Original Paper Acta Anat 1992:143:294-300
" Department of Veterinary Anatomy and h Department of Food Science. Rakuno Gakuen University, Ebetsu. Hokkaido, Japan: c Department of Veterinary Anatomy and Cellular Biology. Ohio State University, Columbus, Ohio. USA: 11 Department of Anatomy. Yamanashi Medical College. Tamaho. Yamanashi, Japan c Aichi Cancer Center Research Institute. Nagoya, Japan
Cytoplasmic Filaments in the Endothelial Cells of the Sheathed Capillary: An Ultrastructural and Immunocytochemical Study in the Pig Spleen
Key Words
Abstract
Intracytoplasmic filament Vimentin Desmin Actin filament Sheathed eapillary endothelial eell Spleen Pig
Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cyto plasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin. and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the intcrendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.
Introduction Since the first report by Billroth [1], it is known that sheathed capillaries are located between the penicilli and the arterial capillaries in the spleen. Sheathed capillaries arc believed to have a filtering function [2,3]. The sheathed capillary has been studied frequently in many species. In reported ultrastructural studies, endothelial cells of sheathed capillaries are elongated with tapered ends and lie parallel to the long axis of the luminal surface [2.4-8]. In all
Received: September 6. 1991 Accepted: November 7, 1991
species, the endothelial cells of sheathed capillaries have the same cytoplasmic filaments, cell organelles, shape and orientation [2. 5-9]. It has been reported that endothelial cells contain a large number of intermediate filaments [2, 4-9], but there arc no reports describing cytoplasmic filaments in the endothelial cells of sheathed capillaries. Generally, cytoplasmic filaments in nonmuscle cells were classified into two categories: thin filaments and inter mediate filaments. A variety of endothelial cells contain fine cytoplasmic filaments which closely resemble the myo-
Kazushige Takehana Department of Veterinary Anatomy Rakuno Gakuen University Ebetsu. Hokkaido 069 (Japan)
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K. Takehana*, M. Abe3, M. Yamaguchic, T. Uchida\ M. Inagakic, K. Yamamotob, J. Masiyc, A. Winnardc, W. Veda3, H. Miyataa
Materials and Methods Sample Source Ten healthy, 6-month-old pigs (100-150 kg), crossbreeds of Landrace. Large White and Hampshire stock, were used in this study. Spleens obtained from the slaughterhouse were excised immediately following death induced by an intravenous injection of sodium pento barbital (40 mg/kg). Preparation o f Permeable Cells The spleen was minced with scissors, and approximately 1 g of minced spleen was homogenized, using a Waring blender, with 20 ml of buffer solution containing 5 milf KCI. 20 mM K-phosphate buffer (pH 6.8). 2 mM dithiothreitol. I mM NaN,. I mM EGTA and I mM EDTA. for 10 sat 4°C. After centrifugation (Beckman J-21. roter 30) at 15.000 rpm for 3 min. the supernatant was discarded, and 20 ml of the buffer solution were added to the precipitate. Homogenization and centrifugation were repeated 5 times at 4 °C. Staining with HMM Rabbit skeletal muscle HMM was obtained by established meth ods [ 13]. A portion of the pellet of the permeable cells was mixed with an equal volume of HMM (4 mg/ml) in 67 mM K-phosphate buffer (pH 7.0). incubated for 18 h at 25 °C. and occasionally gently stirred. Excess HMM was removed by centrifugation and washing 4 times with buffer solution.
Thin-Section Electron Microscopy The permeable cells and HMM-staincd permeable cells were fixed with 3.0% glutaraldehyde in 0.1 M cacodvlate buffer, pH 7.2, for 2 h at 4 °C. Pellets were postfixed in 1.0% osmium tetroxidc for 60 min at room temperature, dehydrated and embedded in Epon 812/Araldite. For conventional electron microscopy, small pieces of spleen were processed as above. Samples were sectioned with an LKB Ultratome 111 with a diamond knife. Sections were stained first in uranyl acetate and then in lead citrate. The sections were then examined under an II300. HU-11DS or JEM-100S electron microscope calibrated with a standard measuring grid at an accelerating voltage of 75 or 80 kV. Tissue Preparation fo r Immunohistochemistry The spleens were cut into small pieces ( l x l x l mm) and fixed in 4.0% paraformaldehyde buffered with 0.067 M Na-phosphate (pH 7.4) for approximately 2 h at room temperature. After washing with the same buffer, the specimens were dehydrated in graded N.N-dimethylformamide at progressively lower temperatures and were embedded in glycol methacrylate (CiMA) at -20 °C [15]. Antisera Antisera to vimentin. desmin and keratin sera were prepared by the method described by Inagaki et al. 116], The vimentin, desmin and keratin antisera that were used recognize only the intermediate fila ment proteins vimentin. desmin and keratin, respectively, and do not cross-reacl with other intermediate filament proteins. The three anti sera were used at dilutions of 1,000 (vimentin). 250 (desmin) and 500 (keratin). The actin antiserum was purchased from Transformation Research Inc. (Framingham. USA) and used at a 500 dilution. As the secondary antibody, gold-labeled immunoglobulins to rab bit were used. Colloidal gold particles (average diameter 5 or 10 nm) were made by the chemical reduction of chloroauric acid by tannic acid and trisodium citrate [ 17|. The linking of colloidal gold particles to immunoglobulins (goat anti-rabbit, rabbit anti-chicken or rabbit anti-mouse IgG) was performed as previously described [ 18].
