British Journal of Dermatology (1979) 100, 531.
la antigens on indeterminate cells of the epidermis: immunoelectronmicroscopic studies of surface antigens G.ROWDEN, T.M.PHILLIPS AND M.G.LEWIS Pathology Department, Georgetown University Medical School, Washington, D.C. 20007, U.S.A. Accepted for publication 9 October 1978
SUMMARY
An antiserum against human B-lymphoblastoid cell membrane alloantigens (la-like antigens) was used to study the presence of such antigens on dendritic cells in human epidermis. Only Langerhans cells and the majority (85',',,) of so-called indeterminate cells were positively stained, as shown by immune-electron microscopy. Fifteen percent of the indeterminate cells were negative and were considered to be immature melanocytes. A relationship exists between the indeterminate cell and the Langerhans cell. A proposal is made concerning emigration of Langerhans cells in response to haptenic stimulation, and the immigration of indeterminate cells to restore the status quo.
Observations reported in the past year concerning surface receptors (Rowden, Lewis & Sullivan, 1977; Stingl et al, 1977) and antigens on Langerhans cells (Rowden, 1977; Klareskog et al, 1977; Rowden et al, 1977; Stingl et al, i977) 1978a) have revived interest in the origins and functions of the cell. In particular, the presence of Fc and C3 receptors (Rowden era/., 1977; Stingl (?r a/., 1977) and Ia antigen(s) (Klareskog et al., 1977; Rowden, 1977; Stingi et al, 1978a) have established the cell as a form of specialized epidermal macrophage. These studies have been carried out in man with heteroantisera against B-cell alloantigens (Rowden, 1977; Rowden et al, 1977) or against HLA-D antigens (Klareskog et al, 1977). In a congenic animal model, such as strain 2 and strain 13 guinea-pigs, Stingl et al (1978a) have confirmed that Ia antigens are expressed only on Langerhans cells in the epidermis. In an associated study, they also confirmed the reports described above for human epidermis, by using different techniques (Stingl et al, 1978b). The situation has recently been carried further in mice using well defined alloantisera (Rowden, Phillips & Delovitch, 1979). Thus, three independent laboratories using different techniques have firmly established that Langerhans cells express antigens coded for by the immune response gene region (Ir) of the major histo-compatibility complex or its equivalent. These observations have considerable significance with respect to suggested functions for the cell This work was supported by BRSG grant Georgetown University, Washington D.C. 20007. 0007-0963/79/0500-0531 $02.00 C 1979 British Association of Dermatologists
532
G.Rowden, T.M.Phillips and M.G.Lewis
and fit well with previous proposals concerning the skin as an important lymphoid organ (Fichetelius, Groth & Liden, 1970). Over the past few years various authors have investigated the involvement of Langerhans cells in contactallergic hypersensitivity reactions (Shelley & Juhlin, 1976,1977; Silberberg, Baer & Rosenthal, 1976; Juhlin & Shelley, 1977; Silberberg-Sinakin et al, 1976,1977). In summary, they have shown in vitro that Langerhans cells bind a range of haptens, transport antigens such as ferritin to the regional nodes, and also have suggested that they act as target cells for the effector T-cell populations resulting from the lymph node sensitization. The previously unknown tissue protein, responsible for binding haptens to the skin, therefore, appears to be related to the surface of Langerhans cells. Since there has been shown to be a traffic of Langerhans cells from the epidermis to the local lymph nodes (Silberberg, Baer & Rosenthal, 1976), it is obvious that if the resident Langerhans cell population in the epidermis is to be maintained, some form of replacement must occur. Two possible explanations for the maintenance of Langerhans cell populations in the human epidermis at around 500-800/mm^ are apparent. Cell loss involved in contact allergic reactions might be balanced by mitosis. Indeed, rare mitoses of Langerhans cells have been reported (Giacometti & Montagna, 1967). However, it is difficult to accommodate the traffic involved with replacement by such activity. The alternative would involve immigration of a stem cell to replace the emigration of Langerhans cells, involved in transporting bound allergens to local lymph nodes. Movement of Langerhans cells from the dermis to the epidermis has been noted previously (Bock, 1972; Wolff, 1972). This study was carried out to determine the surface antigenic composition of the various nonkeratinocytes in the epidermis, in order to establish possible relationships amongst these cells. The major cells present, other than Langerhans cells, are melanocytes, Merkel cells (Breathnach, 1971), lymphocytes and indeterminate or :f-dendritic cells (Zelickson & Mottaz, 1968) (Fig. i). Using antisera against la-like antigens and immunoelectron microscopic analysis, the reactivity of the cells was examined.
