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Somatostatin-like immunoreactivity is found in dendritic guard cells of human sweat ducts
Peptides,Vol. 14, pp. 401-403, 1993
0196-9781/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printedin the USA.
BRIEF COMMUNICATION
Somatostatin-Like Immunoreactivity Is Found in Dendritic Guard Cells of Human Sweat Ducts OLLE JOHANSSON, I M A R I T A HILLIGES A N D LIXIN W A N G
Experimental Dermatology Unit, Department of Histology and Neurobiology, Karolinska Institutet, Stockholm, Sweden Received 26 M a y 1992 JOHANSSON, O., M. HILLIGES AND L. WANG. Somatostatin-likeimmunoreactivityisfound in dendriticguard cellsofhuman sweat ducts. PEPTIDES 14(2) 401-403, 1993.--Somatostatin is reported in a new population of human sweat duct cells. The epithelial content of somatoslatin-like immunoreactivity in normal human skin from various sites of the body was investigated using indirect immunofluorescence. With this methodology we found round-to-oval somatostatin-immunoreactive cells situated outside the lining sweat duct cells at the level of the stratum spinosum. The cells had processes that were clearly directed towards the duct lumen, passing between the lining duct cells. This finding raises new questions and ideas about somatostatin's role in the skin, and could point to a possible involvement of somatostatin in immune defense, sweat secretion modification, antiproiiferation, or other actions. Somatostatin
Sweatglands
Human skin
Dermatology
at 37°C in tetramethylrhodamine-isothiocyanate isomer R (TRITC)-conjugated goat anti-rabbit IgG antiserum diluted 1:80. All antisera contained 0.3% Triton X-100. The sections were thoroughly rinsed in PBS before and after the incubations and mounted in PBS-glycerine l:10 containing paraphenylenediamine. In some sections, collected at random from different volunteers as well as from different biopsy sites, the first antiserum was omitted, or blocked antiserum was used as control. Specificity characteristics of the SOM antiserum has been described in detail elsewhere (5). The material was examined in a Nikon Microphot-FXA fluorescence microscope.
SOMATOSTATIN (SOM) was originally isolated and characterized in 1973 from bovine hypothalamus (1). It has been reported to be widely distributed in the central and the peripheral nervous system, as well as in endocrine cells. It has also been shown in dendritic cells in normal human dermis, testis, thymus, and gingiva (6). Somatostatin is known to have primarily inhibitory effects in many physiological systems, including cell growth, cell proliferation, and secretion. We here report a population of cells, with SOM-like immunofluorescence, in human sweat ducts. METHOD Skin biopsies from shoulder, back, chest, areola, axilla, upper arm, thigh, fingertip, and sole were obtained from 16 healthy volunteers during local anesthesia with lidocaine without epinephrine. The specimens were immersed for 3 h in 4% paraformaldehyde and 14% saturated picric acid in 0.1 M S6rensen's phosphate buffer (pH 7.4) at 4°C and then rinsed in the same buffer containing 10% sucrose for at least 24 h. Cryostat sections (14 am) thawed onto gelatin-coated slides were processed for indirect immunofluorescence (2). As primary antiserum, rabbit anti-SOM diluted 1:400 in phosphate-buffered saline (PBS) was utilized, in which the sections were incubated overnight at 4°C in a humid atmosphere, followed by incubation for 30 rain
RESULTS Somatostatin-like immunofluorescence appeared in cells just outside the lining sweat duct cells in the epidermis of all the areas investigated, but only at the level of stratum spinosum (Fig. l). No ducts without SOM-positive cells were observed. The cells were round-to-oval and had short processes passing between the lining duct cells towards the duct lumen. On the part of the cell directed away from the duct lumen no such processes were seen. The staining, which was strong and even, was located to the cytoplasm, leaving the oval nuclei unstained. In the control sections, no immunoreactivity was found.
Requests for reprints should be addressed to Dr. Olle Johansson, Experimental Dermatology Unit, Department of Histology and Neurobiology, Karolinska Institutet, P.O. Box 60 400, S-104 01 Stockholm, Sweden.
401
402
JOHANSSON, HILLIGES AND WANG
!
V
! ii
i FIG. 1. (A) Low- and (B) high-power micrographs of SOM-like immunofluorescence in cells outside the lining epithelium of a human epidermal sweat duct at the level of stratum spinosum. From male fingertip. Bars = 50 ~m.
