0022-202X/83/8103-0249$02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 81:249-253, 1983 Vol. 81, No.3 Copyright © 1983 by The Williams & Wilkins Co. Printed in U.S.A. A Human Monoclonal Reacting with Merkel Cells: , Immunoperoxidase, and Immunoelectron *

J.-H. 8AURAT, M.D., P. CHAVAZ, M.D., P. CARRAUX, AND L. DIDIERJEAN, PH.D. Department of Dermatology, Hopital Cantonal Uniuersitaire, Geneva, Switzerland

A human monoclonal, mu, kappa, cold agglutinin an­ tiona! heterogeneity of the basal keratinocytes (3) . This inter­ tibody of the rare specificity Pr h (serum and proper pretation has been shown valid for the mucous side of rabbit eluates) was used in immunofluorescence and immuno­ lip where antigenically distinct basal keratinocytes have now peroxidase techniques and in immunoelectron micros­ been demonstrated (4] (also unpublished results). We report copy on rabbit lip specimens. Pr h antibody strongly here that the isolated cells on the OK and PK sides of rabbit reacted with scattered cells in epidermis, which were lip epithelium are actually Merkel cells (MC). Monoclonal Pr demonstrated to be Merkel cells by electron microscopy; h antibody is t herefore, to our knowledge, the first naturally no nerve fibers were stained. In immunoelectron mi­ occurring human antibody shown to react with MC. ' croscopy (IEM), a strong reaction was seen within the cytoplasm and around the granules. This is the first IEM MATERIALS AND METHODS staining of Merkel cells (MC) so far reported; it demon­ Monoclonal A11:tibody to Pr hag strates the expression of a carbohydrate differentiation in MC. The availability of a potent monoclonal This was previously described in detail [2] . Briefly, serum was antibody reacting with MC but not with neighboring obtained from a patient with chronic cold agglutinin disease. Agglutin­ ation titer at +4 ·c against a suspension of RBC was 16,000. Heat epidermal cells in rabbit lip offers a new tool for the eluates were prepared and incubated at +4 ·c with protease-treated and study of several aspects of MC biology, including anti­ untreated RBC and studied in parallel by RBC agglutination and genic properties and kinetics. indirect immunofluorescence (IIF) on RLE specimens as previously described [2,5]. The serum was also subjected to 6 consecutive adsorp­ tions on protease-treated and untreated RBC and the eluates and We have previously described the localization of Pr absorbates were also studied by RBC agglutination and IIF on RLE (Pr ag) within the epidermis; Pr ag are glycoconjugates of the specimens [2]. red blood cell (RBC) membranes-which are traced by human monoclonal cold agglutinin [1]. One of these, Pr h, Rabbit Lip Specimens was found to be expressed in the basal cells of the human Lip specimens were obtained from 38 New Zealand adult rabbits. epidermis [2]. Pr h ag was also detected in animal epidermis, Specimens were processed as previously described [2]. Such a procedure with distinct distributions according to both species and type allows the study of orthokeratinized, parakeratinized, and mucosal of differentiation of the epithelium (2] . The expression of the epithelia in a single lip section: junctional zones between the distinct Pr h ag within rabbit lip epidermis (RLE) specimens was types of differentiation are also well defined [6] . striking! On the mucous side, nearly all basal cells were found Indirect Immunofluorescence Technique to express the antigen; on the parakeratotic (PK) and on t he orthokeratotic (OK) sides, however, the Pr h ag was not uni­ IIF was performed according to standard techniques as previously formly distributed within the basal cells. Only a few scattered described in detail [6]. As soon as the cryostat sections were made, the IIF procedure was conducted at +4· C, +2o· c, and +37· C: incubation cells were strongly positive on the OK side and clusters of for 30 min, with the serum containing Pr h antibodies (Pr h ab), strongly positive cells were observed on the epidermal crests of washing for 30 min in phosphate-buffered saline (PBS) pH 7.2, incu­ PK sides [2]. As the nature of these isolated cells had not been bation for 30 min with the fluorescein isothiocyanate-labeled antisera established and since we were working with a monoclonal to light and heavy chains of human immunoglobulins [goat anti-y antibody which had clearly been demonstrated to react with chains of human immunoglobulin (Hyland Laboratories), fluorescein/ keratinocytes, one possibility was that these isolated cells could molar ratio (F/ P) 2.7, antibody concentration 2 mg/ml, workina be keratinocytes antigenically distinct from t he surrounding dilution 1:40; goat anti-.u chains (Hyland), F/P 2.7, antibody concen~ ones; this would be in agreement with the concept of a func- tration 0.8 mg/ ml, working dilution 1:16; goat anti-a chains (Hyland), F / P 3.1, antibody concentration 2.1 mg/ ml, working dilution 1:46; Manuscript received February 15, 1983; accepted for publication rabbit anti-K chains (Behring), F/P 2.3, antibody concentration 1 mg/ Aprilll, 1983. ml, working dilution 1:20; rabbit anti-!\ chains (Behring), F /P 1.8, This work was supported by a grant from the University of Geneva, antibody concentration 1 mg/ml, working dilution 1:20]. After washing Faculty of Medicine. for 30 min in PBS, t hey were mounted in buffered glycerol and *This work was presented at the SID-ESDR Joint Meeting, April examined on a Zeiss microscope with an optical system for blue narrow 1983, Washington, D.C. band excitation and epi-illumination. Micrographs were taken with Reprint requests to: J .- H. Saurat, M.D., Department of Dermatology, high-speed Ektachrome 400 ASA. Hopital Cantonal Universitaire, 1211 Geneva 4, Switzerland. Abbreviations: I mmunoperoxidase Technique EM: electron microscopy Immunoperoxidase technique was performed on 4 .urn-thick air­ IEM: immunoelectron microscopy dried sections and incubated with Pr h ab for 30 min at optimal IIF: indirect immunofluorescence temperature, washed twice for 15 min in PBS, and treated by peroxi­ liP: indirect immunoperoxidase dase-conjugate fo r 45 min at room temperature (goat anti-.u chain of MC: Merkel cells human I~ On~tit~t Pasteur, Paris) conta inin ~ 1.25 mg of antibody per OK: orthokeratotic ml; workmg d1lutw.n 1:10). Spec•mens were nnsed twice for 10 min in PK: parakeratotic PBS, fo llowed for 10 min in 0.2 M Tris-HCI buffer pH 7.6 and Pr h ab: Pr h antibody incubated in Graham and Karnovsky's cytochemical medium for 30 Pr h ag: Pr h antigen min, again washed in Tris-HCI buffer for 10 min and in PBS for 10 RBC: red blood cell(s) min; finally mounted in buffered glycerol.

