The IDDM (LEW.1AR1/Ztm-iddm) rat is a type 1 diabetic animal model characterized by a rapid apoptotic pancreatic β-cell destruction. Here we have analyzed the time course of islet infiltration, changes in the cytokine expression pattern, and β-cell apoptosis in the transition from the pre-diabetic to the diabetic state. Transition from normoglycemia to hyperglycemia occurred when β-cell loss exceeded 60–70%. At the early stages of islet infiltration, macrophages were the predominant immune cell type in the peripherally infiltrated islets. Progression of β-cell loss was closely linked to a severe infiltration of the whole islet by CD8+ T-cells. With progressive islet infiltration, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were expressed in immune cells but not in β-cells. This proinflammatory cytokine expression pattern coincided with the expression of inducible nitric oxide synthase (iNOS) and procaspase 3 in β-cells and a peak apoptosis rate of 6.7%. Islet infiltration declined after manifestation of clinical diabetes, yielding end-stage islets devoid of β-cells and immune cells without any sign of cytokine expression. The observed coincidence of IL-1β and TNF-α expression in the immune cells and the induction of iNOS and procaspase 3 mRNA expression in the β-cells depicts a sequence of pathological changes leading to apoptotic β-cell death in the IDDM rat. This chain of events provides a mechanistic explanation for the development of the diabetic syndrome in this animal model of human type 1 diabetes.

Type 1 diabetes is an autoimmune disease leading, after a pre-diabetic period, to overt diabetes due to destruction of the pancreatic β-cells (13). Despite many efforts, the mechanisms of interaction between the infiltrating immune cells in the pancreatic islets and pancreatic β-cell death, which finally causes insulin-dependent diabetes, are not well understood (13). The pre-diabetic period is of special interest for understanding the mechanisms responsible for pancreatic β-cell destruction. Animal models of human type 1 diabetes can provide an important contribution to a better understanding of the etiopathology underlying apoptotic β-cell death in type 1 diabetes. The IDDM (LEW.1AR1/Ztm-iddm) rat is a new, particularly attractive autoimmune diabetes animal model (4) for the analysis of the different stages of the immune cell infiltration process because of the short normoglycemic pre-diabetic period with pancreatic islet infiltration of ∼1 week before ending in a state of overt hyperglycemia due to apoptotic β-cell destruction (4,5). In contrast to the bio-breeding (BB) rat and the nonobese diabetic (NOD) mouse as the most studied rodent models, the IDDM rat closely resembles the diabetic syndrome in humans because the animals show a fully developed cellular immune system and no sex bias in diabetes incidence (4,5). In this study, the process of immune cell infiltration in the islets of the pancreas of littermates originating from a homozygous pairing of diabetic parents was investigated. The results allow us to delineate the sequence of pathological changes leading to apoptotic β-cell death in this animal model of human type 1 diabetes.

LEW.1AR1/Ztm-iddm rats were housed in the Institute of Laboratory Animal Science of Hannover Medical School as described previously (4). The animals originated from a breeding colony (4) that was maintained through mating of diabetic female and diabetic male IDDM rats (F7; seventh generation of the IDDM rat; see http://www.mh-hannover.de/institute/clinbiochemistry/iddmrat.htm for details). Homozygous pairing with both parents diabetic resulted in a diabetes incidence of ∼60%. All rats were maintained according to the FELASA (Federation of European Laboratory Animal Science Associations) guidelines. The animals were investigated at days 45, 50, 55, 59, 60, or 65 after birth. On the day the animals were killed, tissue samples of pancreas and spleen were collected for morphologic examination. All analyses were performed on the same set of animals. Blood glucose concentrations were determined using capillary blood samples from the tail vein by the glucose oxidase method (Glucometer Elite; Bayer, Leverkusen, Germany). Serum insulin concentrations were determined by a rat insulin microplate enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden).

Tissue processing.

Tissue specimens of pancreas and spleen were fixed in 4% paraformaldehyde, in 0.15 mol/l PBS, pH 7.3, or rapidly frozen in liquid nitrogen. Fixed tissue was embedded in paraffin, and frozen tissue was stored at −70°C for cryostat histology. Additionally, small pancreatic tissue samples were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH 7.3, postfixed in 1% OsO4, and finally embedded in Epon for electron microscopical analysis (5).

Immunohistochemistry.

Serial paraffin and cryostat sections were stained by the avidin-biotin-complex (ABC) method (6). The slides were incubated overnight with the specific antibodies, followed by a 30-min incubation with biotinylated goat anti-rabbit IgG (1:100) or biotinylated rabbit anti-mouse IgG (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). The slides were then incubated with a mixture of streptavidin (1:100) and biotin-peroxidase (1:1,000) (Jackson Immuno Research, West Grove, IL). The peroxidase reaction was visualized using 0.7 mmol/l diaminobenzidine and 0.002% hydrogen peroxidase in PBS, pH 7.3. The following primary antibodies were used: insulin (1:500) (polyclonal A565; DAKO, Hamburg, Germany), infiltrating macrophages (ED1), tissue macrophages (ED2), dendritic cells (Ox 62), B-cells (IgD), pan–T-cells (R 73), CD4+ T-cells, CD8+ T-cells (β-chain), NK cells (CD 161), major histocompatibility complex class II (Ox 6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) (1:500) (all monoclonal; from Serotec, Oxford, U.K.), inducible nitric oxide synthase (iNOS) (1:50) (polyclonal; Santa Cruz Biotechnology), and activated caspase 3 (1:200) (polyclonal; Biosciences Pharmingen, Brussels, Belgium). All antibodies were certified for immunohistochemistry and showed specific immunostaining in tissue sections of the IDDM rat.

In situ RT-PCR.

Pancreatic sections were fixed on 3-Chamber SuperFrost Plus slides on a heating block at 100°C for 2 min. Subsequently, the slides were treated with proteinase K (20 μg/ml) for 20 min at 37°C. Proteinase K was inactivated thereafter at 95°C for 2 min followed by an overnight incubation with a RNase-free DNase solution (1 unit/ml) at 37°C. The in situ RT-PCR procedure was a modification of different protocols (7,8). After rinsing with PCR-grade water and air drying, reverse transcription was performed in a buffer containing a mixture of all nucleotides (1 mmol/l dATP, dGTP, dCTP, and dTTP) (Genecraft, Lüdinghausen, Germany), oligo-dT primer (Invitrogen, Paisley, U.K.), M-MLV (Moloney murine leukemia virus) reverse transcriptase (0.5 units/μl) (Invitrogen, Paisley, U.K.), RNasin (Promega, Madison, Wisconcin), and ddH2O. Slides were incubated at 37°C for 1 h in a moist chamber, and thereafter reverse transcriptase was inactivated at 92°C for 2 min. PCR amplification was performed in a buffer containing a nucleotide mix (10 μmol/l digoxigenin 11-dUTP, 190 μmol/l dTTP, and 200 μmol/l each of dATP, dCTP, and dGTP), the specific forward and reverse primer (1.25 μmol/l) and Taq biotherm polymerase (0.1 unit/μl) (Genecraft), self-seal reagent (MJ Research, Waltham, MA), and ddH2O. Fifteen microliters of the PCR mix was applied to each chamber of the slide and sealed by cover slips. The slides were then placed in a thermal cycler suitable for in situ amplifications (MJ Research). PCR amplification was performed according to the following protocol: initial denaturation at 95°C for 3 min, followed by 35–40 cycles with a denaturation at 95°C for 45 s, annealing at 57°C for 45 s, and extension at 72°C for 45 s. A final extension was performed for 10 min at 72°C. After removal of the cover slips, the slides were incubated with a blocking reagent for 1 h. Thereafter, the incorporated digoxigenin-labeled nucleotides were detected by an anti-digoxigenin antibody labeled with alkaline phosphatase (1:500) or rhodamine (1:300) at room temperature for 1 h. Alkaline phosphatase activity was detected by the NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt) color reaction (overnight incubation at room temperature). The primer sequences were rat IL-1β 5′-GCATCCAGCTTCAAATCTC-3′ (forward) and 5′-GGTGCTGATGTACCAGTTG-3′ (reverse), rat TNF-α 5′-CTACTGAACTTCGGGGTGATCGGTC-3′ (forward) and 5′-CTGGTATGAAGTGGCAAATCGGCT-3′ (reverse), rat IFN-γ 5′-CATTGAA-AGCCTAGAAAGTCTG-3′ (forward) and 5′-CGACTCCTTTTCCGCTTC-3′ (reverse), rat iNOS 5′-CTTCAACACCAGGTTGT-CTGCAT-3′ (forward) and 5′-ATGTCATGAGCAAAGGCACACAGAAC-3′ (reverse), and rat procaspase 3 5′-TGGTACCGATGTCGATGCAG-3′ (forward) and 5′-TACCGCA-GTCCAGCTCTGTACC-3′ (reverse).

