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The Journal of Immunology, 2004, 173: 6993-7002.
Copyright © 2004 by The American Association of Immunologists

Involvement of Eotaxin, Eosinophils, and Pancreatic Predisposition in Development of Type 1 Diabetes Mellitus in the BioBreeding Rat1

Martin J. Hessner2,*,{dagger}, Xujing Wang*,{dagger}, Lisa Meyer*, Rhonda Geoffrey{dagger}, Shuang Jia*,{dagger}, Jessica Fuller{ddagger}, Ake Lernmark{ddagger} and Soumitra Ghosh*

* The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics at the Medical College of Wisconsin and the Children’s Research Institute of the Children’s Hospital of Wisconsin, Milwaukee, WI 53226; {dagger} The Human and Molecular Genetics Center, The Medical College of Wisconsin, Milwaukee, WI 53226; and {ddagger} Robert H. Williams Laboratory, Department of Medicine, University of Washington, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Allergy and autoimmunity are both examples of deregulated immunity characterized by inflammation and injury of targeted tissues that have until recently been considered disparate disease processes. However, recent findings have implicated mast cells, in coordination with granulocytes and other immune effector cells, in the pathology of these two disorders. The BioBreeding (BB) DRlyp/lyp rat develops an autoimmune insulin-dependent diabetes similar to human type 1 diabetes mellitus (T1DM), whereas the BBDR+/+ rat does not. To better understand immune processes during development of T1DM, gene expression profiling at day (d) 40 (before insulitis) and d65 (before disease onset) was conducted on pancreatic lymph nodes of DRlyp/lyp, DR+/+, and Wistar-Furth (WF) rats. The eosinophil-recruiting chemokine, eotaxin, and the high-affinity IgE receptor (Fc{epsilon}RI) were up-regulated >5-fold in d65 DRlyp/lyp vs d65 DR+/+ pancreatic lymph nodes by microarray (p < 0.05) and quantitative RT-PCR studies (p < 0.05). DR+/+, WF, and d40 DRlyp/lyp animals possessed normal pancreatic histology; however, d65 DRlyp/lyp animals possessed eosinophilic insulitis. Therefore, immunohistochemistry for pancreatic eotaxin expression was conducted, revealing positive staining of d65 DRlyp/lyp islets. Islets of d65 DR+/+ rats also stained positively, consistent with underlying diabetic predisposition in the BB lineage, whereas WF islets did not. Other differentially expressed transcripts included those associated with eosinophils, mast cells, and lymphocytes. These data support an important role for these inflammatory mediators in BB rat T1DM and suggest that the lymphopenia due to the Ian5/(lyp) mutation may result in a deregulation of cells involved in insulitis and {beta} cell destruction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The diabetic prone (DP)3 BioBreeding (BB) rat spontaneously develops an autoimmune insulin-dependent diabetes, characterized by lymphocytic infiltration of the islets of Langerhans (insulitis) and T cell-mediated {beta} cell destruction (1). Clinical presentation and disease progression between BB rat and human type 1 diabetes mellitus (T1DM) are similar including hyperglycemia, glycosuria, weight loss, and decreased plasma insulin (2). The rats die from severe hyperglycemia and ketoacidosis within 1–2 wk of onset unless administered insulin. The diabetes-resistant (DR) BB rat was derived from DP BB/Worchester forebears, and <1% of DR rats develop diabetes (3). However, it is possible to induce diabetes in DR rats through administration of the lymphotoxic RT6 mAb and immune-activating polyinosinic, polycytidylic acid (polyIC) (4, 5, 6) or though viral depletion of CD4+CD25+ regulatory T cells (7), supporting the existence of diabetic predisposition in BB rats that can be phenotypically manifested upon immune perturbation. Such treatment of Wistar-Furth (WF) rats fails to induce disease.