Lc
FIGURE I, Summary of the epidermal dendritic cells. BL = basal lamina; D = dermis; E — epidermis; H = histiocyte; Lc — Langerhans cell; Ly = lymphocyte; Me = melanocyte; Mk = Merkel cell; ? = indeterminatecell. N.B. Langerhanscellsalsomaybenotedinthe basal layer and in the dermis.
la antigens on indeterminate cells
533
MATERIALS AND METHODS
Human skin, obtained at surgery or as punch biopsy specimens obtained under local anaesthesia, was processed as follows: (1) Fixed in half strength Karnovsky fixative at room temperature for i h, for conventional transmission electron microscopy (TEM). Subsequent processing, i.e. osmication, dehydration and embedding, was according to standard schedules. (2) Fixed for 30 min at room temperature, in i-2$y^ paraformaldehyde in 0 1 mol/I phosphate buffer, pH 7-4, for immuno-electron microscopy (IEM). Antisera The antiserum against B-cell alloantigens (la-like) has been described elsewhere (Sullivan et al. 1977). Briefly, it was raised in rabbits against Con A-binding fractions of human B-Iymphoblastoid cell lines. Its characteristics have been described and it has been shown to have a preferential reactivity with B lymphocytes and Langerhans cells (Rowden et al, 1977)IEM The IgG fraction of the rabbit anti-Ia-like antiserum was obtained by affinity chromatography with sepharose-anti-rabbit IgG beads. Fab' fragments were produced by means of papain digestion as described by Porter (i959)Immiinostaining Subsequent to fixation, the tissue strips (1x3 mm long) were washed in phosphate buffered saline (pH 7-4) (PBS), and 25 /im sections were prepared with a Sorval TC-2 tissue chopper. The sections were collected and incubated in o-i mol/I sodium borohydride at pH 72 for i h at room temperature to block free aldehyde groups (Kraehenbuhl & Jamieson, 1974). The sections were exposed to the antisera in an indirect staining schedule, i.e. IgG fraction rabbit anti-la or Fab' fraction rabbit anti-la for 18 h at 0-4 C. The concentration of the IgG fraction was o-i mg Ab N/ml. After vigorous washing for 18 h at 0-4 C. in PBS, the tissue sections were exposed to commercial IgG fraction goat anti-rabbit IgG, conjugated to ferritin (Cappel Labs, Downingtown, Pa.). This reagent was used at o-i mg Ab N/ml. The purity of the commercial reagent, although assured, was checked by immunoelectrophoresis. The second incubation was carried out for 18 h as above, followed by a repeat of the PBS washing step. The sections were post-fixed with osmium and processed for TEM. Thin sections were cut with diamond knives and examined on a Siemens Elmiskop ioi microscope without heavy metal staining. For photographic recording the sections were contrasted with lead citrate and bismuth subnitrate (Ainsworth & Karnovsky, 1972). Controls The following controls, as described previously (Rowden, 1977)5 were carried out: (1) Specific immune blocking: rabbit-anti la IgG, followed by rabbit anti-la IgG coupled to ferritin. Labelling of the IgG was carried out using the method of Kishida et al. (i975)- Subsequent purification and characterization of the direct reagent was as described by the authors. (2) Pre-incubation of the antiserum with the antigen used in its preparation. (3) Omission of the primary reagent, i.e. incubation in labelled goat-anti rabbit IgG. (4) Incubation in pre-immune rabbit serum followed by goat-anti rabbit IgG-ferritin. (5) Incubation in free ferritin.
534
G.Rowden, T M Phillips and M.G.Lewis RESULTS
The staining of Langerhans cells in the epidermis was the same as has been reported previously (Rowden, 1977). The ferritin label, whether with the indirect labelling or direct labelling with either IgG or Fab' reagents, was associated with the cell surface of both the cell body and dendrites of suprabasal Langerhans cells (Fig. 2). No label was ever detected on the surface of melanocytes in the basal layer or on keratinocytes. These findings are a confirmation of the previous report (Rowden, 1977) and do not require illustration here. Special attention was directed at the immigrant lymphocyte population and the so-called indeterminate cell (IDC). Lymphocytes in the epidermis, as identified
FIGURE 2. Supra-basal Langerhans cell with heavy immunoferritin labelling on the plasma membrane (arrows) Langerhans ceil granules (circles) ( x 17,000). Inset—higher magnification of the sxirface of a positively stained Langerhans cell (arrows) ( x 34,000).
Ia antigens on indeterminate cells
535
FIGURE 3. Lymphocyte infiltrating the epidermis (*) near the basal lamina. No ferricin labelling on the cell surface ( x 26,500).
by their nuclear morphology and scant cytoplasm, were never stained (Fig. 3). The IDC's, whether seen at the basal lamina or in the supra basal layers, were almost without exception lightly stained (Fig. 4). These cells were distinguished from the lymphocytes by their infolded nuclei, with a diffuse chromatin pattern similar to that seen in Langerhans cells. They lacked either characteristic Langerhans cell granules or melanosomes. Serial sectioning was carried out on specific examples to ensure that no such organelles were present in the cytoplasm. Over 100 IDC's were examined but the serial sectioning procedure was clearly not practical in more than a few instances. The cells contained abundant cytoplasmic filaments, which are not seen in lymphocytes. They also have a moderately well developed Golgi complex and associated rough membranous system of endoplasmic reticulum. Approximately 15" „ of the cells characterized as IDC's showed no positive staining (Fig. 5). As will be seen from the morphology, these cells were extremely difficult to place in either the lymphocyte or IDC cell category. As was reported previously (Rowden, 1977) histiocytic cells with features reminiscent of those described above were noted in the dermis (Fig. 6). They were often actively phagocytic and their surfaces were strongly stained.