Morphological analysis of islet infiltration.

The cellular composition of the different subsets of infiltrating immune cells was analyzed immunohistochemically using specific antibodies on consecutive cryostat sections. Ten to 15 pancreatic islets were analyzed in each pancreas at the different time points during diabetes development (45–65 days of age). The migration pattern of mononuclear immune cells within the different stages of islet infiltration and the changes in the ratio of ED1+ macrophages to CD8+ T-cells were analyzed by series of 100 consecutive paraffin sections. Pancreatic tissue was scored without knowledge of the glycemic status of the animals. Islet infiltration was graded on a score of 0–4 as follows: stage 0, no immune cell infiltration of the islets (control); stage 1, immune cell infiltration restricted to the periphery of the islets; stage 2, weak invasive immune cell infiltration of the islets; stage 3, large numbers of immune cells within the islets; and stage 4, complete β-cell loss without infiltration (termed “end-stage islets”). A mean insulitis score was calculated from 20 to 30 islets per individual pancreas. The stages of islet infiltration were ultrastructurally confirmed by electron microscopy. The pancreatic β-cell area of the islets was identified by immunostaining for insulin and morphometric quantification by a computer-assisted system as described previously (9). Apoptotic cells were identified by ultrastructural analysis and additionally quantified after TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining (10) using an in situ cell-death detection kit (Roche, Mannheim, Germany). The criteria for β-cell apoptosis were a positive staining for insulin and TUNEL-positive nuclei. The immunostained sections were viewed using bright field illumination with a Zeiss Photomicroscope II (Zeiss, Oberkochen, Germany).

Time course of diabetes development.

Extensive morphologic examinations of pancreata from IDDM rats at all ages up to 45 days revealed no signs of infiltration (data not shown). Between days 50 and 60, diabetes incidence increased from 5 to 60% with a particularly steep increase from 21 to 60% between day 59 and 60 (Table 1). At day 50, 20% of the pancreases from normoglycemic animals already showed signs of a faint infiltration of the islets with an insulitis score of 1.1 (Tables 1 and 2). Normoglycemic animals with pancreas infiltration could be observed until day 59, when 29% of the pancreases were infiltrated with an average insulitis score of 2.4 (Tables 1 and 2). The insulitis score was always higher in the diabetic animals than in the normoglycemic counterparts, with pancreas infiltration at defined time points between day 50 and day 59 (Table 1). Normoglycemic IDDM rats with pancreas infiltration showed a gradual loss of β-cell area approaching 70% at day 59 (Table 2), whereas a diabetic state was usually observed after β-cell loss of >70% (Table 3). Thus, the critical loss of the β-cell mass for onset of overt diabetes in IDDM rats was in the range of 50–70%. In diabetic animals (blood glucose >7 mmol/l) blood glucose concentrations increased from 10.9 mmol/l (day 50) to 19.5 mmol/l (day 60), whereas serum insulin concentrations decreased from 0.8 ng/ml (day 50) to 0.1 ng/ml (day 60) (Table 1).

Time course of pancreas immune cell infiltration.

At day 50, upon immunohistochemical analysis, ED1+ macrophages, which stayed in the range of 60–80 cells/mm2 until day 59 (Table 2), were the predominant immune cell subpopulation within infiltrated islets in normoglycemic rats. A comparable ED1 macrophage infiltration in the range of 70–90 cells/mm2 could also be observed in diabetic animals (Table 3). CD4+ T-cell islet infiltration ranged between 15 and 20 cells/mm2 at day 50 and doubled until day 59 in normoglycemic rats with pancreas infiltration (Table 2) as well as in diabetic rats (Table 3). NK cells and B-cells could be detected in islets of normoglycemic rats with pancreas infiltration as well as in diabetic rats between days 50 and 59 at numbers of <20 and <10 cells/mm2, respectively (Tables 2 and 3). CD8+ T-cell islet infiltration dramatically increased by a factor of five from days 50 to 59 in normoglycemic rats with pancreas infiltration, approaching a value of nearly 150 cells/mm2 (Table 2) in parallel to the increase of the insulitis score (Table 1) and the decrease of β-cell area (Table 2). Diabetic animals manifesting diabetes between ages 50 and 60 days showed constantly high numbers (160–200 CD8+ T-cells per mm2) in the infiltrated islets (Table 3). The decrease of the β-cell area in normoglycemic rats with pancreas infiltration (Table 2) as well as in diabetic animals (Table 3) showed an excellent correlation (r2 = 0.998) with the insulitis score (Table 1). Also, the CD8+ T-cell infiltration showed a high correlation (r2 = 0.996) with the different stages, indicating that this immune cell subpopulation contributes preeminently to the infiltration in the late stages of β-cell destruction in the type 1 diabetes rat model (Table 4). Due to the sharp increase in the number of CD8+ T-cells, the relative percentage of ED1+ macrophages in the total number of immune cells decreased to 30% in stage 3 (Table 4). CD4+ T-cell infiltration was doubled when the insulitis score increased from stage 1 to stage 3, but the relative percentage always remained in the range of 10% of the total immune cell population (Table 4). At the early islet infiltration stage 1, the total number as well as the percentage of the CD4+ T-cells was identical to that of the CD8+ T-cells (Table 4). NK cells and B-cells represented only a minor fraction (<10%) of the infiltrating immune cells in islet stages 1–3 (Table 4).

Pancreas morphology before diabetes development at 45 days of age.

At day 45 of life, all animals were still normoglycemic (Table 1). None of the pancreatic islets and the surrounding pancreas parenchyma of these diabetes-prone IDDM rats showed any signs of insulitis or peri-insulitis (Fig. 1A). Occasionally one to three immune cells, mostly ED1+ macrophages, were observed in the sections of the noninfiltrated islets corresponding to stage 0 in the classification of the islet infiltration stages (Fig. 2A and C and Table 4). There was also no accumulation of ED1+ infiltrating and ED2+ tissue macrophages as well as of major histocompatibility class II -and Ox 62–positive dendritic cells in the connective tissue around the vessels (data not shown). The pancreatic β-cells within the typical mantle islets were well granulated and showed no signs of apoptosis (Fig. 2B). None of the endocrine cell types of the islets as well as none of the different cell types of the exocrine parenchyma showed mRNA expression of the proinflammatory cytokines IL-1β (Fig. 3A) or TNF-α (Fig. 3D) in the cytoplasm. iNOS mRNA expression (Fig. 3G) was also not detectable in pancreatic β-cells, but a few islet cells showed a faint expression of procaspase 3 mRNA (Fig. 4A), confirmed by immunohistochemistry on the protein level (not shown).

Pancreas morphology during early diabetes development at 50 days of age.