Like human T1DM, diabetes in the BB rat is dependent on the MHC localized on rat chromosome 20 (Iddm2). The implicated RT1u haplotype, also shared by the nondiabetic WF and diabetic Tokushima rat, is an orthologue to human HLA-DQ, and many RT1u rat strains can be induced to develop insulitis or diabetes though treatment with polyIC (6, 8, 9). Besides the MHC, there are additional loci that contribute to T1DM in the DP rat, including: Iddm1 on chromosome 4 (10) and Iddm 3 on chromosome 2 (11). The Iddm1/lyp locus, linked to peripheral T cell lymphopenia (<15% normal T cell count) with low representation of CD5, CD4, CD8, and RT6 subsets, is essential for development of the diabetic phenotype, and is inherited in a Mendelian fashion (10, 12, 13). The DP lymphopenia (lyp) gene region on chromosome 4 has been fixed on the BB DR background (14, 15, 16). All DRlyp/lyp animals are lymphopenic, and, unlike the nonobese diabetic (NOD) mouse, 100% of animals have onset of diabetes between 50 and 76 days of age. The DR+/+ and DRlyp/+ animals never develop diabetes nor lymphopenia (11, 15, 16). These nearly genetically identical strains are well suited for comparison because they differ only by the 2-Mb lyp region. The Iddm1/lyp locus has been positionally cloned (12, 17), and the lymphopenic phenotype is attributed to a frame shift deletion in a novel member (Ian5) of the immune-associated nucleotide (IAN)-related gene family, which results in truncation of a significant portion of the encoded protein. Ian5 belongs to an uncharacterized gene family of GTP-binding proteins, is localized to the outer mitochondrial membrane, and is normally present in thymocytes, T cells, and B cells (18). The rat IAN5 consists of 308 amino acid residues and is 52% homologous to its human homologue (17). Other IAN gene family members are expressed in mature T cells and are switched on during thymic T cell development (17). The precise nature by which IAN5 regulates T cell hemostasis remains unclear. However, loss of IAN5 in T cells results in mitochodrial dysfunction, increased mitochondrial stress-inducible chaperonins, and brings about the characteristic DP lymphopenia through T cell-specific apoptosis of recent thymic emigrants (19).

Eosinophilia and eosinophil infiltration into islets of Langerhans before disease onset has been previously observed in the DP BB rat (14, 20) but remains poorly understood. Eosinophil migration is mediated by a number of chemokines, including eotaxin, a potent highly eosinophil-specific CC chemokine (21) that can be expressed many cell types including eosinophils, lymphocytes, macrophages, bronchial smooth muscle cells, endothelial cells, and mast cells. Eotaxin and several other chemokines, including RANTES and macrophage chemoattractant protein-4 (MCP-4), activate a common receptor, CCR3, which is expressed on eosinophils, Th2 cells, basophils, and mast cells (22, 23). Eotaxin has emerged as an important proinflammatory cytokine in the pathogenesis of allergic airway diseases, multiple skin conditions, inflammatory bowel disease, and gastrointestinal allergic hypersensitivity (24), where eosinophils are the central proinflammatory leukocyte. A number of studies evaluating the pathological role of eosinophils during inflammatory responses have demonstrated the necessity of eotaxin for eosinophil recruitment using neutralizing Abs or animal strains genetically deficient for eotaxin (25, 26, 27, 28, 29). Ab-blocking studies have also confirmed that CCR3 is the dominant receptor that mediates chemotactic responses to eosinophils in asthma and other allergic diseases (30). Therefore, effort has been placed on the development of therapeutic small molecule CCR3 antagonists, some of which have been found highly potent against human CCR3 (31, 32, 33).

Microarray technology enables a system-wide analysis through massive simultaneous gene expression profiling, thereby providing the potential to generate new insights into disease processes. To better understand immune-mediated {beta} cell destruction in the BB rat, we conducted gene expression profiling experiments on immune effector cells of the pancreatic lymph node during the prediabetic period (day (d) 40 and d65), using the DRlyp/lyp, DR+/+, and MHC-identical WF lines. Here, we report the overexpression of eotaxin in the pancreatic lymph node of DRlyp/lyp animals and observation of its production by both DR+/+ and DRlyp/lyp islet cells before insulitis. Consistent with recent reports defining a role for mast cells in autoimmunity (reviewed in Ref.34), transcripts associated with these effector cells were also identified. Collectively, these observations suggest that eotaxin, eosinophils, and mast cells may play a role in early disease pathogenesis in this model of T1DM.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

DR+/+ and DRlyp/lyp (15) congenic animals were maintained at the University of Washington or the Medical College of Wisconsin. WF rats were obtained from Harlan Teklad (Madison, WI). Animals raised off-site were shipped after weaning (25–30 days) and allowed to acclimate on-site for 3–7 days. All animals were kept under specific pathogen free conditions with standard light-dark cycles and were fed a regular diet and water ad libitum. Only female animals were selected for analysis to eliminate confounding gender-specific gene expression differences. Before sacrifice at d40 (before macrophage islet infiltration (35, 36)) or d65 (during aggressive insulitis) animals were fasted for 12 h, weighed, and had blood glucose levels measured. Animals with blood glucose levels above 250 mg/dl were disqualified. Animals were anesthetized using a 50 mg/ml intraperitoneal dose of Nembutal. Pancreas and pancreatic lymph nodes were harvested and either snap frozen in liquid nitrogen for RNA extraction or fixed 10% phosphate-buffered formalin for histological analysis. All institutional guidelines for the use and care of laboratory animals were followed.

