Nobuhide Matsuoka

High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice

Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.

Vα14 NK T cell–triggered IFN-γ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets

Pancreatic islet transplantation is a highly promising approach for the treatment of insulin-dependent diabetes mellitus. However, the procedure remains experimental for several reasons, including its low efficiency caused by the early graft loss of transplanted islets. We demonstrate that Gr-1+CD11b+ cells generated by transplantation and their IFN-γ production triggered by Vα14 NKT cells are an essential component and a major cause of early graft loss of pancreatic islet transplants. Gr-1+CD11b+ cells from Vα14 NKT cell–deficient (Jα281−/−) mice failed to produce IFN-γ, resulting in efficient islet graft acceptance. Early graft loss was successfully prevented through the repeated administration of α-galactosylceramide, a specific ligand for Vα14 NKT cells, resulting in dramatically reduced IFN-γ production by Gr-1+CD11b+ cells, as well as Vα14 NKT cells. Our study elucidates, for the first time, the crucial role of Gr-1+CD11b+ cells and the IFN-γ they produce in islet graft rejection and suggests a novel approach to improving transplantation efficiency through the modulation of Vα14 NKT cell function.

Successful islet transplantation to two recipients from a single donor by targeting proinflammatory cytokines in mice.

Background. Currently, the inability to achieve successful islet transplantation from one donor to one recipient is a major obstacle facing clinical islet transplantation. We herein determined whether this limitation could be overcome by targeting pro-inflammatory cytokines with the prevention of immediate islet graft loss in association with engraftment in mice. Methods. Isolated islets were grafted into the liver of streptozotocin-induced diabetic mice and the role of proinflammatory cytokines in the engraftment of islets was evaluated with the use of interferon (IFN)-&ggr;−/−mice and monoclonal antibodies against proinflammatory cytokines. Results. Hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngenic islets, which were isolated from a single mouse pancreas, was ameliorated when IFN-&ggr;−/−, but not wild-type mice, were used as recipients. The treatment with anti-IFN-&ggr; antibody produced normoglycemia in diabetic wild-type mice receiving 200, but not 100 islets. However, when anti–tumor necrosis factor-&agr; and anti-interleukin-1&bgr; antibodies were administered in conjunction with anti-IFN-&ggr; antibody, wild-type diabetic mice receiving 100 islets became normoglycemic after transplantation. In addition, the favorable effect of the combined use of antibodies was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. Conclusions. These findings clearly demonstrate that successful islet transplantation from one donor to two recipients is feasible by targeting pro-inflammatory cytokines in mice, thus suggesting a potential application in clinical islet transplantation if similar mechanisms of islet graft loss could be mediated in humans.

Prevention of early loss of transplanted islets in the liver of mice by adenosine.

Background. The low efficiency of islet transplantation necessitating sequential transplantations with the use of 2 to 3 donors for a recipient has been a major obstacle facing clinical islet transplantation. We determined whether adenosine has any beneficial effects on preventing early loss of transplanted islets in the liver, thereby facilitating successful islet transplantation from one donor to one recipient in mice. Methods. Two hundred islets, the number of islets from a single mouse pancreas, were grafted into the liver of streptozotocin-induced diabetic C57BL/6 mice. Adenosine was administered once at the time of islet transplantation. Mononuclear cells in the liver of mice receiving islets were isolated and examined by flow cytometry. Results. A single injection of adenosine at the time of transplantation ameliorated hyperglycemia of diabetic mice receiving 200 syngenic islets with suppression of interferon (IFN)-&ggr; production of hepatic NKT cells and neutrophils, while that of control did not. The IFN-&ggr; production of NKT cells and neutrophils in the liver of mice treated with &agr;-galactosylceramide, a synthetic ligand of NKT cells was suppressed by adenosine. The beneficial effect of adenosine was also observed for BALB/c islet allografts when alloimmune rejection was prevented by anti-CD4 antibody. Conclusions. Adenosine suppresses the NKT cell-mediated IFN-&ggr; production of neutrophils in the liver of mice receiving islets, thus leading to prevention of early loss of transplanted syngenic and allogenic islets. The findings indicate that adenosine may improve efficiency of clinical islet transplantation.