At day 50, immune cells could be observed migrating from the vessels to the islets (Fig. 1B). The immune cells surrounded the duct structures in the exocrine parenchyma and the pancreatic islets, forming an incomplete rim, and had already started to migrate into the islet periphery, thus representing islet infiltration stage 1 (Fig. 1B). The center of the islets was not infiltrated by immune cells (Fig. 2D–F). At this early infiltration stage, representing an insulitis score of 1.2, the immune cells were composed mainly of ED1+ macrophages (Fig. 2D) and some T-cells, with an equal amount of CD4+ and CD8+ T-cells (Fig. 2F) in normoglycemic rats with pancreas infiltration and a higher amount of CD8+ T-cells in the diabetic animals (Tables 2 and 3). Some apoptotic β-cells could also be detected in the islets, with the typical signs of cell rounding and dense insulin staining (Fig. 2E). Quantitative analysis of TUNEL-positive β-cells revealed an increase of the apoptosis rate from 0.2% in healthy IDDM rats to 1.7% in normoglycemic IDDM rats, with pancreas infiltration at the age of 50 days (Figure 5). In parallel to the infiltration of the islet periphery, mRNA expression of IL-1β (Fig. 3B) and TNF-α (Fig. 3E) could be detected in the immune cells.

Notably, IFN-γ mRNA expression in the immune cell population could not be observed at this early stage of pancreas infiltration and also not in any of the later stages (not shown). ED1+ macrophages in the spleen of these animals, however, stained positively for IFN-γ transcripts (not shown). At variance from the IL-1β–and TNF-α–positive immune cells, in this early infiltration stage with a predominance of macrophages, none of the β-cells in the infiltrated islets expressed mRNA of one of the three cytokines (Fig. 3B and E). Interestingly, the immune cells of the infiltrate in the islet periphery as well as 38 ± 2% of the β-cells in the center of the islets showed a strong iNOS mRNA expression (Fig. 3H). None of the immune cells, but 12 ± 2% of the β-cells, both single cells and groups of 3–5, in the infiltrated islets revealed an increased expression of procaspase 3 mRNA (Fig. 4B), which correlated well with the appearance of activated caspase 3 protein in apoptotic TUNEL-positive β-cells (not shown).

Pancreas morphology during progression of diabetes development at 55 days of age.

At day 55, the migration of immune cells was progressed toward the center of the pancreatic islets in both normoglycemic animals with pancreas infiltration and diabetic rats, resulting in islets with infiltration stage 2 (Fig. 1C) or stage 3 (Fig. 1D). The numbers of infiltrating immune cells within the islets were higher than at day 50 (Fig. 2D, F, G, and I and Tables 2 and 3), leading to a predominance of CD8+ T-cells (Fig. 2I). Severely infiltrated islets (stage 3) showed a decrease of the pancreatic β-cell area with a loss of the architecture of a typical mantle islet, resulting in small residual cell clusters composed of different endocrine cell types (Fig. 2H). In parallel to the severe infiltration of the islets, a massive increase of β-cell apoptosis with a maximal rate of 6.7% was recorded (Fig. 5). Notably, at day 55, infiltration of the islets did not occur at the same time as the increasing percentage of infiltration in stage 1 (20%), stage 2 (40%), and stage 3 (40%) of the affected pancreas. In the transition from stage 1 to stage 3, the number of immune cells, all expressing IL-1β and TNF-α mRNA, increased in parallel to the islet infiltration stages (Fig. 3C and F) without expression in the β-cells, as described in the early infiltration stage (Fig. 3C and F). In contrast to the early islet infiltration stage (stage 1), iNOS (Fig. 3I) and procaspase 3 mRNA expression were no more detectable in pancreatic β-cells in severely infiltrated islets.

Immunohistochemically, IL-1β and TNF-α were localized in both macrophages and T-cells in the islet periphery and center. However, when compared with the mRNA expression results obtained by in situ RT-PCR, fewer immune cells expressed these proinflammatory cytokine proteins immunohistochemically in the cytoplasm, probably due to high functional activity with a quick release of the synthesized cytokines (data not shown).

Pancreas morphology during diabetes manifestation at 59 days of age.

Day 59 marks the transition from a 21% to a 60% diabetes incidence at day 60 (Table 1). Thus, this time point was of particular interest for the documentation of immune cell infiltration, cytokine expression, and pancreatic β-cell destruction during diabetes development. At day 59, the pancreata of IDDM rats showed a severe infiltration with an insulitis score of 2.4 in still normoglycemic animals with pancreas infiltration and a score of 3.3 in diabetic rats characterized by a high rate of β-cell apoptosis, leading to the ultimate diabetes incidence of 60% at day 60 (Table 1). Pancreatic islet infiltration was dominated by CD8+ T-cells (Fig. 2I and Tables 2 and 3). The rate of β-cell apoptosis was slightly lower than at day 55, which can be explained by the progressive decrease of the β-cell area due to cell death (Fig. 5). In the severely infiltrated islets, IL-1β and TNF-α mRNA transcripts in the increasing immune cell subpopulations showed the highest expression level (Fig. 3C and F). After the total loss of β-cells in severely infiltrated islets at stage 3, none of the other endocrine cell types expressed mRNA of any of the three proinflammatory cytokines (Fig. 3C and F) or iNOS (Fig. 3I). Only the immune cells of the islet infiltrate showed a persistent mRNA expression of iNOS (Fig. 3I).

Pancreas morphology after diabetes manifestation at 60 and 65 days of age.

A total of 70% of the islets from diabetic animals showed a severe infiltration (stage 3) with a high pancreatic β-cell apoptosis rate (Fig. 5), finally leading to a complete loss of the β-cell area (Table 3). The composition of severely infiltrated islets (stage 3) at day 60 was not different from that at day 59 with a predominance of CD8+ T-cells, i.e., 60% of the infiltrating immune cells (Table 3). There was no iNOS mRNA expression in other endocrine cell types of these severely infiltrated pancreatic islets (Fig. 3I). In fact, 30% of the islets could be classified as end-stage islets (stage 4) with a complete loss of β-cells (Table 3) and virtually no remaining immune cells (Table 3) increasing to 100% at day 65. However, ED1+ macrophages remained in the exocrine pancreas parenchyma, in particular in the connective tissue around the vessels, with a clear expression of TNF-α mRNA of the proinflammatory cytokine IL-1β, and IFN-γ was not expressed in the remaining ED1+ macrophages.

The IDDM rat is a model of human type 1 diabetes very well suited for the elucidation of the chain of events leading to pancreatic β-cell loss and therefore of crucial importance for the understanding of the etiopathology of the disease process and for a rational design of possible intervention trials. The pre-diabetic period lasts only 1 week in the IDDM rat in contrast to a longer pre-diabetic period of up to 1 month reported for BB rats (11,12), up to 3 months for NOD mice (13), and even years in humans (14,15).

Immune cell infiltration stages during diabetes development.

Macrophages could be detected ultrastructurally and by immunostaining as the first immune cell subpopulation invading the islet periphery, thus acting as initiators of the autoimmune process, as also observed in the BB rat (1619) and in the NOD mouse (2022). However, like in the human situation, but in contrast to these animal models, the IDDM rat showed no signs of peri-insulitis. ED1+ infiltrating macrophages and dendritic cells are accumulated in the periductular and perivascular connective tissue adjacent to noninfiltrated islets in a variable time window in the diabetes-prone BB rat between 10 and 30 days (12,16) and in the NOD mouse 30–50 days before starting to migrate into the islet periphery and initiating the islet infiltration (20,21). The gradual shift to the predominance of CD8+ T-cells in the later infiltration stages was accompanied by a significant decrease of the β-cell area. The proportion of CD4+ T-cells was maintained at a stable level of ∼10% of all infiltrating immune cells during the progression of islet infiltration. CD4+ T-cell infiltration apparently plays a crucial regulatory role in promoting the recruitment of CD8+ T-cells. CD4+ T-cells are critically important apparently also for disease progression in the NOD mouse (23,24). B-cells in small proportion could already be detected in the initial islet infiltration in the IDDM rat and remained low (<5%) during the progression to severe insulitis. B-cells may participate in an initial priming event, as also supported by studies in NOD mice (25), but are apparently not a prerequisite for disease progression. NK cells were highest in the second stage of islet infiltration in the IDDM rat. This coincides with the highest β-cell apoptosis rate. Thus, NK cells are apparently involved in the process of β-cell destruction in this animal model rather than playing an immunoregulatory protective role. This conclusion is also supported by studies in BB rat and NOD mice (2634). The predominance of CD8+ T-cells in severely infiltrated islets of IDDM rats is an intriguing finding of the present study. It is the absolute increase of CD8+ T-cells that correlates well with the apoptotic β-cell destruction and a reduction of the β-cell area in infiltrated islets. The immune cell composition of severely infiltrated pancreatic islets in the IDDM rat corresponds with the late infiltration stages observed in diabetic BB rats and NOD mice (16,17,3537). A predominance of CD8+ T-cells has not been reported in normal NOD mice but in a T-cell–receptor transgenic NOD strain showing a selective recruitment of CD8+ T-cells to the islets and the subsequent rapid β-cell destruction leading to an accelerated diabetes onset (38). Furthermore, NOD mice showed an increase of a lymphoblast subpopulation developing into CD8+ T-cells in the spleen of acutely diabetic animals (39) and an increased priming of autoreactive CD8+ T-cells in lymph nodes after streptozotocin-induced β-cell death (40). CD8+ T-cells were also dominant in islet infiltrates of human pancreas biopsies from patients with recent-onset type 1 diabetes (1,2). Our data thus demonstrate the crucial relationship between macrophages in the initial and CD8+ T-cells in the late phase of infiltration and β-cell destruction in the type 1 diabetes rat, a model of spontaneous type 1 diabetes without inherited developmental defects of the cellular immune system in contrast to BB rat and NOD mouse (41). This model is therefore very well suited for studies on the role of cytokines in the disease process. For this purpose we used in the present study for the first time a systemic approach using a sensitive in situ PCR technique for the analysis of the development of the cytokine gene expression pattern.

