Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies

Regulatory Peptides, 39 (1992) 201-214 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00

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REGPEP 01185

Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies Micaela Pelagi a, Antonia Zanini b, Anna Gasparri c, Laura Ermellino e, Anna M. Giudici b, Stefano Ferrero d, Antonio G. Siccardi c and Roberto Buffa e aIstituto Scientifico Ospedale S. Raffaele, Milano (Italy), b Centro CNR Infrastrutture Cellulari, Dipartimento di Farmacologia, Universitgt di Milano, Milano (Italy), CDipartimento di Biologia e Genetica per le Scienze Mediehe, Milano (Italy), d III Cattedra di Anatomia Patologica, Universitd di Milano, Milano (Italy) and e Dipartimento di Patologia Umana ed Ereditaria dell'Universith di Pavia. Sezione di Anatomia Patologica II, Sede Ospedale Multizonale, Varese (Italk9

(Received 12 September 1991; revised version received 20 January 1992; accepted 4 March 1992)

Key words: Immunoblotting; Immunocytochemistry; Neuroendocrine tissue; Secretogranin II; Secretory product

Summary Secretogranin II (chromogranin C) is an acidic tyrosine-sulfated secretory protein, known to be a marker of neuroendocrine secretory products and of specific neuroendocrine tumours. In order to obtain anti-secretogranin II monoclonal antibodies for cell biology studies and, in particular, for clinical applications, we immunized mice with a secretogranin II-enriched fraction prepared from homogenates of bovine anterior pituitaries. Hybridoma supernatants obtained from the splenocytes of a hyperimmune mouse, screened with an enzyme-linked immunosorbent assay, were analyzed by both immunocytochemistry and two-dimensional immunoblotting. By using this experimental approach, we were able to identify two monoclonal antibodies (8G1 and 5A7) which recognize bovine secretogranin II. Both immunocytochemistry and immunoblotting revealed that one of them, the 5A7 antibody, cross-reacts with the human antigen. The distribution patterns of the immunoreactivity, obtained by immunocytochemistry with the 5A7 antibody in animal and human tissues, partially overlap those, obtained by using a polyclonal antiserum elicited against bovine secretogranin II, previously described. Moreover, the 5A7, but not the polyclonal antibody, reacts with some

Correspondence: M. Pelagi, Dipartimento di Biologia e Genetica per le Scienze Mediche, Via Viotti 3/5, 20133 Milano, Italy.

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duodeno-jejunal cells. In conclusion, both the 5A7 and 8G1 antibodies can be useful for cell biology studies. The 5A7 antibody can be used for the detection of secretogranin II in human tissues and should be of help in clinical and pathological practices.

Introduction

Secretogranin II (SglI) (previously also called chromogranin C, see Ref. 1 for nomenclature) is an acidic tyrosine-sulfated secretory protein present, like chromogranin A (CgA) and chromogranin B (CgB), in secretory granules of a variety of neuroendocrine tissues (for reviews see Refs. 2-5). The function of SgII is still unclear, although recent data on its primary sequence suggest that this protein, like CgA and CgB with which it shares biochemical and biophysical properties, might be the precursor of peptides with possible a hormonal or paracrine role [4,5]. Recently, SgII has been shown to be a useful marker for neoplasms derived from neuroendocrine cells. Its presence in pheochromocytomas [6], C-cell carcinomas [7], bronchial and intestinal carcinoids [8,9], insulinomas, Merkel cell carcinomas and neuroblastomas [ 10] has been detected by immunocytochemistry. However, SgII is not as widely reported in surgical pathology as CgA and CgB [5], possibly because only polyclonal antibodies against bovine or rat SgII have been obtained by different groups [ 11-14] and no monoclonal antibodies have been described. The present paper reports on the preparation of monoclonal antibodies against bovine SgII and their characterization by two-dimensional immunoblotting and immunohistochemistry.

Materials and Methods

Preparation of SglI-enriched fractions An SglI-enriched fraction was prepared from homogenates of bovine anterior pituitaries which were pulse-labeled with [35S]sulfate by means of ion-exchange chromatography on DEAE-Sephadex, as previously reported [ 11 ]. This fraction was used as an immunogen in mice. Anterior pituitary homogenates, used to set up an enzyme-linked immunosorbent assay (ELISA) and as antigen for immunoblotting, were prepared in 0.3 M sucrose containing 20 mM Tris-HC1 (pH 7.4) and 0.5 mM EGTA. An SgII-containing fraction was prepared from human pheochromocytoma by the heat-treatment procedure [6,12]. Pheochromocytoma tissue was frozen in liquid nitrogen immediately after surgical excision, homogenized in distilled water, boiled for 6 min and centrifuged at 120,000 g for 1 h. The supernatant was used as antigen for ELISA and immunoblotting.

