Overexpression of heme oxygenase-1 protects allogeneic thyroid grafts from rejection in naive mice

Overexpression of heme oxygenase-1 protects allogeneic thyroid grafts from rejection in naive mice Masanori Niimi, MD, PhD, Motoi Takashina, MD, Hiroshi Takami, MD, Yoshifumi Ikeda, MD, Tomoo Shatari, MD, Kimikazu Hamano, MD, Kensuke Esato, MD, Kenji Matsumoto, MD, Kaori Kameyama, MD, PhD, Susumu Kodaira, MD, and Kathryn J. Wood, PhD, Tokyo and Yamaguchi, Japan, and Oxford, UK

Background. Endocrine allografts are an option for the treatment of endocrine failure. Methods. One lobe of the thyroid was transplanted under the kidney capsule. Results. C57BL/10 (H2 b) thyroids were rejected in naive CBA (H2 k) mice within 14 days after transplantation. When mice were treated with anti-CD4 monoclonal antibodies (mAb), all grafts survived for more than 60 days. The first grafts still survived after second C57BL/10 or Balb/c (H2d) thyroid grafts that were transplanted into the same recipients were rejected acutely, which suggests that the primary grafts were modified under anti-CD4 mAb treatment. To confirm this hypothesis, C57BL/10 thyroid grafts from anti-CD4 mAb–treated mice were retransplanted. All grafts survived in naive mice; this correlated with the overexpression of heme oxygenase–1 (HO-1) in the grafts. Next, an inhibitor of HO-1 (zinc protoporphyrin) or control compound (copper protoporphyrin) was injected intraperitoneally after transplantation of C57BL/10 thyroid grafts into the primary CBA recipients that had been treated with anti-CD4 mAb. The grafts in mice that had been treated with zinc protoporphyrin, but not copper protoporphyrin, were rejected when retransplanted to naive recipients. Conclusions. Overexpression of HO-1 correlated with the protection of fully allogeneic thyroid grafts from rejection when retransplanted into naive recipients. (Surgery 2000;128:910-7.) From the First Department of Surgery, Teikyo University, Tokyo, Japan; the First Department of Surgery, Yamaguchi University, Yamaguchi, Japan; the Department of Surgery and the Division of Diagnostic Pathology, Keio University, Tokyo, Japan; and the Nuffield Department of Surgery, University of Oxford, Oxford, UK.

SINCE THE FIRST SUCCESSFUL HUMAN renal transplantation in 1954, graft survival rates have improved to between 80% and 90% after the first year. However, chronic immunosuppression of the recipient is required for long-term graft survival. Today, azathioprine, steroids, and cyclosporine A/tacrolimus (FK506) are routinely used in the clinic.1 For acute rejection, more aggressive treatments such as antilymphocyte globulin, antithymocyte globulin, OKT3, and high-dose steroids are available. All these agents have nonspecific Presented at the 21st Annual Meeting of the American Association of Endocrine Surgeons (jointly hosted with the British Association of Endocrine Surgeons), London, United Kingdom, and Lille, France, May 22-25, 2000. Reprint requests: Masanori Niimi, MD, PhD, First Department of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan. Copyright © 2000 by Mosby, Inc. 0039-6060/2000/$12.00 + 0 11/6/109968 doi:10.1067/msy.2000.109968

910 SURGERY

immunosuppressive effects that increase the susceptibility of transplant recipients to opportunistic infections and neoplasms2 and give rise to many other unwanted side effects, such as bone marrow suppression, hypertension, tremor, gingival hypertrophy, hyperlipidemia, diabetes, and osteoporosis. It has long been recognized that these complications could be avoided if it were possible to achieve a condition of donor-specific unresponsiveness or immunologic tolerance to the donor antigens. Endocrine allografts are an option for the treatment of endocrine failure. However, hypofunction of endocrine tissue can be controlled sufficiently (but not completely) with supplements of hormone or other necessary substrates. To apply allotransplantation of endocrine organs clinically, new strategies to prevent rejection are essential. One approach could be to use a short course of immunosuppressive treatment and then to rely on natural mechanisms to maintain graft survival in the long term. If this could be achieved, trans-

