1 Division of Endocrinology, Department of Internal Medicine, University of Ulm, Ulm, Germany
2 Department of Immunology and Medical Microbiology, University of Ulm, Ulm, Germany
3 National Institute of Diabetes and Digestive and Kidney DiseasesNavy Transplantation and Autoimmunity Branch, Bethesda, Maryland
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ABSTRACT |
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INTRODUCTION |
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Evidence from diabetes-prone BB rats (12) and rat insulin promoter (RIP) ovalbumin (Ova)-transgenic (13) mice has shown that treatment with ß-cell antigens may adversely promote diabetes development. Similarly, T-cellmediated experimental autoimmune encephalitis (14) or autoimmune ovarian disease (15) can be induced by injection of myelin basic protein and ZP3 glycoprotein, respectively, both autoantigens expressed in affected target tissues.
For antigen administration, recombinant proteins or synthetic peptides have been used in most experimental studies. DNA vaccination is a more recent and simple strategy of antigen administration to induce T-cell immunity (16). In DNA vaccination, transient in vivo protein expression is induced in the host through injection of a DNA plasmid vector containing cDNA of a desired gene, thus circumventing difficulties with recombinant protein production and purification. We have recently shown that genetic vaccination using GAD65 cDNA is suitable to induce diabetes protection in young NOD mice (17).
To assess further the therapeutic potential of antigen-specific intervention in type 1 diabetes, we extended our DNA vaccination studies to other ß-cell proteins. Instead of disease protection, we observed diabetes induction in both female and male NOD mice after insulin DNA vaccination. Similarly, insulin (but not GAD65) DNA vaccination specifically induced CD4+CD8+ insulitis, ß-cell destruction, and autoimmune diabetes in RIP-B7.1 transgenic mice, even in the absence of diabetes-risk major histocompatibility cell (MHC) alleles. Disease acceleration must therefore be considered as a potential outcome in antigen-specific diabetes prevention. In this process, ß-cell expression of T-cell costimulatory molecules such as B7.1 may be critical. Our novel experimental autoimmune diabetes (EAD) model should allow to address this and other aspects of insulin-induced diabetic autoimmunity.
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RESEARCH DESIGN AND METHODS |
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For vaccination studies, 4- to 6-week-old mice were given an intramuscular injection of 50 µg of DNA plasmid (dissolved in 50 µl of PBS) into each anterior tibialis muscle after shaving and skin desinfection. Injections were repeated once after 1 week (boost), and no adjuvants, other additives, or pretreatment were used. Animals were screened for diabetes development by weekly measurement of glucosuria (Tes-Tape; Lilly, Indianapolis, IN), and diabetes was confirmed (Glucometer Elite; Bayer, Pittsburgh, PA) by blood glucose levels 13.8 mmol/l (
250 mg/dl). Blood glucose was in addition measured weekly in RIP-B7.1 mice for 8 weeks after vaccination. Animal studies were conducted after institutional board approval according to the German Federal Animal Protection Law.
Construction of DNA vaccination vectors.
Complementary DNA of ß-cell antigens was inserted into plasmid vectors containing the cytomegalovirus (CMV) promoter to achieve eukaryotic gene expression. Mouse preproinsulin (ppIns) II cDNA (Genbank X04727, 110 amino acids [aa]) was generated by RT-PCR from murine islet cell RNA (20), followed by ligation into the EcoRI and XbaI sites of pCI (Promega). Similarly, murine proinsulin II cDNA (86 aa, lacking signal peptide) was generated by RT-PCR, with start codon and Kozak consensus incorporated in the sense primer. For the construction of murine insulin II (51 aa, lacking signal peptide and C-peptide), cDNAs encoding B chain (aa position B1-B30) and A chain (aa position A1-A21) were independently RT-PCR amplified, ligated between position B30 and A1, and inserted into the pCI vector as above. Islet cell antigen 69 cDNA (clone is10, 483 aa) (21) was subcloned into XbaI and SmaI sites of pCI (Promega). The construction of the GAD65 cDNA vector (rat GAD65, Genbank M72422, 586 aa) has been described (17). Except for murine proinsulin and insulin cDNA, complete coding sequences and natural translation initiation and termination sites were used in all constructs, and plasmids contained the Ampr gene characterized by CpG-rich immunostimulatory sequences. All vector sequences were confirmed by bidirectional DNA sequencing.
