ARTICLE |
Correspondence to: Lars Grimelius, Pathology Dept., University Hospital, S-75185 Uppsala, Sweden.
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Summary |
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Co-localization of chromogranin (Cg) A, B, and C has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum, and colon) using single-, double-, and triple-immunofluorescence stainings. Virtually all enterochromaffin (EC) cells contained CgA, and those in the luminal two thirds of the antral mucosa and villi of small intestine often also contained CgB. A few EC cells in the duodenal crypts contained CgC. Most gastrin cells harbored both CgB and CgA, although rather more CgB than CgA, but some gastrin cells contained all three types, i.e., also CgC. Some CCK cells also contained all three chromogranins. Enteroglucagon cells in the duodenal villi contained CgA and some CgB. CgA (but not B or C) was found in some secretin, GIP, enteroglucagon/peptide YY, and neurotensin cells. A few somatostatin cells contained CgA but neither CgB nor CgC. CgA and C were found mainly in the basal cell region, whereas CgB occurred more diffusely throughout the cytoplasm. This varying distribution suggests that not all secretory granules contain CgA, or that CgB may occur in a nongranular form. The varying composition of the different chromogranins may reflect their complex functional roles in the widespread neuroendocrine system. (J Histochem Cytochem 45:815-822, 1997)
Key Words: chromogranin A, chromogranin B, chromogranin C, peptides, serotonin, human, gastrointestinal tract
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Introduction |
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The CHROMOGRANIN FAMILY contains several members of glycoproteins: chromogranin (Cg) A, B, and C (secretogranin II), and proteins derived from these. CgA and CgB were first isolated from chromaffin cells in the adrenal medulla and CgC from the anterior pituitary gland. Most of the soluble proteins in the secretory granules of the adrenal chromaffin cells consist of CgA and B, but only to a minor extent of CgC. Chromogranins, especially CgA, also occur in the secretory granules of most neuroendocrine cell types. The main source of circulating CgA appears to be the adrenal medulla, although other neuroendocrine cell systems may also contribute (
The purpose of the present study was, by applying double- or triple-immunofluorescence staining of the same section, to ascertain the extent to which the three main chromogranins (A, B, and C) can occur in different endocrine cell types and to determine their intracellular distribution in relationship to the various neuroendocrine hormones in different parts of the human gastrointestinal tract.
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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 operations for adenocarcinoma. The specimens examined were taken at least 3 to 5 cm from the neoplasm in macroscopically normal mucosa. All were histologically normal.
Fixation of the specimens was either in neutral picric acid-formaldehyde (
The sections were stained with hematoxylin-eosin or were immunostained by different methods to demonstrate various secretory granule products. The sensitive streptavidin-biotin complex (ABC) technique (
In the co-localization studies, immunofluorescence methods were used in single, double, or triple staining. In triple-immunofluorescence staining, the procedure was as follows. The sections were incubated with a cocktail of antibodies [one monoclonal + two polyclonal (anti-rabbit and anti-guinea pig)], overnight incubation at RT &Aelig; biotinylated goat anti-guinea-pig IgG, 30 min at RT &Aelig; a mixture of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG + Texas Red (TXRD)-conjugated goat anti-mouse IgG + aminomethyl coumarin acetic acid (AMCA)-conjugated streptavidin, 30 min at RT. Double immunostaining was performed in the same way except for exclusion of one of the primary antibodies and corresponding secondary antibodies. Before applying the respective primary antibodies, the sections were incubated with nonimmune sera from the animal species producing the secondary antibodies, at a dilution of 1~10.
If two primary polyclonal antisera raised in the same species (rabbit) had to be used, the staining technique was modified as follows: first, primary anti-rabbit antiserum was applied overnight at RT &Aelig; monovalent FITC-conjugated goat Fab anti-rabbit IgG (0.2 mg/ml), overnight at RT &Aelig; second primary anti-rabbit antiserum, overnight at RT &Aelig; biotinylated swine anti-rabbit IgG, 30 min at RT &Aelig; TXRD-labeled streptavidin, 30 min at RT (adapted from
The Fab concentration of 0.2 mg/ml overnight was found to saturate the epitopes of the first-step anti-rabbit antibody.
The primary antibodies are characterized in Table 1.
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Polyclonal antibodies to CgA were raised against a fragment covering amino acids 116-439 of human CgA (
Gastrin antibodies raised against the C-terminal portion were used in antrum and N-terminal gastrin antibodies in the duodenum. The latter antibodies did not crossreact with CCK. The CCK antibodies used did not crossreact with gastrin.
The labeled secondary antisera were biotinylated swine anti-rabbit IgG (Dakopatts; Glostrup, Denmark), biotinylated goat anti-guinea pig IgG, TXRD- and AMCA-labeled streptavidin (Vector Laboratories; Burlingame, CA), TXRD-conjugated goat anti-mouse IgG (Southern Biotechnology Associates; Birmingham, AL), FITC-conjugated goat anti-rabbit IgG, both the monovalent Fab fragment and the whole molecule (Sigma Chemical; St Louis, MO).
