Archhes of
Short communications
N NN]N 9 Springer-Verlag 1992
Arch Dermatol Res (1992) 284:303-306
Basal lamina-like material and hemidesmosome-like structures associated with dermal papilla cells in the normal human anagen hair follicle D. J. Tobin
Department of Dermatology, Room H354, New York University Medical Center, New York, NY 10016, USA Received March 17, 1992
Key words: Hair follicle - Basal lamina papilla cells - Electron microscopy
Dermal
The dermal papilla (DP) is considered a growth-inducing component of the hair follicle [9] occupying the anagen hair bulb cavity and consisting of connective tissue continuous with that ensheathing the follicle. The cells of the vascularized anagen DP lie in an extensive extracellular matrix, rich in basement proteins (laminin, collagen IV), fibronectin and proteoglycans [8]. The DP appears to comprise a constant population of cells [12] which round up and clump during telogen with concomitant vascular collapse. The prominent DP cell type is a specialized fibroblast. Occasional migratory cells are also present including macrophages, mast cells, and Langerhans cells, and their relative numbers reflect hair cycle changes. The anagen DP is supplied with blood vessels consisting of a single continuous layer of low endothelial cells on a distinct, often multilayered, basal lamina surrounded by pericytes. There have been many reports on the structure of the basal lamina separating the DP and hair bulb matrix, and surrounding DP vessels [10, 11] but there is little information on apparently similar material around individual DP cells. In the study reported here the basal lamina-like material in the DP was investigated in more detail with particular emphasis on associated focal membrane attachment plaques in this cell population. Biopsy specimens were excised with a 4-mm punch biopsy instrument after local anesthesia from four normal adult scalps after obtaining informed consent from the subjects. The age range was 15-32 years. A minimum of six anagen follicles from each biopsy were fixed immediately after excision for 4 h in Karnovsky's fixative [5], buffered to pH 7.4 with 0.1 M sodium cacodylate. The biopsies were post-fixed in cacodylate-buffered 1% osmium tetroxide for 1 h, dehydrated in graded ethanol, and Correspondence to: D. J. Tobin
embedded in Taab 812 resin. Using an LKB Ultratome 1-1am and ultrathin sections were cut from each follicle. The ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H300 electron microscope. A total of 26 anagen DP were examined ultrastructurally. DP cells from anagen follicles had high nuclear to cytoplasmic ratios (Fig. l a). Their moderately heterochromatic and irregularly shaped nuclei were without prominent nucleoli. DP cytoplasm was commonly scanty with few organelles, although some dimorphism was apparent, i.e. not all cells displayed similar synthetic activity. While occasional tight junctions were seen between cells, DP cells were usually well dispersed in an extensive extracellular matrix. Basal lamina-like material completely or partially ensheathed some cells in all anagen DP examined (Fig. la, b). However, such material was not apparent around the opposing margins of closely associated DP fibroblasts. With serial sectioning it was apparent that some DP cells had no association with such material . The extent of basement membrane reticulation at the DP/matrix interface and the multiplication of external laminae around the DP vasculature did not correlate with the presence of such material around individual DP fibroblasts, although it was correlated with the presence of clumped basal-lamina material randomly scattered in the extracellular matrix. DP in catagen follicles displayed similar clumped basal lamina-like material unlike the smooth laminae seen around some anagen DP fibroblasts. In the anagen DP this material was associated with accumulations of collagen fibrils (Fig. 1b, c). Points of contact between the lamina-like material and the DP plasma membranes were marked by plaque-like electron densities reminiscent of hemidesmosomes (Fig. l c). Individual cells displayed many such attachment plaques which were present only on the free margins of closely opposed DP fibroblasts (Fig. 1 a). Connecting fibrils were present between the lamina and an electron-dense plaque on the DP cell membrane (Fig. 1c). The contours of the lamina closely followed that of the cell membrane and no plaques were apparent where the lamina was not closely apposed to the membrane. In addition, fine
304
Fig. la-e. Dermal papilla of normal anagen hair follicle,a Dermal papilla fibroblasts associated with extracellular matrix components. b Enlargement of a showing two papilla cells associated with collagen (C) and basal lamina-like material (B). e High power view of basal-lamina material (B), connecting fibrils (F) and hemidesmosome-like plaques (H). Uranyl acetate lead citrate staining. a, x6000; b, • 15000; e, • fibrillar material (less than 10 nm in diameter) projected upward from the 'lamina densa' across the 'lamina lucida' terminating at or within the cell membrane. These were distinct from connecting fibrils prominent only in the regions of the hemidesmosome-like plaques. The laminae exhibited a banding pattern with an approximate periodicity of 15-20 nm. While the diameter of the laminae was mostly uniform and constant (50-75 nm), in some
