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The Journal of Immunology, 2006, 177: 7275-7286.
Copyright © 2006 by The American Association of Immunologists, Inc.

Evidence of a Functional Role for Mast Cells in the Development of Type 1 Diabetes Mellitus in the BioBreeding Rat1

Rhonda Geoffrey*,{dagger}, Shuang Jia*,{dagger}, Anne E. Kwitek{dagger}, Jeffrey Woodliff*, Soumitra Ghosh*,{dagger}, Åke Lernmark{ddagger}, Xujing Wang*,{dagger} and Martin J. Hessner2,*,{dagger}

* Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics at the Medical College of Wisconsin, and Children’s Research Institute of the Children’s Hospital of Wisconsin, Milwaukee, WI 53226; {dagger} Human and Molecular Genetics Center, 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
 Disclosures
 References
 
Human type 1 diabetes mellitus (T1DM) arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the lymphopenic and spontaneously diabetic BioBreeding (BB) DRlyp/lyp rat. Previously, preonset expression profiling of whole DRlyp/lyp pancreatic lymph nodes (PLN) revealed innate immune activity, specifically that of mast cells and eosinophils. Furthermore, we observed that pancreatic islets of DRlyp/lyp rats as well as those of diabetes-inducible BB DR+/+ rats potentially recruit innate cells through eotaxin expression. Here we determine that lifelong eotaxin expression begins before 40 days of life and is localized specifically to β cells. In this report, we find that PLN mast cells are more abundant in DRlyp/lyp compared with related BB DR+/+ rats (2.1 ± 0.9% vs 0.9 ± 0.4% of total cells, p < 0.0001). DRlyp/lyp PLN mast cell gene expression profiling revealed an activated population and included significant overrepresentation of transcripts for mast cell protease 1, cationic trypsinogen, carboxypeptidase A, IL-5, and phospholipase C{gamma}. In the DR+/+ rat, which develops T1DM upon depletion of T regulator cells, mast cells displayed gene expression consistent with the negative regulation of degranulation, including significant overrepresentation of transcripts encoding tyrosine phosphatase SHP-1, lipid phosphatase SHIP, and E3 ubiquitin ligase c-Cbl. To recapitulate the negative mast cell regulation observed in the DR+/+ rats, we treated DRlyp/lyp rats with the mast cell "stabilizer" cromolyn, which significantly (p < 0.05) delayed T1DM onset. These findings are consistent with a growing body of evidence in human and animal models, where a role for mast cells in the initiation and progression of autoimmune disease is emerging.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human type 1 diabetes mellitus (T1DM)3 arises through autoimmune destruction of pancreatic β cells and is modeled in many respects by the BioBreeding (BB) rat, which originally arose as a spontaneous mutation within a Wistar colony (1). The DRlyp/lyp and DR+/+ strains are congenic derivatives of the BB rat that differ only by the 2-megabase lyp (lymphopenia; Iddm2) region on chromosome 4 and have provided important insights into the disease process (2).

The DRlyp/lyp rat develops spontaneous T1DM, which, like human disease, includes hyperglycemia, glycosuria, weight loss, and decreased plasma insulin. Unless insulin is administered, death follows shortly after onset as a result of severe hyperglycemia and ketoacidosis (3). Spontaneous diabetes in the DRlyp/lyp rat is multifactorial; however, MHC class II is the most important genetic factor, as it is in human and NOD mouse T1DM (4). The second key locus for spontaneous T1DM in the DRlyp/lyp rat is the Iddm2/lyp locus, which is linked to peripheral T cell lymphopenia (<15% normal T cell count, with low representation of CD4, CD8, and RT6 subsets) (5). DRlyp/lyp lymphopenia is attributed to a frame shift deletion in the Gimap5 gene that results in truncation of a significant portion of the encoded protein (6, 7). Gimap5 encodes a mitochondrial GTP-binding protein and is normally present in thymocytes, T cells, and B cells (8). Loss of Gimap5 in T cells brings about the characteristic lymphopenia through T cell-specific apoptosis of recent thymic emigrants (9).

The DR+/+ rat possesses a wild-type Gimap5 gene, is not lymphopenic, and does not develop spontaneous diabetes. However, DR+/+ rats are predisposed to T1DM, and disease can be induced through treatment with the lymphotoxic RT6 mAb and immune activating polyinosinic/polycytidylic acid (10) or though viral depletion of CD4+CD25+ TREG cells with Kilham’s rat virus (11). This predisposition is absent in the related MHC-identical Wistar-Furth (WF) rat, because such procedures fail to induce disease. Recently, adoptive transfer experiments of CD4+CD25+ T regulatory (TREG) cells to genetically lymphopenic BB or leukocyte-depleted DR rats have been shown to rescue animals from T1DM, illustrating the importance of this T cell subpopulation in suppressing an autoimmunity to β cells in this model (12, 13).

To better understand the immune processes before disease onset, we previously conducted gene expression profiling of whole pancreatic lymph nodes (PLN) of the DRlyp/lyp, DR+/+, and WF rats (14). We focused on two time points, day 40 and day 65, to capture events before insulitis and before T1DM onset, respectively. Many transcripts indicative of mast cells and eosinophils were overrepresented in day 65 DRlyp/lyp PLN, including the mast cell and eosinophil-recruiting chemokine eotaxin and IgE receptor (Fc{epsilon}RI, which is restricted primarily to mast cells in rodents) (15). Histological examination showed eosinophilic insulitis only in prediabetic day 65 DRlyp/lyp islets of Langerhans, which prompted us to investigate exocrine and/or endocrine expression of eotaxin in the pancreas. Islet cells in both BB strains were found to express eotaxin, whereas exocrine tissue did not (14). These findings fit well with a number of observations that are unique to the BB rat, specifically the ability to induce T1DM in the DR+/+ rat, as well as mast cell and eosinophil recruitment to islets of the DRlyp/lyp rat. Collectively, these observations are consistent with the more general growing recognition that mast cell and granulocyte responses play a role in initiating and propagating autoimmunity (16, 17).

