ARTICLE |
Correspondence to: Anita H. Straus, Dept. of Biochemistry, Universidade Federal de Sao Paulo/EPM, CP 20372, Sao Paulo, SP 04023-900, Brazil.
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Summary |
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We performed immunogold labeling with an ST-1 monoclonal antibody (IgM), specific for intact heparin, to define the subcellular localization of heparin in mast cells. Rat peritoneal mast cells were fixed by a modified Karnovsky method and embedded in Araldite. Ultrathin sections were first treated with sodium periodate and then sequentially incubated with MAb ST-1, rabbit anti-mouse IgM, and protein A-gold. By transmission electron microscopy, gold particles were localized inside cytoplasmic granules of peritoneal mast cells. In contrast, with the same procedure, no labeling was observed in mast cells from rat intestinal mucosa. Control sections of rat peritoneal or intestinal mucosa mast cells treated with an irrelevant MAb (IgM) did not show any labeling. Treatment with nitrous acid abolished the reactivity of MAb ST-1 with peritoneal mast cells. These results show that different mast cells can be identified regarding their heparin content by immunochemical procedures using MAb ST-1. (J Histochem Cytochem 45:231-235, 1997)
Key Words: Electron microscopy, Mast cells, Heparin, Granules, Immunolabeling, Monoclonal antibody
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Introduction |
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Mast cells are defined by their content of cytoplasmic granules that stain metachromatically with basic dyes, indicating the presence of sulfated glycosaminoglycans (GAGs). The concept of mast cell heterogeneity represents a focal point in recent discussions of mast cell biology. Rat and mice mast cells are classified as the "atypical" or "mucosal" mast cells (MMCs) observed in the intestinal lamina propria and the "connective tissue-type" mast cells (CTMCs) of skin, peritoneal cavity, and other sites in rat (
Immunocytochemistry is an accurate method for subcellular localization and characterization of different molecules, and this approach has been used successfully to define the presence of several glycoconjugates (
In this study, using the monoclonal antibody (MAb) ST-1 specific to nonmodified heparin molecules (
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Materials and Methods |
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Animals.
Four male Wistar rats of approximately 300 g were obtained from Central Animal Care of Universidade Federal de São Paulo/EPM.
Mast Cells.
Rat peritoneal cells (n = 4) were harvested by washing the peritoneal cavity with 15 ml of 0.9% NaCl. The peritoneal exudate containing the mast cells was centrifuged at 800 x g for 10 min at 4°C.
The small intestine was removed (n = 4), flushed of fecal material with 0.9% NaCl, and cut into small fragments for ultrastructural studies.
Fixation, Processing, and Embedding for Electron Microscopy.
Cell suspension and small intestine fragments were fixed by immersion in Karnovsky's fixative (2% formaldehyde freshly prepared from paraformaldehyde, 2% glu-taraldehyde, 0.025% CaCl2, 0.1 M sodium cacodylate buffer, pH 7.4) for 1 hr at 25°C, washed in sodium cacodylate buffer twice for 15 min each, dehydrated through a graded series of ethanol, and embedded in Araldite 502 Resin (EMS; Fort Washington, PA). Approximately 90-nm sections were cut on diamond knives on an ultramicrotome (Reichert Ultracut; Leica, Austria) and placed on nickel grids (EMS) for immunogold labeling.
Postembedding Immunogold Labeling.
For murine heparin detection, immunogold staining method using Protein A conjugated to gold (
Nitrous Acid Treatment.
Nickel grids containing mast cell sections were incubated with 0.24 M NaNO2 in 1.8 M acetic acid for 80 min at room temperature as described by
Morphometric Analysis.
Randomly photographed sections of rat mast cells (enlarged to x 34,000) were used. The area and number of gold particles over 146-188 granules in 5-10 representative cells (2-3 cells from each sample) were determined for each labeling protocol. Granule areas were determined with a point-counting method using a square test grid with 87-mm spacing. The number of gold particles was calculated and expressed per µm2 of granule area. Statistical analysis was done with a modified t-test (
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Results |
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Heparin Immunocytochemistry
MAb ST-1, specific for heparin molecules, was used in an immunogold label to define the subcellular localization of heparin in rat peritoneal mast cells. By electron microscopy, ST-1 labeling was restricted to granules of rat peritoneal cells (Figure 1A). A higher magnification of peritoneal mast cell granules immunolabeled with ST-1 is shown in Figure 1B. No gold particles were detected over the nuclei or other organelles, and only low labeling at background level was observed over the cytoplasm. No labeling was observed in cells other than mast cells in the peritoneal exudate.
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In intestinal mucosal mast cells no significant labeling was observed with MAb ST-1 (Figure 1C), indicating the absence of heparin in these cells. In control experiments in which MAb ST-1 was replaced by an irrelevant antibody (IgM), no significant labeling was observed in peritoneal mast cell sections (Figure 1D).
The number of gold particles after ST-1 labeling on granules was determined by morphometric analysis of randomly photographed sections of rat peritoneal and intestinal MMCs. Peritoneal mast cells labeled with MAb ST-1 exhibited an average of 51 gold particles/µm2, whereas intestinal MMCs exhibited 5.1 gold particle/µm2, as shown in Table 1.
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Effect of Heparin Deamination on ST-1 Binding to Peritoneal Mast Cell Granules
As shown by
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Discussion |
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This is the first report of an immunocytochemical study using an MAb against the intact heparin molecule. As reported by
I II III
GlcNSO3(6-OSO3)1-4GlcAß1-4GlcNSO3
IV
(3,6,-di-OSO3)1-4IdoA(2-OSO3)
V
1-4GlcNSO3(6-OSO3)
is not required for MAb ST-1 binding to heparin. The data presented here show that MAb ST-1 can be a potential tool for evaluation of heparin content in mast cells. Immunocytochemical analysis shows positive labeling of rat peritoneal mast cells but not of intestinal MMCs. The labeling was restricted to cytoplasmic granules of mast cells, and no significant labeling was observed in eosinophils or other cells of the peritoneal cavity. Control experiments using an irrelevant antibody were performed and significant labeling of granules was not observed, indicating that the binding is not due to a nonspecific binding of immunoglobulins to cytoplasmic granules. In addition, treatment of mast cells with nitrous acid, which deaminates heparin (
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Footnotes |
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1 Supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundaçao de Amparo à Pesquisa do Estado de Sao Paulo, and Financiadora de Estudos e Projetos.
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