tt 't'.swrt. I i LOCALIZATION OF GOLD CHLORIDE AND ADENOSINE TRIPHOSPHATASE IN HUMAN LANGERHANS CELLS*

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1 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Copyright 1968 by The Williams & Wilkins Co. Vol. 51, No. 5 Printed in U.S.A. LOCALIZATION OF GOLD CHLORIDE AND ADENOSINE TRIPHOSPHATASE IN HUMAN LANGERHANS CELLS* ALVIN S. ZELICKSON, M.D. AND JESS H. MOTTAZ, B.S. At the ultrastructural level, the Langerhans cell is easily distinguished from the surrounding keratinocytes and melanocytes. Its cytoplasm is clear due to a relative absence of filaments. The Langerhans cell is dendritic, lacks desmosomes, contains lysosomes, has a characteristic cytoplasmic granule and a convoluted nucleus. Langerhans cells are found in the superficial portions of the epidermis, in the lower epidermis and significantly as pointed were recently observed by Basset et al. (3, 4) in a case of pulmonary histiocytosis X and eosinophilic granuloma of bone. The presence of Langerhans cells in pathologic conditions and in locations outside of the skin has led to increased interest and numerous studies regarding the histogenesis of this particular cell (5 8). Many of these studies and their interpretations have been based on light microscopic investigations using staining methods to localize the tt 't'.swrt. I i FIG. 1. When stained with gold chloride the dendritic nature of the high level Langerhans cells is clearly visible with the light microscope. out by Zelickson (1) in the dermis as well. Since the first description of the Langerhans cell (2), many theories have been advanced regarding the genesis of this cell but its nature still remains an unsettled question. Cells indistinguishable from epidermal Langerhans cells * From the Division of Dermatology, University of Minnesota Medical School, Minneapolis, Minnesota. Presented at the Twenty-ninth Annual Meeting of the Society for Investigative Dermatology, Inc., June 16 18, 1968, San Francisco, California. This work was supported by Training Grant AM05560 from the TJSPHS. 365 Langerhans cell (9 14). As has been mentioned the Langerhans cell can be specifically identified with the electron microscope. With this in mind an attempt was made, using the electron microscope, to evaluate the accuracy and specificity of the gold chloride and adenosine triphosphatase (ATPase) methods for localizing the Langerhans cell. METHODS AND MATERIALS Tissue for the study was obtained from the lower back and the forearms of Caucasian volunteers. Specimens were removed with a 4mm biopsy

2 366 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY :.-,'4t4's ;-- - Fia. 2. When seen with the electron microscope the Langerhans cells are easily distinguished from the surrounding keratinocytes and melanocytes. Their cytoplasm is laden with gold deposits and their nuclear membrane is sharply demarcated by the dense gold. 5,382 X punch and were divided to provide material for examination with the light and electron microscope. The following technics were performed: hematoxylin and eosin, impregnation with gold chloride as modified by Becker and Zimmerman (11) and ATPase localization using the method of Wachstein and Miesel (15). In the latter an incubation time of 45 minutes was found to yield the most favorable results. The tissue examined with the electron microscope was fixed in buffered glutaraldehyde and embedded in Epon 812. Sections of 500 to 700 Angstroms were taken along with adjoining 2 micron sections for viewing with the phase microscope. The thin sections were stained with uranyl acetate prior to examination. RESULTS Gold chloride localization. In light microscopic sections the gold deposits were localized in dendritic cells (Fig. 1). When viewed with the electron microscope those cells which stained heavily with the gold were identified as Langerhans cells (Fig. 2). Although all of the Langerhans cells were filled with gold not all of them stained as heavily as some of the others. In general, all of the Langerhans cells seen in any particular section were heavily stained and were clearly separable from the