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plantation of endocrine tissue such as parathyroid or thyroid could be used to treat patients with permanent hypoparathyroidism and hypothyroidism. MATERIAL AND METHODS Animals. CBA (H2k), C57BL/10 (H2b), and Balb/c (H2d) mice were purchased from Sankyo Ltd (Tokyo, Japan). All mice were housed in conventional facilities of the Biomedical Services Unit, Teikyo Hospital, Tokyo, and used between 8 and 12 weeks of age in accordance with the Animals Care Guidelines of Teikyo University. Pretreatment of recipients. Mice were treated intraperitoneally with a depleting anti-CD4 mAb (mAb; YTA 3.1,3 100 µg per dose, on days –1, 0, and 1; Fig 1, A). Anti-CD4 mAb was prepared from ascites of nude mice injected with hybridoma of the YTA 3.1 (provided by Professor H. Waldmann, Oxford, UK) with ammonium sulfate precipitation and ion exchange chromatography of DEAE in Yamaguchi University. Heart transplantation. After the recipient received anesthetic, fully vascularized heterotopic hearts were transplanted into the abdomen with the use of microsurgical techniques.4 Graft function was followed by palpation of the grafted hearts at least 3 times a week. Rejection was confirmed by electrocardiogram5 and direct visualization of the graft. Second cardiac allografts into the neck. Some mice were transplanted with a second heart into the neck 30 days after the first transplantation. Briefly, the donor ascending aorta is anastomosed end-to-side to the recipient carotid artery, and the donor pulmonary artery is sutured end-to-side to the recipient external jugular vein. Thyroid transplantation under the right kidney capsule. After the recipient received anesthetic, 1 lobe of thyroid was transplanted under the right kidney capsule of CBA mice. Second thyroid allografts into the left kidney capsule. Some CBA mice were transplanted with second thyroid grafts from naive C57BL/10 or third-party Balb/c mice into the left kidney capsule. Retransplantation of thyroid grafts into naive recipients. Some surviving thyroid grafts in mice that had been treated with anti-CD4 mAb were resected and retransplanted under the kidney capsule of naive CBA mice 30 days after the first transplantation. Morphologic examination. Graft survival was examined by direct visualization and histologic examination at different time points after the transplantation. Morphologic analysis of thyroid grafts was performed on tissue that was prepared by paraffin wax section processing followed by hematoxylin

Niimi et al 911

and eosin staining. Rejection of thyroid grafts was defined as complete loss of thyroid follicles. Statistical analysis. Allograft survival between 2 groups was compared by Mann-Whitney U test with the use of StatView SE + Graphic software (SAS, Cary, NC). Histochemistry of MHC class I, MHC class II, Bcl-2, and heme oxygenase-1 by the grafts. Grafts were flash frozen in O.C.T. embedding medium (Sakura Co, Tokyo, Japan); 6 µm sections were cut and dried overnight. After being fixed in acetone for 10 minutes, serial sections were incubated with endogenous peroxidase blocking solution (2% H2O2, 0.1% sodium azide in phosphate-buffered saline solution) for 15 minutes. Sections were then blocked with avidin D solution (4 drops/mL; Vector, Burlingame, Calif) with 10% mouse and goat serum and in phosphate-buffered saline solution for 15 minutes, followed by biotin solution (4 drops/mL; Vector) for 10 minutes. Sections were incubated at room temperature with either biotinylated monoclonal anti-H2Kb (clone AF6-88.5, 1:100; Pharmingen, San Diego, Calif), biotinylated monoclonal anti-H2IAb (clone AF6-120.1, 1:100; Pharmingen), polyclonal rabbit anti-Bcl-2 (1:100; Pharmingen), or polyclonal rabbit anti-heme oxygenase-1 (OH-1; 1:200; Affinity Bioreagents, Golden, Colo) for 45 minutes. Bound antibody was detected with biotinylated goat anti-rabbit-IgG when the primary antibodies were nonconjugated, and an avidin-biotin-horseradish peroxidase complex was followed by 3,3-diaminobenzidine and enhancing solution (Vector). All sections were then counterstained with hematoxylin and eosin (Sigma-Aldrich, Tokyo, Japan), dehydrated, and mounted in Malinol (Muto Co, Tokyo, Japan). All compared samples were tested at the same time with the same solution and procedure. Expression levels on grafts were scored from (–), (+), (++), (+++), and (++++) by blinded examiners. Prevention of HO-1. To prevent activity of HO1, some mice were injected intraperitoneally with zinc protoporphyrin IX (an inhibitor of HO-1, 30 µmol/L/kg, every day; Sigma-Aldrich) after transplantation of C57BL/10 thyroid grafts into the primary CBA recipients that had been treated with anti-CD4 mAb. A control group was treated with copper protoporphyrin IX (Sigma-Aldrich), which does not inhibit the enzyme in the same dose and period as zinc protoporphyrin. RESULTS First, we determined whether a depleting antiCD4 mAb (YTA 3.1) could be used to induce prolonged survival of fully allogeneic cardiac grafts.

