British Journal of Dermatology {1977) 97) 593-

Clinkai and Laboratory Investigations

Immuno-electron microscopic studies of surface receptors and antigens of human Langerhans cells G.ROWDEN McGill University Cancer Research Unit, Mclntyre Medical Sciences Building, 3655 Drumtnond Street, Montreal, Quebec H3G 1Y6, Canada Accepted for publication 4 July 1977

SUMMARY

A heteroantiserum, prepared in rabbits against fractionated cell membranes of a human B-lymphoblastoid cell line, was used to study the distribution of Ia antigen(s) in human epidermis. Indirect immunofluorescence staining demonstrated specific reactivity of dendritic supra-basal cells, consistent in location with Langerhans cells. Basally located cells were noted in biopsy specimens from vitiliginous skin and from the lcukodermatous regions of halo naevi. The specificity of the reaction was confirmed at the ultrastructural level by means of ferritin labelling methods. Cell surface staining was confined in the epidermis to Langerhans cells. Fc and C j ' receptors were studied by means of rosetting methods. Negative results were obtained on frozen sections, while 2-3% of cells formed rosettes when applied to an epidermal cell suspension.

Recent studies of the reactivity of the skin to various contact sensitizing agents have provided important information on the functions of the epidermal Langerhans cell (Silberberg, i97ia,b, 1973; Silberberg et al., 1975, i976a,b, 1977; Shelley & Juhhn 1977). Despite the earlier proposals concerning a possible immune function for the cell type (Ranvier, 1875; Billingham & Silvers, 1965; Prunieras, 1969), and the growing body of evidence illustrating its resemblance to histiocytes or macrophages (Hashimoto & Tarnowski, 1968; Wolff, 1972), consideration of the nature of possible receptors and antigens expressed at the surface of such cells is only just beginning. Clearly, if the cell is to be accepted as the epidermal equivalent of the reticulo-endothelial system, i.e. 'reticulo-epithelial cell' (Shelley & Juhlin, 1976), information concerning surface receptors or other antigenic properties will be very important. Thus, it was decided to investigate the antigenic properties ofhuman Langerhans cells for the presence or absence of various surface receptors such as the Fc and C3' receptors. Standard immunological methods for identifying such entities were applied. Since there has been much interest in the so-called Ia antigens expressed by various immunologically active cells (Unanue etal., 1968; David, Shreffler& Frelinger, 1973; Gotze, Reisfeld&Klein, 1973; Sachs & Cone, 1973; Hess, 1976), an attempt was made to demonstrate their presence in human epidermis. This was particularly important since, although Ia antigen has been associated mainly with the cell surfaces of lymphocytes of the B lineage, some reports have appeared suggesting its presence on macrophages (Schwartz et al.y 1976). Finally, the report by Hammerling and associates (1975) of the presence of la antigens in animal epidermis, since it relied on a cytotoxic assay performed on cell suspensions and did not distinguish morphologically the cell type(s) involved, required further 'Supported by the National Cancer Institute of Canada. B

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investigation. This then is a report of attempts to obtain further information pertinent to an immune function for the Langerhans cell. A preliminary report in which ultrastructural evidence for Langerhans cells was lacking has been published (Rowden, Lewis & Sullivan, 1977). It goes without saying that there may be other functions for such a cell that may fall outside the normally accepted range of immune phenomena. The possible involvement with control of mitosis and keratinization (Potten & Allen, 1976) will not be considered herein.