Genotyping

Genotyping for simple sequence repeat markers was performed as described previously (17, 37). Animals were also genotyped by allele-specific PCR for the detection of the Ian5 normal and deletion alleles (6C->5C) using primers targeting the deletion point. Reactions possessed either a wild-type reverse primer (5'-ATC TTT GAC TCG AAG ATG GGG tG-3') or a deletion-specific reverse primer (5'-ATC TTT GAC TCG AAG ATG GGG tC-3'), paired with a common forward primer (5'-CCT GAG GAT CCT CCT GGT GG-3'), generating 192-bp and/or 191-bp products, respectively. A second primer pair was included in each reaction for amplification of a 523-bp C-reactive protein gene (CRP) internal positive control product (CRP forward 5'-TCT CTC AGG CTT TTG GTC AT-3'; CRP reverse 5'-TGT AGC AGA CTC CCA GGT G-3'). Allele-specific PCR used 200 ng of genomic DNA in 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTPs, 0.2 µM forward and reverse primers, 0.2 µM CRP forward and reverse primers, and 0.5 U of Taq polymerase (PerkinElmer Cetus, Norwalk, CT) in a reaction volume of 25 µl. Thirty-five cycles of PCR were performed (15 cycles of touchdown PCR: 94°C, 30 s; 70°C, 30 s, decreasing 0.5°C per cycle, to a low of 62.5 after 15 cycles; 72°C, 30 s; followed by 20 cycles of standard PCR: 94°C, 30 s; 63°C, 30 s; 72°C, 30 s). PCRs were terminated after a 10-min extension at 72°C. Products were analyzed by 2% agarose gel electrophoresis.

RNA extractions and GeneChip analysis

Total RNA was extracted from frozen pancreatic lymph nodes using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad CA). A single RNA sample was prepared from each animal with no sample pooling; six female animals per experimental condition were used. First- and second-strand cDNAs were synthesized from 15 µg of total RNA, and cRNA was synthesized, labeled, fragmented, and hybridized to the RG-U34A array in accordance to standard Affymetrix protocols (Affymetrix, Santa Clara, CA). The RG-U34A array allows detection of ~4710 known genes and 4090 expressed sequence tags (ESTs). After hybridization, arrays were washed, stained with PE-conjugated streptavidin (Molecular Probes, Eugene, OR), and scanned. Images were analyzed using Microarray Suite Version 5.0 (MAS 5.0; Affymetrix). The MAS 5.0 statistical algorithms were used to calculate signal intensities, probe set detection, probe set (gene expression) change, and signal log ratio. Hybridization data was analyzed using the commonly used two-profile comparison method with an arbitrary threshold for "significant fold-change" in expression level (38). Because each experimental condition was represented by six biological replicates, each comparison consisted of 36 possible pairings, and the list of differentially expressed genes for a comparison was derived from >1.5-fold changes present in at least 18/36 possible pairings, and the statistical significance of differential gene expression was derived through a t test (p < 0.05). Multiple test adjustments, such as the Bonferroni correction, were not performed because we used the microarray as an exploratory tool (39), with the intention of confirming results of interest with alternative technologies such as quantitative RT-PCR (QRT-PCR). Complex analysis of derived gene lists was performed using hierarchical clustering (Cluster 3.0) and self-organizing maps (SOMs; Genecluster 2) (40), followed by pathway analysis using Expression Analysis Systematic Explorer (EASE) for identification of biologically relevant themes (41). EASE accomplishes this task by conducting an over-representation analysis of the functional gene categories detected relative to the total assayed on the array, using the Gene Ontology and SwissProt databases as references.

Real-time QRT-PCR

Monoplex real-time QRT-PCR was performed using a Cepheid Smart Cycler System (Sunnyvale, CA), QuantumRNA 18s Internal Standards (Ambion, Austin, TX), eotaxin primers (forward, 5'-AGATGCACGCTGAAAGCCATAGTC-3'; reverse: 5'-GGTGCCGATATTCTCCCATAGCAT-3'; Sigma Genosys, The Woodlands, TX), and QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Synthesis of first-strand cDNA from 1 µg of RNA per animal was accomplished with random hexamers (Invitrogen Life Technologies) and Superscript II (Invitrogen Life Technologies) according to the manufacturer’s instructions. Triplicate eotaxin and 18s PCRs were performed for each sample in 25-µl reactions, which included 2 µl of cDNA and 12.5 µl of 2x SYBR QuantiTect SYBR Green PCR Master mix (Qiagen) possessing 1.5 µl of eotaxin (10 µM) or 18s-specific competimers (used as a 3:7 ratio of primer:competimer set; each stock is at 5 µM) and 9 µl of deionized water. Eotaxin reactions were cycled as follows: stage 1, 95°C 900 s; stage 2, 55 cycles 95°C 30 s, 66°C 30 s, 72°C 30 s; stage 3, melt curve 60–95°C. 18s reactions were cycled as follows: stage 1, 95°C 900 s; stage 2, 55 cycles 95°C 30 s, 55°C 30 s, 72°C 30 s, 82°C 6 s; stage 3, melt curve 60–95°C. A pooled and concentrated sample of DRlyp/lyp cDNA was used for both the eotaxin and 18s standard curves at undiluted, 1:5, 1:25, 1:125, and 1:625 concentrations, and a point from the standard curve was used as a positive control in each assay.