As is observed in some human type 1 diabetics, lymphopenic BB rats also develop spontaneous autoimmune thyroiditis (18). Benovac et al., (19) reported significantly higher numbers of MHC class II-positive intrathyroidal mast cells in lymphopenic BB vs normal rats (p < 0.01) and observed increased MHC class II expression before the appearance of thyroid Abs or thyroid lymphocytic infiltration, suggesting that mast cells may be serving as APCs.

The tissue-dwelling mast cell is derived from the CD34+ hemopoietic stem cell in the bone marrow and has the capacity to migrate into virtually all vascularized tissues. Upon activation, mast cells can release numerous immune mediators and cell signaling molecules that are responsible for modulating innate and adaptive immune responses (20). Interactions between mast cells and T cells during the T cell-associated immune responses are still poorly understood. However, within sites of allergic inflammation, parasite infection, and contact hypersensitivity reactions, T cells and mast cells reside in close proximity (21). In vitro evidence shows that activated mast cells influence T cell-mediated responses in a number of ways. First, mast cells can present Ag to T cells through either MHC class I- or class II-restricted and costimulatory molecule-dependent mechanisms in both rodents and humans (22). Second, mast cells can promote T cell migration to inflammatory sites directly through the liberation of chemotactic factors or indirectly by mediating the up-regulation of endothelial cell adhesion molecule expression. Mast cells and their products also influence the maturation, migration, and activity of dendritic cells (21), thereby indirectly influencing the activation and response of T cells. In the BB rat, eosinophila as well as infiltration of the pancreatic islet by eosinophils and other innate cells, including mast cells, have been repeatedly reported before and at onset of T1DM (14, 23, 24, 25, 26, 27, 28); however, their role in disease progression has remained undefined.

In this report we localize islet eotaxin expression in the BB rat specifically to β cells and determine that its expression begins between day 30 and day 40 of life. This is relevant, because both mast cells and eosinophils possess the eotaxin receptor CCR3 and are potentially recruited to the pancreas by eotaxin. Secondly, we refine our initial whole tissue strategy by expression profiling purified day 60 mast cells from DR+/+ and DRlyp/lyp animals, where we identify the gene expression consistent with negative regulation of mast cell activation in the DR+/+ vs the gene expression consistent with an active immune response in the DRlyp/lyp rats. Finally, to recapitulate the negative regulation observed in the DR+/+, we show that treatment of DRlyp/lyp animals with the mast cell inhibitor cromolyn significantly delays onset of T1DM relative to saline-treated controls. Combined, these data present further evidence for a role of mast cells and the innate immune system in the pathogenesis of T1DM in the BB rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals, genotyping, animal monitoring, cromolyn treatment, and serum IgE measurements

Congenic BB rats (2) were maintained at the Medical College of Wisconsin (Milwaukee, WI). DR+/+ and DRlyp/lyp animals were generated through the mating of DRlyp/+ breeder pairs and identified through the genotyping of simple sequence repeat markers as described previously (7). WF rats were obtained from Harlan Teklad and allowed to acclimate onsite for 3–7 days before sacrifice. All institutional guidelines for the use and care of laboratory animals were reviewed by the Medical College of Wisconsin Institutional Animal Care and Use Committee and followed. All animals were kept under specific pathogen-free conditions with standard light/dark cycles and fed a regular diet and water ad libitum. Before sacrifice, animals were fasted for 12 h, weighed, and had blood glucose levels measured with a Bayer Ascensia Elite XL glucometer. Animals with blood glucose levels of >250 mg/dl were disqualified for gene expression and histological analysis. Animals were anesthetized under isoflurane during tissue collection.

Cromolyn (disodium cromoglycate) was delivered to animals by i.p. injection in buffered saline five times per week at a dosage of 200 µg/g body weight. Control littermates were treated with saline only. Animals were monitored 2–3 times per week, during which time they were bled via the tail vein (5–50 µl) for blood glucose and serum IgE measurements. Diabetes onset was defined as the first of two consecutive blood glucose measurements exceeding 250 mg/dl. Serum IgE levels were determined with the Bethyl Laboratories rat IgE ELISA quantitation kit in accordance with the manufacturer’s instructions.

Analysis and isolation of BB rat mast cells

Rat mast cells were identified and isolated from freshly harvested day 60 DR+/+ and DRlyp/lyp PLN using the well-characterized mast cell-specific AA4 mAb (29), which recognizes two derivatives of the ganglioside GD1b that are unique to the surfaces of rat mast cells. Briefly, a single cell suspension from PLN was washed and suspended in 0.1% BSA/PBS. Cells were enumerated and assessed for viability by trypan blue exclusion. Cell suspensions were treated with Fc Block (normal mouse IgG; catalog no.10400; Caltag Laboratories) and then stained with purified mouse anti-rat mast cell AA4 mAb (BD Pharmingen) for 20 min on ice. Cells were washed and then incubated with a 1/20 dilution of FITC-conjugated AffiniPure F(ab')2 goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) as a secondary Ab for 20 min on ice. Cells were recovered by centrifugation and incubated with R-PE-conjugated mouse anti-rat CD45R (BD Pharmingen) and R-PE-conjugated mouse anti-rat CD3 (BD Pharmingen) mAbs to facilitate the sorting of B and T cells, respectively. Next, cells were washed, resuspended in buffer, analyzed, and then recovered on a FACSVantage cytometer (BD Biosciences). It is known that binding of AA4 mAb to mast cells in the presence of extracellular calcium at 37°C rapidly induces morphologic changes and activation of protein kinase C (30); however, such changes are not observed when cell processing is conducted at 4°C or in the absence of extracellular calcium. Therefore, the protocol used calcium-free buffers, and cells were maintained at 4°C. The identity of the recovered AA4+ cell fraction was confirmed through May-Grünwald Giemsa staining and light microscopy as previously described (31). This isolation strategy is illustrated in Fig. 1.


Figure 1
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  		FIGURE 1. FACS analysis strategy for BB rat PLN mast cells using mast cell-specific (AA4 ganglioside GD1b) mAb. Illustrated is an analysis of day 60 DRlyp/lyp PLN mast cells. Forward scatter vs side scatter is plotted in A. Staining with anti-CD45R and anti-CD3 was used to facilitate the separation of mast cells, which are negative for these markers, from B and T cells, respectively (gate R2 in B). Mast cells were sorted with anti-AA4 (gate R3 in C) and collected. May-Grünwald Giemsa was performed on the recovered fraction and consistently showed a homogeneous mast cell population (D).