Immunohistochemical Labeling For the detection of antigenic sites, single- and double-labeling immuno-gold methods were employed. For the single labeling, ultrathin sections mounted on nickel grids were first immersed for 15 min at room temperature in phosphate-buffered saline (PBS. 10 mM Naphosphate. 0.15 M NaCI. pH 7.4) containing 2% normal serum (same species as the second antibody) and 1% bovine serum albumin (PBSNS-BSA). and then incubated overnight at 4 °C with primary antise rum diluted with PBA-NS-BSA. After incubation, the sections were rinsed with PBS and immersed in the 10-nm gold-labeled secondary antibody diluted with PBS-NS-BSA for 40 min at room temperature. For the double labeling, the sections were first stained with either antiactin or anti-desmin antisera, and 5-nm gold-labeled second antibody, as above. After the initial labeling, the sections were rinsed with PBS and stained with anti-vimentin antiserum and 10-nm gold-labeled sec ondary antibody, as above. After immunostaining, the sections were washed with PBS. rinsed with distilled water and air dried. Finally, the sections were stained with uranyl acetate followed by lead citrate, and were then examined. Immunohistochemical specificity was checked by the absorption test. Diluted antisera were preincubated overnight at 4 °C with puri fied vimentin (10 pg/ml) before application to the tissue sections.
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filaments of muscle cells 110, 111. Some of these cytoplas mic filaments can be stained with heavy meromyosin (HMM). indicating the presence of actin filaments 112. 13]. Intermediate filaments have been classified into five types: keratin, neurofilament protein, glial fibrillary acidic pro tein. desmin and vimentin. We have attempted to char acterize the cytoplasmic filaments in the endothelial cells of sheathed capillaries by immune electron microscopy, using antibodies against the major constituent proteins of the fol lowing three different classes of intermediate filaments: (a) vimentin. a protein of ~57.0(K) molecular weight (MW), the major constituent of the intermediate filaments of vari ous mesenchymal and mcscnchymal-derived cells; (b) des min. a protein of 53,000 MW, which is found also in cul tured smooth and cardiac muscle cells, in differentiated myoblasts and in myotubules of skeletal muscle, and (c) keratin, a protein of 47.0(K)-58.0(K) MW. the predominant type of intermediate-sized filament occurring in various epithelial and epithelium-derived cells [14], In this study, we examined the nature of the cytoplasmic filaments in the porcine sheathed capillary, by electron microscopy, using a gold-conjugated antibody to identify intermediate and thin filaments, and HMM staining to identify thin filaments.
Fig. 1. The sheathed capillary endothe lial cells ( E) of a pig spleen containing a large number of intermediate filaments, x 7,600. Fig. 2. A large number of intermediate filaments (diameter: HI nm) filled the cyto plasm. x BO.IXKl. Fig. 3. Thin filaments filled the lateral processes (asterisks). An intercellular junc tion (IJ) on the lateral processes is seen, x 44,000.
Endothelial cells of sheathed capillaries in the pig spleen protruded high into the lumen (fig.l). They contained a large number of intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells (fig.2). These filaments were noted in a narrow zone beneath the plasmalemma, in which mitochondria and rough endoplasmic re
296
ticulum were also detected, and in the perinuclear region located midway through the length of the cell. Thin filaments (diameter: 5 nm) formed a felt-like pat tern which occurred just beneath the plasmalemma and filled most of the lateral cytoplasmic processes. Irregularly shaped patches of matted, thin filaments were scattered in the basal cytoplasm and near intercellular junctions (fig-3).
Takchana/Abc/Yamaguchi/Uchida/Inagaki/ Yamamoto/Masty/Winnard/Ueda/Miyata
Cytoplasmic Filaments of Sheathed Capillary
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Results
Fig. 4. Detail of a permeable sheathed capillary endothelial cell containing a large number of intermediate filaments, x 58,000. Fig. 5. Detail of a permeable sheathed capillary endothelial cell after HMM treat ment. Thin filaments (diameter: 5 nm) occurred in the basal cytoplasm (asterisks). Intermediate filaments do not bind HMM (IF). X58.000. Insert: Thin filaments are stained by HMM (arrows). X 110.000.
