Pancreas Pathology of Latent Autoimmune Diabetes inAdults (LADA) in Patients and in a LADA Rat ModelCompared With Type 1 DiabetesAnne Jörns,1 Dirk Wedekind,2 Joachim Jähne,3 and Sigurd Lenzen1,4

Diabetes 2020;69:624–633 | https://doi.org/10.2337/db19-0865

Approximately 10% of patients with type 2 diabetes suf-fer from latent autoimmune diabetes in adults (LADA).This study provides a systematic assessment of the pa-thology of the endocrine pancreas of patients with LADAand for comparison in a first rat model mimicking the char-acteristics of patients with LADA. Islets in human and ratpancreases were analyzed by immunohistochemistry forimmune cell infiltrate composition, by in situ RT-PCR andquantitative real-time PCR of laser microdissected isletsfor gene expression of proinflammatory cytokines, the pro-liferation marker proliferating cell nuclear antigen (PCNA),the anti-inflammatory cytokine interleukin (IL) 10, and theapoptosismarkers caspase 3andTUNELaswell as insulin.Human and rat LADA pancreases showed differencesin areas of the pancreas with respect to immune cellinfiltration and a changed ratio between the numberof macrophages and CD8 T cells toward macrophagesin the islet infiltrate. Gene expression analyses revealeda changed ratio due to an increase of IL-1b and a de-crease of tumor necrosis factor-a. IL-10, PCNA, andinsulin expression were increased in the LADA situa-tion, whereas caspase 3 gene expression was reduced.The analyses into the underlying pathology in human aswell as rat LADA pancreases provided identical results,allowing the conclusion that LADA is a milder form ofautoimmune diabetes in patients of an advanced age.

Typically, two main types of diabetes are distinguished,namely, type 1 diabetes mellitus (T1DM), with an onset inlife in the younger age-group and a progressive autoimmune-mediatedb-cell loss leading to a complete insulin deficiency,

and type 2 diabetes mellitus (T2DM), characterized byinsulin resistance in the periphery and a preserved b-cellmass in the pancreas at diagnosis in the older age-group(1). However, ;10% of patients with T2DM reveal a di-abetes 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 autoan-tibody, typically against GAD65, and an initial noninsulin-requiring treatment period of ;6 months after diabetesdiagnosis; thereafter, insulin substitution is necessary (4,5).

However, even 30 years after the first description ofthis clinical phenotype in the literature (2,3), a system-atic assessment of the pathology of the endocrine pan-creas of patients with LADA has not been performed.Only a few case reports have been published with a mor-phological assessment of the human LADA pancreas ina pancreatectomized organ (6) or in organ biopsy speci-mens (7,8). Furthermore, a LADA animal model has notbeen available up until now.

In the current study, we therefore defined the biochem-ical and molecular morphology features in a comparativefashion both in a collection of pancreases from patientswith LADA and of pancreases from a first spontaneous ratmodel 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 LADApancreases and in pancreases of adult patients with T1DMwith those in this new ratmodel of LADA, which we describehere for the first time. The results of the current study allowa clear distinction between the biochemical and morpholog-ical characteristics of the two forms of autoimmune diabetes.

1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany2Institute for Laboratory Animal Science, Hannover Medical School, Hannover,Germany3Department of General and Visceral Surgery, Diakovere, Henriettenstift,Hannover, Germany4Institute of Experimental Diabetes Research, Hannover Medical School,Hannover, Germany

Corresponding author: Sigurd Lenzen, lenzen.sigurd@mh-hannover.de

Received 30 August 2019 and accepted 11 January 2020

This article contains Supplementary Data online at https://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0865/-/DC1.

© 2020 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at https://www.diabetesjournals.org/content/license.

624 Diabetes Volume 69, April 2020

ISLETSTUDIES

RESEARCH DESIGN AND METHODS

Human Pancreas SamplesPancreases from Caucasian patients with T1DM (43 67 years; three men, one woman), with LADA (586 7 years;one man, three women), with T2DM (65 6 4 years; twomen, two women), and from healthy donors (446 8 years;three men, one woman) were obtained by organ resectionduring surgical intervention or from organ donors. Theanalyses were approved by the responsible ethics commit-tees (Supplementary Tables 1–4).

Rat Pancreas SamplesCongenic IDDM rats (for details see https://www.mh-hannover.de/3642.html)were bred andmaintained in accordance withthe 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 con-centrations were determined daily (Glucometer Elite; Bayer,Leverkusen, Germany). Serum C-peptide was analyzed witha rat-specific ELISA (Mercodia, Uppsala, Sweden).