3 THE LANGERHANS CELL 367 Fic. 3. At higher magnifications one can see that the cells that are loaded with the gold particles are Langerhans cells. Note the numerous particles which outline the indented nucleus. 12,024 X adjacent keratinocytes and melanocytes. The nuclear membrane of the Langerhans cells stained heavily (Fig. 3), as did the cytoplasm but there was no specific localization of the gold to any cytoplasmic organelle including the Langerhans cell granule. The nuclear membranes of the keratinocytes and nielanocytes were partially stained with gold but iii no instance did their cytoplasm take on the heavy deposits that were noted in the Langerhans cells. The plasma membranes and desmosomes of all cells showed very little staining. The basement membrane did not stain. Fibroblasts and histiocytes in the upper dermis also stained heavily with gold. In the latter, the metal was dispersed throughout the cytoplasm with some localization to the nuclear membrane. There were several other cell types in the upper dermis which stained heavily and which differed somewhat from the fibroblasts. These cells were dendritic in nature, had a markedly convoluted nucleus, and a well developed golgi complex. To some degree they resembled Langerhans cells although specific granules were not noted within their cytoplasm. ATPase localization. Many cells in both the epidermis and dermis demonstrated the ATPase reaction product, lead phosphate. Occasionally ATPa.se reaction particles were seen within the cell cytoplasm but generally the location of these particles was fixed to the cell membrane (Fig. 4). Our results with this method parallel those of Farquhar and Palade (16). The keratinocytes (Fig. 4), melanocytes (Fig. 5), and Langerhans cells (Fig. 6) were all stained to some degree. The Langerhans cells were stained

4 368 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY FIG. 4, Lower magnifications of human epidermis which has been stained for ATFase demonstrate that the lead phosphate particles are localized to the cell membranes of the keratinocytes and dendritic cells. Note the heavy localization in the region of the basement membrane. 5,865 )< regardless of their location in the epidermis but we also found the same to be true of the kerat inocytes and melanocytes. Significantly, not all the Langerhaiis cells showed evidence of ATPase staining. The eccrine sweat glands, fibroblasts, blood cells, capillaries and nerve tissue also showed heavy staining (Fig. 7). DISCUSSION Results with gold impregnation methods have been considered by many to be notoriously inconsistent and variable (5), although Fan and Hunter (12) suggested that the Ferreira- Marques i) modification of the gold chloride technique yielded relatively consistent and clear cut results. Breathnach (5, 10) commented upon the value and limitations of the gold chloride technique for localization of the Langerhans cell. He noted that when dealing with blocks of normal skin one may encounter different degrees of impregnatioli of the Langerhans cells as well as sections in which they are not visible at all. In contrast to this, we have demonstrated that gold chloride staining provides a rather specific method for localizing Langerhans cells in the human epidermis. Every Langerhans cell we examined was stained with gold and in sections with lesser degrees of im

5 rfl S THE LANGERHANS CELL 369 I ' U..., U, 4 'I FIG. 5. ATPase is localized to the cell membranes of the melanocytes and keratinocytes. 16,905 X 4 tfl. pregnation it was still easy to localize the Langerhans cells as they were always stained to a greater degree than the surrounding melanocytes or keratiiocytcs. Many authors have used the histochemical staining techniques originally described by Wachstein and Miesel (15) to localize ATPase activity in skin. It has recently been suggested that ATPase is specific and unique for the Langerhans cell (8, 14). In contrast to this, Bradshaw et al. (17), using ATPase staining, were unable to distinguish one dendritic cell type from another. Farquhar and Palade (16) used the light and electron microscope to study ATPase localization in amphibian epidermis. They demonstrated the presence of ATPase reaction products on all epidermal cell membtanes, including the dendritic cells. Riley (13) used the ATPase method to study the distribution of epidermal cells in pigmented and inpigmented skin and also noted that the stain was not specific for Langerhans cells but stained the melanocytes as well. Similar studies have been reported by Mustakallio (18) in human epidermis. Nakai (19) in an electron microscopic study of ATPase activity in malig-

6 370 THE JOURNAL OF' INVESTIGATIVE DERMATOLOGY r - I Fia. 6. The cell membrane of the Langerhans cell is also stained by the lead phosphate particles. Note the relative absence of stain within the cytoplasm of the Langerhans cell. 15,525 X

7 THE LANGERHANS CELL 371 S 'I 4' 4.. '.4.44, r * (; r Fic. 7. Top, Fibroblasts and nerve tissue are stained by the dense lead phosphate particles denoting the site of the ATPase reaction. 15,525 X Lower left, ATPase is also localized to the cell membrane of red blood cells. 18,975 X Lower right, Both the dark and clear cells of the eccrine sweat duct are stained by the ATPase reaction product. 2,760 X

