Administration of Thrombomodulin (CD141) Could Improve Cardiac Allograft Survival in Mice

Thrombomodulin (TM) is a promising natural anti-coagulant therapeutic protein that is effective in the treatment of disseminated intravascular coagulation. However, the mechanisms by which TM on micro-vessels enable the regulation of intimal hyperplasia remain elusive. We investigated the graft-protective effects of TM in a fully major histocompatibility complex-mismatched murine cardiac allograft transplantation model. CBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of 0.2, 2.0, and 20.0 μg/day of TM for 8 days. Histological staining was conducted to assess the degree of inflammation and infiltration in the transplanted cardiac grafts. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] was 7 days). CBA recipients exposed to the above dosages had significantly prolonged allograft survival (MSTs were 16, 21, and 37.5 days, respectively). Histologic assessments from TM-exposed recipients 2 weeks after grafting showed that the myocardium and vessel structure in their allografts were clearly preserved, and that the infiltration of inflammatory cells around coronary arteries was suppressed. TM can induce the prolongation of fully major histocompatibility complex-mismatched cardiac allograft by exerting graft protective effects within the myocardium and coronary arteries.     

Seven Japanese Herbals Prolonged Cardiac Allograft Survival

Japanese herbal medicines have long been used as alternative therapy because of their immunomodulatory effects. In recent years, use herbal medicines is rapidly increasing worldwide. In this study, we investigated the effect of 17 components of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA mice by using microsurgical techniques. Artemisiae capillaris herba (Inchinko) was given to CBA recipients at a dosage of 1 g/kg/day from the day of transplantation until 7 days afterward. The other 16 components were given at a dosage of 2 g/kg/day for the same time period. Naïve CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST] of 7 days). CBA transplant recipients given 2 g/kg/day of Glycyrrhizae radix (Kanzou), Poria sclerotium (Bukuryo), Pinellia tuber (Hange), Cnidii rhizome (Senkyu), Paeoniae radix (Shakuyaku), and Scutellariae radix (Ogon) had prolonged C57BL/6 allograft survival significantly (MSTs were 18, 18, 17, 14, 12, and 12 days, respectively). Moreover, CBA transplant recipients given 1g/kg/day of Artemisiae capillaris herba had prolonged C57BL/6 allograft survival (MST >100 days); however, none of other 10 components prolonged allograft survival. In conclusion, administration of 7 components of traditional Japanese herbal medicines might induce prolongation of fully major histocompatibility complex–mismatched cardiac allografts.     

Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).     

Artemisiae capillaris herba induces prolonged survival of fully cardiac allografts and generates regulatory cells in mice

Inchingorei-san (TJ-117), a 6-component herbal medicine, is used in Japan for the treatment of vomiting, urticaria, and liver and kidney disorders with few side effects. In this study we investigated the effect of TJ-117 on alloimmune responses in murine cardiac allograft transplantation. CBA (H2^k) mice underwent transplantation of a C57BL/6 (B6, H2^b) heart with oral administration of TJ-117 (or 1 component of TJ-117) from the day of transplantation for 7 days. CBA recipients given 1 g/kg/d of TJ-117 showed prolonged B6 allograft survival (median survival time [MST], 23 days). Naive CBA mice rejected B6 cardiac grafts acutely (MST, 7 days). Moreover, Artemisiae capillaris herba (ACH; 1g/kg/d) 1 component of TJ-117, significantly prolonged B6 allograft survival (MST, > 100 days). However, the other 5 components of TJ-117 were individually less effective than TJ-117 or ACH. ACH also suppressed splenocyte proliferation and interferon-γ production. Secondary CBA recipient showed prolonged survival of B6 hearts after treatments with whole splenocytes from primary ACH-treated CBA recipients carrying B6 cardiac allografts for 30 days (MST, >50 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in transplant recipients given ACH. In conclusion, ACH, 1 component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts with generation of CD4⁺CD25⁺Foxp3⁺ regulatory cells.     

