A barium method for the cytochemical detection of sulfated glycosaminoglycans in mast cells and basophilic leukocytes

Acta histochem. 101, 397-408 (1999) © Urban & Fischer Verlag

acta histochetnica

A barium method for the cytochemical detection of sulfated glycosaminoglycans in mast cells and basophilic leukocytes Juan C. Stockert1, Nora lbaiiez 2, Clara I. Trigoso 1, Magdalena Caiiete 1 and Agustin Tato 3 1 Departamento

de Biologia, Facultad de Ciencias, Universidad Aut6noma de Madrid, Canto Blanco, E-28049 Madrid, Spain, 2 Departamento de Anatomia, Facultad de Agronomia y Veterinaria, Universidad Nacional de Rio Cuarto, 5800 Rio Cuarto, Cordoba, Argentina, and 3Servicio Central de Apoyo a Ia lnvestigaci6n Experimental, Universidad de Valencia, Burjassot, E-461 00 Valencia, Spain Received 16 April 1999; accepted 30 June 1999

Summary Barium ions precipitate inorganic as well as organic sulfate compounds and they can be detected by a reaction with sodium rhodizonate. In this work, we describe the use of a barium method for the selective demonstration of sulfated glycosaminoglycans in cytoplasmic granules of mast cells and basophilic leukocytes. Methanol-fixed smears of mouse peritoneal mast cells and rat bone marrow basophils were treated with 5 % BaC1 2 for 10 min, followed by staining with either 0.2 % sodium rhodizonate in 50% ethanol for 2 h at 60 °C, or 0.01 % brilliant green in distilled water for 1 min. Light microscopic observation revealed a strong staining reaction of the cytoplasmic granules of these cell types, which was more selective when using sodium rhodizonate. Control smears treated with BaC12 or sodium rhodizonate alone, and those subjected to methylation/extraction of sulfate groups before staining remained unstained. The selective binding of barium ions to mast cell granules was established with scanning electron microscopy using a backscattered electron detector, and confirmed by energy dispersive X-ray microanalysis as well as element mapping.

Key words: mast cells - basophils - barium - sulfated glycosaminoglycans sodium rhodizonate- brilliant green- X-ray microanalysis Correspondence to: J. C. Stockert http://www.urbanfischer.de/journals/actahist

0065-1281/99/101/4-397 $ 12.00/0

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Introduction Mast cells and basophilic leukocytes play an important role in diseases such as respiratory and cutaneous allergy, as well as mastocytosis (Marone, 1995). In addition, these cell types are associated with a number of other pathological conditions such as parasitosis, atherosclerosis, and rheumatic, gastrointestinal, cardiac, and neoplastic diseases (Stevens and Austen, 1989; Rothe et al., 1990; Schwartz, 1994; Kitamura et al., 1995). Taking into account the relevance of mast cells and basophils in inflammatory processes, haemostasis, as well as in the pathogenesis of disorders of immediate hypersensitivity (Lagunoff and Chi, 1980; Dvorak et al., 1983), they are of great interest in biomedical research (Stevens, 1989; Valent and Bettelheim, 1990; Eguchi, 1991; Bertolesi et al., 1997). Sulfated glycosaminoglycans (GAGs) are specific components of mast cell and basophilic granules. Mast cell granules mainly contain heparin and chondroitin sulfate, whereas heparan sulfate, chondroitin sulfate, and dermatan sulfate are present in basophilic granules (Parwaresch, 1976; Orenstein et al., 1978; Stevens, 1989). The high content of sulfated GAGs is responsible for both the basophilia and the metachromasia shown by these structures when they are stained by cationic dyes such as toluidine blue (Ackerman, 1963; Thompson, 1966; Blumenkrantz, 1975), alcian blue-safranin (Miyashita et al., 1986), ruthenium red (Lagunoff, 1972; Gutierrez-Gonzalvez et al., 1984), and cuprolinic blue (Juarranz et al., 1987; Landemore et al., 1995). Likewise, several cationic fluorochromes induce selective fluorescence of mast cell and basophilic granules (Hahn von Dorsche and Opitz, 1970; Enerback, 1974; Love, 1979; Bertolesi et al., 1995; Espada et al., 1995). Some cationic reagents (e. g. tetrazonium salts, benzidine, diaminobenzidine) precipitate in the presence of sulfate anions, and this feature has been applied in the histochemical detection of sulfated GAGs (Geyer, 1962; Bussolati, 1971; Hadler et al., 1978). It is also known that soluble barium salts precipitate both inorganic and organic sulfate (Windholz, 1983; Alewell, 1993). At the same time, the barium cation can be detected by means of its chromogenic reaction with sodium rhodizonate (Fig. 1). This compound, first introduced into analytical work by Feigl (1924) and Feigl and Suter (1942) as a spot test reagent for barium and strontium, was later used in histochemistry by several authors (Waterhouse, 1950, 1951; Molnar, 1952; McGee-Russell, 1958; Chaplin and Turner, 1983).

