ARTICLE |
Correspondence to: Michael R. Christie, Dept. of Medicine, Guy's, King's College, and St Thomas' School of Medicine, Bessemer Road, London SE5 9PJ, UK. E-mail: michael.christie@kcl.ac.uk
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Summary |
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A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion. (J Histochem Cytochem 49:767775, 2001)
Key Words: islets, development, secretion, protein tyrosine phosphatase, autoantigen
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Introduction |
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PHOSPHORYLATION OF PROTEINS on serine, threonine, or tyrosine residues is a major mechanism for the regulation of cellular processes, including exocytosis, cell growth, and cell differentiation. Members of a novel family of protein tyrosine phosphatase (PTP)-like proteins, IA-2 (also known as ICA512) and phogrin (IA-2ß /PTP-NP), were originally identified as islet cell autoantigens in Type 1 diabetes (
In addition to their potential role in peptide hormone secretion, other observations have implicated the IA-2 family of PTP-like proteins in pancreatic development. In situ hybridization studies have shown that mRNA for phogrin (PTP-NP) is detected very early in the development of the embryonic pancreas and subsequently is expressed in the developing nervous system (
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Materials and Methods |
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Antibodies
Mouse hybridoma 76F secreting monoclonal antibody to IA-2 was generated after immunization of mice with full-length recombinant human IA-2. An additional five monoclonal antibodies (MAbs) to IA-2 (clones 3C12, 3C11, 4H6, 4C11, 2D8) obtained after immunization of mice with protein representing the cytoplasmic domain of IA-2 were kind gifts from Dr. N. Morgenthaler (Berlin, Germany) and Dr. B. Ziegler (Karlsburg, Germany). A rabbit polyclonal antibody to Pdx-1 (
Animals and Tissues
We examined pancreas sections for IA-2 expression at the following stages of rat pancreas development: at the time of first appearance of the pancreatic bud at 11 days post conception (dpc) but before the appearance of islet hormones; at the time of the first appearance of primitive endocrine cells at 12 dpc; during the appearance of more mature single hormone positive islets at 13, 14, 15, and 17 dpc; during the rapid expansion of islet cell mass and development of normal insulin secretory responses to secretagogues after birth (at 1, 3, 5, 7, and 10 days of age); and in the adult pancreas.
Wistar rats were mated overnight and the day after mating was considered to be 1 dpc. All animals were kept according to the guidelines of the UK Home Office. Pregnant rats at 1117 dpc were sacrificed by cervical dislocation and fetuses fixed by immersion in 4% formaldehyde in 0.85% sodium chloride solution (formal saline) for 18 hr before dehydration and embedding for light microscopy. In some experiments the fetal pancreas was removed by dissection under the microscope and fixed for 4 hr in formal saline or extracted for Western blotting as described below. Pancreata from rats of 110 days of age or from adult rats were dissected and fixed for 4 hr in formal saline or processed for islet isolation as previously described (
Immunohistochemistry
Sensitive detection of IA-2 was achieved by immunoperoxidase labeling with signal amplification using streptavidinbiotin complexes. Paraffin sections 56 µm thick of tissues or rat embryos were dewaxed and rehydrated for immunohistochemistry. Endogenous peroxidase was blocked by treatment with 0.3% hydrogen peroxide in methanol and sections treated with normal swine serum (20% in Tris-buffered saline) to prevent nonspecific labeling. Sections were analyzed for reactivity with antibodies to IA-2, using at least two different MAbs at each stage of pancreatic development. Consecutive sections were labeled with antibodies to Pdx-1, glucagon, insulin, and cytokeratin-20 as markers for endocrine and ductal pancreatic cells. For detection of Pdx-1, sections were cut onto Vectorbond-coated slides (Vector Labs; Peterborough, UK) and heated in a pressure cooker at full pressure for 2 min in 0.01 M sodium citrate, pH 6.0, before incubation with specific antibody. For the detection of cytokeratin-20, sections were treated with trypsin (0.1% in 0.125% CaCl2 for 4 min at 37C) before antibody labeling. Sections were incubated overnight at 4C with monoclonal or polyclonal antibodies as described in the text or figure legends. Antibody reaction was visualized by the peroxidase-labeled streptavidin method with 3,3' diaminobenzidine substrate using a commercial kit (LSAB Plus; Dako, Cambridge, UK). Sections were counterstained with hematoxylin, dehydrated, and mounted for microscopic inspection. In dual-labeling experiments to examine potential co-localization of IA-2 with endocrine cell markers, sections were first labeled with IA-2 antibodies and detected with with 3,3' diaminobenzidine substrate. Sections were then incubated with the second primary antibody (to insulin or glucagon) for 1 hr at room temperature, washed, and antibody binding detected with FITC-conjugated anti-guinea pig or anti-rabbit immunoglobulin (Stratech; Luton, UK).
