Charmaine J. Simeonovic

Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD4+ T cell-depleted mice.

Treatment of CBA/H mice with 5 injections of anti-CD4 (GK1.5 mAb) terminating on day 10 posttransplant resulted in long-term survival (greater than or equal to 6 weeks) of fetal pig proislet (pancreatic islet precursor) xenografts. The GK1.5 mAb dose determined the duration of CD4+ T cell depletion and the extent to which the survival of pig proislet xenografts was prolonged. Sustained depletion of CD4+ T cells (0%, 1%, and 9% of total T cells in peripheral lymph nodes at 2, 4, and 6 weeks, respectively) and survival of proislet xenografts at 6 weeks posttransplant was observed when transplant recipients were treated with 5.4 mg GK1.5 mAb/injection. Treatment of transplanted mice with a suboptimal dose of GK1.5 mAb (0.2 mg/injection) resulted in the same level of depletion at 2 weeks posttransplant but a more rapid recovery of CD4+ T cells in the periphery (24% of total T cells at 4 weeks) and only temporary prolongation in xenograft survival (less than or equal to 4 weeks). Control xenografts showed evidence of graft destruction by as early as 6-7 days posttransplant and were completely rejected by 2 weeks. The rejection reaction consisted predominantly of CD4+ T cells, eosinophils and F4/80-positive macrophages. Only small numbers of CD8+ T cells were identified. CD4+ T cells therefore represented the major T cell component of the cellular infiltrate. In contrast, surviving xenografts in GK1.5 mAb-treated recipient mice showed essentially an absence of CD4+ T cells but presence of CD8+ T cells. This finding may be attributable to the increase (1.7-3.1-fold) in the absolute size of the population of CD8+ T cells in the periphery following GK1.5 mAb treatment in vivo. Compared with isolated fetal pig proislets, which contained only a small population of insulin-producing cells in addition to glucagon- and somatostatin-positive cells, surviving pig proislet xenografts contained mainly insulin-positive beta cells with smaller populations of glucagon- and somatostatin-positive cells. Fetal pig proislets therefore differentiate into insulin-producing islet tissue posttransplant and thus show evidence of normal development of endocrine function.

Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes

The autoimmune type 1 diabetes (T1D) that arises spontaneously in NOD mice is considered to be a model of T1D in humans. It is characterized by the invasion of pancreatic islets by mononuclear cells (MNCs), which ultimately leads to destruction of insulin-producing β cells. Although T cell dependent, the molecular mechanisms triggering β cell death have not been fully elucidated. Here, we report that a glycosaminoglycan, heparan sulfate (HS), is expressed at extraordinarily high levels within mouse islets and is essential for β cell survival. In vitro, β cells rapidly lost their HS and died. β Cell death was prevented by HS replacement, a treatment that also rendered the β cells resistant to damage from ROS. In vivo, autoimmune destruction of islets in NOD mice was associated with production of catalytically active heparanase, an HS-degrading enzyme, by islet-infiltrating MNCs and loss of islet HS. Furthermore, in vivo treatment with the heparanase inhibitor PI-88 preserved intraislet HS and protected NOD mice from T1D. Our results identified HS as a critical molecular requirement for islet β cell survival and HS degradation as a mechanism for β cell destruction. Our findings suggest that preservation of islet HS could be a therapeutic strategy for preventing T1D.

Macrophage-Induced Muscle Pathology Results in Morbidity and Mortality for Ross River Virus-Infected Mice

Ross River virus (RRV) is an Australian alphavirus that is often responsible for chronic epidemic polyarthritis and myalgia in humans. Past studies have shown severe disruption of striated muscle fibers to be prominent in RRV pathology in mice; in the present study, macrophages were directly implicated as the primary mediators of muscle damage. General immunosuppressive therapies had only minor effects on mortality and morbidity in RRV-infected mice, with no inhibition of muscle damage. Treatment of mice with macrophage-toxic agents (e.g., silica) prior to RRV infection completely abrogated disease symptoms without significantly affecting titers of virus in organs. Further studies found that clinical signs of infection and muscle damage correlated with a massive influx of macrophages into hind leg muscle, whereas no such infiltrate or damage was observed for silica-treated mice. These observations are significant for the human disease context, as monocytic cells have been detected in the synovial effusions of persons with epidemic polyarthritis.

Role of CD4+ T-Lymphocytes in Rejection by Mice of Fetal Pig Proislet Xenografts

The role of T-lymphocyte subpopulations in the rejection of fetal pig proislet (islet precursor) xenografts in mice was examined with in vivo administration of monoclonal antibodies (MoAbs) to CD4+ (GK1.5) and CD8+ (49–11.1) T-lymphocyte subsets. The data indicate that the rejection process is T-lymphocyte dependent and mediated by CD4+ T-lymphocytes. The in vivo administration of MoAbs specific for graft-borne pig leukocytes failed to prevent xenog raft rejection in fully immunocompetent transplant recipients.

The role of chemokines and their receptors in the rejection of pig islet tissue xenografts

Solomon MF, Kuziel WA, Mann DA, Simeonovic CJ. The role of chemokines and their receptors in the rejection of pig islet tissue xenografts. Xenotransplantation 2003; 10: 164–177.

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11β-hydroxysteroid dehydrogenase

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11β-HSD) isoforms; 11β-HSD–type-1 and 11β-HSD–type-2. We demonstrated expression of mRNA for 11β-HSD-1 and 11β-HSD-2 and protein for 11β-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11β-HSD-1 and up-regulating 11β-HSD-2. The 11β-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11β-HSD-1 enzyme activity or increased 11β-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

Modulation of tissue immunogenicity by organ culture. Comparison of adult islets and fetal pancreas.