Plasmids were transformed into supercompetent XL1-blue MRF Escherichia coli (Stratagene, La Jolla, CA), grown in selection medium, and isolated using the Plasmid Mega Prep Kit (Qiagen, Hilden, Germany). Endotoxin levels in plasmid preparations (final DNA concentration, 1 µg/µl) were determined by Dr. J. Endl (Roche Diagnostics, Penzberg, Germany), using the Limulus lysate assay (ppIns 0.06 EU/µl, pGAD65 0.12 EU/µl, pCI control vector 0.06 EU/µl, medium 0.003 EU/µl; BioWhittaker, Walkersville, SC).
Vector-induced gene expression.
Protein synthesis of target autoantigens was studied after transient transfection in HEK 293 cells in vitro. Semiconfluent cells were transfected with 0.510 µg of plasmid vector DNA by standard CaCl2 precipitation, grown for 48 h in Dulbeccos modified Eagles medium/10% FCS medium (Gibco, Karlsruhe, Germany) and harvested. Culture supernatants and cell lysates were analyzed as indicated. GAD65 and ICA69 expression was detected by Western blotting using GAD6 (gift of Dr. W. Richter, Heidelberg, Germany) and polyclonal anti-ICA69 (20) as primary antibodies after 10% SDS-PAGE and semi-dry blotting onto Hybond C membranes (Amersham, Freiburg, Germany). Expression of murine ppIns, proinsulin, and insulin was studied using a radioimmunoassay as described below, with an average intra-assay coefficient of variation of 4.5%.
PpIns mRNA in mouse muscle tissue was analyzed by quantitative real-time RT-PCR (TaqMan, Perkin Elmer), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous reference gene. Ratios of ppIns/GAPDH mRNA levels were calculated after triplicate quantifications, and values are expressed as means ± SD. RNA was DNase-treated (Qiagen) before reverse transcription. Mice were DNA-vaccinated with 100 µg of plasmid as a single injection, and groups of two to three mice were subsequently studied at a given time as indicated.
Histology.
Pancreatic cryosections (5 µm thick) from B7.1 mice were treated with the Avidin/Biotin Blocking Kit (Vector Labs) and incubated with primary antibodies to insulin (Biotrend/Linco Research, St. Charles, MO), CD4 (PharMingen, San Diego, CA), or CD8 (PharMingen) at 4°C overnight. Slides for CD4/CD8 staining were incubated with biotin-conjugated goat anti-rat IgG secondary antibody (Jackson Immuno Research, Westgrove, PA), followed by incubation with horseradish peroxidaseconjugated avidin (Vectastain Elite ABC; Vector Labs). Sections for insulin staining were incubated with a peroxidase-conjugated rabbit anti-guinea pig IgG secondary antibody (A5545; Sigma, St. Louis, MO). Finally, slides were developed with diaminobenzidine (DAB Substrate Kit; Vector Labs) and counterstained with hematoxylin. Specificity of immunostaining was confirmed by using irrelevant primary antibody (goat anti-human IgA) as control. Insulitis scores in NOD mice were determined in pancreatic sections as described (22).
Pancreatic insulin contents.
Insulin was isolated from snap-frozen and cryoconserved (-80°C) pancreatic tissue by standard 80% ethanol/0.1 mol/l hydrochloric acid extraction. For measuring extractable insulin, a radioimmunoassay for rat insulin (with 100, 69, and 100% cross-reactivity to murine insulin, human proinsulin, and human insulin, respectively) was used (Linco Research). Insulin contents are expressed as nanograms per milligram of wet-weight tissue.
Statistical analysis.
Statistical analyses were made with SAS software, version 8.2 (SAS Inc., Cary, NC). For comparison of diabetes incidences, Fishers exact test was used. Means were compared using the Wilcoxon or Kruskal-Wallis test, assuming nonparametric statistics. Statistical significance was defined as P < 0.05.
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RESULTS |
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Diabetes development in mice that were vaccinated with murine ppIns (containing insulin B chain, A chain, signal, and connecting peptide) was 88.0% (Fig. 3), i.e., disease development was not reduced in comparison with control DNA-treated or untreated NOD mice. Instead, there was a trend toward higher cumulative diabetes incidences in ppIns-vaccinated mice compared with control groups (P = 0.09). In female NOD mice that were DNA-vaccinated with GAD65 (Fig. 3), however, diabetes development was significantly reduced (P = 0.0013), confirming previous observations (17).
