Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a LADA Rat Model Compared With Type 1 Diabetes

Typically, two main types of diabetes are distinguished, namely, type 1 diabetes mellitus (T1DM), with an onset in life in the younger age-group and a progressive autoimmune-mediated β-cell loss leading to a complete insulin deficiency, and type 2 diabetes mellitus (T2DM), characterized by insulin resistance in the periphery and a preserved β-cell mass in the pancreas at diagnosis in the older age-group (1). However, ∼10% of patients with T2DM reveal a diabetes form called latent autoimmune diabetes in adults (LADA) (2,3), which is defined by a late onset in adult life (>35 years of age), the presence of at least one autoantibody, typically against GAD65, and an initial noninsulin-requiring treatment period of ∼6 months after diabetes diagnosis; thereafter, insulin substitution is necessary (4,5).

However, even 30 years after the first description of this clinical phenotype in the literature (2,3), a systematic assessment of the pathology of the endocrine pancreas of patients with LADA has not been performed. Only a few case reports have been published with a morphological assessment of the human LADA pancreas in a pancreatectomized organ (6) or in organ biopsy specimens (7,8). Furthermore, a LADA animal model has not been available up until now.

In the current study, we therefore defined the biochemical and molecular morphology features in a comparative fashion both in a collection of pancreases from patients with LADA and of pancreases from a first spontaneous rat model of LADA, the so-called LADA IDDM (LEW.1AR1-iddm) rat (9–11), which mimics the characteristics of LADA. We compared the newly identified characteristics in LADA pancreases and in pancreases of adult patients with T1DM with those in this new rat model of LADA, which we describe here for the first time. The results of the current study allow a clear distinction between the biochemical and morphological characteristics of the two forms of autoimmune diabetes.

Pancreases from Caucasian patients with T1DM (43 ± 7 years; three men, one woman), with LADA (58 ± 7 years; one man, three women), with T2DM (65 ± 4 years; two men, two women), and from healthy donors (44 ± 8 years; three men, one woman) were obtained by organ resection during surgical intervention or from organ donors. The analyses were approved by the responsible ethics committees (Supplementary Tables 1–4).

Congenic IDDM rats (for details see https://www.mh-hannover.de/3642.html) were bred and maintained in accordance with the principles of laboratory animal care as previously described (9,11,12). Diabetes was defined as a blood glucose value >10 mmol/L (Supplementary Table 5). Blood glucose concentrations were determined daily (Glucometer Elite; Bayer, Leverkusen, Germany). Serum C-peptide was analyzed with a rat-specific ELISA (Mercodia, Uppsala, Sweden).

Two pancreas tissue biopsies, with removal of 30 mg pancreas each from the pancreas tail, were performed as previously described in detail (13) before diabetes manifestation in the T1DM rats at days 50 and 55 and in the LADA rats at days 70 and 80 of life. Experimental procedures were approved by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), No. 33-42502-05/958 and 509.6-42502-03/684.

Autoimmune diabetes manifestation took place between day 55 and day 65 for the early and quickly developing T1DM form in the IDDM rat, with an average of 59 ± 2 days as previously described (9,11,12). In T1DM animals the prediabetic phase with immune cell infiltration in the islets was between 3 and 7 days before diabetes onset (9,11). Besides the early T1DM form, a late and slowly developing LADA form of autoimmune diabetes was identified in this rat model in a subgroup of animals. In these rats, islet immune cell infiltration started between day 75 and 115, ∼20 days before diabetes manifestation, which could be identified by sequential pancreatic biopsy specimens before diabetes manifestation. The average time of diabetes onset was day 87 ± 4 (n = 25 animals). The rats with the T1DM form became insulin dependent immediately after disease manifestation, requiring the start of an insulin supplementation within the first 3 days (9), while the late form remained insulin independent over at least 1–2 months and thus received no insulin supplementation. C-peptide concentrations in serum obtained at the day of organ collection were higher in the LADA group of animals (376 ± 7 pmol/L; n = 8) than in the T1DM group (168 ± 34 pmol/L; n = 6). Additionally, age-matched, nondiabetic controls were analyzed (controls for T1DM at day 60: 998 ± 41 pmol/L, n = 8; controls for LADA at day 120: 942 ± 38 pmol/L, n = 8).

Pancreas tissue specimens were fixed in 4% paraformaldehyde in 0.15 mol/L PBS and embedded in paraffin (12,14).