 
RNA extractions and GeneChip analysis

Rat mast cells were isolated from freshly harvested, female, day 60 DR+/+ and DRlyp/lyp PLN (n = 8 female animals per group), and total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies). The GeneChip rat genome 230, 2.0 array was selected for this study and possesses >31,000 probe sets representing >17,734 unique UniGenes. Purified RNA (~50 ng) was amplified using an Affymetrix two-cycle cDNA synthesis kit (catalog no. 900432), and cRNA was synthesized, labeled, fragmented, and hybridized to the rat genome 230, 2.0 array in accordance with standard Affymetrix protocols. Each RNA pool was analyzed in duplicate from independent RNA amplifications (four arrays total). After hybridization, arrays were washed, stained with PE-conjugated streptavidin (Molecular Probes), and scanned. Images were analyzed using GeneChip operating software (GCOS) version 1.1.1 (Affymetrix), and its statistical algorithms were used to calculate signal intensities, probe set detection, probe set (gene expression) change, and signal log ratio. Hybridization data were analyzed using the commonly used two-profile comparison method, and the statistical significance of differential gene expression was derived through a Student’s t test (p < 0.05) (32). Pathway analysis of derived gene lists was performed using Onto-Express (33), which conducts an overrepresentation analysis of the functional gene categories detected relative to the total functional gene categories assayed by the array, with the Gene Ontology (GO) project databases used as references (<http://vortex.cs.wayne.edu/projects.htm>).

Real-time quantitative RT-PCR (qRT-PCR)

Specific oligonucleotide primers for the following selected genes were designed with Oligo 6.66 (Molecular Biology Insights): carboxypeptidase A (Cpa1), IL-5, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1), SHIP, SHIP-2, dual specificity phosphatase (Dusp) 1, c-CBL E3 ubiquitin protein ligase, cationic trypsinogen (LOC286911), and chymotrypsin C (Ctrc). Monoplex real-time qRT-PCR was performed using a Rotor-Gene 3000 thermal cycler (Corbett Research), a QuantumRNA 18S internal standards kit (Ambion), locus-specific primers (Sigma Genosys), and QuantiTect SYBR Green PCR Master Mix (Qiagen) according to the manufacturer’s instructions. Synthesis of first strand cDNA from 50 to 100 ng of the pooled, unamplified DRlyp/lyp and DR+/+ RNA was accomplished with random hexamers (Invitrogen Life Technologies) and SuperScript II (Invitrogen Life Technologies) according to the manufacturer’s instructions. Duplicate locus-specific and 18S PCRs were performed for each gene analyzed in 20-µl reactions, which included 2 µl of cDNA and 10 µl of 2x SYBR QuantiTect SYBR Green PCR Master Mix (Qiagen) possessing 1.2 µl of locus-specific (10 µM) or 18S-specific competimers (used as a 3:7 ratio of primer/competimer set; each stock is at 5 µM) and 6.8 µl of deionized water. Reactions were typically cycled as follows: stage 1, 95°C for 900 s; stage 2, 50 cycles at 95°C for 30 s, 50–66°C for 30 s (locus specific), 72°C for 30 s, and fluorescence acquisition at 72–82°C for 20 s (locus specific); stage 3, melt curve at 60–95°C. The 18S reactions were cycled as follows: stage 1, 95°C for 900 s; stage 2, 50 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and 82°C for 15 s; stage 3, melt curve at 60–95°C. A pooled and concentrated sample of DR+/+ or DRlyp/lyp cDNA was used for both the locus-specific and the 18S standard curves at undiluted, 1:5, 1:25, 1:125, and 1:625 concentrations or until the end of linear amplification, and at least two points from the standard curve were used as positive controls in each assay. Specificity for all qRT-PCR was verified by both melting-curve analysis and 1.5% agarose gel detection of a single product of the predicted size. The data were analyzed with the Rotor-Gene 3000 software using the cycle threshold for quantification. Relative gene expression data (fold change) between samples were accomplished using the mathematical model described by Pfaffl (34). Primer designs, product sizes, and reaction performance parameters are provided in Table I.


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  		Table I. qRT-PCR performance parameters and primer designs

 
Immunofluorescent staining of pancreata

Pancreata were subjected to dual immunofluorescence staining using anti-eotaxin-specific Abs in combination with either anti-insulin- or anti-glucagon-specific Abs for the identification of eotaxin-expressing β cells and/or {alpha} cells based upon previously reported protocols (35, 36). Briefly, pancreatic tissues were fixed in 10% phosphate-buffered formalin at 4°C, then serially treated with 10, 20, or 30% sucrose solution, frozen in Tissue-Tek optimum cutting temperature compound (Electron Microscopy Sciences), and 4-µm sections were prepared using a MICROM HM 550 cryostat (Mikron Instruments), mounted onto poly-L-lysine coated slides, and dried for 30 min at 37°C. Sections were heat treated at 95°C in target Ag retrieval (DakoCytomation), rinsed in Tris buffer (pH 7.6), and blocked with 5% donkey serum (Sigma-Aldrich) in Tris buffer (pH 7.6) for 1 h at room temperature. For eotaxin/glucagon dual immunofluorescence, sections were stained with a 1/10 dilution goat anti-mouse eotaxin (R&D Systems) and a 1/2000 dilution mouse monoclonal anti-glucagon (Sigma-Aldrich) in Tris buffer (pH 7.6). Sections were rinsed and then incubated with 1/80 dilutions of Texas Red-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories) and FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories) secondary Abs in 50 mM Tris-HCl (pH 7.6) for 1 h. For eotaxin/insulin dual immunofluorescence, sections were stained with a 1/10 dilution goat anti-mouse eotaxin (R&D Systems) and a 1/8000 dilution mouse monoclonal anti-insulin Ab (Sigma-Aldrich) in Tris buffer (pH 7.6). Sections were rinsed and then incubated with 1/80 dilutions of Texas Red-conjugated donkey anti-goat IgG and FITC-conjugated donkey anti-mouse IgG secondary Abs in 50 mM Tris-HCl (pH 7.6) for 1 h. After staining, slides were washed with PBS and dried by centrifugation; cover slips were mounted with VECTASHIELD mounting medium (Vector Laboratories) and analyzed on a Nikon E600 upright microscope system (Nikon USA, Melville, NY) equipped with infinity optics, a Princeton Instruments MicroMax cooled charged-coupled device camera, and MetaMorph version 4.6 software (Universal Imaging).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Abundance of mast cells in the PLN of BB rats