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Two-dimensional polyacrylamide gel electrophoresis and immunoblotting Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed according to O'Farrell et al. [ 15], with the modifications previously reported [ 16,17], using Mini Protean II Cells (Bio-Rad Laboratories, Richmond, CA). Immunoblotting was performed as previously described [17]. Proteins separated by 2D-PAGE and electrophoretically transferred to nitrocellulose membranes were visualized with Ponceau S. (Serva Feinbiochemica, Heidelberg) and incubated overnight at 4 °C in 8~o bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Membranes were then incubated for 2 h with the first antibodies (anti-bovine SglIenriched fraction mouse antiserum diluted: 1:200; anti-purified bovine SglI rabbit antibodies, purified by affinity chromatography as reported [18], 2 mg/ml; undiluted hybridoma supernatants; ascite fluids, diluted 1:500). After several washings in PBS containing 0.3 ~o Triton X-100 the membranes were incubated for 1 h with horseradish peroxidase conjugated goat anti-mouse IgG and IgM antibodies (Jackson Immuno Research Laboratories, Inc., Avondale, PA), diluted 1:2000 or goat anti-rabbit IgG antibodies (Pasteur Institute, Paris, France), diluted 1:1000. Visualization of the reaction was performed using 3,3'-diaminobenzidine (DAB) or, in the case of anti-SgII rabbit antibodies, 4-chloro-l-naphthol.

Immunizations Balb/c mice, 10 weeks old, were immunized i.p. with 50 mg of the SgII-enriched fraction, prepared from bovine anterior pituitaries, in complete Freund's adjuvant. The second dose, in incomplete Freund's adjuvant, was given on day 15. Doses of 50 mg in PBS were subsequently given every 15 days thereafter.

Fusions and screening of hybridoma supernatants 3 days before killing, mice were boosted i.p. with 50 mg of the immunogen. Spleen cells were fused by standard methods, using 50~o polyethylene glycol (M r 155; Sigma) and the P3-X63 Ag8-NS1 cell line as fusion partner. Hybridomas producing anti-SglI antibodies were revealed by ELISA. 96-well microtitre plates (PBI) were coated with 0.3 mg/well of anterior pituitary homogenates or the SglI-containing fraction prepared from human pheochromocytoma in 50 mM carbonate buffer (pH 9.5) for 90 min at 37 °C. Non-specific binding of antibodies to the plates was then blocked by incubation with 3 ~o bovine serum albumin (B SA) in PB S overnight. The antibody binding assay was performed in three steps: (a) 100 ml of culture fluid was incubated in each well overnight at 4 °C and unbound immunoglobulin was then removed from the well by washing three times with PB S; (b) plates were incubated for 1 h at room temperature with peroxidase conjugated rabbit anti-mouse immunoglobulins diluted 1:1000; (c)after a final wash, a chromogenic substrate (o-phenylenediamine) was added and the reaction was blocked by addition of 2 M n 2 s o 4. Hybridomas producing antibodies with specificity for SglI were cloned by limiting dilution and expanded. Ascitic fluids were obtained from Balb/c mice primed with 2,6,10,11-tetramethyl-

204 pentadecane (Pristane, Aldrich Chem. Co., Steinheim, Germany) and inoculated with hybridoma cells. Anti-serotonin antibodies were purchased from Dakopatts (Glostrup, Denmark).