536
G.Rowden, T.M.Phillips and M.G.Lewis
V V FIGURH 4. IDC(*) near the basal lamina showing light imniuno-lerritin staining on the cell surface (arrows). Wide intercellular space resulting from fixation and prolonged incubations (LS.) ( X 26,500). Inset—higher magnification of the abovt. Immuno-ferritin staining on the cell surface (arrows) (x 53,000).
Ia antigens on indeterminate cells
FIGURE 5. Cell lacking immuno-ferritin surface staining. (*) Situated near the basal lamina (arrow). Cytoplasm lacking melanosomes or Langerhans cell granules ( x 26,500)—higher magnification of the cell surface and intercellular space (LS.) ( x 49,000).
537
538
G.Rowden, T.M.Phillips and M.G.Lewis
FIGURE 6. Dermal histiocyre near the dermoepidermal junction. Heavy surface immuno-ferritin labelling (arrows) ( x 30,000). Inset—higher magnification of surface label ( x 60,000).
la antigens on indeterminate cells
539
Lc
FIGURE 7. Summary of IEM staining. • IgG rabbit anti-human la = ferritin.
The controls all confirmed the specificity of the reactions as described previously (Rowden, 1977). No Merkel cells were identified and thus no comment may be made at this time concerning its Iaantigcn activity. Figure 7 summarizes the IEM results. DISCUSSION
As an introduction to the discussion of the results, it is appropriate to quote a statement made a few years ago by Breathnach (1975) concerning IDC's. He described the cells as dendritic non-keradno~ cytes without specific cytoplasmic identifying features such as Langerhans cell granules, melanosomes or Merkel cell granules. He concluded that they represented variant or immature forms of either melanocytes or Langerhans cells. Further, he felt it to be unfortunate that names such as a-dendritic or indeterminate should have been applied to the cells, since they gave rise to the erroneous idea that there were one or two additional cell types present in the epidermis. It is to be hoped that the findings described here may now accommodate the cells into a functional role or roles in the epidermis. Although we were unfortunate in not sampling Merkel cells, the findings illustrate that the only cells expressing la-like antigen in human epidermis are either Langerhans cells or cells which may be related to them. The only feature they lack is the Langerhans cell granule. Admittedly, serial sectioning might have disclosed such organelles, if it were applied to all of the indeterminate cells examined. Such a technical feat was, however, considered to be unjustified. The cells do not appear to be lymphocytes as judged by morphology and indeed the major lymphocyte in the epidermis appears to be of T-cell origin, where demonstration of la-like antigens would not be expected. The percentage of cells that were difficult to classify and which were negative for la-like antigens might fall in a hinterland between lymphocytes and IDC's, but in our opinion they are more likely to be immature melanocytes. Quantitative studies by previous workers on dendritic cells in the epidermis have apparently demonstrated some relationship of indeterminate cells to the other known cell types (Mishima, Kawasaki & Pinkus, 1972) which requires consideration. The three dendritic cell types were counted in normal skin and in various leukodermatous conditions,
540
G.Rowden, T.M.Phtllips and M.G.Lewis
i.e. vitiligo, halo naevus, and spontaneous regression associated with lentigo maligna (Mishima et al., 1972). These studies appear to demonstrate that as melanocytes disappear, the number of IDC's increases. In later lesions, where melanocytes are absent, the IDC's decrease and Langerhans cells move down to the basal layers. No good explanation has ever been established to explain why Langerhans cells should occupy such sites in leukoderma, other than suggestions that the ratio of keratinocytes/dendritic cells needs to be kept constant in the basal layer (Breathnach, 1978). Thus, absence of melanocytes is compensated for by movement of Langerhans cells. These cytokinetic studies of IDC's do not, of course, provide information that will permit relationships of the IDC's to the other two cell types to be made. Mishima et al. (1972) felt that there is a transformation of melanocytes into inactive IDC's, but they were unable to exclude the possibility that they were derived from or related to cells of a different ontogeny, i.e. the Langerhans cells. The studies presented here would appear to show that the bulk of the IDC's have surface characteristics that indicate an affinity with Langerhans cells. The increase in IDC's in depigmentation may just as likely result from alteration in the kinetics of the Langerhans cell population as solely from degeneration of melanocytes. It must be noted, however, that the observations reported here concern normal human epidermis so comparisons with Mishima et a/.'s (1972) study are not strictly valid. Investigation of leukodermatous epidermis with the immuno-electron microscopic techniques used here will be necessary before final comment may be made. That the bulk of the indeterminate cells described here express surface antigen associated with Langerhans cells, rather than any other epidermal cell type, provides an attractive explanation for the constancy of Langerhans cell populations. The immigration of immature histocytic cells from the dermis to replace Langerhans cells, involved in their function with respect to contact allergic phenomena, would seem to be represented by the indeterminate cell population. The presence of Langerhans cells in the dermis has been repeatedly described (Wolff, 1972) so it need be not only the indeterminate cell repopulating the epidermis, as
Replocement mitosis ('Ore) or immiqrotion
Affererent arc (Allergen comer
Lymphoid pool?
Lvmph node
FIGURE 8. Summary of possible pathways of movement of Langerhans cells and their precursor cells.