Serial human and rat pancreas sections were stained by double immunofluorescence (12,14) for identification of the immune cells by the marker CD45 and the cell types in the islet infiltrate using species specific antibodies (Supplementary Table 6).

The in situ RT-PCR analyses of human and rat pancreas sections placed on 3-chamber slides were performed on a specific thermal cycler (Bio-Rad) as previously described (9) using species-specific primers (Supplementary Tables 7 and 8).

A total of 20–30 pancreatic islets from each individual pancreas and, in the case of the LADA pancreases, 20–30 pancreatic islets each both in the infiltrated and noninfiltrated areas were analyzed after diabetes manifestation for each marker. Quantification of stained and nonstained cells in the islets was performed by counting the positively immunostained cells with an Olympus microscope BX61 (9,14) (Tables 1 and 2). The same approach was used for the TUNEL staining of apoptotic cells (9,14). After the whole pancreas section was scanned, the analyses of the specific mRNA staining were performed with a visual system and a VS-ASW computer-assisted program (Olympus, Hamburg, Germany) manually setting a threshold for the specific mRNA staining and separating and calculating this specifically stained area as a percentage of the measured infiltrated immune cell area or of the whole islet (Tables 3 and 4) comparing LADA with T1DM pancreases (14).

The quality of the RNA isolated from the laser capture microdissected islets (for details see Supplementary Data) was controlled with the Experion electrophoresis system (Bio-Rad); mRNA was quantified in triplicates in quantitative real-time PCR reactions performed with the DNA Engine Opticon fluorescence detection system (Bio-Rad) and confirmed by gel electrophoreses (12). Gene expression results were normalized for all groups against the ribosomal protein L32 (Rpl32) using QGene and LinRegPCR software. Primer sequences designed according specific guidelines (15) and efficiencies are provided in Supplementary Table 9.

Results are presented as mean values ± SEM, and comparisons between the groups were analyzed with the one-way ANOVA followed by Dunnett test using Prism 5 software (GraphPad, San Diego, CA).

The data sets generated and analyzed during this study are available from the corresponding author upon reasonable request.

In autoimmune diabetes, the pancreatic islets are infiltrated with a variety of immune cell types. As documented in the current study, this was the case in the pancreases of patients with LADA (Fig. 1) and with T1DM as well as in the pancreases of the LADA form (Fig. 2) and the adult form of autoimmune diabetes in the IDDM rat model.

A quantification of the immune cell composition of the infiltrates in the human as well as in the rat pancreases revealed that CD8 T cells and CD68 macrophages were by far the most frequent immune cell types present. CD4 T cells, B cells, and natural killer cells were less frequent, with each cell type representing <10% in the islet immune cell infiltrate (Tables 1 and 2). In contrast, the nondiabetic control islets of human and rat pancreases showed only some nonactivated macrophages (Table 1 and Fig. 1), while nonactivated macrophages were doubled in islets of pancreases from patients with T2DM (Table 1).

Islets in certain lobular areas of the pancreatic parenchyma in human (Fig. 1) and rat (Fig. 2) pancreases of the LADA form showed no activated immune cell infiltrate. Rather, it was comparable to the healthy control situation (Figs. 1 and 2) as well as to the T2DM situation, with a few islet-infiltrating nonactivated macrophages (Table 1).

The areas analyzed morphometrically without immune cell infiltration typical for LADA diabetes accounted for approximately one-third of the sections of the pancreas area in contrast to approximately two-thirds of both human and rat LADA pancreases with infiltration (36.8 ± 0.3% vs. 63.2 ± 0.3% [n = 4] and 33.5 ± 1.1% vs. 66.5 ± 1.2% [n = 8]), respectively. Such large areas without autoimmune cell infiltration were not observed in human T1DM pancreases. In none of the analyzed pancreases were end-stage islets without β-cells observed.

When applying the definition of immune cell infiltration in T1DM by Campbell-Thompson et al. (16) with a cutoff threshold value of >15 immune cells per infiltrated islet, the percentage of infiltrated islets in the human LADA pancreases was calculated to be 64.8 ± 2.7% compared with the percentage of 63.4 ± 2.7% in infiltrated islets in the human T1DM pancreases (n = 4 each).

All other islets in the human LADA and T1DM pancreases were infiltrated with <15 immune cells per islet. When adopting a limit of greater than three immune cells per infiltrated islet, all islets in the human T1DM pancreases and in the infiltrated areas in the human LADA pancreases can be classified as infiltrated with activated immune cells. None of these islets contained fewer than three immune cells, which is the upper threshold for the numbers of immune cells infiltrating healthy human control islets and human T2DM islets (Table 1). These immune cells are exclusively nonactivated macrophages (Table 1).