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The degree of pancreatic islet cell inflammation, expressed as the mean insulitis score at 32 weeks of age (22), was similar (P = 0.41, Kruskal-Wallis) in nondiabetic NOD mice after GAD treatment (score 2.39 ± 0.41), ppIns treatment (score 1.92 ± 0.57), and control DNA treatment (score 2.08 ± 0.38).
Notably, diabetes incidences were not different in mice that were treated with control DNA plasmid compared with unvaccinated NOD mice (Fig. 3). Thus, major antigen-independent effects of the DNA plasmid vector backbone carrying the immunostimulatory CpG motif or of contaminating agents in the plasmid DNA preparation, which may affect diabetes development (23), can be ruled out in our studies.
Loss of natural diabetes resistance in male NOD mice after insulin DNA vaccination.
In contrast to female NOD mice, male NOD mice are characterized by a considerable natural (spontaneous) diabetes resistance, despite the presence of autoimmune islet cell infiltration (insulitis) (24). We therefore chose male NOD mice as model system with low spontaneous diabetes probability to test for disease acceleration after insulin DNA vaccination, as hypothesized from our observations in female animals.
Male NOD mice were DNA-vaccinated at 56 weeks of age, using the same protocol as in female mice (Fig. 4). Diabetes incidences after DNA vaccination with ppIns were increased to 63.6% at 32 weeks of age, compared with 12.5% in GAD65-vaccinated mice (P = 0.03). Similarly, in mice that were vaccinated with insulin DNA, a trend toward higher diabetes incidences was observed compared with control or GAD65-treated mice (P = 0.17). Taken together, despite different natural diabetes rates in male and female animals, (prepro)insulin DNA vaccination does not protect but rather promotes diabetes development in both male and female NOD mice.
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We translated these findings from LCMV to insulin as a "natural" diabetes autoantigen. RIP-B7.1 transgenic mice expressing B7.1 in pancreatic ß-cells under control of the RIP were used for DNA vaccination studies (Table 1). Importantly, these mice have a C57BL/6 (H-2b) MHC background, thus lacking genetic diabetes risk alleles required for diabetes development in NOD mice. RIP-B7.1 mice (n = 8) did not develop diabetes spontaneously, even after an extended follow-up of 48 weeks. Similarly, RIP-B7.1 mice that were vaccinated with DNA control plasmid (n = 16) did not develop hyperglycemia or other signs of disease (Table 1). However, DNA vaccination with ppIns was followed by the development of diabetes (Table 1) in 14 of 20 RIP-B7.1 mice (incidence 70%, P < 0.001). Diabetes was diagnosed in these animals after a median of 39 days (range 22134 days). Hyperglycemia was characterized by severe insulin deficiency and heavy lymphocytic infiltration of pancreatic islets by CD4+ and CD8+ T-cells, as described below. In contrast, diabetes did not occur in any of 15 mice that were DNA-vaccinated with GAD65 (incidence 0%; Table 1), despite effective vector-induced gene expression. Similarly, nontransgenic (wt/wt) C57BL/6 mice that were vaccinated with ppIns did not develop diabetes (Table 1). When ppIns DNA vaccination doses for were reduced from 100 to 10 µg, diabetes development was abolished (7 of 13 vs. 0 of 14; P < 0.002). Taken together, diabetes induction in C57BL/6 mice is linked to the ß-cell expression of the B7.1 costimulatory molecule. In addition, the disposition to develop diabetes after DNA vaccination in RIP-B7.1 mice seems limited to particular ß-cell autoantigens.
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Diabetes after DNA vaccination occurred equally in male and female RIP-B7.1 mice. A total of 15 of 23 (65.2%) female mice and 7 of 13 (53.8%) male mice developed diabetes after ppIns, proinsulin, or insulin DNA vaccination (P = 0.72), with identical disease onset after vaccination in both sexes (median 50 days vs. 39 days). Similarly, diabetes rates in mice that were vaccinated at 23 weeks of age were not different from those of animals treated at weeks 46 (P > 0.05). In contrast to NOD mice, EAD after insulin DNA vaccination in RIP-B7.1 mice is characterized by an equal disease susceptibility in male and female individuals without sex bias, thus resembling human type 1 diabetes.
