Murine Cardiac Allograft Transplantation Research Articles

The Immunomodulating Effects of Thalidomide and Dexamethasone in a Murine Cardiac Allograft Transplantation Model.

PurposeThe immunomodulatory effects of thalidomide (TM) and dexamethasone (DX) on immune cells and their co-stimulatory, co-inhibitory molecules in vitro and in vivo have been previously reported. The current study investigated the effects of TM and the combinatorial treatment with DX on immune cells using a murine cardiac allograft transplantation model.Materials and MethodsIntraabdominal transplant of cardiac allografts from BALB/c (H-2d) donors to C57BL/6 (H-2b) recipients was performed. After transplantation, mice were injected daily with TM or DX or a combination of both TM and DX (TM/DX) by intraperitoneal route until the time of graft loss. CD4+ T cell subsets and CD11c+ cells in the peripheral blood mononuclear cells and spleen were examined and quantified with flow cytometry. Serum IL-6 levels were measured by enzyme-linked immunosorbent assay on day 7.ResultsThe mean graft survivals were 6.86 days in the untreated group, and 10.0 days in the TM/DX group (p<0.001). The TM/DX treatment affected the CD4+ T cell subsets without suppressing the total CD4+ T cell population. The CD4+FOXP3+/CD4+CD44hi T cell ratio increased. Increase in cell counts and median fluorescence intensity on CD11c+CD85k+ with TM/DX were observed. The inhibition of pro-inflammatory cytokine interleukin-6 was also observed.ConclusionThese outcomes suggest the immunomodulating effect of the TM/DX combinatorial treatment. In conclusion, TM/DX combination may be a promising immunomodulatory approach for preventing allograft rejection and improving graft survival by inducing tolerance in transplantation.

In vivo Treg expansion under costimulation blockade targets early rejection and improves long-term outcome

CTLA4Ig has been shown to improve kidney allograft function, but an increased frequency of early rejection episodes poses a major obstacle for more widespread clinical use. The deleterious effect of CTLA4Ig on Treg numbers provides a possible explanation for graft injury. Therefore, we aimed at improving CTLA4Ig's efficacy by therapeutically increasing the number of Tregs. Murine cardiac allograft transplantation (BALB/c to B6) was performed under CTLA4Ig therapy modeled after the clinically approved dosing regimen and Tregs were transferred early or late after transplant. Neither early nor late Treg transfer prolonged allograft survival. Transferred Tregs were traceable in various lymphoid compartments but only modestly increased overall Treg numbers. Next, we augmented Treg numbers in vivo by means of IL2 complexes. A short course of IL2/anti‐IL2‐complexes administered before transplantation reversed the CTLA4Ig‐mediated decline in Tregs. Of note, the addition of IL2/anti‐IL2‐complexes to CTLA4Ig therapy substantially prolonged heart allograft survival and significantly improved graft histology on day 100. The depletion of Tregs abrogated this effect and resulted in a significantly diminished allograft survival. The increase in Treg numbers upon IL2 treatment was associated with a decreased expression of B7 on dendritic cells. These results demonstrate that therapy with IL2 complexes improves the efficacy of CTLA4Ig by counterbalancing its unfavorable effect on Tregs.

Graft Protective Effect of HMG-CoA Reductase Inhibitor Pravastatin in Murine Cardiac Allograft Transplantation

The HMG-CoA reductase inhibitor (statin), which reduces serum cholesterol, has been demonstrated in the control of immune responses and may potentially play an important role in the regulation of acute and chronic rejection in organ transplantations. We investigated the graft-protective effect of a kind of statin, pravastatin, in the survival of fully major histocompatibility complex--mismatched murine cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients through microsurgical techniques. CBA recipients transplanted with a C57BL/6 heart received oral administration of 40, 120, or 400 μg/kg/day of pravastatin from the day of transplantation to 7 days afterward. Immunohistochemical staining studies were performed to determine whether intimal formation of coronary arteries in the transplanted cardiac allografts was preserved and also to conduct morphometric analysis. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] 7 days). CBA recipients exposed with 40 and 120 μg/kg/day of pravastatin had a small prolonged allograft survival (MSTs of 10 and 9 days, respectively). However, the MST of CBA recipients exposed to 400 μg/kg/day of pravastatin was significantly effective for allograft survival (MST 50 days). Immunohistochemical staining assessments on 4 weeks after grafting showed suppression of intimal hyperplasia in allograft coronary arteries. Pravastatin could induce the prolongation of fully major histocompatibility complex--mismatched cardiac allograft through the protection of the coronary artery.

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.