After HMM treatment, electron-dense structures were located in the basal cytoplasm and near intercellular junc tions (fig.5). They were marked by characteristic arrow head formations (fig.5 insert). There was no detectable reaction of HMM with the thick filaments. Figures 6-9 show the immunohistochemical results. When the sections were stained using anti-vimentin serum, all the thick filaments in the sheathed capillary showed a positive reaction. The immunoreactivity was located cxclu-
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A large number of thick anti thin filaments were densely packed in the cytoplasm of the endothelial cells in the sheathed capillaries, thus, it was very difficult to analyze or infer any relationships, and distinguish between interme diate and thin filaments by conventional thin-section elec tron microscopy. After permeabilization. the cytoplasm of sheathed capillary endothelial cells was much less electron dense, thus facilitating identification of the intracellular filaments (fig.4).
Fig. 6, 7. An electron micrograph of the immunostained sheathed capillary endothe lial cell, x 53.000 . 6 Vimentin (10-nm gold particles) - all the intermediate filaments in an endothelial cell are positively stained. 7 Desmin - a part of the intermediate fila ments in an endothelial cell is positively stained. Fig. 8. An electron micrograph of the sheathed capillars' endothelial cell: double inununogold labeling for desmin (5-nm gold particles) and vimentin (10-nm gold par ticles). Gold particles (5 nm) for desmin are distributed in part of the thick filaments of the sheathed capillars' (arrosvheads). Gold particles (10 nm) for vimentin arc located mostly in the thick filaments in the sheathed capillary, x 53.000. Fig. 9. The lateral process of the sheathed capillary: double inununogold lab eling for actin (5-nm gold particles) and vimentin (10-nm gold particles). Gold par ticles (5 nm) for actin arc distributed just beneath the cell membrane of lateral process of the sheathed capillary (arrosvheads). Gold particles (10 nm) for vimentin arc located in the center of the lateral process. X66,000.
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the thick filaments in the sheathed capillary (fig.8). After double staining, both the large and small colloidal gold par ticles representing vimentin- and actin-like immunoreactivity were found in the lateral processes of sheathed capillary. The large colloidal gold particles were located in the center of the lateral process, while the small colloidal gold par ticles were located just beneath the cell membrane of the lateral process (fig.9).
Takehana/Abc/Yamaguchi/Uchida/Inagaki/ Yamamoto/Masty/Winnard/Ueda/Miyata
Cytoplasmic Filaments of Sheathed Capillary
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sivelv in the specific electron-dense granules (fig.6). In sec tions that were stained using anti-desmin serum, part of the thick filaments in the sheathed capillary showed a positive reaction (fig. 7). After double staining, both the large and small colloidal gold particles representing immunoreactivity of both vimentin and desmin were found in the sheathed capillary. The large colloidal gold particles were located mostly in the thick filaments of the sheathed capillary, while small colloidal gold particles were located in part of
Immunostaming of the intermediate and thin filaments was abolished or greatly reduced only by the preabsorption of the antisera with their target antigens. Preabsorption with unmatched antigens had almost no effect on the immunocytochcmical reaction.
Discussion Endothelial cells of sheathed capillaries, which contain bundles of cytoplasmic filaments, have been reported by several authors [2.4, 7, 8|. In general, it is well known that filamentous structures in the cell are classified into four types: thick filaments (myosin filament), thin filaments (actin filament), intermediate filaments and microtubules. Nonmuscle cells have thin and intermediate filaments, but usually no thick filaments. In the endothelial cell of sheathed capillaries in the porcine spleen, intermediate filaments predominated, and thin filaments occurred just beneath the cell membrane. This distribution of interme diate and thin filaments in endothelial cells of sheathed capillaries is similar to that of other arterial endothelial cells as described by Chen and Weiss [6]. Yohro and Burnstock [19]. Lauweryns ct al. [20], and Blue and Weiss [5, 9]. Frank ct al. [ 14j suggested that the intermediate filaments in endothelial cells were composed of vimentin. the major protein of intermediate filaments in cells of mesenchymal origin. This is the first study to demonstrate the identification of vcmentin and desmin. and localization of vimentin and
actin in the endothelial cell of the sheathed capillary in the porcine spleen. Intermediate filaments are not contractile; instead they function as part of the cytoskelcton to main tain cell shape and resist mechanical stress [21]. In the endothelium, the intermediate filaments may provide an elastic supporting structure which allows configurational changes, thereby resisting intravascular pressure changes [5,9]. These noncontractile filaments function as the endo thelial cytoskeleton. Huxley [12| reported that actin filaments isolated from skeletal muscle display characteristic arrowhead structures, when treated with HMM. and are negatively stained. Ishikawa ct al. 113] adopted this technique for sectioned prep arations and found HMM-rcactivc filaments in a variety of cell types in the chick embryo. We have observed HMMreactive filaments in endothelial cells of sheathed capillar ies in the porcine spleen. The presence of thin filaments in endothelial cells of sheathed capillaries may be partially responsible for the control of patency of the interendothelial fenestrae and. thus, cell passage from the sheathed cap illary to the reticular meshwork in the red pulp.