DISCUSSION The obvious question to be raised is what kind of function(s) this SOM-like substance may be involved in. Since ducts from the whole body were labeled, it seems reasonable to conclude that they are of eccrine type; however, apocrine ducts cannot be excluded, since axillar sweat ducts also showed immunoreactive cells. From the localization and cell morphology, some speculations can be made. The polarity of the cells and the direction of the dendritic processes point towards an action closely connected to the duct lumen. The sweat duct is a possible path for various substances and agents, as well as microorganisms, to enter the body. One possibility is that these cells are immunologically active guard cells similar to or identical with the Langerhans' cells. Their dendritic appearance and their localization to the spinous layer support this. Earlier studies have also reported SOM-positive dendritic cells, situated both in dermis and epidermis, suggested to be Langerhans' cells (6). On the other hand, the entire Langerhans' cell population is not labeled. No other cells in the spinous layer showed SOM-like immunoreactivity. Furthermore, the shape of the cells was different from the classical Langerhans' cell, e.g., not being spindle-like with long, slender dendrites. Another task for this group of cells could, of course, be modification of the sweat content or sweat secretion, direct or indirect, through modification of the luminar cellular action. It is well known that the chemical content of sweat secretion is changed during the duct passage (l 3). However, the exact localization where these changes are effectuated, as well as the regulatory mechanisms, is not fully known. Recently, prolactin-like material has been demonstrated in cells of the secretory coil and in luminar duct cells of human sweat glands grafted to mice (10). The authors suggested that prolactin may function locally to regulate sweat electrolyte concentration. This could also be the case for the SOM-positive cells, which, however, seem to be another population of cells judged from differences in morphology and position. Somatostatin could act in a paracrine mode on the luminal cells, inducing reabsorption of salts and resulting in the normal hypotonic sweat secretion.
Ishida-Yamamoto and Tohyama (3) most recently have found calcitonin gene-related peptide (CGRP)-like immunoreactivity within single flask-shaped cells of the secretory portions of rat sweat glands from the foot pads. However, no CGRPimmunoreactive cells were seen in the ducts• The immunoreactire cells were located basally and also showed processes projecting up to the lumen• About 10% of the sweat glands contained such cells. In addition, CGRP-containing nerve fibers forming bundles were frequently found around the sweat glands (3,4). The authors concluded that, from a functional point of view, the release of CGRP (or a CGRP-like substance) is probably influenced by information from the luminal contents and that CGRP may be responsible for the contraction of adjacent myoepithelial cells of the sweat glands or for dilating periglandular blood vessels. The SOM-immunoreactive cells, reported in the present study, may also be influenced by information from the luminal content• There are reports that SOM has an antiproliferative effect in endotoxin-induced or acute bacterial leukocytosis in man (12), and that it inhibits T-lymphocyte as well as Molt-4 lymphoblast proliferation in vitro (8). Moreover, a SOM analogue (SMS 201995) has been shown to inhibit the growth in vivo of the Swarm rat chondrosarcoma and a hamster insulinoma (9). Furthermore, SOM has been given to psoriatic patients with reported good antiproliferative results ( I 1). In another study, 85% of the psoriatic patients showed decreased blood levels of SOM (7). It is therefore possible that SOM has an important antiproliferative action even in normal human skin. This function can, of course, be mediated by SOM released from the dermal SOM-positive dendritic cell population earlier described (6) or by a systemic source. ACKNOWLEDGEMENTS This study was supported by grants from IngaBritt och Arne Lundbergs Forskningsstiftelse,Torsten och Ragnar Srderbergs Stifteiser, and funds from the Medical Faculty of the Karolinska Institute. Dr. R. P. Elde, Minneapolis, is gratefullyacknowledged for general support.