Role of cytokines during islet infiltration.

Proinflammatory cytokines play an important role as immune mediators inducing complex signaling pathways finally resulting in β-cell death by apoptosis or necrosis (4244). This prompted us to correlate the expression of the β-cell toxic cytokines IL-1β, TNF-α, and IFN-γ in infiltrated islets of the IDDM rat with the status of immune cell invasion and β-cell destruction. By in situ RT-PCR it could be shown for the first time that IL-1β and TNF-α were expressed in vivo in immune cells at an early infiltration stage. In this IDDM rat model, we could further demonstrate that the expression of these cytokines increased in parallel to the number of the immune cells with the progression of islet infiltration. Cytokine-positive cells were always immune cells, never β-cells or any other endocrine cell type in the islet. In situ RT-PCR is a method that can, with high sensitivity, detect the gene expression of cytokines in infiltrated islets in the in vivo situation. The mRNA detection correlates with protein expression as shown in this study but is independent of the release of the cytokine protein from the immune cell and is therefore a very reliable indicator of the activity status of the immune cell. In the BB rat, IFN-γ transcripts could be detected by RT-PCR in infiltrated islets (45,46). In islets from diabetic NOD mice, the expression of IFN-γ and other Th1 cytokines has also been reported (4750). On the other hand, in a transgenic NOD mouse lacking the IFN-γ receptor on the β-cells, diabetes incidence was not different from that of wild-type mice, suggesting that pancreatic β-cell survival is not crucially affected by a lack of IFN-γ (51). However, the lack of IFN-γ expression in the IDDM rat, as reported here, provides clear evidence for the fact that β-cell destruction in autoimmune diabetes is not critically dependent upon an activation of IFN-γ. A possible explanation for this apparent discrepancy to BB rat and NOD mouse studies could be the fulminant development of β-cell destruction in the IDDM rat without any signs of peri-insulitis. In this IDDM rat model, the classical signs of endothelial cell activation as a prerequisite for immune cell adhesion were absent in the pancreatic parenchyma. Under in vivo conditions IFN-γ plays an important role in mediating adhesion mechanisms by upregulation of molecules such as intracellular adhesion molecule-1 on endothelial or immune cells (51,52). IL-1β and TNF-α, as the dominant proinflammatory cytokines, apparently act directly cytotoxic to pancreatic β-cells or indirectly sensitize T-cell–mediated cytotoxicity by changing the surface protein expression on the β-cell as the target cell in the autoimmune process (46).

Our observation of apoptotic β-cell death is in agreement with biopsy studies (53) in BB rats where infiltrated islets showed an initial increase of β-cell apoptosis at day 68 followed by an accelerated β-cell apoptosis rate at day 85, which coincides with the onset of diabetes, even though the absolute rates of β-cell apoptosis were ∼10 times lower due to the slower disease progression in the BB rat. In the NOD mouse model with its long pre-diabetic period, rates of apoptosis during the time course of diabetes manifestation could be monitored only in transgenic strains with a modulation of the T-cell receptor (54,55) with macrophages expressing IL-1β on the protein level (56). The IDDM rat showed an excellent correlation between islet infiltration, β-cell apoptosis, and onset of diabetes, demonstrating that islet infiltration by ED1+ macrophages and subsequently by CD8+ T-cells is the initial step of a process finally causing β-cell apoptosis. In particular, the parallel induction of iNOS and procaspase 3 in β-cells with the infiltration of immune cells secreting proinflammatory cytokines is strong evidence for a crucial role of proinflammatory cytokines in the process of apoptotic β-cell destruction in vivo, which is in agreement with in vitro data (57,58).

For the first time it was possible in this study to document under in vivo conditions a strong correlation between immune cell infiltration and the gene expression of proinflammatory cytokines, which leads to an upregulation of iNOS and procaspase 3 gene expression in the pancreatic β-cells. The particular, features of the IDDM rat make it possible to depict a chain of events that provides a mechanistic explanation for the development of the diabetic syndrome in this animal model of human type 1 diabetes.

FIG. 1.

Progression of insulitis and pancreatic β-cell destruction during diabetes development in the IDDM rat. Noninfiltrated pancreatic islet of a nondiabetic, control animal representing stage 0 (A). In the early infiltration stage (stage 1) (B), the infiltrate was restricted to the islet periphery of a normoglycemic IDDM rat at day 50. In stage 2 (C), in a normoglycemic animal at day 55, the whole islet was infiltrated to a low degree. Stage 3 (D) represented a severely infiltrated islet of a diabetic animal at day 59. Hematoxylin and eosin staining of pancreatic sections, magnification ×400.

FIG. 1.

Progression of insulitis and pancreatic β-cell destruction during diabetes development in the IDDM rat. Noninfiltrated pancreatic islet of a nondiabetic, control animal representing stage 0 (A). In the early infiltration stage (stage 1) (B), the infiltrate was restricted to the islet periphery of a normoglycemic IDDM rat at day 50. In stage 2 (C), in a normoglycemic animal at day 55, the whole islet was infiltrated to a low degree. Stage 3 (D) represented a severely infiltrated islet of a diabetic animal at day 59. Hematoxylin and eosin staining of pancreatic sections, magnification ×400.

FIG. 2.

Comparison of a noninfiltrated, control pancreatic islet (stage 0) (A–C) with a faintly infiltrated (stage 1) (D–F) and a severely infiltrated (stage 3) (G–I) pancreatic islet in the IDDM rat. Sections were immunostained for ED1+ infiltrating macrophages (A, D, and G), insulin (B, E, and H), and cytotoxic CD8+ T-cells (C, F, and I). The control islet (A–C) showed no signs of immune cell infiltration, and the β-cells exhibited the typical cytoplasmic insulin staining. In the faintly infiltrated islet (DF), the small amount of infiltrating immune cells, mostly macrophages, was restricted to the periphery. Most of the β-cells in the center of the islet showed a moderate insulin immunostaining, whereas β-cells in the islet periphery exhibited a cell rounding and a dense insulin immunostaining as signs of apoptosis. In the severely infiltrated islet (G–I), a large number of ED1+ macrophages and CD8+ T-cells infiltrated the whole islet, thereby destroying the microarchitecture of the islet. Some pancreatic β-cells survived in small cell clusters within the massive immune cell infiltrate. The dashed lines depict the islet boundary. Magnification ×400.

FIG. 2.