胸腺内注射可溶性异基因主要组织相容性复合物抗原诱导皮肤移植特异性耐受

用３ｍｏｌ／ＬＫＣｌ从Ｃ５７ＢＬ／６小鼠脾细胞提取可溶性主要组织相容性复合物（ＭＨＣ）抗原，注射到ＢＡＬＢ／Ｃ受体鼠胸腺内，诱导了成年小鼠对该异基因小鼠皮肤移植物的耐受。除在胸腺注射当天及第３天给予抗Ｔ细胞单克隆抗体外，不使用免疫抑制剂。实验组移植皮肤平均存活时间（ＭＳＴ）为８３天，对照组ＭＳＴ为１１天。诱导耐受的小鼠对第３供体的移植皮肤仍正常排斥（ＭＳＴ为１２天）。单向混合淋巴细胞反应，耐受小鼠脾脏淋巴细胞对特异供体的脾细胞无反应，对第３供体的脾细胞反应正常，对丝裂原刺激的增殖反应正常。显示诱导的耐受是供体特异性的，无非特异性免疫抑制。     

Induction of indefinite survival of fully mismatched cardiac allografts and generation of regulatory cells by sarpogrelate hydrochloride

BACKGROUND: At initiation of the immunologic response, platelets rapidly release chemical mediators such as serotonin (5-hydroxytryptamine, [5-HT]) and cytokines. Sarpogrelate hydrochloride (SH), a selective 5-HT2-receptor antagonist, is used to treat patients with peripheral arterial disease. We investigated the effect of SH on the alloimmune response in a murine cardiac transplantation model. METHODS: CBA mice underwent transplantation of a C57BL/10 heart and received a short course of SH treatment. Survival of the allograft was recorded. An adoptive transfer study was performed to determine whether regulatory cells were generated. Immunohistochemistry studies of intercellular adhesion molecule 1 (ICAM-1), histological, cell-proliferation, and cytokine assessments were performed. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 8 days). In mice given 10 mg/kg of SH, all allografts survived indefinitely (MST, >100 days); these mice also had significantly prolonged survival of donor-specific skin grafts but acute rejection of third-party skin grafts. Secondary CBA recipients given not only whole but also CD4 splenocytes from primary SH-treated CBA recipients with C57BL/10 cardiac allograft had indefinite survival of C57BL/10 hearts (MST, >100 days). SH inhibited upregulation of ICAM-1 on endothelial cells in the allografts. Graft acceptance and hyporesponsiveness were confirmed by the histological and cell-proliferation studies, respectively. Production of interleukin-4 and interleukin-10 from splenocytes of SH-treated transplant recipients increased compared to that from splenocytes of untreated recipients. CONCLUSION: SH induced indefinite survival of fully allogeneic cardiac allografts, generated CD4 regulatory cells, inhibited ICAM-1 expression in the allografts, and upregulated IL-4 and IL-10 production.     

Effect of 34 Kinds of Traditional Japanese Herbal Medicines on Prolongation of Cardiac Allograft Survival

Herbal medicines have been used for over 3,000 years in Asian as alternative therapy for their variety effects and have recently become popular in Europe and the United States. In the last 30 years, Japanese herbal medicines were widely used for treatment of diseases after been recognized officially by Japanese government. In this study, we investigated the effect of 34 kinds of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. CBA mice (H2^k) underwent transplantation of a C57BL/6 (H2^b) heart and received oral administration of 2 g/kg/d of the 34 kinds of herbal medicines from the day of transplantation until 7 days afterward. Naïve CBA mice rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). CBA transplant recipients given 2 g/kg/d of Sairei-to (TJ-114) and Tokishakuyaku-san (TJ-23) had prolonged C57BL/6 allograft survival indefinitely (both MSTs > 100 days). Moreover, CBA transplant recipients given Seisinrensiin (TJ-111), Tokishigyakukagoshuyushokyoto (TJ-38), Rikkunshito (TJ-43), Maobushisaishinto (TJ-127), Ninjin-yoei-to (TJ-108), Ryokan-kyomi-shinge-nin-to (TJ-119), Inchingorei-san (TJ-117), Hochuekkito (TJ-41), Kihi-to (TJ-65), and Sinbu-to (TJ-30) had also prolonged C57BL/6 allograft survival significantly (MSTs of 28, 22, 16, 14, 14, 13, 12, 9.5, 9 and 9 days, respectively). However, none of other 22 kinds of herbal medicines could prolong the allograft survival. Furthermore, oral administration of 2 g/kg/d of Daikenchuto (TJ-100) induced sudden death (within 1 minute) in CBA mice. In conclusion, 12 kinds of Japanese herbal medicines prolonged allograft survival and one showed toxic effect in mice.     