specimens the lamina was considerably thicker (up to 175 nm) (Fig. 2a) where it assumed a more fibrous appearance without a periodic banding pattern. However, laminae of both types were sometimes present in the same D P (Fig. 2a). Many membrane caveolae (containing flocculent material) were present in close association with the fibrous lamina and apparently similar vesicles were also associated with cytoplasmic vacuoles. Basal laminalike material was not present around fibroblasts from the reticular dermis, connective tissue sheath or even from those cells of the D P stalk at the base of the anagen hair bulb. All anagen bulbs examined (n = 26) displayed cells with and without both basal lamina-like material and attachment plaques. Exact quantitation of the proportation of DP cells with these features was difficult as not all follicles were serially sectioned. Basal laminae are sheets of extracellular matrix which have been shown to completely surround adipocytes, cardiac, skeletal and smooth muscle cells, and Schwann cells and are attached to the basal cell layer of all epithelial cells [1]. Recently there have been reports of similar structures around Leydig ceils that have been identified as a basement membrane by immunolabeling [6]. Interactions between fibroblasts and basement membrane are seen during wound healing and in granulation tissue [7]. Indeed, human embryonic fibroblasts possess specific laminin receptors [2]. D P cells in anagen hair follicles exist in an extensive fibronectin-rich extracellular milieu [4]. Fibronectin contributes significantly to basal lamina formation [3] and its content in the DP is reduced with the onset of catagen [4]. The origin of D P basal lamina-like material is unknown. Some, if not all, basal lamina components are secreted by the cells that are attached to them [1]. This is true at least for epithelial, endothelial, muscle, adipose, and Schwann cells. It has been shown recently that D P
305
fibroblasts synthesise basement membrane proteins including collagen type IV and laminin [8]. However, it is possible that the material around anagen DP cells is derived from redundant outer layers of endothelial cell basal laminae. Alternatively, it has been suggested that basal laminae loosely associated with DP cells may be part of a network originating from the basement membrane separating the DP from the hair bulb matrix [10]. This is not strongly supported by observations of laminae surrounding cells in the absence of a connecting network with the DP/matrix junction or multiplication of external laminae surrounding DP vessels. The increased thickness
of some laminae may result from fusing of two or more individual laminae. Hemidesmosomes are recognized in thin sections as electron-dense specializations the plasma membrane of basal epithelial cells [13]. They link keratins of stratified epithelium to collagens of the mesenchyme. Little is known about the formation or function of the hemidesmosomes in biochemical terms except that their biogenesis is dependent on a complex extracellular matrix environment. Their formation can be very rapid and short term [3]. Focal attachment plaques reminiscent of hemidesmosome were observed in this study between
Fig. 2a, b. Basai lamina surrounding dermal papilla cells, a One cell displaying features similar to a Schwann cell (S) is completely surrounded with a basal lamina (B) with hemidesmosome-like plaques (H). Another cell is partially surrounded with thicker lamina material (7). C, Collagen. b Enlargement of a, showing a thickened fibrous lamina (7). Note plasmalemmal caveolae (V) and vesicular budding of cytoplasmic vacuoles. C, Collagen associated with proteoglycan matrix (P). Uranyl acetate lead citrate staining, a, • 10500; b, x21000
306 some D P cells and associated laminae. Similar contacts are also seen when reticular fibroblasts are cultured on Matrigel [3], and here are likely to be involved in positional and developmental signals. As b a s e m e n t m e m branes are n o t static b u t are structurally versatile, it is possible that selective a n d focal d e g r a d a t i o n o f the basal lamina is i m p o r t a n t in m o r p h o g e n e s i s [7]. Multilayering o f endothelial basal laminae is a feature o f the n o r m a l hair follicle a n d some o f the outer layers m a y disassociate with the capillary and b e c o m e functionally associated with the a n a g e n D P cell, perhaps influencing hair cycle control. Basement m e m b r a n e proteins in anagen D P basal lamina-like material and hemidesmosome-like planques m a y influence aspects o f cell and tissue behavior including adhesion, cytoskeletal organization, migration, and differentiation.
Acknowledgements. The author thanks Dr. D. A. Fenton, Prof. M. D. Kendall and Upjohn (UK) for assistance in this study.