The situation in the analyzed rat pancreases is more simple, in that all islets in the rat T1DM pancreases and in the infiltrated areas in the rat LADA pancreases contained >15 immune cells per infiltrated islet (Table 2). The noninfiltrated areas of the rat LADA pancreases contained fewer than three immune cells, in the same way as rat control pancreases (Table 2).

The total number of immune cells infiltrating the islets was four times higher in the rat pancreases than in the human pancreases (numbers in legends to Tables 1 and 2). Interestingly, however, the ratio between the numbers of macrophages and CD8 T cells shifted from 1.0 to 1.1 in human and rat T1DM toward a clear preference for macrophages in the LADA pancreases, with an increase of the ratios to 2.5 and 2.1 in human and rat LADA pancreases, respectively (Tables 1 and 2). These differences in the composition of the immune cell infiltrates were present, although the total numbers of infiltrating immune cells per islet were not significantly different both in the human and rat pancreases of T1DM and LADA.

To identify immune cell activation in the islet infiltrate, the expression of pro- and anti-inflammatory cytokines was analyzed by in situ gene expression of their mRNA transcripts in pancreatic sections. Immune cells in the islet infiltrate expressed the main proinflammatory cytokines interleukin (IL) 1β and tumor necrosis factor-α (TNF-α) in both the human (Table 3) and rat (Table 4) T1DM and LADA pancreases. Quantification of expression documented a shift of the ratio between IL-1β and TNF-α from 1.2 in the human T1DM pancreas to 3.4 in the infiltrated area of the human LADA pancreas (1.21 ± 0.06 vs. 3.38 ± 0.31; n = 4 each; P < 0.01) and from 1.1 in the rat T1DM pancreas to 3.7 in the infiltrated area of the rat LADA pancreas (1.07 ± 0.04 vs. 3.73 ± 0.12; n = 6 and n = 8, respectively; P < 0.01) due to a higher gene expression level in favor of IL-1β compared with TNF-α. Interferon-γ (IFN-γ), the third prominent proinflammatory cytokine, was expressed at a comparatively low level in the infiltrates of both human and rat T1DM and LADA pancreases (Tables 3 and 4 and Supplementary Figs. 1 and 2). At the same time, gene expression levels of the anti-inflammatory cytokine IL-10 were higher in the immune cell infiltrate of the human (Table 3) and rat (Table 4) LADA pancreases compared with human and rat T1DM pancreases. Gene expression of the enzyme inducible nitric oxide synthase (iNOS), which is well known to be induced by IL-1β (17), was high in the immune cell infiltrate of both the human (Table 3) and rat (Table 4) T1DM and LADA pancreases. In the areas of the human and rat LADA pancreases with islets without immune cell infiltration, an expression of the cytokine genes was not detectable, with the exception of a very low IL-1β expression level (Tables 3 and 4 and Supplementary Figs. 1 and 2). The same conclusion refers to the nondiabetic control islets as well as to T2DM islets (Tables 3 and 4).

To evaluate the effects on β-cell destruction, caspase 3 gene expression was analyzed. Caspase 3 expression was more than doubled in human and rat LADA pancreases and was even four to five times higher in the β-cells of human and rat T1DM pancreases compared with the control islets of the human and rat pancreas areas without immune cell infiltration as well as compared with the nondiabetic control islets and T2DM islets (Tables 3 and 4). Gene expression analyses of the proliferation marker proliferating cell nuclear antigen (PCNA) revealed increased expression levels in β-cells of islets of the human and rat LADA pancreases compared with the T1DM pancreases (Tables 3 and 4). Furthermore, insulin gene expression revealed a tendency for increased expression levels in β-cells of infiltrated human and rat LADA islets and a threefold higher expression level in β-cells from noninfiltrated islets of LADA pancreases as well as from nondiabetic control islets compared with T1DM islets (Tables 3 and 4).

To quantify cytokine and cell cycle markers in islets with and without immune cell infiltrate from LADA rat pancreases, the expression of pro- and anti-inflammatory cytokines was analyzed additionally by real-time PCR gene expression of their mRNA transcripts after laser capture microdissection of the islets.