Effects of Glycyrrhizic Acid in Licorice on Prolongation of Murine Cardiac Allograft Survival

Recent evidence has pointed to the promising benefits of using specific immunosuppressive herbal compounds to prolong transplant allograft survival. In this study, we investigated the effects of glycyrrhizic acid (GA), a major component of licorice, in a model of murine heart transplantation. CBA (H2^k) mice were transplanted with a fully-MHC mismatched C57BL/6 (H2^b) heart allograft and subsequently received daily intraperitoneal administration of normal saline or 0.02, 0.2, or 2.0 mg/d of GA for 7 consecutive days. Untreated CBA recipients, with a median survival time (MST) of 7 days, and groups receiving 0.02mg/d (MST, 8 days) or 0.2mg/d (MST, 9 days) of GA acutely rejected C57BL/6 cardiac allografts. But mice treated with 2.0 mg/d of GA demonstrated significant prolongation of allografts (MST, 23 days). Histologic studies showed that cardiac allografts from GA-treated CBA recipients had preserved graft and vessel structure. Moreover, flow cytometric study showed that the percentage of CD4⁺CD25⁺Foxp3⁺ cell (regulatory T cell [Treg]) populations were increased in GA-treated CBA recipients. In a mixed leukocyte culture, splenocytes from GA-treated mice demonstrated suppressed allo-proliferation, in which interleukin (IL)-2 and interferon gamma production were downregulated and IL-10 secretion was upregulated. In conclusion, GA may be a novel promising therapeutic agent to prolong cardiac allograft survival through direct anti-inflammatory effects and induction of Treg populations.     

Administration of donor splenocytes via the respiratory tract generates CD8α+ regulatory dendritic cells and induces hyporesponsiveness to fully allogeneic cardiac grafts

BackgroundWe previously showed that pretreatment with intratracheal delivery (ITD) of alloantigen induced prolonged cardiac allograft survival and generated regulatory T cells (Tregs) in mice. In this study, we examined the role of splenic dendritic cells (DCs) in the ITD model.MethodsCBA mice were treated with ITD from C57BL/10 splenocytes and 7 days later received transplantation of C57BL/10 hearts. In adoptive transfer studies, splenic DCs from ITD-treated mice were transferred into naïve CBA recipients that received C57BL/10 hearts immediately after the transfer. In addition, to determine the role of splenic DCs isolated from ITD-treated mice, the cells were incubated under stimulation with lipopolysaccharide (LPS).ResultsITD-treated CBA recipients had markedly prolonged allograft survival (median survival time [MST], 67 days) while naïve recipients rejected allografts acutely (MST, 8 days). In adoptive transfer studies, CBA recipients of the transfer of splenic DCs from ITD-treated mice had prolonged allograft survival (MST, 85 days), while CBA recipients of the transfer of splenic DCs from naïve mice did not have prolonged allograft survival (MST, 8 days). In another transfer study, CBA recipients of the transfer of splenic CD8α⁺ DCs from ITD-treated mice had prolonged allograft survival (MST, 79 days), while those receiving splenic CD8α⁻ DCs from ITD-treated mice did not have prolonged allograft survival (MST, 8 days). In vitro studies showed that ITD-treated splenic DCs produced more IL-10 and less IL-12 than naïve splenic DCs under stimulation with LPS.ConclusionsITD pretreatment induces regulatory DCs, which produce high amounts of IL-10 resulting in the prolongation of graft survival in our model.     