S O M A T O S T A T I N IN S W E A T D U C T G U A R D CELLS
403
REFERENCES 1. Brazeau, P.; Vale, W.; Burgus, R.; Line~ N.; Butcher, M.; Rivier, J.; Guillemin, R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77-79; 1973. 2. Coons, A. H. Fluorescent antibody methods. In: Danielli, J. F., ed. General cytochemical methods, vol. 1. New York: Academic Press; 1958:399-422. 3. lshida-Yamamoto, A.; Tohyama, M. Calcitonin gene-related peptide-like immunoreactive cells in the secretory portions of rat sweat glands. Cell Tissue Res. 260:621-623; 1990. 4. Johansson, O. Pain, motility, neuropeptides, and the human skin: lmmunohistochemical observations. In: Tiengo, M., et al., eds. Advances in pain research and therapy, vol. 10. New York: Raven Press; 1987:31-44. 5. Johansson, O.; H6kfelt, T.: Elde, R. P. lmmunohistochemical localization of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13:265-339; 1984. 6. Johansson, O.; Vaalasti, A.; Hilliges, M.; Ljungberg, A.; Nordlind, K.; Lid6n, S.: Arver, S.; S&ler, O.; Ritz6n, M.; Luthman, J.; Ahlstr~Sm, U.; Kvint, S. Somatostatin-like immunoreactivity in human dendritic cells with special reference to Langerhans cells of the skin. In: Thivolet, J.; Schmitt, D.. eds. The Langerhans cell. Paris: Colloque INSERM/John Libbey Eurotext Ltd.: 1988:261-271.
7. Neugebauer, D.; Weber, G. Deficiency of somatostatin, hyperinsulinemia, and aggregation of thrombocytes in psoriasis. Z. Hautkr. 63:585-590; 1988. 8. Payan, D. G.; Hess, C. A.; Goetzl, E. J. Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts. Cell. Immunol. 84:433--438; 1984. 9. Reubi, J. C. A somatostatin analogue inhibits chondrosarcoma and insulinoma tumour growth. Acta Endocrinol. (Copenh.) 109:108114; 1985. 10. Robertson, M. T.; Alho, H. R.; Martin, A. A. Localization ofprolactin-like immunoreactivity in grafted human sweat glands. J. Histochem. Cytochem. 37:625-628; 1989. 11. Venier, A.: De Simone, C.; Forni, L.; Ghirlanda, G.; Uccioli, L.; Serri, F.; Frati, L. Treatment of severe psoriasis with somatostatin: Four years of experience. Arch. Dermatol. Res. 280:$51-$54; 1988. 12. Wagner, H.; Hengst, K.; Zierden, E.; Gerlach, U. Investigations of the antiproliferative effect of somatostatin in man and rats. Metabolism, 27(Suppl. 1):1381-1386; 1978. 13. Wechsler, H. L.; Fisher, E. R. Eccrine sweat glands of the rat. Response to induced sweating, hypertension, uremia, and alterations of sodium state. Arch. Dermatol. 97:189-201; 1968.
0196-9781/93 $6.00 + .00 Copyright© 1993PergamonPressLtd.
Printedin the USA.
BRIEF COMMUNICATION
Somatostatin-Like Immunoreactivity Is Found in Dendritic Guard Cells of Human Sweat Ducts OLLE JOHANSSON, I M A R I T A HILLIGES A N D LIXIN W A N G
Experimental Dermatology Unit, Department of Histology and Neurobiology, Karolinska Institutet, Stockholm, Sweden Received 26 M a y 1992 JOHANSSON, O., M. HILLIGES AND L. WANG. Somatostatin-likeimmunoreactivityisfound in dendriticguard cellsofhuman sweat ducts. PEPTIDES 14(2) 401-403, 1993.--Somatostatin is reported in a new population of human sweat duct cells. The epithelial content of somatoslatin-like immunoreactivity in normal human skin from various sites of the body was investigated using indirect immunofluorescence. With this methodology we found round-to-oval somatostatin-immunoreactive cells situated outside the lining sweat duct cells at the level of the stratum spinosum. The cells had processes that were clearly directed towards the duct lumen, passing between the lining duct cells. This finding raises new questions and ideas about somatostatin's role in the skin, and could point to a possible involvement of somatostatin in immune defense, sweat secretion modification, antiproiiferation, or other actions. Somatostatin
Sweatglands
Human skin
Dermatology
at 37°C in tetramethylrhodamine-isothiocyanate isomer R (TRITC)-conjugated goat anti-rabbit IgG antiserum diluted 1:80. All antisera contained 0.3% Triton X-100. The sections were thoroughly rinsed in PBS before and after the incubations and mounted in PBS-glycerine l:10 containing paraphenylenediamine. In some sections, collected at random from different volunteers as well as from different biopsy sites, the first antiserum was omitted, or blocked antiserum was used as control. Specificity characteristics of the SOM antiserum has been described in detail elsewhere (5). The material was examined in a Nikon Microphot-FXA fluorescence microscope.