Comparison of a noninfiltrated, control pancreatic islet (stage 0) (A–C) with a faintly infiltrated (stage 1) (D–F) and a severely infiltrated (stage 3) (G–I) pancreatic islet in the IDDM rat. Sections were immunostained for ED1+ infiltrating macrophages (A, D, and G), insulin (B, E, and H), and cytotoxic CD8+ T-cells (C, F, and I). The control islet (A–C) showed no signs of immune cell infiltration, and the β-cells exhibited the typical cytoplasmic insulin staining. In the faintly infiltrated islet (DF), the small amount of infiltrating immune cells, mostly macrophages, was restricted to the periphery. Most of the β-cells in the center of the islet showed a moderate insulin immunostaining, whereas β-cells in the islet periphery exhibited a cell rounding and a dense insulin immunostaining as signs of apoptosis. In the severely infiltrated islet (G–I), a large number of ED1+ macrophages and CD8+ T-cells infiltrated the whole islet, thereby destroying the microarchitecture of the islet. Some pancreatic β-cells survived in small cell clusters within the massive immune cell infiltrate. The dashed lines depict the islet boundary. Magnification ×400.

FIG. 3.

mRNA expression for IL-1β (A–C), TNF-α (D–F), and iNOS (G–I) during diabetes development in the IDDM rat at different stages of pancreatic islet infiltration: stage 0 (A, D, and G), stage 1 (B, E, and H), and stage 3 (C, F, and J). In comparison with the unaffected control islet (A and D) without any IL-1β mRNA or TNF-α expression, transcripts in the immune cells were markedly increased in the transition from the faint (B and E) to the severe (C and F) infiltration stages (arrows). No sign of iNOS induction was observed in the unaffected control islet (G). In the faintly infiltrated islet (H), the immune cells in the islet periphery (arrows) and the β-cells in the islet center (arrowheads) showed iNOS expression in the cytoplasm. After total loss of β-cells within the severely infiltrated islet (I), only the immune cells (arrows) revealed iNOS expression. ED1+ macrophages, CD8+ T-cells, and pancreatic β-cells were identified by sequential sections immunostained with specific antibodies against the given cell type. The dashed lines depict the islet boundary. Magnification ×300 (A–F), magnification ×650 (G–I).

FIG. 3.

mRNA expression for IL-1β (A–C), TNF-α (D–F), and iNOS (G–I) during diabetes development in the IDDM rat at different stages of pancreatic islet infiltration: stage 0 (A, D, and G), stage 1 (B, E, and H), and stage 3 (C, F, and J). In comparison with the unaffected control islet (A and D) without any IL-1β mRNA or TNF-α expression, transcripts in the immune cells were markedly increased in the transition from the faint (B and E) to the severe (C and F) infiltration stages (arrows). No sign of iNOS induction was observed in the unaffected control islet (G). In the faintly infiltrated islet (H), the immune cells in the islet periphery (arrows) and the β-cells in the islet center (arrowheads) showed iNOS expression in the cytoplasm. After total loss of β-cells within the severely infiltrated islet (I), only the immune cells (arrows) revealed iNOS expression. ED1+ macrophages, CD8+ T-cells, and pancreatic β-cells were identified by sequential sections immunostained with specific antibodies against the given cell type. The dashed lines depict the islet boundary. Magnification ×300 (A–F), magnification ×650 (G–I).

FIG. 4.

Procaspase 3 mRNA expression in a faintly infiltrated pancreatic islet (stage 1) (B) in comparison with a noninfiltrated pancreatic control islet (stage 0) (A). In infiltrated islet (B), a higher level of procaspase 3 mRNA expression could be detected in the cytoplasm of β-cell (arrows) in comparison with a control islet (A) without infiltration. ED1+ macrophages, CD8+ T-cells, and pancreatic β-cells were identified by sequential sections immunostained with specific antibodies against the given cell type. The dashed line in A and B shows the islet boundary. Magnification ×350.

FIG. 4.

Procaspase 3 mRNA expression in a faintly infiltrated pancreatic islet (stage 1) (B) in comparison with a noninfiltrated pancreatic control islet (stage 0) (A). In infiltrated islet (B), a higher level of procaspase 3 mRNA expression could be detected in the cytoplasm of β-cell (arrows) in comparison with a control islet (A) without infiltration. ED1+ macrophages, CD8+ T-cells, and pancreatic β-cells were identified by sequential sections immunostained with specific antibodies against the given cell type. The dashed line in A and B shows the islet boundary. Magnification ×350.

FIG. 5.

Pancreatic β-cell apoptosis during diabetes development in the IDDM rat. Rate of β-cell apoptosis was quantified in pancreatic sections by TUNEL staining and immunostaining for insulin from animals 45, 50, 55, 59, 60, and 65 days of age. Shown are means ± SEM from four normoglycemic rats with pancreas infiltration until 59 days of age and 4 diabetic rats 60 or 65 days of age.

FIG. 5.

Pancreatic β-cell apoptosis during diabetes development in the IDDM rat. Rate of β-cell apoptosis was quantified in pancreatic sections by TUNEL staining and immunostaining for insulin from animals 45, 50, 55, 59, 60, and 65 days of age. Shown are means ± SEM from four normoglycemic rats with pancreas infiltration until 59 days of age and 4 diabetic rats 60 or 65 days of age.

TABLE 1

Characteristics of IDDM rats during diabetes development

Age (days)NNormoglycemic rats without pancreas infiltration
Normoglycemic rats with pancreas infiltration
Diabetic rats
n (%)Blood glucose (mmol/l)Serum insulin (ng/ml)n (%)Blood glucose (mmol/l)Serum insulin (ng/ml)Insulitis Scoren (%)Blood glucose (mmol/l)Serum insulin (ng/ml)Insulitis Score
45 11 11 (100) 4.7 ± 0.2 1.3 ± 0.2 0 (0)    0 (0) — —  
50 20 15 (75) 4.9 ± 0.2 1.2 ± 0.2 4 (20) 5.1 ± 0.2 1.2 ± 0.2 1.1 ± 0.1 1 (5) 10.9 0.8 1.9 
55 43 33 (77) 4.5 ± 0.1 1.2 ± 0.2 4 (9) 5.0 ± 0.1 1.2 ± 0.2 2.0 ± 0.1 6 (14) 13.0 ± 0.2 0.7 ± 0.1 2.5 ± 0.1 
59 14 7 (50) 4.7 ± 0.1 1.0 ± 0.2 4 (29) 5.3 ± 0.2 1.0 ± 0.1 2.4 ± 0.2 3 (21) 17.1 ± 0.1* 0.4 ± 0.2 3.3 ± 0.2 
60 20 8 (40) 4.6 ± 0.1 1.0 ± 0.2 0 (0)    12 (60) 19.5 ± 0.1* 0.10 ± 0.05* 3.3 ± 0.2 
65 12 4 (33) 4.8 ± 0.2 1.0 ± 0.1 0 (0)    8 (67) 22.7 ± 0.3* 0.05 ± 0.01* 4.0 ± 0.1 
Age (days)NNormoglycemic rats without pancreas infiltration
Normoglycemic rats with pancreas infiltration
Diabetic rats
n (%)Blood glucose (mmol/l)Serum insulin (ng/ml)n (%)Blood glucose (mmol/l)Serum insulin (ng/ml)Insulitis Scoren (%)Blood glucose (mmol/l)Serum insulin (ng/ml)Insulitis Score
45 11 11 (100) 4.7 ± 0.2 1.3 ± 0.2 0 (0)    0 (0) — —  
50 20 15 (75) 4.9 ± 0.2 1.2 ± 0.2 4 (20) 5.1 ± 0.2 1.2 ± 0.2 1.1 ± 0.1 1 (5) 10.9 0.8 1.9 
55 43 33 (77) 4.5 ± 0.1 1.2 ± 0.2 4 (9) 5.0 ± 0.1 1.2 ± 0.2 2.0 ± 0.1 6 (14) 13.0 ± 0.2 0.7 ± 0.1 2.5 ± 0.1 
59 14 7 (50) 4.7 ± 0.1 1.0 ± 0.2 4 (29) 5.3 ± 0.2 1.0 ± 0.1 2.4 ± 0.2 3 (21) 17.1 ± 0.1* 0.4 ± 0.2 3.3 ± 0.2 
60 20 8 (40) 4.6 ± 0.1 1.0 ± 0.2 0 (0)    12 (60) 19.5 ± 0.1* 0.10 ± 0.05* 3.3 ± 0.2 
65 12 4 (33) 4.8 ± 0.2 1.0 ± 0.1 0 (0)    8 (67) 22.7 ± 0.3* 0.05 ± 0.01* 4.0 ± 0.1 

Data are means ± SE. Rats were classified as diabetic when blood glucose concentration was >7.5 mmol/l at the respective time point. Pancreas infiltration and insulitis scores were determined by morphological analyses of pancreatic sections. Islet infiltration was graded on a score of 0–4 as follows: stage 0, no immune cell infiltration of the islets (control); stage 1, immune cell infiltration restricted to the islet periphery; stage 2, faint invasive immune cell islet infiltration; stage 3, complete islet infiltration with large numbers of immune cells; and stage 4, complete β-cell loss without any immune cell infiltration (end-stage islets). A mean insulitis score was calculated from 20 to 30 islets per individual pancreas.