MATERIALS AND METHODS

Human skin obtained at surgery or as punch biopsy specimens obtained under local anaesthesia were processed in the following manner: (a) Frozen in isopentane cooled with liquid nitrogen and 5 /(m sections prepared for direct and indirect immunofluorescence (IF) and rosetting methods. (b) Fixed in { strength Karnovsky fixative at room temperature for i h for conventional transmission electron microscopy. Subsequent processing, i.e. osmication, dehydration and embedding, was according to standard schedules. (c) Fixed for 30 min at room temperature in 1-25" ,\ paraformaldehyde in o i M phosphate bufFcr pH 74 for immuno-electron microscopy. Anlisera Human B-Iymphocyte la-like antigen was isolated from a B-Iymphoblastoid cell line (Sullivan ei ai, 1977). The extraction procedure is described extensively elsewhere, but in essence consists of detergent extraction in the presence of a protease inhibitor (Trasylol), followed by gel filtration on Biogel A 1-5 m and affinity chromatography on Sepharose-Con A. The Con A binding fraction ofthe Blymphocyte membranes was purified by electrophoretic separation using polyacrylamide gel electrophoresis. The protein peak utilized for raising the rabbit heteroantiserum had a molecular weight of 33,000. The heteroantiserum produced by hyperimmunizing rabbits was absorbed extensively with a human T cell line (MOLT-4) and was shown to have preferential reactivity against B cells, but not against T cells or thymocytes (Sullivan et al.^ 1976). It was further characterized by precipitin analysis on Ouchterlony plates against a rabbit antiserum to HL-B antigens (DrH.Kunkel, Rockefeller University). A line of immunological identity was evident, indicating a relationship to the la system, Functionally, the heteroantiserum was shown to suppress thymidine incorporation when incubated with lymphocytes in the mixed lymphocyte reaction (MLR). Fluorescence {IF) Sections were incubated in varying dilutions ofthe rabbit monospecific anti-la antiserum and following washing were exposed to monospecific commercial goat anti-rabbit IgG coupled to fluorescein isothiocyanate (Cappel Labs, Warrington, Pa.). The procedure for carrying out the staining was as previously published (Phillips & Lewis, 1970). The sections were examined under a Wild M20 microscope with an FITC interference filter and on BG 38/1 red stop for excitation, with an OG 0-5 mm barrier filter. Photomicrographs were produced on Kodak Tri-X. Rosening EA (IgG) test cells were prepared as outlined by Huber, Douglas & Fudenberg (1969) and Edelson et al. (1973). Fc receptors were demonstrated on frozen sections using EA (IgG) rosettes according to the method of Edelson with minor modifications (Edelson et al., 1973).

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The frozen sections attached to coverslips were air dried and inverted on a cavity slide containing the rosetting solutions. The slides were then inverted to bring the test solutions fully in contact with the sections. After 30 min exposure at room temperature, the slides were again turned over and the coverslips gently removed under buffered saline, after allowing the indicator sheep red blood cells (SRBC's) to settle by gravity for 10 min. Several gentle washes in phosphate-buffered saline (PBS) were followed by fixation in 3'*,, glutaraldehyde for 30 min and staining with H and E. Similarly, C3' receptor assays were carried out on frozen sections using EAC (IgM) rosettes. The EAC rosettes were prepared according to the schedule of Meijer & Lindeman (1975) ^nd exposure of the frozen sections was as described by Dukor, Bianco & Nussenzweig (1970) and by Edelson et al. (1973). Lymph nodes obtained at autopsy were used as control specimens. Fc and C / receptors were also investigated by the standard methods on cells in suspension. EDTA separated epidermis (Scarietta & MacCallum, 1972) was dissociated by 30 min incubation at 37 C in 0-5",, buffered trypsin. Epidermal eell suspensions were checked for viability and for ATPase staining (MacKenzie & Squier, 1975). The EA and EAC rosette tests were carried out on cells in suspension according to the method of Zeylemaker et al. (1974)- Controls involved the use of untreated sheep red blood cells and rosettes consisting of EA (IgM). Immuno-electron microscopy (lEM) Subsequent to fixation the tissue strips (i 3 mm long) were washed in phosphate buffered saline (pH 7-4) and 25 /(m sections were prepared by a non-freezing method on a Sorvall TC-2 Tissue Chopper (Hallowcs & Streek, 1970). The sections were collected and incubated in 01 M sodium borohydride solution at pH 7-2 for i h at room temperature to block any free aldehyde groups (Kraehenbuhl & Jamieson, 1974). The sections were exposed to the IgG fraction of the rabbit anti-la antiserum for 18 h at 0-4 C. The concentration ofthe antiserum was approximately 01 mg Ab N/ml. After 18 h washing in PBS at 4 C with repeated changes, the sections were exposed to the second reagent, i.e. commercial IgG fraction of goat anti-rabbit IgG conjugated to ferritin (Cappel Labs, Downington, Pa.). This reagent was used at a concentration of approximately 0 1 mg Ab N/ml. The specificity of the reagent, although assured by immunoelectrophoretic data provided on purchase, was reassessed prior to use and found to be satisfactory. The second incubation proceeded for 18 hours as above, followed by extensive washing. The sections were post-fixed and processed as for morphological transmission electron microscopy. Thin sections were cut with diamond knives and examined, initially unstained, in a Philips EM 301. For photographic recording the sections were contrasted with lead citrate and bismuth subnitrate (Ainsworth & Karnovsky, 1972). Controls

