THE ANATOMICAL RECORD 232:121-125 (1992)
Anti-Vimentin Monoclonal Antibody Recognizes a Cell With Dendritic Appearance in the Chicken’s Bursa of Fabricius IMRE OLAH, CYNTHIA KENDALL, AND BRUCE GLICK Poultry Science Department, Clemson University, Clemson, South Carolina
ABSTRACT The bursa of Fabricius was studied by immunohistochemical method using anti-vimentin monoclonal antibody (clone 3B4). This monoclonal antibody identified a vimentin positive cell in the medulla of the bursal follicle. During the first 2 weeks of life the vimentin positive cells located along the corticomedullary border and later became prominent in the medulla with the exception of a narrow zone adjacent to the corticomedullary border. After hatching the accumulation of vimentin-type intermediate filaments on one side of the nucleus endowed the vimentin positive cells with a polarized appearance. This “cap-like” vimentin positive area of the cytoplasm determined the position of the major cell process. Within the medulla the Ia positive secretory dendritic cells contained secretory granules in one of the cell processes. The distribution, shape, and polarized appearance of the vimentin positive cells were identical with that of the secretory dendritic cells. Therefore, the anti-vimentin monoclonal antibody proved to be useful for identification of the bursal secretory dendritic cells. During rapid bursal growth the number of secretory dendritic cells increased, possibly, by proliferation of vimentin negative secretory dendritic cell precursors located along the corticomedullary border. Mammalian Langerhans’ cells, a type of dendritic not longer than 2 weeks. After rehydration in phoscell, are located among squamous epithelium. They are phate-buffered saline (PBS), the endogenous peroxiof bone marrow origin (Freilinger et al., 1979; Katz et dase was developed by 3‘ 3’-diaminobenzidine which al., 1979) and contained vimentin type intermediate gave a brown color reaction. Sections were washed in filaments (DeWaal et al., 1984; Mahrhe et al., 1983; PBS for 10 minutes and incubated with primary antiRappensberger e t al., 1990). Previously, we identified body or irrelevant supernatant, e.g., mouse serum conin the medulla of the bursal follicles a novel secretory taining IgG, which served as a control. The unbound cell which was a modified dendritic cell (Olah et al., antibodies were removed by a PBS wash and the sec1979; Olah and Glick, 1978, 1986,1987). The secretory tions were covered with biotinylated secondary antidendritic cells (SDC) differentiated from the bursal body. Phosphate-buffered saline washing was followed mesenchyme (Glick and Olah, 1987; Olah et al., 19861, by incubation with ABC complex. The primary antiwhich was of mesodermal derivative like bone marrow. body binding sites were visualized by 4-chloroTherefore, we speculated that the SDC might possess naphthol, which served as a substrate for the exogevimentin type intermediate filaments (VIF) like the nous peroxidase. This compound produced blue-grey Langerhans’ cells. In this paper we report that vimen- deposits. All incubations were performed in a moisture tin positive cells (ViPC) may be identical with the SDC. chamber at room temperature for 30 minutes and a water soluble mounting media was used to affix cover MATERIALS AND METHODS slips. A New Hampshire breed of chicken, selected for a RESULTS small bursa size, was used in these experiments (Glick Anti-vimentin mAb identified ViPC in the bursa meand Dreesen, 1967). Three chickens per age were sampled a t hatch and 3, 14, 63, and 72 days of age. The dulla at every age studied (Figs. 1,2,3). After the cortex animals were maintained according to institutional was developed the mesenchymal reticular cells expressed VIF (Figs. 3,4). The identification and denguidelines. Anti-vimentin monoclonal antibody (mAb) clone 3B4 dritic shape of the ViPC were made more evident since (Boehringer-Mannheim GmbH, Indianapolis, IN) and medullary and cortical B-lymphocytes failed to express anti chicken B-L (Ia)mAb (gift from Dr. Cooper and Dr. Chen, Birmingham, AL) were used. Biotinylated anti mouse IgG (H + L) and ABC kit were purchased from Vector Laboratories (Burlingame, CA). Received January 17, 1991; accepted June 13, 1991. Bursae were frozen in liquid nitrogen and 10-12 pm Dr. Olah is on leave from the 2nd Department of Anatomy, Semthick cryostat sections were immediately fixed in ace- melweis University Medical School, Budapest, Hungary. tone for 1-2 minutes. The air-dryed sections were Address reprint requests to Dr. Bruce Glick, Department of Poultry stored in a closed container (4”C) until staining, but Science, Clemson University, Clemson, SC 29634-0379. 0 1992 WILEY-LISS, INC.
