.

2. Biliverdin administration suppresses graft cellular infiltration and alloimmune response
To understand the events associated with graft survival prolongation and tolerance induced by biliverdin, we examined histology of grafts 5 days after transplantation. A moderate degree of cellular infiltration, predominantly CD8⁺ T cells and to a lesser extent CD4⁺ T cells, was present in the grafts of control animals. These histological features presumably represented ongoing acute rejection of the hearts. In contrast, treatment with biliverdin reduced both CD4⁺ and CD8⁺ T cell infiltrates in the grafts as compared with the controls. We examined the in vivo immune response of recipients against donor antigens. Splenocytes were taken from the recipient 5 days after transplantation and assessed for their proliferative activity in mixed lymphocyte culture. This response was significantly suppressed in recipients receiving biliverdin for three times per day (11082±3423 cpm) compared with the control (25479±2164 cpm; P<0.01).

3. Biliverdin suppresses T cell proliferation in vitro
To further understand the suppression of the immune response seen in vivo, we studied the effects of biliverdin on T cells in vitro. Proliferation of purified naive B6AF1 T cells in response to anti-CD3 monoclonal antibody (mAb) plus anti-CD28 mAb costimulation, which stimulates T cells in an antigen-presenting cell independent manner, was significantly suppressed by biliverdin (control; 52329±3009 cpm vs. biliverdin at 100 µM; 15995±3049 cpm; P<0.01).

4. Biliverdin suppresses IL-2 production via inhibition of NFAT and NF-B activation
To elucidate the mechanisms underlying T cell suppression by biliverdin, we examined IL-2 secretion in anti-CD3 mAb-activated splenocytes. IL-2 production was significantly suppressed by biliverdin in a dose-dependent manner (Fig. 2 a). This was also the case with interferon- (IFN-) production in vitro (Fig. 2b ), consistent with a suppression of Th1 activity. Addition of recombinant IL-2 (50 U/mL) 6 or 24 h after biliverdin treatment overcame the antiproliferative effect of biliverdin (Fig. 2c ). These data suggest that biliverdin inhibits T cell proliferation directly by interfering with the signal transduction pathway leading to IL-2 synthesis, not by down-regulating IL-2R expression or blocking the signaling pathways involved in IL-2-driven proliferation. We thus hypothesized that these effects of biliverdin related to suppression of transcription factors involved in IL-2 production, NF-B and NFAT. To test this, we stimulated mouse primary T cells with anti-CD3 and CD28 mAbs; nuclear NF-B and NFAT protein levels were studied by measuring the DNA binding activity of nuclear extracts in EMSA. Anti-CD3 and CD28 mAbs induced activation of NF-B and NFAT nuclear translocation and DNA binding as assessed 4 h after stimulation (Fig. 2d, e ). Biliverdin inhibited DNA binding of both NF-B (Fig. 2d ) and NFAT (Fig. 2e ), suggesting that biliverdin suppresses IL-2 production via inhibition of NFAT and NF-B activation.

View larger version (49K): [in this window] [in a new window]   Figure 2. Biliverdin suppresses IL-2 secretion via inhibition of NFAT and NF-B activation in T cells. a, b) IL-2 (a) and IFN- (b) concentrations in the culture supernatants were examined by ELISA. B6AF1 splenocytes was stimulated by anti-CD3 mAb and cultured for 48 h with or without biliverdin. c) Effect of exogenous recombinant IL-2 on B6AF1 splenocyte proliferation under biliverdin treatment. B6AF1 splenocytes were cultured for 48 h with anti-CD3 mAb in the presence of biliverdin (100 µM). Group in which cells were not treated with biliverdin served as controls (). Recombinant IL-2 was added to culture wells 6 () or 24 () h after anti CD3 mAb stimulation and biliverdin treatment, and 3H-thymidine incorporation was measured. Values are expressed as mean ± SE of 3–4 experiments; *P < 0.05, **P < 0.01; n.s., no significance vs. control. d, e) Autoradiograms show EMSA for NF-B (d) and NFAT (e) nuclear DNA binding. Nuclear extracts were prepared from B6AF1 primary T cells after 4 h stimulation with anti-CD3 and CD28 mAbs; NF-B- and NFAT-specific DNA binding activities were examined. The positions of free DNA (F) and specific complexes (C) are indicated. Unlabeled homologous (Homo-competitor) or unrelated heterologous (Hetero-competitor) oligonucleotide was used as competitors in the reactions. Lane 1: probe alone for NF-B or NFAT. Data are representative of 3 experiments.

CONCLUSIONS AND SIGNIFICANCE

We demonstrate that biliverdin facilitates the development of tolerance to cardiac allografts, likely related to the ability of biliverdin to suppress alloimmune responses in vivo and T cell proliferation in vitro. We show that this suppressive effect of biliverdin is mediated by suppression of IL-2 production via inhibition of NFAT and NF-B activation (Fig. 2) .

The known antioxidant activities of biliverdin and bilirubin such as inhibition of protein kinase A and C may relate to the down-regulation of NF-B activation stimulated mainly by hydroxyl radicals. The exact mechanism whereby biliverdin suppresses NFAT activation is still unclear. Further studies are necessary to elucidate the upstream signaling molecules involved in this action of biliverdin, including the possible action on the Ca²⁺/calcineurin pathways. The findings presented here are the first to demonstrate a molecular basis for the action of biliverdin or bilirubin.

It is possible that suppression of alloaggressive T cells by biliverdin, reducing the size of those clones, is important in the ability of biliverdin to induce tolerance. If suppressor cells are involved, the consequence of this effect would be to change the alloaggressive-suppressor cell balance. Indeed, the transient treatment with cyclosporine, a calcineurin/NFAT inhibitor, has been shown to favor the induction of suppressor cells and tolerance to rat heart transplants. It has been demonstrated that the abrogation of NF-B activation suppresses T cell activation and induces permanent acceptance of cardiac allografts by using IB mutant transgenic mice.

In conclusion, we have demonstrated that biliverdin can suppress T cell proliferation and facilitates the development of tolerance to cardiac allografts in mice. Our findings encourage a potential therapeutic evaluation of biliverdin/bilirubin for clinical organ transplantation and other T cell-mediated immune disorders.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram of the principal findings. a) In naive T cells after receiving T cell receptor (TCR) -mediated and costimulatory signals, NFAT and NF-B translocate from cytoplasm to nucleus and bind to the promoter region of the IL-2. These events induce expression and secretion of IL-2, which binds to IL-2R promoting T cell proliferation in an autocrine fashion, leading to alloimmune responses to reject allografts. b) In the presence of biliverdin, NFAT and NF-B nuclear translocations are inhibited upon T cell activation. This results in suppression of IL-2 secretion and T cell proliferation, leading to immunosuppression that prolongs allograft survival.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0839fje; doi: 10.1096/fj.03-0839fje

³ Contributed equally to this paper.

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This Article Full Text (PDF) All Versions of this Article: 18/6/765 03-0839fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by YAMASHITA, K. Articles by BACH, F. H. Search for Related Content PubMed PubMed Citation Articles by YAMASHITA, K. Articles by BACH, F. H.