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Surgery December 2000

A

B

C

Fig 1. A, The protocol for inducing prolonged survival of fully allogeneic cardiac or thyroid grafts. CBA (H2k) mice were treated with 3 doses of a depleting anti-CD4 mAb (100 µg per dose), YTA 3.1, on days –1, 0, and relative to the transplantation of either cardiac or thyroid grafts from C57BL/10 (H2b) mice. B, Survival of fully allogeneic cardiac grafts. CBA recipients were untreated or treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. The recipients underwent transplantation with C57BL/10 cardiac grafts in the abdomen. The 2 groups are statistically different (P < .01). C, Survival of second cardiac grafts. CBA recipients were treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. C57BL/10 cardiac grafts were transplanted in the abdomen of the recipients and survived. Second cardiac grafts from either C57BL/10 or Balb/c mice were transplanted into the neck of the recipients 30 days after the first transplantation. The 2 groups are statistically different (P < .01).

C57BL/10 cardiac allografts were transplanted in the abdomen of CBA recipients. Naive CBA recipients rejected cardiac grafts acutely with a median survival time of 8 days (Fig 1, B). When recipients were treated intraperitoneally with 100 µg of YTA 3.1 on day, –1, 0, and 1 relative to transplantation (Fig 1, A), all recipients accepted their grafts for over 100 days (Fig 1, B). To examine whether donor specific unresponsiveness could be achieved, a second heart from either the same donor strain (C57BL/10) or third party (Balb/c) was transplanted. All of the donor-specific but not the third-party cardiac grafts were accepted in the recipients (median survival time, >100 and 18 days,

respectively; Fig 1, C), which confirmed that the depleting anti-CD4 mAb (YTA 3.1) was able to induce donor-specific unresponsiveness to fully allogeneic cardiac grafts.6 Next, we investigated whether the same protocol could be used to prolong the survival of fully allogeneic thyroid grafts. One lobe of the C57BL/10 thyroid was transplanted under the right kidney capsule of CBA recipients. Grafted thyroid tissue survived under the kidney capsule at 6, 8, and 10 days after transplantation, as assessed by histologic examination (5/5 grafts, 4/5 grafts, and 4/5 grafts, respectively). However, 4 of 5 and all of grafts were rejected by days 12 and 14 after transplantation in

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Niimi et al 913

A

B Fig 2. Histologic examination of the thyroid grafts. CBA recipients were treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. A, One lobe of C57BL/10 thyroid was transplanted in the right kidney capsule of the recipients. B, Some grafts were transplanted into naive syngeneic mice. Grafts were examined histologically 30 days after transplantation.

naive recipients, respectively (Table I). When CBA mice were treated with 100 µg of YTA 3.1 on days –1, 0, and 1 (Fig 1, A), all C57BL/10 thyroid grafts survived for more than 60 days (12/12 grafts on day 30 and 15/15 grafts on day 60; Table I). Histologically, all surviving grafts maintained the structure of thyroid follicle, although moderate leukocyte infiltration was detected in the grafts compared with thyroid tissue grafted in syngeneic recipients (Fig 2). To examine whether donor-specific unresponsiveness had been achieved, second thyroid grafts of either the donor (C57BL/10) or third-party (Balb/c) origin thyroid grafts were transplanted under the left kidney capsule 30 days after transplantation of C57BL/10 thyroid under the right

kidney capsule of CBA recipients that had been treated with YTA 3.1. In contrast with the cardiac grafts, both donor-specific (C57BL/10) and thirdparty (Balb/c) thyroid second grafts were rejected within 30 days. Surprisingly, all of the first C57BL/10 thyroid grafts survived under the right kidney capsule for a further 30 days after the retransplantation (Table II). These data suggested that primary thyroid grafts themselves were modulated after treatment with YTA 3.1 and could survive in the recipients that were still able to mount a rejection response to the first graft. To confirm that the thyroid grafts themselves were modulated, CBA mice that had been treated with YTA 3.1 (100 µg per dose, on days –1, 0, and 1; Fig 1, A), and the thyroid grafts were resected and