Immune cells in the islet infiltrate expressed the main proinflammatory cytokines IL-1β and TNF-α in the rat (Table 5) LADA as well as in the T1DM pancreases. Quantification of expression documented, however, a significant (P < 0.01) shift of the ratio between IL-1β and TNF-α from 0.6 in the T1DM islets to 1.7 in the infiltrated islets of the rat LADA pancreas (0.59 ± 0.09 vs. 1.68 ± 0.74; n = 6 and 8, respectively; P < 0.01). The changed ratio was due to a significantly (P < 0.05) higher gene expression level in favor of IL-1β and to a lesser reduction (P < 0.06) of the TNF-α expression level in the infiltrated rat islets of the LADA pancreases compared with the islets of T1DM pancreases. IFN-γ was expressed at a low level in infiltrated islets of both T1DM and LADA (Table 5). In islets without immune cell infiltration of the LADA pancreases, an expression of cytokine genes was very low (Table 5).

At the same time, gene expression levels of the anti-inflammatory cytokine IL-10 were significantly (P < 0.05) higher in the immune cell infiltrate of the LADA islets with infiltration compared with LADA islets of the same rat pancreases without infiltration. The increase was less (P < 0.07) when the LADA islets with infiltration were compared with T1DM islets (Table 5). The gene of the enzyme iNOS was highly expressed in infiltrated rat T1DM and LADA islets (Table 5).

Caspase 3 expression in rat LADA islets with infiltration was strongly increased by a factor of 10 and even more by a factor of 20 in the β-cells of rat T1DM islets when compared with the very low expression level in the β-cells of the islets of healthy controls and rat LADA pancreases without immune cell infiltration (Table 5). Gene expression analyses of the proliferation marker PCNA revealed a significant increase in β-cells of microdissected islets without infiltration and even a doubling of the expression level in those islets with infiltration of the same rat LADA pancreases compared with islets from T1DM pancreases (Table 5). Furthermore, insulin gene expression revealed a slight tendency for increased expression levels in β-cells of infiltrated rat LADA islets and a more than threefold higher expression level in β-cells from noninfiltrated islets of the rat LADA pancreases as well as from nondiabetic control rat islets as compared with T1DM islets (Table 5).

It has been known for nearly 30 years that a subgroup of ∼10% of patients who develop diabetes at an age of >35 years have a form that differs from T2DM in that patients show positivity for at least one antibody, typically the GAD antibody (18), and develop a dependence on insulin supplementary therapy within 6 months after diagnosis (19,20). This form is known as LADA (2,3,21) and represents a milder, more slowly progressing form of autoimmune diabetes with a manifestation later in life (22–25). However, the morphological, molecular biology, and biochemical characteristics of the affected pancreases have not been explored so far. The current study fills this gap.

The animal model of the LADA form in the IDDM rat, which is an established model of autoimmune diabetes (9–11,26), showed upon characterization in the current study features that were comparable to those of patients with LADA (see points 1–5 in Table 6). The most important observation both in the pancreases of patients with LADA and of the LADA rat model was that not all islets were infiltrated at the moment of disease manifestation but only the islets in approximately two-thirds of the pancreases. Thus, islets in an area of approximately one-third of the pancreases were unaffected, showing no signs of immune cell infiltration and β-cell apoptosis. This was also observed also in an analysis of the LADA pancreas organ in a case report (6). On the other hand, this can explain why infiltrated islets in a human LADA pancreas may not have been found upon analyses of pancreatic islet tissue specimens obtained by pancreas organ biopsy (7,8). The unaffected islets in the areas of the pancreas without infiltration prevent a quick deterioration of the metabolic state. This is at variance from the situation in T1DM pancreases, where all islets containing β-cells are infiltrated with activated immune cells (9,14,27–29).

In the prediabetic phase in the IDDM rat model of human T1DM, areas without infiltrated islets are known to exist (9). These areas decrease in size in the pancreases of rats in the prediabetic phase when analyzed at days 50 and 55 of age (9) as closer the animals approach the time of diabetes manifestation (on average at day 59).

This explains not only the slower disease progression in patients and rats with LADA but also the milder form of autoimmune diabetes as documented by higher serum C-peptide concentrations, an observation that has also been previously reported for patients with LADA (2,3,30,31) as well as a higher level of β-cell insulin gene expression as an explanation for the initial insulin independence.