Two pancreas tissue biopsies, with removal of 30 mgpancreas each from the pancreas tail, were performed aspreviously described in detail (13) before diabetes mani-festation in the T1DM rats at days 50 and 55 and in theLADA rats at days 70 and 80 of life. Experimental proce-dures were approved by the Lower Saxony State Office forConsumer Protection and Food Safety (LAVES), No. 33-42502-05/958 and 509.6-42502-03/684.

Early and Late Autoimmune Diabetes Manifestation inthe IDDM RatAutoimmune diabetes manifestation took place betweenday 55 and day 65 for the early and quickly developingT1DM form in the IDDM rat, with an average of 59 62 days as previously described (9,11,12). In T1DM animalsthe prediabetic phase with immune cell infiltration in theislets was between 3 and 7 days before diabetes onset(9,11). Besides the early T1DM form, a late and slowly de-veloping LADA form of autoimmune diabetes was identifiedin this rat model in a subgroup of animals. In these rats,islet immune cell infiltration started between day 75 and115, ;20 days before diabetes manifestation, which couldbe identified by sequential pancreatic biopsy specimensbefore diabetes manifestation. The average time of diabe-tes onset was day 87 6 4 (n 5 25 animals). The rats withthe T1DM form became insulin dependent immediatelyafter disease manifestation, requiring the start of an in-sulin supplementation within the first 3 days (9), while thelate form remained insulin independent over at least 1–2months and thus received no insulin supplementation. C-peptide concentrations in serum obtained at the day of or-gan collection were higher in the LADA group of animals(3766 7 pmol/L; n 5 8) than in the T1DM group (168 634 pmol/L; n5 6). Additionally, age-matched, nondiabeticcontrols were analyzed (controls for T1DM at day 60: 998641 pmol/L, n 5 8; controls for LADA at day 120: 942 6 38pmol/L, n 5 8).

Tissue FixationPancreas tissue specimens were fixed in 4% paraformal-dehyde in 0.15 mol/L PBS and embedded in paraffin(12,14).

ImmunohistochemistrySerial human and rat pancreas sections were stained bydouble immunofluorescence (12,14) for identification ofthe immune cells by the marker CD45 and the cell types inthe islet infiltrate using species specific antibodies (Sup-plementary Table 6).

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

Analysis of the Infiltrated IsletsA total of 20–30 pancreatic islets from each individualpancreas and, in the case of the LADA pancreases, 20–30pancreatic islets each both in the infiltrated and noninfil-trated areas were analyzed after diabetes manifestationfor each marker. Quantification of stained and nonstainedcells in the islets was performed by counting the positivelyimmunostained cells with an Olympus microscope BX61(9,14) (Tables 1 and 2). The same approach was used forthe TUNEL staining of apoptotic cells (9,14). After thewhole pancreas section was scanned, the analyses of thespecific mRNA staining were performed with a visual sys-tem and a VS-ASW computer-assisted program (Olympus,Hamburg, Germany) manually setting a threshold for thespecific mRNA staining and separating and calculatingthis specifically stained area as a percentage of the mea-sured infiltrated immune cell area or of the whole islet(Tables 3 and 4) comparing LADA with T1DM pancreases(14).

Quantitative Real-time PCRThe quality of the RNA isolated from the laser capturemicrodissected islets (for details see Supplementary Data)was controlled with the Experion electrophoresis system(Bio-Rad); mRNA was quantified in triplicates in quanti-tative real-time PCR reactions performed with the DNAEngine Opticon fluorescence detection system (Bio-Rad)and confirmed by gel electrophoreses (12). Gene expres-sion results were normalized for all groups against theribosomal protein L32 (Rpl32) using QGene and LinRegPCRsoftware. Primer sequences designed according specificguidelines (15) and efficiencies are provided in Supple-mentary Table 9.

StatisticsResults are presented as mean values 6 SEM, and com-parisons between the groups were analyzed with the one-wayANOVA followed by Dunnett test using Prism 5 software(GraphPad, San Diego, CA).

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Data and Resource AvailabilityThe data sets generated and analyzed during this study areavailable from the corresponding author upon reasonablerequest.

RESULTS

Comparison of the Immune Cell Infiltration Pattern inPancreases of Patients With LADA and With T1DMCompared With a Rat Model of LADA and of T1DMIn autoimmune diabetes, the pancreatic islets are infil-trated with a variety of immune cell types. As documentedin the current study, this was the case in the pancreases ofpatients with LADA (Fig. 1) and with T1DM as well as inthe pancreases of the LADA form (Fig. 2) and the adultform of autoimmune diabetes in the IDDM rat model.