Acknowledgments We wish to thank Dr. Kenji Kvasa and Dr. Takeo Hiraga for their kind assistance. This research was supported in part by the American Heart Association, and the Canine Research Fund (M.Y.). Kuribayashi Ikuei Gakujutsu Zaidan (K.T.). Hokkaido. Japan.
References 6 Chen I T. Weiss IF .lec tro n microscopy of the red pulp of human spleen. Am J Altai 1972: 134:425-458. 7 I latac T: Electron microscopic studies on the ellipsoid of the cat spleen with special reference to the filaments in the endothelial cell. Arch llislol Cytol 1978:41:177-186. 8 Tablin F. Weiss L: The equine spleen. An elec tron microscopic analysis. Am .1 Anat 1983; 166:393-416. 9 Blue J. W'eiss L: Electron microscopy of the red pulp of the dog spleen including vas cular arrangements, periarterial macrophage sheaths (ellipsoids) and the contractile inner vated reticular meshwork. Am J Anat 1981: 161:189-218. 10 Burton RP. Kirkland. Wl.: Actin detected in mouse neuroblastoma cells by binding of heavy mcromyosin. Nature 1972:239:244-245.
11 Phelps PC. Luft Jll: Electron microscopical study of relaxation and constriction in frog arterioles. Am .1 Anal 1964:125:399-428. 12 Huxley HE: Electron microscope studies on structure of natural and synthetic protein fila ments from striated muscle. .1 Mol Biol 1963: 7:281-308. 13 Ishikawa H. Bisehoff R. Hollzer H: Formation of arrowhead complexes with heavy meroirtyosin in a variety of the cell types. J Cell Biol 1968:43:312-328. 14 Frank WW. Schmid E. Osborn M. Weber K: Intermediate-sized filaments of human endo thelial cells. J Cell Biol 1979:81:570-580. 15 Uchida T: Serotonin-like immunoreactivity in the taste bud of the mouse circumvallate pa pilla tin Japanese with English abstract). Jpn .1 Oral Biol 1985:27:132-139.
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1 Billroth I : Be it rage zur vcrgleichenden Histo logic der Milz. Virchows Arch |B | 1857:88: 88-108. 2 Scki A. Abe M: Scanning electron microscopic studies on the microvascular system of the spleen in the rat. cat. dog. pig. horse and cow. Jpn J Vet Sci 1985:47:237-249. 3 Imai I : Programs on the sheathed capillary in the spleen: Mainly histological observations. Reports of Blood Discussion Association. 7th ser. Osaka. 1954. pp 60-86 (in Japanese). 4 Abe M. Takchana K. Iwasa K. I tiragaT: Scan ning electron microscopic studies on the red pulp of the mink spleen. Jpn .1 Vet Sci 1989: 51:775-781. 5 Blue .1. Weiss L: Periarterial macrophage sheaths (ellipsoids) in cat spleen - an electron microscope study. Am J Anal 1981:161: 115-134.
19 Yohro T. Burnstock B: Filament bundles and contractility of endothelial cells in coronary arteries. Z Zellforsch 1973:138:85-95. 20 t.auweryns JM. Bear! J. DeLoccker W: Intracytoplasinic filaments in pulmonary lymphatic endothelial cells. Fine structure and reaction after heavy mcromyosin incubation. Cell Tis sue Res 1975:163:111-124.
21 Eriksson A. Thorncll LE: Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cvtoskeietal function. J Cell Biol 1979:80:231—247.
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16 Inagaki M. Nishi Y, Nishizawa K, Matsuyama M, Sato C: Site-specific phosphorylation induced disassembly of vimentin filaments in vitro. Nature 1987:328:649-652. 17 Slot JW. Geuse HJ: A new method of prepar ing gold probes for multiple-labeling cyto chemistry. Eur J Cell Biol 1985:38:87-93. 18 Dc May J: Colloidal gold probes in immunocytochemistry: in Polak JM. Van Noorden S (cd): Immunocytochemistry. Practical Applications in Pathology and Biology. Bristol. Wright. 1983. pp 82-112.