*

P ≤ 0.05 compared with day 55 (ANOVA/Dunnett test).

TABLE 2

Pancreatic β-cell area, islet infiltration, and immune cell composition in normoglycemic IDDM rats with pancreas infiltration in the time course of diabetes development

Age (days)NPancreatic β-cell area
Infiltrated pancreatic islet area
Immune cell subpopulations in the infiltrated pancreatic islets
Total immune cells
ED1-macrophages
CD 4+ T-cells
CD 8+ T-cells
NK-cells
B-cells
mm2%mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%
50 14.8 ± 0.4 89 17.4 ± 0.2 94 114 ± 8* 100 64 ± 7 56 15 ± 3 13 27 ± 2 23 3 ± 1 4 ± 1 
55 8.8 ± 0.5 53 19.3 ± 0.4 104 191 ± 12* 100 84 ± 12 44 21 ± 4 11 69 ± 11 36 12 ± 3 5 ± 1 
59 5.3 ± 0.4 32 20.5 ± 0.2 110 254 ± 21* 100 68 ± 14 28 27 ± 3 11 144 ± 20 57 7 ± 2 6 ± 1 
Age (days)NPancreatic β-cell area
Infiltrated pancreatic islet area
Immune cell subpopulations in the infiltrated pancreatic islets
Total immune cells
ED1-macrophages
CD 4+ T-cells
CD 8+ T-cells
NK-cells
B-cells
mm2%mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%
50 14.8 ± 0.4 89 17.4 ± 0.2 94 114 ± 8* 100 64 ± 7 56 15 ± 3 13 27 ± 2 23 3 ± 1 4 ± 1 
55 8.8 ± 0.5 53 19.3 ± 0.4 104 191 ± 12* 100 84 ± 12 44 21 ± 4 11 69 ± 11 36 12 ± 3 5 ± 1 
59 5.3 ± 0.4 32 20.5 ± 0.2 110 254 ± 21* 100 68 ± 14 28 27 ± 3 11 144 ± 20 57 7 ± 2 6 ± 1 

Data are means ± SE. Pancreatic β-cell area was determined by morphometric analysis of pancreatic sections stained for insulin. The infiltrated islet area and the total number of infiltrated immune cells of different subpopulations were determined by morphometric analysis of hematoxylin and eosin–stained pancreatic sections and specific immunostaining from animals of 50, 55, and 59 days of age, compared with the control situation in noninfiltrated pancreas at 45 days. The β-cell area of islets in the control animals was 16.6 ± 0.2 mm2, and the total islet area of these noninfiltrated islets represented 18.6 ± 0.2 mm2. Both values were set as 100% in comparison with the β-cell area and the total islet area including infiltration in normoglycemic animals with pancreas infiltration. A total of 15 islets from three different regions of the pancreas were examined per animal. The number of immune cells per infiltrate are presented as cells per squared millimeters of islet area and in addition as a percentage of the total immune cell infiltrate at each time point.

*

P ≤ 0.01 compared with day 45

P ≤ 0.05 compared with day 45

P ≤ 0.05 compared with day 50 (all by ANOVA/Dunnett test).

TABLE 3

Pancreatic β-cell area, islet infiltration, and immune cell composition in IDDM rats in the time course of diabetes development

Age (days)NPancreatic β-cell area
Infiltrated pancreatic islet area
Immune cell subpopulations in the infiltrated pancreatic islets
Total immune cells
ED1-macrophages
CD 4+ T-cells
CD 8+ T-cells
NK-cells
B-cells
mm2%mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%
50 7.8 44 12.7 68 278 100 87 31 19 163 59 
55 5.8 ± 0.4* 35 20.7 ± 0.3 111 345 ± 21 100 92 ± 12 27 33 ± 5 10 201 ± 17 58 14 ± 3 5 ± 1 
59 3.8 ± 0.2* 23 10.7 ± 0.4 58 307 ± 13 100 73 ± 9 24 32 ± 3 10 178 ± 20 58 17 ± 2 7 ± 1 
60 12 2.3 ± 0.3 14 7.8 ± 0.3 42 280 ± 12 100 67 ± 10 24 27 ± 4 10 169 ± 13 60 12 ± 3 5 ± 1 
 
Age (days)NPancreatic β-cell area
Infiltrated pancreatic islet area
Immune cell subpopulations in the infiltrated pancreatic islets
Total immune cells
ED1-macrophages
CD 4+ T-cells
CD 8+ T-cells
NK-cells
B-cells
mm2%mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%cells/mm2%
50 7.8 44 12.7 68 278 100 87 31 19 163 59 
55 5.8 ± 0.4* 35 20.7 ± 0.3 111 345 ± 21 100 92 ± 12 27 33 ± 5 10 201 ± 17 58 14 ± 3 5 ± 1 
59 3.8 ± 0.2* 23 10.7 ± 0.4 58 307 ± 13 100 73 ± 9 24 32 ± 3 10 178 ± 20 58 17 ± 2 7 ± 1 
60 12 2.3 ± 0.3 14 7.8 ± 0.3 42 280 ± 12 100 67 ± 10 24 27 ± 4 10 169 ± 13 60 12 ± 3 5 ± 1 
 

Data are means ± SE. Pancreatic β-cell area was determined by morphometric analysis of pancreatic sections stained for insulin. The infiltrated islet area and the total number of infiltrated immune cells of different subpopulations per islet area were determined by morphometric analysis of hematoxylin and eosin–stained pancreatic sections and specific immunostaining from animals of 50, 55, 59, 60, and 65 days of age, compared with the control situation in noninfiltrated pancreas at day 45. The β-cell area of islets in the control animals was 16.6 ± 0.2 mm2, and the total islet area of these noninfiltrated islets represented 18.6 ± 0.2 mm2. Both values were set as 100% in comparison with the β-cell area and the total islet area including infiltration in diabetic animals. A total of 15 islets from three different regions of the pancreas were examined per animal. The number of animals at each time point is given in Table 1. The number of immune cells of the infiltrate are presented as cells per squared millimeters of islet area and in addition as a percentage of the total immune cell infiltrate at each time point.

*

P ≤ 0.05 compared with day 45

P ≤ 0.01 compared with day 45

P ≤ 0.05 compared with day 50 (all by ANOVA/Dunnett test).

