Scherbaum WA, Mirakian R, Pujol-B orrell R, Dean BM, Bottaz- zo GF (1986) Immunocytochemistry ... In: Polak M, Van Noor- den S (eds) Immunocytochemistry ...
Diabetologia (1993) 36:785-790
Diabetologia 9 Springer-Verlag 1993
Human monoclonal islet specific autoantibodies share features of islet cell and 64 kDa antibodies W. Richter 1, T. H. Eiermann 2, J. Endl 3, J. Seigler t, S. Wolfahrt 1, M. Brandt 3, H. Jungfer 3, W. A. Scherbaum 1 1Department of Internal Medicine 1, University of Ulm, Germany 2Department of Transfusion Medicine, University of Ulm, Germany 3Boehringer Mannheim Research Center, Tutzing, Germany
Summary. The first human monoclonal islet cell antibodies of the IgG class (MICA 1-6) obtained from an individual with Type 1 (insulin-dependent) diabetes mellitus were cytoplasmic islet cell antibodies selected by the indirect immunofluorescence test on pancreas sections. Surprisingly, they all recognized the 64 kDa autoantigen glutamate decarboxylase. In this study we investigated which typical features of cytoplasmic islet cell antibodies are represented by these monoclonals. We show by double immunofluorescence testing that MICA 1-6 stain pancreatic beta cells which is in agreement with the beta-cell specific expression of glutamate decarboxylase. In contrast an islet-reactive IgM monoclonal antibody obtained from a pre-diabetic individual stained all islet cells but lacked the tissue specificity of MICA 1-6 and must therefore be considered as a polyreactive IgM-antibody. We further demonstrate that MICA 1~5 revealed typical features of epitope sensitivity to biochemical treatment of the target tissue which has been demonstrated for islet cell antibodies,
and which has been used to argue for a lipid rather than a protein nature of target antigens. Our results provide direct evidence that the epitopes recognized by the MICA are destroyed by methanol/chloroform treatment but reveal a high stability to Pronase digestion compared to proinsulin epitopes. Conformational protein epitopes in glutamate decarboxylase therefore show a sensitivity to biochemical treatment of sections such as ganglioside epitopes. MICA 1-6 share typical features of islet cell and 64 kDa antibodies and reveal that glutamate decarboxylase-reactive islet cell antibodies represent a subgroup of islet cell antibodies present in islet cell antibody-positive sera.
The humoral autoimmune response to islet specific antigens in Type 1 (insulin-dependent) diabetes mellitus comprises a variety of islet-specific autoantibodies circulating in the serum of affected individuals. Most target molecules of the autoimmune response to islets were unknown for a long time. Therefore the variant islet cell autoantibodies were classified according to the method by which they were detected. Cytoplasmic islet cell antibodies (ICA) have been defined by a cytoplasmic staining pattern of all islet cells in histochemical tests [1,2] and 64 kDa antibodies were defined by immunoprecipitation experiments on in vitro labelled islet cells [3]. Whereas 64 kDa antibodies were shown to recognize the beta-cell specific enzyme protein glutamate decarboxylase (GAD) [4] ICA have been suggested to recognize gangliosides and not proteins [5, 6]. Evidence supporting this claim included the similarity in biochemical properties of antigens recognized by anti-ganglioside monoclonal antibodies with that of ICA-positive sera [5], and the observation that ICA