Immunohistochemistry Fresh samples from the pituitary, thyroid, parathyroid, adrenals, pancreas and gastrointestinal tract were taken from oxen, guinea-pigs, rats, rabbits, cats and dogs. Fragments from human adrenals, pancreas, thyroid, parathyroid and gastrointestinal mucosa were obtained from surgical specimens or during endoscopic examination; human pituitary, thymus and lung were collected at necroscopy. The following fixatives were used: PAF (neutral picric acid-formaldehyde [ 19]), AAF (70 ~o ethanol- 5 ~o acetic acid-4~o formaldehyde, modified from Bodian [20]), Bouin's solution and buffered formaldehyde (4~o in 0.1 M (pH 7.3) phosphate). After dewaxing, paraffin sections were immunostained with an avidin-biotin technique (ABC reagent from Vector, Burlingame, CA, USA). The primary reagents were applied overnight in 0.15 M Tris buffer (pH 7.3)-0.145 M saline (TBS). Peroxidase activity was detected with either DAB or 4 CI-1 naphthol (0.5 mM in TBS supplemented with 0.005 mM H202). Pertinent specificity tests (controls included the use of antibodies added with related and unrelated antigens and substitution of the primary reagent with non immune sera) were done according to Van Noorden [21].

Results

Anti-bovine SgII-enriched fraction antisera The antisera obtained by immunizing two mice with the SgII-enriched fraction prepared from bovine anterior pituitaries [ 11 ] were analyzed by direct-binding ELISA, using anterior pituitary homogenates as antigen. As shown in Fig. 1, a strong reaction, significantly different from the one obtained with the preimmune sera, was obtained with both antisera. Two-dimensional immunoblotting of anterior pituitary homogenates revealed that the two antisera (Fig. 2a) recognized a component corresponding to SgII, as demonstrated by comparison with the pattern obtained by using previously characterized polyclonal antibodies anti-purified bovine SgII raised in rabbit [11,18] (Fig. 2b). Other components (SgII breakdown products and, probably, CgB and its breakdown products) were also recognized. When a heat stable fraction obtained from human pheochromocytoma (Fig. 2c,d) was used as antigen, a component corresponding to SgII was recognized by both the polyclonal antiserum raised in mice and the polyclonal antibodies raised in rabbit. SgII breakdown components in the human pheochromocytoma were also recognized by both antibodies. These products were more abundant in the case of the mouse serum which also recognized putative CgB. At immunohistochemistry (not shown), the polyclonal antisera reacted with several endocrine/paracrine cells scattered in different tissues obtained from seven mammalian species. In PAF-fixed human tissues immunoreactivity was also found in non-

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Fig. I. Reactivity of mouse preimmune ([3) and immune sera ( 0 , O) revealed by ELISA performed as described in Materials and Methods. Bovine anterior pituitary homogenate was used as substrate.

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Fig. 2. Immunoblots of proteins separated by 2D-PAGE from bovine anterior pituitary homogenate (a,b) or from the human pheochromocytoma heat-stable fraction (c,d) and incubated with anti-bovine SglIenriched fraction mouse serum (a,c), or anti-purified bovine SglI rabbit serum (b,d). Arrows point to SglI.

206

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b

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Fig. 3. Immunoblots of proteins separated by 2D-PAGE from bovine anterior pituitary homogenate and incubated with three differenttypes of hybridoma supernatants which recognizedpredominantlyunidentified components and reacted only slightly(a,b) or did not recognize SgII (c). Arrows: SgII.

endocrine cells. In intestinal and gastric epithelia immunostaining was restricted to a paranuclear halo. Hybridomas Hybridomas were obtained from the splenocytes of a hyperimmune mouse. Of the 500 cultures obtained, 102 were ELISA-positive when an absorbance of 0.300 was used as a cut-off value. All the ELISA-positive supernatants were then screened by indirect immunocytochemistry on a panel of PAF-fixed human non-neoplastic tissues (both fundic and antral gastric mucosa, duodenal and colonic mucosa, pancreas and pituitary). The 102 supernatants could be divided into two groups. Group 1 consisted of 15 supernatants which reacted with most of the pituitary cells and some of the antral and insular cells; group 2 consisted of 87 supernatants showing no reactivity with human endocrine cells. Reactivity of non-endocrine epithelia, which was morphologically identical to that of polyclonal antisera, was sometimes expressed by some of the supernatants of group 2. 26 supernatants, chosen from the most representative of the two groups, were characterized by the immunoblotting of proteins separated from bovine anterior pituitary homogenates. The majority of them recognized components different from SgII (Fig. 3). Only two of the analyzed supernatants (8G1 and 5A7) recognized SgII and its breakdown products (Fig. 4a and c). 8G1 and 5A 7 supernatant characterization When characterized by the immunoblotting of heat-stable proteins obtained from human pheochromocytoma, the 8G1 supernatant did not recognize any component (Fig. 4b), whereas the 5A7 supernatant recognized SgII and its degradation products (Fig. 4d). At immunohistochemistry, immunoreactivity for the 8G 1 supernatant was detectable in a consistent number of pituitary cells (ox, rat, guinea-pig, cat and dog) (Fig. 5a), in