QRT-PCR for Fc{epsilon}RI was conducted using specific forward (5'-GGACGACATTGCTTTCAAGTACTCT-3') and reverse (5'-ATCAGATTTACATTCAACCTTGTTC-3') primers and methodology as described for eotaxin QRT-PCR except reactions were performed in a Rotor-Gene 3000 (Corbett Research, Morelake, Australia). Again a pooled and concentrated sample of DRlyp/lyp cDNA was used for both the Fc{epsilon}RI and 18s standard curves at undiluted, 1:3.5, 1:12.25, 1:42.9, 1:150, and 1:5, 1:25, 1:125, 1:625 concentrations, respectively, and a point from the standard curve was used as a positive control in each assay.

Specificity for all QRT-PCR was verified by both melting curve analysis and 2% agarose gel detection of single product. The data was analyzed with the Smart Cycler or Rotor-Gene 3000 software using the cycle threshold for quantification. Relative gene expression data (fold-change) between samples was accomplished using the mathematical model described by Pfaffl (42).

Histological and immunohistochemical analysis

Pancreatic tissues were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and duplicate 5-µm sections were mounted on each slide. Sections were then stained with H&E, reviewed by light microscopy, and scored for pancreatic insulitis as previously described (43). Immunohistochemistry was conducted with goat anti-mouse eotaxin (R&D Systems, Minneapolis, MN) on paraffin sections. Tissue sections were deparaffinized with xylene, washed with 100% ethanol, and rehydrated. Endogenous peroxidase activity was blocked with hydrogen peroxide, and sections were heat treated at 95°C in target Ag retrieval (Dako, Carpinteria, CA) and rinsed in Tris buffer, pH 7.6. Tissues were blocked using biotin/serum blocking system (Dako), rinsed, and incubated with a 1:50 dilution of goat anti-mouse eotaxin Ab in diluent (Dako). Sections were rinsed in Tris buffer, pH 7.6, incubated with biotinylated anti-goat IgG secondary Ab (Vector Laboratories, Burlingame, CA), followed by incubation with avidin/HRP conjugate (Vectastain ABC/DAB Elite kit) as prescribed by the manufacturer. Sections were rinsed in Tris buffer, pH 7.6, stained with 3'3'diaminobenzidine (Dako), washed with distilled water, dehydrated, mounted (permanent mounting medium; Dako), and viewed by light microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Analysis of WF, DR+/+, and DRlyp/lyp gene expression profiles

To better understand immune processes occurring before insulitis and before onset of diabetes, gene expression profiling of the pancreatic lymph node was conducted on female d40 and d65 DRlyp/lyp, DR+/+, and WF animals. A total of 36 animals, six per experimental condition, were analyzed independently, and no RNA pooling was performed. The expression profiles of the WF, DR+/+, and DRlyp/lyp strains were analyzed by hierarchical clustering to evaluate the overall relatedness of the three lines at d40 and d65. Expression values of d40 and d65 DR+/+ and DRlyp/lyp were normalized against the mean of the corresponding WF as a common reference, and the log ratios of genes that were labeled "present" or "marginal" on all arrays (n = 1232 genes) were clustered (Fig. 1). The WF animals distinctly grouped at both time points, while DRlyp/lyp and DR+/+ animals were less distinct. Analysis of the d65 data also pointed to underlying differences between the BB lines vs the WF, though the two BB strains were not obviously separable, possibly due to their nearly identical genetic background, shared underlying disease predisposition, or both.



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  		FIGURE 1. Hierarchical clustering of the three rat strains at (A) d40 and (B) d65. At both time points, the WF rats form a distinct group from the BB rats, while DRlyp/lyp and DR+/+ rats are not distinct. The difference between the BB strains and the Wistar strain widens at d65. The results indicate that there are similar responses at the level of pancreatic lymph node gene expression in BB DRlyp/lyp and DR+/+ strains.

 
We further studied the microarray data using the SOM function of GeneCluster 2, restricting the analysis to the mean log ratios showing significant (p < 0.05) differential expression in at least one of the four comparisons: d40 DR+/+ vs WF, d65 DR+/+ vs WF, d40 DRlyp/lyp vs WF, and d65 DRlyp/lyp vs WF. Six distinct groups were identified when clustering the 752 remaining genes (Fig. 2). Consistent with the hierarchical clustering (Fig. 1), most genes in the DR+/+ and DRlyp/lyp that were differentially expressed relative to the WF, showed a similar trend of change in the two BB strains (Fig. 2, A, C–E). However, the degree of change distinguished the two BB strains from one another. For clusters 2A–2E, the coordinate changes in gene expression between d40 DRlyp/lyp and d65 DRlyp/lyp was significantly greater than the corresponding changes in DR+/+ (paired t test, p < 0.002). The top 30 genes (excluding ESTs), ranked in the order of their confidence within the corresponding cluster, are tabulated in Fig. 2.