" Department of Veterinary Anatomy and h Department of Food Science. Rakuno Gakuen University, Ebetsu. Hokkaido, Japan: c Department of Veterinary Anatomy and Cellular Biology. Ohio State University, Columbus, Ohio. USA: 11 Department of Anatomy. Yamanashi Medical College. Tamaho. Yamanashi, Japan c Aichi Cancer Center Research Institute. Nagoya, Japan
Cytoplasmic Filaments in the Endothelial Cells of the Sheathed Capillary: An Ultrastructural and Immunocytochemical Study in the Pig Spleen
Key Words
Abstract
Intracytoplasmic filament Vimentin Desmin Actin filament Sheathed eapillary endothelial eell Spleen Pig
Cytoplasmic filaments of the endothelial cells of sheathed capillaries in the pig spleen were identified and their ultrastructure was studied. Two types of cyto plasmic filaments were found: intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells, and thin filaments (diameter: 5 nm) which were located just beneath the cell membrane and filled the lateral cytoplasmic processes. In immunocytochemical preparations, the intermediate filaments were positive for vimentin and desmin. and were negative for keratin. Staining of the thin filaments with heavy meromyosin resulted in arrowhead formations. These observations suggest that the intermediate filaments maintain the cytoarchitecture, possibly protecting the cell from structural alterations induced by blood pressure changes. Concurrently, thin filaments may facilitate the passage of red blood cells and blood platelets through the intcrendothelial fenestrae of the sheathed endothelial cell to the reticular meshwork in the capillary sheath.
Introduction Since the first report by Billroth [1], it is known that sheathed capillaries are located between the penicilli and the arterial capillaries in the spleen. Sheathed capillaries arc believed to have a filtering function [2,3]. The sheathed capillary has been studied frequently in many species. In reported ultrastructural studies, endothelial cells of sheathed capillaries are elongated with tapered ends and lie parallel to the long axis of the luminal surface [2.4-8]. In all
Received: September 6. 1991 Accepted: November 7, 1991
species, the endothelial cells of sheathed capillaries have the same cytoplasmic filaments, cell organelles, shape and orientation [2. 5-9]. It has been reported that endothelial cells contain a large number of intermediate filaments [2, 4-9], but there arc no reports describing cytoplasmic filaments in the endothelial cells of sheathed capillaries. Generally, cytoplasmic filaments in nonmuscle cells were classified into two categories: thin filaments and inter mediate filaments. A variety of endothelial cells contain fine cytoplasmic filaments which closely resemble the myo-
Kazushige Takehana Department of Veterinary Anatomy Rakuno Gakuen University Ebetsu. Hokkaido 069 (Japan)
© 1992 Karger AG. Basel (MXU-5180/92/1434-4)294 $ 2.75/0
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K. Takehana*, M. Abe3, M. Yamaguchic, T. Uchida\ M. Inagakic, K. Yamamotob, J. Masiyc, A. Winnardc, W. Veda3, H. Miyataa
Materials and Methods Sample Source Ten healthy, 6-month-old pigs (100-150 kg), crossbreeds of Landrace. Large White and Hampshire stock, were used in this study. Spleens obtained from the slaughterhouse were excised immediately following death induced by an intravenous injection of sodium pento barbital (40 mg/kg). Preparation o f Permeable Cells The spleen was minced with scissors, and approximately 1 g of minced spleen was homogenized, using a Waring blender, with 20 ml of buffer solution containing 5 milf KCI. 20 mM K-phosphate buffer (pH 6.8). 2 mM dithiothreitol. I mM NaN,. I mM EGTA and I mM EDTA. for 10 sat 4°C. After centrifugation (Beckman J-21. roter 30) at 15.000 rpm for 3 min. the supernatant was discarded, and 20 ml of the buffer solution were added to the precipitate. Homogenization and centrifugation were repeated 5 times at 4 °C. Staining with HMM Rabbit skeletal muscle HMM was obtained by established meth ods [ 13]. A portion of the pellet of the permeable cells was mixed with an equal volume of HMM (4 mg/ml) in 67 mM K-phosphate buffer (pH 7.0). incubated for 18 h at 25 °C. and occasionally gently stirred. Excess HMM was removed by centrifugation and washing 4 times with buffer solution.