Prevention of high-mobility group box 1-mediated early loss of transplanted mouse islets in the liver by antithrombin III.

Background The low efficiency of pancreatic islet transplantation mainly because of the early loss of transplanted islets hampers its clinical application. Previously, we have shown in mice that the early loss of transplanted islets in the liver is caused by innate immune rejection in concert with dendritic cells, natural killer T cells, and neutrophils to produce interferon (IFN)-&ggr;, which is triggered by high-mobility group box 1 (HMGB1) released from transplanted islets. We herein determined whether the HMGB1-mediated early loss of transplanted mouse islets is prevented by antithrombin (ATIII). Methods The effect of ATIII on in vitro and in vivo HMGB1-stimulated IFN-&ggr; production of hepatic mononuclear cells was examined. Then, the effect of ATIII on amelioration of hyperglycemia in streptozotocin-induced diabetic mice receiving 200 syngeneic islets from a single donor was determined. Results In vitro and in vivo IFN-&ggr; production of mononuclear cells in the liver of mice in response to HMGB1 was suppressed by ATIII. Hyperglycemia of streptozotocin-induced diabetic mice receiving 200 syngeneic islets into the liver from a single donor was ameliorated with down-regulation of IFN-&ggr; production of natural killer T cells and neutrophils in the liver when ATIII but not vehicle was administered once at the time of islet transplantation. The favorable effect of ATIII was similarly achieved in mice receiving islet allografts when rejection was prevented with anti-CD4 antibody treatment. Conclusions These findings demonstrate that ATIII prevents HMGB1-mediated early loss of transplanted islets caused by innate immune rejection, suggesting a potential application of ATIII to improve efficiency of clinical islet transplantation.

Inhibitory Effect of Thrombomodulin on HMGB1-Stimulated IFN-γ Production of Hepatic NKT and Gr-1+ Cells, Facilitating to Prevent Early Loss of Transplanted Islets in the Liver of Mice: 1512

Currently, insulin independence in patients with IDDM has been hardly achieved after pancreatic islet transplantation (tx) from a single donor mainly due to early loss of transplanted islets. Previously, we have shown in mice that pancreatic islets contain abundant HMGB1, released into circulation and triggering NKT cell-dependent IFN-γ production of Gr-1+ cells (neutrophils) in the liver receiving islets (JCI 2010) which is an essential component of early loss of transplanted islets (JEM 2005). In the present study, we hypothesize that the beneficial effect of thrombomodulin (TM) on engraftments of islets in the liver might be mediated through inhibition of HMGB1-NKT-Gr-1+cell pathways since TM has been reported to produce sequestration of HMGB1 (JCI 2005). Hyperglycemia of STZ-diabetic mice (C57BL/6) receiving 200 syngenic islets from a single donor into the liver via the portal vein was ameliorated when TM was administered IV for 3 times (0, 12 and 24hrs, 200μg/ injection/mouse, n=5), while those of mice (n=5) treated with saline did not. Morphologically, intact islet grafts with well granulated β cells were seen in the liver of normoglycemic recipients treated with TM, while in contrast, degenerated islets with de-granulated β cells were seen in hyperglycemic control mice. IPGTT (1g/kg glucose) at 60 days after tx revealed that the glucose tolerance of TM-treated mice receiving 200 islets (n=5) was superior to that of normoglycemic mice receiving 400 islets without TM treatment (n=5). FACS analysis showed that IFN-γ production of NKT cells and Gr-1+ cells accumulated in the liver of mice receiving islets and treated with saline was up-regulated at 6 hours after tx as reported previously, while in contrast, that in mice receiving 200 islets and treated with TM was down-regulated with reduction in number of infiltrating Gr-1+ cells. IFN-γ production of NKT cells and Gr-1+ cells accumulated in the liver of mice at 2 hours after the IV injection of HMGB1 (100 μg/injection/mouse) without islet transplantation was up-regulated, while in marked contrast, that in mice treated with TM (500μg, iv) prior to the HMGB1 injection was down-regulated with reduced number of infiltrated Gr-1+ cells. These findings indicate that TM prevents the early loss of transplanted islets in the liver of mice through inhibition of stimulatory effects of HMGB1. Importantly, recombinant TM has already been used in clinics with great impact on its efficacy for the treatment of sepsis with disseminated intravascular coagulation in Japan. Thus, the safety issue regarding the clinical use of TM has been cleared and it seems ready to apply this to clinical islet transplantation to improve the efficiency of intraportal islet transplantation. 1290

Impact of short hepatic vein reconstruction in living donor adult liver transplantation using a left liver plus caudate lobe graft.

