ARTICLE |
Correspondence to:
F. Anders Karlsson, Dept. of Medical Sciences, Internal Medicine, University Hospital, S-751 85 Uppsala, Sweden. E-mail:
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Summary |
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Activated immune cells contribute to the development of diabetes mellitus in multiple low-dose streptozotocin-treated mice. However, a role in the process for MHC Class I restricted T-cells remains a matter of debate. In this study, we examined by confocal microscopy the pancreatic expression of MHC Class I protein, insulin, and ICA 512 protein tyrosine phosphatase in C57BL/Ks mice given 40 mg/kg bw streptozotocin IP on 5 consecutive days. All animals were hyperglycemic from Day 7 and onwards. A loss of ICA 512 from the central portions of the islets was noted on Day 3. On Day 7, an increase in MHC Class I expression, confined primarily to immune cells in the exocrine pancreas and the periinsular areas, was detected. Later, several MHC class I/glucagon and some MHC class I/insulin double-positive cells were found. The insulitis was maximal on Day 14 and declined thereafter. The induction of MHC Class I expression in endocrine cells, occuring only after the cellular infiltration and when the animals were diabetic, indicates that the immune component of the disease does not depend on MHC Class I-restricted cytotoxic T-cells but rather comprises a non-antigen-specific process. (J Histochem Cytochem 48:761767, 2000)
Key Words: diabetes, ICA 512, MHC class I, insulin, pancreatic islets, streptozotocin
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Introduction |
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Streptozotocin given at a high dose causes massive ß-cell destruction and permanent hyperglycemia in various experimental animals (
In human autoimmune diabetes, exogenous factors, e.g., viruses, toxic substances, are believed to trigger an immunological attack on ß-cells ( (
In this study we examined the islet expression of MHC Class I, insulin, and ICA 512/IA-2 protein tyrosine phosphatase, key proteins of a putative antigen-specific process in the development of multiple low-dose streptozotocin-induced diabetes mellitus.
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Materials and Methods |
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Animal Treatment
Inbred 1216-week-old C57BL/Ks male mice (Biomedical Centre; Uppsala, Sweden), originally obtained from the Jackson Laboratory (Bar Harbor, ME) were used. The animals had free access to tapwater and pelleted food (R34; AnaLyzen, Lindköping, Sweden). The mice were treated either with IP injections of saline (0.2 ml) for 5 days or with IP injections of streptozotocin (40 mg/kg bw) dissolved in saline for 5 consecutive days. Some animals were sacrified by cervical dislocation before any injections (Day 0).
Blood glucose determinations (ExacTech blood glucose meter; Baxter Travenol, Deerfield, IL) were performed on blood samples taken from the tail tip on Day 0 before any injection, and on Days 3, 7, 10, 14, 21, and 28 after the first injection. On these days groups of mice (n=5) were sacrificed for morphological examination of the pancreatic glands.
Morphological Examinations
After sacrifice, the pancreata were removed and one half of each gland was fixed in 10% formalin solution and the other half rapidly frozen in liquid nitrogen for confocal microscopy. For light microscopy, the fixed glands were embedded in paraffin and sections 5-µm thick were cut and stained using the PAP technique to demonstrate ß-cells (guinea pig anti-porcine insulin, 1:100; DAKO, Carpinteria, CA). The sections were counterstained with Mayer's hematoxylin for observation of mononuclear cell infiltration. Frozen tissues were cut in 5-µm sections and kept at -70C before immunostaining.
The immunofluorescence stainings were performed on cryosections for MHC Class I (purified mouse anti-mouse H-2Kd monoclonal antibody, 1:80; Pharmingen, San Diego, CA) and ICA 512 (rabbit antiserum (8959) against the cytoplasmic domain of the human ICA 512 protein, diluted 1:800; a generous gift from Bayer, Elkhart, IN).
To investigate the expression of MHC Class I in islet cells, double immunofluorescence stainings were used separately for MHC Class I/insulin, MHC Class I/glucagon (rabbit anti-porcine glucagon serum, 1:200; DAKO), and MHC Class I/ICA 512. Sections were fixed in acetone for 7 min and incubated with mixtures of primary antibodies at 4C overnight, then incubated with the biotinavidinTexas Red color system for MHC Class I and FITC-conjugated secondary antibodies for other reagents. Confocal microscopy (Zeiss LSM 410 invert Laser Scan Microscope; Carl Zeiss Jena, Jena, Germany) was used with excitations of 488 nm and 543-nm wavelength laser.
The average intensity of ICA 512 staining per islet area was evaluated by confocal microscopy at different time points after saline or streptozotocin injections. This was performed by measurements of >2000 µm2 islet area selected from >20 islets/pancreas. For each pancreas three to five sections (>50-µm apart) were examined. To standardize the parameters for the digitized images, brightness and contrast were set for normal islets and then used for all other experimental islets during that session on the confocal microscope.
To control the specificities of the immunostaining procedures, the primary antibodies were replaced by normal serum from the appropriate species or by PBS.