In our previous analysis of whole PLN, the overrepresentation of mast cell transcripts in normoglycemic, prediabetic day 65 DRlyp/lyp rats relative to their DR+/+ counterparts was unexpected because mast cells are normally thought to reside in tissues, raising the question of whether the observed gene expression differences were due to increased numbers of mast cells, differences in the activity of mast cells, or a combination of both. Therefore, the abundance of mast cells was investigated by flow cytometry using the mast cell-specific AA4 mAb. These studies used 12 female DR+/+ and 12 female DRlyp/lyp animals. On average, approximately half as many total cells per animal were observed in the lymphopenic DRlyp/lyp vs DR+/+ PLN (2.4 x 106 ± 1.6 x 106 vs 4.3 x 106± 5.2 x 105, p < 0.005; two-tailed t test). In either case, >90% of the total events in either DRlyp/lyp or DR+/+ animals were identified as T and B cells through staining with anti-CD3 and anti-CD45R mAbs (93.4 ± 1.7 and 98.1 ± 0.8%, respectively). Mast cells, detected using anti-AA4, comprised 2.1 ± 0.9% of the total cells in the DRlyp/lyp vs 0.9 ± 0.4% of the total cells in the DR+/+ animals (2.3-fold less; p < 0.0001; two-tailed t test).

Expression profiling of DR+/+ and DRlyp/lyp PLN mast cells

To investigate transcriptional differences between day 60 DR+/+ and DRlyp/lyp PLN mast cells, 4.1 x 105 and 7.2 x 105 mast cells from eight pooled DR+/+ and eight pooled DRlyp/lyp animals, respectively, were isolated by FACS. RNA was extracted, amplified, and hybridized to the Affymetrix GeneChip rat genome 230, 2.0 array. Each sample pool was independently amplified in duplicate, hybridized to two arrays, and data were analyzed with Affymetrix GeneChip operating software, version 1.1.1. The raw data files have been submitted to the Gene Expression Omnibus database (accession number GSE4990) as files GSM112464 (DRlyp/lyp replicate 1), GSM112487 (DRlyp/lyp replicate 2), GSM112488 (DR+/+ replicate 1), and GSM112489 (DR+/+ replicate 2). After data filtering, 3567 probe sets (11.5%) exhibited differential gene expression (|log2 ratio| > 0.5; p < 0.05, Student’s t test). More stringent statistical criteria were not applied, as relevant genes and pathways would be investigated further by qRT-PCR. Genes overrepresented in the DRlyp/lyp (n = 1,647 probe sets; supplemental table A)4 and those overrepresented in the DR+/+ (n = 1920 probe sets; supplemental table B) were independently evaluated for biological themes using Onto-Express. This analysis resulted in the identification of 291 significant up-regulated GO molecular functions in the DRlyp/lyp (supplemental table C) and 398 up-regulated GO molecular functions in the DR+/+ (supplemental table D). To focus on activities representing the largest functional differences between the DRlyp/lyp and DR+/+, the analysis was restricted to those GO molecular functions possessing more than three unique detected UniGenes. The top 25 categories meeting this criterion are shown in Table II.


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  		Table II. Top 25 DRlyp/lyp up-regulated (upper section) and top 25 down-regulated DR+/+ (lower section) GO molecular functionsa

 
GO molecular functions detected in DRlyp/lyp mast cells

Protein tyrosine and lipid phosphorylations are critical, early events in mast cell activation through Fc{epsilon}R1 and other cell surface receptors (i.e., c-kit, IL-3R, and TLR). Aggregation of multiple IgE-occupied Fc{epsilon}RI by polyvalent Ag leads to phosphorylation of Fc{epsilon}RI by Lyn, a protein tyrosine kinase (PTK), and the association of other PTKs, including Syk, as well as the recruitment of adaptor molecules such as LAT (linker for activation of T cells; recently reviewed in Ref. 21). Syk activation leads either directly or indirectly to the phosphorylation of numerous cytoplasmic signaling molecules that do the following 1) activate/phosphorylate other proteins (i.e., protein kinase C and other serine, threonine, and tyrosine kinases); 2) modify membrane phospholipids (i.e., phospholipase C{gamma} isoforms, PI3K isoforms, and others); and 3) activate small GTPases, Ras, Rac, and Rho, which regulate the activation of MAPK, JNK, ERK, and p38. These, in turn, regulate transcription factors necessary for cytokine production and the activity of phospholipase A2 to generate arachidonic acid metabolites (21). This activation cascade is reflected in the DRlyp/lyp gene expression profile by the identification of up-regulated GO molecular functions that included GTPase binding, phospholipase C activity, protein kinase C activity, signal transducer activity, protein serine/threonine kinase activity, and chymotrypsin activity (Table II, upper section). The up-regulated genes in the DRlyp/lyp that gave rise to the GO molecular functions related to mast cell signal transduction (and their associated fold increase relative to the DR+/+) included phospholipase C{gamma} (5.8-fold), LAT (3.0-fold), protein kinase C, β1 (3.9-fold), protein kinase C, Theta (10.2-fold), MEK kinase kinase 4 (2.4-fold), and numerous others summarized in Table III (and supplemental tables A and C). Consistent with the activation of the DRlyp/lyp mast cells is the overrepresentation of transcripts encoding mast cell mediators or enzymes involved in mediator synthesis, including cationic trypsinogen (44.5-fold), elastase 2 (36.7-fold), eosinophil cationic protein (14.5-fold), arachidonate 12-lipoxygenase (4.5-fold), chondroitin sulfate proteoglycan 2 (4.4-fold), vascular endothelial growth factor C (4.2-fold), mast cell protease 1 (3.7-fold); mast cell peptidase 2 (2.5-fold); mast cell protease 4 (2.1-fold) and others (Table III and supplemental tables A and C).


