ARTICLE |
Correspondence to: Ann M. Dvorak, Dept. of Pathology, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215.
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Summary |
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We performed ultrastructural immunogold localization of osteopontin in the mucosa of human stomach. This adhesive glycoprotein was present in mucous and chief cells of the epithelial layer and in macrophages in the lamina propria. Parietal and endocrine cells of the epithelial layer and mast cells and plasma cells in the lamina propria did not contain osteopontin, serving as internal negative controls. Subcellular localizations of osteopontin included secretory granules and synthetic organelles in mucous and chief cells and phagolysosomes in macrophages. Extracellular concentrations of osteopontin were present in the glycocalyx and in an electron-lucent band between epithelial surface cells and the gastric lumen. Paracellular edema between the epithelium of the same cells was devoid of osteopontin. Immunogold localization of pepsinogen II was done to identify cells with mixed granule populations and contents of multicompartmental secretory granules. These studies revealed mucous cell granules and chief cell granules, each containing compartmentalized storage products, which included osteopontin and mucigen in mucous cells and osteopontin and pepsinogen II in chief cells. Cytochemical controls for the immunogold localizations were negative. The subcellular distribution of osteopontin in human gastric mucosa suggests possible roles for this glycoprotein in barrier function, host defense, and/or secretion. (J Histochem Cytochem 45:21-33, 1997)
Key Words: Osteopontin, Stomach, Mucigen, Pepsinogen II, Immunoelectron microscopy
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Introduction |
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Osteopontin (OPN) is an adhesive glycoprotein with the peptide sequence glycine-arginine-glycine-aspartate-serine (GRGDS) that promotes cell attachment (
To gain a better understanding of the biology of OPN in epithelia, we have embarked on a systematic examination of subcellular localizations of OPN in the gastrointestinal tract, using postembedding immunogold preparations of human tissues (
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Materials and Methods |
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Case Material
Material for the ultrastructural immunogold analysis of gastric mucosal epithelium was obtained either from surgical biopsy specimens or from gastric specimens removed at the time of surgery from four patients. The electron microscopic samples were obtained from either chronically inflamed or histologically normal areas of gastric resection margins or biopsies, remote from areas of primary disease. The diagnoses for each patient follow: reflux esophagitis, adenocarcinoma arising in Barrett's esophagus, adenocarcinoma of the stomach, and malignant gastric stromal tumor. Small blocks of tissue were excised and fixed by immersion for 2 hr at room temperature (RT) in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde buffered at pH 7.4 with 0.1 M sodium cacodylate containing 0.025% calcium chloride, and were further processed for electron microscopy as described (
Tissue Processing
After primary fixation, tissues were washed in 0.1 M sodium cacodylate buffer (twice for 5 min) and stored in the same buffer at 4C for 1 or 2 days. Tissues were then postfixed in 1.33% OsO4 in 0.2 M Sym-collidine buffer, pH 7.4, for 2 hr at RT, washed with 0.05 M sodium maleate buffer, pH 5.2 (three times for 2 min), and stained with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6, for 2 hr at RT. Finally, tissues were washed again with 0.05 M sodium maleate buffer, pH 5.2 (three times for 2 min), and dehydrated through graded ethanols. The samples were infiltrated in a propylene oxide-Epon 812 sequence and embedded and polymerized in Epon 812 at 60C for 16 hr. Gold to silver thin sections were cut on an LKB IV ultratome (LKB; Bromma, Sweden). The sections were picked up on uncoated gold grids (Pelco; Tustin, CA) and air-dried for 30 min.
Immunogold Labeling for Osteopontin
Grids were stained by submerging them (section side up) in 30-µl drops of reagents on Teflon-surfaced glass slides (Roboz Surgical Instruments; Washington, DC) in the following sequence at RT: (a) 0.1% Triton X-100 in 0.1 M Tris-buf-fered saline (TBS), pH 7.6, 10 min; (b) TBS containing 0.1% bovine serum albumin (TBS-BSA), three washes, 10 min each; (c) TBS-BSA containing 5% normal goat serum, 30 min; (d) primary antibody (affinity-purified rabbit anti-human OPN antibody, as described previously (Brown et al. 1992), 1:20 dilution in TBS-BSA containing 1% normal goat serum and 0.1% Tween-20, 60 min; (e) TBS-BSA, three washes, 10 min each; (f) secondary antibody (10-nm gold-labeled goat anti-rabbit IgG) (Energy Beam Sciences; Agawam, MA), 1:40 dilution in the same diluent as for primary antibody, 60 min; (g) TBS-BSA, two washes, 5 min each; and (h) two washes with double-distilled water, 5 min each. The grids were air-dried overnight.