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Table I. Survival of fully allogeneic thyroid grafts* Days after transplantation Donor

Recipient

Treatment

C57BL/10 C57BL/10 C57BL/10

C57BL/10 CBA CBA

None None YTA3.1

6

8

10

12

14

30

60

ND 5/5 ND

ND 4/5 ND

ND 4/5 ND

ND 1/5 ND

ND 0/5 ND

ND ND 12/12

5/5 ND 15/15

ND, Not done. *CBA recipients were untreated or treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. One lobe of C57BL/10 thyroid was transplanted in the right kidney capsule of either the CBA recipients or syngeneic C57BL/10 mice. Graft survival was confirmed by histologic examination.

Table II. Survival of the first and second thyroid grafts* Graft 1st C57BL/10 graft in the right kidney capsule 2nd C57BL/10 graft in the left kidney capsule 2nd Balb/c graft in the left kidney capsule

30

60

29/29†

29/29 0/15 0/14

Grafting Grafting

*CBA recipients were treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. One lobe of C57BL/10 thyroid was transplanted in the right kidney capsule of the recipients. Second cardiac grafts from either C57BL/10 or Balb/c mice were transplanted into the left kidney capsule of the recipients 30 days after the first transplantation. †Examined by direct visualization.

Table III. Survival of the retransplanted graft* Days after transplantation Treatment for primary recipient None YTA3.1 alone YTA3.1 + zinc protoporphyrin YTA3.1 + copper protoporphyrin

Primary recipient

15

30

C57BL/10 CBA CBA CBA

0/5 ND ND ND

ND 15/15 4/20 10/10

ND, Not done. *CBA recipients were treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb (100 µg per dose), YTA 3.1, on days –1, 0, and 1 relative to transplantation. One lobe of C57BL/10 thyroid was transplanted in the right kidney capsule of the CBA recipients or naive C57BL/10 mice. Some mice were given additional intraperitoneal treatment of either zinc protoporphyrin (HO-1 inhibitor) or copper protoporphyrin (control) every day for 30 days after the transplantation (30 µmol/L/kg per dose). The thyroid grafts were resected and retransplanted under the kidney capsule of naive CBA mice 30 days after the transplantation.

retransplanted into naive CBA mice 30 days after transplantation. All of the retransplanted grafts survived for 30 days in naive CBA mice; all retransplanted grafts that had survived for 30 days in naive syngeneic C57BL/10 mice were rejected within 15 days (Table III). To examine the role of candidate molecules that might play a role in allowing the grafts to escape from rejection, C57BL/10 thyroid grafts in CBA mice that had been treated with YTA 3.1 were stained for expression of major histocompatibility complex (MHC) class I, MHC class II, Bcl-2, and HO-1. Expression levels of the molecules were compared with those in C57BL/10 thyroid grafts that were transplanted in syngeneic recipients. HO1 was strongly expressed on C57BL/10 thyroid

grafts in CBA mice that had been treated with YTA 3.1 compared with that on control C57BL/10 thyroid grafts in syngeneic recipients. The other 3 molecules showed no significant difference between the 2 groups (Table IV). This result suggested that overexpression of HO1 may augment the ability of the graft to survive when retransplanted to a naive recipient. To confirm the pivotal role of overexpression of HO-1 in allowing graft survival after retransplantation, an inhibitor of HO-1 (zinc protoporphyrin) or control (copper protoporphyrin) that did not inhibit the enzyme was injected intraperitoneally for 30 days after the transplantation of C57BL/10 thyroid grafts into the primary CBA recipients that had been treated with YTA 3.1. The grafts were then

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Surgery Volume 128, Number 6 Table IV. Expression of MHC class I and II, Bcl-2, and HO-1 on the grafts* Recipient Naive C57Bl/10 mice CBA mice treated with YTA 3.1

MHC class I

MHC class II

Bcl-2

HO-1

++ ++

++ ++

+ +

+ ++/+++

Expression levels, –, +, ++, +++, and ++++ (determined by blinded examiners). *CBA recipients were treated intraperitoneally with 100 µg of a depleting anti-CD4 mAb, YTA 3.1, on days –1, 0, and 1 relative to transplantation. One lobe of C57BL/10 thyroid was transplanted in the right kidney capsule of the CBA recipients or naive C57BL/10 mice. Thirty days after the transplantation, expression of MHC class I and II, Bcl-2, and HO-1 on the grafts was examined by histochemistry.