Thin-Section Electron Microscopy The permeable cells and HMM-staincd permeable cells were fixed with 3.0% glutaraldehyde in 0.1 M cacodvlate buffer, pH 7.2, for 2 h at 4 °C. Pellets were postfixed in 1.0% osmium tetroxidc for 60 min at room temperature, dehydrated and embedded in Epon 812/Araldite. For conventional electron microscopy, small pieces of spleen were processed as above. Samples were sectioned with an LKB Ultratome 111 with a diamond knife. Sections were stained first in uranyl acetate and then in lead citrate. The sections were then examined under an II300. HU-11DS or JEM-100S electron microscope calibrated with a standard measuring grid at an accelerating voltage of 75 or 80 kV. Tissue Preparation fo r Immunohistochemistry The spleens were cut into small pieces ( l x l x l mm) and fixed in 4.0% paraformaldehyde buffered with 0.067 M Na-phosphate (pH 7.4) for approximately 2 h at room temperature. After washing with the same buffer, the specimens were dehydrated in graded N.N-dimethylformamide at progressively lower temperatures and were embedded in glycol methacrylate (CiMA) at -20 °C [15]. Antisera Antisera to vimentin. desmin and keratin sera were prepared by the method described by Inagaki et al. 116], The vimentin, desmin and keratin antisera that were used recognize only the intermediate fila ment proteins vimentin. desmin and keratin, respectively, and do not cross-reacl with other intermediate filament proteins. The three anti sera were used at dilutions of 1,000 (vimentin). 250 (desmin) and 500 (keratin). The actin antiserum was purchased from Transformation Research Inc. (Framingham. USA) and used at a 500 dilution. As the secondary antibody, gold-labeled immunoglobulins to rab bit were used. Colloidal gold particles (average diameter 5 or 10 nm) were made by the chemical reduction of chloroauric acid by tannic acid and trisodium citrate [ 17|. The linking of colloidal gold particles to immunoglobulins (goat anti-rabbit, rabbit anti-chicken or rabbit anti-mouse IgG) was performed as previously described [ 18].
Immunohistochemical Labeling For the detection of antigenic sites, single- and double-labeling immuno-gold methods were employed. For the single labeling, ultrathin sections mounted on nickel grids were first immersed for 15 min at room temperature in phosphate-buffered saline (PBS. 10 mM Naphosphate. 0.15 M NaCI. pH 7.4) containing 2% normal serum (same species as the second antibody) and 1% bovine serum albumin (PBSNS-BSA). and then incubated overnight at 4 °C with primary antise rum diluted with PBA-NS-BSA. After incubation, the sections were rinsed with PBS and immersed in the 10-nm gold-labeled secondary antibody diluted with PBS-NS-BSA for 40 min at room temperature. For the double labeling, the sections were first stained with either antiactin or anti-desmin antisera, and 5-nm gold-labeled second antibody, as above. After the initial labeling, the sections were rinsed with PBS and stained with anti-vimentin antiserum and 10-nm gold-labeled sec ondary antibody, as above. After immunostaining, the sections were washed with PBS. rinsed with distilled water and air dried. Finally, the sections were stained with uranyl acetate followed by lead citrate, and were then examined. Immunohistochemical specificity was checked by the absorption test. Diluted antisera were preincubated overnight at 4 °C with puri fied vimentin (10 pg/ml) before application to the tissue sections.
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filaments of muscle cells 110, 111. Some of these cytoplas mic filaments can be stained with heavy meromyosin (HMM). indicating the presence of actin filaments 112. 13]. Intermediate filaments have been classified into five types: keratin, neurofilament protein, glial fibrillary acidic pro tein. desmin and vimentin. We have attempted to char acterize the cytoplasmic filaments in the endothelial cells of sheathed capillaries by immune electron microscopy, using antibodies against the major constituent proteins of the fol lowing three different classes of intermediate filaments: (a) vimentin. a protein of ~57.0(K) molecular weight (MW), the major constituent of the intermediate filaments of vari ous mesenchymal and mcscnchymal-derived cells; (b) des min. a protein of 53,000 MW, which is found also in cul tured smooth and cardiac muscle cells, in differentiated myoblasts and in myotubules of skeletal muscle, and (c) keratin, a protein of 47.0(K)-58.0(K) MW. the predominant type of intermediate-sized filament occurring in various epithelial and epithelium-derived cells [14], In this study, we examined the nature of the cytoplasmic filaments in the porcine sheathed capillary, by electron microscopy, using a gold-conjugated antibody to identify intermediate and thin filaments, and HMM staining to identify thin filaments.
Fig. 1. The sheathed capillary endothe lial cells ( E) of a pig spleen containing a large number of intermediate filaments, x 7,600. Fig. 2. A large number of intermediate filaments (diameter: HI nm) filled the cyto plasm. x BO.IXKl. Fig. 3. Thin filaments filled the lateral processes (asterisks). An intercellular junc tion (IJ) on the lateral processes is seen, x 44,000.
Endothelial cells of sheathed capillaries in the pig spleen protruded high into the lumen (fig.l). They contained a large number of intermediate filaments (diameter: 10 nm) which filled most of the interior of the cells (fig.2). These filaments were noted in a narrow zone beneath the plasmalemma, in which mitochondria and rough endoplasmic re
296
ticulum were also detected, and in the perinuclear region located midway through the length of the cell. Thin filaments (diameter: 5 nm) formed a felt-like pat tern which occurred just beneath the plasmalemma and filled most of the lateral cytoplasmic processes. Irregularly shaped patches of matted, thin filaments were scattered in the basal cytoplasm and near intercellular junctions (fig-3).