OBJECTIVE To investigate the impact of short hepatic vein reconstruction in the transplanted left liver plus caudate lobe graft. METHODS Six left liver plus caudate lobe grafts used for living donor adult liver transplantation were included in this study. The liver grafts were divided into two groups: those with (V1 group; n = 4) or without (control group; n = 2) short hepatic vein reconstruction. The changes in the transplanted left lobe (segments II-IV) and caudate lobe were compared between the two groups at 1 month after transplantation. RESULTS The addition of the caudate lobe increased the graft volume by 15 mL, which corresponded to a 4.3% gain of graft volume at the time of transplantation. Although the graft volume/standard liver volume ratio of the whole grafts after transplantation showed no difference between the two groups, the regeneration rate of the caudate lobe in the V1 group was significantly greater than that in the control group (p= 0.04). CONCLUSION Although no definite advantage from the V1 reconstruction was demonstrated, hepatic vein reconstruction with a significantly-sized short hepatic vein might provide an additional margin of safety for marginally-sized liver grafts during the early phase of graft regeneration.

A NOVEL APPROACH TO PREVENT EARLY LOSS OF TRANSPLANTED ISLETS BY TARGETING DONOR ISLETS WITH A PPARγ AGONIST PRIOR TO TRANSPLANTATION: 1267

Introduction: The limited success in achieving insulin-independence of IDDM recipients from a single donor hampers clinical application of islet transplantation. A major reason for this includes early loss of transplanted islets in the liver, the site of transplantation, in association with engraftments. Previously, we have shown that NKT cell-dependent IFN-Î3 production of Gr-1+CD11b+ cells in the liver triggered by HMGB1 released from islets themselves plays an essential role in early loss of transplanted islets in mice (JEM 2005, JCI 2010). Here we demonstrate that HMGB1 release from islets is induced by hypoxia and that the in vitro pretreatment of islets with a PPARÎ3 agonist (pioglitazone; Pio) alleviates islet cell damage in hypoxia facilitating to prevent early loss of transplanted islets. Methods and Results: we first confirmed the expression of PPARÎ3 on pancreatic islet Î2 cells immunohistochemically. Islet cell damage was assessed by fluorescent microscopy to evaluate islet cell damage with PI and HO342 stain. To examine the effect of hypoxia on islet cell damage , isolated mouse islets (C57BL/6) were placed in a chamber at 37C filled with hypoxic gas (1%O2+5%CO2+94%N2) in which O2 concentration dropped to 20mmHg within 30 min and maintained the same level for more than 24 hours. Islets cultured in CO2 incubator (5%CO2+95% air) served as controls in which O2 concentration was 130-150mmHg (normoxia). When islets were cultured in normoxia, islets remained intact morphologically by 24 hours. In contrast, islets became degenerated with central necrosis when cultured in hypoxia for more than 6 hours. Importantly, the central necrosis at 6 hrs was not seen in islets pretreated with Pio (10Î1⁄4M, 3hrs) prior to the hypoxic culture. The beneficial effect of Pio was abolished when anti-PPARÎ3 was added to the culture medium with Pio. The amount of HMGB1 (ng/ml) in the medium of islets in hypoxia was increased with time, which was significantly decreased in the culture medium of islets pretreated with Pio at 12 and 24 hours (6 hrs; 2.1+-0.7 (n=5) vs 2.0+-1.1, 12 hrs; 7.5+-2.7 vs 4.5+-0.9*, 24hrs; 10.6+-1.6 vs 7.5+-1.2*). The amount of HMGB1 in the culture medium of islets in normoxia remained low by 24 hours (6hrs; 0.5+-0.4 (n=5), 12hrs; 1.1+-0.4, 24hrs; 0.1+-0.2). When 200 syngenic islets, the number of islets from one donor, were grafted into the liver of STZ-diabetic mice (C57BL/6), recipient mice remained hyperglycemic after transplantation. In contrast, diabetic mice became normoglycemic when donor islets were pretreated with Pio prior to transplantation and grafted into the liver. Diabetic mice receiving 200 islets pretreated with pio and anti-PPARÎ3 antibody remained hyperglycemic and those receiving islets pretreated with Pio and control ab became normoglycemic after transplantation. Conclusions: These findings clearly demonstrate that hypoxic islet cell death is alleviated by the pretreatment of islets with Pio and afford a novel approach to prevent early loss of transplanted islets by targeting donor islets prior to transplantation. The molecular mechanism involved in the effect by PPAR-Î3 on hypoxic islet cell death is currently under investigation.

