Yonago Acta medica 1999;42:153–161

Some New Aspects of Langerhans Cells in the Human Epidermis:
Light and Electron Microscopic Observations on the Swelling Sites
Seen in the Process Terminals of the Dendritic Cells Described byLangerhans in 1868
Shigeo Oota
First Department of Physiology, Faculty of Medicine, Tottori University, Yonago 683-0826,
Japan
In 1868, Langerhans discovered the dendritic cells now generally called Langerhans
cells (LCs) in the human epidermis using the gold impregnation method. He pointedout that the terminals of the upward running processes of the cells end with button-likeswellings under the cornified layer. In 1951, Ferreira-Marques classified the shapes of
the terminals into 7 types, named the largest one Organum trompiforme and reckoned it
to be a sense receptor. However, the swellings of the process terminals seem to havebeen entirely forgotten along with the negation of the nerve cell theory of LCs. Theauthor of this study recovered some of the actual forgotten terminals, the button-shaped(knopfförmig) swellings of the process terminals of the cells, beneath the cornified layerof a healthy volunteer epidermis using the methylene blue staining method. Furthermore, the ultrastructures of the swellings were captured for the first time using theThin-Section-Reembedding and Ultrathin-Sectioning (TRUS) technique named by theauthor. Investigation of the swellings by electron microscopy showed that there were anumber of Birbeck granules and vacuoles of various sizes together with elements ofcytoskeletons in the cytoplasm. Possibly this shows that LCs play an essential role inthe differentiation of the epidermis at the process terminals.
Key words: electron microscopy; epidermis; Langerhans cells; methylene blue

In 1868, Langerhans described and illustrated
the dendritic cells in the human epidermis now
generally designated as Langerhans cells (LCs),and he gave his view of the process terminals of
the cells as follows: Sie enden mit einer leichten
aber deutlichen knopfförmigen Anschwellung
unmittelbar unter der Grenze zwischen Reteund Hornschicht; that is, they end with a slightbut distinct button-shaped swelling directly under the border between the rete and horny layers(Langerhans, 1868). In 1951, Ferreira-Marques
reported his widely pursued studies of the terminals of upward running processes of LCs(Ferreira-Marques, 1951). He depicted and classified the shapes of the terminals into 7 types

and named each one, especially the largest one,
Organum trompiforme. This seems to be thesame as the knopfförmige Anschwellung which
Langerhans described in his original paper. Although both Langerhans and Ferreira-Marques
identified the dendritic cells in the human epidermis using the gold impregnation method, theauthor in this study observed the same characteristic features expressed as knopfförmig or
trompiform beneath the horny layer of a healthy
volunteer epidermis including the hair canal withlight and electron microscopes using methylene
blue staining and reembedding methods.
This study could contribute to a precise understanding of the structure of the LCs and also

Abbreviations: EM, electron microscopy; LC, Langerhans cell; LM, light microscopy; TRUS, Thin-SectionReembedding and Ultrathin-Sectioning

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S. Oota

Table 1. Types of fixative solutions and number of materials
Fixative solution

Number of materials
Fixation via
Directphysiological saline fixation

Total

2.5% Glutaraldehyde + 0.05 mol/L cacodylate buffer
3 ( 2)
2.5% Glutaraldehyde + 0.1 mol/L cacodylate buffer
5 ( 3)
5 (2)2.5% Glutaraldehyde + 0.05 mol/L phosphate buffer
3 ( 2)
2.5% Glutaraldehyde + 0.1 mol/L phosphate buffer
3 ( 2)
2.5% Glutaraldehyde + Kindaly 2
3 ( 2)Total
17 (11)5 (2)22 (13)( ), number of finished materials; Kindaly 2, acetate dialysate for hemodialysis (Fuso Pharmaceutical Indust.,Ltd.).

could suggest the LCs’ function. Special attention has been paid to the terminal swellings beneath the cornified layer of the epidermis and to
those beneath the same layer of hair canals.