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  		FIGURE 2. Legend continues

SOM analysis of six major clusters were identified. The four data points connected by a blue line in each graph represent gene group values of DR+/+ at d40, DR+/+ at d65, DRlyp/lyp at d40, and DRlyp/lyp at d65, respectively. The red lines illustrate the variance associated with each group. In four of the six clusters (A, C, D, and E), genes show similar trends of change in both BB DRlyp/lyp and DR+/+ rats from d40 to d65, though the degree of change is different. Paired t test indicate that in the groups A-E, the measured differences are significant at p < 0.002. Listed for each cluster are associated genes (top 30) in the order of the their confidence; the left and right columns under each cluster plot show the GenBank accession number and gene name, respectively. ESTs are not shown.

 
Lists of significantly (p < 0.05) differentially expressed genes (d65 DRlyp/lyp vs WF and DR+/+ vs WF) were analyzed with the EASE software for identification of biologically relevant themes (41). The three most significant (p < 0.0001) functional categories identified in the DRlyp/lyp vs WF comparison, 1) response to biotic stimulus, 2) response to stress, and 3) defense response, were also significantly (p < 0.01) identified as the top three categories in the DR+/+ vs WF comparison, underscoring the high degree of genetic homology between these two congenic strains, as well as pointing to common predisposing factor(s) in the BB line. Consistent with the presence of an underlying predisposition in the BB lineage relative to the WF (4) was significantly (p < 0.05) increased gene expression (1.5- to 2.0-fold) between d40 and d65 of the secretory stress proteins pancreatitis-associated protein (PAP) I (Fig. 2D) and PAP III (Fig. 2A). These Reg (regeneration) family proteins were detected in seven of the 10 top functional categories in the DRlyp/lyp vs WF and six of the 10 top DR+/+ vs WF functional categories. The chemokine eotaxin and high-affinity IgE receptor, Fc{epsilon}RI, were detected in eight and five of the top 10 EASE functional categories when comparing DRlyp/lyp and WF expression profiles, respectively. Eotaxin and Fc{epsilon}RI expression were only detected in the d65 DRlyp/lyp group, where they were 6.78-fold and 8.11-fold overexpressed relative to the d65WF group, respectively. When directly comparing the d65 DRlyp/lyp group to the d65 DR+/+ group (Table I), these two genes were the most highly differentially expressed genes unique to the d65 time point.


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  		Table I. Summary of the top 20 differentially expressed genes between d65 DRlyp/lyp and d65 DR+/+ not observed at d40; ESTs are not shown

 
Confirmation of eotaxin and FcRI differential expression by QRT-PCR

Given the high d65 DRlyp/lyp expression of eotaxin in the pancreatic lymph node, the role this chemokine has in inflammatory disorders, and the previously observed eosinophilia in DP rats before disease onset (14, 20), eotaxin was initially selected for follow-up studies. Sufficient pancreatic lymph node RNA was available after the microarray studies for independent QRT-PCR analysis of five of the six d40 DRlyp/lyp, five of the six d65 DRlyp/lyp, five of the six d40 DR+/+, five of the six d65 DR+/+, six of the six d40 WF, and six of the six d65 WF animals. Therefore, the same animals/RNA samples were analyzed by both microarray and QRT-PCR analysis. Differences between RNA quality and quantity were normalized against the 18s rRNA as an internal housekeeping transcript. Standard curves for eotaxin and 18s rRNA had slopes of –3.719 (r2 = –0.992) and –4.879 (r2 = –0.999), respectively.

QRT-PCR provided confirmation of increased eotaxin transcript in the d65 DRlyp/lyp group; however, pancreatic lymph node eotaxin transcript was also detected in a single d40 DR+/+ and a single d40 DRlyp/lyp animal (Fig. 3A), due perhaps to the greater sensitivity of QRT-PCR. Variability in eotaxin expression was observed among the d65 DRlyp/lyp group likely arising from differences in disease progression between animals (11, 15, 16). More importantly, we observed a 5.2 ± 3.8-fold increase of the eotaxin transcript in the d65 DRlyp/lyp group relative the d65 WF (p < 0.05; two-tailed t test), a 0.43 ± 0.38-fold change in the d40 DRlyp/lyp group relative the d40 WF (p = 0.41), a 0.25 ± 0.06-fold change in the d65 DR+/+ group relative the d65 WF (p < 0.001), and a 0.56 ± 0.50-fold change in the d40 DR+/+ group relative the d40 WF (p = 0.59). At day 65, the relative difference in eotaxin expression by QRT-PCR between the two BB rat strains is ~20-fold, while the microarray measured a 7.69-fold difference. Although the relative decrease in eotaxin expression between the nondiabetic d65 DR+/+ group relative to the d65 WF group reaches statistical significance, it is likely not biologically relevant because the absolute expression in both of these groups is very low (Fig. 3A).