Characteristics of EAD in RIP- B7.1 transgenic mice.
To analyze mechanisms of diabetes development in RIP-B7.1 mice after DNA vaccination, we first studied islet morphology in serial pancreatic sections after hematoxylin and eosin staining. Pancreatic islets in mice that were vaccinated with insulin or ppIns DNA showed a dense, homogeneously distributed mononuclear cell infiltration in the islets of Langerhans, affecting all islets at similar cell density. In contrast, sections from mice after GAD65 or control DNA vaccination did not exhibit cellular infiltration in the islets (data not shown). For further characterizing islet infiltration in these animals, immunostaining for CD4 and CD8 was used. In mice that were treated with insulin DNA, a dense accumulation of CD4+ (Fig. 5D) and CD8+ cells (Fig. 5E) was observed, whereas such cells were absent in the islets of GAD65-treated (Fig. 4G and H) or control DNA-treated (Fig. 4A and B) mice. Some CD8+ cells were detectable in surrounding exocrine pancreatic tissue, although at low density (Fig. 4E). In mice that were vaccinated with ppIns (data not shown), identical immunostaining was observed as in insulin-treated mice. Taken together, insulin and ppIns DNA-vaccinated animals are characterized by a prominent CD4+CD8+ insulitis.
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DISCUSSION |
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Diabetes occurred specifically after DNA vaccination with (prepro)insulin, whereas GAD65 treatment conferred partial diabetes protection, confirming previous findings in NOD mice (17). From these observations, diabetes induction upon DNA vaccination with ß-cell antigens is not a general feature in RIP-B7.1 and NOD mice but seems limited to particular autoantigens. High gene expression levels and nearly exclusive tissue specificity may in this context set apart insulin from other ß-cell proteins. The potential effect of autoantigens other than insulin and GAD65 in diabetes intervention will require additional study.
Prevention of NOD mouse diabetes after DNA vaccination with hsp60 has been described (23), but treatment with control plasmid (not containing antigen cDNA) was equally effective in that report. In contrast, in our studies, treatment with empty DNA vector neither promoted nor prevented diabetes development, so major antigen-independent treatment effects of DNA vaccination (e.g., mediated by plasmid CpG motifs or contaminants) may be excluded in our work.
In a recent study (26), DNA vaccination with insulin ß chain conferred diabetes protection in young NOD mice, whereas DNA constructs encoding the entire (prepro)insulin protein were not studied in that work. Insulin ß chain contains major diabetes-associated T-cell epitopes, and it is conceivable that treatment outcomes reflect the differential choice of antigenic determinants expressed by the DNA vaccine. In addition, plasmid design and vaccination modalities may affect the clinical outcome of DNA vaccination in NOD mice (26,27).
To map diabetes induction to functional domains of the insulin molecule, we constructed and tested in vivo deletion-mutant vectors for DNA vaccination. Insulin and its precursor molecules proinsulin and ppIns all were effective to induce autoimmune diabetes in our experimental system. Therefore, insulin (A121) and ß (B130) chain domains seem sufficient to confer diabetogenic effects of the DNA vaccine. Ongoing vaccination studies using insulin minigene constructs will help to define T-cell epitopes that mediate in vivo diabetogenicity in this novel EAD model.
Diabetes development in RIP-B7.1 mice after insulin treatment was characterized by a diffuse infiltration of pancreatic islets with CD4+ and CD8+ T-cells, whereas insulitis was absent after GAD65 or control DNA vaccination. In RIP-LCMV/B7.1 transgenic mice, we previously observed insulitis and diabetes development upon DNA vaccination with LCMV glycoprotein, accompanied by the induction of CD8+ cytotoxic T-cell (CTL) responses against the immunodominant LCMV epitope gp33 (K. Pechhold et al., unpublished observations). T-cell proliferative responses to proinsulin were not significantly different in NOD mice that were vaccinated with ppIns or control plasmid (data not shown), reminiscent of T-cell proliferative responses in GAD65-vaccinated animals (17). A recent workshop report (28) concluded that instead of proliferation, more sensitive assays (including ELISPOT or tetramer technology) may be preferred to study autoreactive T-cells. Thus, the exact contribution of individual T-cell subsets to diabetes induction in NOD and B7.1 mice after (prepro)insulin DNA vaccination remains to be clarified.
