ARTICLE |
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
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Summary |
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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 411424), while PP cells were stained with four CgA region-specific antibodies between amino acid sequences 63195. 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 49:483490, 2001)
Key Words: chromogranin A, chromogranin A fragments, immunocytochemistry, pancreas, human
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Introduction |
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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 (
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Materials and Methods |
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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 bodytail region and two from the head (processus uncinatus).
The pancreatic specimens were fixed in 10% buffered neutral formalin for 1820 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 hematoxylineosin as a routine staining.
Immunocytochemistry
Other sections were immunostained to demonstrate the various CgA fragments. The streptavidinbiotin complex (ABC) technique (
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 streptavidinTexas 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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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 114), 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 116439 (
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 475555 nm for Texas Red (filter no. 32821, dichroic mirror BH2-DMG), 453488 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 550570 nm and 480495 nm, respectively). Photographs were taken with Fujicolor 400 film.
Antibody Production
Deduced from the amino acid sequences of human CgA (
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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 CgA1738 peptide was coupled to KLH, using glutaraldehyde as previously described (
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Results |
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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 CgA361372 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 1337 (Fig 2), with the exception of CgA116130. 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 CgA115 and 1738).
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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., CgA116130, 238247, and 361372.
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 CgA411422 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 CgA6376 and 176195. CgA116130 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 CgA116437, a faint staining reaction with CgA250301, but moderate to strong staining with CgA284301. These neutralization tests point to a localization of the epitopes to the N-terminal region of pancreastatin, between the sequence CgA250283.
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.
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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 (
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 proteinprotein interactions (
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 CgA1113, corresponding to vasostatin in human tissue) and WE-14 (porcine sequence CgA316329, corresponding to human sequence CgA352372) immunoreactivities has been reported in the human GI tract and pancreas (
The presence of CgA411424 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 63195. Of interest is the visualization of immunoreactivity to CgA116130 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 (CgA250301), as the immunostaining was almost completely inhibited after pre-incubation with this peptide but was not affected by pre-incubation with the CgA284301 peptide. Therefore, the MAb CgA probably binds somewhere in the CgA sequence 250284, 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 (
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.
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Acknowledgments |
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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.
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