Journal of Histochemistry and Cytochemistry, Vol. 49, 483-490, April 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Selective Processing of Chromogranin A in the Different Islet Cells in Human Pancreas

Guida Maria Portela–Gomesa,c and Mats Stridsbergb
a Departments of Genetics and Pathology, Unit of Pathology, University of Lisbon, Portugal
b Medical Sciences, Clinical Chemistry, University of Lisbon, Portugal
c Uppsala University Hospital, Sweden, and the Centres of Gastroenterology and of Nutrition, University of Lisbon, Portugal

Correspondence to: Mats Stridsberg, Assoc. Prof., Dept. of Medical Sciences, Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. E-mail: mats.stridsberg@klinkem.uas.lul.se


  Summary
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Materials and Methods
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We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the four major neuroendocrine cell types of the human pancreas by using double immunofluorescence techniques. The antibodies raised to the N-terminal and midportions of CgA showed, on the whole, stronger immunoreactivity than did the C-terminal antibodies, with a few exceptions. Often the immunoreactivity was stronger in glucagon cells. Insulin cells expressed immunoreactivity to all region-specific antibodies, but glucagon cells were nonreactive to two antibodies. Somatostatin cells reacted only with the C-terminal antibodies (amino acid sequences CgA 411–424), while PP cells were stained with four CgA region-specific antibodies between amino acid sequences 63–195. The cause of these differences may be that the CgA molecule is cleaved, partly masked, or partly translated from CgA mRNA. Microwave treatment improved only the staining with the CgA 361-372 antibodies, which indicates that masking is not the sole or entire cause. Our findings may indicate that the CgA molecule is cleaved in different ways in the various pancreatic endocrine cell types, giving rise to a variety of biologically functional fragments. (J Histochem Cytochem 49:483–490, 2001)

Key Words: chromogranin A, chromogranin A fragments, immunocytochemistry, pancreas, human


  Introduction
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Introduction
Materials and Methods
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CHROMOGRANIN A (CgA) is an acidic glycoprotein consisting of 439 amino acids and having a molecular mass of 48 kD. Commercial antibodies to CgA give rise to immunocytochemical staining in most neuroendocrine (NE) cell types and have therefore been used as a reliable NE immunocytochemical marker. Electron microscopic studies have shown that immunoreactivity appears in the secretory granules (Varndell et al. 1985 ). The primary amino acid sequence of human CgA contains 10 pairs of basic amino acids which are potential cleavage sites for specific endogenous proteases, but other sites in the molecule can also be split. Several CgA-related peptides have been identified in biological tissue from humans and other species. These are, following their localization from the N-terminal to the C-terminal region of the molecule, vasostatins (amino acid sequences 1-17/113; Helle et al. 1990 ; Drees et al. 1991 ), chromostatin (124–143; Galindo et al. 1991 ), chromacins (173–194; Strub et al. 1997 ), pancreastatin (250–301; Tatemoto et al. 1986 ), WE-14 (316–329; Curry et al. 1992 ), catestatin (344–364; Mahata et al. 1997 ), parastatin (347–419; Fasciotto et al. 1993 ), and GE-25 (367–391; Kirchmair et al. 1995 ). Posttranslational processing of CgA has been reported in bovine and rat endocrine tissues and in endocrine cells of human adrenal glands and pancreas (Hutton et al. 1987 ; Wohlfarter et al. 1988 ; Curry et al. 1991 ; Watkinson et al. 1991 ). Chromatographic separation of tissue extracts has shown that CgA processing varies in different NE tissues, more marked in pancreatic islets than in adrenal medulla (cf. Iacangelo and Eiden 1995 ). However, our knowledge of the extent to which the CgA molecule and its fragments are expressed in the different islet cell types is limited. In the present work we have studied CgA immunoreactivity in the four major NE cells of the human pancreas, using antibodies raised to 12 regions of the human CgA molecule, which may contribute to increasing our understanding about the functional role of the CgA-related peptides in this endocrine pancreatic organ.