Takchana/Abc/Yamaguchi/Uchida/Inagaki/ Yamamoto/Masty/Winnard/Ueda/Miyata
Cytoplasmic Filaments of Sheathed Capillary
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Results
Fig. 4. Detail of a permeable sheathed capillary endothelial cell containing a large number of intermediate filaments, x 58,000. Fig. 5. Detail of a permeable sheathed capillary endothelial cell after HMM treat ment. Thin filaments (diameter: 5 nm) occurred in the basal cytoplasm (asterisks). Intermediate filaments do not bind HMM (IF). X58.000. Insert: Thin filaments are stained by HMM (arrows). X 110.000.
After HMM treatment, electron-dense structures were located in the basal cytoplasm and near intercellular junc tions (fig.5). They were marked by characteristic arrow head formations (fig.5 insert). There was no detectable reaction of HMM with the thick filaments. Figures 6-9 show the immunohistochemical results. When the sections were stained using anti-vimentin serum, all the thick filaments in the sheathed capillary showed a positive reaction. The immunoreactivity was located cxclu-
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A large number of thick anti thin filaments were densely packed in the cytoplasm of the endothelial cells in the sheathed capillaries, thus, it was very difficult to analyze or infer any relationships, and distinguish between interme diate and thin filaments by conventional thin-section elec tron microscopy. After permeabilization. the cytoplasm of sheathed capillary endothelial cells was much less electron dense, thus facilitating identification of the intracellular filaments (fig.4).
Fig. 6, 7. An electron micrograph of the immunostained sheathed capillary endothe lial cell, x 53.000 . 6 Vimentin (10-nm gold particles) - all the intermediate filaments in an endothelial cell are positively stained. 7 Desmin - a part of the intermediate fila ments in an endothelial cell is positively stained. Fig. 8. An electron micrograph of the sheathed capillars' endothelial cell: double inununogold labeling for desmin (5-nm gold particles) and vimentin (10-nm gold par ticles). Gold particles (5 nm) for desmin are distributed in part of the thick filaments of the sheathed capillars' (arrosvheads). Gold particles (10 nm) for vimentin arc located mostly in the thick filaments in the sheathed capillary, x 53.000. Fig. 9. The lateral process of the sheathed capillary: double inununogold lab eling for actin (5-nm gold particles) and vimentin (10-nm gold particles). Gold par ticles (5 nm) for actin arc distributed just beneath the cell membrane of lateral process of the sheathed capillary (arrosvheads). Gold particles (10 nm) for vimentin arc located in the center of the lateral process. X66,000.
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the thick filaments in the sheathed capillary (fig.8). After double staining, both the large and small colloidal gold par ticles representing vimentin- and actin-like immunoreactivity were found in the lateral processes of sheathed capillary. The large colloidal gold particles were located in the center of the lateral process, while the small colloidal gold par ticles were located just beneath the cell membrane of the lateral process (fig.9).
Takehana/Abc/Yamaguchi/Uchida/Inagaki/ Yamamoto/Masty/Winnard/Ueda/Miyata
Cytoplasmic Filaments of Sheathed Capillary
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sivelv in the specific electron-dense granules (fig.6). In sec tions that were stained using anti-desmin serum, part of the thick filaments in the sheathed capillary showed a positive reaction (fig. 7). After double staining, both the large and small colloidal gold particles representing immunoreactivity of both vimentin and desmin were found in the sheathed capillary. The large colloidal gold particles were located mostly in the thick filaments of the sheathed capillary, while small colloidal gold particles were located in part of
Immunostaming of the intermediate and thin filaments was abolished or greatly reduced only by the preabsorption of the antisera with their target antigens. Preabsorption with unmatched antigens had almost no effect on the immunocytochcmical reaction.
Discussion Endothelial cells of sheathed capillaries, which contain bundles of cytoplasmic filaments, have been reported by several authors [2.4, 7, 8|. In general, it is well known that filamentous structures in the cell are classified into four types: thick filaments (myosin filament), thin filaments (actin filament), intermediate filaments and microtubules. Nonmuscle cells have thin and intermediate filaments, but usually no thick filaments. In the endothelial cell of sheathed capillaries in the porcine spleen, intermediate filaments predominated, and thin filaments occurred just beneath the cell membrane. This distribution of interme diate and thin filaments in endothelial cells of sheathed capillaries is similar to that of other arterial endothelial cells as described by Chen and Weiss [6]. Yohro and Burnstock [19]. Lauweryns ct al. [20], and Blue and Weiss [5, 9]. Frank ct al. [ 14j suggested that the intermediate filaments in endothelial cells were composed of vimentin. the major protein of intermediate filaments in cells of mesenchymal origin. This is the first study to demonstrate the identification of vcmentin and desmin. and localization of vimentin and
actin in the endothelial cell of the sheathed capillary in the porcine spleen. Intermediate filaments are not contractile; instead they function as part of the cytoskelcton to main tain cell shape and resist mechanical stress [21]. In the endothelium, the intermediate filaments may provide an elastic supporting structure which allows configurational changes, thereby resisting intravascular pressure changes [5,9]. These noncontractile filaments function as the endo thelial cytoskeleton. Huxley [12| reported that actin filaments isolated from skeletal muscle display characteristic arrowhead structures, when treated with HMM. and are negatively stained. Ishikawa ct al. 113] adopted this technique for sectioned prep arations and found HMM-rcactivc filaments in a variety of cell types in the chick embryo. We have observed HMMreactive filaments in endothelial cells of sheathed capillar ies in the porcine spleen. The presence of thin filaments in endothelial cells of sheathed capillaries may be partially responsible for the control of patency of the interendothelial fenestrae and. thus, cell passage from the sheathed cap illary to the reticular meshwork in the red pulp.
