Specific gene expression in pancreatic beta-cells: cloning and characterization of differentially expressed genes.

Identification and characterization of genes expressed preferentially in pancreatic beta-cells will clarify the mechanisms involved in the specialized properties of these cells, as well as providing new markers of the development of type 1 diabetes. Despite major efforts, relatively few beta-cell-specific genes have been characterized. We applied representational difference analysis to identify genes expressed selectively in the pancreatic beta-cell line betaTC1 compared with the pancreatic alpha-cell line alphaTC1 and isolated 26 clones expressed at higher levels in the beta-cells than in the alpha-cells. DNA sequencing revealed that 14 corresponded to known genes (that is, present in GenBank). Only four of those genes had been shown previously to be expressed at higher levels in beta-cells (insulin, islet amyloid polypeptide, neuronatin, and protein kinase A regulatory subunit [RIalpha]). The known genes include transcription factors (STAT6) and mediators of signal transduction (guanylate cyclase). The remaining 12 genes are absent from the GenBank database or are present as expressed sequence tag (EST) sequences (4 clones). Some of the genes are expressed in a highly specific pattern-expression in betaTC1 and islet cells and in relatively few of the non-beta-cell types examined; others are expressed in most cell types tested. The identification of these differentially expressed genes may aid in attaining a clearer understanding of the mechanisms involved in beta-cell function and of the possible immunogens involved in development of type 1 diabetes.

Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and downregulates autoimmunity to various beta-cell antigens.

A peptide of the human 60-kDa heat-shock protein (hsp60), designated p277, was found to be useful as a therapeutic agent to arrest the autoimmune process responsible for diabetes in nonobese diabetic (NOD) mice. The effectiveness of peptide treatment was associated with the induction of peptide-specific antibodies of the IgG1 but not of the IgG2a isotype, suggesting the possibility that a Th2-type response may have been induced. We now report that the effectiveness of p277 treatment is associated with the transient activation of anti-p277 splenic T-cells that produce the Th2 cytokines interleukin-4 (IL-4) and IL-10. The Th2 response to p277 was associated with reduced Th1-type autoimmunity to hsp60 and to two other target antigens associated with diabetes: GAD and insulin. The Th2 shift appeared to be relatively specific; spontaneous T-cell reactivity to a bacterial antigen peptide remained in the Th1 mode in the p277-treated mice. Moreover, treatment with the bacterial peptide did not induce a change in cytokine profile, and it did not affect progression of the disease. Thus, effective peptide treatment of the diabetogenic process associated with the induction of antibodies may be explained by selective and transient activation of Th2 autoimmune reactivity.

In vitro inhibition of insulin release by blood mononuclear cells from insulin-dependent diabetic and healthy subjects: synergistic action of IL-1 and TNF.

Previous studies have demonstrated that peripheral blood mononuclear cells (BMC) from type 1 (insulin-dependent) diabetic patients inhibit insulin release (IR) from rat or mouse islet cells in vitro. This phenomenon is of great interest as a model for islet graft rejection. We found that lipopolysaccharide (LPS)-stimulated BMC of healthy donors and type 1 diabetic patients suppress both basal and stimulated insulin secretion. To study whether this inhibition was due to soluble mediators we added supernatants of LPS-stimulated BMC or recombinant human interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) at concentrations comparable to those found in the supernatants to rat islet cells. The inhibitory effect of BMC on islet cells could be transferred by supernatants of LPS-stimulated BMC. We found that neither IL-1 nor TNF alone inhibit IR from dispersed adult rat islet cells. However, the combination of IL-1 and TNF was highly effective. Ultrafiltration of supernatants of LPS-stimulated BMC through a PM-10 membrane (10 kDa cutoff) deprived the supernatants of the inhibitory activity indicating that only intact IL-1 and TNF (m.w. about 17 kDa), but not smaller IL-1 and TNF fragments, were responsible for the effects on islet cells. These data suggest that activation of BMC and cytokine release at islet graft site may result in an early loss of graft function. Islet transplantation using microcapsules not permeable for molecules with m.w. > 10 kDa would be preferable.