Fig. 1. Three-day-old bird. The interfollicular connective tissue was highly positive for vimentin. The ViPC were dispersed within the medulla. X 140. Fig. 2. Fourteen-day-old bird. The increased number of ViPC was localized parallel with the corticomedullary border. The weakly stained cortex can be recognized on several follicles. x 140.
Fig. 3. Sixty-three-day-old bird. The ViPC occupied the medulla of epithelial origin which was surrounded by the cortex of mesenchymal origin. The cortex was “disrupted’ at the follicular associated epithelium (FAE). x 90.
A DENDRITIC CELL IN THE BURSA
Fig. 4. Seventy-two-day-old birds. The area adjacent to the corticomedullary epithelial cells was free of ViPC. The cortex showed a well-developed vimentin positive mesenchymal network. X 140. Fig. 5. Three-day-old bird. The vimentin positive intermediate filaments appeared in the perinuclear region. They accumulated on one side of the nucleus, which endowed the cells with a polarized appearance. x 560.
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Fig. 6. Sixty-three-day-old bird. The majority of the ViPC produced one and/or two vimentin positive cell processes. X 380. Fig. 7. Seventy-two-day-oldbird. ViPC contained one, two or three cell processes. In general, one of the cell processes was thinner and contained much less VIF than the other. x 560.
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Fig. 8. Sixty-three-day-old bird. The SDC were scattered over the medulla (arrow). The secretory granules occupy one of the cell processes. Some SDC showed a heavily ruffled surface membrane (arrowhead). x 1,200. Fig. 9. Seventy-two-day-old bird. Anti Ia (class 11,B-L) mAb stained the cortical cells while in the medulla only scattered positive cells
occur. Many Ia positive cells appeared parallel to the corticomedullary border. x 140. Fig. 10. Seventy-two-day-old bird. The medullary location of the Ia positive cells was identical to the ViPC. x 380.
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vimentin. Control sections did not stain with mouse serum and biotinylated secondary antibody. At day 3, the ViPC were scattered over the medullary area (Fig. 1). The accumulation of VIF on one side of the nucleus endowed the ViPC with a polarized appearance (Fig. 5). By day 14 the number of ViPC increased markedly and the majority were arranged parallel with the corticomedullary border (Fig. 2). Extending from the vimentin positive cytoplasmic cap was a long cell process which also contained VIF. The number of ViPC continued to increase after 2 weeks of age. There was a zone free of cells adjacent to the corticomedullary border (Fig. 4). At 2 weeks of age the mesenchymal reticular cells of the cortex stained poorly with anti-vimentin mAb (Fig. 2). The network of cortical mesenchymal cells in older birds expressed vimentin (Fig. 4).The ViPC assumed three different cell shapes (Fig. 6). The most frequent cell shape expressed one long process (Figs. 6,7) apparently originating from the early vimentin accumulation (Fig. 5). The other ViPC types possessed cell processes on opposites sides of the nucleus or occasionally three processes (Figs. 6,7). Secretory dendritic cells, like ViPC, produced one or two cell processes and only one of the processes contained cytoplasmic granules (Fig. 8). The bursal cortex and medulla were heavily and sparsely populated, with Ia positive cells, respectively (Fig. 9). The medullary location of the heavily stained Ia positive cells was identical to the ViPC (Fig. 10). DISCUSSION
Anti-vimentin mAb identified a cell with dendritic appearance in the medulla of bursal follicles. After hatching the VIF accumulated on one side of the nucleus endowing the cells with a highly polarized appearance. During the first 2 weeks of life the ViPC produced one to three cell processes. These cell processes were unequal in size and variable in their vimentin expression. The frequency, location, shape, and polarized appearance of the ViPC were very similar to that of the Ia positive SDC which suggests that antivimentin mAb identified SDC in the bursal follicle. Therefore, anti-vimentin mAb proved to be a useful reagent for identification of the SDC. While it may be possible that a n occasional bursal macrophage could be vimentin positive in older birds, 63 and 72 days of age, no macrophages, based on light microscopy, associated with ViPC in the bursal medulla of younger birds, 3 and 14 days of age. The tissue-specific VIF (Klymkowsky et al., 1989; Lazarides, 1982; Steinert and Liem, 1990; Steinert and Roop, 1988) supported the mesenchymal origin of the SDC as previously suggested (Glick and Olah, 1987; Olah e t al., 1986). The vimentin and Ia positive SDC in the epithelial environment of the follicular medulla might be comparable with the mammalian Langerhand cell, which is one type of dendritic cell.