Uncultured mouse islet allografts (BALB/c to CBA) are rejected 2 to 4 weeks after transplantation. Allografts, cultured in 95% O2 and 5% CO2 for 7 days before transplantation, show no sign of rejection up to 3 months post-transplantation. However, the cultured allografts are rejected if the CBA recipient is given an i.v. injection of 10(5) peritoneal cells at the time of transplantation. Organ culture of BALB/c fetal pancreas (16 to 17 days gestation) under the same conditions failed to prevent allograft rejection. The immunogenicity of fetal pancreas is reduced if this tissue is cultured in 95% O2 and 5% CO2 for 17 days before transplantation.

Immune mechanisms associated with the rejection of fetal murine proislet allografts and pig proislet xenografts: comparison of intragraft cytokine mrna profiles1

BACKGROUND Previous in vivo depletion studies of CD4 and CD8 T cells indicated that different rejection mechanisms operate for proislet allografts and xenografts. The cellular and molecular mechanisms of acute proislet allograft and xenograft rejection have therefore been characterized and directly compared. METHODS The intragraft cytokine mRNA profile in rejecting BALB/c (H-2d) proislet allografts was analyzed in control, CD4 T cell-depleted, and CD8 T cell-depleted CBA/H (H-2k) recipient mice using semi-quantitative reverse transcriptase-assisted polymerase chain reaction (RT-PCR). The cytokine profiles for proislet allografts and pig proislet xenografts at 3-10 days posttransplant were directly compared and correlated with graft histopathology. RESULTS Allograft rejection was protracted (2-3 weeks), characterized by infiltrating CD8 T cells and CD4 T cells (no eosinophils) and was associated with a Th1-type CD4 T cell response (IL-2, IFN-gamma, and IL-3 mRNA) and a CD8 T cell-dependent spectrum of cytokine gene expression (IL-2, IFN-gamma, IL-3, and IL-10 mRNA). Xenograft rejection was rapid (6-8 days), involved predominantly CD4 T cells and eosinophils, and in contrast to allografts, exhibited intragraft mRNA expression for the Th2 cytokines IL-4 and IL-5. CONCLUSIONS Proislet allograft and xenograft rejection differ in the tempo of destruction, phenotype of the cellular response and intragraft profile of cytokine mRNA. The recruitment of eosinophils only to the site of xenorejection correlates with IL4 and IL-5 mRNA expression. These findings suggest that different anti-rejection strategies may need to be developed to optimally target the allograft and the xenograft response.

Analysis of the Th1/Th2 paradigm in transplantation: interferon-gamma deficiency converts Th1-type proislet allograft rejection to a Th2-type xenograft-like response

The rejection mechanisms for fetal proislet allografts and pig proislet xenografts in mice are characterized by different intragraft cytokine mRNA profiles and cellular responses. Allograft rejection is predominantly CD8 T-cell-dependent and is associated with a Th1-type cytokine pattern (i.e., IFN-γ, IL-2 but no IL-4 or IL-5 mRNA). In contrast, xenograft rejection is CD4 T-cell-dependent and is accompanied by a strong Th2-type response (i.e., enhanced expression of IL-4 and IL-5 mRNA) and by marked eosinophil accumulation at the graft site. We have now examined and compared the regulatory role of IFN-γ in both proislet allograft and xenograft rejection processes. The histopathology and intragraft cytokine mRNA profile of BALB/c (H-2d) proislet allografts were examined in IFN-γ-deficient and wild-type C57BL/6J recipient mice. The survival of pig proislet xenografts was also assessed in IFN-γ –/– and wild-type hosts. Both proislet allografts and xenografts were acutely rejected in IFN-γ –/– and wild-type mice. Unlike the conventional allograft reaction, which lacks eosinophil infiltration, the rejection of proislet allografts in IFN-γ-deficient hosts correlated with intragraft expression of IL-4 and IL-5 mRNA (i.e., a Th2-type response) and eosinophil recruitment. The rejection of proislet allografts and xenografts can therefore occur by IFN-γ-independent pathways; IFN-γ, however, regulates the pathology of the allograft reaction but not the xenograft response. The immune destruction of proislet allografts is not prevented by Th2 cytokine gene expression; instead, the latter correlated with the recruitment of unconventional inflammatory cells (eosinophils), which may play an accessory role in effecting graft injury. Significantly, the Th1-to-Th2-like switch resulted in the novel conversion of an allograft rejection reaction into a xenograft-like rejection process.

Antibody-induced rejection of pig proislet xenografts in CD4+ T cell-depleted diabetic mice.

Reversal of diabetes in mice was achieved following in vivo depletion of host CD4+ T cells and transplantation of xenogeneic fetal pig proislets (pancreatic islet precursors). These procedures resulted in xenograft tolerance since established pig proislet xenografts were not rejected by antipig antibodies produced in the host, and rejection was not induced following the administration of donor major histocompatibility complex--specific pig lymphocytes. Proislet xenografts were rejected following the administration of donor MHC-specific hyper-immune antipig PBL serum raised in normal mice. Although established proislet xenografts in anti-CD4-treated mice are sensitive to antibody-mediated destruction, such hosts are unable to produce an antibody response that leads to graft rejection. The study indicates that the mechanism of preventing xenograft rejection by anti-CD4 treatment in vivo involves not only initial CD4+ T cell depletion but also quantitative and/or qualitative modulation of a CD4+ T cell-dependent antibody response. As a consequence, an apparent state of xenograft tolerance is produced.