  Materials and Methods
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Materials and Methods
Results
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Tissue specimens from six adult human pancreata were obtained from surgical samples removed at operation for pancreatic adenocarcinoma. The specimens examined were taken from macroscopically and microscopically normal glandular regions at least 3 cm from the neoplasm. Four specimens were taken from the body–tail region and two from the head (processus uncinatus).

The pancreatic specimens were fixed in 10% buffered neutral formalin for 18–20 hr at room temperature (RT) and routinely processed to paraffin. Sections 5 µm thick were cut and attached to poly-L-lysine-coated or to positively charged (Superfrost+; Menzel, Braunschweig, Germany) glass slides. Some sections were stained with hematoxylin–eosin as a routine staining.

Immunocytochemistry
Other sections were immunostained to demonstrate the various CgA fragments. The streptavidin–biotin complex (ABC) technique (Hsu et al. 1981 ), with diaminobenzidine as chromogen, was applied as a single immunostain mainly to reveal the distribution pattern of endocrine cells in the pancreas and to perform the control stainings specified below. Some sections were pretreated in a microwave oven (Philips Whirlpool; Stockholm, Sweden) twice for 5 min at 750 W, using a citrate buffer, pH 6.0, as retrieval solution.

Co-localization studies were performed with the present CgA region-specific antibodies and either a commercial monoclonal antibody to CgA (MAb CgA) or antibodies to various secretory granule hormones. In these co-localization studies, immunofluorescence methods were used in single or double staining. For double immunofluorescence staining, the sections were incubated with a cocktail of two antibodies, either one monoclonal and one polyclonal antibody or two polyclonal antibodies (anti-rabbit, anti-guinea pig, and/or anti-chicken), overnight at RT. Then the sections were incubated with biotinylated swine anti-rabbit IgG for 30 min at RT and then transferred to a mixture of streptavidin–Texas Red and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse, anti-guinea pig, or anti-chicken IgG. Before applying the respective primary antibodies, the sections were incubated with non-immune sera from the animal species producing the secondary antibodies, at a dilution of 1:10. The secondary antibody in question was pre-incubated overnight at 4C with 10 µl/ml normal serum, both from the animal species recognized by the other secondary antibody and from the species producing the other secondary antibody. Between each staining step, the sections were carefully washed with PBS.

The primary antibodies used are characterized in Table 1. The labeled secondary antisera were biotinylated swine anti-rabbit IgG (DAKO; Glostrup, Denmark), Texas Red-labeled streptavidin (Vector Laboratories; Burlingame, CA), FITC-conjugated goat anti-mouse, anti-guinea pig, and anti-chicken IgG (Sigma Chemical; St Louis, MO).


 
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Table 1. Antibodies used

The control stainings entailed (a) omission of the primary antisera, (b) replacement of the first layer of antibody by non-immune serum diluted 1:10 and by the diluent alone, (c) preincubation (24 hr) of primary antiserum with the relevant antigen (10 nmol per ml diluted antibody solution, respectively) before application to the sections. The secondary antibodies were tested in relation to the specificity of the species in which the primary antibodies had been raised, the secondary antibody in question being replaced by secondary antibodies from different animal species. These control tests were performed with ABC (single staining) and immunofluorescence techniques (co-localization studies). Neutralization tests were performed to exclude crossreactivity among the CgA region-specific antibodies, as well as between these and chromogranin B, insulin, glucagon, somatostatin (amino acid sequence 1–14), and pancreatic polypeptide. Particular attention was paid to specificity tests on the commercial MAb CgA to reveal the amino acid sequence to which it was raised. This monoclonal antibody was pre-incubated with antigens corresponding to the amino acid sequences CgA 116–439 (Stridsberg et al. 1993 ), CgA 250–301 (Peninsula Laboratories; Belmont, CA), and the synthesized peptide covering CgA 284–301, at concentrations of 10 and 20 nmol per ml diluted antibody solution. The other CgA fragment antigens used were the present synthesized peptides. The other antigens used were obtained from Sigma.