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  		FIGURE 3. Confirmation of microarray results by QRT-PCR. A, Eotaxin expression relative to WF determined by QRT-PCR (n = 5 animals per BB group; n = 6 animals per WF group). Inset, 2% agarose gel analysis of QRT-PCR products showing specificity of reactions: lane 1, 18s negative control, water; lane 2, d65 DRlyp/lyp 315-bp 18s rRNA product; lane 3, 100-bp ladder; lane 4, eotaxin negative control, water; lane 5, d65 DRlyp/lyp 219-bp eotaxin product. B, Fc{epsilon}RI expression of d65 DRlyp/lyp and DR+/+ groups relative to d65 WF group determined by QRT-PCR (n = 5 animals per BB group; n = 6 animals per d65 WF group). Inset, 2% agarose gel analysis of QRT-PCR products showing specificity of reactions: lane 1, 18s negative control, water; lane 2, d65 DRlyp/lyp 315-bp 18s rRNA product; lane 3, 100-bp ladder; lane 4, Fc{epsilon}RI negative control, water; lane 5, d65 DRlyp/lyp 124-bp Fc{epsilon}RI product.

 
After eotaxin, the Fc{epsilon}RI (high-affinity IgE receptor I) was the second most highly over-represented gene in the DRlyp/lyp group relative to the DR+/+ among those genes unique to the d65 time point (Table I). In rodents, this receptor is restricted primarily to mast cells and basophils (44). Sufficient RNA was available from the d65 animals to perform QRT-PCR for Fc{epsilon}RI using the same samples used for the GeneChip analysis (Fig. 3B). Again differences between RNA quality and quantity were normalized against the 18s rRNA as an internal housekeeping transcript and standard curves for Fc{epsilon}RI and 18s rRNA had slopes of –3.698 (r2 = –0.995) and –5.899 (r2 = –0.999), respectively. We observed an 11.6 ± 7.0-fold induction of the Fc{epsilon}RI transcript in the d65 DRlyp/lyp group relative the d65 WF group (p < 0.05; two-tailed t test) and a 1.96 ± 0.9-fold in the d65 DR+/+ group relative the d65 WF group. The relative QRT-PCR difference in Fc{epsilon}RI expression between the two BB rat strains was ~7-fold, again confirming the microarray result. The gene expression profile of the pancreatic lymph node of the d65 DRlyp/lyp group further indicated presence of mast cells because transcripts for mast cell protease 5 precursor, mast cell carboxypeptidase A, and IL-4 (45) were also detected. These observations are consistent with previous studies implicating mast cells in BB rat autoimmunity (46, 47).

Histological and immunohistochemical analysis

Pancreatic histology was examined through H&E staining and light microscopy in d40 and d65 DRlyp/lyp (n = 5 and 5, respectively), DR+/+ (n = 5 and 5, respectively), and WF (n = 4 and 4, respectively) animals. On average, >15 islets per section were analyzed and scored for insulitis (43). Islets in d40 and d65 DR+/+, d40 and d65 WF, as well as d40 DRlyp/lyp animals were normal. Conversely, four of the five of the d65 DRlyp/lyp animals analyzed possessed +3 insulitis defined as clumped mantle endocrine cells, leukocyte infiltration into the islet core, and presence of {beta} cell remnants (43). Immune infiltration into the islet included eosinophils, lymphocytes, and macrophages. One of the d65 DRlyp/lyp animals did not yet show insulitis at the time of sacrifice. These results, consistent with previous observations (35, 36, 43), are illustrated in Fig. 4, A, B, C, and G. A correlation between pancreatic lymph node eotaxin or Fc{epsilon}RI gene expression with degree of insulitis could not be made, at least in the sections analyzed.



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  		FIGURE 4. H&E staining of pancreata (A–C and G). A, B, and C, show d65 DRlyp/lyp, d65 DR+/+, and d65 WF, respectively, imaged with a x40 dry objective lens. Insulitis is detected only in d65 DRlyp/lyp animals, while DR+/+ and WF are histologically normal. Boxed area in A, d65 DRlyp/lyp, is imaged under higher magnification using a x60 oil immersion objective lens in G. Eosinophils are indicated with arrows. Anti-eotaxin immunohistochemical staining of pancreata (D, E, F, H, I, and J). D, E, F, and J show d65 DRlyp/lyp, d65 DR+/+, d65 WF, and d65 DRlyp/lyp lacking anti-eotaxin primary Ab (specificity control), respectively, imaged with a x40 dry objective lens. Positive staining of islets is detected in DRlyp/lyp and DR+/+ animals, but not WF. Boxed areas in D and E (d65 DRlyp/lyp and d65+/+) are imaged under higher magnification using a x60 oil immersion objective lens in H and I, respectively.

