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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We studied the immunoreactivity of 12 different region-specific antibodies to the chromogranin A (CgA) molecule in the various neuroendocrine cell types of the human gastrointestinal (GI) tract by using double immunofluorescence techniques. These staining results were compared with others obtained with a commercial monoclonal CgA antibody (LK2H10). G (gastrin)-cells showed immunoreactivity to virtually all region-specific antibodies, but with varying frequency. Most intestinal EC (enterochromaffin)- and L (enteroglucagon)-cells were immunoreactive to the antibodies to the N-terminal and mid-portion of the CgA molecule, whereas the EC-cells in the stomach reacted with fewer region-specific antibodies. D (somatostatin)-cells reacted to the CgA 411424 antibody and only occasionally showed immunoreactivity to the other CgA antibodies. A larger cytoplasmic area was stained with the antibodies to CgA 1738 and 176195 than with the other antibodies tested. These differences in staining pattern may reflect different cleavage of the CgA molecule in different cell types and at different regions of the GI tract.
(J Histochem Cytochem 50:14871492, 2002)
Key Words: chromogranin A, chromogranin A fragments, immunocytochemistry, gastrointestinal tract, human, neuroendocrine cells
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Introduction |
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HUMAN CHROMOGRANIN A (CgA) is an acidic glycoprotein with 439 amino acids. In a previous work, CgA immunoreactivity was studied in the different neuroendocrine (NE) cell types of the human gastrointestinal (GI) tract by using a commercial monoclonal CgA antibody (MAb CgA) (
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Materials and Methods |
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Tissue specimens from adult human gastric corpus and antrum, proximal duodenum, distal ileum, and sigmoid colon were obtained from surgical samples removed at surgeries for adenocarcinoma. The specimens examined were taken from macroscopically normal mucosa at least 35 cm from the neoplasm; all showed normal microscopic structure.
The tissue specimens were fixed in 10% buffered neutral formalin for 1820 hr at RT, followed by routine processing to paraffin. Sections 5 µm thick were cut and attached to poly-L-lysine-coated or to positively charged (Superfrost+; Menzel Glässer, Braunschweig, Germany) glass slides. Some sections were pretreated in a microwave oven (Philips Whirlpool; Nordic AB, Stockholm, Sweden) twice for 5 min at 750 W, using a citrate buffer, pH 6.0, as retrieval solution.
The sections were stained with hematoxylineosin or immunostained to demonstrate the presence of various parts of the CgA molecule. The streptavidinbiotin complex (ABC) technique (
Co-localization studies were performed with the present CgA region-specific antibodies and either antibodies to various secretory granule hormones or a commercial MAb CgA. In these co-localization studies, immunofluorescence methods were used. For double immunofluorescence staining, the sections were incubated with a cocktail of two antibodies, either one MAb and one polyclonal antibody or two polyclonal antibodies (anti-rabbit and/or anti-guinea pig), overnight at RT, followed by incubations in biotinylated swine anti-rabbit IgG, 30 min at RT, and a mixture of streptavidinTexas Red and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse or anti-guinea pig 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 CgA region-specific antibodies used are characterized in Table 1. The selected sequences for the respective peptides are shown in Fig 1. For details of the production and characterization of the CgA region-specific antibodies see
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The labeled secondary antisera were biotinylated swine anti-rabbit IgG (DAKO; Glostrup, Denmark), Texas Red-labeled streptavidin (Vector Laboratories; Burlingame, CA), and FITC-conjugated goat anti-mouse and anti-guinea pig IgG (Sigma Chemical; St Louis, MO).
The control stainings included (a) omission of the primary antiserum, (b) replacement of the first layer of antibody by non-immune serum 1:10 and by the diluent alone, and (c) pre-incubation (24 hr) of primary antiserum with the relevant antigen (10 nmol per ml diluted antibody solution) 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).
For co-localization studies, the sections were examined in a Vanox AHBS3 fluorescence microscope (Olympus; Tokyo, Japan) equipped with filters (Olympus) giving excitation at wavelengths of 475555 nm for Texas Red (filter no. 32821, dichroic mirror BH2-DMG), and 453488 nm for FITC (no. 32822, BH2-DMIB), 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.
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Results |
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The antibodies to the different CgA antigens gave rise to various staining patterns in the GI tract. The intensity of the immunoreactivity was strong with antibodies to CgA 1738, 176195, and 238247, and weak to moderate with the others. Furthermore, a larger cytoplasmic area was stained with the antibodies corresponding to the sequences 1738 and 238247 in the antrum and to 176195 in most cells at all GI levels, compared with the other region-specific antibodies, which immunostained mainly the infranuclear region. This difference was most apparent with the antibody to 176195 in the serotonin (enterochromaffin, EC) cells of the antrum and duodenum. The staining intensity with the region-specific antibodies was enhanced by microwave pretreatment but the frequency was not affected except for the CgA 361372 antibody. The latter visualized few or no cells without this pretreatment but a moderate number afterwards.
Immunoreactivity of the Different CgA Region-specific Antibodies in the Different Endocrine Cell Types (Table 2)
G-cells.