SOMATOSTATIN (SOM) was originally isolated and characterized in 1973 from bovine hypothalamus (1). It has been reported to be widely distributed in the central and the peripheral nervous system, as well as in endocrine cells. It has also been shown in dendritic cells in normal human dermis, testis, thymus, and gingiva (6). Somatostatin is known to have primarily inhibitory effects in many physiological systems, including cell growth, cell proliferation, and secretion. We here report a population of cells, with SOM-like immunofluorescence, in human sweat ducts. METHOD Skin biopsies from shoulder, back, chest, areola, axilla, upper arm, thigh, fingertip, and sole were obtained from 16 healthy volunteers during local anesthesia with lidocaine without epinephrine. The specimens were immersed for 3 h in 4% paraformaldehyde and 14% saturated picric acid in 0.1 M S6rensen's phosphate buffer (pH 7.4) at 4°C and then rinsed in the same buffer containing 10% sucrose for at least 24 h. Cryostat sections (14 am) thawed onto gelatin-coated slides were processed for indirect immunofluorescence (2). As primary antiserum, rabbit anti-SOM diluted 1:400 in phosphate-buffered saline (PBS) was utilized, in which the sections were incubated overnight at 4°C in a humid atmosphere, followed by incubation for 30 rain
RESULTS Somatostatin-like immunofluorescence appeared in cells just outside the lining sweat duct cells in the epidermis of all the areas investigated, but only at the level of stratum spinosum (Fig. l). No ducts without SOM-positive cells were observed. The cells were round-to-oval and had short processes passing between the lining duct cells towards the duct lumen. On the part of the cell directed away from the duct lumen no such processes were seen. The staining, which was strong and even, was located to the cytoplasm, leaving the oval nuclei unstained. In the control sections, no immunoreactivity was found.
Requests for reprints should be addressed to Dr. Olle Johansson, Experimental Dermatology Unit, Department of Histology and Neurobiology, Karolinska Institutet, P.O. Box 60 400, S-104 01 Stockholm, Sweden.
401
402
JOHANSSON, HILLIGES AND WANG
!
V
! ii
i FIG. 1. (A) Low- and (B) high-power micrographs of SOM-like immunofluorescence in cells outside the lining epithelium of a human epidermal sweat duct at the level of stratum spinosum. From male fingertip. Bars = 50 ~m.
DISCUSSION The obvious question to be raised is what kind of function(s) this SOM-like substance may be involved in. Since ducts from the whole body were labeled, it seems reasonable to conclude that they are of eccrine type; however, apocrine ducts cannot be excluded, since axillar sweat ducts also showed immunoreactive cells. From the localization and cell morphology, some speculations can be made. The polarity of the cells and the direction of the dendritic processes point towards an action closely connected to the duct lumen. The sweat duct is a possible path for various substances and agents, as well as microorganisms, to enter the body. One possibility is that these cells are immunologically active guard cells similar to or identical with the Langerhans' cells. Their dendritic appearance and their localization to the spinous layer support this. Earlier studies have also reported SOM-positive dendritic cells, situated both in dermis and epidermis, suggested to be Langerhans' cells (6). On the other hand, the entire Langerhans' cell population is not labeled. No other cells in the spinous layer showed SOM-like immunoreactivity. Furthermore, the shape of the cells was different from the classical Langerhans' cell, e.g., not being spindle-like with long, slender dendrites. Another task for this group of cells could, of course, be modification of the sweat content or sweat secretion, direct or indirect, through modification of the luminar cellular action. It is well known that the chemical content of sweat secretion is changed during the duct passage (l 3). However, the exact localization where these changes are effectuated, as well as the regulatory mechanisms, is not fully known. Recently, prolactin-like material has been demonstrated in cells of the secretory coil and in luminar duct cells of human sweat glands grafted to mice (10). The authors suggested that prolactin may function locally to regulate sweat electrolyte concentration. This could also be the case for the SOM-positive cells, which, however, seem to be another population of cells judged from differences in morphology and position. Somatostatin could act in a paracrine mode on the luminal cells, inducing reabsorption of salts and resulting in the normal hypotonic sweat secretion.