Ia antigens on indeterminate cells
541
mature Langerhans cells have been shown to do (Wolff, Lessard & Winkelmann, 1971; Breathnach, 1978). The fact that histiocytes in the dermis are also Ia positive suggests the presence of such a pool of cells capable of immigration. Whether the pool of immature Langerhans cells is actually resident in the dermis or originates from other lymphoid sources such as the bone marrow, spleen, or nodes, requires testing. Kinetic studies using isolated macrophage populations as well as Langerhans cells are underway and will provide important insights into this question. Figure 8 summarizes the pathways involving Langerhans cells and their precursor cells. It will be noted that the suggestions made imply that the granule which is so characteristic of Langerhans cells develops only subsequent to the arrival of immature indeterminate cells in the epidermis. Studies on the repopulation of the epidermis by Langerhans cells after tape stripping have shown that in the guinea-pig this seems to be the case. Granules appear only after cells arrive in the upper layers of the epidermis (Wolff, et al, 1971). There is no evidence to rule out the possibility that Langerhans cells with their granules are not capable of returning to the epidermis after migrating to the local nodes during their response to contact allergens. This possibility also requires testing with labelled Langerhans cells. Breathnach (1978) has very recently published a description of mesenchymal cells of human embryonic and fetal dermis that might be ancestral for the Langerhans cell. These cells might be carried over into the postnatal dermis. Quiescent histiocytes under certain circumstances might also, as he suggests, be transformed into Langerhans cells. ACKNOWLEDGMENTS
Dr A.K.Sullivan is to be thanked for providing the antiserum against human la-like antigen(s). The skilled technical assistance of Mr N.More and Miss E.Rusnock is acknowledged. Mrs M.Davis is to be thanked for typing the manuscript. REFERENCES AiNSWOBTH, S.K. & KARNOVSKY, M.J. (1972) An ultrasrruciural staining method for enhancing the size and electron opacity of ferritin in thin sections. Journal of Histochemistry and Cytochemistry, 20, 225. BOCK, P. (1972) The mesenchymal origin of Langerhans ccW?.. Journal of Siihnicroscopic Cytology, 4, 271. BREATHNACH, A.S. (1971) An Atlas of rhe Ultrastructure of Human Skin. Churchill, London. BBEATHNACH, A.S. (1975) Aspects of epidermal ultrastructure. ^^''Hnia/ of Investigative Dermatology, 65, 2. BREATHNACH, A.S. (1978) Development and differentiation of dermal cells in man. Journal of Investigative Dermatology, 71, 2. FiCHELTELius, K.E., GROTH, O. & LiDEN, S. C1970) The skin: a first level lymphoid organ. International Archives of Allergy, 37, 607. GlACOMETTi, L. & MONTAGNA, W. (1967) Langcrhans cells: uptake of tritiated thymidine. Science, 157, 438. JUHLIN, L. & SHELLEY, W.B. (1977) New staining techniques for the Langerhans cells. Acta dermato-venereologica, 57, 289. KiSHiDA, Y., OLSEN, B.R., BERG, R.A. & PROCKOP, D.J. (1975) Two improved methods for preparing ferritinprotein conjugates for electron microscopy. Journal of Cell Biology, 64, 331. KLARESKOG, L., TJERNLUND, U.M., FORSUM, U . & PETERSON, P.A. (1977) Epidermal Langerhans cells express Ia
antigens. Nature, 268, 248. KRAEHENBUHL, J.P. & JAMIESON, J.D. (1974) Localization of intracellular antigen by immuno-electron microscopy. International Reviezvs of Experimental Pathology, 13, i. MisHiMA, Y., KAWASAKI, H . & PINKUS, H , (1972) Dendritic cell dynamics in progressive depigmentations. Archiv far Dermatologische Porschung, 243, 67. PORTER, R.R. (1959) The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochemical Journal, 83, 119. ROWDEN, G. (1977) Immuno-electron microscopic snidies of surface receptors and antigens in human Langerhans cells. British Journal of Dermatology, 97, 592.
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ROWDEN, G., LEWIS, M.G. & SULLIVAN, A.K. (1977) Ia antigen expression on human epidermal Langerhans cells. Nature, 268, 247. ROWDEN, G . , PHILLIPS, T.M. & DELOVITCH, T.L. (1979) Visualization of Ia antigen localization in murine keratinizing epithelia. Immunogenetics, in press. SHELLEY, W.G. & JUHLIN, L . (1976) Langerhans cells from a reticulo-epithelial trap for external contact allergens. Nature, 261, 46. SHELLEY, W.B. & JUHLIN, L . (1977) Selective uptake of contact allergens by Langerhans cells. Archives of Dermatology, 113, 187. SILBERBERG, E., BAER, R.L. & ROSENTHAL, S.A. (1976) The role of Langerhans cells in allergic contact hypersensitivity. A review of the findings in man and guinea-pigs. Journal of Invcstiga'.ive Dermatology, 66, 210. SlLBERBERG-SlNAKlN, L , F E D O R K C , M . E . , B A E R , R . L . , RoSENTHAL, S.A., B E R E Z O W S K Y , V. & T H O R B E C K E ,
G.J.