EARLY LOSS OF TRANSPLANTED ISLETS IN THE LIVER OF MICE TRIGGERED BY HMGB1 IS PREVENTED BY ANTITHROMBIN III: 1263

Introduction: Currently, insulin independence in patients with IDDM has been hardly achieved after pancreatic islet transplantation (tx) from a single donor mainly due to early loss of transplanted islets, and therefore, sequential transplantations with the use of 2-3 donor pancreases are required for a single recipient. Previously, we have shown in mice that pancreatic islets contain abundant HMGB1 triggering NKT cell-dependent IFN-Î3 production of Gr-1+ cells (neutrophils) (JCI 2010) which is an essential component of early loss of transplanted islets (JEM 2005). In the present study, we hypothesize that antithrombin III (AT III) might modify the HMGB1-NKT cell-Gr-1+cell-mediated pathways to prevent early loss of transplanted islets since AT III has been reported to have anti-inflammatory as well as anti-coagulatory effect. Methods and Results: Hyperglycemia of STZ-diabetic mice (C57BL/6) receiving 200 syngenic islets from a single donor into the liver via the portal vein was ameliorated by a single injection of AT III at the time of islet transplantation (10U, iv, n=5), while that was not by saline (control, n=5). Morphologically, intact islet grafts with well granulated Î2 cells were seen in the liver of normoglycemic recipients, while in contrast, degenerated islets with de-granulated Î2 cells were seen in the liver of hyperglycemic recipient mice. IPGTT (1g/kg glucose) at 60 days after tx revealed that the glucose tolerance of normoglycemic mice receiving 200 islets and treated with AT III (n=5) was similar to that of naà ̄ve untreated mice (n=5). FACS analysis revealed that IFN-Î3 production of NKT cells and Gr-1+ cells accumulated in the liver of mice receiving islets and treated with saline was up-regulated at 6 hours after transplantation, while in contrast, that in mice receiving 200 islets and treated with AT III was down-regulated. IFN-Î3 production of NKT cells and Gr-1+ cells accumulated in the liver of mice treated with saline without islet transplantation was up-regulated at 2 hours after the iv injection of HMGB1 (100 Î1⁄4g/injection/mouse), while that in mice treated with AT III (10U) in conjunction with the HMGB1 injection was down-regulated. The beneficial effect on prevention of early loss of transplanted islets by AT III was also the case in diabetic mice (C57BL/6) receiving allogeneic islets (BALB/c) when alloimmune rejection was prevented by anti-CD4 antibody (200Î1⁄4g/ip injection/mouse, once at day 0). These findings show that AT III produces prevention of early loss of transplanted islets in the liver through inhibition of stimulatory effects of HMGB1. It is currently under investigation to determine whether DC, NKT and/or Gr-1+ cells in the liver are direct targets for AT III. Conclusion: AT III has a potential to increase the efficiency of clinical islet transplantation when its beneficial effect holds true in human. Importantly, AT III has already been introduced into clinics for the treatment of AT III deficiency and DIC and therefore, the issue of safety has been cleared and it seems ready to apply this to clinical islet transplantation.