A quantification of the immune cell composition of theinfiltrates in the human as well as in the rat pancreasesrevealed that CD8 T cells and CD68 macrophages were byfar the most frequent immune cell types present. CD4T cells, B cells, and natural killer cells were less frequent,with each cell type representing,10% in the islet immunecell infiltrate (Tables 1 and 2). In contrast, the nondiabeticcontrol islets of human and rat pancreases showed onlysome nonactivated macrophages (Table 1 and Fig. 1), whilenonactivated macrophages were doubled in islets of pan-creases from patients with T2DM (Table 1).

Islets in certain lobular areas of the pancreatic paren-chyma in human (Fig. 1) and rat (Fig. 2) pancreases of theLADA 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 fewislet-infiltrating nonactivated macrophages (Table 1).

The areas analyzed morphometrically without immunecell infiltration typical for LADA diabetes accounted forapproximately one-third of the sections of the pancreasarea in contrast to approximately two-thirds of both hu-man and rat LADA pancreases with infiltration (36.8 60.3% vs. 63.2 6 0.3% [n5 4] and 33.5 6 1.1% vs. 66.561.2% [n 5 8]), respectively. Such large areas without au-toimmune cell infiltration were not observed in humanT1DM pancreases. In none of the analyzed pancreases wereend-stage islets without b-cells observed.

When applying the definition of immune cell infiltra-tion in T1DM by Campbell-Thompson et al. (16) witha cutoff threshold value of .15 immune cells per infil-trated islet, the percentage of infiltrated islets in the hu-man LADA pancreases was calculated to be 64.8 6 2.7%compared with the percentage of 63.46 2.7% in infiltratedislets in the human T1DM pancreases (n 5 4 each).

All other islets in the human LADA and T1DM pan-creases were infiltrated with ,15 immune cells per islet.When adopting a limit of greater than three immune cellsper infiltrated islet, all islets in the human T1DMpancreasesand in the infiltrated areas in the human LADA pancreasescan be classified as infiltrated with activated immune cells.None of these islets contained fewer than three immunecells, which is the upper threshold for the numbers ofimmune cells infiltrating healthy human control islets andhuman T2DM islets (Table 1). These immune cells areexclusively nonactivated macrophages (Table 1).

The situation in the analyzed rat pancreases is moresimple, in that all islets in the rat T1DM pancreases and in

Table 2—Quantification of the absolute numbers of immunecells in the rat pancreatic islets

Immune cell type(per islet) Rat control Rat T1DM Rat LADA

CD4 T cells 0.0 6 0.0 1.9 6 0.2 1.7 6 0.2

CD8 T cells 0.0 6 0.0 18.2 6 0.4 12.5 6 0.3*

CD68 macrophages 0.5 6 0.2 19.4 6 0.4 25.8 6 0.5

CD20 B cells 0.0 6 0.0 2.1 6 0.3 2.6 6 0.2

CD161 natural killercells 0.0 6 0.0 1.0 6 0.2 1.3 6 0.1

The quantitative analysis of the frequency in the immune cellswas performed in each of 4 nondiabetic pancreases, of 6 pan-creases in T1DM, and of 8 pancreases in the LADA group (120 or160 islets for each immune cell type) and expressed as numberof immune cells. The total number of immune cells per islet was42.66 6.8 in T1DM vs. 44.66 1.6 in LADA, obtained by stainingwith the pan immune cell marker CD45. The ratio of CD68macrophages to CD8 T cells was 1.16 0.0 in T1DMand 2.16 0.1in LADA (P , 0.01). Results are presented as mean values 6SEM. *P , 0.05 for LADA vs. T1DM analyzed with one-wayANOVA followed by Dunnett test.

Table 1—Quantification of the absolute numbers of immune cells in human pancreatic islets

Immune cell type (per islet) Human control Human T1DM Human LADA Human T2DM

CD4 T cells 0.0 6 0.0 0.5 6 0.0 0.6 6 0.1 0.0 6 0.0

CD8 T cells 0.0 6 0.0 7.1 6 0.3 3.8 6 0.1* 0.0 6 0.0

CD68 macrophages 0.3 6 0.1 7.2 6 0.2 9.3 6 0.4 1.3 6 0.0

CD20 B cells 0.0 6 0.0 1.0 6 0.1 1.2 6 0.2 0.0 6 0.0

CD161 natural killer cells 0.0 6 0.0 0.5 6 0.1 0.7 6 0.1 0.0 6 0.0

The quantitative analysis of the frequency in the immune cells was performed in each of 4 pancreases in all groups (80 islets for eachimmune cell type) and expressed as number of immune cells. The total number of immune cells per islet was 15.96 0.2 in T1DMvs. 16.360.3 in LADA, obtained by staining with the pan immune cell marker CD45. The ratio of CD68 macrophages to CD8 T cells was 1.06 0.0 inT1DM and 2.56 0.2 in LADA (P, 0.01). Results are presented as mean values6 SEM. *P, 0.05 for LADA vs. T1DM analyzed with one-way ANOVA followed by Dunnett test.