8 372 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY nant melanoma cells also demonstrated ATPase activity in melanocytes. The enzyme has also been located in blood cells (2), kidney and liver cells (21), nerve tissue (22), capillaries (23), and in cells in the urinary bladder (24). Our findings demonstrate the variability and non-specificity of this stain in human epidermis. This indicates to us that one cannot rely on ATPase localization to specifically identify the Langerhans cell. Most likely ATPase staining in human skin, as well as in other tissue, is membrane specific but not cell specific. SUMMARY At present the best method for localizing Langerhans cells is by direct observation with the electron microscope. The gold chloride technic offers a great deal of specificity and accuracy at the light microscope level while adenosine triphosphatase staining is non-specific and should not be used as a means of localizing Langerhans cells. REFERENCES 1. Zelickson, A. S.: The Langerhans cell. J. Invest. Derm., 44: 201, Langerhans, P.: Uber die nerven der rnenschlichen Haut. Virchows. Arch. Path. AnaL, 44: 325, Basset, F., Nezelof, C., Mallet, R. and Turiaf, J.: Nouvel1e mise en óvidence par la microscopic électronique, de pargicules d'allure virale dans une seconde forme clinique de l'histiocytose X le granulome éosinophile de l'os. CR. Acad. Sci., Paris, 261: 5719, Basset, R. and Turiaf, J.: Indentification par la microscopie lectronique de particules de nature probablernent virale dans les liaisons granulornaeuses d'une histiocytose X pulmonaire. C.R. Acad. Sci,, Paris, 261: 3701, Breathnach, A. S.: The cell of Langerhans. Tnt. Rev. Cytol., 18: 1, Hashimoto, R. and Tarnouski, W. M.: Some new aspects of the Langerhans cell. Arch. Derm., 97: 450, Kiistala, U. and Mustakallio, K.: The presence of Langerhans cells in human dennis with special reference to their potential mesenchymal origin. Acta Dermatovener., 48: 115, Wolff, K. and Winkelrnann, R. K.: Quantitative studies on the Langerhans cell population of guinea pig epidermis. J. Invest. Derm., 48: 504, Ferreira-Marques, J.: Systema sensitivurn intra-epiderrnicum. Die Langerlianschen Zellen als Rezeptopen des hellen Schmerzes: Doloriceptores. Arch. Derm. Syph., (Berlin) 193: 191, Breathnach, A. S., Birbeck, M. S. and Everall, J. D.: Observations on Langerhans cells in leprosy. Brit. J. Derm., 74: 243, Becker, S. W., Jr. and Zimmerrnaijn, A. A.: Further studies on melanocytes and melanogenesis in the human fetus and new born. J. Invest. Derm., 25: 105, Fan, J. and Hunter, R.: Langerhans cells and the Modified Technic of Gold Impregnation by Ferreira-Marques, J. Invest. Derm., 31: 115, Riley, P. A.: A study of the distribution of epidermal dendritic cells in pigmented and unpigmented skin. J. Invest. Derm., 48: 28, Wolff, K. and Winkelmann, R. K.: Ultrastructural localization of nucleoside tn phosphatase in Langerhans cells. J. Invest. Derm., 48: 50, : Wachstein, M. and Meisel, E.: Histochemistry of hepatic phosphatases at a physiologic ph with special reference to the demonstration of bile canaliculi. Amer. J. Clin. Path., 27: 13, Farquhar, M. G. and Palade, G. E.: Adenosine triphosphatase localization in amphibian epidermis. J. Cell Biol., 30: 259, Bradshaw, M., Wachstein, M., Spence, J., and Elias, J. M.: Adenosine triphosphatase activity in melanocytes and epidermal cells of human skin. J. Histochem., 11: 465, Mustakallio, K.: Adenosine triphosphatase activity in neural elements of human epidermis: Exp. Cell Res., 28: 449, Nakai, T.: Electron microscopic study of "adenosine triphosphatase" activity in malignant melanoma cells of the Syrian Golden hamster. J. Invest. Derm., 44: 264, White, J. G. and Krivit, W.: Fine structural localization of adenosine triphosphatase in human platelets and other blood cells, blood, 26: 554, Ashworth, C. T., Luibel, F. J., and Stewart, S. C.: The fine structural localization of adenosine triphosphatase in the small intestine, kidney and liver of the rat. J. Cell Biol., 17: 1, Torack, R. and Barrnett, R. J.: Nucleoside phosphatase activity in membranous fine structures of neurons and glia. J. Histocliem. Cytochem., 11: 763, Manthesi, V. T. and Barrnett, R. J.: Th demonstration of enzymatic activity in pinocytic vesicles of blood capillaries with the electron microscope. J. Cell Biol., 17: 547, Bartoszewicz, W. and Barrnett, R. J.: Fine structural localization of nucleoside phos phatase activity in the urinary bladder of the toad. J. Ultrastruct. Res., 10: 599, 1964.