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  		Table III. Genes overrepresented in the DRlyp/lyp relative to DR+/+ associated with detected GO molecular functions

 
GO molecular functions detected in DR+/+ mast cells

Despite the fact that the DR+/+ strain does not develop spontaneous T1DM, kinase activity, protein kinase activity, protein serine/threonine kinase activity, and protein-tyrosine kinase activity were significantly detected GO molecular functions, suggesting that mast cells in this strain are also activated (Table II, lower section, and supplemental table C). Genes annotated under these GO molecular functions (and their associated DR+/+:DRlyp/lyp fold change) included Lyn Src-related tyrosine kinase (3.9-fold), Janus kinase 3 (1.6-fold), Janus kinase 1 (1.8-fold), MAPK kinase kinase kinase 1 (4.2-fold), and others. More importantly, pathway analysis of the DR+/+ revealed numerous activities related to the attenuation of signal transduction. Phosphatases play an important antagonistic role to kinases in regulating mast cell activation/degranulation. Accordingly, the Onto-Express analysis of the DR+/+ mast cell expression profile detected both protein phosphatase type 2A activity and MAPK phosphatase activity as significant GO molecular functions, which were identified due to overrepresentation of Dusp1 (4.4-fold), Dusp5 (2.0-fold), and Dusp6 (1.8-fold), which reverse MAPK activation by dephosphorylating critical phosphotyrosine and phosphothreonine residues (37), as well as to the detection of protein phosphatase 1, 2, and 3 subunits (Table IV and supplemental tables B and D).


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  		Table IV. Genes overrepresented in the DR+/+ relative to DRlyp/lyp associated with detected GO molecular functions

 
Although tyrosine phosphatases provide an essential mechanism of negative regulation for tyrosine kinase-coupled receptors (38), protein ubiquitylation of activated PTKs and other crucial early signaling intermediates results in directing them to the proteasome or lysosome and represents another mechanism of negatively regulating signal transduction (39, 40). These are multistep processes that include E2 ubiquitin-conjugating enzymes and E3 ubiquitin protein ligases. The Onto-Express analysis identified GO molecular functions associated with ubiquitylation and proteosomal degradation up-regulated in the day 60 DR+/+ mast cell expression profile; these functions included endopeptidase activity and ubiquitin-protein ligase activity (Table II, lower section). Genes overexpressed in the DR+/+ that lead to the identification of these categories included the ring finger protein (C3H2C3 type) 6 (2.2-fold), ring finger protein 4 (2.4-fold), ubiquitin-conjugating enzyme E2D 3 (1.9-fold), ubiquitin-conjugating enzyme E2D 2 (1.9-fold), proteasome (prosome, macropain) subunit, {alpha} type 1 (1.9-fold), and others (Table IV).

Although evidence for activation was observed in DR+/+ mast cells and two molecular functions consistent with attenuation of signal transduction were observed in the DRlyp/lyp (protein phosphatase type 2A activity and ubiquitin-conjugating enzyme activity; Table II, upper section), in general terms the Onto-Express analysis indicated that PLN mast cells of the DRlyp/lyp animals were terminally activated whereas those of the DR+/+ animals were negatively regulated. This conclusion is supported by the number of GO molecular functions identified (and their p values) as well as the genes annotated under these GO categories.

qRT-PCR of genes relevant to mast cell function

To further investigate the activity levels of DRlyp/lyp and DR+/+ mast cells, qRT-PCR studies were conducted on genes identified as being differentially expressed by the GeneChip studies as well on annotated genes important to mast cell function. These studies focused on mediators and negative regulators (summarized in Table V) and used the same pools of unamplified DR+/+ and DRlyp/lyp mast cell total RNA that were used in the microarray analysis. The expression of carboxypeptidase A, an exopeptidase mediator and major protein component in secretory granules, begins during the mast cell progenitor stage and increases as immature mast cells mature (41, 42). Because carboxypeptidase–/– mast cells fail to develop a fully mature phenotype, carboxypeptidase may also play an important intracellular function (43). Carboxypeptidase A was found to be overrepresented in the DRlyp/lyp relative to the DR+/+ by >30-fold in both array and qRT-PCR analyses. Cationic trypsinogen as well as chymotrypsin C (caldecrin), which stimulates autoactivation of cationic trypsinogen (44), were respectively overrepresented 44.5 and 4.4-fold by array analysis in the DRlyp/lyp. Confirmatory qRT-PCR for these transcripts detected changes of 18.6- and 2.1-fold, respectively. Mast cells are also capable of liberating numerous chemokines and cytokines including IL-5, which promotes eosinophil differentiation and release from the bone marrow (45, 46, 47). Both the array and the qRT-PCR studies found the IL-5 transcript overrepresented in the DRlyp/lyp >4-fold, which is consistent with the eosinophilia observed in BB rats before T1DM onset (14, 27, 48).


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  		Table V. DRlyp/lyp:DR+/+ qRT-PCR of genes relevant to mast cell function

 
Several negative intracellular regulators of signaling events initiated by Fc{epsilon}RI were also investigated by qRT-PCR studies (Table V). SHP-1, which dephosphorylates/inactivates Syk, was found overrepresented in DR+/+ mast cells by both GeneChip and qRT-PCR analysis (1.7- and 2.7-fold, respectively). Dusp1 (also known as Ptpn16 or MAPK phosphatase 1) was found ~4-fold overrepresented in DR+/+ by both types of analysis. The lipid phosphatases SHIP and SHIP2, which act as negative regulators of myeloid cell proliferation, survival, and activation by hydrolyzing the PI3K-generated second messenger PtdIns(3, 4, 5)P3(PI-3, 4, 5-P3) to PtdIns(3, 4)P2 (21, 49), were also investigated. SHIP was overrepresented in DR+/+ mast cells (2.7-fold). SHIP2 was found 1.3-fold overrepresented; however, it did not reach statistical significance in either qRT-PCR or microarray analysis. Likewise, the E3 ubiquitin ligase c-Cbl, which facilitates ubiquitination of Fc{epsilon}RI, Syk, and Lyn (21), was overrepresented in DR+/+ mast cells 1.7-fold.

