ARTICLE |
Correspondence to: Guida Maria PortelaGomes, Dept. of Genetics and Pathology, Unit of Pathology, University Hospital, 75185 Uppsala, Sweden. E-mail: portela_gomes@yahoo.com
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Summary |
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Chromogranin (Cg) B is an acidic glycoprotein present in neuroendocrine tissue. The sequence shows several dibasic amino acid positions susceptible to proteolytic cleavage. The purpose of this study was to elucidate the expression of CgB epitopes in the human endocrine pancreas. Tissue sections of six human pancreata were immunostained with 16 different region-specific antibodies to the CgB molecule, using double immunofluorescence techniques. The CgB epitope pattern varied in the four major islet cell types. B (insulin)-cells expressed immunoreactivity to all region-specific antibodies. The antibodies to the N-terminal and mid-portions of CgB showed moderate immunoreactivity, the C-terminal antibodies weak. A (glucagon)-cells were reactive only to the N-terminal and mid-portion antibodies but, after microwave pretreatment, to all antibodies, whereas D (somatostatin)-cells expressed only the sequence CgB 244255 and a subpopulation CgB 580595. PP (pancreatic polypeptide) cells were immunostained with antibodies between CgB 1417 and a few with CgB 580593. The fragment CgB 244255 was expressed in all four cell types. The cause of these differences may be cell-specific cleavage or masking of the molecule, but varying translation of CgB mRNA is also possible. The extent to which these epitopes reflect fragments having biological functions remains to be evaluated.
(J Histochem Cytochem 50:10231030, 2002)
Key Words: chromogranin B, chromogranin B fragments, immunohistochemistry, pancreas, human
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Introduction |
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CHROMOGRANIN (Cg) B is a glycoprotein of 657 amino acids with a molecular mass of about 100 kD. Like CgA, CgB has been localized in secretory granules of most neuroendocrine (NE) cell types. These proteins share similar structure elements, such as multiple pairs of basic amino acids, which are potential cleavage sites, an internal disulfide bridge, and high interspecies amino acid homology in the N-terminal part. In CgA several cleavage products, i.e., peptides with biological activity, have been identified (for an overview see
Most studies on CgB have been focused on biochemical analysis but only a few studies have centered on its cellular localization and function. Post-translational processing of CgB has been reported in various NE tissues from humans and other species. Biochemical characterization of plasma and tissue extracts has shown that CgB processing varies in brain and in adrenal and pituitary glands (
We have recently studied the IHC expression and processing of CgA in the different cell types in human pancreatic islets by using 12 region-specific antibodies (
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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 surgery for pancreatic adenocarcinoma. The specimens were taken from both macro- and microscopically normal glandular regions at least 3 cm distant from the neoplasm. Four of the specimens were taken from the bodytail region and two from the head (processus uncinatus).
The pancreatic specimens were fixed for 1820 hr at room temperature (RT) in 10% buffered neutral formalin, then processed routinely 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. Hematoxylineosin was used for routine staining.
Immunohistochemistry
The streptavidinbiotin complex (ABC) technique (
Double immunofluorescence techniques were used to identify the CgB epitopes in the various cell types of the endocrine pancreas. The sections were incubated overnight at RT with a cocktail of two antibodies, either one monoclonal and one polyclonal antibody or two polyclonal antibodies raised in different animal species (anti-rabbit, anti-guinea pig, and/or anti-chicken). Then the sections were incubated for 30 min at RT with biotinylated swine anti-rabbit IgG 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 application of the respective primary antibodies, the sections were incubated with non-immune serum 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 that antibody. Between each staining step, the sections were carefully washed with PBS.
The CgB region-specific antibodies used are characterized in Table 1. The other primary antibodies were as follows: mouse monoclonal antibodies against human somatostatin (Novo Nordisk, Bagsvaerd, Denmark; clone Som-018); sheep polyclonal antibodies against human glucagon (The Binding Site, Birmingham, UK; code no. PH519); guinea pig antibodies against human insulin (P. Westermark, Dept. Gen. Pathology, Unit of Pathology, Uppsala, Sweden; code no. Ma 47); and chicken antibodies against human pancreatic polypeptide (A. Larsson, Dept. Med Sci., Clin. Chemistry; Uppsala, Sweden). The working dilutions for immunofluorescence were 1:80, 1:10, 1:80, 1:200, and 1:80, respectively.
