Journal of Clinical Laboratory Analysis 6:410-416 (1992)
lmmunoelectron Microscopic Localization of Antigens Which React With Islet Cell Cytoplasmic Antibodies Within Human Pancreatic Beta Cells Hirofumi Takino,’ Toshiro Yoshimura,’ Eiji Kawasaki,’ Nobuhiro Chikuba,’ Atsushi Yokota,’ Shoich Akazawa,’ Mitsuhiro Tsujihata,* Paul K. Nakane,3 and Shigenobu Nagataki’ ’The First Department of Internal Medicine, ’School of Allied Medical Sciences, and 3the Third Department of Anatomy, Nagasaki University School of Medicine, Nagasaki 852, Japan ~
The presence of circulating autoantibody to islet cell cytoplasm is considered to be an important marker of Type 1 (insulin-dependent) diabetes mellitus. In the present study using islet cell cytoplasmic antibody positive patient sera as the first antibody, we studied the intracellular distribution of its antigen at the electron microscopic level using the pre-embedding Key words:
0 1992 Wiley-Liss, Inc.
ICA, Type 1 diabetes, pre-embedding method, imrnunoelectron microscopy, insulinsecretory granules
INTRODUCTION Circulating antibody to cytoplasm of pancreatic islet cells (ICA) was found first by Bottazzo and Florian-Christensen (1) in sera of diabetic patients associated with autoimmune polyendocrine disease, and its presence is an important factor in the diagnosis of Type 1 (insulin-dependent) diabetes mellitus. However, intracellular localization of the antigen which reacts with ICA has not been clarified. By capitalizing on the experiment acquired during the ICA Proficiency Test and ICA Serum Exchange Program of Immunology and Diabetes Workshop (2,3), we proceeded to define cellular structures which react with ICA at the ultrastructural level utilizing the peroxidase-labeled antibody method.
MATERIALS AND METHODS Pancreatic Tissues Normal human pancreatic tissues (blood group 0) were obtained from two gastric cancer patients and one insulinoma patient at the time of surgery. A portion of each pancreatic tissue was quick frozen in O.C.T. compound and kept frozen at - 80°C until used. Another portion was fixed with shaking in 4% periodate-lysine-paraform-aldehyde(PLP) (4) at 4°C for 6 h. Then these were subsequently washed in 0.1 M phosphate buffered saline (PBS) containing 10% sucrose for 4 h, then cryoprotected in 0.1 M PBS containing 15% sucrose for 4 h and in 0.1 M PBS containing 20% sucrose for 4 h. The tissues were then embedded in O.C.T. compound (Lab@ 1992 Wiley-Liss, Inc.
~~
immunoperoxidase method. Specific immunoreactivity was found in the membranes of beta cell-secretory granules and cytoplasmic membranes. This result is compatible with the interpretationthat the antigen@)on the membranes of beta cell secretory granules is (are) the target of islet cell cytoplasmic antibody.
Tek), quick frozen in liquid nitrogen, and kept in the liquid nitrogen until used.
Antisera Sera were obtained from 2 patients with Type 1 diabetes and from 2 patients with Type 1 diabetes accompanied with autoimmune thyroid disease (AITD). Titering of ICA was first carried out by our conventional method (5) and converted into Juvenile Diabetes Foundation (JDF) units using the ICA Proficiency Test standard serum, Autoantibodies to insulin (IAA) (6) were measured by radioimmunoassay-polyethyleneglycol . Autoantibodies to 64 kDa protein (7) were determined by the radioimmunoprecipitationmethod using 35S-methioninelabeled rat islet cells. Autoantibodies to microsome (AMA) and thyroglobulin (ATA) were measured by the hemagglutination test using commercial kits (Seroidia AMC, Seroidia ATG; Fujirebio, Inc., Japan). Two sera obtained from patients with Type 1 diabetes were positive for ICA, 160 JDF units and 320 JDF units, respectively. Both sera were also positive for 64 kD protein but were free from other autoantibodies. Serum from one of the patients with Type 1 diabetes accompanied with AITD contained ICA with a titer of 1,280 JDF units, anti-64 kD protein autoantibody, and AMA with a titer of Received June 5 , 1992; accepted June 10, 1992. Address reprint requests to Dr. Shigenobu Nagataki, The First Department of Internal Medicine, Nagasaki University School of Medicine, 7-1 Sakamotomachi, Nagasaki 852, Japan.
lmmunoelectron Microscopic Localization of Antigen Against ICA
TABLE 1. Serum Characteristicsof Q p e 1 Diabetic Patients Used in This StudP
Case 1 Case 2 Case 3 Case 4
ICA(JDF)
IAA
1 60 320 1,280 2,560
-
64KA
-
+ + + +
AMA
ATA
-
-
-
-
6,400 25,600
25,600
-
aICA, islet cell cytoplasmic antibody; IAA, insulin autoantibody; 64KA, 64 kD protein autoantibody; AMA, antimicrosome antibody; ATA, antithyroglobulin antibody.