References 1. Abrahamson DR (1986) Recent studies on the structure and pathology of basement membranes. J Pathol 149:257-278 2. Couchman JR, Hook M, Rees DA, Timpl R (1983) Adhesion, growth and matrix production by fibroblasts on laminin substrates. J Cell Biol 96:177-183 3. Emonard H, Calle A, Grimaud J-A, Peyrol S, Castronova V, Noel A, Lapiere C, Kleinman HK, Foidart J-M (1987) Interactions between fibroblasts and a reconstituted basement membrane matrix. J Invest Dermatol 89:156 163
4. Jahoda CAB, Oliver RF (1990) The dermal papilla and growth of hair. In: Orfanos CE, Happle R (eds) Hair and hair diseases. Springer, Berlin Heidelberg New York, pp 19 44 5. Karnovsky MJA (1965) Formaldehyde glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27: 137-138a 6. Kuopio T, Pelliniemi L (1989) Patchy basement of rat Leydig cells shown by ultrastructural immunolabeling. Cell Tissue Res 256:45-51 7. Kurkinen M, Vaheri A, Roberts PJ, Stenman S (1980) Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest 43:47-51 8. Messenger AG, Elliot K, Temple A, Randall VA (1991) Expression of basement membrane proteins and interstitial collagens in dermal papillae of human hair follicles. J Invest Dermatol 96:93-97 9. Oliver RF (1970) The induction of follicle formation in the adult hooded rat by vibrissa dermal papilla. J Embryol Exp Morph 23:219-236 10. Puccinelli V, Caputo R, Ceccarelli B (1968) Changes in the basement membrane of the papilla and wall of the human hair follicle. Arch Klin Exp Dermatol 233:173-183 11. Sato S, Nishijima A, Hiraga K (1976) Changes in basal lamina of blood vessels within hair dermal papilla: a possible relation to the hair cycle. In: Kobori T, Montagna W (eds) Biology and disease of the hair. University of Tokyo Press, Tokyo, pp 87-102 12. Silver AF, Chase HP (1977) The incorporation of tritiated thymidine in hair germ and dermal papilla during dormancy (telogen) and activation (early anagen). J Invest Dermatol 68: 201-205 13. Thacher SM, Malone KL, Dave K (1991) Localisation of the 290-kD bullous pemphigoid antigen in cultured keratinocytes: formation of a prehemidesmosome. Exp Cell Res 194: 2; 238-247
Short communications
N NN]N 9 Springer-Verlag 1992
Arch Dermatol Res (1992) 284:303-306
Basal lamina-like material and hemidesmosome-like structures associated with dermal papilla cells in the normal human anagen hair follicle D. J. Tobin
Department of Dermatology, Room H354, New York University Medical Center, New York, NY 10016, USA Received March 17, 1992
Key words: Hair follicle - Basal lamina papilla cells - Electron microscopy
Dermal
The dermal papilla (DP) is considered a growth-inducing component of the hair follicle [9] occupying the anagen hair bulb cavity and consisting of connective tissue continuous with that ensheathing the follicle. The cells of the vascularized anagen DP lie in an extensive extracellular matrix, rich in basement proteins (laminin, collagen IV), fibronectin and proteoglycans [8]. The DP appears to comprise a constant population of cells [12] which round up and clump during telogen with concomitant vascular collapse. The prominent DP cell type is a specialized fibroblast. Occasional migratory cells are also present including macrophages, mast cells, and Langerhans cells, and their relative numbers reflect hair cycle changes. The anagen DP is supplied with blood vessels consisting of a single continuous layer of low endothelial cells on a distinct, often multilayered, basal lamina surrounded by pericytes. There have been many reports on the structure of the basal lamina separating the DP and hair bulb matrix, and surrounding DP vessels [10, 11] but there is little information on apparently similar material around individual DP cells. In the study reported here the basal lamina-like material in the DP was investigated in more detail with particular emphasis on associated focal membrane attachment plaques in this cell population. Biopsy specimens were excised with a 4-mm punch biopsy instrument after local anesthesia from four normal adult scalps after obtaining informed consent from the subjects. The age range was 15-32 years. A minimum of six anagen follicles from each biopsy were fixed immediately after excision for 4 h in Karnovsky's fixative [5], buffered to pH 7.4 with 0.1 M sodium cacodylate. The biopsies were post-fixed in cacodylate-buffered 1% osmium tetroxide for 1 h, dehydrated in graded ethanol, and Correspondence to: D. J. Tobin