For co-localization studies, the sections were examined in a Vanox AHBS3 fluorescence microscope (Olympus; Tokyo, Japan) equipped with filters (Olympus) giving excitation at a wavelength of 475–555 nm for Texas Red (filter no. 32821, dichroic mirror BH2-DMG), 453–488 nm for FITC (no. 32822, BH2-DMIB), respectively, and a double-band filter set (no. 39538, BH2-DFC5) for simultaneous visualization of Texas Red- and FITC-labeled cells was also used (excitation at 550–570 nm and 480–495 nm, respectively). Photographs were taken with Fujicolor 400 film.

Antibody Production
Deduced from the amino acid sequences of human CgA (Benedum et al. 1987 ), 12 polypeptides were synthesized by a solid-phase system using Fmoc chemistry (Applied Biosystems model 430A; Foster City, CA). The peptides were purified by reverse-phase chromatography and analyzed by plasma desorption mass spectrometry (PDMS, Bioion 20; Bioion Nordic AB, Uppsala, Sweden). The sequences were selected to be specific for human CgA and the homology was less than 48% to any other known protein sequence in the data-band MPsrch Protein, version 1.5 (Shane S. Surrock and John F. Collins 1993; Biocomputing Research Unit, University of Edinburgh, Scotland), except for the respective sequences of CgA from other species. The selected sequences for the respective peptides are shown in Fig 1 and Table 2. To facilitate the coupling to the carrier protein (see below), a cysteine residue was added to either the C-terminal or the N-terminal of the synthesized peptides. To facilitate labeling of the peptides with iodine, a tyrosine residue was added in the position between the cysteine residue and the peptide if the CgA–peptide sequence lacked a tyrosine residue. The peptide corresponding to amino acid sequence CgA17–38, which includes cysteine residues at both the N-terminal and the C-terminal, was treated with acid to induce cyclization of the peptide. This peptide was also modified by replacing the glutamine residue at position 36 with a tyrosine residue. The peptide corresponding to amino acid sequence CgA1–17 was modified by substituting the lysine residue at position 16 with a tyrosine residue.



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Figure 1. Schematic representation of the human chromogranin A molecule, with the amino acid sequences of the twelve peptides used for raising antibodies placed in the appropriate position (A 1–17 to A 411–424). The disulfide bridge (S) is shown on the N-terminal. The 10 potential cleavage sites (first amino acid of the dibasic pair) are indicated by vertical lines (the figures indicate positions 77–437).


 
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Table 2. Co-localization of chromogranin A-related peptides in human pancreatic endocrine cells using region-specific antibodiesa

Before immunization, the peptides were coupled to a carrier protein. Peptides (1 mg) were dissolved in 100 µl DMSO and thereafter 100 µl (1 mg) of Imject maleimide activated keyhole limpet hemocyanin (aKLH, Pierce; Boule Nordic AB, Huddinge, Sweden) was added. This mixture was allowed to react for 2 hr at RT. The coupled peptides were then purified on a PD-10 column (Pharmacia Biotech; Uppsala, Sweden) with PBS as the moving phase. The cyclizated CgA17–38 peptide was coupled to KLH, using glutaraldehyde as previously described (Stridsberg et al. 1995 ). Aliquots of 200 µg (n=5) coupled peptide were frozen and stored at -20C until immunization. The peptide complexes were injected into New Zealand White rabbits, using the intradermal injection technique to produce polyclonal antibodies (Stridsberg et al. 1995 ).


  Results
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Materials and Methods
Results
Discussion
Literature Cited

The antibodies to the different CgA fragments gave rise to various staining patterns in the endocrine pancreas. Microwave pretreatment did not influence the staining results of the different CgA region-specific antibodies, except for the CgA361–372 antibody. The latter stained weakly without microwave pretreatment but moderate to strong staining was apparent after microwave pretreatment.