Effects of immunosuppressants on induction of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery (ITD) of alloantigen generated regulatory cells in mice. Here, we examined the effect of various doses of conventional immunosuppressants (FK506, cyclosporine A, azathioprine, mycophenolate mofetil, and rapamycin) on inducing regulatory cells in our model. METHODS: CBA mice (primary recipients) were given C57BL/6 splenocytes by ITD and either no additional treatment or various doses of an immunosuppressant. Seven days later, splenocytes from these mice were adoptively transferred into naive secondary CBA recipients that underwent C57BL/6 cardiac grafting the same day. RESULTS: Adoptive transfer from primary recipients given ITD of splenocytes alone induced prolonged allograft survival in secondary recipients (median survival time [MST], 50 days), suggesting that regulatory cells were generated. When ITD of alloantigen was combined with daily administration of 0.1 mg/kg FK506 or 0.2 mg/kg rapamycin, graft survival was similarly prolonged (MST 55 and 50 days, respectively). When combined with 20 or 40 mg/kg MMF or 0.4 mg/kg rapamycin, the majority of recipients demonstrated indefinite survival (MST, >100 days in all groups). When ITD of alloantigen was combined with 0.3, 0.5, or 1.0 mg/kg FK506; 5, 10, or 25 mg/kg cyclosporine A; or 1.0 or 2.0 mg/kg azathioprine, allografts were rejected acutely (MST 7-13 days). CONCLUSION: Generation of regulatory cells by ITD of alloantigen was facilitated by mycophenolate mofetil and high doses of rapamycin but abrogated by cyclosporine A, azathioprine, and high doses of FK506. Low doses of rapamycin and of FK506 did not interfere with generation of regulatory cells.     

Induction of Regulatory CD4+ Cells and Prolongation of Fully Major Histocompatibility Complex Mismatched Murine Cardiac Allograft by Shigyakusan

Shigyakusan (also known as Tsumura Japan [TJ]-35) is composed of peony, bitter orange, licorice, and Bupleuri radix is used for cholecystitis and gastritis as an anti-inflammatory agent. We investigated the effect of TJ-35 on alloimmune response in a murine heart transplantation model. CBA mice that underwent transplantation of a C57BL/6 (B6) heart were assigned to four groups: no treatment, TJ-35–exposed, each component–exposed, or each component missing–exposed. The four groups above each received oral administration of TJ-35, each component, or TJ-35 with each component missing from the day of transplantation until 7 days, respectively. Untreated CBA recipients rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). TJ-35–exposed CBA recipients had significantly prolonged B6 allograft survival (MST, 20.5 days). However, MSTs of CBA recipients that had been administered each component and TJ-35 with each component missing did not reach that of TJ-35–exposed recipients. Adoptive transfer of CD4⁺ splenocytes from TJ-35–exposed primary allograft recipients resulted in significant prolonged allograft survival in naïve secondary recipients (MST, 63 days). Flow cytometry studies showed that the percentage of CD4⁺CD25⁺Foxp3⁺ cell population was increased in TJ-35–exposed CBA recipients. In conclusion, TJ-35–induced prolongation of fully allogeneic cardiac allografts and may generate regulatory CD4⁺CD25⁺Foxp3⁺ cells in our model. The effect seemed to require all components of TJ-35.     