During the rapid period of bursal growth, the first 2 weeks of life, the SDC were localized along the corticomedullary border and later when their number significantly increased they covered the entire medulla. This finding supports the possibility previously suggested (Olah, et al., 1979) that the corticomedullary border is a generative zone for the SDC. ACKNOWLEDGMENTS
We thank Ms. Gloria Freeman for typing the manuscript. The research was supported, in part, by USDA grant 38-34116-3652 and the Animal Biotechnology Program, “Functional Enhancement of the Immune System During Embryonic Development” of the College of Agricultural Sciences. This is Technical Contribution number 3142 of the South Carolina Agricultural Experiment Station, Clemson University. LITERATURE CITED DeWaal, R.M.W., J.T. Semeiju, I.M.H. Cornelissen, and F.C.S. Ramaekers 1984 Epidermal Langerhans cells contain intermediatesized filaments of the vimentin type: An immunocytologic study. J . Invest. Dermatol., 82:602. Freilinger, J.G., L. Hood, S. Hill, and J.A. Frailinger 1979 Mouse epidermal Ia molecules have a bone marrow origin. Nature, 282: 321-323. Glick, B., and L.J. Dreesen 1967 The influence of selecting for large and small bursa size on adrenal, spleen, and thymus weights. Poultry Sci., 46t396-402. Glick, B., and I. Olah 1987 Contribution of a specialized dendritic cell, the secretorv cell. to the microenvironment of the bursa of Fabriclus. In: A”vian Immunology. W.T. Weber and D.L. Ewert, eds. Alan R. Liss Inc., New York, pp. 53-66. Katz, S.I., K. Tamaki, and D.H. Sachs 1979 Epidermal Langerhans cells are derived from cells originating in the bone marrow. Nature, 282:324-326. Klymkowsky, M.W., J.B. Bachant, and A. Doming0 1989 Functions of intermediate filaments. Cell Motil. Cytoskeleton, f4t309-311. Lazarides, E. 1982 Intermediate filaments: A chemically heterogeneous, developmentally regulated class of proteins. Annu. Rev. Biochem., 51:219-250. Mahrhe, G., R. Bolling, M. Osborn, and K. Weber 1983 Intermediate filaments of the vimentin and prekeratin type in human epidermis. J. Invest. Dermatol., 81:46-48. Olah, I., and B. Glick 1978 Secretory cell in the medulla of the bursa of Fabricius. Experientia, 34:1642-1643. Olah, I., and B. Glick 1986 Effect of carrageenan on the histology of bursa of Fabricius and the humoral immune response to Salmonella 0 antigen. Anat. Rec., 2f4r398-404. Olah, I., and B. Glick 1987 Bursal secretory cells: An electron microscope study. Anat. Rec., 219:268-274. Olah, I., B. Glick, F. McCorkle, and R. Stinson 1979 Light and electron microscopic structure of secretory cells in the medulla of bursal follicles of normal and cyclophosphamide treated chickens. Dev. Comp. Immunol., 3:101-115. Olah, I., B. Glick, and I. Tor0 1986 Bursal development in normal and testosterone-treated chick embryos. Poultry Sci., 65574-588. Rappensberger, K., M. Binder, E. Zonzits, U. Hornick, and K. Wolff 1990 Immunogold staining of intermediate-sized filaments of the vimentin type in human skin: A postembedding immunoelectron microscopic study. J . Invest. Dermatol., 94:700-705. Steinert, P.M., and D.R. Roop 1988 Molecular and cellular biology of intermediate filaments. Annu. Rev. Biochem.. 57.593-625. Steinert, P.M., and R.K.H. Liem 1990 Intermediate filament dynamics. Cell, 60521-523.