The following controls were carried out at both IF and IEM levels: (a) Specific immune blocking: rabbit anti-la IgG followed by rabbit anti-la IgG coupled either to fluorescein isothiocyanate or to ferritin (Polyscience Inc., Warrington, Pa.). Ferritin conjugation to the IgG was achieved by the method of Kishida et al. (1975). Subsequent purification and characterization was as described by these authors. (b) Pre-incubation ofthe antiserum with the antigen preparation used in its preparation. (c) Omission ofthe primary reagent, i.e. incubation in labelled goat anti-rabbit IgG. (d) Incubation in preimmune rabbit serum followed by labelled goat anti-rabbit IgG. (e) Incubation with Fab' fraction ofthe IgG ofthe rabbit anti-la antiserum followed by labelled goat anti-rabbit IgG. Fab' fragments were prepared according to the method of Porter (1959) and were purified as outlined by Avrameas & Ternynck (1971). (f) Incubation in free fluoreseein isothiocyanate or ferritin.

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Fluorescence

Positive staining of dendritic cells was demonstrated in a supra-basal location in normal epidermis (Fig. i). Both dendrites and the cell bodies were intensely stained. Positive staining was noted in isolated instances in cells in the upper dermis. These cells appeared to be histiocytes. The positive reaction titred out at approximately 1000. The controls were negative, i.e. specific immune blocking, incubation with antigen, omission of primary reagent and incubation with free label. Incubation with the Fab' fragment of the anti-la antiserum as the primary reagent produced similar staining reactions to the whole antibody preparations, although some reduction in intensity was noted. Positively stained dendritic cells were also noted in the outer root sheaths of hair follicles. Staining results in leukodermatous skin samples were similar with respect to both positive and negative controls. The distribution of the stained dendritic cells, however, varied from the normal epidermal pattern. Dendritic cells were noted not only in the supra-basal position, but also in the basal layer (Fig. 2).

