Some new aspects of LCs in the human epidermis

A1

B1

A2

B2A3*Fig. 5. The process terminal swellings in a round fashion and contacts with the horny layer, but theconnection between the process and the cell body can not be confirmed. A1–3: Serial thin section. × 750 (A2oil). A2 shows the process and terminal button, but the connection between the process and the cell body can
not be found. B1 and 2: In the button, some mitochondria and vacuoles are seen, but Birbeck granules are notobserved. The outlines of these buttons are not rugged. × 6,750 (bar = 1 µm).

Morphology
The whole appearance of the button corresponds well to the image of LM. However, it isnecessary to make a careful observation, because the features at the start and the end of
ultrathin-sectioning differed somewhat fromeach other (Figs. 5B1 and B2).

Birbeck granules and vacuoles
On one occasion, plenty of Birbeck granules(Birbeck et al., 1961) and many vacuoles ofvarious sizes were observed in the process terminal (button) and, on another occasion, noBirbeck granules were observed at all. (Figs. 2and 3 versus Figs. 4 and 5).

159

S. Oota

Discussion
It is very important to start with light microscopic observation for the study of skin specimens where the view of every section changes
panoramically. For a detailed examination of a
specific cell or tissue in the skin, the section
containing the object should first be selected by
LM, then the proper object for the electron
microscopic study can be carefully investigatedon the ultrathin sections taken from the section
viewed by LM. The author accomplished thepurpose by the Thin-Section-Reembedding andUltrathin-Sectioning (TRUS) technique (Fig.
1). This TRUS technique is simple, and moreover, trustworthy for anyone. It was usedthroughout the author’s studies. The rate of error was less than 1%. This technique was instructed to the author from Researcher N. Shindo(Shindo et al., 1984) at the Clinical ResearchInstitute, National Medical Center, Tokyo, Japan,
and the author drew a picture of the detailed
manner of this procedure in Fig. 1. The technique was called the TRUS technique by theauthor for brevity. In this study, because the“No Cover Glass (NCG)” object lens could not
be employed in the light microscopic examination, the micrographs, except for the “oil immersion” views, were taken from the sections
covered with a dry coverglass on the slide. Theoil immersion micrographs were taken from thesections affixed on the surfaces of the reembedded Epon blocks. Then, a drop of water was put
between the section (Epon block surface) andthe coverglass in order that the section surface
woul not get oil on it. The hardness of the Epon
used in this study was relatively hard. The ratioof mixture A to mixture B was 3 to 7, according
to Luft’s method (1961). This Epon hardness
seems to be the key to success.
As was stated above, in some cases a great
many Birbeck granules were found in the buttons of LCs, but in the other cases were notfound at all. These different results could be
due to the different stage of interactions between LCs and the adjacent keratinocytes; one
would be in a progressive stage of exocytosis ofBirbeck granules, while another may be in theend state of this function. Nevertheless, the exact role of the process terminals (button) and thenature of Birbeck granules remains for furtherstudy. The observations of the process terminals (having buttons) of intraepidermal LCs
were achieved first by Langerhans, and succeeded by Ferreira-Marques’ elaborate examination. Both Langerhans and Ferreira-Marques
identified the cells by using the gold impregnation method in the human epidermis. The present author used the methylene blue staining
method and identified the cells easily and precisely according to numerous studies by otherworkers (Breathnach, 1965; Wolff, 1972): dendritic cell shapes, a clear cytoplasm (tonofilaments free), no desmosome, indented nuclei,etc. The methylene blue staining method has
the advantage of detailed description of the sections on the slide. To the large swelling of theprocess terminals Ferreira-Margues gave thename trompiform; the author thinks that theword knopfförmig (that is “button-shaped”) by
Langerhans and the word trompiform (that is“trumpet-shaped”) by Ferreira-Marques are
identical entities, and possibly produced fromthe exocytosis of Birbeck granules. Since
Ferreira-Margues’ time, the swellings of theprocess terminals of dendritic cells in the epidermis have been observed with the ATPasestaining method of Bradshaw and colleagues(1963) in the human epidermis and were expressed as “buttons” or “caps”, but their significance was not discussed at all.
This study describes the first time that electron microscopic examination of Langerhans’“button” has ever been done. Langerhans, whoinvestigated this button in 1868 with a lightmicroscope of the 19th century and recorded hisfindings as an important matter, was a man ofinsight. Ferreira-Marques who took notice of
these process terminals was also a man of great
ability. According to the formation of “buttons” of the process terminals of the intraepidermal LCs and their ultrastructure in theauthor’s findings, it may be assumed that LCs
play an essential role in the differentiation of
the human epidermis at the process terminals.

160

Some new aspects of LCs in the human epidermis

Acknowledgments: The author gratefully acknowledges the support of Emeritus Prof. Tokichi Yumotoand Prof. Yasutake Hiji who gave him the opportunity to carry out this study in the First Dept. ofPathology and then in the First Dept. of Physiology,respectively. The author further expresses his gratitude to Prof. Yasutake Hiji for the reading of thismanuscript. The author also expresses his appreciation to Prof. Motoyuki Mihara for the kind help in
obtaining biopsy specimens and to Dr. Toku
Kanaseki for his cordial help in EM.

References
1 Birbeck MS, Breathnach AS, Everall JD. An
electron microscope study of basal melanocytes
and high-level clear cells (Langerhans cells) invitiligo. J Invest Derm 1961;37:51–64.
2 Bradshaw M, Wachstein M, Spence J, Elias JM.

Adenosine triphosphatase activity in melanocytes
and epidermal cells of human skin. J Histochem
Cytochem 1963;11:465–473.
3 Breathnach AS. The cell of Langerhans. Int Rev
Cytol 1965;18:1–28.
4 Ferreira-Marques J. Systema sensitivum intraepidermicum: Die Langerhansschen Zellen als
Rezeptoren des hellen Schmerzes: Doloriceptores. Arch Dermat Syph 1951;193:191–250.
5 Langerhans P. Über die Nerven der menschlichen Haut. Virchows Arch Pathol Anat 1868;
44:325–337.
6 Luft JH. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 1961;9:409–414.
7 Shindo N, Kobayashi E, Okada M. Immunoelectron microscopic (IEM) studies on glutaraldehyde-fixed renal specimen. J Histochem
Cytochem 1984;32:501–509.
8 Wolff K. The Langerhans cell. Curr Prob Derm
1972;4:79–145.

(Received March 31, Accepted April 13, 1999)

161