Western Blotting
Tissues or cultured cells were gently homogenized in lysis buffer (50 mM Tris, pH 7.5, 10 mM sodium phosphate, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM NaF, 5 mM iodoacetate, 1 mM benzamidine, 5 µg/ml leupeptin, 5 µg/ml aprotinin) and incubated for 30 min on ice. The lysate was centrifuged at 10,000 x g for 20 min to remove insoluble material and the protein concentration determined. Aliquots of tissue lysates representing 10 µg of protein or 20 ng of recombinant protein representing the cytoplasmic domains of IA-2 or phogrin, purified as previously described (
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Results |
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Specificity of Antibodies for IA-2
Six mouse MAbs recognizing the cytoplasmic domain of IA-2 were available for analysis of IA-2 expression. The antibodies were initially tested for their specificity by Western blotting against extracts of rat pituitary and ßTC cells, and against purified recombinant proteins representing the cytoplasmic domains of IA-2 and phogrin. All MAbs gave a similar pattern of reactivity against proteins in pituitary and ß-TC cell extracts, with a predominant doublet or multiple bands at Mr 60,00065,000 (Fig 1). All antibodies reacted strongly to the recombinant IA-2 preparation and only antibodies from clones 4H6 and 2D8 (weakly) crossreacted with the closely related protein phogrin. Antibody reactivity was confirmed by immunoprecipitation of recombinant protein (data not shown). Antibodies showing specificity for IA-2 (clones 3C12, 3C11, 4C11, and 76F) were selected for further studies of IA-2 expression by immunohistochemistry.
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Expression of IA-2 During Fetal Pancreas Development
Consecutive sections of formalin-fixed rat fetuses of 1117 dpc were labeled with antibodies to Pdx-1, cytokeratin 20, glucagon, and insulin to identify the developing pancreas and primitive endocrine cells, and with two or more MAbs to IA-2. No IA-2 labeling was detected in the pancreatic primordia appearing as Pdx-1-positive structures at 11 dpc before the appearance of endocrine cells (Fig 2A and Fig 2B). However, weak IA-2 labeling was observed in primitive glucagon-positive cells appearing early at 12 dpc (Fig 2C and Fig 2D). These primitive endocrine structures were negative for Pdx-1, whereas surrounding pancreatic epithelium forming the major part of the pancreatic bud was positive (Fig 2E). Larger islet-like structures appearing at 12 dpc, positive for both glucagon and insulin, showed strong positivity for IA-2 immunoreactivity (Fig 2F2H). IA-2 expression was confirmed with two different MAbs to the protein, whereas controls performed by omitting the primary antibody step showed no labeling on any of the sections. In contrast to the observations at 12 dpc, examination of sections from fetal rats at 14 dpc onwards revealed very little IA-2 labeling in the fetal islets, with only sporadic cells positive (Fig 2I and Fig 2K). Single-hormone positive cells, predominantly glucagon rather than insulin, were visible at this stage (Fig 2J and Fig 2L). No labeling with IA-2 antibodies was detected outside of the pancreas, including neuronal tissue, at any gestational age in the sections analyzed. Therefore, throughout fetal development, IA-2 expression appears restricted to the primitive Pdx-1-negative islet-like structures appearing at 12 dpc.
