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Immunohistochemical study of the pancreas of patients with long termed diabetes mellitus type 1

Alexandra Proshchina1, Yuliya Krivova1*, Valeriy Barabanov1 and Sergey Saveliev1
  • 1 FSBI Human Morphology SRI RAMS, Russia

There has been a concept of the autoimmune pathogenesis of DM1 developed in the diabetology. The basic notion of the concept is that a root cause of a death of the insulin containing B-cells is in an autoimmune aggression against them (1, 2). There is a significant destruction of B-cells observed in patients with DM1 prior to diagnosis. In patients with recently diagnosed DM1 the autoimmune processes has manifested in a form of insulitis - chronic inflammatory infiltration of mononuclear cells in the residual cells of the pancreatic islets (3). Although considered typical for recent onset disease, the insulitis has been described only in approximately 150 cases per 100 years (4).
It is known that DM1 is accompaniedby peripheral nervous system lesions wich leads to a reduction of a life span and mortality enlargement. It has been suggested that peripheral nervous system lesions, as well as B-cell destruction, in DM1 patients also has an autoimmune nature. Moreover, experiments on DM1 animal models (NOD mice) shows the autoimmune destruction of pancreatic nerve terminals at a beginning of the DM1 progression (5-11). According to this model the autoimmune reaction on B-cells develops after T-cells infiltration into the pancreatic islets as a result of similarities between B-cells and neurons in some synthezied proteins and intracellular processes, and high sensitivity of B-cells to the cytokine induced destruction (5, 6, 12,13).

The present study was perfomed on autopsied pancreatic speciments from 5 patients with clinically diagnosed DM1 (men, aged from 27 to 66 years) and 9 patients not suffering from carbohydrate metabolism and pancreatic disorders (men, aged from 24 to 70 years). All of the speciments were collected at Moscow hospitals during 2007 – 2011 years. The study has been approved by a local ethical committee.
The speciments from the pancreatic tail region were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned (10 mkm). A hematoxylin and eosin staining of paraffined sections were used for histological analysis.
An analysis of immunohistochemistry with antibodies against endocrine cell hormones (mouse monoclonal antibodies for insulin, glucagon, “Sigma”, working dilutions 1:1000 – 1:2000) and immune cells surface markers (mouse monoclonal antibodies for CD16, CD20, CD25, CD71 and rabbit epitopspecific antibodies for CD8, “Lab Vision Corp”, Ready to use) was applied in order to estimate the level of damaged endocrine cells and inflammation in the pancreas.
Reactions were detected with Ultra Vision Detection System Anti-polyvalent (Lab Vision Corp) with Diaminobenzidine as a chromogen.
Negative and positive (paraffin sections of normal human amygdala or pancreas) control speciments were made for each antibody applied in this study.
All slides were visually analysed with Leica DMLS microscope. Capturing of images was perfomed with Sony SSc–Dc50P camera.

The imuunohistochemical detection of pancreatic hormones has shown that insulin-containing B-cells were almost absent in the pancreas of 2 patients with clinically diagnosed DM1. Single insulin immunoreactive cells were indicated among the pancreatic duct cells. An immunopositive reaction to glucagon was retained in the pancreatic islet cells.
In the pancreas of 3 other patients with DM1 and 9 control patients a positive reaction to insulin and glucgon was detected in B- and A-cells located in the pancreatic islets and as a single cells and their small clusters in the acinar parenchyma.
Inflammatory infiltration was observed in the pancreas of 2 patients with severe DM1 with the absence of B-cells (figure 1) and 1 other DM1 patient having positive reaction to insulin retained. However, infiltration of immune cells in the pancreatic islets, typical for insulitis or perinsulitis, was not observed. Inflammatory cells were regullary distributed in the intra- and extralobular pancreatic connective tissue, exocrine parenchyma and around pancreatic blood vessels (figure 1).
The immunohistochemical labeling has indicated that in 3 cases of DM1 with inflammation the CD16- and CD25-positive cells have made the largest contribution to this reaction (figure 2, 3). Besides that the amount of cells positive to transferring receptor (CD71) in these 3 cases was increased comparing to the control speciments. The number of cells positive to antibodies to the CD8 and CD20 in these cases was very small.
In 2 cases of DM1 as well as in 9 control speciments the CD16-, CD25-, CD8-, CD20- and CD71-positive cells were presented only in a small number within major pancreatic blood vessels.