These findings, combined with the enumeration data, show that mast cells within the PLN of the DRlyp/lyp and the DR+/+ differ both in their relative abundance and activity. Furthermore, these data suggest that day 60 PLN mast cells of both BB strains are activated; however, the DR+/+ are negatively regulated whereas the DRlyp/lyp mast cells are not.

Measurement of serum IgE levels in BB and WF rats

Because the gene expression studies showed evidence of mast cell activation in both BB strains, serum IgE levels were monitored longitudinally from day 30 to day 70 in DRlyp/lyp, DR+/+, and WF rats. These studies further solidified a relationship between mast cells and T1DM in the DRlyp/lyp rat, because significant increases in measurable serum IgE levels were observed by day 50 in all DRlyp/lyp animals whereas serum IgE levels were undetectable in the DR+/+ and WF strains (Fig. 2).


Figure 2
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  		FIGURE 2. Longitudinal monitoring of serum IgE levels. Serum IgE of DRlyp/lyp (n = 17, dashed line), DR+/+ (n = 10, solid line), and WF (n = 14, dotted line) rats determined with the Bethyl Laboratories rat IgE ELISA quantitation kit.

 
Immunofluorescence staining of pancreata

Consistent with other studies (24, 25, 26), we previously observed normal pancreatic histology in DR+/+ and WF rats at either day 40 or day 65 (14). We also found DRlyp/lyp rats to be histologically normal at day 40; however, day 65 DRlyp/lyp animals possessed insulitis that included numerous eosinophils, as well as lymphocytes and macrophages (14, 24). Because eotaxin plays an important in vivo role in the recruitment of CCR3-bearing cells, which include both eosinophils and mast cells (50, 51, 52), we investigated whether either endocrine or exocrine cells of the pancreas were expressing eotaxin by using immunohistochemistry. Pancreatic islets of both BB rat strains were positive for eotaxin expression at days 40 and 65, consistent with the existence of an underlying pancreas-specific diabetic predisposition in BB rats and the ability to induce diabetes in DR+/+, whereas WF islets did not express eotaxin at either time point. For this study, determining when islet eotaxin expression begins in life was a prerequisite for therapeutically targeting mast cells to functionally implicate them in diabetes pathogenesis, because the use of mast cell inhibitors to delay or prevent disease onset would be more likely to succeed if they were administered before the initiation of immune signaling.

Pancreata from day 20 to day 60 WF, DR+/+, and DRlyp/lyp animals were harvested for analysis, and dual immunofluorescence staining protocols were optimized so that islet eotaxin expression could be specifically localized. Pancreatic islets contain four endocrine cell types, {alpha}, β, {gamma}, and {delta}, which respectively produce glucagon, insulin, pancreatic polypeptide, and somatostatin and possess respective distributions of ~21, ~68, ~5, and ~6% (53). Dual immunofluorescence staining was performed on pancreatic sections of WF, DR+/+, and DRlyp/lyp animals at days 20, 30, 40, 50, and 60 using anti-eotaxin-specific Abs in combination with anti-insulin or anti-glucagon-specific Abs for identification of eotaxin-expressing β cells and/or {alpha} cells. At least three animals were evaluated for each time point, and between 18 and 51 islets per animal were examined under each of the two staining protocols. All WF animals (n = 20) possessed negative eotaxin staining under either staining protocol (Fig. 3, columns A and C), whereas 100% of BB rats at day 40 or older (n = 14 DR+/+ and n = 14 DRlyp/lyp) exhibited positive islet eotaxin staining (Fig. 3, columns B and D). A single DR+/+ animal (n = 1/4) showed weak eotaxin staining at day 30. Islet eotaxin expression in the DRlyp/lyp (not shown) paralleled that of the DR+/+ with the exception that autoimmune β cell loss was evident in some day 60 animals as evidenced by a loss of insulin (and eotaxin) staining cells. Finally, this study revealed that islet eotaxin expression in DR+/+ and DRlyp/lyp rats is localized to β cells and not to {alpha} cells (Fig. 3, columns B and D; day 40 through day 60) and is consistent with the concept that β cells are active participants in the autoimmune process (54, 55, 56).


Figure 3
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  		FIGURE 3. Immunofluorescent staining of pancreata. Columns A and B, staining of WF and DR+/+ sections with anti-glucagon (FITC; green) and anti-eotaxin (Texas Red). Columns C and D, staining of WF and DR+/+ sections with anti-insulin (FITC; green) and anti-eotaxin (Texas Red). Time points from day 20 through day 60 are indicated by row; the bottom row (–C) lacks primary anti-eotaxin Ab as a negative (specificity) control. All imaging was conducted under a x40 dry objective lens.

 
Delaying onset of T1DM with the mast cell inhibitor cromolyn

The expression profiles of day 60 DRlyp/lyp and DR+/+ mast cells were consistent with activated and negatively regulated states, respectively. Therefore we set out to pharmacologically regulate DRlyp/lyp mast cell activity by treating animals with the mast cell inhibitor cromolyn (disodium cromoglycate), which has been used to implicate a functional role for mast cells in other autoimmune models through a reduction in disease severity (57, 58).

A total of 16 animals were treated with 200 µg/g cromolyn (in PBS) administered five times per week by i.p. injection. Control animals (n = 14) were treated with saline. In either case, treatment was initiated by day 30, which is before islet eotaxin expression, and animals had fasting blood glucose measured three times per week. The study duration was defined as 130 days, twice the normal time to onset, and disease onset was defined as the first of two consecutive blood glucose measurements >250 mg/dl. A survival plot is illustrated in Fig. 4, where the cromolyn-treated group survived an average of 82 ± 30 days (range 52–130), with 25% of the animals remaining disease-free until the study endpoint (130 days). The saline-injected controls (n = 14) survived an average of 63 ± 6 days (range 53–75), with 100% of the animals developing T1DM. A log-rank test finds significant delay in disease onset between treated and control animals (p = 0.045), whereas the hazard ratio is 0.49, indicating that cromolyn treatment reduces the risk for diabetes by about one-half. Histological examination of pancreata at day 130 after cromolyn treatment showed normal islet histology (data not shown).