6,400 times and ATA with a titer of 25,600 times but was negative for IAA. The serum from the other patient with Type 1 diabetes and AITD was positive for ICA, with a titer of 2,560 JDF units, anti-64 kD protein autoantibody, and AMA with a titer of 25,600 times but was negative for ATA and IAA. These results are summarized in Table 1 . Eight sera from normal individuals were also analyzed similarly and were found to be free from all autoantibodies examined and were used for control sera.
Immunohistochemistry The fresh frozen tissues were sectioned in a cryostat at 4-6 p m in thickness, placed on egg albumen coated glass slides, and air dried. The tissue sections were then fixed for 10 min in one of following solutions by immersion: cold acetone, cold ethanol, 4%paraformaldehyde in distilled water, 3% glutaraldehyde in cacodylate buffer at pH 7.2, and PLP. All slides with fixed tissue sections were washed three times with 0.01 M PBS containing 2% bovine serum albumin (BSA, Sigma). Fifty microliters of diluted patient sera (diluted 1:32 with PBS and with and without 50 pU/ml insulin [Actrapid human insulin, Novo] and 5% BSA) was applied to the tissue sections and incubated in a moist chamber for 1 h at room temperature. The insulin and BSA were added in order to rule out the possibility of cross-reaction of the insulin and BSA. The slides were washed 3 times in 0.01 M PBS. Fifty microliters of peroxidase-labeled protein A (POPA, Sigma) was added to the sections and incubated for 30 min at room temperature. The sections were washed three times in PBS. These were incubated with diaminobenzidine (DAB) with H202 for 7 min and washed 3 times in 0.01 M PBS. The stained sections were dehydrated in increasing concentrations of ethanol and cleared in xylene and mounted in Paramount.
lmmunoelectron Microscopy The tissues fixed in PLP were frozen and sectioned at 4-6 pm in thickness, and the sections were placed on ovoalbumincoated microscope glass slides and air dried. All slides were washed 3 times with 0.01 M PBS containing 2% BSA. Fifty microliters of diluted patient sera or control sera (diluted 1:32 with PBS which contained 50 pU/ml insulin and 5% BSA) was applied to the tissue sections and incubated in a moist
41 1
chamber for 3 h at 37°C. The tissue sections were washed 3 times in 0.01 M PBS. Fifty microliters POPA was added to the sections and incubated for 2 h at room temperature. After washing 3 times with 0.01 M PBS, the sections were fixed in 1% glutaraldehyde at 4°C for 20 min. The sections were washed 5 times in 0.01 M PBS. These were soaked with Karnovsky’s DAB solution without H202 for 30 min at room temperature and then reacted with DAB with H202for 5 min. They were washed 3 times in 0.01 M PBS. At this point, some stained sections were dehydrated in increasing concentrations of ethanol and mounted in Paramount for the light microscopic observation. Other tissue sections were incubated with 2% osmium tetroxide for 1 h at room temperature. These were dehydrated in graded alcohol solutions (70, 8 0 , 9 0 , 9 5 , 100% x 3 ethanol), and gelatin capsules containing Epon 8 12 were placed upside down over the tissue section and were polymerized at 60°C. After the Epon was polymerized, the capsules with the tissue sections were removed from the glass slide by heating over a Bunsen burner. Ultrathin sections were made from the embedded tissue section and placed on grids coated with carbon. The ultrathin sections were observed by JEM- 1200 electron microscope (JEOL) without counterstaining.
RESULTS Light Microscopy The intensity of immunostaining varied considerably depending upon fixatives. The most intense staining was observed in islets of those tissue sections fixed in PLP (Fig. 1) and weak staining was observed in those fixed in cold ethanol, 4% paraformaldehyde, or cold acetone. No positive staining was seen in those fixed in 3% gIutaraIdehyde. In the islets of sections fixed in PLP, intense staining was observed in cytoplasm as well as along the plasma membrane and was seen in the islets homogeneously. On the other hand, in sections fixed in cold acetone, cytoplasmic staining was faint while the staining near or on the plasma membrane was as strong as in those fixed with PLP. In sections fixed in cold ethanol, no significant islet cell staining was seen when the diluted serum (1:32 dilution) was used as the first antibody, but some islet cells were stained when the concentrated serum ( 1 :8 dilution) was used as the first antibody. No staining was observed when the control sera from normal individuals were used as the first antibody (Fig. 2). The immunostaining was not affected in sections which were reacted with patient antibody which was pre-mixed with insulin and BSA.