 
Given that the microarray and RT-PCR analysis showed presence of eotaxin transcript in the pancreatic lymph node of the d65 DRlyp/lyp rat, and histological examination of these animals revealed eosinophilic insulitis, immunohistochemistry assays with anti-eotaxin Ab was used to investigate the presence of eotaxin-expressing cells in the pancreas. Pancreata of d40 and d65 DRlyp/lyp (n = 6 and 5, respectively), DR+/+ (n = 5 and 5, respectively), and WF (n = 4 and 4, respectively) animals were analyzed. Two 5-µm paraffin-embedded sections per animal were reviewed and, on average, >15 islets per section were analyzed. Furthermore, negative controls lacking primary Ab were included to rule out nonspecific staining. Strong islet staining was observed in d65 DRlyp/lyp and DR+/+ animals (Fig. 4, D, E, H, and I). Examination of d65 WF islets did not show anti-eotaxin staining (Fig. 4F), and the intensity of staining in the WF was comparable to that observed in the negative controls lacking primary Ab (Fig. 4J). Evaluation of d40 animals revealed strong anti-eotaxin staining in two of the six DRlyp/lyp and two of the five DR+/+ animals but not in the WF, indicating that eotaxin expression within the islets of Langerhans occurs before onset of insulitis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, gene expression profiling of pancreatic lymph nodes was conducted on WF, DR+/+, and DRlyp/lyp rats with the objective of capturing the gene expression changes associated with the events before (d40) and at the initiation (d65) of diabetes within immune cells trafficking to/from the pancreas. Up-regulated eotaxin and Fc{epsilon}RI expression was detected in the pancreatic lymph node of only the d65 DRlyp/lyp animals. Consistent with previous studies, immune cell infiltration of the endocrine pancreas was not observed in d40 DRlyp/lyp animals, nor in DR+/+ or WF animals; however, insulitis-possessing eosinophils was observed in d65 DRlyp/lyp animals (35, 36). Eosinophilia coinciding with diabetes, along with increases in circulating neutrophils, lymphocytes, and monocytes, has been previously observed in the BB rat (14). Before the establishment of insulitis, the progression of cellular infiltrations is poorly understood; however, previous studies have indicated that the infiltration T and B lymphocytes is preceded by macrophages (48, 49, 50). The mechanisms behind immune cell infiltration into the islets is not clear; however, cytokine expression by infiltrating immune cells or islet cells themselves are likely involved because expression of IFN-{gamma}, IL-6, IL-1B, and TNF-{alpha} have been previously detected at or before diabetes onset (51, 52). The gene expression studies presented here implicated eotaxin, eosinophils, and mast cells in DRlyp/lyp disease progression; therefore, we performed eotaxin immunohistochemical staining to determine whether eotaxin-expressing cells could be detected in the either the endocrine or exocrine pancreas. We detected positive staining of BB rats islets and no staining of WF islets. Because eotaxin can be produced by leukocytes (eosinophils, macrophages, and T cells) (21, 22, 23), contribution to staining by these cell types cannot be excluded from islets possessing insulitis; however, no detectable difference in staining intensity between d65 DRlyp/lyp islets, and d65DR+/+ islets, which did not possess insulitis, was observed. The fact that eotaxin staining was detected in the both BB lines at d40 indicates eotaxin expression precedes insulitis and therefore is being produced by islet cells themselves. Because the insulin-producing {beta} cell is the most abundant islet cell type (~70%) (53) along with the degree and homogeneity of the anti-eotaxin immunohistochemical staining observe in the islets of BB rats, we speculate that {beta} cells themselves are producing eotaxin. We are currently investigating this through the use of dual immunofluorescence staining techniques.

The observation of eotaxin production by islets of both BB rat stains seems at first paradoxical, because only the DRlyp/lyp strain proceeds to develop diabetes. However, the fact that the DR+/+ strain produces eotaxin by its islets is consistent with previous studies where diabetes can be induced in DR rats through lympho-depletion with RT6 mAb (4, 5, 6), or though viral depletion of CD4+CD25+ regulatory T cells (7). In the WF rat, where we observe no anti-eotaxin staining of islets, these protocols fail to induce disease. Together, these data support the existence of a pancreas-specific diabetic predisposition that is represented, at least by islet eotaxin expression in BB rats. Development of insulitis and the transcription of eotaxin and Fc{epsilon}RI by immune cells of the pancreatic lymph node by DRlyp/lyp animals represents a different response to this underlying predisposition in the presence of the lymphopenia brought about by the Ian5 mutation.