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Fig. 4. Immunoblotsof proteins separated by 2D-PAGE from bovine anterior pituitary homogenate (a,c) or from the human pheochromocytomaheat-stable fraction (b,d) and incubated with the hybridoma supernatants 8G1 (a,b) and 5A7 (c,d). Antibodies from 8G1 hybridoma recognized SglI and its degradation products in the bovine anterior pituitary (a) but not in the human pheochromocytoma(b); antibodies from 5A7 hybridomarecognized SglI both in the bovine and in the human tissue (c,d). Arrows: SglI.

a few cells of the antral mucosa of the stomach (ox, rat, rabbit, guinea-pig and cat) (Fig. 5b), in a minority of pancreatic endocrine cells located at the periphery of the islets (possibly glucagon cells, ox, rabbit, cat and dog) (Fig. 5c). No reactivity was detected in the adrenals, thyroid, fundic gastric mucosa, intestines (small and large bowel), on the nervous structures of any of the all animal species under study. Moreover, this supernatant failed to react with human tissues. The 5A7 supernatant stained endocrine cells in all of the animal species investigated. It reacted with a fair number of cells of the pituitary anterior lobe although the intensity of the immunoreaction was variable, the most intense staining being detectable in cells of the lateral wings (Fig. 6a). Some species specificity was found in the distribution patterns of immunoreactivity. Most of the cells of the islets (probably the glucagon and insulin cells) (Fig. 6b) proved to be reactive in ox, cat, dog and human pancreas, while only relatively few (glucagon cells?) were stained in rabbit and guinea-pig. Numerous immunoreactive cells were found in the middle third of the antral mucosa of ox, cat and dog stomach (Fig. 6c,d),

208

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Fig. 5. lmmunocytochemistry pattern obtained with 8G1 antibody': (a) guinea-pig pituitary; (b) cat gastric antral mucosa; (c) dog pancreas islet cells. Nomarski optic, x 450. their distribution patterns overlapping that of gastrin cells. Conversely, few cells were detectable in human and guinea-pig antral mucosa. N o immunoreactive cells were found in fundic gastric mucosa. Ox, rat and human adrenal medulla cells showed an immunoreactivity which was absent in guinea-pig and cat. C-cells of guinea-pig, cat, dog and human thyroid proved to react with the 5A7 supernatant which failed to stain their equivalent in rabbit. A faint stain was found in cat and human parathyroid cells.

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Fig. 6. 5A7 antibody stains a number of cells of the lateral wings of guinea-pig pituitary (a), most cat p a n c r e a s islet cells (b) and the endocrine cells ofox and dog stomach antral m u c o s a (c,d). In o x stomach antral m u c o s a (c), intramucosal nerve terminals also react with 5A7 antibody. Nomarski optic, x 450.

Moreover, the 5A7 supernatant detected a number of endocrine cells in duodenal, ileal and colonic mucosa of all animal species under study. These cells were generally located at the bottom of the mucosal crypts and, in adjacent histological sections, showed no reactivity with anti-serotonin antibodies (Fig. 7a). Few cells were identifed in bronchial mucosa of human foetuses. The 5A7 super-

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Fig. 7. Endocrine cells of the h u m a n duodenal mucosa (a), as well as nervous bodies and terminals of submucosal plexus (b), react with 5A7 antibody. In a, hematoxylin was used as nuclear stain. Nomarski optic, × 450.

natant stained axons and nerves of the intramural plexuses of the gastrointestinal tract (Fig. 7b). AAF fixation proved to be the best treatment for retaining the immunoreactivity of endocrine cells and the worse for retaining the immunoreactivity of neurons. PAF, buffered formaldehyde and Bouin provided good, but not excellent, results with both endocrine and nervous cells. 8G1 and 5A 7 subclones and ascite fluids The two anti-SglI hybridomas were subcloned and, at immunoblotting, their products showed the same reactivity as that of the original hybridomas (compare Fig. 4a, c and Fig. 8a,b).

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Fig. 8. lmmunoblots of proteins separated by 2 D - P A G E from bovine anterior pituitary homogenate and incubated with antibodies from 8G1 subclones (a) or from 5A7 subclones (b). Arrows: SgII.