Distribution of Immunoreactivity to CgA Region-specific Antibodies
In the pancreatic islets, the number of immunoreactive cells with the different CgA region-specific antibodies varied from a few to virtually all cells. The staining intensity also varied among the different antibodies. On the whole, the staining intensity decreased with the antibodies raised to epitopes from the N- to the C-terminal sequences of the CgA molecule. The results are summarized in Table 2.

Immunoreactivity to CgA Region-specific Antibodies in Different Endocrine Cell Types
Insulin Cells. Virtually all insulin cells were immunostained with the antibodies corresponding to sequences between amino acids 1–337 (Fig 2), with the exception of CgA116–130. From CgA361 to the C-terminus, only a minority of insulin cells were immunoreactive. The intensity of the immunoreactivity was weak, with the exception of two N-terminal antibodies (sequences CgA1–15 and 17–38).



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Figure 2. Human pancreatic islet immunostained for (A) insulin (FITC) and (B) chromogranin (Cg)A17–38 (Texas Red). This CgA fragment is present in insulin cells and also in other islet cell types. Bar = 80 µm.

Figure 3. Human pancreatic islet double immunostained for glucagon (FITC) and CgA17–38 (Texas red). The microphotograph taken using a double filter demonstrates that the glucagon cells display immunoreactivity to this CgA fragment (yellow). The other islet cells also contain this CgA fragment (red). Bar = 24 µm.

Figure 4. Human pancreatic islet double immunostained for glucagon (FITC) and CgA411–424 (Texas red). Co-localization of this CgA fragment was seen in only some glucagon cells (yellow). Bar = 24 µm.

Figure 5. Human pancreatic islet double immunostained for glucagon (FITC) and CgA324–337 (Texas Red). This CgA fragment was co-localized in almost all glucagon cells (yellow). Only one cell displays green fluorescence, indicating the absence of the CgA fragment. Bar = 24 µm.

Figure 6. Human pancreatic islet from the PP-rich processus uncinatus, double immunostained for PP (FITC) and CgA116–130 (Texas Red). The CgA fragment is demonstrated only in PP cells (yellow). Its immunoreactivity is located mainly in the cellular area facing the exocrine parenchyma. PP cells located in a subperipheral zone were nonreactive. Bar = 80 µm.

Figure 7. Human pancreatic islet double immunostained for (A) somatostatin (FITC) and (B) CgA411–424 (Texas Red). (C) The microphograph exposed with a double-band filter shows that somatostatin and CgA411–424 appear in the same cells (yellow), with one exception (green). Bar = 53 µm.

Figure 8. Consecutive sections from normal human endocrine pancreas stained with the streptavidin–biotin complex method with diaminobenzidine as chromogen and counterstained with Mayer's hematoxylin using (A) antibodies to CgA176–195 and (B) the commercial monoclonal CgA antibody. (A) All cells are stained with the CgA176–195 antibodies, but those with a localization corresponding to glucagon cells show stronger immunoreactivity. (B) With the monoclonal CgA antibody, only a fraction of the islet cells display immunoreactivity, apparently corresponding to the intensely stained cell in A. Bars = 44 µm.

Glucagon Cells. The frequency of the glucagon cells displaying immunoreactivity to the different region-specific antibodies agrees, with some exceptions, with that of insulin cells (Fig 3 Fig 4 Fig 5). No immunoreactivity was observed with three of the region-specific antibodies, i.e., CgA116–130, 238–247, and 361–372.

The intensity of the immunoreactivity was generally strong, not only to the N-terminal antibodies but also to antibodies to the middle portion of the CgA molecule, but weak to the C-terminal antibodies.

Somatostatin Cells. Somatostatin cells were non-immunoreactive to all CgA region-specific antibodies, with the exception of CgA411–422 antibodies, which stained virtually all somatostatin cells (Fig 6).