The control stainings included (a) omission of the primary antisera, (b) replacement of the first layer of antibody by nonimmune serum diluted 1:10 and by the diluent alone, (c) preincubation (24 hr) of primary antiserum with the relevant antigen (0.5, 2.5, and 7 mmol per ml diluted antibody solution, respectively) before application to the sections. These control tests were performed with both immunofluorescence and ABC techniques.
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 TXRD (filter no. 32821, dichroic mirror BH2-DMG), 453-488 nm for FITC (no. 32822, BH2-DMIB), and 340-375 nm for AMCA (no. 32817, BH2-DMU), and a double-band filter set (no. 39538, BH2-DFC5) for simultaneous visualization of TXRD- and FITC-labeled cells was also used (excitation at 550-570 nm and 480-495 nm, respectively). Photographs were taken with Fujicolor 400 film; the triple stainings were photographed by double-exposing the film first through the double band and then with the AMCA filter sets.
Co-localization of two or three of the fluorochromes was revealed by the resulting additive colors: the co-localization of green and red is perceived as yellow, blue (AMCA) and red as magenta, blue and green as cyan, and all three fluorochromes as white.
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Results |
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Distinctly immunoreactive (IR) cells were revealed by all tested antisera in the relevant regions of the gastrointestinal tract when ABC and immunofluorescence methods were used with both fixatives. In double- and triple-immunofluorescence stainings, omission of one or two of the primary antibodies gave a staining pattern corresponding to the remaining primary antibody or antibodies. The other staining controls were all negative.
Distribution of CgA, B, and C in the Gastrointestinal Tract
CgA-IR cells were observed in all parts of the gastrointestinal tract and were more numerous than CgB- and CgC-IR cells. They were seen at all levels of the mucosa, although mainly in the middle third portion. The staining intensity of the CgA-IR cells was strong and even in all regions except for the antrum, where it varied from strong to very weak; the weak IR cells predominated in the antrum.
CgB-IR cells were most numerous in the antrum, where they occurred at almost the same frequency as CgA-IR cells. In duodenum they were sparse, in the ileum they were sporadic, and in the corpus and colon they were virtually nonexistent. In the antral mucosa, CgB-IR cells were observed mainly in the midportion, in the regions of the small intestine in the various levels of the villi, but only occasionally in the crypts, where they were seen in the upper half. A few CgB-IR cells were also observed in Brunner's glands.
CgC-IR cells were few in number, being present mainly in the antrum, where they were seen in patches in the middle third of the mucosa. A few cells were present in the duodenum (mainly in the crypts, occasionally in the villi) but were completely absent in Brunner's glands and the other gastrointestinal regions.
Co-localization of CgA, B, and C in Endocrine Cells
The results are summarized in Table 2.
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Corpus. Serotonin (enterochromaffin, EC) cells but not somatostatin cells displayed CgA immunoreactivity. Most of the CgA-IR cells were probably enterochromaffin-like (ECL) cells, a type predominating in this region but which could not be identified immunocytochemically because available histamine antibodies are unsuitable for use with formalin-fixed tissue.
Antrum. All EC cells were CgA-immunoreactive, and a subpopulation thereof also contained either CgB or CgC. Those cells harboring CgB and the few with CgC were located in the middle and upper parts of the mucosa. Only occasionally did the antral EC cells display immunoreactivity for all three chromogranins.
Most gastrin cells were immunoreactive for CgB and a fraction of them also for CgA, but a few contained all three chromogranins. CgA immunoreactivity in gastrin cells varied in intensity, being weak in most cells and sometimes virtually absent.
Immunoreactivity for CgB was stronger than for CgA or C (Figure 1A-D and Figure 2). A few gastrin cells were also serotonin-reactive and somatostatin cells were occasionally CgA immunoreactive (Figure 3).
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DuodenumMucosa.
The EC cells located in the crypts were CgA-IR, but only some were located in the villi. CgB immunoreactivity was observed in a subpopulation of EC cells in the villi, but not in EC cells in the crypts (Figure 4A and Figure 4B); occasional EC cells in the crypts contained CgC. Gastrin and CCK cells reacted to CgB and CgA antibodies, and some also to CgC. Some secretin and GIP cells and virtually all enteroglucagon cells were also immunoreactive to CgA, but somatostatin cells only occasionally so. A few enteroglucagon cells in this region were also CgB-immunoreactive.
Brunner's Glands. The identified EC, gastrin, and CCK cells (but not somatostatin or enteroglucagon cells) were CgA-IR. Gastrin and CCK cells also contained CgB.
Ileum. The EC cells were CgA-positive and some of those located in the villi also contained CgB. The somatostatin cells proved negative for all three chromogranins. Some enteroglucagon/PYY and neurotensin cells were CgA-IR, whereas CgB and C staining was absent.