binding to islet cells is inhibited by glycolipid extracts from human pancreas [6]. Our group recently isolated the first human monoclonal ICA of the lgG-class (MICA 1-6) derived from a patient with newly-diagnosed Type 1 diabetes [7]. M I C A 1-6, which represent five different circulating primary B-lymphocyte clones from blood [7, 8], were selected by the histochemical ICA test and surprisingly recognized G A D as a target antigen. In addition, blocking studies with ICA-positive sera provided clear evidence that G A D reactivity of ICA was not limited to the single individual from whom the M I C A were derived [7]. Our data were controversial to the suggested ganglioside nature of target antigens of ICA [5, 6]. In this study we, therefore, addressed the question which of the typical features of ICA or 64 kDa antibodies are represented by M I C A 1-6. We analysed the reactivity of the M I C A within the islet by double immunofluorescence staining with anti-hormone-specific antibodies and compared their tissue specificity with that of FAE7, a new
Key words. Islet cell antibodies, 64 kDa antibodies, human monoclonal antibodies, glutamate decarboxylase, gangliosides, Type 1 (insulin-dependent) diabetes mellitus, islet cell antigens
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W. Richter et al.: Human monoclonal islet specific autoantibodies
Table 1, Reactivity of MICA 14~ and FAE7 with various human tissues and islets of different mammalian species Human tissue a
MICA 1-6
FAE7
Islet cellsb
MICA FAE7 1-6
Pancreatic Islets Thyroid Adrenal cortex Adrenal medulla Pituitary Stomach Intestine
+ -
+ + + -
Human Rat Pig Cow Monkey
+ + + + +
+ -
"Reactivity assessed by peroxidase conjugated pre-formed immune complexes. b Reactivity assessed by indirect immunofluorescence staining. MICA, Human monoclonal IgG-islet cell antibody. FAE 7, Human monoclonal islet cell antibody of the IgM-class
h u m a n m o n o c l o n a l I C A of the IgM-class which we d e r i v e d f r o m a p r e - d i a b e t i c individual. W e f u r t h e r a n a l y s e d w h e t h e r t h e M I C A e p i t o p e s in G A D show the typical sensitivity of I C A e p i t o p e s r e p o r t e d p r e v i o u s l y for a p a n e l of I C A - p o s i t i v e s e r a a n d for a n t i - g a n g l i o s i d e antib o d i e s [5]. C o m p l e t e l y d i f f e r e n t b i o c h e m i c a l p r o p e r t i e s between GAD and antigens recognized by ICA from sera w o u l d suggest t h a t G A D - r e a c t i v e I C A a r e a m i n o r subg r o u p of I C A , n o t r e l e v a n t for t h e h i s t o c h e m i c a l staining p a t t e r n o b t a i n e d with I C A - p o s i t i v e sera f r o m p a t i e n t s with T y p e 1 d i a b e t e s .
washed for 10 min in PBS and incubated with Pronase 0.1 mg/ml in PBS (Boehringer, Mannheim, Mannheim, Germany) for 10 rain at 20~ or with neuraminidase 0.5 U/ml in 50 mmol/1 sodium citrate pH 5.5 (Boehringer) for 60 rain at 37 ~ A mouse monoclonal antihuman proinsulin antibody (1:10 in PBS; Novo) was used to optimize the duration of the Pronase incubation step. The monoclonal antibody A2B5, recognizing a ganglioside antigen in islets [12] was used as a control in the neuraminidase incubation step. After pretreatment sections were washed for 15 rain in PBS and staining of the antibodies was assessed by the indirect immunofluorescence test.
Immunoblotting. GAD was purified from the cytosolic fraction of pig brain homogenates (10% weight/volume in 20 mmol/l KHzPO4 pH 7.4, 1 retool/1 phenyl-methyl-sulphonyl-fluoride, i mmol/1 amino-ethyl-isothiouronium-bromide, 0.02 retool/1 pyridoxal phosphate). The 33.000 x g supernatant was purified by gelfiltration on a Sepharose Q column and fractions with GAD enzyme activity were pooled and spotted on nitrocellulose membranes (30 btl/slot of dilutions 1:10, 1:100 and 1:500) (Hybond; Amersham, Amersham, Bucks., UK). Part of the blots were incubated in methanol/chloroform (1:2) for 5 rain (20 ~ or in Pronase (0.1 mg/m110 rain, 20 ~ After washing the nitrocellulose membranes were immunostained with supernatant of MICA 4 (2 gg/ml), a pool ofafl MICA (2 [tg/ml), an irrelevant human monoclonal antibody (2 gg/ml), or the GAD 65-specific rabbit antiserum 1267 (donated by J.Peterson, Copenhagen, Denmark). Reactivity was detected in an enhanced chemiluminescence assay (Amersham) on Kodak XO Mat film. Membranes were then washed in PBS and stained with Coomassie brilliant blue.