Electron Microscopy Preservation of the ultrastructure was insufficient to identify cell types in islets by Lacy’s criteria (8), however in sections reacted with ICA positive sera, the immunostained cells were judged to be beta cells from their frequency and distri-
412
Takino et al.
Fig. 1. Light microscopic section of human pancreas fixed in PLP fixative. Arrows show the islets of the pancreas. Section incubated with ICA positive serum. Pancreatic islets were clearly stained. (Magnification: X 200.)
Fig. 2. Section incubated with ICA negative serum. No immunostaining was observed. (Magnification: x 200.)
Immunoelectron Microscopic Localization of Antigen Against ICA
413
Fig. 3. Electron microscopic section of pancreatic beta cells incubated with ICA positive serum. Specific immunostaining was observed in the membranes of secretory granules (A) and cytoplasmic membranes (B) (arrows). (Magnification: x 10,000.)
bution and the shape of secretory granules. In these cells, most of secretory granules were void of the usual osmophilic central core and instead the area usually occupied by the core appeared as empty space and portions of cytoplasmic membranes were lost. In these beta cells, specific immunostaining was constantly observed on the membranes of secretory granules (Fig. 3A) and on portions of the plasma membrane of beta cells (Fig. 3B). On the other hand, those non-beta cells contained secretory granules which were larger than those found in the Beta cells and were filled with amorphous material (Fig. 4) and their membranes were unstained immunocytochemically. In the sections incubated with control sera, no specific immunoreactivity was observed (Fig. 5). Naturally osmophilic subcellular materials such as celioid bodies and lipid droplets were stained regardless of type of antibody used.
DISCUSSION In the current study, we showed that the antigen(s) against ICA is (are) located on the membranes of beta cell secretory granules and cytoplasmic membranes. We used the so-called pre-embedding method (4)in this study, because the antigens in question were extremely fragile and their immunore-
activity was lost easily in routine electron microscopic fixatives (e.g., glutaraldehyde and routine PLP fixative) and could not withstand the plastic polymer embedding. The latter is a prerequisite for the post-embedding method. To maintain the antigenicity, it was necessary to reduce the duration of PLP fixation at the expense of morphology. And to minimize the diffusion of the antigen during incubation with antibodies, the time was shortened even though the antibody may not have completely penetrated through the tissue sections. In spite of the suboptimal tissue preservation, we were able to localize the antigen in the cytoplasmic membrane and the membranes of beta cell secretory granules. At the onset of Type 1 diabetes, several autoantibodies which also react with pancreatic islets are known to be present in the sera of patients, such as ICA (I), IAA (6), and anti-64 kD antibody (7). To avoid the immunostaining from IAA, the sera were first incubated with excess insulin to absorb out IAA. The reactions to pancreatic tissues were not affected with the absorption and the results indicated that the antisera which were used in this study were free from the IAA. As for the anti-64 kD autoantibody, since we were unable to obtain sufficient human 64 kD antigen to perform the absorption, we opted to demonstrate somewhat indirectly that our pres-
414
Takino et al.
Fig. 4. Electron microscopic section of non-beta cell incubated with ICA positive serum. No specific immunostaining was observed. (Magnification: X 10,000.)