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Fig. 1. Chemical structure of sodium rhodizonate.

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In the present work, we describe the microscopic application of a barium method for the demonstration of sulfated GAGs in the cytoplasmic granules of mouse mast cells and rat basophils, which is based on the reaction of barium ions with either sodium rhodizonate (Chaplin and Turner, 1983) or brilliant green (Gurr, 1971). In addition to light microscopic observations, results from scanning electron microscopy, energy dispersive X-ray microanalysis, and element mapping after application of the barium method to mast cells are also described.

Material and methods This study was carried out on bone marrow and peritoneal cell smears, which are rich in basophilic leukocytes and mast cells, respectively. Smears of bone marrow cells were made from adult male Wistar rats, whereas mast cells were obtained from the peritoneal cavity of adult male BALB/c mice (Bertolesi et al., 1995). Briefly, 3 ml of Tris-A-EDTA buffer, pH 7.6 [containing 0.025 M Tris-HCl (Aldrich, Steinheim/Albuch, Germany), 0.12 M NaCl (Merck, Darmstadt, Germany), 0.01 M EDTA (Aldrich) and 0.1 mg/ml bovine serum albumin (Sigma, St. Louis, MO, USA)] were injected into the peritoneal cavity. The abdomen was gently massaged for 1 min and the peritoneal washing solution was recovered and centrifuged at 1000 rpm (rotor radius: 7 em) for 10 min. The cell pellet was resuspended in Tris-A-EDTA buffer and the concentration of the cells was adjusted to 4-8 X 105 cells/mi. Both bone marrow and mast cell smears were fixed in 100 % methanol (Merck) for 2 min and air dried for 5 min. Cell smears were treated with a 5 % barium chloride (BaC12 ; Probus, Badalona, Spain) solution for 10 min at room temp, washed in distilled water and air dried for 5 min. Staining was performed with a freshly made 0.2% solution of sodium rhodizonate (Sigma) in 50% ethanol (Merck) for 2 h at 60 °C. The staining procedure is a modification of previously described methods (Thompson, 1966; Chaplin and Turner, 1983; Pearse, 1985). Smears were washed for 5 min in the same solvent, air dried for 5 min and mounted in DePeX (Serva, Heidelberg, Germany) or directly examined under immersion oil (Zeiss, Oberkochen, Germany) in a Zeiss photomicroscope III using bright field illumination. To analyze the optimal rhodizonate reaction, other solvent (distilled water), different temperatures (room temp or 37 oq and staining times (0.5, 1, or 1.5 h) were tested as well. Likewise, the specific detection of barium by the rhodizonate reaction was controlled using cell smears treated with sodium rhodizonate or BaCh alone. To confirm the selectivity of the barium reaction for sulfated GAGs, some mast cell smears were subjected to methylation/extraction of sulfate groups with 0.1 N HCl (Merck) in 100% methanol at 60 °C for 24 h. After this blocking procedure, smears were briefly washed in 50% methanol, air dried for 5 min, treated with BaCh and stained with sodium rhodizonate as described above. On the other hand, BaClrtreated cell smears were also stained with 0.01 % brilliant green (Gurr, London, UK) in distilled water for 1 min. Precipitation tests were made by mixing equal volumes of the BaC12 solution with either sodium rhodizonate or brilliant green staining solutions. The occurrence of barium in BaClrtreated mast cells was also studied by scanning electron microscopy (SEM). For this purpose, peritoneal cell smears were made on aluminum sheets and then fixed and treated with BaC12 as described above. These samples and others not treated with BaC12 (controls) were directly observed at 10 kV in a field emission SEM S 4100 (Hitachi, Naka, Japan), equipped with the usual secondary electron (SE) detector and a backscattered electron (BSE) detector (model: Autrata, Hitachi; mode: compo-

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sttwn, working distance: 5 mm). The normal signal polarity for BSE detection was employed to visualize structures with high atomic number (Z) contrast, which appeared as bright images. To determine the elements present in BaClrtreated and untreated mast cells, smears were also observed at 10 kV and subjected to energy dispersive X-ray microanalysis (XRMA) in a SEM JSM 6300 (Jeol, Tokyo, Japan), equipped with a solid state BSE detector (mode: composition, working distance: 15 mm) and an XRMA device (Link-ISIS; Oxford Instruments, Bucks, UK) using the X-ray spectrometer Link Pentafet ATW (Oxford Instruments; working distance: 15 mm, tilt: 0°, system resolution: 90 eV, counting time: 400 s). The ZAF method (Goldstein et al., 1981; Morgan, 1985) (2 iterations) was used for the quantitative analysis of phosphorus, sulfur and barium by means of the K, K and L lines of these elements, respectiveiy. Element mapping was performed using the program Speedmap (incorporated into the Link-ISIS software) and a recording time of 30 min.