Rosetiing

EA (IgG) and EAC (IgM) rosetting on frozen sections were uniformly negative, despite the fact that the reagents and methods produced positive localization as previously indicated on lymph node sections (Edelson et al., 1973). The controls for the rosetting were similarly negative. Rosetting on the cell suspensions were, however, positive, i.e. both EA (IgG) and EAC (IgM) rosettes were formed between 2-3",j of the viable epidermal cells. The central cell of the rosettes (surrounded by 5-^ SRBCs) did not have a dendritic form. Thus, although results on frozen sections suggest the absence of Fc and C3' receptors, those obtained on cell suspensions were not in agreement. IEM The ultrastructural morphology of the Langerhans cells was well preserved by the method of fixation applied for IEM studies. In all respects the cells were as previously reported (Breathnach, 1965; Wolff, 1972). Transmission electron microscopic observations of both the vitiliginous skin and halo naevi samples confirmed previous reports (Birbeck, Breathnach & Everall, 1961; Jacobs et al., 1975; Rowden & Lewis, 1975; Gauthier et ai, 1975) of an absence of melanocytes in the basal layer and their replacement by a population of Langerhans cells. The localization of ferritin to the surface of both the cell bodies (Figs 3 & 4) and the dendritic process of Langerhans cells (Fig. 5) was specific and easy to recognize. No association of ferritin with the plasma membranes of melanocytes or keratinocytes was noted. No internalization of ferritin into the cytoplasm of Langerhans cells or into the characteristic granules was noted. In the dermis, the only cells to show any cell surface localization of ferritin were histiocytes (Fig. 6) and small mononuclear cells on the rare occasion these were seen in the blood vessel lumens (Fig. 7). These were presumably lymphocytes of the B-lineage. The controls, as for fluorescent microscopy, were negative and in particular the specificity of the reactions from cell to cell was assured in areas where positively stained histiocytes were noted adjacent to other identifiable cell types, i.e. naevus cells (Fig. 8), which were invariably negative. Specific immune blocking abolished the reaction with Langerhans cell surfaces, and there was no non-specific affinity of either goat anti-rabbit IgG or free ferritin for such sites.

Surface antigens on Langerhans cells

FIGURE I. (a) Normal skin—fluorescent antibody staining with anti-la andserum (indirect). Suprabasal dendritic cells positively stained (arrows) ( •' iooo). Basal dendritic cells positively stained (arrows) ( -' iooo). (b) Detail of a positive Langerhans cell ( x iooo).

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FIGURE 2. (a and b) vitiliginous skin—fiuorescent antibody staining with anti-la antiserum (indirect), stained dendritic cells in supra-basal {*), and basal positions (arrows) ( •• rooo).

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FIGURE 3. Langerhans cell body with distinctive granules (*) and a segment of the nucleus (N). {••crritin-antibody labelling of the cell surface (arrows) (.•-. 85,000).

DISCUSSION

The fluorescent antibody staining results clearly and unequivocally demonstrate the existence within human epidermis of a population of cells with a specific affinity for antibodies directed against components of B-lymphocyte cell membranes. The characteristics ofthe antigen used in raising the heteroantiserum are similar to those published elsewhere for Ia antigen in animal models (Vitetta, Klein & Uhr, 1974; Schwartz, Paul & Shevach, 1976; Balner, van Vreeswijk & Roger, 1976) and in man, both in physical and functional terms (Winchester^ra/., 1975jWernetera/., 1975;Winchester(3? a/., 1976; Wernet, 1976). Confirmation that the cells displaying this surface antigen are indeed Langerhans cells, however, must rest on the ultrastructural demonstration ofthe characteristic granule ofthe cell type. Thus, melanocytes being dendritic might be capable of producing similar staining patterns. The supra-basal location, however, speaks against such a possibility as does the staining seen in leuko-

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FIGURE 4. Langerhans cell body with a granule (*). Ferritin-antibody labelling on the cell surface (arrows) (x 112,500).

Surface antigens on Langerhans cells

FIGURE 5. Detidritic process of a Langerhans cell with prominent surface labelling with ferritin antibody (arrows). Desmosome (D) and keratin filaments (Kf) ( < 112,500).

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FIGURE 6. Dermal macrophage with prominent phagocytic vacuoles. Heavy surface labelling with ferritin-antibody (arrows) ( •: 17,750).

Surface antigens on Langerhans celb

FIGURE 7. Lymphocyte with a prominent nuclear cleft and an intracytoplasmic 'Gall' body (•). Heavy surface labelling of ferritin-antibody (arrows) ( •' 37,500).

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FIGURE 8. Macrophage with phagocytosed material adjacent to a naevus cell with a prominent nucleus (N) ( x 22,750), Inset. Surface labelling (arrows) with ferritin antibody on the macrophage but not on the naevus cell (*) ( \ 47,500).

