ARTICLE |
Correspondence to: Robert J. Kayton, Dept. of C.R.O.E.T., Oregon Health Sciences Univ., 3181 SW Sam Jackson Park Rd., Portland, OR 97201..
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Summary |
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We previously reported that mast cells (MCs) serve as a source of basic fibroblast growth factor (bFGF), a potent angiogenic and mitogenic polypeptide, suggesting that bFGF may mediate MC-related neovascularization and fibroproliferation. Unlike many other growth factors, bFGF lacks a classic peptide sequence for its secretion, and the mechanism(s) for its release remains controversial. Because MCs release a wide spectrum of bioactive products via degranulation, we hypothesized that MC degranulation may be a mechanism of bFGF release and used ultrastructural immunohistochemistry to test the hypothesis. We reasoned that if bFGF is released through degranulation, it should be localized to MC secretory granules. Human tissues with chronic inflammation and rat/mouse tissues with anaphylaxis were studied. In all tissue samples examined, positive staining (or immunogold particle localization) for bFGF in MCs was predominantly in the cytoplasmic granules. Moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles exhibited minimal immunogold particle localization. In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells. In rat/mouse lingual tissue undergoing anaphylaxis, immunogold particle localization for bFGF was found not only in swollen cytoplasmic granules but also in the extruded granules of MCs. Three different anti-bFGF antibodies gave similar immunogold particle localization patterns, whereas all controls were negative. These results provide morphological evidence suggesting that, despite the lack of a classic secretory peptide in its structure, bFGF is localized to the secretory granules in MCs and may be released through degranulation. (J Histochem Cytochem 46:11191128, 1998)
Key Words: mast cell, basic fibroblast growth factor (bFGF), electron microscopy, degranulation
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Introduction |
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Basic fibroblast growth factor (bFGF) is a member of a heparin binding growth factor family (fibroblast growth factor family) that includes at least eight other members sharing 2040% homology in their gene structure (
Several lines of evidence indicate that the release of bFGF at both the cellular and the tissue level is tightly controlled and that loss of such control may be deleterious. First, increased free bFGF has been linked to neoplasms. For example, increased levels of bFGF were found in cerebral spinal fluid (CSF) in 62% patients with brain tumors and correlated well with the vascular density of the tumors. In contrast, bFGF was not detectable in the normal controls (
It has been well documented that cell injury results in bFGF release. However, injury also results in release of many other intracellular components and growth factors that are normally secreted, such as transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF). Although the exact role(s) of bFGF remains unknown, its wide tissue distribution, broad spectrum of target cells, and mutation lethality in transgenic animals strongly suggest its important roles in normal homeostasis. It is reasonable to believe that release mechanisms other than cell injury play the major role in controlling the export of bFGF in normal homeostasis. A study by
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Materials and Methods |
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Tissue Processing
Fresh synovium (n = 4), lung (n = 3), and nasal mucosal membrane (n = 2) samples were obtained during surgery from patients with rheumatoid arthritis, pulmonary fibrosis, and nasal polyposis, respectively. All samples were divided into two parts and processed for light and electron microscopy separately. For light microscopy (LM), the samples were fixed in neutralized buffered formalin for 2430 hr at 4C, dehydrated, and embedded in paraffin.
For electron microscopy (EM), tissue samples were sliced into 13-mm3 pieces and fixed with 4% paraformaldehyde0.5% glutaraldehyde in 0.1 M Sörensen's phosphate buffer for 4 hr at 4C. From each case, some sample pieces were also postfixed in 1% OsO4 for 1 hr at room temperature (RT). After extensive washes in the phosphate buffer, the samples underwent ethanol dehydration to propylene oxide and then were embedded in Araldite 502 (Electron Microscopic Sciences; Ft Washington, PA).
In Vivo Mast Cell Degranulation
To induce degranulation in vivo, direct lingual anaphylactic assay was performed on female Brown Norway rats weighing 250300 g (n = 4). Rats were anesthetized with isoflurane/O2, delivered with an anesthetic vaporizer, and about 15 µg goat anti-rat IgE antibody in 100 µl PBS was injected into the tongue along the midline. The control rats (n = 3) were injected with an equal amount of nonimmune goat IgG. Anesthetized rats were sacrificed 1 or 2 hr after the injection by IV perfusion with 0.1 M Na-citrate containing 15% glycerol at 37C, pH 7.4, followed by 4% paraformaldehyde0.2% glutaraldehyde in 0.1 M PBS at 4C. The rats were kept at 4C overnight. The following day, lingual tissue was dissected out and processed for both LM and EM as described above.