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Fig. 9. Immunoblots of proteins separated by 2D-PAGE from bovine anterior pituitary homogenate (a) and from the human pheochromocytoma heat-stable fraction (b), and incubated with ascite fluid from the 5A7 subclone. Arrows: SglI.

Finally, ascite fluids obtained from these subclones were as reactive as the original subclones both with anterior pituitary homogenate and h u m a n p h e o c h r o m o c y t o m a (Fig. 9 and data not shown). The subclones of the 8G1 and 5A7 hybridomas were also tested by E L I S A on the solid phase of anterior pituitary homogenates and the SglI-containing fraction prepared from h u m a n pheochromocytoma. As shown in Fig. 10, the 5A7 subclone was reactive with both preparations, whereas the 8G1 subclone only reacted with anterior pituitary homogenate.

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Fig. 10. Reactivity of 801 (I-3, A) and 5A7 (©, 0 ) antibodies with bovine anterior pituitary homogenate (I-q, ©) and human pheochromocytoma heat-stable fraction (A, O) at ELISA. Negative control was the unrelated mouse antibody which gave similar results with both bovine anterior pituitary homogenate (A) and human pheocromocytoma heat-stable fraction (not shown) as substrate.

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Discussion SglI has a well defined distribution in neuroendocrine cells, which does not always overlap that of CgA and CgB [4,5,12,22-24]. The importance of SgII, as a neuroendocrine marker in cell biology and, in particular, as a marker of specific neuroendocrine tumors in pathology, has been established [1-10]. Therefore, the development of monoclonal antibodies against human SgII would provide additional tools for the diagnosis of such tumors. As far as we know, neither polyclonal nor monoclonal antibodies against human SgII have yet been described. However, polyclonal antisera raised against bovine SgII have been reported to cross-react apparently well with their human analogs [25] and so it was decided to raise monoclonal antibodies against bovine SgII. The selection of tissues to be tested with supernatants in immunocytochemistry was done on the basis of previously reported results [22,26]. Some insular, antral and colonic cells were expected to show some reactivity for SgII antibodies that did not stain endocrine cells of fundic mucosa. This working hypothesis has been confirmed by experimental results. In fact, by screening the supernatants with these selected tissues, we were able to reveal two monoclonal antibodies (8G 1 and 5A7) which, on immunocytochemistry, stain several endocrine cells but do not stain endocrine cells of fundic mucosa and, on immunoblotting, recognize SgII (and its degradation products) in bovine anterior pituitary, the tissue in which SgII was first characterized and where it is present in the largest amounts [ 11,27]. Moreover, the 5A7 antibody, cross-reacts with the human antigen on both immunocytochemistry and immunoblotting. Immunocytochemistry revealed that the 5A7 antibody also cross-reacts with human SgII stored in non-neoplastic endocrine cells. Previous reports [6-10] refer to the immunoreactivity of some human endocrine tumours. The distribution patterns of the immunoreactivity obtained with the 5A7 antibody in human and in animal tissues only partially overlap those previously described [22,26,28]. So, the 5A7 but not the 8G1 antibody reacts with some duodenojejunal cells which were not found to be immunoreactive by Rindi et al. [22] or Buffa et al. [26] who used a polyclonal antiserum elicited against bovine SgII. The precise identification of the endocrine cells reacting with 8G1 and 5A7 antibodies on the basis of their hormonal content is in progress. The 8G 1 antibody recognizes an epitope which is more species specific than that of the 5A7 and cannot be used for the detection of SgII in human tissues. Moreover, the reactivity for 5A7 is detectable in several endocrine cells lacking reactivity for 8G1, which also fails to react with nerves. The fact that the reactivity of 8G 1 is not consistent with that of 5A7 in the same animal species (e.g., ox antral mucosa) is possibly due to a different sensitivity of their epitopes to fixatives. In conclusion, antibody 5A7 can be used for the detection of SgII in human tissues and should be of help in clinical and pathological practices. Both antibodies, 5A7 and 8G1, can be useful for cell biology studies, such as those in which SgII is used as a marker of secretory products.

213

Acknowledgements The authors wish to thank G. Goglione and P. Tinelli for printing the photographs. This work was supported by grants from the Italian National Research Council, Target Project on Biotecnology and Bioinstrumentation.

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