Results Samples treated with BaC1 2 and stained with sodium rhodizonate showed a strong reddish brown colour of the cytoplasmic granules from both peritoneal mast cell smears (Fig. 2 a) and bone marrow basophils (Fig. 2 b). Other

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Fig. 2. Bright field micrographs of mouse mast cells (a, c) and rat basophils (b, d) after staining with BaCI 2-sodium rhodizonate (a, b) or BaCirbrilliant green (c, d). A strong and selective staining reaction in cytoplasmic granules of both mast cells and basophils is observed. Bar: 10 11m.

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cell structures such as nuclei and the cytoplasm from lymphocytes, macrophages and erythropoietic elements, as well as neutrophil and eosinophil precursor cells were either unstained or scarcely stained in pale yellow. A similar deep green staining pattern was found in cell smears treated with BaC12 and then with brilliant green (Fig. 2 c, d). Lymphocyte nuclei and basophilic cytoplasm stained slightly green, whereas the remaining cell types remained unstained. The reactivity in vitro of both sodium rhodizonate and brilliant green with barium ions was confirmed by direct mixing of these reagents, which rendered deep red-brown and green precipitates, respectively. Best results for the barium rhodizonate reaction on cell smears were obtained when a 50 % ethanol sodium rhodizonate solution was applied for 2 h at 60 °C. The use of distilled water as solvent, a lower staining temperature or a shorter staining time resulted in less specific and/or diminished staining reactions. Mast cells and basophils in control preparations which lacked either BaC12 or sodium rhodizonate treatment, as well as those subjected to methylation/extraction of sulfate groups before the barium rhodizonate method remained unstained. Likewise, granules in mast cells and basophils that were not treated with BaClz stained in a light green colour only after brilliant green treatment. Both mounting media (DePeX and immersion oil) were similarly adequate for microscopic observation. When observed by SEM using the SE detector for topographic contrast, untreated mast cells had morphological features which allowed simple identification. As critical point drying and metal coating were not used, the surface of mast cells was not well preserved. In BaC12 -treated samples mast cells could be identified by SE imaging (Fig. 3, top), but internal barium deposits could not be recognized. In contrast, BSE images of mast cells clearly showed increased brightness (high Z contrast) due to selective deposition of barium in the cytoplasmic granules (Fig. 3, bottom). Mast cell nuclei could be discriminated from the cytoplasm because they showed a weak BSE signal (dark image). These BSE features were not observed in smears that had not been treated with BaC12 . X-ray emission spectra confirmed the presence of elements P, S and Ba in BaC12 -treated mouse mast cells (Fig. 4 a). Similar peaks of P and S were also found in XRMA of untreated smears, whereas the signal for Ba was not present (data not shown). Due to the close proximity of the Kal and Ka2 lines of P (2.014 keV and 2.013 keV, respectively) and S (2.308 keV and 2.307 keV, respectively), the two lines of each element appeared as a single peak. However, the Ba signal showed two peaks corresponding to its separate Lal (4.466 keV) and Lfil (4.828 keV) lines, which are characteristic features of X-ray emission of this element. The X-ray signal of P was quite similar in all mast cells (about 1.2 cps), whereas that of S and Ba appeared to be lower in some mast cells (Fig. 4 b), possibly indicating degranulation.

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f Fig. 3. Scanning electron micrographs showing SE (top) and BSE (bottom) images of a mast cell after BaCirtreatment. Note the enhanced contrast of the cytoplasmic granules in the BSE image, which appears bright due to its high Z contrast. Bar: 1 Jlm.

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Fig. 4. X-ray emission spectra of BaCirtreated mast cells showing the peaks of elements C, N, 0, P, S and Ba. The signals for AI and Si originate from the support sheet and usual Si contamination, respectively. P and S show the peaks from their close Ka1 and Ka2 lines, whereas for Ba the two peaks corresponding to its separate La1 and LP1 lines are detected. cps: counts per second.

Mast cells treated with BaCh and subjected to element mapping showed that the X ray image of P was localized over the nuclear area, while accumulation of S and Ba was found in cytoplasmic regions (Fig. 5). Although X-ray emission images did not show well-defined cell boundaries and internal structures, element mapping confirmed co-localization of Ba and S in the granular cytoplasm of mast cells. As expected, Ba was not found in untreated samples.