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dermatous skin where melanocytes have been shown to be absent. Positive staining of dendritic cells in such samples being present not only in the supra-basal position but also in the basal layer is in accordance with the previously reported observations concerning the behaviour and disposition of Langerhans cells. Immuno-electron microscopic demonstration of specific localization of ferritin-labelled antibodies on the surface of cells bearing Langerhans granules is the clearest demonstration of their unique affinity in the epidetmis for such reagents. Neither melanocytes nor keratinocytes showed any positive staining. Merkel cells were not observed in this study, so it is not possible to comment on the possible expression of la antigens on such a cell type. The fact that the IEM was carried out on fixed material by a diffusion technique makes it extremely unlikely that any possible intracellular sites of la antigen in Langerhans, or indeed any other cell type, might be demonstrated. It is well documented that ultrastructural demonstrations of antigens in an intracelluiar location requires other approaches which are beyond the scope of this presentation (Kraehenbuhl & Jamieson, 1974; Sternberger, 1974). The observations made on cells in the dermis arc of interest in several ways. Staining ofthe surface of identifiable macrophages (i.e. containing phagocytosed material) is in accordance with other workers' observations concerning la. The fact that cells in the same vicinity, i.e. mast cells, fibroblasts, endothelial cells, naevus cells, remain unstained serves as a reliable control. At this stage it is not possible to comment on the numbers/percentage of histiocytes present within the dermis that are stained. Since it has been shown that only a percentage (8-15%) of murine peritoneal exudate macrophages express la antigens (Schwartz et al., 1976), it may well be that a similar situation exists in the population seen in the dermis. Clearly there are resident histiocytes and a percentage of cells of the macrophage lineage that may be considered as transient even in normal skin. Bearing in mind the previously proposed relationship between Langerhans cells and macrophages, it is interesting to speculate on the possibility that the proportion of macrophages/histiocytes that express la antigen might be the precursors ofthe positive Langerhans cells. The objection might be raised in considering the fluorescence data, that what is being stained in the epidermis is a population of imniigrant macrophages. This is unlikely in normal skin and indeed need not be disagreed with provided that the IEM evidence demonstrates unequivocally that these macrophages arc indeed Langerhans cells! Concerning the surface receptor studies, the results show some disparities. Demonstration of Fc and Cj' receptors by means of rosetting techniques on frozen sections has been shown to be valuable in investigation of various human skin conditions involving extensive mononuclear infiltrates in the dermis and epidermis (Edelson et al., 1973; Edelson et al., 1975; Tannenbaum, Pinkus & Schur, 1976). Provided that appropriate controls are carried out to ensure the efficacy ofthe procedures and the reliability ofthe reagents, there is no reason to suppose that, if it is possible to demonstrate such receptors in sections of lymph nodes, cells which might be classified as histiocytes or macrophages in the skin will not be demonstrable. However, little is known about the efficiency ofthe frozen sectionrosetting technique, so the possibility that Langerhans cells express such receptors on their surface, but at levels below that amenable to detection, must be admitted. It is interesting that other workers have been similarly unable to demonstrate cells that might be interpreted as Langerhans cells using frozen section rosetting methods (Edelson—personal cotnmunication). The results with cell suspensions clearly demonstrate that it is possible to detect a small percentage of epidermal cells expressing such receptors. The numbers present certainly fall within the expected values for Langerhans cells. Recently a report has appeared in abstract (Stingl et aj., 1977) ofthe successful demonstration of Fc and C,' receptors on epidermal cell suspensions and the results expressed here are essentially a confirmation of these findings. As yet the ultrastructural confirmation that the central cells in such rosettes are Langerhans cells is not at hand although they do stain for ATPase. The apparent disparity in the results may result from variations in technique. Clearly, production of a cell suspension exposes more surface area for interaction with rosetting cells and thus increases