Three specificity controls were employed, substituting for primary antibody as follows: (a) primary antibody to OPN that had been absorbed with OPN purified from human milk, as described (
Immunogold Labeling for Pepsinogen II
Grids were stained in a similar fashion in the following sequence at RT: (a) freshly made saturated sodium metaperiodate (Fisher Scientific; Medford, MA) in double-distilled water, 30 min; (b) double-distilled water, two washes, 5 min each; (c) TBS, two washes, 10 min each; (d) TBS-BSA containing 5% normal donkey serum, 30 min; (e) primary antibody (sheep anti-human pepsinogen II IgG) (Biodesign International; Kennebunk, ME), 1:20 dilution in TBS-BSA containing 1% normal donkey serum and 0.1% Tween 20, 60 min; (f) TBS-BSA, three washes, 10 min each; (g) secondary antibody (20-nm gold-labeled donkey anti-sheep IgG) (Goldmark Biologicals; Phillipsburg, NJ), 1:20 dilution in the same diluent as for primary antibody, 60 min; (h) TBS-BSA, two washes, 5 min each; and (i) two washes with double-distilled water, 5 min each. The grids were air-dried overnight.
Three specificity controls were employed, substituting for primary antibody as follows: (a) normal sheep serum or irrelevant sheep IgG at equivalent concentration; (b) omission of the primary antibody; and (c) absorption of primary antibody with solid-phase or soluble pepsinogen II (both from Sigma; St Louis, MO).
Before observation the grids were stained with lead citrate for 4 min and viewed in a CM-10 electron microscope (North American Philips; Mahwah, NJ).
Quantitation of gold particles that labeled mucigen granules and appropriate specificity controls were done on randomly obtained photographs of labeled cells. The number of gold particles/µm2 of granule area was analyzed for significance with Kruskal-Wallis nonparametric ANOVA, Dunn's Multiple Comparison, and Mann-Whitney tests.
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Results |
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The mucosa of chronically inflamed or microscopically normal human stomach biopsies, or surgical specimens, was examined to define cellular, extracellular, and subcellular sites of OPN using a postembedding immunogold procedure. Cell lineages that were examined in detail included mucous epithelial cells, chief cells, parietal cells, and endocrine cells in the epithelial layer, and macrophages, plasma cells, and mast cells in the subepithelial area of the mucosa. Of these cells, we found OPN in mucous epithelial cells, chief cells, and macrophages. Other cells examined did not contain OPN.
Mucous Cells
Subcellular and Extracellular Sites of Osteopontin.
Mucigen granules, Golgi structures, small apical vesicles, the glycocalyx of microvilli, and an electron-lucent extracellular layer between the apical cell surface and luminal contents of the stomach contained gold particles, indicating the presence of OPN (Figure 1 Figure 2 Figure 3 Figure 4). Background label of cytosol, nucleus, and mitochondria was minimal in OPN-positive mucous cells. The Golgi structures that contained OPN were located on the mucigen granule-forming side oriented towards the apical membrane of mucous cells (Figure 4). Both immature small and mature large mucigen granules contained OPN. OPN was also present in granules undergoing secretion at the apical cell surface. Small apical cytoplasmic vesicles contained OPN (Figure 2 and Figure 3A). Gold particles indicating OPN were often located near microvillous surfaces, in the glycocalyceal area (Figure 3A, inset). In some areas adjacent to the luminal surface of gastric epithelium, a broad electron-lucent band with little to no detectable substructure bound large amounts of gold, indicating an OPN-rich layer overlying the gastric epithelium (Figure 3A).