Results Immunohistochemical characterization. F A E 7 , a h u m a n
Subjects and methods Generation of the human monoclonal IgM-ICA FAE7. Blood was obtained after informed consent from an ICA-posifive pre-diabetic individual who acquired Type 1 diabetes 1 year after blood donation. B lymphocytes were immortalized by Epstein-Barr virus infection as described [9] and supernatants of the cultured cells were screened for ICA after 3-4 weeks by the classic indirect immunofluorescence test using fiuorescein isothiocyanate (FITC)-labelled goat anti-human IgG and IgM antibodies (Dianova, Hamburg, Germany) [10]. Single cell cloning and stabilisation of the monoclonal line was performed as described for the MICA [7]. Immunohistochemical characterization. Species specificity and cross-reactivity of the monoclonal antibodies with different human tissues was assessed on cryostat sections using either preformed complexes of the individual MICA with peroxidase conjugated antihuman Ig antibodies [11] or indirect immunofluorescence testing [10]. Double immunofluorescence staining was performed by incubating a mouse monoclonal anti-proinsulin specific antibody (Novo, Bagsvaerd, Denmark) diluted 1:5 in supernatant of the different MICA on cryostat sections of human pancreas (1 h, 20~ After washing in phosphate buffered saline (PB S) (30 rain) the presence of antibodies was assessed by FITC-conjugated goat anti-human IgG (preabsorbed with mouse serum proteins, Dianova) and rhodamineconjugated goat anti-mouse IgG (preabsorbed with human serum proteins, Dianova) diluted 1:30 in PBS (60 rain, 20~ For glucagon- or somatostatin-specific staining a rabbit anti-glucagon antibody or rabbit anti-somatostatin antibody (Dako, Glostrup, Denmark) and donkey anti-rabbit conjugate (Dianova) were used.
Pre-treatment of pancreas sections. Cryostat sections of human pancreas from a donor with blood group 0 were processed as described [5]. Sections were fixed in acetone (5 rain, 20~ or incubated in methanol/chloroform (1:2) (5 min, 20 ~ or, after acetone fixation,
m o n o c l o n a l I C A o f t h e I g M class was d e r i v e d f r o m a p r e d i a b e t i c i n d i v i d u a l using t h e a b o v e - m e n t i o n e d m e t h o d s . R e a c t i v i t y of F A E 7 a n d M I C A 1-6 was a n a l y s e d on diff e r e n t h u m a n o r g a n s a n d o n p a n c r e a s s e c t i o n s of d i f f e r e n t m a m m a l i a n species. I n c o n t r a s t to t h e M I C A , F A E 7 was n o t r e s t r i c t e d to islet cells a n d d i d n o t c r o s s - r e a c t with p a n c r e a s sections of t h e o t h e r m a m m a l i a n species tested. T h e r e f o r e , o n l y M I C A 1-6 e x h i b i t e d a r e a c t i v i t y typical for I C A (Table 1).