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Results |
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Blood Glucose and Insulitis
The mice treated with multiple low doses of streptozotocin (STZ) had normal blood glucose levels on Day 3, but from Day 7 the animals displayed hyperglycemia (Fig 1). Infiltration into the pancreas of lymphocyte-like cells was first seen on Day 7, at which time many mononuclear cells were noted in the exocrine tissue (Fig 2b). On Day 10 and onwards, the cells were homed to the peripheries of the islets and the number of infiltrating cells in the exocrine tissue diminished. The islet infiltration was most pronounced on Day 14 (Fig 2d). Occasionally, islets with no lymphocytes were also found at this time point (not shown).
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MHC Class I
In the normal pancreas (Day 0), MHC Class I expression was low and was found only in a few dendritic-like cells in the exocrine tissue and in the islets (Fig 3a). In one of the five normal pancreata examined, MHC Class I-positive dendrite-like and capillary cells were abundant (Fig 3b), an unexplained observation. In none of the five normal tissues was MHC Class I staining of endocrine cells detected. With STZ treatment, an increase in pancreatic MHC Class I protein was observed on Day 7 (Fig 4b). At this time point, the staining was associated with lymphocyte-like cells in the exocrine pancreas and in the peri-insular regions (Fig 4b, Fig 5c, and Fig 5d). The MHC Class I expression then became more intense and was concentrated to the islets, also involving endocrine cells (Fig 4c4f and Fig 5e5g). On Day 14, both insulin- (Fig 5f) and glucagon-containing cells (not shown) stained positive for MHC Class I, after which the staining in the islet areas gradually weakened. On Day 28, some MHC Class I staining associated with lymphocyte-like and dendritic cells (Fig 4f and Fig 5h) remained.
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ICA 512, Insulin, and Glucagon
ICA 512 was found as an even staining of all endocrine cells before STZ treatment (not shown). The islet ICA 512 content, as quantified by confocal microscopy, was reduced by 36% on Day 3 (control group 124 ± 12; STZ 80 ± 13; p<0.05, unpaired Student's t-test) and by 67% on Day 7 (STZ 41 ± 6; p<0.001). In particular, the ICA 512 disappeared from the central portions of the islets. Endocrine cells positive for both ICA 512 and MHC Class I were not detected on Day 7 but were present from Day 10 onwards. On Day 21, several strongly ICA 512 and MHC Class I-positive cells were observed (Fig 6).
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Insulin reactivity was reduced on Day 7 but was detectable in most of the islets (Fig 5c and Fig 5d). Occasionally, cells staining for both insulin and MHC Class I were found (Fig 5d). On Days 10 and 14, few ß-cells could be detected in the islets, and most of them showed double staining for insulin and MHC Class I (Fig 5e and Fig 5f).
Glucagon/ICA 512-containing cells were localized to the islet periphery before treatment. At later time points, several strongly double-positive cells were found localized throughout the islets (not shown). Glucagon-staining cells that were also positive for MHC Class I were found on Days 1021. The number of -cells increased during the course of the disease (
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Discussion |
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In this study we observed that the multiple low-dose STZ treatment caused ß-cell loss and hyperglycemia, with the occurrence of lymphocyte-like cells in the exocrine pancreas and the peri-insular areas before the appearance of MHC Class I in endocrine cells. Because cytotoxic T-cells recognize antigens in the context of MHC Class I protein complexes on the target cell surfaces (
Macrophages are of key importance for the occurrence of diabetes in multiple low-dose STZ-treated mice. The toxic destruction of ß-cells triggers recruitment of macrophages, single-cell insulitis (, which have a cytopathic action on the islet cells (
, the cytokine most likely responsible for the induction of MHC Class I expression in the endocrine cells (
has been reported to induce islet cell MHC antigens and to enhance STZ-induced diabetes in the CBA mouse (
The ICA 512 protein tyrosine phosphatase, an intrinsic membrane protein of secretory granules of peptide-secreting endocrine cells (
The presence of some MHC Class I/insulin double-positive cells on Day 14, when the lymphocytic infiltration of the islets was most pronounced, argues against the existence of insulin-specific cytotoxic T-cells in the infiltrate. Likewise, no ICA 512-specific cytotoxic T-cells appear to be induced, because many ICA 512/MHC Class I-positive cells (including ICA 512/glucagon-positive cells) survive. The selective ß-cell damage rather may be due to a direct toxic effect of STZ and to the effects of cytokines released from macrophages and activated T-helper cells. The disease appears to represent a form of immunological, non-antigen-specific diabetes mellitus. Absence of antigen-specific response is also supported by the survival of syngenic pancreatic islets transplanted to multiple low-dose STZ diabetic mice (
In human autoimmune diabetes, insulin, ICA 512, and glutamate decarboxylase are important autoantigens and, according to common belief, cytotoxic T-cells reacting with peptides of these proteins in the context of MHC Class I are pathogenic. However, there is no direct evidence for the occurrence of such cytotoxic T-cells in the human disease (
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Acknowledgments |
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Supported by grants from the Swedish Medical Research Council, the Novo Nordic Fund, the Swedish Diabetes Association, the Söderberg Foundation, the Family Ernfors Fund, and the Juvenile Diabetes FoundationWallenberg Fund.
We thank Margareta Ericson and Astrid Nordin for excellent technical assistance.
Received for publication January 28, 2000; accepted February 2, 2000.
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