Pancreatic Polypeptide (PP) cells. PP cells were stained with four of the region-specific antibodies, whose epitopes are located between the amino acid sequences CgA63–76 and 176–195. CgA116–130 stained only PP cells (Fig 7).

Identification of the Epitope for MAb CgA
The commercially available MAb CgA gave no staining reaction when preincubated with the antigen with amino acid sequence CgA116–437, a faint staining reaction with CgA250–301, but moderate to strong staining with CgA284–301. These neutralization tests point to a localization of the epitopes to the N-terminal region of pancreastatin, between the sequence CgA250–283.

MAb CgA-immunoreactive Cells. Glucagon cells were strongly immunoreactive to MAb CgA. Some insulin cells showed weak immunostaining, while somatostatin and PP cells were virtually nonreactive to MAb CgA.

MAb CgA antibodies stained fewer cells than did most of the region-specific antibodies (Fig 8A and Fig 8B).

Control Stainings
In double immunofluorescence stainings, our control tests showed that omission of one of the primary antibodies gave a staining pattern corresponding to the remaining primary antibody. The other staining controls performed were all negative.


  Discussion
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The present study is, thus far, the most comprehensive investigation using human CgA region-specific antibodies in human endocrine pancreas.

All the region-specific antibodies demonstrated immunoreactive cells in the human pancreas, although frequencies and distribution patterns differed. On the whole, the antibodies raised against the N-terminal and mid-parts of CgA stained more pancreatic endocrine cells than did the C-terminal antibodies, and the intensity of the immunoreactivity was also stronger. The immunoreactivity to the different region-specific antibodies was often stronger in glucagon than cells in the other endocrine cell types.

These differences in staining pattern reflect the number of available epitopes of the CgA molecule. This may indicate that the molecule is cleaved on the epitope(s) in question, giving rise to fragments that do not react with the antibodies used. This is consistent with the biochemical finding of a predominant processing of CgA towards the C-terminal end (Metz-Boutigue et al. 1993 ; Eskeland et al. 1996 ).

An additional explanation is that the antibody binding epitopes may be masked, perhaps by the hormone in question, by other granule proteins, or by the histological processing, thus preventing the immunocytochemical staining. However, although it is possible that microwave treatment may not unmask all protein–protein interactions (Frost et al. 2000 ), this latter hypothesis is contradicted by the fact that this pretreatment did not significantly affect the staining results except for the CgA361–372 antibodies for which this explanation may be valid. The negative or weak immunoreactivity found with some of the region-specific antibodies may also indicate that the CgA fragments are present at such a low concentration that they cannot be detected with the immunostaining technique used.

Within one and the same cell type, there are variations in the numbers of immunoreactive cells, as well as in the intensity of immunoreactivity, which reflect the availability of the epitopes. This, in turn, may indicate differing degrees of cleavage of the CgA molecule.

Immunocytochemical studies in human endocrine tissue with region-specific antibodies to CgA are sparse and are mainly related to the adrenal chromaffin cells. The presence of ß-granin (rat sequence CgA1–113, corresponding to vasostatin in human tissue) and WE-14 (porcine sequence CgA316–329, corresponding to human sequence CgA352–372) immunoreactivities has been reported in the human GI tract and pancreas (Hutton et al. 1988 ; Gleeson et al. 1996 ). Other immunocytochemical studies in the human pancreas demonstrated the presence of pancreastatin (porcine sequence; Schmidt et al. 1988 ) and chromostatin (bovine sequence; Cetin et al. 1993 ). These immunocytochemical studies show only that these CgA epitopes exist in the pancreas, but they have not shown whether they were as isolated small fragments or as epitopes of either larger fragments or of the whole CgA molecule. Cetin et al. 1993 reported pancreastatin immunoreactivity in glucagon cells and chromostatin immunoreactivity in insulin cells, whereas these immunoreactivites were not found in somatostatin cells. Regarding pancreastatin, different immunostaining patterns were found with the C- and N-terminal antibodies; our antibodies directed to the mid- and C-terminal pancreastatin (sequence CgA 284–301) showed a similar staining pattern when compared with that described by Cetin et al. 1993 , with stronger immunoreactivity in glucagon cells than in insulin cells, while the antibody to the N-terminal pancreastatin (sequence CgA238–247) showed a similar immunostaining in both these cell types. Regarding chromostatin (sequence CgA116–130), our results differed from those of Cetin et al. 1993 because we found only weak immunostaining in a few cells. This discrepancy is probably related to species differences of the peptides used for antibody production. We have used antibodies to human CgA, whereas Schmidt et al. 1988 used antibodies to bovine sequences and Cetin et al. 1993 against porcine sequences.

