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Induction of Regulatory CD4� Cells and Prolongation of Survival ofully Allogeneic Murine Cardiac Grafts by Danazol

M. Uchiyama, X. Jin, Q. Zhang, A. Amano, T. Watanabe, and M. Niimi

ABSTRACT

Danazol, a modified testosterone, has been used to treat endometriosis and pretreatmentbefore in vitro fertilization and embryo transfer, although its reproductive mechanismsremain unclear. We investigated the effect of danazol on alloimmune responses in murineheart transplantation. CBA male mice (H2k) that underwent transplantation of C57BL/6(B6, H2b) hearts received danazol (0.4 and 4 mg/kg/d) by intraperitoneal injection from theday of transplantation to days 6. We performed an adoptive transfer study to determineregulatory cells as well as cell proliferation, cytokine, and flow cytometry assessments.Danazol-treated (4 mg/kg/d) CBA mice showed prolonged allograft survival (mediansurvival time [MST], 63 days). Moreover, secondary CBA recipients of whole splenocytesand CD4� cells from primary danazol-treated (4 mg/kg/d) CBA recipients at 30 days aftertransplantation displayed prolonged allograft survival (MSTs, 29 and 60 days, respec-tively). Cell proliferation, interleukin (IL)-2, and interferon-� were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directlysuppressed alloproliferation in mixed leukocyte cultures. Flow cytometry studies showedan increased CD4�CD25�Foxp3� cell population among splenocytes from danazol-treated mice. In conclusion, danazol induced prolonged cardiac allograft survival and

generation of regulatory CD4� cells.

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DANAZOL, A 17-ETHINYLTESTOSTERONE DE-RIVATIVE, has been widely used as a therapeutic

agent for endometriosis. Recently, a new formulation ofdanazol has been developed for first-line therapy in patientswith aplastic anemia.1,2 Additionally, danazol exerts inhib-itory effects on the growth of mammary tumors in rats.3

Although these has been considerable research on danazoltherapy in gynecology and hematology, its use in thetransplantation field, remains controversial, and little isknown about its effects on alloimmune responses. Thecurrent study investigated the impact of danazol on murinecardiac transplantation.

METHODSHeart Transplantation and in Vitro Study

Hearts from male C57BL/6 (H2b) were transplanted into maleBA (H2k) recipients as described previously.4

Treatment With Danazol

Transplant CBA recipients were given one dose of 0.4 or 4 mg/kg/dof danazol (Mitsubishi Pharma, Osaka, Japan) by intraperitoneal

injection from the day of to 6 days after transplantation. Danazol

© 2012 by Elsevier Inc. All rights reserved.360 Park Avenue South, New York, NY 10010-1710

Transplantation Proceedings, 44, 1067–1069 (2012)

(0.4 and 4 mg) was first dissolved in saline (39 mL) plus ethanol (1mL; Kanto Chemical, Tokyo, Japan). Transplant recipients incontrol groups were given either no injections (no treatmentgroup) or intraperitoneal injections of normal saline with orwithout ethanol.

Adoptive Transfer Studies

Adoptive transfer studies were performed to determine whetherregulatory cells were generated in danazol-treated mice. Thirtydays after transplantation of B6 hearts in primary CBA recipientstreated with danazol for 7 days after grafting yielded splenocytes(5.0 � 107) from hosts with functioning allografts. They weredoptively transferred into naïve secondary CBA recipients by

From the Department of Cardiovascular Surgery (M.U., A.A.),Juntendo University Hospital, Tokyo, Japan; Department ofSurgery (M.U., X.J., Q.Z., T.W., M.N.), Teikyo University, Tokyo,Japan; and Department of Cardiovascular and Thoracic Surgery(X.J.), The 4th Affiliated Hospital to Harbin Medical University,Harbin, China.