Anti-Vimentin Monoclonal Antibody Recognizes a Cell With Dendritic Appearance in the Chicken’s Bursa of Fabricius IMRE OLAH, CYNTHIA KENDALL, AND BRUCE GLICK Poultry Science Department, Clemson University, Clemson, South Carolina
ABSTRACT The bursa of Fabricius was studied by immunohistochemical method using anti-vimentin monoclonal antibody (clone 3B4). This monoclonal antibody identified a vimentin positive cell in the medulla of the bursal follicle. During the first 2 weeks of life the vimentin positive cells located along the corticomedullary border and later became prominent in the medulla with the exception of a narrow zone adjacent to the corticomedullary border. After hatching the accumulation of vimentin-type intermediate filaments on one side of the nucleus endowed the vimentin positive cells with a polarized appearance. This “cap-like” vimentin positive area of the cytoplasm determined the position of the major cell process. Within the medulla the Ia positive secretory dendritic cells contained secretory granules in one of the cell processes. The distribution, shape, and polarized appearance of the vimentin positive cells were identical with that of the secretory dendritic cells. Therefore, the anti-vimentin monoclonal antibody proved to be useful for identification of the bursal secretory dendritic cells. During rapid bursal growth the number of secretory dendritic cells increased, possibly, by proliferation of vimentin negative secretory dendritic cell precursors located along the corticomedullary border. Mammalian Langerhans’ cells, a type of dendritic not longer than 2 weeks. After rehydration in phoscell, are located among squamous epithelium. They are phate-buffered saline (PBS), the endogenous peroxiof bone marrow origin (Freilinger et al., 1979; Katz et dase was developed by 3‘ 3’-diaminobenzidine which al., 1979) and contained vimentin type intermediate gave a brown color reaction. Sections were washed in filaments (DeWaal et al., 1984; Mahrhe et al., 1983; PBS for 10 minutes and incubated with primary antiRappensberger e t al., 1990). Previously, we identified body or irrelevant supernatant, e.g., mouse serum conin the medulla of the bursal follicles a novel secretory taining IgG, which served as a control. The unbound cell which was a modified dendritic cell (Olah et al., antibodies were removed by a PBS wash and the sec1979; Olah and Glick, 1978, 1986,1987). The secretory tions were covered with biotinylated secondary antidendritic cells (SDC) differentiated from the bursal body. Phosphate-buffered saline washing was followed mesenchyme (Glick and Olah, 1987; Olah et al., 19861, by incubation with ABC complex. The primary antiwhich was of mesodermal derivative like bone marrow. body binding sites were visualized by 4-chloroTherefore, we speculated that the SDC might possess naphthol, which served as a substrate for the exogevimentin type intermediate filaments (VIF) like the nous peroxidase. This compound produced blue-grey Langerhans’ cells. In this paper we report that vimen- deposits. All incubations were performed in a moisture tin positive cells (ViPC) may be identical with the SDC. chamber at room temperature for 30 minutes and a water soluble mounting media was used to affix cover MATERIALS AND METHODS slips. A New Hampshire breed of chicken, selected for a RESULTS small bursa size, was used in these experiments (Glick Anti-vimentin mAb identified ViPC in the bursa meand Dreesen, 1967). Three chickens per age were sampled a t hatch and 3, 14, 63, and 72 days of age. The dulla at every age studied (Figs. 1,2,3). After the cortex animals were maintained according to institutional was developed the mesenchymal reticular cells expressed VIF (Figs. 3,4). The identification and denguidelines. Anti-vimentin monoclonal antibody (mAb) clone 3B4 dritic shape of the ViPC were made more evident since (Boehringer-Mannheim GmbH, Indianapolis, IN) and medullary and cortical B-lymphocytes failed to express anti chicken B-L (Ia)mAb (gift from Dr. Cooper and Dr. Chen, Birmingham, AL) were used. Biotinylated anti mouse IgG (H + L) and ABC kit were purchased from Vector Laboratories (Burlingame, CA). Received January 17, 1991; accepted June 13, 1991. Bursae were frozen in liquid nitrogen and 10-12 pm Dr. Olah is on leave from the 2nd Department of Anatomy, Semthick cryostat sections were immediately fixed in ace- melweis University Medical School, Budapest, Hungary. tone for 1-2 minutes. The air-dryed sections were Address reprint requests to Dr. Bruce Glick, Department of Poultry stored in a closed container (4”C) until staining, but Science, Clemson University, Clemson, SC 29634-0379. 0 1992 WILEY-LISS, INC.
