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Journal of the Anatomical Society of India

Light and Electron Microscopic Appearance of Langerhans Cell (LC) in Human Tonsil Surface Epithelium: A Zinc Iodide Osmium (ZIO) Study

Author(s): Indrasingh, I., Chandi, G., & Vettivel, S.

Vol. 51, No. 2 (2002-07 - 2002-12)

Department of Anatomy, Christian Medical College, Vellore, INDIA


Four human palatine tonsils were used to study light and electron microscopic structure of Langerhans cell in the surface epithelium. A modified zinc iodide osmium (ZIO) staining was used as a marker for dendritic cells. In the epithelial sheet wholemount preparation, dendritic cell was ZIO positive and light microscopy showed a dendritic cell body, long dendritic processes, pale nucleus, and single nucleolus. Electron microscopy showed that the Langerhans cell was located suprabasally and had a lobulated or indented nucleus and free ribosomes, rough endoplasmic reticulum, mitochondria, Golgi bodies, lysosomes, Birbeck granules and an absence of tonofilaments in the cytoplasm. The Langerhans cell had no desmosomes with other epithelial cells.

Key words: Dendritic cell, Langerhans cell, surface epithelium, tonsil, ultrastructure, wholemount, zinc iodide osmium


Langerhans cell (LC) was observed in human skin with gold chloride. Birbeck, Breathnach and Everall (1961) ultrastructurally demonstrated the characteristic rod-like granules in the cytoplasm of LC. Dendritic cell (DC) with Birbeck's granules is an LC. LC represents effete melanocytes (Breathnach 1963) and controls the proliferation of keratinocytes (Potten and Allen, 1976). LC belongs to the monocyte-macrophage histiocyte family (Shelly and Juhlin 1976). LC is a phagocyte, belonging to a system of antigen presenting cells, binding the antigen that penetrates the epidermis and presenting it to the lymphocytes, which trigger a sequence of immune responses (Silberberg 1973). LC migrates from skin to the lymph nodes under normal conditions. The migration may be accelerated in an immune reaction (Silberberg et al. 1976). Recognition of LC as a member of the immune system has initiated studies on it. During the last two decades, immunological and biochemical techniques provided more information of LC.

Zinc iodide osmium (ZIO) was originally used to demonstrate nerve fibres (Malliet, 1959). It was applied to demonstrate LC by light and electron microscopy LC has ZIO reactive sites (Niebauer, Krawczy, Kidd and Wilgram 1969). The location of palatine tonsil at the gateway to the respiratory and digestive tracts suggests its functional role in generating an immune response to inhaled or swallowed antigens. Our ealier ZIO stained human palatine tonsil (paraffin impregnated) sections show DC with a selective affinity for ZIO in the tonsil epithelium (Chandi, Inbam and Sridharan 1988). Wholemount preparation of tonsillar epithelium enables a view of the whole profile of a DC. Birbeck's granules can be seen only by electron microscopy. Earlier, we demonstrated, by conventional electron microscopy, the presence of LC in tonsil epithelium (Chandi, Indrasingh and Chandi 1989). and in crypt epithelium (Indrasingh 2000). ZIO-stained tissue may exhibit well the ultrastructure of LC, particularly, Birbeck's granule. Therefore, tonsil, wholemount, ZIO technique and electron microscopy were selected. The aim of the study was, using ZIO technique, to show the light microscopic wholemount appearance of DC and electron microscopic appearance of LC in the surface epithelium of tonsil.

Materials and Methods:

Four human palatine tonsils were collected from patients, who underwent tonsillectomy (age 6 to 20 years) at the Christian Medical College Hospital, Vellore. One tonsil was used to study the light microscopic appearance of DC in epithelial sheet wholemounts and three tonsils to study the electron microscopic appearance of LC using ZIO.

Zinc iodide osmium (ZIO) mixture preparation

2.4 gm of zinc powder and 1 gm of iodine bisublimate were dissolved in 40 ml of 0.2 M Veronal-sodium HCI buffer, pH 7.4 (V.7.4 ZIO). The solution was shaken for 10 minutes, filtered immediately and mixed with 2% OSO4 at a ratio of 3:1 to obtain the ZIO mixture (V.7.4-ZIO). Light microscopic study