Induction of indefinite survival of fully allogeneic cardiac grafts and generation of regulatory cells by intratracheal delivery of alloantigens under blockade of the CD40 pathway

BACKGROUND: The authors previously showed that intratracheal delivery (ITD) of donor splenocytes induced prolonged survival of fully allogeneic cardiac grafts in mice. In this study, this treatment protocol was combined with blockade of the CD40 pathway in an attempt to induce operational tolerance. METHODS: CBA mice were given donor splenocytes (1x107) or Kb peptide (100 microg) by ITD with or without antibody specific for mouse CD40 ligand (MR1, 200 microg) 7 days before transplantation of a C57BL/10 heart. Also, splenocyte (5 x 107) from primary recipient CBA mice given ITD of donor splenocytes or Kb peptide plus MR1 were adoptively transferred into naive CBA secondary recipients 7 days after the pretreatment and C57BL/10 hearts were transplanted into those recipients the same day. RESULTS: ITD of donor splenocytes and Kb peptide induced prolonged survival of cardiac grafts (median survival time [MST], 74 and 56 days, respectively), whereas naive control mice and mice pretreated with syngeneic splenocytes had acute graft rejection (MST in both groups, 7 days). When MR1 was included, all grafts survived indefinitely (>200 days), but mice pretreated with MR1 alone had graft rejection (MST, 54 days). Mice bearing cardiac grafts had acceptance of skin grafts from C57BL/10 but not BALB/c mice, demonstrating that operational tolerance was induced. Secondary recipients given adoptive transfer of splenocytes from primary recipients of the combined treatment had acceptance of C57BL/10 grafts, suggesting that regulatory cells were generated within 7 days of pretreatment. CONCLUSIONS: ITD of donor splenocytes or Kb peptide under blockade of the CD40 pathway induced operational tolerance and generated regulatory cells.     

Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen

BACKGROUND: We previously reported that intratracheal delivery of alloantigen-induced regulatory cells in mouse heart-transplantation model. Here, we investigated roles of interleukin (IL)-10 and transforming growth factor (TGF)-beta in induction and effector phases of the regulatory cells. METHODS: CBA mice were pretreated with intratracheal delivery of C57BL/10 splenocytes and administration of neutralizing anti-IL-10 or anti-TGF-beta monoclonal antibody (mAb). Seven days after the pretreatment, naive CBA mice (secondary recipients) were given adoptive transfer of splenocytes from the pretreated mice and underwent heart grafting from C57BL/10 mice. To determine roles of these cytokines in the effector phase of the regulatory cells, anti-IL-10 or anti-TGF-beta mAb was administered weekly into the secondary recipients after the adoptive transfer. RESULTS: Adoptive transfer of splenocytes from CBA mice that had been pretreated with intratracheal delivery of C57BL/10 splenocytes significantly prolonged the survival of C57BL/10 allograft (median survival time [MST] 68 days) as compared with adoptive transfer from untreated CBA mice (MST 12 days). In the induction phase, anti-IL-10 mAb abrogated development of the regulatory cells that afforded prolonged allograft survival in the secondary recipients (MST 20 days), whereas anti-TGF-beta mAb did not abrogate it (MST 88 days). In the effector phase, anti-IL-10 mAb abrogated prolonged allograft survival afforded by adoptive transfer of the regulatory cells in the secondary recipients (MST 27 days), whereas anti-TGF-beta mAb did not abrogate suppressor function of the regulatory cells (MST 53 days). CONCLUSION: IL-10 but not TGF-beta was required for generation and suppressor function of the regulatory cells induced by intratracheal delivery of alloantigen.     

CD27/CD70, CD134/CD134 ligand, and CD30/CD153 pathways are independently essential for generation of regulatory cells after intratracheal delivery of alloantigen

BACKGROUND: We investigated whether blockade of tumor necrosis factor receptor-ligand pathways could generate regulatory cells induced by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of splenocytes (1x10(7)) from C57BL/10 (H-2b) mice and intraperitoneal administration of monoclonal antibody (mAb) specific for CD70, CD134 ligand (CD134L), CD153, or CD137L. Seven days later, C57BL/10 hearts were transplanted into pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes (5x10(7)) from pretreated CBA mice and transplantation of a C57BL/10 heart on the same day. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST] 12 days). Pretreatment with intratracheal delivery of C57BL/10 donor splenocytes prolonged graft survival significantly (MST 84 days). Mice given intratracheal delivery of alloantigen plus anti-CD70, anti-CD134L, or anti-CD153 mAb, but not those given intratracheal delivery of alloantigen plus anti-CD137L mAb, rejected their graft acutely (MST 16, 14, 10, and 65 days, respectively). Adoptive transfer of splenocytes from mice pretreated with intratracheal delivery of alloantigen plus anti-CD70, CD134L, or CD153 mAb did not prolong survival of C57BL/10 cardiac grafts in naive secondary CBA recipients (MST 14, 11, and 11 days, respectively), whereas adoptive transfer of splenocytes from mice given intratracheal delivery of alloantigen plus anti-CD137L mAb did (MST 75 days). CONCLUSION: The CD27/CD70, CD134/CD134L, and CD30/CD153 pathways are independently required for generation of regulatory cells in our model.     