C1977) Langerhans cells: target cells in immune complex reactions. Cell Immunology, 32, 400. SiLBERBERG-SiNAKiN, L, THORBECKE, G.J., BAER, R.L., ROSENTHAL, S.A. & BEREZOWSKY, V. (1976) Antigen
binding Langerhans cells in the skin, dermal lymphatics and in lymph nodes. Cell Immunology, 25, 137. STINGL, G., KATZ, S.L, SHEVACH, E.M., WOLFE-SCHREINER, E . C . & GREEN, L (1978a) Detection of Ia antigens
on Langerhans cells in guinea-pig skin. Journal of Immunology, 120, 570. STINGL, G., KATZ, S.I., ABELSON, L.D. & MANN, D.L. (1978b) Immunofluorescent detection of human B cell
alloantigen on S-Ig-positive lymphocytes and epidermal Langerhans ccWs. Journal of Immunology, 120, 661. STINGL, G . , WOLFF-SCHREINER, E.C., PINCHLER, W.J., SCHMAIT, F . , KNAPP, W . & WOLFE, K . (1977) Epidermal
Langerhans cells bear Fc and C3 receptors. Nature, 268, 245. SULLIVAN, A.K., JERRY, L.M., ROWDEN, G . & SHEA, M . C1977") Expression of a B-lymphocyte antigen in chronic
lymphocytic and other leukemias. Clinical Immunology and Immunopathology, 8, 64. WOLFF, K . (1972) The Langerhans cell. Current Problems in Dermatology, 4, 79. WOLFE, K., LESSARD, R.J. & WINKELMANN, R.K. (1971) Electron microscopic observations on Langerhans cells after epidermal trauma. Dermatology Digest, lo, 35. ZELICKSON, A.S. & MOTTAZ, J.H. (1968) Epidermal dendritic cells: a quantitative study. Archives of Dermatology, 98, 652.
la antigens on indeterminate cells of the epidermis: immunoelectronmicroscopic studies of surface antigens G.ROWDEN, T.M.PHILLIPS AND M.G.LEWIS Pathology Department, Georgetown University Medical School, Washington, D.C. 20007, U.S.A. Accepted for publication 9 October 1978
SUMMARY
An antiserum against human B-lymphoblastoid cell membrane alloantigens (la-like antigens) was used to study the presence of such antigens on dendritic cells in human epidermis. Only Langerhans cells and the majority (85',',,) of so-called indeterminate cells were positively stained, as shown by immune-electron microscopy. Fifteen percent of the indeterminate cells were negative and were considered to be immature melanocytes. A relationship exists between the indeterminate cell and the Langerhans cell. A proposal is made concerning emigration of Langerhans cells in response to haptenic stimulation, and the immigration of indeterminate cells to restore the status quo.
Observations reported in the past year concerning surface receptors (Rowden, Lewis & Sullivan, 1977; Stingl et al, 1977) and antigens on Langerhans cells (Rowden, 1977; Klareskog et al, 1977; Rowden et al, 1977; Stingl et al, i977) 1978a) have revived interest in the origins and functions of the cell. In particular, the presence of Fc and C3 receptors (Rowden era/., 1977; Stingl (?r a/., 1977) and Ia antigen(s) (Klareskog et al., 1977; Rowden, 1977; Stingi et al, 1978a) have established the cell as a form of specialized epidermal macrophage. These studies have been carried out in man with heteroantisera against B-cell alloantigens (Rowden, 1977; Rowden et al, 1977) or against HLA-D antigens (Klareskog et al, 1977). In a congenic animal model, such as strain 2 and strain 13 guinea-pigs, Stingl et al (1978a) have confirmed that Ia antigens are expressed only on Langerhans cells in the epidermis. In an associated study, they also confirmed the reports described above for human epidermis, by using different techniques (Stingl et al, 1978b). The situation has recently been carried further in mice using well defined alloantisera (Rowden, Phillips & Delovitch, 1979). Thus, three independent laboratories using different techniques have firmly established that Langerhans cells express antigens coded for by the immune response gene region (Ir) of the major histo-compatibility complex or its equivalent. These observations have considerable significance with respect to suggested functions for the cell This work was supported by BRSG grant Georgetown University, Washington D.C. 20007. 0007-0963/79/0500-0531 $02.00 C 1979 British Association of Dermatologists
532
G.Rowden, T.M.Phillips and M.G.Lewis
and fit well with previous proposals concerning the skin as an important lymphoid organ (Fichetelius, Groth & Liden, 1970). Over the past few years various authors have investigated the involvement of Langerhans cells in contactallergic hypersensitivity reactions (Shelley & Juhlin, 1976,1977; Silberberg, Baer & Rosenthal, 1976; Juhlin & Shelley, 1977; Silberberg-Sinakin et al, 1976,1977). In summary, they have shown in vitro that Langerhans cells bind a range of haptens, transport antigens such as ferritin to the regional nodes, and also have suggested that they act as target cells for the effector T-cell populations resulting from the lymph node sensitization. The previously unknown tissue protein, responsible for binding haptens to the skin, therefore, appears to be related to the surface of Langerhans cells. Since there has been shown to be a traffic of Langerhans cells from the epidermis to the local lymph nodes (Silberberg, Baer & Rosenthal, 1976), it is obvious that if the resident Langerhans cell population in the epidermis is to be maintained, some form of replacement must occur. Two possible explanations for the maintenance of Langerhans cell populations in the human epidermis at around 500-800/mm^ are apparent. Cell loss involved in contact allergic reactions might be balanced by mitosis. Indeed, rare mitoses of Langerhans cells have been reported (Giacometti & Montagna, 1967). However, it is difficult to accommodate the traffic involved with replacement by such activity. The alternative would involve immigration of a stem cell to replace the emigration of Langerhans cells, involved in transporting bound allergens to local lymph nodes. Movement of Langerhans cells from the dermis to the epidermis has been noted previously (Bock, 1972; Wolff, 1972). This study was carried out to determine the surface antigenic composition of the various nonkeratinocytes in the epidermis, in order to establish possible relationships amongst these cells. The major cells present, other than Langerhans cells, are melanocytes, Merkel cells (Breathnach, 1971), lymphocytes and indeterminate or :f-dendritic cells (Zelickson & Mottaz, 1968) (Fig. i). Using antisera against la-like antigens and immunoelectron microscopic analysis, the reactivity of the cells was examined.