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the infiltrated areas in the rat LADA pancreases contained.15 immune cells per infiltrated islet (Table 2). The non-infiltrated areas of the rat LADA pancreases containedfewer than three immune cells, in the same way as ratcontrol pancreases (Table 2).

The total number of immune cells infiltrating the isletswas four times higher in the rat pancreases than in thehuman pancreases (numbers in legends to Tables 1 and 2).Interestingly, however, the ratio between the numbers ofmacrophages and CD8 T cells shifted from 1.0 to 1.1 inhuman and rat T1DM toward a clear preference for macro-phages in the LADA pancreases, with an increase of theratios 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 perislet were not significantly different both in the human andrat pancreases of T1DM and LADA.

Cytokine and Cell Cycle Marker Gene Expression by InSitu RT-PCR in Areas With and Without Immune CellInfiltration in Human and Rat LADA Pancreases and forComparison in T1DM PancreasesTo identify immune cell activation in the islet infiltrate, theexpression of pro- and anti-inflammatory cytokines wasanalyzed by in situ gene expression of their mRNA tran-scripts in pancreatic sections. Immune cells in the isletinfiltrate expressed the main proinflammatory cytokines

Table 3—Gene expression quantification of pro- and anti-inflammatory cytokines and cell cycle markers as well as of insulin inhuman pancreatic islets by in situ RT-PCR

Human LADA

Parameter (%) Human control Human T1DM Area with infiltration Area without infiltration Human T2DM

IL-1b 0.3 6 0.1 22.5 6 1.3 29.2 6 1.5 0.1 6 0.1 1.3 6 0.1

TNF-a 0.0 6 0.0 18.6 6 0.9 8.8 6 0.6* 0.0 6 0.0 0.0 6 0.0

IFN-g 0.0 6 0.0 5.1 6 0.4 2.1 6 0.3 0.0 6 0.0 0.0 6 0.0

IL-10 0.3 6 0.2 3.6 6 0.2 10.1 6 0.6* 0.0 6 0.0 0.0 6 0.0

iNOS 0.1 6 0.1 10.7 6 3.0 7.7 6 0.6 0.0 6 0.0 0.0 6 0.0

Caspase 3 0.1 6 0.1 0.9 6 0.1 0.5 6 0.1 0.2 6 0.0 0.2 6 0.1

PCNA 0.8 6 0.1 0.6 6 0.1 1.6 6 0.1 1.2 6 0.2 1.2 6 0.0

Insulin 19.1 6 0.9 5.4 6 0.6 8.7 6 0.4 15.2 6 0.4** 16.6 6 1.3

Gene expression for the cytokines IL-1b, TNF-a, and IFN-g, for IL-10, and for iNOS, caspase 3 (CPP32), PCNA, and insulin. Thequantitative analysis of the expression frequency in the immune cells was performed in 4 pancreases in all groups (a total 80 islets for allparameters in each column) and calculated as a percentage of the positive mRNA transcript staining of the immune cells in the infiltrationarea for the cytokines and of the islet area for caspase 3, PCNA, and insulin. Analyses of TUNEL-positive b-cells revealed 0.56 0.1% inT1DM and 0.26 0.1% in LADA with infiltration and 0.16 0.0%without infiltration. *P, 0.05 and **P, 0.01 for LADA vs. T1DM analyzedwith one-way ANOVA followed by Dunnett test (n 5 4 in each group).

Table 4—Gene expression quantification of pro- and anti-inflammatory cytokines and cell cycle markers as well as of insulin inthe rat pancreatic islets by in situ RT-PCR

Rat LADA

Parameter (%) Rat nondiabetic Rat T1DM Area with infiltration Area without infiltration