The frequency of the antral G-cells displaying immunoreactivity to the different region-specific antibodies varied. The two N-terminal and the two C-terminal antibodies, as well as two mid-portion antibodies (CgA 176195 and 238247) stained a majority of G-cells (Fig 2), whereas the remaining antibodies stained either few or no cells.
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EC-cells. The serotonin-producing EC-cells also showed a varying immunoreactive pattern with the region-specific antibodies. In the corpus, virtually all EC-cells displayed immunoreactivity to CgA 100113 and 176195. In the antrum, only six antigens were detected, i.e., CgA 117, 1738, 100113, 176195, 284301, and 324337 (Fig 3). The CgA 1738 antibody was detected in virtually all EC-cells, the others only in a few to a moderate number of cells. In the duodenum, ileum, and colon, vitually all EC-cells were immunoreactive to the N-terminal and mid-portion antibodies. However, no immunoreactive cells were observed at any GI region with the region-specific antibodies to CgA 361 to the C-terminus, with one exceptiona few cells in the corpus showed immunostaining with the antibody to 375384.
D-cells. Virtually all D-cells in the corpus, a moderate number in the duodenum, but very few at the remaining GI levels, were stained with the CgA 411424 antibody. Few D-cells in the duodenum displayed immunoreactivity with four antibodies, i.e., CgA 1738, 100113, 176195, and 324337, whereas this cell type was virtually nonreactive to all the other region-specific antibodies in the GI tract (Fig 4 Fig 5 Fig 6 Fig 7).
L-cells. L-cells in all regions of the intestinal tract were immunoreactive to all region-specific antibodies except two, whose epitopes are localized between the sequence 361384. The C-terminal antibody stained fewer cells compared with the other antibodies.
Immunoreactivity to MAb CgA
MAb CgA immunostained G-, EC-, and L-cells but only exceptionally D-cells (Fig 8).
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Discussion |
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The human GI tract contains several NE cell types, most of which display CgA immunoreactivity with the commercial MAb CgA (LK2H10; PortelaGomes et al. 1977). The present study is the first investigation of the human GI tract regarding the expression of various regions of the CgA molecule related to the different NE cell types.
The immunoreactive patterns varied markedly in the different NE cell types. A similar variation with our region-specific antibodies had also been found in the human endocrine pancreas (
The G-cell type displayed immunoreactivity to all but one of the CgA region-specific antibodies (116130), but the frequency of the immunostained cells with the various region-specific antibodies varied from virtually all to very few. It is unknown whether these variations may be related to different molecular forms of gastrin or to different functional states of the cell. However, these variations may reflect a different expression of PCs. PC2 is responsible for the generation of gastrin-17, but the cleavage specificities of PC1/3 and PC5, also found in G-cells, are not known (
D-cells expressing CgA 411424 occurred at all regions of the human GI tract, but the immunoreactive cell fraction varied; this cell type was mostly unreactive with the other antibodies. These results differed from those reported by
EC-cells displayed a different staining pattern in the stomach and intestine, with the stomach expressing fewer CgA antigens than the intestine. Most EC-cells in the corpus were immunostained by only two antigens (100113 and 176195) and in the antrum by one (1738). In the intestine, most EC-cells were stained with the N-terminal and mid-portion antibodies, whereas the C-terminal antibodies did not give rise to any immunoreactivity.
The intestinal L-cells showed a pattern similar to the EC-cells at the same level of the intestinal tract. However, the expression of CgA epitopes in the L-cells did not exactly match the CgA expression in pancreatic glucagon (A)-cells (
ECL (enterochromaffin-like)-cells are a type predominating in the corpus, not specifically identified in the present study. However, the immunoreactive cells that were visualized by the region-specific antibodies and nonreactive to the serotonin antibodies were most likely ECL-cells. On the whole, the frequency of CgA-immunoreactive cells not showing serotonin immunoreactivity parallels that of EC-cells.
The variations, both in number of immunoreactive cells and in intensity of immunoreactivity, reflect the available epitopes and raise the possibility that the pattern of CgA epitopes might be the result of cell function. The negative or weak immunoreaction may indicate that the CgA antigens were too few to be detected or that the epitopes were masked either by other granule-related proteins or by the histological processing. A certain masking effect was apparent, because microwave pretreatment increased the staining intensity but did not influence the quantitative results except for CgA 361372. Staining with the latter antibody appeared positive only after this pretreatment. A further explanation for the various staining patterns could be that the CgA molecule is cleaved by different processing enzymes, particularly prohormone convertases (PC2, 1/3), but also by carboxypeptidases and amidating enzymes (
In conclusion, this study shows that different cell types at various regions of the GI tract demonstrate to various extents epitopes of the 12 CgA region-specific antibodies, which may indicate processing of the CgA molecule, giving rise to several peptides of possible functional significance.
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Acknowledgments |
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Supported by a grant from the Swedish Cancer Foundation and the Ihres Fund.
We thank Professor Lars Grimelius, Uppsala, for fruitful discussions and laboratory facilities.
Received for publication March 27, 2002; accepted June 7, 2002.
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