Cell staining within islets. A s j u d g e d b y d o u b l e i m m u n o f l u o r e s c e n c e staining of t h e m o n o c l o n a l a n t i b o d i e s a n d a n t i - h o r m o n e specific a n t i b o d i e s t h e M I C A s h o w e d a b e t a - c e l l r e s t r i c t e d staining p a t t e r n in m a n y islets which o v e r l a p p e d a l m o s t c o m p l e t e l y w i t h that of t h e a n t i - p r o insulin antibody. H o w e v e r , s o m e single cells lying at t h e p e r i p h e r y of s o m e islets, which w e r e n e g a t i v e with the p r o i n s u l i n a n t i b o d y w e r e w e a k l y s t a i n e d b y the M I C A , e s p e c i a l l y w h e n a p o o l e d s u p e r n a t a n t of M I C A 1 - 6 was a p p l i e d (Fig. 1 a - c ) . Cells positive for a n t i - g l u c a g o n o r a n t i - s o m a t o s t a t i n a n t i b o d i e s w e r e n e g a t i v e for t h e M I C A when judged within the same section (Fig.ld, e and results n o t shown). F A E 7 , in contrast, r e a c t e d with all cells of t h e islet a n d in a d d i t i o n with single cells of the e x o c r i n e p a n c r e a s as well as with p a n c r e a t i c ducts (Fig. 1 f). Biochemical stability of the antigenic epitopes of M I C A 16. W e c o m p a r e d t h e b i o c h e m i c a l stability of t h e M I C A e p i t o p e s with t h a t o f e p i t o p e s r e c o g n i z e d b y I C A f r o m serum. M I C A 3 a n d M I C A 4 w e r e s e l e c t e d b e c a u s e t h e y w e r e s h o w n to r e c o g n i z e distinct e p i t o p e s in G A D which
W. Richter et al.: Human monoclonal islet specific autoantibodies
]pig.l(a-0. Double-fluorochrome immunofluorescence staining of a pool of MICA 1-6 and a mouse monoclonal proinsulin-specific antibody within the same section of human pancreas (a-e). MICA 16 were detected with FITC-conjugated anti-human IgG (a) and the anti-proinsulin antibody with rhodamine-conjngated anti-mouse IgG (b). Double exposure photography of picture a and b is shown in c. The yellow colour in c is given by the superimposed green and red colour in beta cells. Note that, in addition, there are some greencoloured cells visible at the periphery of the islets (indicated by the arrow) which are weakly stained by the MICA 1~5,but are negative for proinsulin. An islet double stained with MICA 1~5 (green in d) and an anti-glucagon-cell specific antibody (red in e) indicates glucagon-positive cells negative for the MICA (compare arrows in d and e). FAE7 gives a whole islet cell staining pattern in islets (f). Scale bar corresponds to 100 gm in a, b, e, and f and to 62 gm in d and e
are both common for sera from patients with Type 1 diabetes [8]. The anti-proinsulin specific monoclonal antibody and the monoclonal antibody A2B5 were included as examples of anti-protein and anti-ganglioside specific monoclonal antibodies, respectively. Binding of the ICApositive serum and of M I C A 3 and MICA 4 was not affected by treatment of the sections with acetone or Pronase. However, reactivity with islets was completely abolished when sections were pre-treated with methanol/chloroform. Neuraminidase treatment of the tissue optimized to ablate binding of the anti-ganglioside antibody A2B5 decreased the staining of ICA-positive serum and of M I C A 3 and 4. This reduction of binding, however, was non-specific since the same result was achieved when the
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sections were pre-incubated with the control buffer alone (50 retool/1 sodium citrate, 37~ p H 5.5). In contrast, A2B5 staining was not affected by the buffer control incubation (Table 2). The biochemical stability of the epitopes recognized by M I C A 3 and 4 was in line with the biochemical stability of the epitopes recognized by ICA from serum. Except for neuraminidase treatment, M I C A 3 and 4 behaved similarly to the anti-ganglioside antibody A2B5. Despite recognizing protein epitopes, M I C A 3 and 4 behaved differently from the anti-proinsulin antibody.