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The labeled secondary antisera were as follows: biotinylated swine anti-rabbit IgG (DAKO; Glostrup, Denmark); Texas Red-labeled streptavidin, FITC-conjugated goat anti-sheep (Vector Laboratories; Burlingame, CA); FITC-conjugated goat anti-mouse, anti-guinea pig, and anti-chicken IgG (Sigma Chemical; St Louis, MO).
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, or (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 also tested in relation to the specificity of the species in which the primary antibodies had been raised, by using single staining, the secondary antibody in question being replaced by secondary antibodies from different animal species, e.g., a primary antibody raised in guinea pig was tested with an anti-rabbit-labeled secondary antibody. These control tests were performed with ABC (single staining) and immunofluorescence techniques (co-localization studies). Neutralization tests were performed to exclude crossreactivity among the CgB region-specific antibodies and between these and chromogranin A, insulin, glucagon, somatostatin (amino acid sequence 114), and pancreatic polypeptide. The CgB fragment antigens used were the present synthesized peptides (see below). 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) producing excitation at wavelengths 475555 nm for Texas Red (filter no. 32821, dichroic mirror BH2-DMG), and 453488 nm for FITC (no. 32822, BH2-DMIB), respectively, while a double-band filter set (no. 39538, BH2-DFC5) was also used for simultaneous visualization of Texas Red- and FITC-labeled cells (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 CgB (
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Before immunization, the peptides were coupled to a carrier protein. Peptides (1 mg) were dissolved in 100 µl DMSO, after which 100 µl (1 mg) of ImjectÆ Maleimide Activated Keyhole Limpet Hemocyanin (aKLH; Pierce, Boule Nordic) 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. 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 to produce polyclonal antibodies, as described earlier (
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Results |
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The immunostaining pattern with the different CgB region-specific antibodies in the endocrine pancreas varied among the different cell types (see below). Some minor variations in the staining intensity were noted among the various cases. No immunostaining was elicited in the exocrine tissue. Microwave pretreatment affected the intensity of the staining, whereby faint staining without such pretreatment became moderate after such pretreatment, especially the antibodies to CgB 153167 and 259270, which stained faintly without microwave pretreatment. The distribution pattern of the endocrine cells was not affected by microwave pretreatment, except for glucagon cells (see below).
In the pancreatic islets, the number of immunoreactive cells with the different CgB 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 with the antibodies to epitopes in the C-terminal sequences was weaker than that of those in the N-terminal and mid-portion sequences. Because the staining intensity with the antibodies to CgB 1637 was too weak to allow double staining, these results are not presented. The immunostaining results are summarized in Table 2.
Immunoreactivity to the CgB Region-specific Antibodies in Different Endocrine Cell Types
B (Insulin)-cells.
Virtually all B-cells expressed immunoreactivity to all CgB region-specific antibodies (Fig 2 Fig 3 Fig 4). The intensity of the immunoreactivity was moderate with most N-terminal and mid-portion antibodies but weak with the C-terminal antibodies.
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A (Glucagon)-cells. The frequency of the A-cells displaying immunoreactivity to the different region-specific antibodies varied. Some N-terminal and mid-portion antibodies stained from a few to virtually all (Fig 5 and Fig 6). The intensity of the immunoreactivity was moderate to strong. No immunoreactivity was observed with the region-specific antibodies from CgB 555 to the C-terminus unless microwave pretreated, when moderate to strong staining became evident.
D (Somatostatin)-cells. D-cells were non-immunoreactive to all CgB region-specific antibodies except the 244255 antibody, which stained virtually all D-cells (Fig 7). A subpopulation of D-cells was also stained by the 580595 antibody (Fig 8). The intensity of the immunoreactivity with both antibodies was strong.
PP (Pancreatic Polypeptide)-cells. PP cells were stained with antibodies raised to epitopes located in sequences 1 and 417 (Fig 9). A few PP cells were also immunostained by antibodies raised to 506517 and 568593 (Fig 10). The staining intensity with the different antibodies varied from weak to strong.
Control Stainings
In double immunofluorescence stainings, our control tests showed that omission of one of the primary antibodies gave a staining pattern corresponding to that of the remaining primary antibody. The other staining controls performed were all negative (Fig 11).