TABLE 4

Quantitative morphometric analysis of the pancreatic islet infiltration stages of IDDM rats

Pancreatic islet infiltration stages
Stage 0
Stage 1
Stage 2
Stage 3
Stage 4
mm2 (%)           
    β-Cell area 16.6 ± 0.2 100 ± 2 12.0 ± 0.1 75 ± 3 7.5 ± 0.3* 47 ± 5 4.2 ± 0.2* 25 ± 3 
    Infiltrated islet area 18.6 ± 0.2 100 ± 3 16.5 ± 0.5 91 ± 4 20.6 ± 0.6 114 ± 5 21.0 ± 0.4 116 ± 6 
Cells/mm2 (%)           
    Total immune cells 1 ± 1 100 98 ± 8 100 188 ± 12 100 301 ± 17 100 
    ED1 macrophages 1 ± 1 100 67 ± 15 69 87 ± 22 46 78 ± 23 26 
    CD 4+ T-cells 12 ± 7 12 23 ± 12 12 27 ± 5 
    CD 8+ T-cells 13 ± 5 13 61 ± 20 33 183 ± 48 61 
    NK-cells 2 ± 1 17 ± 3 7 ± 2 
    B-cells 4 ± 1 6 ± 1 
Pancreatic islet infiltration stages
Stage 0
Stage 1
Stage 2
Stage 3
Stage 4
mm2 (%)           
    β-Cell area 16.6 ± 0.2 100 ± 2 12.0 ± 0.1 75 ± 3 7.5 ± 0.3* 47 ± 5 4.2 ± 0.2* 25 ± 3 
    Infiltrated islet area 18.6 ± 0.2 100 ± 3 16.5 ± 0.5 91 ± 4 20.6 ± 0.6 114 ± 5 21.0 ± 0.4 116 ± 6 
Cells/mm2 (%)           
    Total immune cells 1 ± 1 100 98 ± 8 100 188 ± 12 100 301 ± 17 100 
    ED1 macrophages 1 ± 1 100 67 ± 15 69 87 ± 22 46 78 ± 23 26 
    CD 4+ T-cells 12 ± 7 12 23 ± 12 12 27 ± 5 
    CD 8+ T-cells 13 ± 5 13 61 ± 20 33 183 ± 48 61 
    NK-cells 2 ± 1 17 ± 3 7 ± 2 
    B-cells 4 ± 1 6 ± 1 

Data are means ± SE. β-Cell area and immune cell infiltration were determined by quantitative morphometry in pancreata from IDDM rats (45–65 days of age). Islet infiltration was graded on a score of 0–4 as follows: stage 0, no immune cell infiltration of the islets (control); stage 1, immune cell infiltration restricted to the periphery of the islets; stage 2, weak invasive immune cell infiltration of the islets; stage 3, large numbers of immune cells within most islets; and stage 4, complete β-cell loss without infiltration (end-stage islets). A mean insulitis score was calculated from 10 islets per individual pancreas. The numbers of analyzed rats were 12 in stage 0, 5 in stage 1, 9 in stage 2, 7 in stage 3, and 12 in stage 4.

*

P < 0.01 compared with stage 0 of islet infiltration

P < 0.05

P < 0.01 compared with stage 1 of islet infiltration (all by ANOVA/Dunnett test).

This work has been supported by a grant from the Deutsche Forschungsgemeinschaft (Jo 395/1-1/2) to A.J. and by a National Institutes of Health grant (1R21AI55464-01) to S.L. and H.-J.H. Some of the results were obtained during thesis work by A.G.

The technical assistance of D. Lischke and U. Sommerfeld is gratefully acknowledged.