ent results were not due to the contaminating anti-64 kD autoantibody. We immunostained the sections of pancreas with serum which was positive for anti-64 kD antibody and negative for ICA and compared with sections reacted with ICA positive sera. With the anti-64 kD antibody positive and ICA negative serum as the first antibody, no definite structure in human pancreas was stained (data not shown), although there was slightly higher background staining. These results suggest that the staining observed with our ICA positive sera was not the result of the immune reaction with anti-64 kD protein. The ICA positive sera reacted antigens on the cytoplasmic membranes and the membranes of beta cell secretory granules, whereas Reetz et al. (9) found that antigens which react with sheep anti-rabbit glutamic acid decarboxylase (GAD), i.e., 64 kD protein (lo), reacted with membranes of pleiomorphic microvesicles and of tubular and cisternal elements of rat pancreatic beta cells (9). This difference in the distributions of antigens at the electron microscopic level further suggests that the antigens reacting with the ICA positive sera were something other than 64 kD protein. Even though, why our ICA positive sera failed to localize intra-islet 64 kD protein should be addressed. A most probable reason is GAD or 64 kD protein is a soluble cytosolic protein and dissolved
out of the tissues during fixation, since we opted for suboptimal fixation conditions in order to maintain the antigenicity of antigens reactive with the ICA positive sera. If this is the case, then the antigens reacted with the ICA positive sera, on the other hand, may be categorized as insoluble elements of the membranes and not 64 kD protein. Another possible reason for the differences in the results of Reetz et al. and ours may have been differences of immunoreactivity of 64 kD protein between species. In any event, this aspect requires further investigation. Although we are able to determine that the antigen for ICA localized in our study was not insulin and probably not 64 kD antigen, we could not ascertain what is (are) antigen(s) for ICA. However, the behavior of ICA antigen in various fixatives suggests what the antigen might be. Our histochemical results suggest that the antigen(s) is (are) not an ordinal trans-membrane proteinaceous antigen since the antigen was readily lost in cold acetone or in organic solvents. In addition, that the antigen can be fixed somewhat by carbohydrate cross linking fixative (a main chemical reaction of PLP is to cross link carbohydrates) suggests that the antigen contains a carbohydrate moiety. These characteristics are similar to the immunohistochemical findings of Nayak et al. (1 l), who
lmmunoelectronMicroscopic Localization of Antigen Against ICA
415
Fig. 5. Electron microscopic section incubated with ICA negative control serum. No specific immunostaining was observed. (Magnification: x l0,OOO.)
reported that the antigens for ICA had the properties of sialic acid containing glycolipid because of decreased antigenicity of ICA following incubation of pancreatic sections with neuraminidase and exposure to ch1oroform:methanol or methanol. A difference between our results and those of Nayak et al. was that they found the antigen was stable in acetone whereas we found the decrease of antigen after fixation in acetone. The differences may have resulted from immunohistochemical procedural differences; however, Nayak et a1.did not report the detailed procedure and we were unable to directly compare the results. As another autoantigen of ICA, Powers and Bowen (12) suggested that insulin secretion granule membrane associated soluble carboxypeptidase H (CPH) was the antigen for ICA. The behavior of CPH epitope during immunohistochemical procedures is yet undefined in pancreatic islets; however, CPH being glycoprotein, it is expected that routine immunohistochemical procedures used in our study should have been applicable for the localization of CPH in pancreatic islets. The failure to stabilize the autoantigen of ICA by protein precipitating fixatives such as organic solvents in our study suggests that either CPH might have behaved unorthodoxly from other glycoproteins in immunohistochemical procedures or the antisera used in the current study recognized autoantigens of ICA
other than CPH. This aspect also requires further investigation using the same human autoantibodies since the characterization of autoantibodies used to identify CPH in pancreatic islets was not reported. In conclusion, using pre-embedding immunoelectron microscope techniques, we showed that the antigen(s) against ICAs was (were) located in the membranes of beta cell-secretory granules and cytoplasmic membranes.
ACKNOWLEDGMENTS We are gratefully thankful to A. Nakashima, M. Morimoto, and T. Suematsu for skillful technical assistance.
REFERENCES 1. Bottazzo GF, Florian-Christensen A: Islet cell antibodies in diabetes
mellitus with autoimmune polyendocrine deficiency. Lancer ii: 12791282, 1974. 2 . Boitard C, Bonifacio E, Bottazo GF, Gleichmann H , Molenaar J: Immunology and diabetes work shop: Report on the third international (Stage 3) workshop on the standardization of cytoplasmic islet cell antibodies. Diabetologia 31:451-452, 1988. 3. Lernmark A, Molenaar JL, van Beers WAM, Yamaguchi Y, Nagataki S, Ludvigsson J , and Maclaren NK on behalf of the Immunology and Diabetes Workshops and participating laboratories: The fourth interna-
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4.
5.
6.
7.
8.