IL-1b 0.1 6 0.0 18.8 6 0.5 34.8 6 0.7**# 0.1 6 0.1

TNF-a 0.0 6 0.0 17.6 6 0.6 9.4 6 0.4**# 0.0 6 0.0

IFN-g 0.0 6 0.0 1.7 6 0.2 1.0 6 0.1* 0.0 6 0.0

IL-10 0.1 6 0.1 3.5 6 0.2 9.1 6 0.5**## 0.2 6 0.1

iNOS 0.1 6 0.1 15.2 6 0.9 20.1 6 0.7** 0.0 6 0.0

Caspase 3 0.2 6 0.0 1.7 6 0.1 0.8 6 0.1**# 0.3 6 0.1

PCNA 1.1 6 0.2 1.3 6 0.1 2.9 6 0.2*# 1.2 6 0.1

Insulin 20.1 6 1.1 5.8 6 0.5 9.5 6 0.7 18.2 6 0.7**

Gene expression for the proinflammatory cytokines IL-1b (Il1b), TNF-a (Tnf), and IFN-g (Ifn-g), for IL-10 (Il10), and for iNOS (Nos2),caspase 3 (Cpp32), PCNA (Pcna), and insulin (Ins2). The quantitative analysis of the expression frequency in the immune cells wasperformed in 4 nondiabetic pancreases, 6 pancreases in the T1DM, and 8 in the LADA groups (total 120 for T1DM and 160 islets for eachLADA group per parameter) and calculated as a percentage of the positive mRNA transcript staining of the immune cells in the infiltrationarea for the cytokines and of the islet area for caspase 3, PCNA, and insulin. Analyses of TUNEL-positive b-cells revealed 2.86 0.2% inT1DM and 1.6 6 0.2% in LADA with infiltration and 0.1 6 0.1% without infiltration. *P , 0.05; **P , 0.01 for LADA from areas withinfiltrated islets against LADA from areaswith isletswithout infiltration analyzedwith one-wayANOVA followed byDunnett test; #P, 0.05;##P , 0.01 for LADA from areas with infiltrated islets against T1DM analyzed with one-way ANOVA followed by Dunnett test.

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Figure 1—Heterogeneous immune cell infiltration pattern in the lobular areas of the pancreas and in pancreatic islets of the same humanLADA pancreas compared with the nondiabetic situation and the T1DM form. A–H: b-Cells (green) and immune cells (red) [CD8 T cells (A andC) and CD68 macrophages (B and D)] were examined in islets from the same human LADA pancreas in different lobular areas without isletinfiltration (A) or with islet infiltration (C). The islets from LADA with infiltration resembled more the T1DM situation (E and F ), whereas isletswithout infiltration those of the control subjects (G and H). Islets were immunostained for insulin (Ins; green), CD68 macrophages (red), andCD8 T cells (red) and counterstained with DAPI (blue). Arrowsmark the immune cells in the infiltrate. Erythrocytes were identified by yellow toorange color through autofluorescence in the red and green channel. In each of the 4 pancreases 40–80 islets were analyzed. Scale bar 525 mm.

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Figure 2—Heterogeneous immune cell infiltration pattern in the lobular areas of the pancreas and in pancreatic islets of the same rat LADApancreas comparedwith the nondiabetic situation and the T1DM form.A–H: b-Cells (green) and immune cells (red) [CD8 T cells (A andC) andCD68 macrophages (B and D)] were examined in islets from the same rat LADA pancreas in different lobular areas (A and C) without isletinfiltration (B) or with islet infiltration (D). The islets from LADA with infiltration resembled more the T1DM situation (E and F), whereas isletswithout infiltration those of the controls (G and H). Islets were immunostained for insulin (Ins; green), CD8 T cells (red), and CD68macrophages (red) and counterstained with DAPI (blue). Arrows mark the immune cells in the infiltrate. Erythrocytes were identified by yellowto orange color through autofluorescence in the red and green channel. In each of the 8 pancreases 80–160 islets were analyzed. Scale bar525 mm.

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interleukin (IL) 1b and tumor necrosis factor-a (TNF-a) inboth the human (Table 3) and rat (Table 4) T1DM andLADA pancreases. Quantification of expression documenteda shift of the ratio between IL-1b and TNF-a from 1.2 inthe human T1DM pancreas to 3.4 in the infiltrated area ofthe human LADA pancreas (1.21 6 0.06 vs. 3.38 6 0.31;n 5 4 each; P , 0.01) and from 1.1 in the rat T1DMpancreas to 3.7 in the infiltrated area of the rat LADApancreas (1.07 6 0.04 vs. 3.73 6 0.12; n 5 6 and n 5 8,respectively; P , 0.01) due to a higher gene expressionlevel in favor of IL-1b compared with TNF-a. Interferon-g(IFN-g), the third prominent proinflammatory cytokine,was expressed at a comparatively low level in the infil-trates of both human and rat T1DM and LADA pancreases(Tables 3 and 4 and Supplementary Figs. 1 and 2). At thesame time, gene expression levels of the anti-inflammatorycytokine IL-10 were higher in the immune cell infiltrate ofthe human (Table 3) and rat (Table 4) LADA pancreasescompared with human and rat T1DM pancreases. Geneexpression of the enzyme inducible nitric oxide synthase(iNOS), which is well known to be induced by IL-1b (17),was high in the immune cell infiltrate of both the human(Table 3) and rat (Table 4) T1DM and LADA pancreases. Inthe areas of the human and rat LADA pancreases withislets without immune cell infiltration, an expression ofthe cytokine genes was not detectable, with the exceptionof a very low IL-1b expression level (Tables 3 and 4 andSupplementary Figs. 1 and 2). The same conclusion refers tothe nondiabetic control islets as well as to T2DM islets(Tables 3 and 4).