Fig. 1. Three-day-old bird. The interfollicular connective tissue was highly positive for vimentin. The ViPC were dispersed within the medulla. X 140. Fig. 2. Fourteen-day-old bird. The increased number of ViPC was localized parallel with the corticomedullary border. The weakly stained cortex can be recognized on several follicles. x 140.
Fig. 3. Sixty-three-day-old bird. The ViPC occupied the medulla of epithelial origin which was surrounded by the cortex of mesenchymal origin. The cortex was “disrupted’ at the follicular associated epithelium (FAE). x 90.
A DENDRITIC CELL IN THE BURSA
Fig. 4. Seventy-two-day-old birds. The area adjacent to the corticomedullary epithelial cells was free of ViPC. The cortex showed a well-developed vimentin positive mesenchymal network. X 140. Fig. 5. Three-day-old bird. The vimentin positive intermediate filaments appeared in the perinuclear region. They accumulated on one side of the nucleus, which endowed the cells with a polarized appearance. x 560.
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Fig. 6. Sixty-three-day-old bird. The majority of the ViPC produced one and/or two vimentin positive cell processes. X 380. Fig. 7. Seventy-two-day-oldbird. ViPC contained one, two or three cell processes. In general, one of the cell processes was thinner and contained much less VIF than the other. x 560.
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Fig. 8. Sixty-three-day-old bird. The SDC were scattered over the medulla (arrow). The secretory granules occupy one of the cell processes. Some SDC showed a heavily ruffled surface membrane (arrowhead). x 1,200. Fig. 9. Seventy-two-day-old bird. Anti Ia (class 11,B-L) mAb stained the cortical cells while in the medulla only scattered positive cells
occur. Many Ia positive cells appeared parallel to the corticomedullary border. x 140. Fig. 10. Seventy-two-day-old bird. The medullary location of the Ia positive cells was identical to the ViPC. x 380.
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vimentin. Control sections did not stain with mouse serum and biotinylated secondary antibody. At day 3, the ViPC were scattered over the medullary area (Fig. 1). The accumulation of VIF on one side of the nucleus endowed the ViPC with a polarized appearance (Fig. 5). By day 14 the number of ViPC increased markedly and the majority were arranged parallel with the corticomedullary border (Fig. 2). Extending from the vimentin positive cytoplasmic cap was a long cell process which also contained VIF. The number of ViPC continued to increase after 2 weeks of age. There was a zone free of cells adjacent to the corticomedullary border (Fig. 4). At 2 weeks of age the mesenchymal reticular cells of the cortex stained poorly with anti-vimentin mAb (Fig. 2). The network of cortical mesenchymal cells in older birds expressed vimentin (Fig. 4).The ViPC assumed three different cell shapes (Fig. 6). The most frequent cell shape expressed one long process (Figs. 6,7) apparently originating from the early vimentin accumulation (Fig. 5). The other ViPC types possessed cell processes on opposites sides of the nucleus or occasionally three processes (Figs. 6,7). Secretory dendritic cells, like ViPC, produced one or two cell processes and only one of the processes contained cytoplasmic granules (Fig. 8). The bursal cortex and medulla were heavily and sparsely populated, with Ia positive cells, respectively (Fig. 9). The medullary location of the heavily stained Ia positive cells was identical to the ViPC (Fig. 10). DISCUSSION
Anti-vimentin mAb identified a cell with dendritic appearance in the medulla of bursal follicles. After hatching the VIF accumulated on one side of the nucleus endowing the cells with a highly polarized appearance. During the first 2 weeks of life the ViPC produced one to three cell processes. These cell processes were unequal in size and variable in their vimentin expression. The frequency, location, shape, and polarized appearance of the ViPC were very similar to that of the Ia positive SDC which suggests that antivimentin mAb identified SDC in the bursal follicle. Therefore, anti-vimentin mAb proved to be a useful reagent for identification of the SDC. While it may be possible that a n occasional bursal macrophage could be vimentin positive in older birds, 63 and 72 days of age, no macrophages, based on light microscopy, associated with ViPC in the bursal medulla of younger birds, 3 and 14 days of age. The tissue-specific VIF (Klymkowsky et al., 1989; Lazarides, 1982; Steinert and Liem, 1990; Steinert and Roop, 1988) supported the mesenchymal origin of the SDC as previously suggested (Glick and Olah, 1987; Olah e t al., 1986). The vimentin and Ia positive SDC in the epithelial environment of the follicular medulla might be comparable with the mammalian Langerhand cell, which is one type of dendritic cell.