[In vitro phenomenon of beta cell migration from fetal calf pseudoislets]. / Fenomen migratsii beta-kletok iz psevdoostrovkov podzheludochnoi zhelezy plodov krupnogo rogatogo skota in vitro.

Pseudoislets--pancreatic microfragments containing a lot of beta-cells and capable of insulin secretion in vitro-were obtained from 12 fetal calf pancreata by the use of collagenase. Morphological and functional changes of the pseudoislets were studied during culture. We found a rapid migration of beta-cells out of the pseudoislets to the bottom of plastic tissue culture plates. This process was accompanied by a significant decrease of insulin-secreting capacity of the floating microfragments. This should be taken into consideration in cases when pseudoislets are prepared for transplantation in order to avoid beta-cell loss.

[Neoislets obtained from rat pancreas do not contain passenger leukocytes]. / Neoostrovki, poluchennye iz podzheludochnoi zhelezy krysy, ne soderzhat leikotsitov-passazhirov.

Reaggregation of pancreatic islet cells during their cultivation in stationary culture leads to formation of neoislets--compact insulin secreting clusters. By the means of indirect immunofluorescence it has been shown that neoislets have no leukocytes-passengers, common for normal islets and responsible for allograft rejection.

[Interactions of mononuclear cells of peripheral blood with islet cells in vitro]. / Vzaimodeistvie mononuklearov perifericheskoi krovi s ostrovkovymi kletkami in vitro.

Peripheral blood mononuclear cells from Type I diabetic patients health donors or Wistar rats were cultured with rat islet cells for 18 h. Then TNF content of the medium and basal and stimulated insulin release were determined. Mononuclear cells from both healthy donors and diabetic patients could inhibit the insulin release with no correlation to TNF content. Addition of lipopolysaccharide resulted in a 5-7 times increase of TNF content of the medium followed by a more pronounced inhibition of insulin release. Rat mononuclear cells inhibited the beta-cell function almost completely and initially produced large amounts of TNF. The data indicate that inhibition of insulin release by blood mononuclear cells in vitro does not reflect anti beta-cell specific cellular immunity, involves cytokines and, probably depends upon the initial properties of the cells.

The pathogenicity of islet-infiltrating lymphocytes in the non-obese diabetic (NOD) mouse.

The aim of the present study was to investigate the pathogenic properties of islet-infiltrating lymphocytes related to the severity of the autoimmune destruction of islet beta-cells in the NOD mouse. We analysed the development of insulin-dependent diabetes mellitus (IDDM) produced by adoptive transfer of islet lymphocytes from NOD into NOD.scid mice. Here we show that the transfer was most effective when both CD4+ and CD8+ T cells were present in the infiltrate, but CD4+ T cells alone were sufficient to cause the disease. Islet lymphocytes from both females and males transferred diabetes effectively, but the severity of IDDM was higher when female islet lymphocytes were used. Unexpectedly, the sensitivity of male islets to beta-cell damage was greater than that of female islets. Treatment of NOD females with a peptide of heat shock protein (hsp)60, p277, known to protect NOD mice from IDDM, reduced the pathogenicity of the islet lymphocytes. In contrast, administration of cyclophosphamide to males, a treatment that accelerates the disease, rendered the islet lymphocytes more pathogenic. More severe disease in the recipient NOD.scid mice was associated with more interferon-gamma (IFN-gamma)-secreting islet T cells of the NOD donor. The disease induced by islet lymphocytes was strongly inhibited by co-transfer of spleen cells from prediabetic mice, emphasizing the regulatory role of peripheral lymphocytes. Thus, the cellular characteristics of the islet infiltrate and the pathogenicity of the cells are subject to complex regulation.

Islet T cells secreting IFN-gamma in NOD mouse diabetes: arrest by p277 peptide treatment.