Acknowledgments We wish to thank Dr. Kenji Kvasa and Dr. Takeo Hiraga for their kind assistance. This research was supported in part by the American Heart Association, and the Canine Research Fund (M.Y.). Kuribayashi Ikuei Gakujutsu Zaidan (K.T.). Hokkaido. Japan.
References 6 Chen I T. Weiss IF .lec tro n microscopy of the red pulp of human spleen. Am J Altai 1972: 134:425-458. 7 I latac T: Electron microscopic studies on the ellipsoid of the cat spleen with special reference to the filaments in the endothelial cell. Arch llislol Cytol 1978:41:177-186. 8 Tablin F. Weiss L: The equine spleen. An elec tron microscopic analysis. Am .1 Anat 1983; 166:393-416. 9 Blue J. W'eiss L: Electron microscopy of the red pulp of the dog spleen including vas cular arrangements, periarterial macrophage sheaths (ellipsoids) and the contractile inner vated reticular meshwork. Am J Anat 1981: 161:189-218. 10 Burton RP. Kirkland. Wl.: Actin detected in mouse neuroblastoma cells by binding of heavy mcromyosin. Nature 1972:239:244-245.
11 Phelps PC. Luft Jll: Electron microscopical study of relaxation and constriction in frog arterioles. Am .1 Anal 1964:125:399-428. 12 Huxley HE: Electron microscope studies on structure of natural and synthetic protein fila ments from striated muscle. .1 Mol Biol 1963: 7:281-308. 13 Ishikawa H. Bisehoff R. Hollzer H: Formation of arrowhead complexes with heavy meroirtyosin in a variety of the cell types. J Cell Biol 1968:43:312-328. 14 Frank WW. Schmid E. Osborn M. Weber K: Intermediate-sized filaments of human endo thelial cells. J Cell Biol 1979:81:570-580. 15 Uchida T: Serotonin-like immunoreactivity in the taste bud of the mouse circumvallate pa pilla tin Japanese with English abstract). Jpn .1 Oral Biol 1985:27:132-139.
299
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1 Billroth I : Be it rage zur vcrgleichenden Histo logic der Milz. Virchows Arch |B | 1857:88: 88-108. 2 Scki A. Abe M: Scanning electron microscopic studies on the microvascular system of the spleen in the rat. cat. dog. pig. horse and cow. Jpn J Vet Sci 1985:47:237-249. 3 Imai I : Programs on the sheathed capillary in the spleen: Mainly histological observations. Reports of Blood Discussion Association. 7th ser. Osaka. 1954. pp 60-86 (in Japanese). 4 Abe M. Takchana K. Iwasa K. I tiragaT: Scan ning electron microscopic studies on the red pulp of the mink spleen. Jpn .1 Vet Sci 1989: 51:775-781. 5 Blue .1. Weiss L: Periarterial macrophage sheaths (ellipsoids) in cat spleen - an electron microscope study. Am J Anal 1981:161: 115-134.
19 Yohro T. Burnstock B: Filament bundles and contractility of endothelial cells in coronary arteries. Z Zellforsch 1973:138:85-95. 20 t.auweryns JM. Bear! J. DeLoccker W: Intracytoplasinic filaments in pulmonary lymphatic endothelial cells. Fine structure and reaction after heavy mcromyosin incubation. Cell Tis sue Res 1975:163:111-124.
21 Eriksson A. Thorncll LE: Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cvtoskeietal function. J Cell Biol 1979:80:231—247.
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16 Inagaki M. Nishi Y, Nishizawa K, Matsuyama M, Sato C: Site-specific phosphorylation induced disassembly of vimentin filaments in vitro. Nature 1987:328:649-652. 17 Slot JW. Geuse HJ: A new method of prepar ing gold probes for multiple-labeling cyto chemistry. Eur J Cell Biol 1985:38:87-93. 18 Dc May J: Colloidal gold probes in immunocytochemistry: in Polak JM. Van Noorden S (cd): Immunocytochemistry. Practical Applications in Pathology and Biology. Bristol. Wright. 1983. pp 82-112.