Rikkunshito (TJ-43) Improved Reduction of Food Intake in a Murine Cardiac Transplantation Model

Rikkunshito (TJ-43), an eight-component traditional Japanese herbal medicine, has been used in clinics for gastritis, vomiting, and appetite loss. We investigated the effects of TJ-43 on the amelioration of appetite loss in the surgical-exposed model of murine cardiac allograft transplantation. CBA mice underwent transplantation of a CBA (syngeneic group) or C57BL/6 heart (allogeneic group) and received oral administration of 2 g/kg/d of TJ-43 from the day of transplantation until 7 days afterward. The amount of food intake (FI) and weight change after operation were recorded from 1 to 28 postoperative days. The allogeneic group had less average amounts of FI for 1 week compared with the syngeneic group (FI was 1.90 ± 0.43 g and 2.66 ± 0.46 g, respectively). Average FIs between the syngeneic and allogeneic groups with TJ-43 for 1 week were 2.36 ± 0.44 g and 2.30 ± 0.13 g, respectively, and those with distilled water were 2.66 ± 0.46 g and 1.90 ± 0.43 g, respectively, suggesting that exposure with TJ-43 tended to ameliorate the reduction of FI. Similarly, the effect on the amelioration of average FI in syngeneic and allogeneic groups exposed for 2 weeks was confirmed. However, exposure to with TJ-43 had no effects on FI after 4 weeks. TJ-43 could prevent reduction of average FI induced by the surgical-exposed model of murine cardiac allograft transplantation.

The Limits of Linked Suppression for Regulatory T Cells.

We have previously found that CD4(+)CD25(+) regulatory T cells (Tregs) can adoptively transfer tolerance after its induction with costimulatory blockade in a mouse model of murine cardiac allograft transplantation. In these experiments, we tested an hypothesis with three components: (1) the Tregs that transfer tolerance have the capacity for linked suppression, (2) the determinants that stimulate the Tregs are expressed by the indirect pathway, and (3) the donor peptides contributing to these indirect determinants are derived from donor major histocompatibility complex (MHC) antigens (Ags). First heart transplants were performed from the indicated donor strain to B10.D2 recipients along with costimulatory blockade treatment (250 μg i.p. injection of MR1 on day 0 and 250 μg i.p. injection of CTLA-4 Ig on day 2). At least 8 weeks later, a second heart transplant was performed to a new B10.D2 recipient who had been irradiated with 450 cGy. This recipient was given 40 × 106 naive B10.D2 spleen cells + 40 × 106 B10.D2 spleen cells from the first (tolerant) recipient. We performed three different types of heart transplants using various donors. (1) Tregs suppress the graft rejection in an Ag-specific manner. (2) Tregs generated in the face of MHC disparities suppress the rejection of grafts expressing third party MHC along with tolerant MHC. The limits of linkage appear to be quantitative and not universally determined by either the indirect pathway or by peptides of donor MHC Ags.

Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell.

Exercise therapy has been associated with improvement in functional capacity and quality of life. The role of exercise therapy in heart transplant recipients is of great interest for the transplant society, although concerning the effect of exercise therapy, there is little knowledge at present. We analyzed the effects of exercise on alloimmune responses in murine cardiac allograft transplantation. CBA mice (H2k) underwent transplantation of C57Bl/6 (H2b) hearts and exercised on a treadmill. Untreated CBA recipients rejected C57Bl/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients treated with treadmill for 1 week after transplantation, and for 1 week both before and after transplantation prolonged allograft survivals (MSTs, 35 and 18 days, respectively). However, treadmill exercise recipients for 1 week before transplantation were not effective to allograft survival (MST, 8 days). Adoptive transfer of whole splenocytes and CD4+ cells from treadmill exercise recipients significantly prolonged allograft survival in naive secondary recipients (MSTs, 30 and 52 days, respectively), suggesting that regulatory cells was generated after treadmill exercise. Moreover, flow cytometry studies showed that CD4+CD25+Foxp3+ cell population increased in treadmill exercise recipients. Therefore, postoperative but not pre-operative exercise could induce prolongation of survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory T cells.