Ishida-Yamamoto and Tohyama (3) most recently have found calcitonin gene-related peptide (CGRP)-like immunoreactivity within single flask-shaped cells of the secretory portions of rat sweat glands from the foot pads. However, no CGRPimmunoreactive cells were seen in the ducts• The immunoreactire cells were located basally and also showed processes projecting up to the lumen• About 10% of the sweat glands contained such cells. In addition, CGRP-containing nerve fibers forming bundles were frequently found around the sweat glands (3,4). The authors concluded that, from a functional point of view, the release of CGRP (or a CGRP-like substance) is probably influenced by information from the luminal contents and that CGRP may be responsible for the contraction of adjacent myoepithelial cells of the sweat glands or for dilating periglandular blood vessels. The SOM-immunoreactive cells, reported in the present study, may also be influenced by information from the luminal content• There are reports that SOM has an antiproliferative effect in endotoxin-induced or acute bacterial leukocytosis in man (12), and that it inhibits T-lymphocyte as well as Molt-4 lymphoblast proliferation in vitro (8). Moreover, a SOM analogue (SMS 201995) has been shown to inhibit the growth in vivo of the Swarm rat chondrosarcoma and a hamster insulinoma (9). Furthermore, SOM has been given to psoriatic patients with reported good antiproliferative results ( I 1). In another study, 85% of the psoriatic patients showed decreased blood levels of SOM (7). It is therefore possible that SOM has an important antiproliferative action even in normal human skin. This function can, of course, be mediated by SOM released from the dermal SOM-positive dendritic cell population earlier described (6) or by a systemic source. ACKNOWLEDGEMENTS This study was supported by grants from IngaBritt och Arne Lundbergs Forskningsstiftelse,Torsten och Ragnar Srderbergs Stifteiser, and funds from the Medical Faculty of the Karolinska Institute. Dr. R. P. Elde, Minneapolis, is gratefullyacknowledged for general support.
S O M A T O S T A T I N IN S W E A T D U C T G U A R D CELLS
403
REFERENCES 1. Brazeau, P.; Vale, W.; Burgus, R.; Line~ N.; Butcher, M.; Rivier, J.; Guillemin, R. Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179:77-79; 1973. 2. Coons, A. H. Fluorescent antibody methods. In: Danielli, J. F., ed. General cytochemical methods, vol. 1. New York: Academic Press; 1958:399-422. 3. lshida-Yamamoto, A.; Tohyama, M. Calcitonin gene-related peptide-like immunoreactive cells in the secretory portions of rat sweat glands. Cell Tissue Res. 260:621-623; 1990. 4. Johansson, O. Pain, motility, neuropeptides, and the human skin: lmmunohistochemical observations. In: Tiengo, M., et al., eds. Advances in pain research and therapy, vol. 10. New York: Raven Press; 1987:31-44. 5. Johansson, O.; H6kfelt, T.: Elde, R. P. lmmunohistochemical localization of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13:265-339; 1984. 6. Johansson, O.; Vaalasti, A.; Hilliges, M.; Ljungberg, A.; Nordlind, K.; Lid6n, S.: Arver, S.; S&ler, O.; Ritz6n, M.; Luthman, J.; Ahlstr~Sm, U.; Kvint, S. Somatostatin-like immunoreactivity in human dendritic cells with special reference to Langerhans cells of the skin. In: Thivolet, J.; Schmitt, D.. eds. The Langerhans cell. Paris: Colloque INSERM/John Libbey Eurotext Ltd.: 1988:261-271.
7. Neugebauer, D.; Weber, G. Deficiency of somatostatin, hyperinsulinemia, and aggregation of thrombocytes in psoriasis. Z. Hautkr. 63:585-590; 1988. 8. Payan, D. G.; Hess, C. A.; Goetzl, E. J. Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts. Cell. Immunol. 84:433--438; 1984. 9. Reubi, J. C. A somatostatin analogue inhibits chondrosarcoma and insulinoma tumour growth. Acta Endocrinol. (Copenh.) 109:108114; 1985. 10. Robertson, M. T.; Alho, H. R.; Martin, A. A. Localization ofprolactin-like immunoreactivity in grafted human sweat glands. J. Histochem. Cytochem. 37:625-628; 1989. 11. Venier, A.: De Simone, C.; Forni, L.; Ghirlanda, G.; Uccioli, L.; Serri, F.; Frati, L. Treatment of severe psoriasis with somatostatin: Four years of experience. Arch. Dermatol. Res. 280:$51-$54; 1988. 12. Wagner, H.; Hengst, K.; Zierden, E.; Gerlach, U. Investigations of the antiproliferative effect of somatostatin in man and rats. Metabolism, 27(Suppl. 1):1381-1386; 1978. 13. Wechsler, H. L.; Fisher, E. R. Eccrine sweat glands of the rat. Response to induced sweating, hypertension, uremia, and alterations of sodium state. Arch. Dermatol. 97:189-201; 1968.