Colon. The EC cells and a subpopulation of EG/PYY cells were found immunoreactive to CgA, but not to B or C. The somatostatin cells reacted negatively to all the Cg antibodies. The few CgB cells visualized could not be correlated to any of the endocrine cell types identified.
Intracellular Localization of Chromogranins and Corresponding Hormones
CgA and C were concentrated mostly in the basal (infranuclear) region of the cells, whereas CgB was more diffusely spread in the cytoplasm, as was serotonin immunoreactivity, involving the cell processes occasionally seen in some of these cells. CgA immunoreactivity in antral EC cells in the lower third of the mucosa was located mainly adjacent to the basal cell membrane. In gastrin cells, CgA was found mainly in the infranuclear region, although occasionally occupying the entire cytoplasm. CgA immunoreactivity was mainly basal in CCK, secretin, GIP, enteroglucagon, enteroglucagon/PYY, and neurotensin cells, whereas the entire cytoplasm was stained in the (few) CgA-IR somatostatin cells.
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Discussion |
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For co-localization studies, the present multiple immunostaining technique using fluorochromes proved superior to single immunostaining in consecutive sections, because the substances could be determined more exactly in the individual cells. A similar techniquebut with chromogens for light microscopy
is excellent if the substances occur in different cells, but difficulties may arise in determining the resulting color if they are located in the same cells. Immunoelectron microscopy can also be used in co-localization studies by using various sizes of gold particles, although the more complicated staining steps are a disadvantage compared with the present fluorescence methods. Furthermore, only a small number of cells can be studied at one time. With single-band filter sets, the different substances can be located individually. With the present technique, using a double-band filter, co-localization of two of these substances can be studied simultaneously, and with a further single-band filter and double exposure of the film it was possible to identify a third substance at the same time and even to determine its location in relationship to the other two. A triple-band filter for the three fluorochromes has also been tested, but it did not help us assess the stainings.
With double or triple immunostaining, crossreactivity of the antibodies must be avoided. In our investigation we used both monoclonal and polyclonal antibodies raised in different animal species. Only exceptionally did we use antibodies raised in the same animal species, and in these cases we blocked the epitopes of the first antibody by using monovalent Fab fragments of IgG. Appropriate control stainings, necessary to evaluate the staining specificity, were carried out. Furthermore, staining specificity was evaluated for single immunostaining, not only with fluorescence but also with the ABC technique.
The distribution of chromogranin immunoreactivity along the gastrointestinal tract is fairly similar to that demonstrated in previous reports (
As mentioned earlier, the ECL cells were indirectly identified, because the commercially available histamine antibodies tested were ineffective in our tissue. These antibodies need a special fixative containing carbodiimide. ECL cells predominate in the corpus and their frequency and distribution are as for the CgA cells, i.e., at least the majority of ECL cells must be CgA-positive, which would be consistent with other reports (
EC cells displayed CgA immunoreactivity in all gastrointestinal regions examined, as in other reports (Varndell et al. 1989;
The antral gastrin cells were immunoreactive for both CgA and B, and some also for CgC. The latter glycoprotein has not been reported earlier in this cell type in humans, but was found in guinea pig (
With very few exceptions, somatostatin cells were the only cell type in which we were unable to find CgA. The reason for this is unclear, but one possible explanation may be that CgA is present at such a low concentration that it cannot be detected with the immunostaining used, although
Regarding the secretin cells and CgA, our results differ somewhat from those reported by
The co-localization of enteroglucagon and CgA varied in the different intestinal regions. In the duodenal mucosa, enteroglucagon cells reacted to CgA, which agrees with the results reported by
It has been demonstrated ultrastructurally that CgA and B are present in secretory granules in the gastrointestinal tract (
Our knowledge of the physiological functions of chromogranins is limited. Most studies have concentrated on CgA and its proteolytic fragments, which may have several biological roles, among others an intracellular hormone-binding function, as precursors to various smaller fragments that may be biologically active, and/or an inhibitory effect on the secretion of different hormones belonging to the neuroendocrine system, e.g., parathyroid hormone, parathyroid hormone-related protein, insulin, calcitonin, and catecholamines (cf.
The function of CgB and CgC is still virtually unknown. In addition to having a role as precursors of smaller peptides (
Immunoelectron microscopic studies have shown that serotonin (
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Acknowledgments |
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Supported by a grant from the Swedish Medical Research Council (project no. 102), by the Lion's Foundation, Uppsala, and by Hässle Läkemedel AB, Mölndal, Sweden.
We thank Ms Birgitta Vagnhammar for excellent technical assistance.
Parts of this work were presented at the 11th International Symposium on Regulatory Peptides, Copenhagen, Denmark, 3-7 September, 1996.
Received for publication October 25, 1996; accepted February 13, 1997.
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