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the efficiency ofthe process. However, the trypsinization used to effect this end almost certainly alters the normal cell surface and probably exposes receptors and antigenic structures that might be hidden in vivo. Since it has been shown recently that Langerhans cells m vivo do have an affinity for immune complexes in the form of peroxidase-antiperoxidase (Silberbcrg et al., 1977)^ it would seem that receptors are normally available and functional and the results on frozen sections are due to lack of sensitivity. Much still remains to be determined, however, concerning variations within the population of epidermal Langerhans cells with respect to expression of surface receptors, since there is a growing awareness of variations within the more extensively studied macrophage populations with respect to Fc receptors, etc. (Askenase & Hayden, 1974; Unkeless, 1977)The observations reported here provide further evidence to support the contention that the epidermal Langerhans cell is a specialized macrophage and the presence of Ia antigens on its surface raises various possibilities concerning its function. Since it is known from the work on Ia antigens in animal systems that this gene product is involved in a variety of immune phenomena (Shreffler& David, 1975; Bach er a/., 1972; Klein & Park, 1973; Klein, Hauptfeld & Hauptfeld, I974)> it may be a little early to speculate on the situation for Langerhans cells. Clearly the fact that Ia antigens are involved in the ability of certain immune cells to respond to particular antigens may have implications with respect to Silberberg's findings on reactivity of Langerhans cells with various allergens (Silberberg et al.., 1977) as well as in pathological conditions such as mycosis fungoides where Langerhans cells have been shown to have similar anatomical relationships to activated lymphocytes (Rowden & Lewis, 1976). Other areas where Ia antigens have been shown to play a role are in the MLC, in graft-versus-host reactions and in skin graft rejection. Thus, a spectrum of phenomena involving cell contact and cell-to-cell co-operation appears to be mediated via this glycoprotein. Hopefully, it may now be possible to pursue research into the known function of Ia antigens with respect to their particular situation on Langerhans cells. ACKNOWLEDGMENTS

Dr A.K.Sullivan is to be thanked for the gift ofthe anti-la antiserum. The expert technical assistance of Mr Eric Hcdderson is gratefully acknowledged. Professor Martin Lewis read the manuscript and his suggestions and encouragement cannot be estimated. REFERENCES AiNSWORTH, S.K. & KARNOVSKY, M.J. (1972) An ultrastructural staining method for enhancing the size and electron opacity of ferritin in thin sections. Journal of Hisrochemistry anii Cytochemistry, 20, 225. AsKANASE, P.W. & HAYDEN, B.J. (1974) Cytophilic antibodies in mice contact sensitized with oxalozone. Immunology, z-j, S63.

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SHELLEY, W . B . & JUHLIN, L . (1976) Langerhans cells form a reticulo-epithelial trap for extemal contact allergens. Nature {London), 261, 46, SHELLEY, W.B. & JUHLIN, L . (1977) Selective uptake of contact allergens by the Langerhans cell. Archives of Dermatology, 113, 187. SHKEFFLER, D.C. & DAVID, C.S. (1975) The H-2 major histocompatibility complex and the I immune response region: Genetic variation, function and organization. Advances in Immunology., 20, 125. SILBERBERG, I. (1971a) Ultrastructural studies of Langerhans cells in contact sensitivity and primary irritant reactions to mercuric chloride. Clinical Research, 19, 715. SILBERBERG, L (1971b) Studies by electron microscopy on epidermis after topical application of mercuric chloride: Morphologic and histochemical findings in epidermal cells of human subjects who do not show allergic or primary irritant reactions to mercuric chloride (0,1%), Journal of Investigative Dermatology, 56, 147. SILBERBERG, I. (1973) Apposition of mononuclear cells to Langerhans cells in contact allergic reactions. An ultrastructural study. Acta dermato-venereologica, 53, i. SILBERBERG, I., BAER, R.L., ROSENTHAL, S.A., THORBECKE, G J . & BEREZOWSKY, V. (1975) Dermal and intravas-

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Immuno-electron microscopic studies of surface receptors and antigens of human Langerhans cells.

British Journal of Dermatology {1977) 97) 593- Clinkai and Laboratory Investigations Immuno-electron microscopic studies of surface receptors and an...
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