Programmed death-1-programmed death-L1 interaction is essential for induction of regulatory cells by intratracheal delivery of alloantigen

BACKGROUND: Programmed death (PD)-1 has been implicated in peripheral tolerance. The authors investigated the roles of PD-1 and its ligands, PD-L1 and PD-L2, in the induction of regulatory cells by intratracheal delivery of alloantigen. METHODS: CBA (H-2k) mice were pretreated with intratracheal delivery of C57BL/10 (H-2b) splenocytes and administration of monoclonal antibody (mAb) specific for PD-1, PD-L1, or PD-L2. Seven days later, C57BL/10 hearts were transplanted into the pretreated CBA mice. Some naive CBA mice underwent adoptive transfer of splenocytes from the pretreated CBA mice and transplantation of C57BL/10 heart. RESULTS: Untreated CBA mice rejected C57BL/10 cardiac grafts acutely (median survival time [MST], 7 days). Pretreatment with intratracheal delivery of C57BL/10 splenocytes prolonged graft survival significantly (MST, 65 days). Administration of control immunoglobulin (Ig) G or anti-PD-L2 mAb did not significantly affect the prolongation (MST, 72 and 68 days, respectively). In contrast, anti-PD-1 or anti-PD-L1 mAb abrogated the prolongation (MST, 18 and 17 days, respectively). Adoptive transfer from mice pretreated with intratracheal delivery of alloantigen plus control IgG or anti-PD-L2 mAb prolonged survival of C57BL/10 grafts in secondary CBA recipients (MST, 72 and 56 days, respectively). However, concurrent administration of anti-PD-1 or anti-PD-L1 mAb abrogated prolonged survival after the adoptive transfer (MST, 14 and 20 days, respectively). CONCLUSIONS: PD-1-PD-L1 interaction was essential for induction of regulatory cells by intratracheal delivery of alloantigen.     

The smell of Tokishakuyaku-san (TJ-23) induces generation of regulatory T cells and prolongation of survival of fully allogeneic cardiac grafts in mice

Oral administration of Tokishakuyaku-san (TJ-23), a Japanese herbal medicine, induces prolongation of cardiac allograft survival and generates regulatory cells in mice. Because herbal medicines usually have unique odor, and because smell is supposed to modulate the immune system, we examined whether the odor of TJ-23 induced prolonged allograft survival and regulatory cell generation. Naïve CBA mice (H2^k) and olfactory-dysfunctional CBA mice after a stereotaxic operation underwent transplantation of C57BL/6 (B6, H2^b) hearts, receiving fumigated water only or TJ-23 until rejection. Untreated or treated with water fumigation CBA mice rejected B6 cardiac grafts acutely (median survival times [MSTs], 7 and 8.5 days). When CBA mice were treated with fumigation of TJ-23, allograft survival was significantly prolonged (MST, 48 days). Olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 rejected grafts acutely (MST, 7 days). Treatment with fumigation of TJ-23 also suppressed splenocytes proliferation and interferon-γ production. Secondary CBA recipients of whole splenocytes or CD4⁺ cells from primary TJ-23-treated CBA recipients of B6 cardiac allografts at 30 days after grafting showed prolonged survival of B6 hearts (MST, >60 days). Flow cytometry studies showed increased CD4⁺CD25⁺Foxp3⁺ regulatory cells in recipients given fumigation of TJ-23. In conclusion naïve but not olfactory-dysfunctional CBA mice treated with fumigation of TJ-23 displayed prolonged survival of fully allogeneic cardiac allografts and generation of regulatory cells.     