To evaluate the effects on b-cell destruction, caspase3 gene expression was analyzed. Caspase 3 expression wasmore than doubled in human and rat LADA pancreases andwas even four to five times higher in the b-cells of humanand rat T1DM pancreases compared with the control islets

of the human and rat pancreas areas without immune cellinfiltration as well as compared with the nondiabetic con-trol islets and T2DM islets (Tables 3 and 4). Gene expres-sion analyses of the proliferation marker proliferating cellnuclear antigen (PCNA) revealed increased expression levelsin b-cells of islets of the human and rat LADA pancreasescompared with the T1DM pancreases (Tables 3 and 4). Fur-thermore, insulin gene expression revealed a tendency forincreased expression levels in b-cells of infiltrated humanand rat LADA islets and a threefold higher expression levelin b-cells from noninfiltrated islets of LADA pancreases aswell as from nondiabetic control islets compared with T1DMislets (Tables 3 and 4).

Cytokine and Cell Cycle Marker Gene Expression byReal-time PCR in Areas With and Without Immune CellInfiltration in Laser Capture Microdissected Islets ofRat LADA Pancreases and for Comparison in Rat T1DMPancreasesTo quantify cytokine and cell cycle markers in islets withand without immune cell infiltrate from LADA rat pan-creases, the expression of pro- and anti-inflammatory cyto-kines was analyzed additionally by real-time PCR geneexpression of their mRNA transcripts after laser capturemicrodissection of the islets.

Immune cells in the islet infiltrate expressed the mainproinflammatory cytokines IL-1b and TNF-a in the rat(Table 5) LADA as well as in the T1DM pancreases. Quan-tification of expression documented, however, a significant(P , 0.01) shift of the ratio between IL-1b and TNF-afrom 0.6 in the T1DM islets to 1.7 in the infiltrated isletsof the rat LADA pancreas (0.596 0.09 vs. 1.686 0.74; n56 and 8, respectively; P , 0.01). The changed ratio wasdue to a significantly (P , 0.05) higher gene expressionlevel in favor of IL-1b and to a lesser reduction (P, 0.06)

Table 5—Quantification of gene expression of pro- and anti-inflammatory cytokines, iNOS, and cell cycle markers as well as ofinsulin by real-time PCR in laser capture microdissected islets in rats

Rat LADA

Group Rat nondiabetic Rat T1DM Area with infiltration Area without infiltration

IL-1b 0.01 6 0.01 0.72 6 0.14 1.57 6 0.68**# 0.01 6 0.01

TNF-a 0.01 6 0.00 1.36 6 0.43 0.82 6 0.18** 0.06 6 0.02

IFN-g 0.00 6 0.00 0.37 6 0.14 0.23 6 0.12** 0.01 6 0.00

IL-10 0.02 6 0.01 0.91 6 0.20 1.50 6 0.20* 0.64 6 0.23

iNOS 0.01 6 0.01 0.85 6 0.18 1.12 6 0.27** 0.08 6 0.03

Caspase 3 0.01 6 0.01 0.57 6 0.15 0.29 6 0.12**# 0.02 6 0.01

PCNA 0.49 6 0.27 0.72 6 0.18 1.34 6 0.39# 1.01 6 0.37

Insulin 5.57 6 2.19 1.62 6 0.32 2.12 6 0.45* 5.42 6 1.41

Relative gene expression for the cytokines IL-1b (Il1b), TNF-a (Tnf), and IFN-g (Ifn-g), for IL-10 (Il10), and for iNOS (Nos2), caspase3 (Casp3), PCNA (Pcna), and insulin (Ins2) normalized against Rpl32 as housekeeper gene. The quantitative analysis of the relativeexpressionwas performed in 6 pancreases of the nondiabetic and T1DMgroup and in 8 of the LADA groups. The islet equivalents (IE) werefor the nondiabetic situation 40.4 6 1.3 IE (n 5 6), for T1DM 51.0 6 5.6 IE (n 5 6) and for LADA with infiltration 41.8 6 2.1 IE (n 5 8) andwithout 41.46 2.7 IE (n5 8). *P, 0.05; **P, 0.01 for LADA from areas with infiltrated islets against LADA from areas with islets withoutinfiltration analyzed with one-way ANOVA followed by Dunnett test; #P , 0.05 for LADA from areas with infiltrated islets against T1DManalyzed with one-way ANOVA followed by Dunnett test.