resected and retransplanted into naive CBA mice 30 days later. Most of the grafts in mice that had been treated with zinc protoporphyrin were rejected by naive mice after retransplantation; however, all grafts in mice that had been treated with copper protoporphyrin survived in the secondary recipients (Table III). DISCUSSION CD4 is expressed by MHC class II–restricted T cells.7 It interacts with the β2 domain of the MHC class II molecule present on the antigen-presenting cells during antigen recognition through T-cell receptor.8 Depletion of the CD4+ T-cell subset was first shown to result in skin allograft (full MHC mismatch) prolongation by Cobbold et al9 in 1984. Subsequently perioperative treatment with antiCD4 mAb has been shown to induce the prolonged survival of renal,10,11 cardiac,6 islet,12 and skin allografts.13 The efficacy of a particular anti-CD4 mAb as an immunosuppressive agent in vivo is dependent on its molecular properties, including epitope specificity, affinity, and framework region characteristics.14 First, the effect of anti-CD4 mAb was thought to be dependent on its ability to deplete CD4+ cells. However, in a mouse model, nondepleting anti-CD4 mAb also induced indefinite prolongation of fully allogeneic cardiac grafts,15 which suggests that the elimination of CD4+ cells was not essential to induce graft prolongation. In some studies, CD4 mAb has been shown to induce donor-specific tolerance, as confirmed for cardiac allografts here (Fig 1, C). The short-course treatment with anti-CD4 mAb in our study was also able to induce the prolonged survival of fully allogeneic thyroid grafts, with all of the first thyroid grafts surviving for over 60 days when recipients were treated with the same protocol of anti-CD4 mAb as was the cardiac study (Table I). Interestingly, the ability to reject not only thirdparty but also donor-type first thyroid grafts had recovered by 30 days after the operation (Table II), which suggested that this therapeutic strategy did not lead to donor-specific tolerance, rather the grafts themselves may be modulated to escape from

rejection after anti-CD4 mAb treatment. This hypothesis was confirmed when thyroid graft, which had been accepted in mice that had been treated with anti-CD4 mAb, were retransplanted into naive CBA recipients (Table III). One possibility to explain why the thyroid grafts escaped from rejection was that the immunogenicity of grafts decreased after anti-CD4 treatment. However, this did not appear to be the case because the expression of MHC class I and II on the grafts transplanted in allogeneic mice that had been treated with anti-CD4 mAb was the same as that on grafts transplanted in syngeneic mice (Table IV). An alternative explanation for this observation was that the grafts were “protected” from rejection by the induced expression of a protection molecules.16 HO-1 was one of the potential candidates of protective molecules. We found that HO-1 was overexpressed in the grafts in mice that had been treated with anti-CD4 mAb compared with the level of expression in syngeneic transplanted graft (Table IV). Moreover, the grafts in mice that had been treated with zinc protoporphyrin, but not copper protoporphyrin, were rejected when retransplanted to naive recipients (Table III). However, because 4 of 20 grafts survived in naive recipients even when the anti-CD4 mAb protocol was combined with an intraperitoneal injection of zinc protoporphyrin, overexpression of HO-1 by the grafts may not be the only mechanism involved in graft survival (Table III). In the field of xenotransplantation, activation of complement by antigraft antibodies at the graft itself is implicated in the pathogenesis of organ graft rejection.16 However, in some situations, a graft can survive in the presence of both antigraft antibodies and complement, a situation referred to as “accommodation.” To explain accommodation, some researchers hypothesize that the graft “protects” itself from the reactions elicited by antibodies and complement, which normally leads to rejection. They found that HO-1,17 Bcl-2,18 and A2019 were overexpressed in “accommodated” xenografts. They have also shown that accommodation cannot be induced in HO-1 knockout mice.17