Danazol induces prolonged survival of fully allogeneic cardiac grafts and maintains the generation of regulatory CD4(+) cells in mice

Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2(k) ) underwent transplantation of C57BL/6 male (H2(b) ) hearts and received a single dose of danazol (0.4, 1.2 or 4mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4mg/kg/day) mice was 28 and 63days, respectively, compared with 7days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4(+) cells from primary danazol-treated (4mg/kg/day) CBA recipients 30days after transplantation had prolonged allograft survival (MSTs, 29 and 60days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4(+) CD25(+) Foxp3(+) cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4(+) cells.     

Effects of Each Domain in Recombinant Human Soluble Thrombomodulin on Prolongation of Murine Cardiac Allograft Survival

Thrombomodulin is used to manage disseminated intravascular coagulation. In our murine heart transplantation model, the administration of recombinant human soluble thrombomodulin (rTM) could induce the prolongation of cardiac allograft survival. However, there are limited data on the graft protective effects of each r domain (D1, D2, and D3). In this study, we investigated the effects of each domain of rTM on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients using microsurgical techniques. CBA mice that underwent transplantation of C57BL/6 cardiac allografts were assigned to 4 groups: no treatment and each domain-exposed group. The dosage of each domain was determined based on our previous experiments. Flow cytometry and histologic studies were performed to determine whether Foxp3⁺ regulatory T cells were generated. Untreated and D2-exposed CBA recipients acutely rejected C57BL/6 cardiac allografts within 9 days. Administration of D3 resulted in modest prolongation of allograft survival, and administration of D1 significantly prolonged allograft survival. Histologic studies showed that myocardial damage of allografts from D1- and D3-exposed CBA recipients was controlled compared with that of untreated recipients. In particular, the CD4⁺CD25⁺Foxp3⁺ cell population in the splenocytes of D1-exposed CBA recipients was increased. In conclusion, D1 in rTM could help prolong cardiac allograft survival through regulatory T cell induction and graft protective effects.     

Role of the CTLA4 pathway in hyporesponsiveness induced by intratracheal delivery of alloantigen

BACKGROUND: The authors previously reported that intratracheal delivery (ITD) of donor alloantigen induced donor-specific hyporesponsiveness to C57BL/10 cardiac allografts in CBA recipients and that blockade of the B7 pathways abrogated that hyporesponsiveness. In this study, the authors used a CD28-deficient model to evaluate which signal, either through CD28 or cytotoxic T-lymphocyte-associated antigen (CTLA4), is involved in the induction of hyporesponsiveness. METHODS: Seven days before transplantation of hearts from C3H/HeJ (H2k) mice into C57BL/6 (H2b) or CD28-deficient (C57BL/6 background) mice, the transplant recipients were given ITD of donor splenocytes (1 x 10(7)), alone or in combination with human CTLA4-immunoglobulin (Ig) (200 microg). RESULTS: ITD of C3H splenocytes induced donor-specific hyporesponsiveness to C3H cardiac grafts in C57BL/6 recipients (graft median survival time [MST], 40 days). Administration of CTLA4-Ig concurrently with ITD abrogated the prolonged allograft survival (MST, 12 days). Interestingly, ITD of C3H splenocytes induced prolonged survival of C3H allografts in CD28-deficient recipients (MST, 55 days). Furthermore, administration of CTLA4-Ig combined with ITD of C3H splenocytes abrogated the prolonged survival of C3H allografts in CD28-deficient recipients (MST, 7 days), whereas recipients given isotype-control antibody in combination with ITD of splenocytes had prolonged survival of C3H allografts (MST, 58 days). CONCLUSIONS: Taken together, the authors' findings indicate that a signal through CTLA4, rather than through CD28, plays an important role in the induction of hyporesponsiveness by ITD of alloantigen in this model.