Lc
FIGURE I, Summary of the epidermal dendritic cells. BL = basal lamina; D = dermis; E — epidermis; H = histiocyte; Lc — Langerhans cell; Ly = lymphocyte; Me = melanocyte; Mk = Merkel cell; ? = indeterminatecell. N.B. Langerhanscellsalsomaybenotedinthe basal layer and in the dermis.
la antigens on indeterminate cells
533
MATERIALS AND METHODS
Human skin, obtained at surgery or as punch biopsy specimens obtained under local anaesthesia, was processed as follows: (1) Fixed in half strength Karnovsky fixative at room temperature for i h, for conventional transmission electron microscopy (TEM). Subsequent processing, i.e. osmication, dehydration and embedding, was according to standard schedules. (2) Fixed for 30 min at room temperature, in i-2$y^ paraformaldehyde in 0 1 mol/I phosphate buffer, pH 7-4, for immuno-electron microscopy (IEM). Antisera The antiserum against B-cell alloantigens (la-like) has been described elsewhere (Sullivan et al. 1977). Briefly, it was raised in rabbits against Con A-binding fractions of human B-Iymphoblastoid cell lines. Its characteristics have been described and it has been shown to have a preferential reactivity with B lymphocytes and Langerhans cells (Rowden et al, 1977)IEM The IgG fraction of the rabbit anti-Ia-like antiserum was obtained by affinity chromatography with sepharose-anti-rabbit IgG beads. Fab' fragments were produced by means of papain digestion as described by Porter (i959)Immiinostaining Subsequent to fixation, the tissue strips (1x3 mm long) were washed in phosphate buffered saline (pH 7-4) (PBS), and 25 /im sections were prepared with a Sorval TC-2 tissue chopper. The sections were collected and incubated in o-i mol/I sodium borohydride at pH 72 for i h at room temperature to block free aldehyde groups (Kraehenbuhl & Jamieson, 1974). The sections were exposed to the antisera in an indirect staining schedule, i.e. IgG fraction rabbit anti-la or Fab' fraction rabbit anti-la for 18 h at 0-4 C. The concentration of the IgG fraction was o-i mg Ab N/ml. After vigorous washing for 18 h at 0-4 C. in PBS, the tissue sections were exposed to commercial IgG fraction goat anti-rabbit IgG, conjugated to ferritin (Cappel Labs, Downingtown, Pa.). This reagent was used at o-i mg Ab N/ml. The purity of the commercial reagent, although assured, was checked by immunoelectrophoresis. The second incubation was carried out for 18 h as above, followed by a repeat of the PBS washing step. The sections were post-fixed with osmium and processed for TEM. Thin sections were cut with diamond knives and examined on a Siemens Elmiskop ioi microscope without heavy metal staining. For photographic recording the sections were contrasted with lead citrate and bismuth subnitrate (Ainsworth & Karnovsky, 1972). Controls The following controls, as described previously (Rowden, 1977)5 were carried out: (1) Specific immune blocking: rabbit-anti la IgG, followed by rabbit anti-la IgG coupled to ferritin. Labelling of the IgG was carried out using the method of Kishida et al. (i975)- Subsequent purification and characterization of the direct reagent was as described by the authors. (2) Pre-incubation of the antiserum with the antigen used in its preparation. (3) Omission of the primary reagent, i.e. incubation in labelled goat-anti rabbit IgG. (4) Incubation in pre-immune rabbit serum followed by goat-anti rabbit IgG-ferritin. (5) Incubation in free ferritin.