916 Niimi et al

HO degrades heme and generates bilirubin, free iron (Fe2+), and carbon monoxide. Bilirubin is a potent antioxidant; free iron upregulates the transcription of the cytoprotective gene, ferritin, and carbon monoxide is thought to be essential in the regulation of vascular relaxation in a manner similar to nitric oxide. HO exists in 2 forms: HO-1 and HO-2. A third isozyme with relatively lower catalytic activity, HO-3, has been described recently. HO-1, also known as heat shock protein 32, is induced by stressors that include cytokines, the intake of heavy metals, hypoxia, and oxygen free radicals that result from a variety of disease conditions, including ischemia reperfusion. By contrast, HO-2 is constitutive; it is abundant in the brain, the testis, and the unstimulated liver. The differing regulation of the 2 isozymes is caused by their respective promoter regions. Thyroid stroma is exceptionally rich in blood vessels that form extensive capillary plexuses, lying in close proximity to the follicular basement membrane. HO-1 is expressed on these capillary vessels and thyroid follicular cells.20 The reason that the overexpression of HO-1 induced unresponsiveness to alloantigen is not clear in our study. Some investigators have also shown that the overexpression of HO-1 correlates with the suppression of immune responses in vitro and in vivo. Woo et al21 showed that upregulation of HO-1 by cobalt-protoporphyrin suppressed T-cell–mediated and natural killer–cell– mediated cytotoxicity in vitro. Hancock et al 22 demonstrated that vascular expression of the “protective” genes HO-1, Bcl-xL, and A20 correlated with freedom from chronic rejection in mouse cardiac grafts induced by the blockade of CD4 or CD40-ligand plus donor cells. DeBruyne et al 23 suggested that HLA class I sequences increased HO-1 activity and extended survival of nonvascularized heart grafts. The reason that anti-CD4 mAb can induce overexpression of HO-1 in the thyroid grafts is also unclear. Both cardiac and thyroid grafts survived after anti-CD4 mAb treatment. However, moderate leukocyte infiltration was detected in the thyroid grafts, but not cardiac grafts, which suggests that thyroid grafts suffered from the stress of rejection, which may induce the overexpression of HO-1.24,25 In contrast to the thyroid grafts, 3 doses of antiCD4 mAb treatment may be able to suppress the immune response to cardiac grafts because cardiac grafts are less immunogenic than thyroid grafts. Therefore, we propose that some immunogenic or nonimmunogenic stress against the graft upregu-

Surgery December 2000

lates the expression of HO-1 on the thyroid grafts, which may protect grafts from rejection in primary and secondary hosts. Allogeneic thyroid transplantation will not be introduced into clinical practice until new strategies to prevent rejection are identified. Donor-specific tolerance is a holy grail of transplantation and one of the major goals in transplant immunology. Anti-CD4 mAb is a candidate to induce this condition. In this study, we have demonstrated a new mechanism by which anti-CD4 mAb may allow thyroid grafts to escape from graft rejection by inducing the overexpression of HO-1. This observation may be applicable for clinical endocrine grafts with the gene transfer of HO-1.

REFERENCES 1. Brinker KR, Dickerman RM, Gonwa TW, Hull AR, Langley JW, Long DY, et al. A randomized trial comparing doubledrug and triple drug therapy in primary cadaveric renal transplants. Transplantation 1990;50:43-9. 2. London NJ, Farmery SM, Will EJ, Davison AM, Lodge JPA. Risk of neoplasia in renal transplant patients. Lancet 1995;346:403-6. 3. Cobbold SP, Martin G, Qin S, Waldmann H. Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 1986;323:164-6. 4. Niimi M, Hara M, Bushell A, Madsen JC, Morris PJ, Wood KJ. Results of heart transplantation in mice. In: Timmermann W, Gassel H-J, Ulrichs K, Zhong R,Thiede A, editors, Organ transplantation in rats and mice. Berlin: Springer; 1998. p. 637-47. 5. Superina RA, Peugh WN, Wood KJ, Morris PJ. Assessment of primarily vascularized cardiac allografts in mice. Transplantation 1986;42:226-7. 6. Madsen JC, Peugh WN, Wood KJ, Morris PJ. The effect of anti-L3T4 monoclonal antibody treatment on first-set rejection of murine cardiac allografts. Transplantation 1987;44:849-52. 7. Swain SL. T cell subsets and the recognition of MHC class. Immunol Rev 1983;74:129-42. 8. Cammarota G, Scheirle A, Takacs B, Doran DM, Knorr R, Bannwarth W, et al. Identification of a CD4 binding site on the *2 domain of HLA-DR molecules. Nature 1992;356:799801. 9. Cobbold SP, Jayasuriya A, Nash A, Prospero TD, Waldmann H. Therapy with monoclonal antibodies by elimination of Tcell subsets in vivo. Nature 1984;312:548-51. 10. Cosimi AB, Delmonico FL, Wright KJ, Wee SL, Preffer FI, Jolliffe LK, et al. Prolonged survival of nonhuman primate renal allograft recipients treated with only anti-CD4 monoclonal antibody. Surgery 1990;108:406-13. 11. Wood KJ, Pearson TC, Darby C, Morris PJ. CD4: a potential target molecule for immunosuppressive therapy and tolerance induction. Transplant Rev 1991;5:150-64. 12. Shizuru JA, Gregory AK, Chao CTB, Fathman CG. Islet allograft survival after a single course of treatment of recipients with antibody to L3T4. Science 1987;237:278-80. 13. Cobbold S, Waldmann H. Skin allograft rejection by L3T4+ and LYT-2+ T cell subsets. Transplantation 1986;41:634-9.