249 250 SAURAT .ET AL Vol. 81, No. 3

Semithin Sections and Electron Microscopy described by Farr and Nakane [8], and then quick-frozen in liquid RLE specimens were fixed in 3% phosphate-buffered glutaraldehyde, nitrogen and sectioned at 12 J.Lm in a cryostat. The sections were postfixed in 1.33% collidine-buffered OsO. aqueous solution, dehy­ mounted on plastic coverslips and incubated with the P r h antiserum drated in acetone and embedded in Araldite. Semithin sections 0.5-1 for 19 h at room temperature. After washing in phosphate-buffered sucrose they were incubated for 5 h with the goat antihuman IgM J.L m were stained by methylene blue or Giemsa stain. Ultrathin sections were cut with a Reichert OMU3 ultramicrotome, contrasted with uranyl serum peroxidase labeled as described above. After washing in phos­ acetate and lead citrate, and observed with a Philips EM 300 at 80 kV. phate-buffered sucrose and 0.25 M Tris-HCL pH 7.6, the reaction was revealed with DAB Graham and Karnowsky's medium for 30 min at room temperature. A postfixation in 1% Os0 in phosphate buffer was Immunoelectron Microscopy (!EM) 4 performed at room temperature for 20 min. The specimens were de­ RLE specimens were sliced in 2 mm-thick pieces with a razor blade, hydrated in alco hol and embedded in Araldite. Controls included spec­ and immediately fixed for 10 min in periodate-lysine-paraformaldehyde imens incubated for 19 h with normal human serum instead of Pr h ab fixative at room temperature according to the method of McLean and as well as specimens incubated only in phosphate-buffered sucrose Nakane [7] . The specimens were washed in 0.1 M phosphate buffer pH prior to incubation with anti-lgM antiserum. Osmification was also 7.4 with increasing concentrations of sucrose and finally of glycerol as omitted in one experiment in both experimental and control specimens.