Suppressor of cytokine signaling 3 (SOCS3) gene transfer prolongs the survival of the murine cardiac allograft by attenuating interleukin-17-producing alloreactive T-cell responses

Suppressor of cytokine signaling 3 (SOCS3) is the main negative feedback regulator of cytokine signals. We investigated the hypothesis that overexpression of SOCS3 in a murine cardiac allograft transplantation model may result in a survival advantage of the allograft by attenuating alloreactive T-cell responses. With the use of BALB/c (H-2(d) ) donor mice and C57Bl/6j (H-2(b) ) recipient mice, the murine cardiac transplantation model was established. Recipient mice received a tail intravenous injection with eukaryotic expression plasmid pEF-FLAG-I/mSOCS3 before and after transplantation. The heart beat of the grafts and immune responses were monitored. Overexpression of SOCS3 within grafts and spleens can prolong the survival time of cardiac allografts by attenuating infiltration of inflammatory cells such as T cells and macrophages in the grafts, decreasing the number of CD4(+) IL-17(+) cells and CD8(+) IL-17(+) cells in spleens, as well as reducing the expression of STAT3 in grafts and phosphorylation of STAT3 in both grafts and spleens. Overexpression of SOCS3 significantly delays cardiac allograft acute rejection, which is associated with reduced allograft proinflammatory leukocyte infiltration and impaired alloreactive IL-17(+) T cell immunity.

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 (H2k) mice underwent transplantation of a C57BL/6 (B6, H2b) 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.

Inchingorei-san (TJ-117) and Artemisiae Capillaris Herba Induced Prolonged Survival of Fully Mismatched Cardiac Allografts and Generated Regulatory Cells in Mice

We investigated Inchingorei-san (TJ-117), a 6-component Japanese herbal medicine, on alloimmune responses in murine cardiac allograft transplantation. CBA mice underwent transplantation of a C57BL/6 (B6) heart and received oral administration of TJ-117 or each component of TJ-117 from the day of transplantation until 7 days afterward. Naive CBA mice rejected B6 cardiac grafts acutely (median survival time (MST), 7 days). CBA recipients given 1 g/kg/day of TJ-117 had prolonged B6 allograft survival (MST, 37 days). Moreover, given 1 g/kg/day of Artemisiae Capillaris Herba (ACH), one component of TJ-117, indefinitely prolonged B6 allograft survival (MST, >100 days). However, other five components of TJ-117 were less effective than TJ-117 and ACH. Secondary CBA recipients given whole splenocytes, CD4+, and CD4+CD25+ cells from primary ACH-treated CBA recipients with B6 cardiac allografts 30 days after grafting had prolonged survival of B6 hearts (MSTs, 57, >100, and >100 days, resp.). Flow cytometry studies showed that the CD4+CD25+Foxp3+ regulatory cell population was increased in transplant recipients given ACH. Cell proliferation, interleukin-2, and interferon-γ were suppressed in ACH-treated mice, whereas interleukin-4 and interleukin-10 were upregulated. In conclusion, ACH, one component of TJ-117, as well as TJ-117 induced hyporesponsiveness to fully allogeneic cardiac allografts and may generate CD4+CD25+Foxp3+ regulatory cells.

Effective induction of immune tolerance by portal venous infusion with IL-10 gene-modified immature dendritic cells leading to prolongation of allograft survival.

Dendritic cells (DC) not only initiate T cell responses, but are also involved in the induction of tolerance. The functional properties of DC are strictly dependent on their state of maturation. It has been shown that immature DC can induce immune tolerance and prolong allograft survival. Interleukin-10 (IL-10) is an important immunosuppressive cytokine which inhibits maturation and function of DC. In order to improve the tolerogenicity of DC, we and others showed that adenovirus vectors can effectively mediate IL-10 genetic modification of DC, and IL-10 genetic modification can inhibit MHC II, B7.2, and CD40 expression, IL-12 secretion and the T cell stimulatory capacity of DC. The primary aim of this study is to examine the in vivo effects of this approach on allograft survival in a murine cardiac allograft transplantation model. To our surprise,we observed that infusion of immature DC genetically modified to express IL-10 (DC-IL-10) via the tail vein could not prolong allograft survival in the recipients, but shortened their survival. More interestingly, portal venous infusion of DC-IL-10 markedly prolonged allograft survival. The diverse effects of DC-IL-10 infusion through different routes may be due to the different immune responses to alloantigens in recipients that received DC-IL-10 via either the portal or the tail vein. Decreased cytotoxicity, polarization of Th2 response, poor T cell stimulating activity of liver DC and enhanced incidence of donor DC in the recipients may contribute to the more efficient prolongation of allograft survival observed after portal venous infusion of DC-IL-10. These results suggest that portal venous infusion may be an effective approach for immature DC to induce immune tolerance or hyporesponsiveness against donor antigens, and prolong allograft survival.

In vivo differentiation of alloreactive CD8+ T cells after murine cardiac allograft transplantation

In vivo differentiation of alloreactive CD8+ T cells after murine cardiac allograft transplantation