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14. Waldmann H. Manipulation of T-cell responses with monoclonal antibodies. Annu Rev Immunol 1989;7:407-44. 15. Darby CR, Morris PJ, Wood KJ. Evidence that long-term cardiac allograft survival induced by anti-CD4 monoclonal antibody does not require depletion of CD4+ T cells. Transplantation 1992;54:483-90. 16. Bach FH, Ferran C, Hechenleitner P, Mark W, Koyamada N, Miyatake T, et al. Accommodation of vascularized xenografts: Expression of “protective gene” by donor endothelial cells in a host TH2 cytokine environment. Nature Med 1997;3:196-204. 17. Soares MP, Lin Y, Anrather J, Csizmadia E, Takigami K, Sato K, et al. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med 1998;4:1073-7. 18. Badrichani AZ, Stroka DM, Bilbao G, Curiel DT, Bach FH, Ferran C. Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-kappaB. J Clin Invest 1999;103:543-53. 19. Grey ST, Arvelo MB, Hasenkamp W, Bach FH, Ferran C. A20 inhibits cytokine-induced apoptosis and nuclear factor kappaB- dependent gene activation in islets. J Exp Med 1999;190:1135-46. 20. Suematsu M, Ishimura Y. The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function. Hepatology 2000;31:3-6. 21. Woo J, Iyer S, Cornejo MC, Mori N, Gao L, Sipos I, et al. Stress protein-induced immunosuppression: inhibition of cellular immune effector functions following overexpression of haem oxygenase (HSP 32). Transpl Immunol 1998;6:84-93. 22. Hancock WW, Buelow R, Sayegh MH, Turka LA. Antibodyinduced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat Med 1998;4:1392-6. 23. DeBruyne LA, Magee JC, Buelow R, Bromberg JS. Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival. Transplantation 2000; 69:120-8. 24. Keyse SM, Applegate LA, Tromvoukis Y, Tyrrell RM. Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts. Mol Cell Biol 1990;10:4967-9.

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25. Applegate LA, Luscher P, Tyrrell RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res 1991;51:974-8.

DISCUSSION Dr Collin Weber (Atlanta, Ga). What mechanisms do you think might be operative in the thyroid? Dr Niimi. Thyroid is more immunogenic than cardiac grafts. Anti-CD4 mAb can suppress the immune response against thyroid grafts, but not completely. Therefore, we can detect some inflammation in thyroid grafts. The inflammation may upregulate HO-1. Dr Weber. Have you tried culturing thyroid cells from these mice and adding the antibody to them? Dr Niimi. No. Dr Weber. Are you suggesting a direct effect of the antibody on the donor tissue or the necessity of having a host in some in vivo third-party reaction? Dr Niimi. I think that antibody may protect grafts from rejection. Dr Norman Thompson (Ann Arbor, Mich). Have you tried an allogeneic parathyroid tissue? Dr Niimi. I tried a human parathyroid. This is a thyroid study, but I transplanted a human parathyroid into the same mice. Dr Michael Demeure (Milwaukee, Wis). Can you speculate a little on a mechanism for how an anti-CD4 antibody would increase your HO-1? Dr Niimi. The anti-CD4 is not sufficient to stop the immunogenic response. The stress induced HO-1. Dr John Farndon (Bristol, UK). Human thyroid tissue often contains quite a bit of lymphoid tissue. Do the mice thyroids have any lymphoid tissue? Could that account for the increase? Dr Niimi. Mice thyroid also has lymphoid tissue. After transplantation, we can see some lymphocyte infiltration. I am not sure whether this accounts for the increase.