534
G.Rowden, T M Phillips and M.G.Lewis RESULTS
The staining of Langerhans cells in the epidermis was the same as has been reported previously (Rowden, 1977). The ferritin label, whether with the indirect labelling or direct labelling with either IgG or Fab' reagents, was associated with the cell surface of both the cell body and dendrites of suprabasal Langerhans cells (Fig. 2). No label was ever detected on the surface of melanocytes in the basal layer or on keratinocytes. These findings are a confirmation of the previous report (Rowden, 1977) and do not require illustration here. Special attention was directed at the immigrant lymphocyte population and the so-called indeterminate cell (IDC). Lymphocytes in the epidermis, as identified
FIGURE 2. Supra-basal Langerhans cell with heavy immunoferritin labelling on the plasma membrane (arrows) Langerhans ceil granules (circles) ( x 17,000). Inset—higher magnification of the sxirface of a positively stained Langerhans cell (arrows) ( x 34,000).
Ia antigens on indeterminate cells
535
FIGURE 3. Lymphocyte infiltrating the epidermis (*) near the basal lamina. No ferricin labelling on the cell surface ( x 26,500).
by their nuclear morphology and scant cytoplasm, were never stained (Fig. 3). The IDC's, whether seen at the basal lamina or in the supra basal layers, were almost without exception lightly stained (Fig. 4). These cells were distinguished from the lymphocytes by their infolded nuclei, with a diffuse chromatin pattern similar to that seen in Langerhans cells. They lacked either characteristic Langerhans cell granules or melanosomes. Serial sectioning was carried out on specific examples to ensure that no such organelles were present in the cytoplasm. Over 100 IDC's were examined but the serial sectioning procedure was clearly not practical in more than a few instances. The cells contained abundant cytoplasmic filaments, which are not seen in lymphocytes. They also have a moderately well developed Golgi complex and associated rough membranous system of endoplasmic reticulum. Approximately 15" „ of the cells characterized as IDC's showed no positive staining (Fig. 5). As will be seen from the morphology, these cells were extremely difficult to place in either the lymphocyte or IDC cell category. As was reported previously (Rowden, 1977) histiocytic cells with features reminiscent of those described above were noted in the dermis (Fig. 6). They were often actively phagocytic and their surfaces were strongly stained.
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G.Rowden, T.M.Phillips and M.G.Lewis
V V FIGURH 4. IDC(*) near the basal lamina showing light imniuno-lerritin staining on the cell surface (arrows). Wide intercellular space resulting from fixation and prolonged incubations (LS.) ( X 26,500). Inset—higher magnification of the abovt. Immuno-ferritin staining on the cell surface (arrows) (x 53,000).
Ia antigens on indeterminate cells
FIGURE 5. Cell lacking immuno-ferritin surface staining. (*) Situated near the basal lamina (arrow). Cytoplasm lacking melanosomes or Langerhans cell granules ( x 26,500)—higher magnification of the cell surface and intercellular space (LS.) ( x 49,000).
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G.Rowden, T.M.Phillips and M.G.Lewis
FIGURE 6. Dermal histiocyre near the dermoepidermal junction. Heavy surface immuno-ferritin labelling (arrows) ( x 30,000). Inset—higher magnification of surface label ( x 60,000).
la antigens on indeterminate cells
539
Lc
FIGURE 7. Summary of IEM staining. • IgG rabbit anti-human la = ferritin.
The controls all confirmed the specificity of the reactions as described previously (Rowden, 1977). No Merkel cells were identified and thus no comment may be made at this time concerning its Iaantigcn activity. Figure 7 summarizes the IEM results. DISCUSSION
As an introduction to the discussion of the results, it is appropriate to quote a statement made a few years ago by Breathnach (1975) concerning IDC's. He described the cells as dendritic non-keradno~ cytes without specific cytoplasmic identifying features such as Langerhans cell granules, melanosomes or Merkel cell granules. He concluded that they represented variant or immature forms of either melanocytes or Langerhans cells. Further, he felt it to be unfortunate that names such as a-dendritic or indeterminate should have been applied to the cells, since they gave rise to the erroneous idea that there were one or two additional cell types present in the epidermis. It is to be hoped that the findings described here may now accommodate the cells into a functional role or roles in the epidermis. Although we were unfortunate in not sampling Merkel cells, the findings illustrate that the only cells expressing la-like antigen in human epidermis are either Langerhans cells or cells which may be related to them. The only feature they lack is the Langerhans cell granule. Admittedly, serial sectioning might have disclosed such organelles, if it were applied to all of the indeterminate cells examined. Such a technical feat was, however, considered to be unjustified. The cells do not appear to be lymphocytes as judged by morphology and indeed the major lymphocyte in the epidermis appears to be of T-cell origin, where demonstration of la-like antigens would not be expected. The percentage of cells that were difficult to classify and which were negative for la-like antigens might fall in a hinterland between lymphocytes and IDC's, but in our opinion they are more likely to be immature melanocytes. Quantitative studies by previous workers on dendritic cells in the epidermis have apparently demonstrated some relationship of indeterminate cells to the other known cell types (Mishima, Kawasaki & Pinkus, 1972) which requires consideration. The three dendritic cell types were counted in normal skin and in various leukodermatous conditions,