Histological changes observed in the pancreas of patients with DM1, such as the inflammation and the absence of isulin, are typical and repeatedly described (14-16). The insulitis, the lymphocytic infiltration of the islet cells, was described at autopsy of the pancreas in patients with recently diagnosed type 1 diabetes in 1965 (17) and further was confirmed in studies with animals.The infiltrate consists predominantly of activated T-cells (CD4 +, CD8 +), i. e. with receptor for interleukin 2, but may also contains B-lymphocytes and macrophages (1). The selective destruction of pancreatic B-cells is observed at the insulitis, while other endocrine cells remain intact. It is believed that the insulitis is a characteristic for the islets, which have retained a significant part of the B-cells. Therefore the insulitis is considered to be typical for children and adolescents with newly diagnosed DM1 (4, 17) and rarely detected in adults with a long duration of diabetes (4, 15, 18). However, there are described cases of insulitis after 8 years of DM1 (19).
Unfortunately, we have no information about a duration of DM1 in the investigated patients. However, histological and immunohistochemical changes observed in the pancreas of these patients such as the absence of B-cells and inflammation suggests a long course of DM1. In our study, we have not revealed a pattern of insulitis in any of the samples. The inflammation was deteced only in the exocrine part of the pancreas of 2 DM1 patients with B-cells lost and 1 other DM1 patient with B-cells retained. The CD16+ and CD25+ cells have made a largest contribution to the detected reaction.
The CD16 (also known as a low affinity Fc gamma receptor III for IgG (FcgammaRIII) or Leu 11) is an antigen of NK-cells and of cells myelomonocytic origin. In addition, the CD16 is a receptor of an antibody - dependent cellular cytotoxicity and cells expressing interleukin-2. The CD25 is a marker of lymphocyte activation and growth factor receptor of T cells. It is expressed on activated T - and B - lymphocytes, and according to some sources in the NK-cells and macrophages. An increased expression of the CD71 in these samples indicates that the inflammatory response involves cells, which have passed a recent proliferation. The T (CD8+) and B- lymphocytes (CD20) took an insignificanct part in the observed reaction. Thus, the activated cytotoxic T lymphocytes and NK cells play a leading role in this reaction that corresponds to literature data (2, 20).
The inflammation of the exocrine pancreas in patients with different duration of DM1 was identified also by other authors, for example, in a nationwide study of DM1 in Japan (15). This allowed the authors to suggest a fundamental difference between the course of DM1 in the Japanese in contrast to the Caucasian. Therefore, the exocrine tissue as well as beta cells are a target of immune reactions. A precise mechanism of the lymphocytic infiltration of the exocrine gland is unknown and requires further investigation.
We have described a general reduction in the number of islets in the pancreas of diabetics in our previous article (21). A total area occupied by both insulin and glucagon positive cells was also reduced. Similar findings have been reported by other researchers (15). Probably these data can be interpreted as follows: New islets do not form while existing islets gradually degrades with a course of DM1 disease. Thus a key question is why a population of insulin positive cells does not restore rather then why it dies.
Experiments on NOD (nonobese diabetic) mice have revealed an autoimmunization against neuronal and glial cell antigens (S-100, GFAP, GAD) at early stages of DM1 (5,9). Moreover, the same lymphocytes autoreative to S-100 and GFAP were detected in blood of the humans with probable prediabetes (9). In our previous study we had shown a decreased number of fine nerve fibers in the pancreatic parenchyma of DM1 patients compared to the control group (22). Thus, immune reaction in DM1 may be aimed not only on B-cells but also on other components of the pancreas. The observed localization of immune cells in the pancreatic mesenchyma and exocrine parenchyma in the investigated cases of DM1 accompanied by literature data confirms this hypothesis.
The pancreatic mesenchyma, blood vessels and nerves play an important role in a normal islet morphogenesis. It is possible that autoimmune attack on these components of the pancreas leads to the diturbed islet morphogenesis resulting in the loss of B-cells and decrease in islet mass in DM1.

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Figure 2
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Acknowledgements

This study was supported by a special fund for target capital management in order to support scientific investigations in the area of biology and medicine «Fundamental».

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Keywords: Pancreas, Diabetes Mellitus, Type 1, pancreatic islets, insulitis, B-cells

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Immune-mediated disease pathogenesis

Citation: Proshchina A, Krivova Y, Barabanov V and Saveliev S (2013). Immunohistochemical study of the pancreas of patients with long termed diabetes mellitus type 1. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00978

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Received: 28 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Dr. Yuliya Krivova, FSBI Human Morphology SRI RAMS, Moscow, 117418, Russia, homulkina@gmail.com