G A D was less sensitive to Pronase than proinsulin. To test whether G A D is insensitive to Pronase digestion, various concentrations and incubation times of Pronase were applied on tissue sections. Titration studies revealed a rapid degradation of proinsulin by 0.1 mg/ml Pronase in 1015 rain, whereas G A D epitopes recognized by the M I C A remained stable during that time. A 1-h incubation with this Pronase concentration was necessary to degrade epitopes recognized by the M I C A (Fig. 2). Alternatively a 10fold increase in Pronase concentration degraded the M I C A epitopes within 15 min and proinsulin epitopes within 2 rain. G A D localized in the tissue, therefore, was much less sensitive to unspecific proteolysis by Pronase than proinsulin, but was not completely stable. Methanol~chloroform treatment destroyed the M I C A epitopes. We investigated whether the conformational G A D epitopes recognized by MICA 3 and M I C A 4 were destroyed by incubation with methanol/chloroform or whether elution of G A D from the tissue contributed to the
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W. Richter et al.: Human monoclonal islet specific autoantibodies
Table 2. Sensitivity of islet cell epitopes to treatment with fixatives and enzymes: evaluation of islet cell staining on pre-treated sections Treatment of sections
Immunostaining with a
Untreated
MICA 3
MICA 4
ICApositive serum
Antiproinsulin
A2B5
+
+
+ +
+ +
+ +
Acetone
+
+
++
+ +
+ +
Methanol/ Chloroform
-
-
-
+ +
-
Pronase
+
+
++
-
+ +
Neuraminidase
-
(+)
+
+ +
-
Control buffer b
-
(+)
+
+ +
+ +
a Staining was performed with the indirect immunofiuorescence test on cryostat sections of human pancreas. b Buffer control incubation of the neuraminidase experiment: 50 mmol/1 sodium citrate buffer pH 5.5 for 1 h at 37 ~ MICA, Human monoclonal IgG-islet cell antibody. ICA, cytoplasmic islet cell antibody. Semiquantitativeevaluation of staining: + + very strong; + strong, ( + ) weak, - no staining
1:128t 1: 64-
Fig.3. Immunoblotting of GAD purified from pig brain. GAD (1 mg/ml) was spotted on nitrocellulose at dilutions 1 : 10, 1:100 and 1:500 (top to bottom) and blots left untreated or incubated with methanol/chloroform (5 min, 20~ or Pronase (0.1 mg/ml, 10 min, 20 ~ After washing blots were immunostained by MICA 4 or a polyclonal serum raised against a linear epitope in GAD (1267). An enhanced chemiluminescence assay was used to detect the peroxidase staining on an X-ray film
a l i n e a r e p i t o p e in G A D . A n i d e n t i c a l staining intensity with m e t h a n o l / c h l o r o f o r m - t r e a t e d a n d u n t r e a t e d G A D blots was o b s e r v e d c o n f i r m i n g t h a t no e l u t i o n of G A D occurred. T h e r e f o r e , t h e c o n f o r m a t i o n a l e p i t o p e s r e c o g nized by MICA 3 and 4 must have been destroyed by metha n o l / c h l o r o f o r m t r e a t m e n t w h e r e a s the l i n e a r 1267 epit o p e in G A D r e m a i n e d u n a f f e c t e d .
1:32o 1:16p- 1:84-a
1:41:2-
Discussion I
5
I
15
I
30
I
60
Time of Treament with Pronase (min) Fig.2. Sensitivity of proinsulin and glutamate decarboxylase epitopes to Pronase digestion of the tissue section was assessed. Cryostar sections of human pancreas were incubated with Pronase (0.1 mg/ml) and after washing an indirect immunofluorescence test was performed on pre-treated sections. The titre of a monoclonal anti-human proinsulin antibody ( 9 and of a pool of the human monoclonal islet cell IgG antibodies MICA 3 and 4 ([]) was assessed in relation with the time of Pronase digestion of the tissue. Antibody titres were not affected by the buffer control incubations without Pronase (not shown)