In RIP-B7.1 mice, diabetes developed on a wild-type C57BL/6 (H-2b) genetic background, not carrying diabetes-associated MHC alleles or other established disease risk loci, thus resembling most cases of sporadic human type 1 diabetes. As nontransgenic C57BL/6 mice remained healthy after DNA vaccination with insulin, diabetes susceptibility is directly linked to the expression of B7.1 in pancreatic ß-cells. In mice and humans, professional APCs upregulate B7.1 upon various activation signals, including viral and bacterial products, CD40:CD40L interaction, or interferon- (IFN-
) and other cytokines. We showed previously that functional B7.1 expression can be induced on nonprofessional APCs after treatment with IFN-
(29). It is conceivable that ongoing ß-cell inflammation (e.g., through enterovirus infection) thus may increase diabetes susceptibility independent of MHC risk alleles. Indeed, enteroviral infection has been implicated frequently (e.g., 30) as a relevant environmental risk factor for human type 1 diabetes. In this scenario, T-cell sensitization to insulin after its release from ß-cell damage or through exogenous insulin administration may adversely promote insulitis and autoimmune diabetes, as shown in our work.
In a variety of animal studies (15), local or systemic application of insulin or insulin fragments using different treatment protocols has been shown to be effective to reduce diabetes development in NOD mice, with antigen therapy commonly initiated at early stages of autoimmunity to achieve disease protection. Protection was associated with T-cell regulation or tolerance induction in some of these studies. However, oral administration of an Ova to mice expressing Ova as a transgene in pancreatic ß-cells led to the induction of cytotoxic T-cells and autoimmune diabetes (13). Similarly, insulin treatment in conjunction with bacterial adjuvant resulted in diabetes acceleration in diabetes-prone BB rats (12). This effect was attributed to increased enteral expression in the gut of IFN-, a potent enhancer of Th1 and CTL immunity and T-cell costimulation (12). In other T-cellmediated autoimmune disorders, experimental autoimmune encephalitis (14) and autoimmune ovarian disease (15) have been induced by myelin basic protein and ZP3 glycoprotein, respectively, both autoantigens expressed in affected tissues. We now demonstrate for the first time the induction of EAD by insulin in naturally diabetes-resistant mice, as well as disease promotion in the classic NOD diabetes model. Taken together, these observations suggest that treatment of autoimmune disorders with autoantigen may confer either disease protection or acceleration, depending on factors (e.g., T-cell costimulation, cytokine environment) other than the antigenic determinant itself.
Our work may have significant clinical implications. Insulin is being used in a large clinical trial (11) to prevent autoimmune diabetes in high-risk individuals with detectable islet cell antibodies and impaired insulin secretion, marking progressive diabetic autoimmunity. In advanced insulitis preceding overt diabetes in NOD mice, secretion of IFN- (a potent inducer of B7.1 costimulation) prevails (31). We have shown that co-vaccination with B7.1 cDNA abrogates GAD65-mediated diabetes protection in NOD mice (17). Antigen treatment in the presence of B7.1 upregulation, e.g., during advanced diabetic autoimmunity, may result in a substantially different outcome compared with treatment in unprimed individuals. When therapeutically given at time of diabetes manifestation, insulin has indeed been shown to be ineffective to arrest ß-cell destruction in patients with type 1 diabetes (32). In a clinical setting (33), antigen treatment seems particularly problematic during episodes of systemic or viral infections or other states of increased IFN-
secretion.
In conclusion, clinical deterioration of autoimmune disease may be a potential adverse outcome after antigen-specific immune therapy. Additional study of its dual role in disease prevention and acceleration should be considered before insulin (and other autoantigens) may be fully accepted as immunologically safe for the prevention of human type 1 diabetes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication 17 October 2001 and accepted in revised form 8 August 2002.
aa, amino acids; APC, antigen-presenting cell; CMV, cytomegalovirus; CTL, cytotoxic T-cell; EAD, experimental autoimmune diabetes; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-, interferon-
; LCMV, lymphocytic choriomeningitis virus; MHC, major histocompatibility cell; ppIns, preproinsulin; Ova, ovalbumin; RIP, rat insulin promoter.
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REFERENCES |
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