Address reprint requests to Masanori Niimi, MD, PhD, Depart-ment of Surgery, Teikyo University, 2-11-1 Kaga, Itabashi-ku,

Tokyo 173-8605, Japan. E-mail: mniimi@medikyo-u.ac.jp

0041-1345/–see front matterdoi:10.1016/j.transproceed.2012.01.103

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1068 UCHIYAMA, JIN, ZHANG ET AL

intravenous injection into the penile vein. Each secondary recipientunderwent transplantation of a B6 or BALB/c heart immediatelyafter completion of the adoptive transfer. In some experiments,CD4� cells were purified from the spleens of primary transplantrecipients by positive selection using a magnetically activated cellsorter and CD4 microbeads (Miltenyi Biotec, Auburn, Calif, USA;purity � 98%). CD4� (2.0 � 107) cells were adoptively transferredinto naïve secondary recipients, which immediately underwenttransplantation of a B6 heart.

Flow Cytometry Analysis of CD4, CD25, andFoxp3 Expression

Splenocytes were obtained from naïve CBA mice and fromdanazol-treated and untreated cardiac allograft transplant re-cipients at 4 weeks after transplantation. The cells were stainedwith fluorochrome-conjugated anti-CD4 or anti-CD25 mAb(RM4–5 and PC61, respectively; BD Biosciences) or anti-mouseFoxp3 mAb (FJK-16s; eBioscience, San Diego, Calif, USA), as wellas their isotype controls (eBioscience). The stained cells wereanalyzed using a FACS Canto2 system (BD Biosciences). Wedetermined the percentage of CD4�CD25�Foxp3� in CD4� cells.

Statistical Analysis

Cardiac allograft survival was compared in two experimentalgroups by log-rank tests. For cell proliferation, cytokine, and flowcytometry studies, differences between two groups were assessedusing unpaired Student t tests. A P value � .05 was consideredsignificant.

RESULTSSurvival of Fully Mismatched Cardiac Allografts in MiceTreated With Danazol

CBA recipients of B6 cardiac allografts that were given 0.4mg/kg/d danazol or no treatment, or intraperitoneal injec-tions of normal saline with or without ethanol rejected theirgrafts acutely: median survival times (MSTs) of 9, 7, 9 and7 days respectively (Table 1). In contrast, CBA allograftrecipients treated with danazol (4 mg/kg/d) for 7 daysshowed significantly prolonged survival of B6 grafts (MST,63 days; Table 1).

Generation of Regulatory Cells in Mice TreatedWith Danazol

We previously observed that some anti-inflammatory orimmunomodulatory agents induce hyporesponsiveness tofully allogeneic grafts by generation of regulatory cells.5–7

Table 1. Cardiac Allograft Survival in Control Mice andDfanazol-Treated Mice

Group n Individual STs (d) MST (d)

No treatment 5 6, 7, 7, 7, 8 7Normal saline with ethanol 5 8, 8, 9, 10, 22 9Normal saline without ethanol 5 6, 7, 7, 7, 7 7Danazol

4 mg/kg/d for 7 d 5 36, 42, 63, 63, 100� 630.4 mg/kg/d for 7 d 5 7, 7, 9, 10, 100� 9

STs, allograft survival times; MST, median allograft survival time.

In the present investigation, we performed adoptive trans-fer studies to determine whether generation of regulatorycells was involved in the induction of hyporesponsiveness indanazol-treated mice. We noted that compared with trans-fer of splenocytes and CD4� cells from naïve CBA mice

aïve secondary CBA allograft recipients given an adoptiveransfer of splenocytes (5 � 107) and CD4� (2 � 107) cellsrom danazol-treated primary CBA recipients at 30 daysfter transplantation showed significantly prolonged sur-ival of B6 hearts (MSTs, 29 and 60 days respectively; P �05 and P � .005; Table 2). In contrast, naïve secondaryBA recipients adoptively transferred splenocytes andD4� cells from naïve CBA mice rejected C57BL/6 heartscutely (MSTs, 10 and 8 days respectively; Table 2). More-ver, when whole splenocytes from danazol-treated primaryBA transplant recipients with functioning B6 allograftsere adoptively transferred into naïve secondary CBA

ecipients that immediately underwent transplantation of aALB/c heart, the BALB/c allografts were rejected acutely

MST, 7 days). These data indicated that danazol treatmentenerated regulatory cells in primary allograft recipientsnd that one of the regulatory populations consisted ofD4� cells, which seemed to be donor specific.Flow cytometry studies showed that the population of

D4�CD25�Foxp3� cells among the CD4� cells was in-creased in the spleens of danazol-treated compared with notreatment CBA mice at 4 weeks after transplantation:11.99% � 0.47% versus 6.43% � 0.14% (P � .001).