Non-obese diabetic (NOD) mice spontaneously develop insulin-dependent (type 1) diabetes mellitus (IDDM) caused by T cells which destroy the insulin-producing islet beta-cells. Since cytokines are involved in this auto-immune beta-cell damage, we used an ELISPOT assay to enumerate the islet-associated T cells that secreted interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) or interleukin-4 (IL-4). We used mitogenic anti-CD3 antibody to activate all the T cells capable of responding, irrespective of their antigen specificity. We found that NOD females, more susceptible than males to IDDM, accumulated islet IFN-gamma producers more rapidly with age than did the males. Acceleration of male IDDM by cyclophosphamide led to a marked increase in IFN-gamma secreting islet T cells. In contrast, a decrease in IFN-gamma-producing islet T cells was associated with arrest of IDDM by administration of peptide p277 of the 60 kDa heat-shock protein (hsp60) to 12-week-old female NOD mice. The p277-treated mice later manifested a greater number of islets and fewer leukocytes per islet than did the mice treated with a bacterial hsp60 peptide. Thus, the development of diabetes could be correlated with the accumulation in the islets of T cells producing IFN-gamma, and destructive insulitis could be downregulated by the administration of a single peptide.

Induction of diabetes in standard mice by immunization with the p277 peptide of a 60-kDa heat shock protein.

We previously reported that immunity to the p277 peptide of the human 60-kDa heat shock protein (hsp60) was a causal factor in the diabetes of non-obese diabetic (NOD) mice, which are genetically prone to develop spontaneous autoimmune diabetes. The present study was done to test whether immunization with the p277 peptide could cause diabetes in standard strains of mice. We now report that a single administration of the p277 peptide conjugated to carrier molecules such as bovine serum albumin or ovalbumin can induce diabetes in C57BL/6 mice and in other strains not genetically prone to develop diabetes. The diabetes was marked by hyperglycemia, insulitis, insulin autoantibodies, glucose intolerance and low blood levels of insulin. The diabetes could be transferred to naive recipients by anti-p277 T cell lines. Similar to other experimentally induced autoimmune diseases, the autoimmune diabetes remitted spontaneously. After recovery, the mice were found to have acquired resistance to a second induction of diabetes. Susceptibility to induced diabetes in C57BL/6 mice was influenced by sex (males were much more susceptible than were females) and by class II genes in the major histocompatibility complex (B6.H-2bm12 mice with a mutation in the MHC-II molecule were relatively resistant). Other strains of mice susceptible to induced diabetes were C57BL/KSJ, C3HeB/FeJ, and NON/Lt. BALB/c and C3H/HeJ strains were relatively resistant. Immunization to p277-carrier conjugates could also induce transient hyperglycemia in young NOD mice, but upon recovery from the induced diabetes, the NOD mice were found to have acquired resistance to later development of spontaneous diabetes. Thus, T cell immunity to the p277 peptide can suffice to induce diabetes in standard mice, and a short bout of induced diabetes can affect the chronic process that would otherwise lead to spontaneous diabetes in diabetes-prone NOD mice.

[Cultures of islet cells obtained from the fetal bovine pancreas]. / Kul'tury ostrovkovykh kletok, poluchennye iz podzheludochnoi zhelezy plodov krupnogo rogatogo skota.

The pancreas from bovine fetuses of 27-35 cm crown-rump length were used as a source of islet cell cultures. Pancreatic tissue was treated by collagenase, filtered through the metal sieve and incubated in MEM for 24 h. Using this method, cultures similar to so-called pseudo-islets were obtained. Aldehyde-fuchsin staining and electron microscopy revealed a significant number of beta-cells within the pseudo-islets, the insulin-producing activity of which was confirmed by RIA.

Acceleration of autoimmune diabetes by cyclophosphamide is associated with an enhanced IFN-gamma secretion pathway.

Cyclophosphamide (CY), an alkylating cytostatic drug, is known for its ability to accelerate a number of experimental autoimmune diseases including spontaneous diabetes in NOD mice. The mechanism(s) by which CY renders autoreactive lymphocytes more pathogenic is largely unknown, but it has been postulated that the drug preferentially depletes regulatory (suppressor) T cells. It has been suggested that in cell-mediated autoimmune diseases, Th2-like lymphocytes secreting IL-4 and/or IL-10 provide protection, while Th1-like cells secreting IFN-gamma are pathogenic. In this study, we analysed the effects of CY on autoimmune diabetes and cytokines in two mouse models: the spontaneous diabetes of NOD mice and the diabetes induced in C57BL/KsJ mice by multiple injections of low dose streptozotocin (LD-STZ). In both models, CY induced severe lymphopenia and accelerated the progression to hyperglycemia. This was associated with changes in splenic cytokine patterns indicating a shift towards the IFN-gamma-secreting phenotype. We provide here evidence that IFN-gamma producers are relatively resistant to depletion by CY and that Th0 clones can be shifted towards Th1. However, direct exposure of T lymphocytes to CY may not be a necessary condition for exacerbation of diabetes; NOD.scid mice treated with CY before adoptive transfer of NOD splenocytes developed diabetes at a higher rate than did controls. Thus, the acceleration of diabetes by CY seems to be a complex event, which includes the relatively high resistance of IFN-gamma producers to the drug, their rapid reconstitution, and a Th1 shift of surviving T cell clones.