The milder syndrome is convincingly documented also by the results of the molecular morphology analyses both in the pancreases of patients with LADA and of the LADA rat model in this study. A shift of the immune cell infiltration from CD8 T cells toward CD68 macrophages and a shift of proinflammatory cytokine gene expression from TNF-α toward IL-1β (see points 6–7 in Table 6) are major elements of the alleviation of the disease process. In addition, the higher number of macrophages may support a faster removal of apoptotic β-cells from the infiltrated islets.

Macrophages are known to produce more IL-1β than other immune cell types (32), whereas T cells are the main site of TNF-α production (33). A high IL-1β release is known to recruit preferentially macrophages into the area of infiltration (34). Since the β-cell toxic potential of IL-1β is known to be lower (35) than that of TNF-α, it is plausible that the predominance of IL-1β in the immune cell infiltrate in the LADA pancreas compared with the predominance of TNF-α in the immune cell infiltrate in the T1DM pancreas, both in humans as well as in the rat model, is a major reason for the milder and more slowly progressing form of autoimmune diabetes with a manifestation later in life.

In addition, protective mechanisms, such as increased β-cell proliferation capacity (increase of PCNA gene expression) and anti-inflammatory capacity (increase of IL-10 gene expression) to alleviate proapoptotic activities (lower caspase-3 gene expression along with reduced TUNEL positivity) mediating the autoimmune β-cell destruction process through the proinflammatory cytokines, could be documented under LADA conditions (see points 8–10 in Table 6).

The pathological changes in the pancreas of the LADA rat model mirror the changes in the human LADA pancreas, making this new LADA animal model well suited for future in vivo and in vitro analyses into the pathogenesis of β-cell dysfunction and death in the LADA form of autoimmune diabetes.

The present results provide a solid basis for execution of LADA treatment schedules with curative potential based on the improved understanding of the underlying pancreatic islet pathology (23,25). The slower progression of the disease process in LADA makes this autoimmune diabetes form an ideal model for translation of new effective curative antibody combination therapies from the T1DM animal model (10,12) to patients with autoimmune diabetes (10,23). The results document conclusively that LADA is without any doubt a form of autoimmune diabetes, which was categorized by Ahlqvist et al. (36) in subgroup 1. The characteristics are all not features of T2DM. In human T2DM pancreases, the number of macrophages was doubled compared with healthy control subjects, confirming earlier data from Richardson et al. (37). It is noteworthy to menton that the macrophages in human T2DM pancreases are clearly less than in the infiltrate of human T1DM and LADA pancreases (Tables 1 and 2). In this context, our observation that the macrophages in the T2DM and control pancreases show no signs of immune cell activation is important (38). Thus, our results do not provide support for the view that LADA might be a diabetes form in the transition between T1DM and T2DM as previously considered by some authors (21,39).

In parallel, gene association studies in LADA patients revealed more susceptibility loci for immune cells and β-cells associated with T1DM than with T2DM loci (40,41). The same was true for the proinflammatory cytokine profile analyzed in peripheral blood of patients with LADA showing similarities to the profile in T1DM rather than in patients with T2DM (42). In addition, the shift from CD8 T cells to macrophages in the islet infiltrate of both human and rat LADA pancreases, as shown in the current study, went along with a decreased frequency of islet effector CD8 T cells in the peripheral blood of patients with LADA compared with patients with T1DM, as previously reported (43). A recent cohort study comparing patients with T1DM and those with T2DM identified >20% of patients with T2DM as late onset patients with T1DM (44).

We can conclude, therefore, that LADA is a milder form of autoimmune diabetes in patients of an advanced age. This confirms the conclusion by Buzzetti and colleagues (24) that “compared with young onset diabetes, LADA represents the other extreme of the autoimmune diabetes spectrum.” That is a phenomenon which is characteristic of many diseases that progress more slowly at an increasing age.

Nevertheless, using the term LADA is justified in clinical medicine to identify a late form of autoimmune diabetes in patients with adult-onset diabetes of an older age and distinguish this form from T2DM, because this diagnosis has important implications for the prognosis and treatment as well as for the well-being of the affected patients. So autoimmune diabetes (21–25) is just another example of a disease with a broad spectrum along the course of life (45).

Acknowledgments. The authors thank R. Chucholl and D. Lischke (Institute of Clinical Biochemistry, Hannover Medical School) for skillful technical assistance.

Funding. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to A.J. (JO 395/2-2).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. A.J. designed the study, performed experiments, analyzed and interpreted data, and wrote the manuscript. D.W. provided materials and reviewed the manuscript. J.J. provided materials. S.L. designed the study, analyzed and interpreted data, and wrote the manuscript. A.J. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.