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G.Rowden, T.M.Phtllips and M.G.Lewis
i.e. vitiligo, halo naevus, and spontaneous regression associated with lentigo maligna (Mishima et al., 1972). These studies appear to demonstrate that as melanocytes disappear, the number of IDC's increases. In later lesions, where melanocytes are absent, the IDC's decrease and Langerhans cells move down to the basal layers. No good explanation has ever been established to explain why Langerhans cells should occupy such sites in leukoderma, other than suggestions that the ratio of keratinocytes/dendritic cells needs to be kept constant in the basal layer (Breathnach, 1978). Thus, absence of melanocytes is compensated for by movement of Langerhans cells. These cytokinetic studies of IDC's do not, of course, provide information that will permit relationships of the IDC's to the other two cell types to be made. Mishima et al. (1972) felt that there is a transformation of melanocytes into inactive IDC's, but they were unable to exclude the possibility that they were derived from or related to cells of a different ontogeny, i.e. the Langerhans cells. The studies presented here would appear to show that the bulk of the IDC's have surface characteristics that indicate an affinity with Langerhans cells. The increase in IDC's in depigmentation may just as likely result from alteration in the kinetics of the Langerhans cell population as solely from degeneration of melanocytes. It must be noted, however, that the observations reported here concern normal human epidermis so comparisons with Mishima et a/.'s (1972) study are not strictly valid. Investigation of leukodermatous epidermis with the immuno-electron microscopic techniques used here will be necessary before final comment may be made. That the bulk of the indeterminate cells described here express surface antigen associated with Langerhans cells, rather than any other epidermal cell type, provides an attractive explanation for the constancy of Langerhans cell populations. The immigration of immature histocytic cells from the dermis to replace Langerhans cells, involved in their function with respect to contact allergic phenomena, would seem to be represented by the indeterminate cell population. The presence of Langerhans cells in the dermis has been repeatedly described (Wolff, 1972) so it need be not only the indeterminate cell repopulating the epidermis, as
Replocement mitosis ('Ore) or immiqrotion
Affererent arc (Allergen comer
Lymphoid pool?
Lvmph node
FIGURE 8. Summary of possible pathways of movement of Langerhans cells and their precursor cells.
Ia antigens on indeterminate cells
541
mature Langerhans cells have been shown to do (Wolff, Lessard & Winkelmann, 1971; Breathnach, 1978). The fact that histiocytes in the dermis are also Ia positive suggests the presence of such a pool of cells capable of immigration. Whether the pool of immature Langerhans cells is actually resident in the dermis or originates from other lymphoid sources such as the bone marrow, spleen, or nodes, requires testing. Kinetic studies using isolated macrophage populations as well as Langerhans cells are underway and will provide important insights into this question. Figure 8 summarizes the pathways involving Langerhans cells and their precursor cells. It will be noted that the suggestions made imply that the granule which is so characteristic of Langerhans cells develops only subsequent to the arrival of immature indeterminate cells in the epidermis. Studies on the repopulation of the epidermis by Langerhans cells after tape stripping have shown that in the guinea-pig this seems to be the case. Granules appear only after cells arrive in the upper layers of the epidermis (Wolff, et al, 1971). There is no evidence to rule out the possibility that Langerhans cells with their granules are not capable of returning to the epidermis after migrating to the local nodes during their response to contact allergens. This possibility also requires testing with labelled Langerhans cells. Breathnach (1978) has very recently published a description of mesenchymal cells of human embryonic and fetal dermis that might be ancestral for the Langerhans cell. These cells might be carried over into the postnatal dermis. Quiescent histiocytes under certain circumstances might also, as he suggests, be transformed into Langerhans cells. ACKNOWLEDGMENTS
Dr A.K.Sullivan is to be thanked for providing the antiserum against human la-like antigen(s). The skilled technical assistance of Mr N.More and Miss E.Rusnock is acknowledged. Mrs M.Davis is to be thanked for typing the manuscript. REFERENCES AiNSWOBTH, S.K. & KARNOVSKY, M.J. (1972) An ultrasrruciural staining method for enhancing the size and electron opacity of ferritin in thin sections. Journal of Histochemistry and Cytochemistry, 20, 225. BOCK, P. (1972) The mesenchymal origin of Langerhans ccW?.. Journal of Siihnicroscopic Cytology, 4, 271. BREATHNACH, A.S. (1971) An Atlas of rhe Ultrastructure of Human Skin. Churchill, London. BBEATHNACH, A.S. (1975) Aspects of epidermal ultrastructure. ^^''Hnia/ of Investigative Dermatology, 65, 2. BREATHNACH, A.S. (1978) Development and differentiation of dermal cells in man. Journal of Investigative Dermatology, 71, 2. FiCHELTELius, K.E., GROTH, O. & LiDEN, S. C1970) The skin: a first level lymphoid organ. International Archives of Allergy, 37, 607. GlACOMETTi, L. & MONTAGNA, W. (1967) Langcrhans cells: uptake of tritiated thymidine. Science, 157, 438. JUHLIN, L. & SHELLEY, W.B. (1977) New staining techniques for the Langerhans cells. Acta dermato-venereologica, 57, 289. KiSHiDA, Y., OLSEN, B.R., BERG, R.A. & PROCKOP, D.J. (1975) Two improved methods for preparing ferritinprotein conjugates for electron microscopy. Journal of Cell Biology, 64, 331. KLARESKOG, L., TJERNLUND, U.M., FORSUM, U . & PETERSON, P.A. (1977) Epidermal Langerhans cells express Ia
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