Mast cells are emerging as important initiators and propagators of autoimmune diseases. In multiple sclerosis or its mouse model, experimental allergic encephalomyelitis, development of disease has been correlated with the number and/or distribution of mast cells, and in animal models drugs inhibiting mast cell degranulation have reduced disease severity (34, 54). Mast cells and granulocytes have also been implicated in joint and skin autoimmune disorders, as well as their mouse models where again mast cell-inhibiting agents have been found to have therapeutic effects (reviewed in Ref.34). Consistent with these reports, the Fc{epsilon}RI transcript was found highly over-represented in the d65 DRlyp/lyp group relative the d65 DR+/+ group. Additionally, our studies detected differential expression of other mast cell transcripts including mast cell protease 5 precursor, mast cell carboxypeptidase A, and IL-4 (Fig. 2, A and D) (45), which is consistent with previous studies implicating mast cells in BB rat autoimmunity (46, 47). Both mast cells and eosinophils possess the eotaxin receptor, CCR3, and both have the capacity to express eotaxin. Furthermore, mast cells can be activated by major basic protein liberated by eosinophils (55), and eosinophil degranulation and cellular adhesion capacity are regulated and promoted by mast cell mediators (56), illustrating the cooperative activities between mast cells and eosinophils.

In addition to islet eotaxin expression, EASE analysis of pancreatic lymph node gene expression profiles indicated an underlying pathology in the BB compared with the WF rats. This is consistent with the ability to induce diabetes in the DR BB but not the WF rats, as discussed above (4, 5, 7, 57). Consistent with underlying pancreatic predisposition in BB rats, is the increased Reg family gene expression. PAP I and PAP III are coordinately up-regulated in rat exocrine pancreas during induced acute pancreatitis (58). Furthermore, these proteins have been detected in newly diagnosed T1DM in human and NOD mouse, are up-regulated in response to injury, and are mitogenic to islet cells (59, 60, 61).

Serum triglycerides of BB rats are significantly higher than WF controls (62), and an association between hyperlipidemia and pancreatitis has been recognized for over a century, though poorly understood (63). An accepted and experimentally supported mechanism by which hypertriglyceridemia leads to pancreatitis involves hydrolysis of triglycerides in and around the pancreas by pancreatic lipase, leading to locally high concentrations of toxic free fatty acid and causing acinar cell and capillary damage (64, 65). In this study, we observed differences in lipid metabolism between the BB and WF rats, including increased BB rat pancreatic lipase expression (Fig. 2D). This condition may contribute to underlying pancreatic pathology present in the BB lineage, which combined with an immune system lacking suppressive activity of regulatory T cells (brought about through the Ian5 mutation, anti-RT6/polyIC, or viral induction) contributes to the development of diabetes.

Taken together, we conclude that comparison of the prediabetic DRlyp/lyp and DR+/+ rats has been useful in detecting gene expression differences related to disease progression/immune response, such as pancreatic lymph node expression of eotaxin. Inclusion of the WF has been valuable in identifying characteristics related to underlying autoimmune/diabetic predisposition that are shared between the DRlyp/lyp and DR+/+ lines, such as Reg family gene expression in the pancreatic lymph node and the expression of eotaxin in the islets of Langerhans. These studies point to involvement of eotaxin, eosinophils, and mast cells in BB rat diabetes pathogenesis as well as support the involvement of predisposing factors outside of Iddm1 and Iddm2 in this animal model.


   Acknowledgments
 
We thank G. R. Slocum and C. A. Bobrowitz for excellent technical assistance in the histological studies. The authors also thank Parthav Jailwala for assisting in data analysis, Daniel H. Moralejo for tissue collection, as well as Gareth Davies and Howard Jacob for assisting in the development of the study design.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work has been supported by Grant EB001421 awarded to M.J.H. and X.W. by the National Institute of Biomedical Imaging and Bioengineering and by a special fund from the Children’s Hospital of Wisconsin Foundation. Back

2 Address correspondence and reprint requests to Martin J. Hessner, The Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, The Medical College of Wisconsin and Children’s Hospital of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail address: mhessner{at}mcw.edu Back

3 Abbreviations used in this paper: DP, diabetic prone; BB, BioBreeding; T1DM, type 1 diabetes mellitus; DR, diabetes-resistant; polyIC, polyinosinic, polycytidylic acid; WF, Wistar-Furth; NOD, nonobese diabetic; IAN, immune-associated nucleotide; MCP, macrophage chemoattractant protein; d, day; CRP, C-reactive protein; EST, expressed sequence tag; QRT-PCR, quantitative RT-PCR; SOM, self-organizing map; EASE, Expression Analysis Systematic Explorer; PAP, pancreatitis-associated protein. Back

Received for publication May 6, 2004. Accepted for publication September 1, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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