Islet heparan sulfate but not heparan sulfate proteoglycan core protein is lost during islet isolation and undergoes recovery post-islet transplantation.

Islet beta cells in situ express intracellular heparan sulfate (HS), a property previously shown in vitro to be important for their survival. We report that HS levels inside islet beta cells correlate with the novel intracellular localization of the HSPG core proteins for collagen type XVIII (Col18), a conventional extracellular matrix component. Syndecan-1 (Sdc1) and CD44 core proteins were similarly localized inside beta cells. During isolation, mouse islets selectively lose HS to 11-27% of normal levels but retain their HSPG core proteins. Intra-islet HS failed to recover substantially during culture for 4 days and was not reconstituted in vitro using HS mimetics. In contrast, significant recovery of intra-islet HS to ∼40-50% of normal levels occurred by 5-10 days after isotransplantation. Loss of islet HS during the isolation procedure is independent of heparanase (a HS-degrading endoglycosidase) and due, in part, to oxidative damage. Treatment with antioxidants reduced islet cell death by ∼60% and increased the HS content of isolated islets by ∼twofold compared to untreated islets, preserving intra-islet HS to ∼60% of the normal HS content of islets in situ. These findings suggest that the preservation of islet HS during the islet isolation process may optimize islet survival posttransplant.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: impact for allograft rejection.

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet ß-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores ß-cell-matrix attachment, a recognized requirement for ß-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

Unexpected new roles for heparanase in Type 1 diabetes and immune gene regulation.

Heparanase (Hpse) is an endo-ß-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

Molecular composition of the peri-islet basement membrane in NOD mice: a barrier against destructive insulitis.

AIMS/HYPOTHESIS: This study examined whether the capsule which encases islets of Langerhans in the NOD mouse pancreas represents a specialised extracellular matrix (ECM) or basement membrane that protects islets from autoimmune attack. METHODS: Immunofluorescence microscopy using a panel of antibodies to collagens type IV, laminins, nidogens and perlecan was performed to localise matrix components in NOD mouse pancreas before diabetes onset, at onset of diabetes and after clinical diabetes was established (2-8.5 weeks post-onset). RESULTS: Perlecan, a heparan sulphate proteoglycan that is characteristic of basement membranes and has not previously been investigated in islets, was localised in the peri-islet capsule and surrounding intra-islet capillaries. Other components present in the peri-islet capsule included laminin chains alpha2, beta1 and gamma1, collagen type IV alpha1 and alpha2, and nidogen 1 and 2. Collagen type IV alpha3-alpha6 were not detected. These findings confirm that the peri-islet capsule represents a specialised ECM or conventional basement membrane. The islet basement membrane was destroyed in islets where intra-islet infiltration of leucocytes marked the progression from non-destructive to destructive insulitis. No changes in basement membrane composition were observed before leucocyte infiltration. CONCLUSIONS/INTERPRETATION: These findings suggest that the islet basement membrane functions as a physical barrier to leucocyte migration into islets and that degradation of the islet basement membrane marks the onset of destructive autoimmune insulitis and diabetes development in NOD mice. The components of the islet basement membrane that we identified predict that specialised degradative enzymes are likely to function in autoimmune islet damage.

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-gamma, 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-gamma 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-gamma-deficient and wild-type C57BL/6J recipient mice. The survival of pig proislet xenografts was also assessed in IFN-gamma -/- and wild-type hosts. Both proislet allografts and xenografts were acutely rejected in IFN-gamma -/- and wild-type mice. Unlike the conventional allograft reaction, which lacks eosinophil infiltration, the rejection of proislet allografts in IFN-gamma-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-gamma-independent pathways; IFN-gamma, 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.

Immune mechanisms associated with the rejection of fetal murine proislet allografts and pig proislet xenografts: comparison of intragraft cytokine mRNA profiles.

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.

Role of anti-donor antibody in the rejection of pig proislet xenografts in mice.