Takino et al.
tional serum exchange work shop to standardize cytoplasmic islet cell antibodies. Diaberologia 34534-535, 1991. Mclean IW, Nakane PK: Periodate-lysine-paraformaldehyde fixative, a new fixative for immunoelectron microscopy. J Histochem Cytochem 22( 12):1077-1083, 1974. Takahashi A, Tsujihata M, Yokota A, Yarnaguchi Y, Miyake S, Nagataki S: A new method of detection of islet cell antibodies (ICA) using peroxidase-labeled protein A, and incidence of ICA in Type I (insulin dependent) diabetes. Diabetologia 29:378-382, 1986. Palmer P, Asplin CM, Clernona P, Lyen K, Tatpati 0,Raghu PK, Paquette ZT: Insulin antibodies in insulin dependent diabetes before insulin treatment. Science 222:1237-1239, 1982. Colman PG, Campbell IL, Kay TWH, Harrison LC: 64,000-Mr. autoantigen in Type 1 diabetes. Diabetes 36: 1432-1440, 1987. Lacy P, Greider M: Ultrastructural organization of mammalian pancreatic islets. Handbook of Physiology, Section 7: Endocrinology 1: 77-89
9. Reetz A, Solimena M, Matteoli M, Folli F, Takei K, De Camilli P: GABA and pancreatic B-cells: Colocalization of glutarnic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA-storage and secretion. EMBO J 1012751284, 1991. 10. Baekkeskov S, Nielsen JH, Mamer B, Bilde T, Ludvigsson J, Lernmerk A: Autoantibodies in newly diagnosed diabetic children immunoprecipitate human pancreatic cell protein. Nature (Lond) 298: 167-169, 1982. 11. Nay& RC, Omar MAK, Rabizadeh Z, Strikanta S, Eisenbarth GS: Cytoplasmic islet cell antibodies: Evidence that the target antigen is a sialoglycoconjugate. Diabetes 34:617-619, 1985. 12. Power AC, Bowen S: Carboxypeptidase H is an autoantigen of the ICA and is expressed on the cell surface of islet cells. Diabetes 40:lA (Abstract), 1991.
lmmunoelectron Microscopic Localization of Antigens Which React With Islet Cell Cytoplasmic Antibodies Within Human Pancreatic Beta Cells Hirofumi Takino,’ Toshiro Yoshimura,’ Eiji Kawasaki,’ Nobuhiro Chikuba,’ Atsushi Yokota,’ Shoich Akazawa,’ Mitsuhiro Tsujihata,* Paul K. Nakane,3 and Shigenobu Nagataki’ ’The First Department of Internal Medicine, ’School of Allied Medical Sciences, and 3the Third Department of Anatomy, Nagasaki University School of Medicine, Nagasaki 852, Japan ~
The presence of circulating autoantibody to islet cell cytoplasm is considered to be an important marker of Type 1 (insulin-dependent) diabetes mellitus. In the present study using islet cell cytoplasmic antibody positive patient sera as the first antibody, we studied the intracellular distribution of its antigen at the electron microscopic level using the pre-embedding Key words:
0 1992 Wiley-Liss, Inc.
ICA, Type 1 diabetes, pre-embedding method, imrnunoelectron microscopy, insulinsecretory granules
INTRODUCTION Circulating antibody to cytoplasm of pancreatic islet cells (ICA) was found first by Bottazzo and Florian-Christensen (1) in sera of diabetic patients associated with autoimmune polyendocrine disease, and its presence is an important factor in the diagnosis of Type 1 (insulin-dependent) diabetes mellitus. However, intracellular localization of the antigen which reacts with ICA has not been clarified. By capitalizing on the experiment acquired during the ICA Proficiency Test and ICA Serum Exchange Program of Immunology and Diabetes Workshop (2,3), we proceeded to define cellular structures which react with ICA at the ultrastructural level utilizing the peroxidase-labeled antibody method.
MATERIALS AND METHODS Pancreatic Tissues Normal human pancreatic tissues (blood group 0) were obtained from two gastric cancer patients and one insulinoma patient at the time of surgery. A portion of each pancreatic tissue was quick frozen in O.C.T. compound and kept frozen at - 80°C until used. Another portion was fixed with shaking in 4% periodate-lysine-paraform-aldehyde(PLP) (4) at 4°C for 6 h. Then these were subsequently washed in 0.1 M phosphate buffered saline (PBS) containing 10% sucrose for 4 h, then cryoprotected in 0.1 M PBS containing 15% sucrose for 4 h and in 0.1 M PBS containing 20% sucrose for 4 h. The tissues were then embedded in O.C.T. compound (Lab@ 1992 Wiley-Liss, Inc.
~~
immunoperoxidase method. Specific immunoreactivity was found in the membranes of beta cell-secretory granules and cytoplasmic membranes. This result is compatible with the interpretationthat the antigen@)on the membranes of beta cell secretory granules is (are) the target of islet cell cytoplasmic antibody.
Tek), quick frozen in liquid nitrogen, and kept in the liquid nitrogen until used.