lack of r e a c t i v i t y after m e t h a n o l / c h l o r o f o r m t r e a t m e n t . G A D p u r i f i e d f r o m p i g b r a i n was i m m o b i l i z e d on n i t r o c e l lulose a n d a n i m m u n o b l o t t i n g a s s a y w a s p e r f o r m e d . P a r t o f the blots w e r e p r e - t r e a t e d with m e t h a n o l / c h l o r o f o r m o r P r o n a s e . G A D p r e - t r e a t e d w i t h m e t h a n o l / c h l o r o f o r m was no l o n g e r r e c o g n i z e d b y M I C A 3 a n d 4, w h e r e a s u n t r e a t e d o r P r o n a s e - t r e a t e d G A D was still r e a c t i v e (Fig. 3). A n irr e l e v a n t h u m a n m o n o c l o n a l a n t i b o d y of t h e I g G class did n o t r e a c t with a n y of t h e slots ( n o t shown). To e x c l u d e t h a t G A D was n o t e l u t e d f r o m t h e n i t r o c e l l u l o s e m e m b r a n e during methanol/chloroform treatment, parallel blots w e r e s t a i n e d with a p o l y c l o n a l s e r u m (1267) r a i s e d against
H u m a n m o n o c l o n a l a n t i b o d i e s a r e a n i n v a l u a b l e t o o l with w h i c h to s t u d y t h e n a t u r e of t h e h u m o r a l i m m u n e res p o n s e a s s o c i a t e d with p a n c r e a t i c b e t a - c e l l d e s t r u c t i o n a n d d e v e l o p m e n t of T y p e 1 diabetes. W e r e c e n t l y obt a i n e d the first h u m a n m o n o c l o n a l I C A of the I g G - c l a s s ( M I C A 1~5) d e r i v e d f r o m a p a t i e n t with T y p e 1 diabetes. M I C A 1-6, s e l e c t e d b y t h e classic I C A test o f i n d i r e c t imm u n o f l u o r e s c e n c e all r e c o g n i z e d t h e T y p e 1 d i a b e t e s specific 64 k D a a u t o a n t i g e n G A D [7]. T h e e p i t o p e s recogn i z e d b y t h e M I C A w e r e m u l t i p l e a n d c o m m o n for I C A f r o m s e r u m [7, 8]. W e h e r e a s k e d t h e q u e s t i o n w h e t h e r t h e islet-reactive I g G m o n o c l o n a l s M I C A 1-6 d e m o n s t r a t e all t y p i c a l f e a t u r e s of I C A . W e f u r t h e r i n v e s t i g a t e d w h e t h e r F A E 7 , a n e w h u m a n m o n o c l o n a l I g M - I C A isol a t e d f r o m a p r e - d i a b e t i c i n d i v i d u a l fits in this p a n e l of disease-related ICA. F A E 7 was d e r i v e d f r o m b l o o d o b t a i n e d 1 y e a r b e f o r e o n s e t of T y p e 1 d i a b e t e s , b u t t h e a n t i b o d y d i d n o t r e p r e s e n t the t y p i c a l f e a t u r e s k n o w n for I C A - p o s i t i v e sera. D u e to its c r o s s - r e a c t i v i t y with h u m a n p i t u i t a r y cells, thyroid, e x o c r i n e p a n c r e a t i c epithelia, a n d p a n c r e a s ducts, F A E 7 m u s t b e c o n s i d e r e d as a p o l y r e a c t i v e I g M a n t i b o d y like t h e p r e v i o u s i s l e t - r e a c t i v e h u m a n m o n o c l o n a l I g M a n t i b o d i e s d e r i v e d f r o m p a t i e n t s with T y p e 1 d i a b e t e s [13]. Such p o l y r e a c t i v e I g M - a n t i b o d i e s also o c c u r in n o r -
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W. Richter et al.: Human monoclonal islet specific autoantibodies mal individuals unrelated to autoimmune diseases [14]. Therefore, FAE7 can give no hints on ICA-reactive targets during earlier stages of beta-cell destruction in Type 1 diabetes. Although M I C A 1-6 were isolated using the conventional test for detection of ICA, they demonstrate all the known features of G A D antibodies. Beside the precipitation of a 64 k D a protein from islets and the immunotrapping of G A D - e n z y m e activity from brain extracts shown previously [7] M I C A 1-6 revealed