FIG 1. Orthokeratotic side of a RLE specimen. A, Semithin section: clear ce lls clustering in the epidermal crest are MC as demonstrated clear cells in basal position are MC as demonstrated by EM (see Fig by EM (see Fig 3) . Methylene blue stain. x 500. B, IIF with Pr h 3) . Methylene blue stain, X 500. B, IIF with Pr h monocl onal antibody diluted 1:20. Note the bright staining of cells diluted 1:20. Note the bright staining of scattered cells in basal position. clustering in the epidermal crest. x 500. C, liP with Pr h monoclonal x 500. C, liP with Pr h monoclonal antibody diluted 1:10. Note the antibody diluted 1:10. Note the peroxidase reaction within cells clus­ peroxidase reaction within scattered basal cells. x 500. tering in the epidermal crest. FIG 2. Parakeratotic side of a RLE specimen. A, Semithin section: Sept. 1983 MONOCLONAL ANTIBODY TO MERKEL CELLS 251

RESULTS of RLE (Figs lA, 2A). EM demonstrated that these clear cells Distribution of Pr h Positive Cells Within Basal Cell Layers of were MC (Fig 3). OK and PK Sides of RLE Immunoelectron Microscopy (Fig 4) Fig 1 shows the IIF and liP staining of the OK side of RLE: Strong electron-dense deposits were observed in the cyto­ scattered cells in basal position, the longitudinal axis of which plasm of t hese cells. The overall aspect was that of regular is often parallel to the basal membrane zone, were seen in the staining of the cytoplasm with disposition around granular interfollicular areas. Neighboring basal cells (keratinocytes) structures. Although it seemed that there was a regular concen­ were not stained. No staining was observed in the superficial tration of the electron-dense material around the specific MC dermis. granules, we could not establish whether this appearance indi­ Fig 2 shows the IIF and liP staining of the PK side of RLE: cated that the antigen was a localized component of the granule clusters of cells in basal and suprabasal position in epidermal surface or was diffusely distributed in the cytoplasm. Neigh­ crests were strongly positive for Pr h ag. No staining was boring keratinocytes did not show deposits. No staining of the detectable in the adjacent superficial dermis. superficial nerves in the underlying dermis was observed. Con­ Similar aspects were observed with both the serum and trol specimens incubated with or without normal human serum proper eluates and absorbates; the activity responsible for the prior to incubation with anti-lgM antiserum did not show staining in IIF was found in the eluates from untreated RBC electron-dense deposits within MC. but not in the eluates from RBC treated with papain. Similarly the activity was present in the absorbate from RBC treated DISCUSSION with papain but not in the absorbate from untreated RBC. These observations were consistently reproduced in the 38 We have demonstrated that a monoclonal antibody of the rabbits we studied. No staining was observed when unrelated rare specificity Pr h obtained from a patient with a chronic antisera were used, i.e., anti-!', a nti-e~, and anti-A chains. cold agglutinin disease [2,9] reacted with MC. That the immunologic reaction occurred with MC is based Histologic and Electron Microscopy (EM) Analysis of OK and on the following observations: (1) The distribution of Pr h PK Sides of RLE specimens positive cells was exactly that of MC within the RLE as Microscopic analysis of semi thin sections revealed that areas described by several authors who routinely use this specimen, containing Pr h reactive basal cells were rich in clear cells which is particularly rich in MC, for the study of MC biology distinguishable from other cells. Characteristically, this type of [10] . (2) The areas in which Pr h positive cells were observed cell was found to cluster in the epidermal crests in the PK side were also found to contain MC in a similar position, as dem-