A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation.

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6-8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.

Changing patterns of cell surface mono (ADP-ribosyl) transferase antigen ART2.2 on resting versus cytopathically-activated T cells in NOD/Lt mice.

AIMS/HYPOTHESIS: ART2.2 is a mouse T-cell surface ectoenzyme [mono (ADP-ribosyl) transferase] shed upon strong activation. We analysed temporal changes in ART2.2 expression in unmanipulated and cyclophosphamide-treated NOD/Lt mice compared with diabetes-resistant control strains. We used NAD, the ART2.2 substrate, to test whether ART-mediated ADP-ribosylation could retard diabetogenic activation of islet-reactive T cells in vitro. METHODS: ART2.2 and CD38, another NAD-utilizing enzyme, were measured by flow cytometry. ADP-ribosylation from ethano-NAD was followed by flow cytometry using a reagent specific for etheno-ADP ribose. RESULTS: Although mature NOD CD4 + and C D8 + T cells expressed ART2.2, this expression was delayed in young NOD mice when compared with control strains. This ontological delay at 3 weeks of age correlated with an early burst of CD25 expression unique to NOD splenic T cells. This pattern was reproduced in cyclophosphamide-accelerated diabetes in young NOD/Lt males, wherein a retarded repopulation of ART2.2 T cells in spleen and islets correlated with development of heavy insulitis and diabetes. NAD inhibited anti-CD3 induced activation of splenic T cells in vitro and also retarded killing of beta-cell targets by NOD islet-reactive CD8 effectors in vitro at concentrations equal to or greater than 1 micromol/l. Evidence suggested that CD38 on B lymphocytes competes with ART2.2 for substrate needed by B lymphocytes for ADP ribosylation. CONCLUSIONS: ART2.2 on T cells may not simply mark the resting state, but could also contribute to it via ADP-ribosylation.

[Aggregates of islet cells, their structure and insulin-producing activity during cultivation in vitro]. / Agregaty ostrovkovykh kletok, ikh struktura i insulinprodutsiruiushchaia aktivnost' v usloviiakh kul'tivirovaniia in vitro.

Purified rat islets were dissociated into single-cell suspension with an EDTA-Trypsin treatment. During a stationary culture in vitro the islet cells reassociated forming aggregates (neoislets). Electron microscopy revealed that the aggregates consisted mostly of beta-cells and not numerous alpha-cells. They showed a good insulin-secreting capacity and were able to increase insulin release in response to glucose plus theophylline. The lack of passenger leukocytes makes the neoislets particularly suited for experimental and clinical transplantation.

A shared TCR CDR3 sequence in NOD mouse autoimmune diabetes.

T cells involved in autoimmune diseases have been characterized by the genetic elements used to construct their autoimmune TCR. In the present study, we sequenced the alpha and beta chains of the TCR expressed by a CD4(+) T cell clone, C9, functional in NOD mouse diabetes. Clone C9 can adoptively transfer diabetes or, when attenuated, C9 can be used to vaccinate NOD mice against diabetes. Clone C9 recognizes a peptide epitope (p277) of the 60 kDa heat shock protein (hsp60) molecule. We now report that the C9 TCR beta chain features a CDR3 peptide sequence that is prevalent among NOD mice. This CDR3 element is detectable by 2 weeks of age in the thymus, and later in the spleen and in the autoimmune insulitis. Thus, a TCR CDR3beta sequence appears to be a common idiotope associated with mouse diabetes.