Materials and Methods
A skin specimen sized 5 mm × 15 mm was biopsied under local anesthesia from the lateralside of the left forearm of a healthy volunteer(64-year-old male) who gave informed consent.
The biopsy specimen was immediately divided
in 2, a large and a small piece. The large one
was soaked in physiological saline at the dermatological biopsy-operation room at this Faculty. After 20 min, the specimen was dissected
into 5 small pieces in the laboratory, then thesesmall pieces were steeped in 5 types of fixativesolution as shown in Table 1. The small specimen was soaked directly into any one of thefixatives as a control in the biopsy-operationroom.
The fixative material was 2.5% glutaraldehyde in all fixative solutions, buffered with
0.05 mol/L and 0.1 mol/L cacodylate and phosphate, controled at pH 7.4. In addition, Kindaly
2 (Fuso Pharmaceutical Indust., Ltd., Osaka,Japan), acetate dialysate for hemodialysis, wasused tentatively to evaluate its effect. (The mitotic LCs in the prophase were detected only inthe sections fixed in this fixative solution. Thedata will be published elsewhere.)All specimens in the fixative were preserved
at 4˚C. After 1 h fixation, each specimen wasdissected again into smaller pieces (less than 2mm3) (Table 1). The continual fixation for 24h, postfixation with 1% OsO4 for 2 h and dehydration were performed in the usual manner.
Then they were embedded in Epon 812 withinverted polyethylene capsules (TAAB Lab.
Equip. Ltd., Reading, Berks., United Kingdom)on the slide glass. Polymerization of the epoxy
resin was carried out at 37˚C, 45˚C and 60˚C,respectively for 24 h. A polymerized Epon
containing skin specimen in a capsule can beeasily detached on an 80˚C hotplate by hand.
The above procedures are Step 1 in Fig. 1.
Step 2 is as follows: The Epon block takenout of the capsule was trimmed largely along
the outline of the specimen and was cut into
1.5–2.0 µm slices with glass knives on a Sorvall
ultramicrotome MT-1. All sections in thisstudy were cut perpendicular to the surface.
Four serial sections each were taken on a slideand dried on the hotplate at 80˚C.
In Step 3, after the sections on the slide werestained with a 1% methylene blue alkaline solution of 50% alcohol, the most important sections for electron microscopy (EM) were selected by light microscope and these were noted bymarking on the underside of the slide.
In Step 4, the marked sections were reembedded in epoxy resin with inverted polyethylene capsules on the slide and were polymerized at 45˚C and then at 60˚C, each for 24 h.
The polymerized epoxy resin containing thesection for EM in the polyethylene capsules
was easily removable by hand on the 80˚C hotplate. The sections for EM were affixed to the154

Some new aspects of LCs in the human epidermis

Step

1

Illustration of the procedure
BiopsyFixation

Dehydration

Embedding
Detaching2

Trimming
Thin
sectioning

Staining

Examination toselect the cellLM

3

4

Reembedding
Detaching5

Trimming
Ultrathinsectioning

Doubleelectron staining

EMFig. 1. The procedure of the Thin-Section-Reembedding and Ultrathin-Sectioning (TRUS) technique. Step
1: From biopsy to specimen embedding in Epon 812. Step 2: Thin sectioning (1.5–2.0 µm thick). Step 3:
Staining with methylene blue and light microscopic examination followed by selection of the objects. Step 4:
Reembedding, the same as Step 1. Step 5: Ultrathin-sectioning (60–70 nm thick) and electron microscopic
examination. EM, electron microscopy; LM, light microscopy.

top of each epoxy resin block. Thereafter, it
was necessary to check the sections by using alight microscope for their preservation in handling until this step.
In Step 5, the final step, the well-prepared
Epon blocks were trimmed for EM under thedissecting microscope. The trimmed Eponblocks were cut as thick as 60–70 nm with adiamond knife on the Sorvall MT-7000 ultramicrotome. Then, it was necessary that the surface of the block moves in a strictly tangential
direction to the knife edge. The ultrathin sections were placed serially on the copper grid,
stained with uranyl acetate and lead citrate, andobserved with a Hitachi electron microscope
H-800 or H-500, at 75 or 80 kV, respectively.

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