Figure 4
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  		FIGURE 4. Longitudinal monitoring of cromolyn-treated and saline-treated DRlyp/lyp animals. Survival of animals treated with 200 µg/g cromolyn group (n = 16) is shown by the solid line, survival of the control group is shown by the dashed line. Treated animals survived 82 ± 30 days (range 52–130), whereas the saline-injected controls (n = 14) survived 63 ± 6 days (range 53–75) (p = 0.045; log rank test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
T cells are considered the primary mediators of rodent and human T1DM (55, 59, 60, 61, 62). In rodent models, this concept is supported by the fact that early thymectomy in both the NOD mouse and the BB rat prevents disease (55, 59, 60, 61, 62); however, precisely how β cell-specific adaptive immune responses arise remains unresolved. Naive T cells are typically thought to circulate through the blood and lymphoid organs, normally lacking the ability to infiltrate organ tissue. A growing body of evidence supports that events initiating T1DM occur at the β cell level (reviewed in Ref. 55). Under various stresses (infection, hyperglycemia, and hyperlipidemia), β cell apoptosis and/or cytokine production may result in the recruitment of APCs (dendritic cells, macrophages, and mast cells) (54, 55, 56, 63, 64, 65, 66, 67). Within the islet, APCs likely become activated, take up β cell Ags, and then migrate to draining lymph nodes where Ag is presented via the MHC to T cells, enabling them to enter tissues where they reencounter Ag and ultimately initiate an adaptive immune response (55, 59, 60, 62). Our studies presented here and previously (14) are consistent with this overall model in that we have determined that BB rat β cells begin producing eotaxin early in life (by day 40), which is before insulitis (24, 25, 26). Islet eotaxin expression provides, at least in part, a possible explanation for the observed recruitment of CCR3-bearing mast cells and eosinophils to the endocrine pancreas (14, 50, 51, 52) in the lymphopenic DRlyp/lyp rat. This underlying pancreatic predisposition is also consistent with the ability to induce diabetes in DR+/+ rats through Ab-mediated leukocyte depletion or viral depletion of CD4+CD25+ TREG cells, whereas such treatment of the MHC-identical WF rat, which lacks islet eotaxin expression, fails to induce disease.

Activated mast cells can influence T cell-mediated responses in a number of ways, such as through the presentation of Ag to T cells through either MHC class I- or class II-restricted and costimulatory molecule-dependent mechanisms, as well as by promoting T cell migration to inflammatory sites through the release of chemotactic factors and by mediating the up-regulation of endothelial cell adhesion molecule expression (21, 22). Among the numerous mechanisms described for mast cell activation, cross-linking of cell surface Fc{epsilon}RI through IgE bound to Ag is best characterized (68). Such aggregation leads to the phosphorylation of Fc{epsilon}RI by Lyn and the association of other protein tyrosine kinases that phosphorylate numerous targets and activate signaling pathways leading to degranulation, as well as cytokine and chemokine production. In this study, mast cell gene expression in the day 60 DRlyp/lyp was reflective of an activated mast cell population, showing overrepresentation of transcripts for numerous genes encoding the components of signal transduction pathways as well as known mast cell mediators (Tables I–V), including mast cell protease 1, cationic trypsinogen, carboxypeptidase A, phospholipase C{gamma}, and IL-5. IL-5 plays an important role in regulating the production, differentiation, activation, survival, and recruitment of eosinophils (69). Eosinophilia is often associated with high IL-5 expression. The observed overrepresentation of IL-5 transcript in day 60 DRlyp/lyp mast cells is consistent with the eosinophila and eosinophilic insulitis observed before and at the onset of BB rat T1DM (14, 23, 24, 25, 26, 27, 28, 70). Perhaps of greater interest is the contrasting day 60 DR+/+ mast cell gene expression profile, which shows overrepresentation of transcripts for genes encoding known negative regulators of signal transduction through Fc{epsilon}RI, including tyrosine phosphatase SHP-1, lipid phosphatase SHIP, E3 ubiquitin ligase c-Cbl, and Dusp1. Like the DRlyp/lyp mast cells, pathway analysis of the DR+/+ mast cell expression profile revealed overrepresentation of GO molecular functions related to activation/signal transduction (kinase activity, protein kinase activity, and protein serine/threonine kinase activity, protein-tyrosine kinase activity; Table II, lower section), within which the protein tyrosine kinase Lyn was annotated. Consistent with overexpression of Lyn in DR+/+ mast cells (3.8-fold relative to the DRlyp/lyp) is its important role in negatively regulating mast cell activation through the recruitment of inhibitory signaling molecules such as SHIP as well as through the negative regulation of Gab2 phosphorylation events (reviewed in Ref. 21). Furthermore, Lyn knockout mast cells and mice are hyperresponsive to IgE and Ag stimulation, underscoring its role as a potent negative regulator (21).

It is unlikely that the isolation process may have partially or fully activated the DRlyp/lyp and DR+/+ mast cells, as can happen when the AA4 mAb is bound to mast cells in the presence of extracellular calcium at 37°C (30), because caution was taken to maintain cells at 4°C, and all of the buffers used lacked calcium. Furthermore, the fact that distinct expression profiles consistent with known biology were observed supports that at least some, if not all, of the original in vivo profile was conserved. For either BB rat strain, these observations are consistent with activation through Fc{epsilon}RI; in the DRlyp/lyp the inflammatory response appears unchecked, whereas in the DR+/+ it appears attenuated.

Although development of the DRlyp/lyp rat has resulted in an animal that is no longer thought to develop islet autoantibodies before clinical onset (71) as was described for outbred BB rats (72), we investigated the presence of high IgE titers in DRlyp/lyp and DR+/+ rats. This line of investigation was pursued not only because of the differential expression of many genes involved in mast cell signal transduction through Fc{epsilon}RI but because of the numerous parallels that the DRlyp/lyp phenotype shares with the human immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX). In humans, IPEX is characterized by T1DM, enteropathy, thyroiditis, high IgE levels, and frequent eosinophila, as well as other pathologies (73). IPEX arises through mutations in the X-linked Forkhead box P3 (FOXP3) gene, which encodes a transcription factor that is used and is crucial for the development of CD4+CD25+ TREG cells. The IPEX phenotype in many ways is represented, in a milder form, by the BB rat. This is not to state that BB rats possess a defect in Foxp3; rather, the lymphopenia brought about through functional loss of Gimap5 includes the CD4+CD25+ TREG cell subpopulation. Parallels between IPEX and DRlyp/lyp rats include the development of enteropathy shortly after weaning (74), followed by T1DM, which is associated with eosinophila. Furthermore, if BB rats survive the onset of T1DM through daily treatment with insulin, they are susceptible to other inflammatory and autoimmune disorders such as collagen-induced arthritis and autoimmune thyroid disease (75). Because IgE Abs to IA2 and GAD65 have been identified in T1DM patients (76, 77), studies to determine whether the detected IgE in DRlyp/lyp animals is β cell-specific are ongoing. Because specific as well as nonspecific IgE (78) can augment mast cell activation and degranulation, the detected IgE may be an important contributing factor to β cell destruction in this model.