Cell Proliferation and Cytokine Production in Mice TreatedWith Danazol

Proliferation of splenocytes from transplanted CBA recip-ients treated with danazol was markedly suppressed com-pared with that of those from untreated recipients: mean0.517 optical density (OD) � standard error of the mean0.194 versus 0.795 OD � 0.197 (P � .05). Moreover, theddition of danazol to an allogeneic MLC inhibited prolif-ration of CBA responder cells against B6 stimulator cellsn a dose-dependent manner. Compared with untreatedBA mice the levels of interleukin (IL)-4 and IL-10 were

ignificantly increased in splenocytes from danazol-treatedice: 1.59 � 0.10 ng/mL versus 1.19 � 0.05 ng/mL, and

8.84 � 11.10 ng/mL versus 17.69 � 1.37 ng/mL (P � .001and P � .05 respectively). In contrast compared withuntreated CBA mice the levels of IL-2 and interferon-�were considerably decreased: 2.29 � 0.48 ng/mL versus.79 � 2.04 ng/mL and 42.49 � 4.14 ng/mL versus 83.60 �0.76 ng/mL (P � .001 and P � .001 respectively).

DISCUSSION

In our murine model, 1 week of danazol treatment signifi-cantly prolonged the survival of fully mismatched cardiacallografts. The treatment also generated CD4� regulatorycells in allograft recipients. These cells displayed suppres-sive activity in MLC. Furthermore, IL-2 and interferon-�

were suppressed whereas IL-4 and IL-10 were up-regulated

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in danazol-treated hosts. Flow cytometry studies showed anincreased CD4�CD25�Foxp3� cell population in spleno-ytes.

There are several possible mechanisms by which treat-ent with danazol might have increased allograft survival in

ur model: first, danazol might have prolonged allografturvival by generation of regulatory cells. In adoptiveransfer studies, naïve secondary CBA transplant recipientsiven whole splenocytes and CD4� cells from danazol-reated primary CBA recipients with functioning B6 cardiacllografts displayed significantly prolonged survival of B6ardiac grafts.

A second possible mechanism of danazol-induced hypo-esponsiveness is the nature of danazol as a syntheticnabolic steroid with unique properties similar to cortico-teroids. Danazol has a corticosteroid-sparing effect, in-reasing platelet counts even among patients refractory tother therapeutic approaches. In this sense, danazol haseen used successfully in autoimmune diseases such as

diopathic thrombocytopenic purpura.8 In our study, dana-ol inhibited alloproliferation in a dose-dependent mannern MLC. These results indicated that danazol may inhibitctivation of alloreactive T cells directly, either throughltering the balance of Th-1/Th-2 cytokines or other un-nown mechanisms.A third possible mechanism is the possibility of function

s a sex hormone. As a modified testosterone, danazol hasproperty similar to testosterone that is hypoestrogenemic.

Table 2. Cardiac Allograft Survival After Adoptive Transfer ofWhole Splenocytes or CD4� Cells

Group n Individual STs (d) MST (d)

Whole splenocytes fromprimary recipients

5 9, 13, 29, �100, �100 29

D4� cells from primaryrecipients

5 31, 46, 60, �100, �100 60

ControlsWhole splenocytes

from naïve CBA6 8, 8, 8, 12, 14, 14 10

CD4� from naïve CBA 5 7, 7, 8, 8, 9 8

STs, allograft survival times; MST, median allograft survival time.

any studies have related to testosterone as a sex hormone.mr

n particular, two previous studies reported testosterone torolong skin graft survival in rats9 and as a novel factor in

the differentiation of regulatory T cells.10 In our currenttudy, danazol may indirectly function as testosterone,nducing prolonged allograft survival and generation ofegulatory CD4� cells.

In conclusion, treatment with danazol induced prolongedallograft survival and the generation of regulatory CD4�

cells, enabled inhibition of alloimmune responses directly,and functioned indirectly much like testosterone.

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