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Discussion |
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CgB and CgA are two members of the same family. They are structurally closely related proteins present in most NE cell types. There is often co-expression of these two glycoproteins, although CgA frequently predominates in NE cells. In the present study, all region-specific antibodies to CgB revealed immunoreactive cells in the human pancreatic islets but the distribution patterns varied demonstrably. The antibodies raised against the N-terminal and mid-parts of the CgB molecule stained more cells and with a generally stronger immunoreactivity than did the C-terminal antibodies. B-cells reacted with all our region-specific antibodies, although the immunoreactivity was usually weak. A-cells showed strong immunoreactivity to the antibodies directed towards the N-terminal and mid-portion of the molecule. Without microwave pretreatment, no immunoreactivity to the C-terminal antibodies was seen, but after such treatment intense immunoreactivity was apparent. The presence of CgB immunoreactivity in human D-cells is reported for the first time. However, these cells reacted only with the mid-portion antibody (CgB 244255), although a subpopulation of the cells was also immunostained with the C-terminal antibody (580593). The N-terminal and mid-portion antibodies, as well as some C-terminal antibodies, immunostained the PP cells. Our results show similarities and differences in expression of both CgB and CgA (
The observed differences in staining pattern reflect the number of existing or available CgB epitopes. This indicates that, in some cells, the CgB molecule is cleaved in such a way that some of our antibodies do not bind to the molecule. This would be consistent with biochemical data of variable CgB processing in different NE cells, being more marked in brain than in adrenal glands but less marked in the pituitary glands (
Previous IHC studies using region-specific antibodies to CgB are sparse. GAWK (amino acid sequence CgB 420493) immunoreactivity was detected by electron microscopy in A-cells by
The physiological functions of CgB and its fragments are still largely unknown. CgB has been reported to bind calcium (
In conclusion, this study has shown a variable IHC expression pattern of CgB epitopes available in the endocrine pancreatic cell types, with different CgB epitopes available in the various cell types. This may be the result of different post-translational processing of the CgB molecule or different expression at the mRNA level, indicating that the fragments probably have different roles in the various cell types. Studies of the expression of CgB region-specific antibodies in different NE tumors and diabetic islets may provide some insight into the functional importance of these CgB fragments.
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Acknowledgments |
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Supported by a grant from the Swedish Cancer Foundation and by the Ihres Fund.
We thank Prof Lars Grimelius for fruitful discussions and laboratory facilities and Prof Henry Johansson for comments on the manuscript. We are also grateful for the PP antibodies raised in chicken, kindly provided by Assoc Prof Anders Larsson.
Received for publication December 19, 2001; accepted March 6, 2002.
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Literature Cited |
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Benedum UM, Lamouroux A, Konecki DS, Rosa P, Hille A, Baeuerle PA, Frank R et al. (1987) The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region. EMBO J 6:1203-1211[Abstract]
Benjannet S, Leduc R, Advouche N, Falgueyret JP, Marcinkiewicz M, Seidah NC, Mbikay M et al. (1987) Chromogranin B (secretogranin 1), a putative precursor of two novel pituitary peptides through processing at paired basic residues. FEBS Lett 224:142-148[Medline]
Bishop AE, Sekiya K, Salahuddin MJ, Carlei F, Rindi G, Fahey M, Steel JH, Hedges M, Domoto T, FischerColbrie R et al. (1989) The distribution of GAWKlike immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B. Histochemistry 90:475-483[Medline]
Buffa R, Gini A, Pelagi M, Siccardi AG, Bisiani C, Zanini A, Solcia E (1989) Immunoreactivity of hormonally-characterized human endocrine cells against three novel anti-human chromogranin B (B11 and B13) and chromogranin A (A11) monoclonal antibodies. Arch Histol Cytol 52(suppl):99-105[Medline]