Antisera Sera were obtained from 2 patients with Type 1 diabetes and from 2 patients with Type 1 diabetes accompanied with autoimmune thyroid disease (AITD). Titering of ICA was first carried out by our conventional method (5) and converted into Juvenile Diabetes Foundation (JDF) units using the ICA Proficiency Test standard serum, Autoantibodies to insulin (IAA) (6) were measured by radioimmunoassay-polyethyleneglycol . Autoantibodies to 64 kDa protein (7) were determined by the radioimmunoprecipitationmethod using 35S-methioninelabeled rat islet cells. Autoantibodies to microsome (AMA) and thyroglobulin (ATA) were measured by the hemagglutination test using commercial kits (Seroidia AMC, Seroidia ATG; Fujirebio, Inc., Japan). Two sera obtained from patients with Type 1 diabetes were positive for ICA, 160 JDF units and 320 JDF units, respectively. Both sera were also positive for 64 kD protein but were free from other autoantibodies. Serum from one of the patients with Type 1 diabetes accompanied with AITD contained ICA with a titer of 1,280 JDF units, anti-64 kD protein autoantibody, and AMA with a titer of Received June 5 , 1992; accepted June 10, 1992. Address reprint requests to Dr. Shigenobu Nagataki, The First Department of Internal Medicine, Nagasaki University School of Medicine, 7-1 Sakamotomachi, Nagasaki 852, Japan.
lmmunoelectron Microscopic Localization of Antigen Against ICA
TABLE 1. Serum Characteristicsof Q p e 1 Diabetic Patients Used in This StudP
Case 1 Case 2 Case 3 Case 4
ICA(JDF)
IAA
1 60 320 1,280 2,560
-
64KA
-
+ + + +
AMA
ATA
-
-
-
-
6,400 25,600
25,600
-
aICA, islet cell cytoplasmic antibody; IAA, insulin autoantibody; 64KA, 64 kD protein autoantibody; AMA, antimicrosome antibody; ATA, antithyroglobulin antibody.
6,400 times and ATA with a titer of 25,600 times but was negative for IAA. The serum from the other patient with Type 1 diabetes and AITD was positive for ICA, with a titer of 2,560 JDF units, anti-64 kD protein autoantibody, and AMA with a titer of 25,600 times but was negative for ATA and IAA. These results are summarized in Table 1 . Eight sera from normal individuals were also analyzed similarly and were found to be free from all autoantibodies examined and were used for control sera.
Immunohistochemistry The fresh frozen tissues were sectioned in a cryostat at 4-6 p m in thickness, placed on egg albumen coated glass slides, and air dried. The tissue sections were then fixed for 10 min in one of following solutions by immersion: cold acetone, cold ethanol, 4%paraformaldehyde in distilled water, 3% glutaraldehyde in cacodylate buffer at pH 7.2, and PLP. All slides with fixed tissue sections were washed three times with 0.01 M PBS containing 2% bovine serum albumin (BSA, Sigma). Fifty microliters of diluted patient sera (diluted 1:32 with PBS and with and without 50 pU/ml insulin [Actrapid human insulin, Novo] and 5% BSA) was applied to the tissue sections and incubated in a moist chamber for 1 h at room temperature. The insulin and BSA were added in order to rule out the possibility of cross-reaction of the insulin and BSA. The slides were washed 3 times in 0.01 M PBS. Fifty microliters of peroxidase-labeled protein A (POPA, Sigma) was added to the sections and incubated for 30 min at room temperature. The sections were washed three times in PBS. These were incubated with diaminobenzidine (DAB) with H202 for 7 min and washed 3 times in 0.01 M PBS. The stained sections were dehydrated in increasing concentrations of ethanol and cleared in xylene and mounted in Paramount.
lmmunoelectron Microscopy The tissues fixed in PLP were frozen and sectioned at 4-6 pm in thickness, and the sections were placed on ovoalbumincoated microscope glass slides and air dried. All slides were washed 3 times with 0.01 M PBS containing 2% BSA. Fifty microliters of diluted patient sera or control sera (diluted 1:32 with PBS which contained 50 pU/ml insulin and 5% BSA) was applied to the tissue sections and incubated in a moist
41 1
chamber for 3 h at 37°C. The tissue sections were washed 3 times in 0.01 M PBS. Fifty microliters POPA was added to the sections and incubated for 2 h at room temperature. After washing 3 times with 0.01 M PBS, the sections were fixed in 1% glutaraldehyde at 4°C for 20 min. The sections were washed 5 times in 0.01 M PBS. These were soaked with Karnovsky’s DAB solution without H202 for 30 min at room temperature and then reacted with DAB with H202for 5 min. They were washed 3 times in 0.01 M PBS. At this point, some stained sections were dehydrated in increasing concentrations of ethanol and mounted in Paramount for the light microscopic observation. Other tissue sections were incubated with 2% osmium tetroxide for 1 h at room temperature. These were dehydrated in graded alcohol solutions (70, 8 0 , 9 0 , 9 5 , 100% x 3 ethanol), and gelatin capsules containing Epon 8 12 were placed upside down over the tissue section and were polymerized at 60°C. After the Epon was polymerized, the capsules with the tissue sections were removed from the glass slide by heating over a Bunsen burner. Ultrathin sections were made from the embedded tissue section and placed on grids coated with carbon. The ultrathin sections were observed by JEM- 1200 electron microscope (JEOL) without counterstaining.