During the rapid period of bursal growth, the first 2 weeks of life, the SDC were localized along the corticomedullary border and later when their number significantly increased they covered the entire medulla. This finding supports the possibility previously suggested (Olah, et al., 1979) that the corticomedullary border is a generative zone for the SDC. ACKNOWLEDGMENTS
We thank Ms. Gloria Freeman for typing the manuscript. The research was supported, in part, by USDA grant 38-34116-3652 and the Animal Biotechnology Program, “Functional Enhancement of the Immune System During Embryonic Development” of the College of Agricultural Sciences. This is Technical Contribution number 3142 of the South Carolina Agricultural Experiment Station, Clemson University. LITERATURE CITED DeWaal, R.M.W., J.T. Semeiju, I.M.H. Cornelissen, and F.C.S. Ramaekers 1984 Epidermal Langerhans cells contain intermediatesized filaments of the vimentin type: An immunocytologic study. J . Invest. Dermatol., 82:602. Freilinger, J.G., L. Hood, S. Hill, and J.A. Frailinger 1979 Mouse epidermal Ia molecules have a bone marrow origin. Nature, 282: 321-323. Glick, B., and L.J. Dreesen 1967 The influence of selecting for large and small bursa size on adrenal, spleen, and thymus weights. Poultry Sci., 46t396-402. Glick, B., and I. Olah 1987 Contribution of a specialized dendritic cell, the secretorv cell. to the microenvironment of the bursa of Fabriclus. In: A”vian Immunology. W.T. Weber and D.L. Ewert, eds. Alan R. Liss Inc., New York, pp. 53-66. Katz, S.I., K. Tamaki, and D.H. Sachs 1979 Epidermal Langerhans cells are derived from cells originating in the bone marrow. Nature, 282:324-326. Klymkowsky, M.W., J.B. Bachant, and A. Doming0 1989 Functions of intermediate filaments. Cell Motil. Cytoskeleton, f4t309-311. Lazarides, E. 1982 Intermediate filaments: A chemically heterogeneous, developmentally regulated class of proteins. Annu. Rev. Biochem., 51:219-250. Mahrhe, G., R. Bolling, M. Osborn, and K. Weber 1983 Intermediate filaments of the vimentin and prekeratin type in human epidermis. J. Invest. Dermatol., 81:46-48. Olah, I., and B. Glick 1978 Secretory cell in the medulla of the bursa of Fabricius. Experientia, 34:1642-1643. Olah, I., and B. Glick 1986 Effect of carrageenan on the histology of bursa of Fabricius and the humoral immune response to Salmonella 0 antigen. Anat. Rec., 2f4r398-404. Olah, I., and B. Glick 1987 Bursal secretory cells: An electron microscope study. Anat. Rec., 219:268-274. Olah, I., B. Glick, F. McCorkle, and R. Stinson 1979 Light and electron microscopic structure of secretory cells in the medulla of bursal follicles of normal and cyclophosphamide treated chickens. Dev. Comp. Immunol., 3:101-115. Olah, I., B. Glick, and I. Tor0 1986 Bursal development in normal and testosterone-treated chick embryos. Poultry Sci., 65574-588. Rappensberger, K., M. Binder, E. Zonzits, U. Hornick, and K. Wolff 1990 Immunogold staining of intermediate-sized filaments of the vimentin type in human skin: A postembedding immunoelectron microscopic study. J . Invest. Dermatol., 94:700-705. Steinert, P.M., and D.R. Roop 1988 Molecular and cellular biology of intermediate filaments. Annu. Rev. Biochem.. 57.593-625. Steinert, P.M., and R.K.H. Liem 1990 Intermediate filament dynamics. Cell, 60521-523.