here a predominantly beta-cell specific staining pattern in islets which is consistent with the beta-cell specific expression of G A D [15, 16]. In some islets, a few single cells lying in the periphery were negative for proinsulin, but were weakly stained by the M I C A . As glucagon or somatostatin staining did not overlap with the M I C A staining, these single cells may be identical with the GAD-containing neural cells identified recently by Sorenson et al. [17]. These neurons were reported to be localized in the islet mantle and to extend nerve cell processes into the islet. We did not, however, test the PP-cell population and cannot exclude that PP cells contribute to this staining pattern. Only some features typical for I C A were observed for the M I C A . Whereas the specificity for islet cells and the cross-reactivity with islets of different m a m m a l i a n species was typical for ICA, M I C A 1-6 did not show the whole islet cell staining pattern reported for m a n y ICA-positive sera [2]. Beta-cell specific G A D - r e a c t i v e ICA, therefore, exist in at least some ICA-positive sera. Since m a n y sera from newly-diagnosed patients with Type 1 diabetes are positive for both I C A and 64 k D a antibodies [3,18], G A D - r e a c t i v e I C A may be very c o m m o n i n ICA-positive sera. N a y a k and co-workers [5] have suggested that the islet antigen responsible for the I C A staining is a monosialoganglioside. When we analysed the biochemical properties of the M I C A 3 and M I C A 4 epitopes in G A D they revealed the same sensitivity to chemical pre-treatment of the tissue as epitopes recognized by I C A from serum. In addition, the stability of the M I C A epitopes, except for the neuraminidase sensitivity, was identical to the anti-ganglioside antibody A2B5 and to the panel of I C A positive sera analysed by N a y a k and colleagues [5]. The anti-protein control antibody directed to proinsulin behaved differently. We were able to show that G A D was very stable to Pronase digestion of tissue sections. Using G A D immobilized on nitrocellulose m e m b r a n e s we provided clear evidence that the conformational M I C A epitopes in G A D were destroyed by treatment with methanol/chloroform. No elution of G A D from nitrocellulose m e m b r a n e s due to methanol/chloroform treatment was evident. However, it cannot be excluded that under normal conditions in the tissue sections elution of G A D may occur as an additional effect of methanol/chloroform treatment beside epitope destruction. Together our data demonstrate that conformational protein epitopes can share sensitivity to chemical pretreatment of the target tissue with ganglioside epitopes. Therefore, chemical pre-treatment of the tissue used in the past was not suitable to characterize the chemical nature of target antigens recognized by ICA. The I g G m o n o clonal antibodies M I C A 1-6 share features of both I C A and 64 k D a antibodies and must be considered as beta-
cell specific G A D - r e a c t i v e ICA. These data together with data from others [19, 20] strongly suggest a heterogeneous composition of I C A in ICA-positive sera from patients with Type 1 diabetes. We demonstrated with the M I C A that the protein G A D is one of the autoantigens recognized by I C A and we suggest that proteins as well as gangliosides are potential candidates for additional target antigens of I C A beside G A D .
Acknowledgement. We thank Ms. E. Erne for technical assistance. Antibody 1267 was kindly provided by Dr. J. Peterson and antibody A2B5 by Dr. G. Eisenbarth. This work was supported by grants from the DeutscheForschungsgemeinschaft Sche 225/3-2 andSche 225/6-2.