embedded in Taab 812 resin. Using an LKB Ultratome 1-1am and ultrathin sections were cut from each follicle. The ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Hitachi H300 electron microscope. A total of 26 anagen DP were examined ultrastructurally. DP cells from anagen follicles had high nuclear to cytoplasmic ratios (Fig. l a). Their moderately heterochromatic and irregularly shaped nuclei were without prominent nucleoli. DP cytoplasm was commonly scanty with few organelles, although some dimorphism was apparent, i.e. not all cells displayed similar synthetic activity. While occasional tight junctions were seen between cells, DP cells were usually well dispersed in an extensive extracellular matrix. Basal lamina-like material completely or partially ensheathed some cells in all anagen DP examined (Fig. la, b). However, such material was not apparent around the opposing margins of closely associated DP fibroblasts. With serial sectioning it was apparent that some DP cells had no association with such material . The extent of basement membrane reticulation at the DP/matrix interface and the multiplication of external laminae around the DP vasculature did not correlate with the presence of such material around individual DP fibroblasts, although it was correlated with the presence of clumped basal-lamina material randomly scattered in the extracellular matrix. DP in catagen follicles displayed similar clumped basal lamina-like material unlike the smooth laminae seen around some anagen DP fibroblasts. In the anagen DP this material was associated with accumulations of collagen fibrils (Fig. 1b, c). Points of contact between the lamina-like material and the DP plasma membranes were marked by plaque-like electron densities reminiscent of hemidesmosomes (Fig. l c). Individual cells displayed many such attachment plaques which were present only on the free margins of closely opposed DP fibroblasts (Fig. 1 a). Connecting fibrils were present between the lamina and an electron-dense plaque on the DP cell membrane (Fig. 1c). The contours of the lamina closely followed that of the cell membrane and no plaques were apparent where the lamina was not closely apposed to the membrane. In addition, fine
304
Fig. la-e. Dermal papilla of normal anagen hair follicle,a Dermal papilla fibroblasts associated with extracellular matrix components. b Enlargement of a showing two papilla cells associated with collagen (C) and basal lamina-like material (B). e High power view of basal-lamina material (B), connecting fibrils (F) and hemidesmosome-like plaques (H). Uranyl acetate lead citrate staining. a, x6000; b, • 15000; e, • fibrillar material (less than 10 nm in diameter) projected upward from the 'lamina densa' across the 'lamina lucida' terminating at or within the cell membrane. These were distinct from connecting fibrils prominent only in the regions of the hemidesmosome-like plaques. The laminae exhibited a banding pattern with an approximate periodicity of 15-20 nm. While the diameter of the laminae was mostly uniform and constant (50-75 nm), in some
specimens the lamina was considerably thicker (up to 175 nm) (Fig. 2a) where it assumed a more fibrous appearance without a periodic banding pattern. However, laminae of both types were sometimes present in the same D P (Fig. 2a). Many membrane caveolae (containing flocculent material) were present in close association with the fibrous lamina and apparently similar vesicles were also associated with cytoplasmic vacuoles. Basal laminalike material was not present around fibroblasts from the reticular dermis, connective tissue sheath or even from those cells of the D P stalk at the base of the anagen hair bulb. All anagen bulbs examined (n = 26) displayed cells with and without both basal lamina-like material and attachment plaques. Exact quantitation of the proportation of DP cells with these features was difficult as not all follicles were serially sectioned. Basal laminae are sheets of extracellular matrix which have been shown to completely surround adipocytes, cardiac, skeletal and smooth muscle cells, and Schwann cells and are attached to the basal cell layer of all epithelial cells [1]. Recently there have been reports of similar structures around Leydig ceils that have been identified as a basement membrane by immunolabeling [6]. Interactions between fibroblasts and basement membrane are seen during wound healing and in granulation tissue [7]. Indeed, human embryonic fibroblasts possess specific laminin receptors [2]. D P cells in anagen hair follicles exist in an extensive fibronectin-rich extracellular milieu [4]. Fibronectin contributes significantly to basal lamina formation [3] and its content in the DP is reduced with the onset of catagen [4]. The origin of D P basal lamina-like material is unknown. Some, if not all, basal lamina components are secreted by the cells that are attached to them [1]. This is true at least for epithelial, endothelial, muscle, adipose, and Schwann cells. It has been shown recently that D P
305