1.
Imagawa A, Hanafusa T, Itoh N, Miyagawa J, Nakajima H, Namba M, Kuwajima M, Tamura S, Kawata S, Matsuzawa Y, Harlan DM: Islet-infiltrating T lymphocytes in insulin-dependent diabetic patients express CD80 (B7-1) and CD86 (B7-2).
J Autoimmun
9
:
391
–396,
1996
2.
Imagawa A, Hanafusa T, Tamura S, Moriwaki M, Itoh N, Yamamoto K, Iwahashi H, Yamagata K, Waguri M, Nanmo T, Uno S, Nakajima H, Namba M, Kawata S, Miyagawa JI, Matsuzawa Y: Pancreatic biopsy as a procedure for detecting in situ autoimmune phenomena in type 1 diabetes: close correlation between serological markers and histological evidence of cellular autoimmunity.
Diabetes
50
:
1269
–1273,
2001
3.
Eisenbarth GS:
Type I Diabetes. Molecular, Cellular and Clinical Immunology.
New York, Oxford, Oxford University Press,
2003
4.
Lenzen S, Tiedge M, Elsner M, Lortz S, Weiss H, Jörns A, Klöppel G, Wedekind D, Prokop CM, Hedrich HJ: The LEW. 1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus.
Diabetologia
44
:
1189
–1196,
2001
5.
Jörns A, Kubat B, Tiedge M, Wedekind D, Hedrich H, Klöppel G, Lenzen S: Pathology of the pancreas and other organs in the diabetic LEW.1AR1/Ztm-iddm rat, a new model of spontaneous insulin-dependent diabetes mellitus.
Virchows Arch
444
:
183
–189,
2004
6.
Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures.
J Histochem Cytochem
29
:
577
–580,
1981
7.
Morel G, Berger M, Ronsin B, Recher S, Ricard-Blum S, Mertani HC, Lobie PE: In situ transcription-polymerase chain reaction: applications for light and electron microscopy.
Biol Cell
90
:
137
–150,
1998
8.
Bagasra O, Sehamma T, Pomerantz R, Hansen J: In situ PCR and hybridization to detect low-abundance nucleic acid targets. In
Current Protocols in Molecular Biology
. Vol. 2 (Suppl. 31). Chanda VB, Ed. New York, John Wiley and Sons,
1995
, p. 14.18.11–14.18.23
9.
Jörns A, Tiedge M, Lenzen S: Nutrient-dependent distribution of insulin and glucokinase immunoreactivities in rat pancreatic beta cells.
Virchows Arch
434
:
75
–82,
1999
10.
Sgonc R, Wick G: Methods for the detection of apoptosis.
Int Arch Allergy Immunol
105
:
327
–332,
1994
11.
Logothetopoulos J, Valiquette N, Madura E, Cvet D: The onset and progression of pancreatic insulitis in the overt, spontaneously diabetic, young adult BB rat studied by pancreatic biopsy.
Diabetes
33
:
33
–36,
1984
12.
Mordes JP, Desemone J, Rossini AA: The BB rat.
Diabetes Metab Rev
3
:
725
–750,
1987
13.
Buschard K: Diabetic animal models.
APMIS
104
:
609
–614,
1996
14.
Klöppel G, Clemens A: Insulin-dependent diabetes mellitus: islet changes in relation to etiology and pathogenesis.
Endocr Pathol
8
:
273
–282,
1997
15.
Lally FJ, Bone AJ: Animal models of type 1 diabetes. In
Textbook of Diabetes
. Pickup JC, Williams G, Eds. Oxford, Blackwell Scienific Publications, p. 19.11–19.17,
2002
16.
Walker R, Bone AJ, Cooke A, Baird JD: Distinct macrophage subpopulations in pancreas of pre-diabetic BB/E rats: possible role for macrophages in pathogenesis of IDDM.
Diabetes
37
:
1301
–1304,
1988
17.
Voorbij HA, Jeucken PH, Kabel PJ, De Haan M, Drexhage HA: Dendritic cells and scavenger macrophages in pancreatic islets of pre-diabetic BB rats.
Diabetes
38
:
1623
–1629,
1989
18.
Oschilewski U, Kiesel U, Kolb H: Administration of silica prevents diabetes in BB rats.
Diabetes
34
:
197
–199,
1985
19.
Lee KU, Pak CY, Amano K, Yoon JW: Prevention of lymphocytic thyroiditis and insulitis in diabetes-prone BB rats by the depletion of macrophages.
Diabetologia
31
:
400
–402,
1988
20.
Jansen A, Homo-Delarche F, Hooijkaas H, Leenen PJ, Dardenne M, Drexhage HA: Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and β-cell destruction in NOD mice.
Diabetes
43
:
667
–675,
1994
21.
Lee KU, Amano K, Yoon JW: Evidence for initial involvement of macrophage in development of insulitis in NOD mice.
Diabetes
37
:
989
–991,
1988
22.
Christianson SW, Shultz LD, Leiter EH: Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice: relative contributions of CD4+ and CD8+ T-cells from diabetic versus pre-diabetic NOD.NON-Thy-1a donors.
Diabetes
42
:
44
–55,
1993
23.
Bendelac A, Carnaud C, Boitard C, Bach JF: Syngeneic transfer of autoimmune diabetes from diabetic nod mice to healthy neonates: requirement for both L3t4+ and Lyt-2+ T-cells.
J Exp Med
166
:
823
–832,
1987
24.
Charlton B, Mandel TE: Progression from insulitis to β-cell destruction in NOD mouse requires L3T4+ T-lymphocytes.
Diabetes
37
:
1108
–1112,
1988
25.
Charlton B, Zhang MD, Slattery RM: B lymphocytes not required for progression from insulitis to diabetes in non-obese diabetic mice.
Immunol Cell Biol
79
:
597
–601,
2001
26.
Kagi D, Ledermann B, Burki K, Zinkernagel RM, Hengartner H: Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo.
Annu Rev Immunol
14
:
207
–232,
1996
27.
Sobel DO, Newsome J, Ewel CH, Bellanti JA, Abbassi V, Creswell K, Blair O: Poly I:C induces development of diabetes mellitus in BB rat.
Diabetes
41
:
515
–520,
1992
28.
Sobel DO, Azumi N, Creswell K, Holterman D, Blair OC, Bellanti JA, Abbassi V, Hiserodt JC: The role of NK cell activity in the pathogenesis of poly I:C accelerated and spontaneous diabetes in the diabetes prone BB rat.
J Autoimmun
8
:
843
–857,
1995
29.
Todd DJ, Forsberg EM, Greiner DL, Mordes JP, Rossini AA, Bortell R: Deficiencies in gut NK cell number and function precede diabetes onset in BB rats.
J Immunol
172
:
5356
–5362,
2004
30.
Iwakoshi NN, Greiner DL, Rossini AA, Mordes JP: Diabetes prone BB rats are severely deficient in natural killer T cells.
Autoimmunity
31
:
1
–14,
1999
31.
Jacobson JD, Markmann JF, Brayman KL, Barker CF, Naji A: Prevention of recurrent autoimmune diabetes in BB rats by anti-asialo-gm1 antibody.
Diabetes
37
:
838
–841,
1988
32.
Carnaud C, Gombert JM, Donnars O, Garchon HJ, Herbelin A: Protection against diabetes and improved NW/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex.
J Immunol
166
:
2404
–2411,
2001
33.
Laloux V, Baudoin L, Jeske D, Carnaud C, Lehuen A: NK T cell-induced protection against diabetes in V alpha 14-J alpha 281 transgenic nonobese diabetic mice is associated with a Th2 shift circumscribed regionally to the islets and functionally to islet autoantigen.
J Immunol
166
:
3749
–3756,
2001
34.
Wang KS, Ritz J: Identification of IL-12 induced genes in human T cells and NK cells by gene expression profiling.
FASEB J
15
:
A348
–A348,
2001
35.
Homo-Delarche F: Beta-cell behaviour during the pre-diabetic stage. Part II. Non-insulin-dependent and insulin-dependent diabetes mellitus.
Diabetes Metab
23
:
473
–505,
1997
36.
Mathis D, Benoist C: Beta cell death during progression to diabetes.
Nature
414
:
792
–798,
2001
37.
Nagata M, Santamaria P, Kawamura T, Utsugi T, Yoon JW: Evidence for the role of CD8+ cytotoxic T cells in the destruction of pancreatic beta-cells in nonobese diabetic mice.
J Immunol
152
:
2042
–2050,
1994
38.
Jun HS, Santamaria P, Lim HW, Zhang ML, Yoon JW: Absolute requirement of macrophages for the development and activation of β-cell cytotoxic CD8+ T-cells in T-cell receptor transgenic NOD mice.
Diabetes
48
:
34
–42,
1999
39.
Kurner T, Burkart V, Kolb H: Large increase of cytotoxic/suppressor T-lymphoblasts and eosinophils around manifestation of diabetes in BB rats.
Diabetes Res
3
:
349
–353,
1986
40.
Zhang YQ, O’Brien B, Trudeau J, Tan RS, Santamaria P, Dutz JP: In situ beta cell death promotes priming of diabetogenic CD8 T lymphocytes.
J Immunol
168
:
1466
–1472,
2002
41.
Rossini AA: From beast to bedside: a commentary.
Diabetologia
47
:
1647
–1649,
2004
42.
Rabinovitch A, SuarezPinzon W, ElSheikh A, Sorensen O, Power RF: Cytokine gene expression in pancreatic islet-infiltrating leukocytes of BB rats: expression of Th1 cytokines correlates with β-cell destructive insulitis and IDDM.
Diabetes
45
:
749
–754,
1996
43.
Kolb H, Worz-Pagenstert U, Kleemann R, Rothe H, Rowsell P, Scott FW: Cytokine gene expression in the BB rat pancreas: natural course and impact of bacterial vaccines.
Diabetologia
39
:
1448
–1454,
1996
44.
Rabinovitch A: Immunoregulation by cytokines in autoimmune diabetes.
Adv Exp Med Biol
520
:
159
–193,
2003
45.
Rabinovitch A, Sorensen O, Suarezpinzon WL, Power RF, Rajotte RV, Bleackley RC: Analysis of cytokine messenger-RNA expression in syngeneic islet grafts of NOD mice: interleukin-2 and interferon-gamma messenger-RNA expression correlate with graft-rejection and interleukin-10 with graft-survival.
Diabetologia
37
:
833
–837,
1994
46.
Rabinovitch A, Suarez-Pinzon WL: Cytokines and their roles in pancreatic islet beta-cell destruction and insulin-dependent diabetes mellitus.
Biochem Pharmacol
55
:
1139
–1149,
1998
47.
Muir A, Peck A, Claresalzler M, Song YH, Cornelius J, Luchetta R, Krischer J, Maclaren N: Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-gamma transcription.
J Clin Invest
95
:
628
–634,
1995
48.
Hirai H, Kaino Y, Ito T, Kida K: Analysis of cytokine mRNA expression in pancreatic islets of nonobese diabetic mice.
J Pediatr Endocrinol Met
13
:
91
–98,
2000
49.
Campbell IL, Kay TWH, Oxbrow L, Harrison LC: Essential role for interferon-gamma and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice.
J Clin Invest
87
:
739
–742,
1991
50.
Kovarik J, Koulmanda M, Mandel TE: Expression of both Th1 and Th2 cytokines correlates with the histological rejection of MHC-matched and MHC-mismatched foetal pancreas allografts in mice.
Immunol Cell Biol
75
:
303
–309,
1997
51.
Thomas HE, Parker JL, Schreiber RD: IFN-γ action on pancreatic beta cells causes class I MHC upregulation but not diabetes.
J Clin Invest
102
:
516
–526,
1998
52.
Savinov AY, Wong FS, Chervonsky AV: IFN-gamma affects homing of diabetogenic T cells.
J Immunol
167
:
6637
–6643,
2001
53.
Lally FJ, Ratcliff H, Bone A: Apoptosis and disease progression in the spontaneously diabetic BB/S rat.
Diabetologia
44
:
320
–324,
2001
54.
Kurrer MO, Pakala SV, Hanson HL, Katz JD: Beta cell apoptosis in T cell-mediated autoimmune diabetes.
Proc Natl Acad Sci U S A
94
:
213
–218,
1997
55.
Augstein P, Dunger A, Heinke P, Wachlin G, Berg S, Hehmke B, Salzsieder E: Beta-cell apoptosis in an accelerated model of autoimmune diabetes.
Mol Med
4
:
495
–501,
1998
56.
Reddy S, Young M, Ginn S: Immunoexpression of interleukin-1 beta in pancreatic islets of NOD mice during cyclophosphamide-accelerated diabetes: co-localization in macrophages and endocrine cells and its attenuation with oral nicotinamide.
Histochem J
33
:
317
–327,
2001
57.
Aktan F: iNOS-mediated nitric oxide production and its regulation.
Life Sci
75
:
639
–653,
2004
58.
Eizirik DL, Darville MI: β-Cell apoptosis and defense mechanisms: lessons from type 1 diabetes.
Diabetes
50 (Suppl. 1)
:
S64
–S69,
2001