The presence of CgA411–424 in human somatostatin cells is reported here for the first time, while these cells were mostly unreactive with antibodies to all the other CgA fragments tested. Somatostatin cells thus show a selective processing of CgA but contain only this part of the CgA molecule. PP cells also contain a limited number of CgA epitopes, located between amino acids 63–195. Of interest is the visualization of immunoreactivity to CgA116–130 antibodies in PP cells only. The explanation for the expression of this limited number of epitopes may be that the CgA molecule is cleaved, or that only these parts of the CgA molecule are translated from the CgA mRNA. The first alternative implies an extensive cleavage of the CgA molecule, which weakens this hypothesis.

The commercial MAb CgA (clone LK2H10) is often used to visualize neuroendocrine cells in routine histopathology. To our knowledge it has not been reported earlier to which part of the CgA molecule the antibody binds. Our results show that the binding epitopes are located within the pancreastatin region (CgA250–301), as the immunostaining was almost completely inhibited after pre-incubation with this peptide but was not affected by pre-incubation with the CgA284–301 peptide. Therefore, the MAb CgA probably binds somewhere in the CgA sequence 250–284, i.e., in the N-terminal part of pancreastatin.

Different fragments of CgA have been reported to have specific biological functions. Thus, both autocrine and paracrine inhibition of hormone secretion have been associated with pancreastatin on insulin release (Tatemoto et al. 1986 ), with parastatin and vasostatins on parathyroid hormone secretion (Drees et al. 1991 ; Fasciotto et al. 1993 ), with vasostatin on calcitonin and endothelin release (Deftos et al. 1990 ; Aardal et al. 1993 ), and with chromostatin and catestatin on catecholamine release (Galindo et al. 1991 ; Mahata et al. 1997 ). In addition, vasodilatory functions have been associated with the vasostatins (Helle et al. 1990 ) and antibacterial effects have been shown for chromacin (Strub et al. 1996 ). Therefore, different CgA fragments may exercise an autocrine or paracrine regulatory function on the secretion of different hormones belonging to the neuroendocrine system. Recently, antibacterial and antifungal functions have also been shown for vasostatin (Lugardon et al. 2000 ). No biological function has yet been attributed to the WE-14 and GE-25 fragments, although its involvement in the release of histamine has been proposed for WE-14 (Forsythe et al. 1997 ).

In conclusion, this study has clearly shown that different CgA epitopes are available in various pancreatic endocrine cell types. PP cells, and especially somatostatin cells, express few available. These findings may mean that the CgA molecule is cleaved in different ways in different cell types, giving rise to biologically functional fragments.


  Acknowledgments

Supported by a grant from the Swedish Medical Research Council (project no. 102), and by the Swedish Cancer Foundation and Ihres Fund.

We thank Professor Lars Grimelius for fruitful discussions and laboratory facilities, and Professor Henry Johansson for comments on the manuscript. We are also grateful for the PP antibodies raised in chicken kindly provided by Associate Professor Anders Larsson.

Received for publication June 22, 2000; accepted December 12, 2000.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
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