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of the TNF-a expression level in the infiltrated rat islets ofthe LADA pancreases compared with the islets of T1DMpancreases. IFN-g was expressed at a low level in infil-trated islets of both T1DM and LADA (Table 5). In isletswithout 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 withinfiltration compared with LADA islets of the same ratpancreases without infiltration. The increase was less (P,0.07) when the LADA islets with infiltration were com-pared with T1DM islets (Table 5). The gene of the enzymeiNOS was highly expressed in infiltrated rat T1DM andLADA islets (Table 5).

Caspase 3 expression in rat LADA islets with infiltrationwas strongly increased by a factor of 10 and even more bya factor of 20 in the b-cells of rat T1DM islets whencompared with the very low expression level in the b-cellsof the islets of healthy controls and rat LADA pancreaseswithout immune cell infiltration (Table 5). Gene expres-sion analyses of the proliferation marker PCNA revealeda significant increase in b-cells of microdissected isletswithout infiltration and even a doubling of the expressionlevel in those islets with infiltration of the same rat LADApancreases compared with islets from T1DM pancreases(Table 5). Furthermore, insulin gene expression revealeda slight tendency for increased expression levels in b-cellsof infiltrated rat LADA islets and a more than threefoldhigher expression level in b-cells from noninfiltrated isletsof the rat LADA pancreases as well as from nondiabeticcontrol rat islets as compared with T1DM islets (Table 5).

DISCUSSION

It has been known for nearly 30 years that a subgroup of;10% of patients who develop diabetes at an age of .35years have a form that differs from T2DM in that patientsshow positivity for at least one antibody, typically the GADantibody (18), and develop a dependence on insulin sup-plementary therapy within 6months after diagnosis (19,20).This form is known as LADA (2,3,21) and represents amilder, more slowly progressing form of autoimmune di-abetes with a manifestation later in life (22–25). However,the morphological, molecular biology, and biochemical char-acteristics of the affected pancreases have not been ex-plored 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 currentstudy features that were comparable to those of patientswith LADA (see points 1–5 in Table 6). The most importantobservation both in the pancreases of patients with LADAand of the LADA rat model was that not all islets wereinfiltrated at the moment of disease manifestation butonly the islets in approximately two-thirds of the pan-creases. Thus, islets in an area of approximately one-thirdof the pancreases were unaffected, showing no signs of

immune cell infiltration and b-cell apoptosis. This was alsoobserved also in an analysis of the LADA pancreas organ ina case report (6). On the other hand, this can explain whyinfiltrated islets in a human LADA pancreas may not havebeen found upon analyses of pancreatic islet tissue speci-mens obtained by pancreas organ biopsy (7,8). The unaf-fected islets in the areas of the pancreas without infiltrationprevent a quick deterioration of the metabolic state. This isat variance from the situation in T1DM pancreases, whereall islets containing b-cells are infiltrated with activatedimmune cells (9,14,27–29).

In the prediabetic phase in the IDDM rat model ofhuman T1DM, areas without infiltrated islets are known toexist (9). These areas decrease in size in the pancreases ofrats in the prediabetic phase when analyzed at days 50 and55 of age (9) as closer the animals approach the time ofdiabetes manifestation (on average at day 59).

This explains not only the slower disease progression inpatients and rats with LADA but also the milder form ofautoimmune diabetes as documented by higher serum C-peptide concentrations, an observation that has also beenpreviously reported for patients with LADA (2,3,30,31) aswell as a higher level of b-cell insulin gene expression as anexplanation for the initial insulin independence.

The milder syndrome is convincingly documented alsoby the results of the molecular morphology analyses bothin the pancreases of patients with LADA and of the LADArat model in this study. A shift of the immune cell infil-tration from CD8 T cells toward CD68 macrophages anda shift of proinflammatory cytokine gene expression fromTNF-a toward IL-1b (see points 6–7 in Table 6) are majorelements of the alleviation of the disease process. In addition,the higher number of macrophages may support a fas-ter removal of apoptotic b-cells from the infiltrated islets.