fibroblasts synthesise basement membrane proteins including collagen type IV and laminin [8]. However, it is possible that the material around anagen DP cells is derived from redundant outer layers of endothelial cell basal laminae. Alternatively, it has been suggested that basal laminae loosely associated with DP cells may be part of a network originating from the basement membrane separating the DP from the hair bulb matrix [10]. This is not strongly supported by observations of laminae surrounding cells in the absence of a connecting network with the DP/matrix junction or multiplication of external laminae surrounding DP vessels. The increased thickness
of some laminae may result from fusing of two or more individual laminae. Hemidesmosomes are recognized in thin sections as electron-dense specializations the plasma membrane of basal epithelial cells [13]. They link keratins of stratified epithelium to collagens of the mesenchyme. Little is known about the formation or function of the hemidesmosomes in biochemical terms except that their biogenesis is dependent on a complex extracellular matrix environment. Their formation can be very rapid and short term [3]. Focal attachment plaques reminiscent of hemidesmosome were observed in this study between
Fig. 2a, b. Basai lamina surrounding dermal papilla cells, a One cell displaying features similar to a Schwann cell (S) is completely surrounded with a basal lamina (B) with hemidesmosome-like plaques (H). Another cell is partially surrounded with thicker lamina material (7). C, Collagen. b Enlargement of a, showing a thickened fibrous lamina (7). Note plasmalemmal caveolae (V) and vesicular budding of cytoplasmic vacuoles. C, Collagen associated with proteoglycan matrix (P). Uranyl acetate lead citrate staining, a, • 10500; b, x21000
306 some D P cells and associated laminae. Similar contacts are also seen when reticular fibroblasts are cultured on Matrigel [3], and here are likely to be involved in positional and developmental signals. As b a s e m e n t m e m branes are n o t static b u t are structurally versatile, it is possible that selective a n d focal d e g r a d a t i o n o f the basal lamina is i m p o r t a n t in m o r p h o g e n e s i s [7]. Multilayering o f endothelial basal laminae is a feature o f the n o r m a l hair follicle a n d some o f the outer layers m a y disassociate with the capillary and b e c o m e functionally associated with the a n a g e n D P cell, perhaps influencing hair cycle control. Basement m e m b r a n e proteins in anagen D P basal lamina-like material and hemidesmosome-like planques m a y influence aspects o f cell and tissue behavior including adhesion, cytoskeletal organization, migration, and differentiation.
Acknowledgements. The author thanks Dr. D. A. Fenton, Prof. M. D. Kendall and Upjohn (UK) for assistance in this study.
References 1. Abrahamson DR (1986) Recent studies on the structure and pathology of basement membranes. J Pathol 149:257-278 2. Couchman JR, Hook M, Rees DA, Timpl R (1983) Adhesion, growth and matrix production by fibroblasts on laminin substrates. J Cell Biol 96:177-183 3. Emonard H, Calle A, Grimaud J-A, Peyrol S, Castronova V, Noel A, Lapiere C, Kleinman HK, Foidart J-M (1987) Interactions between fibroblasts and a reconstituted basement membrane matrix. J Invest Dermatol 89:156 163
4. Jahoda CAB, Oliver RF (1990) The dermal papilla and growth of hair. In: Orfanos CE, Happle R (eds) Hair and hair diseases. Springer, Berlin Heidelberg New York, pp 19 44 5. Karnovsky MJA (1965) Formaldehyde glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27: 137-138a 6. Kuopio T, Pelliniemi L (1989) Patchy basement of rat Leydig cells shown by ultrastructural immunolabeling. Cell Tissue Res 256:45-51 7. Kurkinen M, Vaheri A, Roberts PJ, Stenman S (1980) Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest 43:47-51 8. Messenger AG, Elliot K, Temple A, Randall VA (1991) Expression of basement membrane proteins and interstitial collagens in dermal papillae of human hair follicles. J Invest Dermatol 96:93-97 9. Oliver RF (1970) The induction of follicle formation in the adult hooded rat by vibrissa dermal papilla. J Embryol Exp Morph 23:219-236 10. Puccinelli V, Caputo R, Ceccarelli B (1968) Changes in the basement membrane of the papilla and wall of the human hair follicle. Arch Klin Exp Dermatol 233:173-183 11. Sato S, Nishijima A, Hiraga K (1976) Changes in basal lamina of blood vessels within hair dermal papilla: a possible relation to the hair cycle. In: Kobori T, Montagna W (eds) Biology and disease of the hair. University of Tokyo Press, Tokyo, pp 87-102 12. Silver AF, Chase HP (1977) The incorporation of tritiated thymidine in hair germ and dermal papilla during dormancy (telogen) and activation (early anagen). J Invest Dermatol 68: 201-205 13. Thacher SM, Malone KL, Dave K (1991) Localisation of the 290-kD bullous pemphigoid antigen in cultured keratinocytes: formation of a prehemidesmosome. Exp Cell Res 194: 2; 238-247