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Discussion Besides the sodium rhodizonate reaction for the histochemical demonstration of barium (Waterhouse, 1951; Molnar, 1952; Chaplin and Turner, 1983), various microscopic methods are based on the use of this heavy cation. Barium ions have been used in Dziewiatkowski's fixative (4% formaldehyde saturated with Ba(OH)z) to preserve sulfated GAGs, in the Kruszynski's barium nitrate method for the visualization of the Golgi apparatus, and in the Saxena's method for the improved preservation of lipids (Thompson, 1966). The alkaline solution of Ba(OH)z is used in C-banding methods for chromosomes (e. g. Bella and Gosalvez, 1991), and for blocking RNA-dependent basophilia in tissue sections (Clark and Meischen, 1978). Since treatment with BaC12 is ineffective, this blocking mechanism of basophilia by Ba(OH)z may be rather

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alkaline hydrolysis, which selectively removes RNA but not DNA (Geyer and Scheibner, 1970; Stockert and Pelling, 1992). lt is well known that addition of sodium, potassium or barium salts to cationic dye solutions abolishes the metachromatic reaction. Ba2 + is the most effective cation to prevent metachromasia of chromotropic (polyanionic) substrates (Lison, 1935, 1960). DNA in chromatin remains well stained by thiazine dyes in the presence of barium nitrate (Mello, 1980), and Ba2 + is a weak cation for precipitation of isolated chromatin (Walker et al., 1989). Taken together, the reactivity of barium with cellular polyanions are in agreement with our present results showing selective binding of this heavy cation to sulfated GAGs of mast cells and basophils. The sodium rhodizonate reaction gives a reddish brown precipitate with barium and strontium salts (Feigl and Suter, 1942; Waterhouse, 1951), although other cations (Zn, Cd, Cu, Sn, Pb, Bi, Hg, Tl, Ag, U) also produce coloured precipitates (Feigl, 1924; Feigl and Suter, 1942; Lillie, 1977). Because these metal cations are not present in normal cells, with the exception of zinc and copper, the rhodizonate reaction with metals other than barium does not seem to be relevant. Although zinc is present in mast cell granules (Pihl and Falkmer, 1967), it does not seem to interfere with our results, since no reaction was found after treatment of samples with sodium rhodizonate alone. Therefore, it is possible that zinc ions have been removed from mast cells by EDTA in the buffer that is used during the isolation procedure. Sodium rhodizonate is a useful reagent for the histochemical detection of barium (Waterhouse, 1951; Thompson, 1966; Lillie, 1977; Chaplin and Turner, 1983). The present results show that after BaClrtreatment, deposition of barium in mast cell and basophilic granules can be visualized easily by means of sodium rhodizonate. Different conditions for the microscopic use of this reagent have been recommended by several authors (Waterhouse, 1950; McGee-Russell, 1958; Chaplin and Turner, 1983). In our case, a slight modification of the staining procedure of Chaplin and Turner (1983) was found to be optimal for cell smears. It must be taken into account that solutions of sodium rhodizonate are unstable (Windholz, 1983). Moreover, not all batches of this reagent may be suitable for the detection of barium deposits (Chaplin and Turner, 1983). In addition to light microscopic observations using the barium rhodizonate reaction, the barium-brilliant green method was also useful to demonstrate specific granules of mast cells and basophils but it showed a lower selectivity. After heavy metal deposition in samples for SEM analysis with a BSE detector (Ogura and Hasegawa, 1980; Ushiki and Fujita, 1986; Trigoso et al., 1992), the presence of barium in mast cells largely increased the Z contrast of BSE images. Likewise, the selective binding of barium to sulfated GAGs in mast cells is clearly confirmed in SEM preparations by using XRMA and element mapping. In conclusion, the observations described here indicate that barium uptake followed by the sodium rhodizonate reaction is a reliable and

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selective method for the cytochemical demonstration of sulfated GAGs in mast cell and basophil granules, which could be useful in studies of these cells under normal or pathological conditions. Acknowledgements. We are greatly indebted toM. Planes (Servicio de Microscopfa Electr6nica, Universidad Politecnica de Valencia, Spain) for his technical assistance, and to J. Blanco and A. Juarranz for valuable comments. This work was supported by the Direcci6n General de Investigaci6n Cientffica y Tecnica, Spain (Grants: PM95-0027, PM98-0017-C0201). J. C. S. is a scientific member of the Consejo Superior de Investigaciones Cientfficas, Spain.

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