In vivo mast cell degranulation in mice was induced using a passive cutaneous anaphylactic assay previously described by
Histochemical and Immunohistochemical Staining
Paraffin-embedded tissues were cut into 23-µm sections and laid on poly-L-lysine-coated slides. The sections were deparaffinized in xylene and rehydrated through graded alcohol. To visualize tissue mast cells, the sections were stained with 0.1% toluidine blue (Sigma) in 0.1 N HCl for 1 hr at RT. Adjacent sequential sections were stained for bFGF as described previously (
Postembedding Immunogold Labeling for Electron Microscopy
Ultrathin sections were cut at 5070 nm, collected on 200-mesh nickel grids coated with Coat-Quick "G" (Electron Microscopic Sciences), and immunolabeled with three different antibodies specific for bFGF, using a two-step procedure. All immunogold steps were carried out at RT in 70-µl reagent drops on moisture-resistant parafilm (American National Can; Greenwich, CT). The grids were first treated with hyaluronidase at 2 mg/ml in 0.1 M acetate buffer, pH 5.2, for 15 min to unmask antigens (
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Results |
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Localization of bFGF in Rat and Mouse Tissue After Cutaneous Anaphylaxis
On intralingual injection of anti-Ig E antibody or IV challenge of antigen-sensitized rats or mice, the site of antigen injection became blue within 30 min. The local coloration by Evan's blue remained unaffected by IV perfusion with glycerol/citrate buffer and fixative solution, suggesting extravasation of Evan's blue dye into the extravascular compartment. No such change was observed in nonsensitized animals or sensitized animals challenged with Evan's blue only (data not shown). When the Evan's blue dye-containing tissues were examined microscopically, extrusion of mast cell granules was readily observed (Figure 1A and Figure 1B). Strong immunohistochemical staining for bFGF was found in both mast cells and their extruded granules (Figure 1A), which were identified by toluidine blue stain on an adjacent section (Figure 1B). In contrast, bFGF immunoreactivity was not found to be associated with neutrophils, lymphocytes, or plasma cells. Similar findings were observed in the lingual tissue after local injection of anti-IgE antibody. Increased extracellular staining for bFGF was also found in areas near the degranulating mast cells. In the lingual tissues from control rats, positive staining for bFGF was mainly associated with the cytoplasmic granules confined within the cells that were also labeled by toluidine blue dye (Figure 1C and Figure 1D), indicating that they are mast cells. No apparent granule extrusion or increased extracellular staining for bFGF were present in the control animals. In all tissues from rats and mice examined, no tissue necrosis was seen.
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To identify the subcellular compartment associated with bFGF-like immunoreactivity, tissue samples were examined by EM. Tissue mast cells were identified by metachromasia after toluidine blue staining of semi-thin sections and by characteristic ultrastructure, such as typical plicated membrane folds, conspicuous membrane-bound cytoplasmic granules, and nonlobed nucleus (Figure 2). Immunogold particle localization of bFGF was found predominantly in the cytoplasmic granules of MCs (Figure 2). However, no particular distribution pattern of the bFGF immunoreactivity within the cytoplasmic granules was observed. Similarly, moderate bFGF immunoreactivity was also found in the nucleus, whereas the cytosol and other subcellular organelles were essentially devoid of immunogold particles for bFGF in MCs. Only background level of immunogold particle localization was found in cytoplasm and mitochondria (Figure 3A). Moderate to little immunogold particle localization was present in the cytoplasm and nucleus of fibroblasts and endothelial cells (data not shown). In contrast, no immunogold particle localization for bFGF was observed in lymphocytes or plasma cells.
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After degranulation, mast cells in the anaphylactic tissue exhibited morphological changes characteristic of degranulation, such as degranulation channel formation, swollen granules, shed surface folds, and granule extrusion (Figure 2 and Figure 3A). The extruded granules were membrane-free and exhibited reduced matrix density. Extensive granule extrusion was observed (Figure 3A). Immunogold particle localization of bFGF was present in cytoplasmic secretory granules of the degranulating mast cells. Notably, bFGF immunogold particle localization was also present in the extruded granules (Figure 2 and Figure 3A).
Localization of bFGF in Human Tissues
In all samples examined, immunogold particle localization for bFGF was present in mast cells. Specific bFGF staining in the human tissues at the LM level was reported in detail our previous article (
Controls to Confirm the Specificity of bFGF Staining
To confirm the specificity of bFGF staining present in mast cells, cornu ammon 1 and cornu ammon 2 regions from primate and rat hippocampus known to contain bFGF were used as a positive control tissue. Previous studies by several independent research groups showed that both mRNA for bFGF and immunoreactivity, as well as biological activity, were present in neurons in these regions (
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Discussion |
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In this article we present morphological evidence suggesting that mast cell-derived bFGF can be released through degranulation, a classical exocytotic process characteristic of noninjured, properly stimulated mast cells. Because bFGF is often found extracellularly and may be readily available to mast cells, one can argue that mast cells could take up extracellular bFGF and store it in their granules. Two findings appear to refute such an explanation. First, unlike epithelial cells of the tongue and airways that express both bFGF and FGF receptor-1, -2, and -3 (
Mast cells have been implicated in a wide spectrum of diseases characterized by fibroproliferation and neovascularization (for review see
Although the PCA animal model was used to demonstrate the release of bFGF through mast cell degranulation in the present study, extensive studies by Dvorak et al. have shown that a slow degranulation process, piecemeal degranulation, is also present and is clinically relevant to diseases characterized by fibroproliferation and neovascularization (for review see
In addition to cell injury, bFGF release has been described in two other experimental systems in which noninjury release is also contemplated (
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Acknowledgments |
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Supported by NIH grant EY 10572, by a grant from the Gerlinger Fund, and by an unrestricted grant from Research to Prevent Blindness, New York. JTR is a senior scholar supported by Research to Prevent Blindness, New York. SRP is a recipient of an RPB Special Scholar Award, New York.
We wish to acknowledge the helpful critical review and important insights freely given this manuscript by Dr Ann Dvorak.
Received for publication December 1, 1997; accepted June 22, 1998.
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