RESULTS Light Microscopy The intensity of immunostaining varied considerably depending upon fixatives. The most intense staining was observed in islets of those tissue sections fixed in PLP (Fig. 1) and weak staining was observed in those fixed in cold ethanol, 4% paraformaldehyde, or cold acetone. No positive staining was seen in those fixed in 3% gIutaraIdehyde. In the islets of sections fixed in PLP, intense staining was observed in cytoplasm as well as along the plasma membrane and was seen in the islets homogeneously. On the other hand, in sections fixed in cold acetone, cytoplasmic staining was faint while the staining near or on the plasma membrane was as strong as in those fixed with PLP. In sections fixed in cold ethanol, no significant islet cell staining was seen when the diluted serum (1:32 dilution) was used as the first antibody, but some islet cells were stained when the concentrated serum ( 1 :8 dilution) was used as the first antibody. No staining was observed when the control sera from normal individuals were used as the first antibody (Fig. 2). The immunostaining was not affected in sections which were reacted with patient antibody which was pre-mixed with insulin and BSA.
Electron Microscopy Preservation of the ultrastructure was insufficient to identify cell types in islets by Lacy’s criteria (8), however in sections reacted with ICA positive sera, the immunostained cells were judged to be beta cells from their frequency and distri-
412
Takino et al.
Fig. 1. Light microscopic section of human pancreas fixed in PLP fixative. Arrows show the islets of the pancreas. Section incubated with ICA positive serum. Pancreatic islets were clearly stained. (Magnification: X 200.)
Fig. 2. Section incubated with ICA negative serum. No immunostaining was observed. (Magnification: x 200.)
Immunoelectron Microscopic Localization of Antigen Against ICA
413
Fig. 3. Electron microscopic section of pancreatic beta cells incubated with ICA positive serum. Specific immunostaining was observed in the membranes of secretory granules (A) and cytoplasmic membranes (B) (arrows). (Magnification: x 10,000.)
bution and the shape of secretory granules. In these cells, most of secretory granules were void of the usual osmophilic central core and instead the area usually occupied by the core appeared as empty space and portions of cytoplasmic membranes were lost. In these beta cells, specific immunostaining was constantly observed on the membranes of secretory granules (Fig. 3A) and on portions of the plasma membrane of beta cells (Fig. 3B). On the other hand, those non-beta cells contained secretory granules which were larger than those found in the Beta cells and were filled with amorphous material (Fig. 4) and their membranes were unstained immunocytochemically. In the sections incubated with control sera, no specific immunoreactivity was observed (Fig. 5). Naturally osmophilic subcellular materials such as celioid bodies and lipid droplets were stained regardless of type of antibody used.
DISCUSSION In the current study, we showed that the antigen(s) against ICA is (are) located on the membranes of beta cell secretory granules and cytoplasmic membranes. We used the so-called pre-embedding method (4)in this study, because the antigens in question were extremely fragile and their immunore-
activity was lost easily in routine electron microscopic fixatives (e.g., glutaraldehyde and routine PLP fixative) and could not withstand the plastic polymer embedding. The latter is a prerequisite for the post-embedding method. To maintain the antigenicity, it was necessary to reduce the duration of PLP fixation at the expense of morphology. And to minimize the diffusion of the antigen during incubation with antibodies, the time was shortened even though the antibody may not have completely penetrated through the tissue sections. In spite of the suboptimal tissue preservation, we were able to localize the antigen in the cytoplasmic membrane and the membranes of beta cell secretory granules. At the onset of Type 1 diabetes, several autoantibodies which also react with pancreatic islets are known to be present in the sera of patients, such as ICA (I), IAA (6), and anti-64 kD antibody (7). To avoid the immunostaining from IAA, the sera were first incubated with excess insulin to absorb out IAA. The reactions to pancreatic tissues were not affected with the absorption and the results indicated that the antisera which were used in this study were free from the IAA. As for the anti-64 kD autoantibody, since we were unable to obtain sufficient human 64 kD antigen to perform the absorption, we opted to demonstrate somewhat indirectly that our pres-
414
Takino et al.