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Mucigen granules in mucous epithelial cells were readily identified by a reticular distribution of electron-dense strands in an electron-lucent background (Figure 1). In general, mucous cells were filled with reticular granules, and these granules contained OPN (Figure 1). In addition, mucous granules with central, rounded, electron-dense structures within the reticular meshwork were present. In these granules, OPN was confined to reticular portions of the granule substance, and the electron-dense areas were devoid of OPN (Figure 1, Figure 2, Figure 3A, 5A, 6A, and 7A). The electron-dense areas within mucigen granules of mucous cells did, however, label for pepsinogen II (Figure 7B), and most of these mucous cells were in the necks of gastric glands. Mucigen granules devoid of these electron-dense structures did not contain pepsinogen II (Figure 6B), and most of these mucous cells were surface-related. Some mucous cells contained mucigen granules displaying reticular and dense nucleoid domains, as well as peripheral elongated electron-lucent domains that were rich in OPN (Figure 1 and Figure 3B). Rarely, large zymogen granules with entirely electron-dense contents were present within mucous neck cells. These granules were not labeled for OPN. The densities of gold label indicating the presence of OPN in mucigen granules (and appropriate specificity controls) are listed in Table 1. These counts show that mucigen granules have a significantly greater label of reticular areas (285.5/µm2) compared to electron-dense, rounded areas (32.6/µm2, p<0.001) or to specificity controls (0/µm2, p<0.001). There was no significant difference in OPN label of electron-dense granule areas (32.6/µm2) compared to controls (0/µm2, p = NS).
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Chief Cells
Subcellular Sites of Osteopontin.
Chief cells, located primarily in the depths of gastric glands, contained distinctive large, membrane-bound, electron-dense secretory zymogen granules (Figure 6C and Figure 6D). These homogeneously electron-dense granules contained pepsinogen II, as detected with postembedding immunogold stains (Figure 6D). Some zymogen granules in chief cells characteristically were bizonal, with approximately one half of the granule electron-dense and the remaining half electron-lucent. The proportions between these two compartments in zymogen granules were variable among chief cells, but granules that were completely electron-lucent or completely filled with a reticular mucigen meshwork were not present in chief cells. The electron-lucent compartment of zymogen granules in chief cells contained OPN, but the electron-dense, pepsinogen II-containing compartment was devoid of OPN (Figure 7C and Figure 8). Therefore, chief cells contained pepsinogen II-positive granules (Figure 6D) and granules with mixtures of pepsinogen II and OPN in readily identified different subcompartments of their bizonal granules (Figure 8). Golgi structures in chief cells contained some label for OPN (Figure 7C). Background label in cytosol, nucleus, and mitochondria was minimal.
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Macrophages
Macrophages, present beneath the epithelial layer of the gastric mucosa in chronically inflamed samples, often contained large numbers of phagolysosomes with heterogeneous contents. These structures contained OPN, as indicated by gold label over electron-lucent components of the heterogeneous contents (Figure 9). Electron-dense components did not label with gold. Other subcellular structures in these phagocytes also did not label with the immunogold stain to detect OPN. Background labeling of cytosol, nucleus, and mitochondria was minimal.
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Specificity Controls for Immunogold Stains
Substitution of irrelevant primary antibodies (Figure 5B) or omission of primary antibodies (Figure 5D) revealed no gold-labeled structures in gastric mucosal epithelial cells or macrophages. Absorption of the OPN-specific primary antibody with OPN (Figure 5C) or of the pepsinogen II-specific primary antibody with pepsinogen II (data not shown) resulted in marked diminution of specific gold-labeled structures.
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Discussion |
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We report here the first subcellular localization of OPN, an adhesive glycoprotein (
On the basis of the presence of the GRGDS sequence [the peptide sequence of OPN that promotes cell adhesion (
Classical synthesis, granulogenesis, and regulated secretion (reviewed in
The immunolocalization of OPN in human gastric mucosa also provides support for the existence of mixed-function epithelial cells, and compartmentalization of individual secretory granules, in this epithelial cell layer. The epithelium of gastric mucosa contains four epithelial cell categories: mucous cells, parietal cells, chief cells, and endocrine cells (
In summary, an ultrastructural immunogold study of the human gastric mucosa shows that OPN is a prominent component of this epithelium. We found it in the glycocalyx, in an overlying electron-lucent layer, and in apical cytoplasmic vesicles of epithelial cells as well as in Golgi structures, immature, mature, and secreting mucigen granules of mucous cells, in multicompartmental granules in mucous cells and chief cells, and in phagolysosomes of macrophages. These locations suggest possible roles for this glycoprotein in barrier function, secretion, and host defense in the human gastric epithelium.
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Acknowledgments |
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We thank Peter K. Gardner for assistance in the preparation of the manuscript and Jodi A. Wood for assistance with the photographic prints.
Supported by USPHS grants AI-33372 and DK-34854.
Received for publication June 19, 1996; accepted September 10, 1996.
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