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Expression of IA-2 After Birth
Islet development continues after birth and, in the rat, glucose-induced insulin secretion is not apparent until a few days after birth (
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In the neonatal rat, some endocrine cells, predominantly located at the periphery of islets, co-express the epithelial marker cytokeratin-20, suggestive of cells that are transitional in the differentiation from duct cells to islets (
Within each islet there was considerable heterogeneity among cells in the intensity of IA-2 labeling at all ages, and this was particularly evident in the adult pancreas (Fig 3J). Although less evident than for IA-2, there was also heterogeneity in insulin labeling within individual islets, with strongest labeling occurring in ß-cells at the periphery of the islet (Fig 3I and Fig 3K). Double labeling of sections to co-localize IA-2 (by peroxidase labeling) and insulin (by immunofluorescence) in ß-cells revealed that cells expressing highest levels of IA-2 did not necessarily have high insulin content (compare Fig 3I and Fig 3J). In case of potential artifacts caused by quenching of immunofluorescence by the DAB precipitate, the discordance between high IA-2 and insulin expression was confirmed by labeling consecutive mirror sections with antibodies to insulin and IA-2 (Fig 3K and Fig 3L). IA-2 was present predominantly in ß-cells (e.g., the cell labeled by large arrow in Fig 3K and Fig L) but also non-ß-cells (small arrow) of the endocrine pancreas.
Western Blotting Analysis of IA-2 in Tissues
To confirm the presence of IA-2 in the fetal and neonatal pancreas, Western blotting studies were performed with tissue extracts (10 µg total extracted protein) prepared from dissected pancreata of 12 and 14 dpc rat fetuses, isolated islets from 5- and 10-day-old newborn rats, and adult rat islets. Predominant reactivities of the MAbs to IA-2 were to multiple bands of Mr 60,00065,000 (Fig 4). These multiple bands were visible in the 12 dpc fetal rat pancreas but were weak in the 14 dpc pancreas. Increased levels of staining of these bands were observed in isolated islets from 5 days of age to adult. These results, taken together with the histochemical observations, are consistent with a transient expression of IA-2 in the 12 dpc fetal pancreas and increased islet expression of the protein after birth.
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Discussion |
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In this study, immunohistochemical techniques were used to visualize IA-2 expression during rat pancreas development using selected MAbs raised to the cytoplasmic domain of the molecule. Analysis of reactivity to deletion mutants of IA-2 indicated that these antibodies recognize different epitopes within either the PTP-like domain or the juxta-membrane domain of the molecule (data not shown). We selected for our study antibodies that did not crossreact with recombinant phogrin on Western blots. The MAbs showed similar patterns of binding to proteins in extracts of adult rat pituitary and ß-TC cells, with predominant reactivity towards multiple bands of Mr 60,00065,000 and minor bands of Mr 110,000, 130,000, and 40,000. Similar multiple forms of IA-2 have been reported in Western blotting studies of bovine pituitary (
Pancreatic islets develop from progenitor cells localized within duct-like structures formed by proliferation of epithelial cells in the pancreatic bud (
Expansion of islet cell mass continues throughout fetal and early neonatal life and is believed to occur both as a result of growth of pre-formed islets and by neo-islet formation from ductal epithelium (
The transient appearance of IA-2 in a subpopulation of pancreatic endocrine cells contrasts with a different pattern of expression of the structurally related phogrin (PTP-NP) reported by
In fetal life, IA-2 expression was only transient; from 14 dpc, IA-2 expression in islets was very weak or absent. However, IA-2 labeling was again detectable 1 day after birth, and a progressive increase in islet IA-2 expression was observed during the first few days of life. In the neonate, IA-2 expression was restricted to mature endocrine cells. Cytokeratin-20-positive cells at the periphery of islets, which may represent immature endocrine cells at a transitional stage of differentiation from ductal precursors to islets (
Within individual islets in the neonatal and adult pancreas, there was considerable heterogeneity among cells in levels of expression of IA-2. In vitro observations have established that individual ß-cells show heterogeneity in a number of characteristics, including insulin biosynthesis, insulin release, activity of ATP-sensitive K+ channels, glucose utilization and oxidation, and morphological appearance of insulin secretory granules by electron microscopy (
IA-2 is a major target for autoimmunity in human Type 1 diabetes. The finding that IA-2 is expressed only at low levels throughout most of fetal and early neonatal life, whereas phogrin expression may be more persistent, may be relevant to the initiation of autoimmune responses to these proteins in Type 1 diabetes. It has long been recognized that neonatal mice are more susceptible to tolerance induction than adult mice (
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Acknowledgments |
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Supported by grants from the British Diabetic Association (RD96/0001250) and the European Union (TMR CT 970142). GR was supported by a Medical Research Council Clinical Training Fellowship.
We thank Dr Nils Morgenthaler, Dr Brigitte Ziegler, and Dr Jan Jensen for gifts of antibodies.
Received for publication January 4, 2001; accepted January 11, 2001.
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