References 1. Bottazzo GF, Florin-Christensen A, Doniach D (1974) Islet cell antibodies in diabetes mellitus with autoimmune poIyendocrine deficiencies. Lancet II: 127%1283 2. Bottazzo GF, Doniach D (1978) Islet-cell antibodies (ICA) in diabetes mellitus. Evidence of an autoantigen common to all cells in the islet of Langerhans. La Ricerca Clin Lab 8:29-38 3. Baekkeskov S, Nielsen JH, Marner B, Bilde T, Ludvigsson J, Lernmark _A.(1982) Autoantibodies in newly diagnosed diabetic children immunoprecipitate pancreatic islet cell proteins. Nature 298:167-169 4. Baekkeskov S, Aanstoot HJ, Christgau Set al. (1990) Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 347:151-156 5. Nayak RC, Omar AK, Rabizadeh A, Srikanta S, Eisenbarth GS (1985) Cytoplasmic islet cell antibodies: evidence that the target antigen is a sialoglycoconjugate. Diabetes 34:617-619 6. Colman PG, Nayak RC, Campbell IL, Eisenbarth GS (1988) Binding of cytoplasmic islet cell antibodies is blocked by human pancreatic glycolipid extracts. Diabetes 37:645-652 7. Richter W, Endl J, Eiermann TH et al. (1992) Human monoclonal islet cell antibodies from a patient with insulin-dependent diabetes mellitus reveal glutamate decarboxylase as the cytoplasmic islet cell antigen. Proc Natl Acad Sci USA 89: 84678471 8. Richter W, Shi Y, Baekkeskov S (1993) Autoreactive epitopes defined by diabetes associated human monoclonal antibodies. Proc Natl Acad Sci USA: 90:2832-2836 9. Richter W, Eiermann TH, Graf G, Glueck M, Scherbaum WA, Pfeiffer EF (1989) Isolation of IgG islet cell antibody-producing B-lymphocytes from the peripheral blood of type I diabetic patients and an ICA-positive nondiabetic individual. Horm Metabol Res 21:686-688 I0. Scherbaum WA, Mirakian R, Pujol-B orrell R, Dean BM, Bottazzo GF (1986) Immunocytochemistry in the study and diagnosis of organ-specific autoimmune diseases. In: Polak M, Van Noorden S (eds) Immunocytochemistry modern methods and applications. Wright, Bristol, pp 456-476 11. Tuson R J, Pascoe EW, Jacob DA (1990) A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections. J Histochem Cytochem 38:923-926 12. Eisenbarth GS, Shimizu K, Bowring MA, Wells S (1982) Expression of receptors for tetanus toxin and monoclonal antibody A2B5 by pancreatic islet cells. Proc Natl Acad Sci USA 79: 50665070 13. Satoh J, Prabhakar BS, Haspel MM Ginsberg-Feltner F, Notkins AL (1983) Human monoclonal autoantibodies that react with multiple endocrine organs. New Engl J Med 309:217-220 14. Casali R Notkins AL (1990) Probing the human B-cell repertoire with EBV: polyreactive antibodies and CD5 + B lymphocytes. Ann Rev Immunol 7:513-535
790 15. Christie MR, Pipeleers DG, Lernmark .~, Baekkeskov S (1990) Cellular and subcellular localization of an Mr 64.000 protein autoantigen in insulin-dependent diabetes. J Biol Chem 265:376-381 16. Garry DJ, Sorenson RL, Elde RR Maley BE, Madsen A (1986) Immunohistochemical colocalization of G A B A and insulin in ]3cells of rat islets. Diabetes 35:1090-1095 17. Sorenson RL, Garry DG, Brelje TC (1991) Structural and functional considerations of G A B A in islets of Langerhans. Diabetes 40:1365-1374 18. Atkinson MA, MacLaren NK, Scharp DW, Lacy PE, Riley WJ (1990) 64.000 Mr autoantibodies as predictors of insulin-dependent diabetes. Lancet 335:1357-1360 19. Gianani R, Pugliese A, Bonner-Weir S e t al. (1992) Prognostically significant heterogeneity of cytoplasmic islet cell antibodies in relatives of patients with type 1 diabetes. Diabetes 41:347-353
W. Richter et al.: Human monoclonal islet specific autoantibodies 20. Genovese S, Bonifacio E, McNally JM (1992) Distinct cytoplasmic islet cell antibodies with different risks for type l (insulindependent) diabetes mellitus. Diabetologia 35:385-388 Received: 27 January 1993 and in revised form: 8 April 1993 Dr. W. Richter Department of Internal Medicine 1 University of Ulm Robert-Koch-Strasse 8, 023/3527 D-89081 Ulm Germany