In this study, we observed that cromolyn treatment significantly delayed onset in DRlyp/lyp rats and enabled one-fourth of treated animals to survive the entire duration of the study (130 days, two-times normal time to onset) without a single hyperglycemic episode. Unfortunately, cromolyn has a short duration of action (~6 h) and does not efficiently cross membranes, complicating oral and i.p. delivery. We attribute our variable response to these features. Upon sacrifice, the day 130 animals exhibited normal islet histology and their granulocyte counts did not display the typical DRlyp/lyp eosinophilia, rather their granulocyte counts mirrored those of nondiabetic DR+/+ animals (M. J. Hessner, unpublished data). Activated mast cells release mediators, including IL-5, that influence eosinophil biology. Consistent with our observations, cromolyn has been clinically demonstrated to reduce eosinophila associated with allergic conditions (79). IgE-dependent activation of mast cells is characterized by an influx of extracellular Ca2+, which is necessary for the subsequent release of granule-derived as well as newly synthesized mediators and cytokines. The β2-adrenergic agonist salbutamol inhibits the intermediate conductance Ca2+-activated K+ channel (iKCa1) expressed by mast cells (80) and, when delivered i.p., is a potent in vivo suppressor of mast cells in murine collagen-induced arthritis (81). We have initiated treatment of DRlyp/lyp animals with this agent, and preliminary results also show delay of T1DM onset (data not shown).

Mast cells have been found to be potent effector cells in a number of autoimmune disorders, including Sjogren’s syndrome, systemic lupus erythematosus, multiple sclerosis (MS) and rheumatoid arthritis (reviewed in Ref. 16). The most extensive evidence of mast cell involvement in an autoimmune process has been observed in MS and its rodent model, experimental allergic/autoimmune encephalomyelitis. In this disorder mast cells have been associated with sites of demyelination, and mast cell abundance has been associated with the degree of CNS inflammation (16). Gene expression studies of brain lesions isolated from MS patients have revealed an increased abundance of mast cell-specific transcripts relative to controls, including Fc{epsilon}RI (82). Cromolyn and related drugs that inhibit mast cell degranulation have been found to be effective in reducing disease severity in both humans and mice (16, 58). Recently, mast cells have been shown to play a multifaceted role in the autoreactive T cell response of experimental allergic/autoimmune encephalomyelitis, being necessary in both initial peripheral CD4 and CD8 T cell activation as well as in exerting potent effects after initiation of the initial T cell response (83), thus adding to a growing body of evidence that supports a role for mast cells in adaptive autoimmune responses.

We have found that both DRlyp/lyp and DR+/+ show evidence of a β cell pathology in that eotaxin expression begins in either strain before the time at which spontaneous insulitis takes place in the DRlyp/lyp. These studies have found day 60 mast cells of the DRlyp/lyp to be terminally activated, whereas those in the DR+/+ appear negatively regulated. We speculate that despite early events that may mobilize innate immune cells in both BB rat strains, DR+/+ rat islets are protected by a peripheral regulatory mechanism, whereas in the lymphopenic DRlyp/lyp rat innate immune cells proceed to terminal activation and facilitate islet destruction. Thymically derived CD4+CD25+ TREG cells are potent immunosuppressive cells that play a pivotal role in peripheral self-tolerance and negative control of immune responses. Although loss of Gimap5 does not impact the numbers of functional CD4+CD25+ thymocytes, post-thymically these cells fail to thrive, rendering DRlyp/lyp animals deficient in this essential T cell subpopulation (13). The fact that DRlyp/lyp rats can be rescued through adoptive transfer of CD4+CD25+ regulatory T cells (12, 13) whereas depletion of these cells in the DR+/+ induces disease (11) supports the possibility that T1DM disease progression in BB rats is dependent on the absence of T cell regulation. Whether CD4+CD25+ TREG cells act upon mast cells and whether lack of such regulation is primary to diabetes progression remains unanswered.

In conclusion, these observations are consistent with recent findings in humans and in animal models where mast cells and other innate immune cells are being identified as key effectors in the initiation and progression of autoimmune disease. Combined, not only do these observations continue to diminish the distinction between allergy and autoimmunity, but they offer new opportunities for intervention and therapy.


   Acknowledgments
 
We thank S. Muheisen, G. R. Slocum, and C. A. Bobrowitz for excellent technical assistance in the histological studies, as well as L. Meyer and S. Holmes for excellent technical assistance in the animal, Ab, and gene expression studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 was supported by National Institute of Biomedical Imaging and Bioengineering Grant EB001421 (to M.J.H.), National Institute of Allergy and Infectious Diseases Grant P01-AI-42380 (to Å.L.), and a special fund from the Children’s Hospital of Wisconsin Foundation. Back

2 Address correspondence and reprint requests to Dr. Martin J. Hessner, Max McGee National Research Center for Juvenile Diabetes, Department of Pediatrics, 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: T1DM, type 1 diabetes mellitus; BB, BioBreeding (rat); Dusp, dual specificity phosphatase; GO, Gene Ontology (project); IPEX, immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome; MS, multiple sclerosis; PLN, pancreatic lymph node; qRT-PCR; quantitative RT-PCR; SHP, Src homology 2-containing protein tyrosine phosphatase; TREG, regulatory T cell. Back

4 The online version of this article contains supplemental material. Back

Received for publication June 9, 2006. Accepted for publication August 15, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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