CBA/H mice produced serum anti-pig IgG1, IgG2a, and IgG2b following xenotransplantation of pig proislets beneath the kidney capsule; anti-pig IgM was present as pre-existing antibody in the serum of normal CBA/H mice and was also produced in response to pig proislet xenografts. Serum anti-pig IgG3 was not detected post-xenotransplantation. Rejection of pig proislet xenografts and the production of anti-pig IgG1, IgG2a, and IgG2b isotypes were CD4 T cell-dependent. The capacity for adoptively transferred hyperimmune CBA/H mouse anti-pig PBL serum to induce the rejection of intact pig proislet xenografts in CD4 T cell-depleted mice was dose dependent and correlated with markedly elevated levels of serum anti-pig IgG3. Levels of anti-pig IgG1, IgG2a, IgG2b, and IgM comparable to control mice acutely rejecting pig proislet xenografts and achieved following adoptive transfer of hyperimmune serum did not correlate with induced xenograft rejection. These findings suggest that anti-pig Ig isotypes produced during the conventional process of acute proislet xenograft rejection do not play a major role in mediating graft damage. The acute rejection of pig proislet xenografts in the absence of serum anti-pig Ig in microMT-/- hosts confirmed that anti-pig antibody is not essential for proislet xenograft destruction.

Eosinophils are not required for the rejection of neovascularized fetal pig proislet xenografts in mice.

The rejection of neovascularized pig proislet (islet precursor) xenografts in mice is a CD4 T cell-dependent process involving invasion of the graft site mainly by host CD4 T cells and eosinophils. We previously identified CD4 T cell-dependent enhancement of intragraft IL-3, IL-4, and IL-5 mRNA expression during acute xeno-rejection in CBA/H recipient mice. In the present study we investigated the role of each cytokine and the involvement of eosinophils in the rejection of pig proislet xenografts using cytokine gene knockout mice (IL-4 -/- and IL-5 -/-) and the treatment of transplant recipients with anti-IL-3 mAb. In IL-4 -/- mice, IL-5 -/- recipient animals, and anti-IL-3 mAb-treated CBA/H mice, eosinophil accumulation at the transplant site was inhibited or ablated, but the kinetics of xenograft rejection was unaltered. Prolonged xenograft survival was only achieved in anti-CD4 mAb-treated mice and consistently correlated with the absence of intragraft IL-3, IL-4, and IL-5 mRNA enhancement. Together these findings indicate that neither IL-3, nor IL-4, nor IL-5 individually plays an obligatory role in the rejection process. The cytokine mRNA profile correlating with the lack of eosinophil recruitment was variable; the data suggest that IL-4 regulates eosinophil involvement in the xeno-rejection reaction indirectly via effects on IL-5 and IL-3 transcript expression. There is also suggestive evidence that IL-5 may influence IL-3 and IL-4 mRNA expression via feedback inhibition. Eosinophils, therefore, do not play an essential role in the rejection of neovascularized pig proislet xenografts in mice.

Differences in the contribution of CD4+ T cells to proislet and islet allograft rejection correlate with constitutive class II MHC alloantigen expression.