Fig. 4. Electron microscopic section of non-beta cell incubated with ICA positive serum. No specific immunostaining was observed. (Magnification: X 10,000.)
ent results were not due to the contaminating anti-64 kD autoantibody. We immunostained the sections of pancreas with serum which was positive for anti-64 kD antibody and negative for ICA and compared with sections reacted with ICA positive sera. With the anti-64 kD antibody positive and ICA negative serum as the first antibody, no definite structure in human pancreas was stained (data not shown), although there was slightly higher background staining. These results suggest that the staining observed with our ICA positive sera was not the result of the immune reaction with anti-64 kD protein. The ICA positive sera reacted antigens on the cytoplasmic membranes and the membranes of beta cell secretory granules, whereas Reetz et al. (9) found that antigens which react with sheep anti-rabbit glutamic acid decarboxylase (GAD), i.e., 64 kD protein (lo), reacted with membranes of pleiomorphic microvesicles and of tubular and cisternal elements of rat pancreatic beta cells (9). This difference in the distributions of antigens at the electron microscopic level further suggests that the antigens reacting with the ICA positive sera were something other than 64 kD protein. Even though, why our ICA positive sera failed to localize intra-islet 64 kD protein should be addressed. A most probable reason is GAD or 64 kD protein is a soluble cytosolic protein and dissolved
out of the tissues during fixation, since we opted for suboptimal fixation conditions in order to maintain the antigenicity of antigens reactive with the ICA positive sera. If this is the case, then the antigens reacted with the ICA positive sera, on the other hand, may be categorized as insoluble elements of the membranes and not 64 kD protein. Another possible reason for the differences in the results of Reetz et al. and ours may have been differences of immunoreactivity of 64 kD protein between species. In any event, this aspect requires further investigation. Although we are able to determine that the antigen for ICA localized in our study was not insulin and probably not 64 kD antigen, we could not ascertain what is (are) antigen(s) for ICA. However, the behavior of ICA antigen in various fixatives suggests what the antigen might be. Our histochemical results suggest that the antigen(s) is (are) not an ordinal trans-membrane proteinaceous antigen since the antigen was readily lost in cold acetone or in organic solvents. In addition, that the antigen can be fixed somewhat by carbohydrate cross linking fixative (a main chemical reaction of PLP is to cross link carbohydrates) suggests that the antigen contains a carbohydrate moiety. These characteristics are similar to the immunohistochemical findings of Nayak et al. (1 l), who
lmmunoelectronMicroscopic Localization of Antigen Against ICA
415
Fig. 5. Electron microscopic section incubated with ICA negative control serum. No specific immunostaining was observed. (Magnification: x l0,OOO.)
reported that the antigens for ICA had the properties of sialic acid containing glycolipid because of decreased antigenicity of ICA following incubation of pancreatic sections with neuraminidase and exposure to ch1oroform:methanol or methanol. A difference between our results and those of Nayak et al. was that they found the antigen was stable in acetone whereas we found the decrease of antigen after fixation in acetone. The differences may have resulted from immunohistochemical procedural differences; however, Nayak et a1.did not report the detailed procedure and we were unable to directly compare the results. As another autoantigen of ICA, Powers and Bowen (12) suggested that insulin secretion granule membrane associated soluble carboxypeptidase H (CPH) was the antigen for ICA. The behavior of CPH epitope during immunohistochemical procedures is yet undefined in pancreatic islets; however, CPH being glycoprotein, it is expected that routine immunohistochemical procedures used in our study should have been applicable for the localization of CPH in pancreatic islets. The failure to stabilize the autoantigen of ICA by protein precipitating fixatives such as organic solvents in our study suggests that either CPH might have behaved unorthodoxly from other glycoproteins in immunohistochemical procedures or the antisera used in the current study recognized autoantigens of ICA
other than CPH. This aspect also requires further investigation using the same human autoantibodies since the characterization of autoantibodies used to identify CPH in pancreatic islets was not reported. In conclusion, using pre-embedding immunoelectron microscope techniques, we showed that the antigen(s) against ICAs was (were) located in the membranes of beta cell-secretory granules and cytoplasmic membranes.
ACKNOWLEDGMENTS We are gratefully thankful to A. Nakashima, M. Morimoto, and T. Suematsu for skillful technical assistance.
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