Preparation of epithelial sheet and staining with ZIO : The tonsil was divided into fragments ranging in size between 6 and 12 mm2. The fragments were incubated in 20 mM phosphate buffered saline ethylene diamine tetra acetic acid (PBS-EDTA), pH 7.4 for 2 hours at 37°C (Scaletta and MacCallum 1972). This treatment resulted in the detachment of epithelial sheets. The sheets were incubated in V.7.4 ZIO for 18 hrs at 4° C with continuous agitation (Figueroa and Caorsi, 1980). The epithelial sheets were then thoroughly washed in distilled water, dehydrated in graded ethanol, cleared in xylene and mounted in resin medium on slides. Electron microscopic study

Tonsils were incubated in V.7.4-ZIO for 18 hrs at 4°C, washed in distilled water, dehydrated in graded ethanol, cleared in propylene oxide and embedded in araldite. Ultra thin sections of the ZIO blocks were cut with a diamond knife, stained with lead citrate only (Figueroa and Caorsi 1980). and viewed using a Philips E.M. 201 at 60 kV.


Light microscopy of epithelial sheet wholemount preparation showed a ZIO-positive DC having a dendritic cell body, long dendritic processes, pale nucleus, and single nucleolus (Fig. 1).

Electron microscopy of surface epithelium revealed suprabasally situated LC (Fig. 2) having a lobulated or indented nucleus and free ribosomes, rough endoplasmic reticulum, mitochondria, Golgi bodies, lysosomes, Birbeck granules and an absence of tonofilaments in the cytoplasm (Fig. 3). LC had no desmosomes with other epithelial cells (Fig. 3). LC had one or two dendritic processes (Figs. 2, 3) and the cytoplasm contained tennis racket-shaped Birbeck granule (Fig. 4).


LCs are present in mouse epidermis (Mackenzie and Squiere 1975) and human eosophagus (Al Yassin and Toner 1976). LCs are demonstrated electron microscopically in human tonsillar epithelium (Chandi et al, 1989). LCs are found in cervix and vagina of rhesus macaques (Millar, McChesney and Moore 1992). Pulmonary LCs are ZIO positive (Zeid and Miller 1995). Ultrastructure of LC was revisited (Rodriguez and Carosi 1978) and studied (Figueroa and Caorsi 1980).

ZIO method was modified to investigate the morphology of LC and the histochemical reactivity and macromolecular arrangement of the "so-called" Langerhans cell granules (Rodriguez and Caorsi 1978). Veronal buffer and a pH of 7.4 enhanced the selectivity of disposition of zinc at the reactive sites on LC. Thus, the cells heavily impregnated with zinc are identifiable as LC.

Application of V.7.4-ZIO procedure to the human palatine tonsil surface epithelial sheet wholemount preparation, for the first time in this study, gives a better visualization of DC with its branched dendritic processes, distinct nucleus and nucleolus, LC is also characterized by a suprabasal location in the epidermis or epithelium. These characteristics are shared by melanocytes, which, however, contain only melanin granules, and stain only feebly with Veronal buffered ZIO at pH 7.4 (Rodriguez and Caorsi 1978). Tonsillar epithelium, stained by the DOPA method, failed to show any melanocytes (Mausle, Mootz and Schondorf 1971).

Presence of ZIO-stained DC in the subepithelial connective tissue and lymphoid tissues suggests a migration and that they may present antigens to the sub-epithelial lymphoid tissue (Chandi et al, 1988). Distribution of DC in the tonsillar epithelium was patchy and irregular. Probably, that was why others (Weinberg, Pinkus and Murphy 1987; Perry 1994). were unable to identify an LC electron microscopically in tonsillar epithelium.

The ultrastructural observation of ZIO stained racket-shaped Birbeck granule in the cytoplasm of suprabasally situated DC in human tonsil surface epithelium establishes those dendritic cells as Langerhans cells. Compared to the conventional electron microscopic appearance (Chandi et al, 1989), the appearance of tennis-racket-shaped granule in ZIO-treated tissue is enhanced. There is, however, no heavy metal deposit on the cell membrane of LC.


The authors thank the Fluid Research Committee of the Christian Medical College, Vellore for funding this study. Permission to use the electron microscope in the Welcome Research Unit of Christian Medical College Hospital and expert technical assistance by Mr. Swamy Nathan are gratefully acknowledged.


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Fig. 1: ZIO positive dendritic cell (arrow) with four dendritic processes, a plane nucleus and a single nucleolus in the epithelial sheet whole mount. X 400.

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Fig. 2: ZIO stained section showing a pale stained basally situated dendritic cell (arrow X 3010.

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Fig. 3: Pale stained dendritic cell showing the characteristic Berbeck granules (arrow) in its cytoplasm. X 6705.

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Fig. 4: ZIO positive tennis racket-shaped Birbeck granule (arrow) in the cytoplasm of the LC. X 40950.

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