Allografts of BALB/c (H-2d) fetal proislets facilitated long-term (> 100 days) reversal of streptozotocin-induced diabetes in CBA/H (H-2k) mice treated with a combination of anti-CD4 and anti CD8 mAbs. Anti-CD8 monotherapy was partially effective in restoring normoglycemia but anti-CD4 mAb treatment of host animals failed to promote allograft function. In contrast, allografts of BALB/c adult islets demonstrated indefinite reversal of diabetes in recipient mice treated only with anti-CD8 mAb. Anti-CD4 monotherapy resulted in only transient restoration of normoglycemia. These findings clearly demonstrate (1) a critical role for CD8 T cells in the acute rejection of pancreatic islet tissue allografts and (2) tissue-specific differences in the participation of CD4 T cells as primary effectors in the rejection reaction. Immunohistochemical studies showed that the capacity for CD4 T cells to initiate the rejection of proislet but not adult islet allografts correlates with the presence/absence, respectively, of graft parenchymal cells that constitutively express Class II MHC alloantigens. Proislet grafts, unlike transplants of purified adult islets, contain heterogeneous tissue components including Class II MHC+ve duct epithelium. Thus, the participation of CD8 and CD4 T cells as primary effectors of graft rejection depends on which class or classes of MHC antigens are constitutively expressed on graft parenchymal cells and are available for recognition. Islet tissue in both rejecting proislet and islet allografts showed de novo induction of Class II MHC alloantigens only after severe disruption to islet architecture had been achieved by infiltrating mononuclear cells. Thus, at this stage of advanced allograft injury, CD4 T cells have the potential to act as secondary effectors, possibly by amplifying the inflammatory reaction and thus accelerating graft destruction. The capacity for antirejection mAb therapy to establish transplant tolerance was facilitated in the islet allograft model where it was necessary to target only the CD8 T cell subpopulation.

Intragraft expression of cytokine transcripts during pig proislet xenograft rejection and tolerance in mice.

The rejection of pig proislet (islet precursor) xenografts in CBA/H mice is a CD4+ T cell-dependent process. The molecular mechanisms of xenograft rejection, xenograft survival during anti-CD4 mAb therapy and xenograft tolerance post-withdrawal of anti-CD4 mAb administration, were examined by using a semiquantitative PCR method. Temporal analysis of intragraft cytokine mRNA demonstrated a Th0-like pattern of expression (IL-2, IFN-gamma, IL-3, IL-4, IL-5, and IL-10) on day 4 of the acute xenograft rejection process. From day 5, however, only Th2-associated transcripts (IL-3, IL-4, IL-5, and IL-10) were enhanced in xenografts compared with isograft controls. Immunohistochemistry showed that the principal participants in the rejection infiltrate were CD4+ T cells and eosinophils, with smaller numbers of CD8+ T cells. In vivo depletion of CD4+ T cells prevented xenograft rejection but had minimal effect on the peak levels of IL-2, IFN-gamma, and IL-10 mRNA; in contrast, the enhanced expression of IL-3, IL-4, and IL-5 transcripts seen in rejecting xenografts was abrogated. This established a positive correlation between acute xenograft rejection, presence of CD4+ T cells, and enhanced intragraft expression of mRNA for the Th2-type cytokines IL-3, IL-4, and IL-5. In tolerant hosts, long-term proislet xenograft survival and function (> 190 days) was accompanied by intragraft expression of IL-2 and IL-10 mRNA; IFN-gamma, IL-3, IL-4, and IL-5 mRNA were either undetected or not enhanced. The induced rejection of long-term functioning xenografts (> 170 days) in nontolerant hosts resulted in selective enhancement of IL-4 transcript expression. This study suggests that Th2-like CD4+ T cells are differentially activated in response to xenoantigen and that xenograft tolerance is associated with lack of expression of the Th2 cytokine, IL-4.

Induction of class II major histocompatibility antigens on thyroid, adult pancreatic islet, and fetal proislet allografts.

Cultured BALB/c (H-2d) thyroid, adult pancreatic islet and fetal proislet tissues can be accepted permanently in CBA/H (H-2k) recipients until rejection is triggered by the transfer of antidonor strain activated immune T cells. Class II MHC antigens are not expressed on the accepted grafts and are induced following immune cell transfer, but expression is quantitatively and qualitatively different for the different tissues. Thyroid allografts show intense class II MHC antigen expression early after immune cell transfer and before extensive tissue destruction has occurred. In contrast, adult islet and fetal proislet allografts show patchy and cytoplasmic staining usually associated with areas of tissue destruction. Gamma-interferon treatment of tissues in vitro showed that proislet tissue has a greater capacity for class II MHC antigen induction than adult islet tissue. The differences in the capacity for class II MHC induction by fetal and adult islet tissue may be important in relation to the pathogenesis of autoimmune disease.