Chen M, Tempst P, Yankner BA (1992) Secretogranin I/chromogranin B is a heparin-binding adhesive protein. J Neurochem 58:1691-1698[Medline]
Flanagan T, Taylor L, Poulter L, Viveros OH, Diliberto EJ, Jr (1990) A novel 1745-dalton pyroglutamyl peptide derived from chromogranin B is in the bovine adrenomedullary chromaffin vesicle. Cell Mol Neurobiol 10:507-523[Medline]
Gill BM, Barbosa JA, Dinh TQ, Garrod S, O'Connor D (1991) Chromogranin B: isolation from chromocytoma, N-terminal sequence, tissue distribution and secretory granule processing. Regul Pept 33:223-235[Medline]
Gorr SU, Shioi J, Cohn DV (1989) Interaction of calcium with porcine adrenal chromogranin A (secretory protein I) and chromogranin B (secretogranin I). Am J Physiol 257:E247-257
Hsu SM, Raine T, Fanger H (1981) Use of avidin-biotin-peroxidase complex (ABC) in immmunoperoxidase techniques: a comparison between ABC and unlabeled (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Iguchi H, Natori S, Kato K-I, Nawata H, Chrétien M (1988) Different processing of chromogranin B into GAWK-immunoreactive fragments in the bovine adrenal medulla and pituitary gland. Life Sci 43:1945-1952[Medline]
Karlsson E, Stridsberg M, Sandler S (2000) Chromogranin-B regulation of IAPP and insulin secretion. Regul Pept 87:33-39[Medline]
Kimura N, Pilichowska M, Okamoto H, Kimura I, Aunis D (2000) Immunohistochemical expression of chromogranins A and B, prohormone convertases 2 and 3, and amidating enzyme in carcinoid tumors and pancreatic endocrine tumors. Mod Pathol 13:140-146[Medline]
Kroesen S, Marksteiner J, Leitner B, HogueAngeletti R, FisherColbrie R, Winkler H (1996) Rat brain: distribution of immunoreactivity of PE-11, a peptide derived from chromogranin B. Eur J Neurosci 8:2679-2689[Medline]
Laslop A, Doblinger A, Weiss U (2000) Proteolytic processing of chromogranins. Adv Exp Med Biol 482:155-166[Medline]
Laslop A, Weiss C, Savaria D, Eiter C, Tooze SA, Seidah SA, Winkler H (1998) Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases. J Neurochem 70:374-383[Medline]
Marksteiner J, Bauer R, Kaufmann WA, Weiss E, Barnas U, Maier H (1999) PE-11, a peptide derived from chromogranin B, in the human brain. Neuroscience 91:1155-1170[Medline]
MetzBoutigue MH, Goumon Y, Strub JM, Aunis D (1998) Antibacterial peptides are present in chromaffin cell secretory granules. Cell Mol Neurobiol 18:249-266[Medline]
Mouland AJ, Bevan S, White JH, Hendy GN (1994) Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression. J Biol Chem 269:6918-6926
Natori S, Huttner WB (1996) Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor. Proc Natl Acad Sci USA 93:4431-4436
Nielsen E, Welinder BS, Madsen OD (1991) Chromogranin B, a putative precursor of eight novel rat glucagonoma peptides through processing at mono-, di- or tribasic residues. Endocrinology 129:3147-3156[Abstract]
PortelaGomes GM, Stridsberg M (2001) Selective processing of chromogranin A in the different islet cells in human pancreas. J Histochem Cytochem 49:483-490
Schmid KW, Weiler R, Xu RW, HogueAngeletti R, FischerColbrie R, Winkler H (1989) An immunological study on chromogranin A and B in human endocrine and nervous tissue. Histochem J 21:365-373[Medline]
Seidah NG, Day R, Marcinkiewicz M, Chrétien M (1998) Precursor convertases: an evolutionary ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and proteins. Ann NY Acad Sci 839:9-24
Stridsberg M, Öberg K, Li Q, Engström U, Lundqvist G (1995) Measurements of chromogranin A, chromogranin B (secretogranin I), chromogranin C (secretogranin II) and pancreastatin in plasma and urine from patients with carcinoid tumours and endocrine pancreatic tumours. J Endocrinol 144:49-59[Abstract]
Strub JM, GarciaSablone P, Lonning K, Taupenot L, Hubert P, Van Dorsselaer A, Aunis D et al. (1995) Processing of chromogranin B in bovine adrenal medulla. Identification of secretolytin, the endogenous C-terminal fragment of residues 614-626 with anti-bacterial activity. Eur J Biochem 229:356-368[Abstract]
Winkler H, Laslop A, Leitner B, Weiss C (1998) The secretory cocktail of adrenergic large dense-core vesicles: the functional role of the chromogranins. Adv Pharmacol 42:257-259[Medline]
Wu HJ, Rozansky DJ, Parmer RJ, Gill BM, O'Connor DT (1991) Structure and function of the chromogranin A gene. Clues to evolution and tissue-specific expression. J Biol Chem 266:13130-13134