Table 6—Ten features distinguishing the LADA form ofdiabetes from T1DM at the time point of diseasemanifestation in both human and rat LADA pancreases

Points Differences in characteristics

1 Slower disease progression and later onset of disease

2 Initial insulin independence of therapy after diagnosis

3 Higher serum C-peptide concentrations

4 Higher islet b-cell insulin gene expression

5 Distinct areas with and without islet infiltration in theaffected pancreas

6 Shift of immune cell infiltration from CD8 T cells →CD68 macrophages

7 Shift of the ratio of the proinflammatory cytokine geneexpression of TNF-a/IL-1b toward IL-1b

8 Increased gene expression of the proliferation markerPCNA

9 Increased gene expression of the anti-inflammatorycytokine IL-10

10 Lower expression of the apoptosis markers caspase3 and TUNEL

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Macrophages are known to produce more IL-1b thanother immune cell types (32), whereas T cells are the mainsite of TNF-a production (33). A high IL-1b release isknown to recruit preferentially macrophages into the areaof infiltration (34). Since the b-cell toxic potential of IL-1bis known to be lower (35) than that of TNF-a, it is plausiblethat the predominance of IL-1b in the immune cell in-filtrate in the LADA pancreas compared with the predom-inance of TNF-a in the immune cell infiltrate in the T1DMpancreas, both in humans as well as in the rat model, isa major reason for the milder and more slowly progressingform of autoimmune diabetes with a manifestation later inlife.

In addition, protective mechanisms, such as increasedb-cell proliferation capacity (increase of PCNA gene expres-sion) and anti-inflammatory capacity (increase of IL-10 geneexpression) to alleviate proapoptotic activities (lower cas-pase-3 gene expression along with reduced TUNEL positiv-ity) mediating the autoimmune b-cell destruction processthrough the proinflammatory cytokines, could be documentedunder LADA conditions (see points 8–10 in Table 6).

The pathological changes in the pancreas of the LADArat model mirror the changes in the human LADA pan-creas, making this new LADA animal model well suited forfuture in vivo and in vitro analyses into the pathogenesisof b-cell dysfunction and death in the LADA form ofautoimmune diabetes.

The present results provide a solid basis for execution ofLADA treatment schedules with curative potential basedon the improved understanding of the underlying pancre-atic islet pathology (23,25). The slower progression of thedisease process in LADA makes this autoimmune diabetesform an ideal model for translation of new effective cu-rative antibody combination therapies from the T1DManimal model (10,12) to patients with autoimmune di-abetes (10,23). The results document conclusively that LADAis 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 humanT2DM pancreases, the number of macrophages was dou-bled compared with healthy control subjects, confirmingearlier data from Richardson et al. (37). It is noteworthy tomenton that the macrophages in human T2DM pancreasesare clearly less than in the infiltrate of human T1DM andLADA pancreases (Tables 1 and 2). In this context, ourobservation that the macrophages in the T2DM and con-trol pancreases show no signs of immune cell activation isimportant (38). Thus, our results do not provide supportfor the view that LADA might be a diabetes form in thetransition between T1DM and T2DM as previously con-sidered by some authors (21,39).

In parallel, gene association studies in LADA patientsrevealed more susceptibility loci for immune cells andb-cells associated with T1DM than with T2DM loci (40,41).The same was true for the proinflammatory cytokine profileanalyzed in peripheral blood of patients with LADA showingsimilarities to the profile in T1DM rather than in patients

with T2DM (42). In addition, the shift from CD8 T cells tomacrophages in the islet infiltrate of both human and ratLADA pancreases, as shown in the current study, went alongwith a decreased frequency of islet effector CD8 T cells inthe peripheral blood of patients with LADA compared withpatients with T1DM, as previously reported (43). A recentcohort study comparing patients with T1DMand those withT2DM identified.20% of patients with T2DMas late onsetpatients with T1DM (44).

We can conclude, therefore, that LADA is a milder formof autoimmune diabetes in patients of an advanced age.This confirms the conclusion by Buzzetti and colleagues(24) that “compared with young onset diabetes, LADA rep-resents the other extreme of the autoimmune diabetes spec-trum.” That is a phenomenonwhich is characteristic ofmanydiseases that progress more slowly at an increasing age.

Nevertheless, using the term LADA is justified in clinicalmedicine to identify a late form of autoimmune diabetes inpatients with adult-onset diabetes of an older age and dis-tinguish this form from T2DM, because this diagnosis hasimportant implications for the prognosis and treatment aswell as for the well-being of the affected patients. So au-toimmune diabetes (21–25) is just another example of a dis-ease with a broad spectrum along the course of life (45).

Acknowledgments. The authors thank R. Chucholl and D. Lischke (In-stitute of Clinical Biochemistry, Hannover Medical School) for skillful technicalassistance.Funding. This work was supported by a grant from the DeutscheForschungsgemeinschaft to A.J. (JO 395/2-2).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.J. designed the study, performed experiments,analyzed and interpreted data, and wrote the manuscript. D.W. provided materialsand 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 ofthis work and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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