FIG 3. Electron microscopy of the PK side of a RLE specimen, sections. Note the specific granules of MC shown in the inset. s howing the characteristic features of the clear cells seen on semi thin 252 SAURAT ET AL Vol. 81, No. 3 onstrated by semithin sections and EM. (3) Finally, a strong in the rabbit. They did not study the distribution of Pr h ag. reaction was demonstrated by IEM within these cells. We have previously found that Pr h ag were expressed in It seems that this is the first example of a monoclonal stratified squamous epithelia of mice, rats, guinea pigs, rabbits, antibody of human origin reacting with MC. Immunotracing of and humans with distinct distribution according to both species MC has been described to date only in cats and rats with two and differentiation of the epithelium [2]. Pr h ag were also types of polyclonal antisera raised in animals. Hartschuh et al found in several other tissues in rabbit, including adrenal [11] have reported that metenkephalin-like immunoreactivity medulla (unpublished observations). From this study it appears was detectable in MC of rat sinus hair follicles. Recently, that Pr h ag are not expressed in the endings of cutaneous neuron-specific enolase, an found in neurons and in a nerves. number of APUD cells, was traced in MC and satellite nerves This ubiquitous but variable distribution of defined carbo­ by immunoperoxidase [12] . In these two studies IEM was not hydrate sequences, as traced by monoclonal antibodies, is now used and MC were recognized by their distribution and shape. a well-established concept [14] . The biochemical definition of Pr ag is relatively well estab­ Within MC, Pr h glycoconjugate was expressed within the lished. Hemagglutination inhibition studies of crude or chemi­ cytoplasm; such a cytoplasmic expression of glycoconjugates of cally modified RBC glycoproteins have shown that Pr deter­ the RBC surface has also been observed in keratinocytes [2] . minants are represented by alkali labile oligosaccharides with, The aspect of the electron-dense deposits in IEM was compat­ except for Pr h, N-acetyl neuraminic acid as terminal amino ible with a diffuse cytoplasmic distribution but an expression sugar [1]. The distribution of Pr ag within tissues has been of Pr h ag at the surface of the MC granule was also possible. studied by Romer et al [13]; these authors found that Pr ag We cannot at present speculate on the significance of the was present in several tissues including nearly all cells of brain expression of Pr h glycoconjugate on MC. The changing anti-

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FIG 4. Immunoelectron microscopy of the PK side of a RLE speci­ A, Detail of the cell showing the aspect and distribution of the peroxi­ men reacted with Pr h ab. Note the peroxidase deposits within the dase deposits. Inset B, Control specimen incubated with normal human cytoplasm and around the MC granules. Postfixation with Os04• Inset serum instead of Pr h ab. Postfixation with Os04• Sept. 1983 MONOCLONAL ANTIBODY TO MERKEL CELLS 253 genicity in the glycoproteins of cells is linked to successive REFERENCES developmental stages and may be produced by sequential ad­ 1. Roelcke D: Cold agglutination: antibodies and ant igens. A review. dition or deletion of monosaccharide residues [1 4]. To deter­ Clin Immunopathol 2:266- 280, 1974 mine whether this concept is applicable to the developing MC, 2. Saurat JH, Didierjean L, Habibi B: Pr antigens in the skin: distinct locali zation linked to t he stage and t he type of keratinocyte the so-called transitional MC [10] is currently under study differentiation. Br J Dermatol 105:25-38, 1981 [15]. 3. Bickenbach JR, Mackenzie IC: Slowly cycling cell s in the skin and It is also of interest to note t hat t he surrounding keratino­ oral mucosa (Abstr) . J Invest Dermatol 72:284, 1979 cytes were not found to express the Pr h glycoconjugate al­ 4. Saurat JH, Didierjean L: The immunotracing of keratinocyte sub­ sets, Stratum Corneum. Edited by R Marks. Heidelberg, Spring­ though this is expressed in basal keratinocytes in areas where Verlag, 1983, pp 135-143 MC are not present, such as the mucosal side of RLE [2] (the 5. Habibi B, Cregut R, Brossard Y, Veron P, Salmon C: Auto-anti­ finding of such areas in human skin would make it possible to Pr•: a "second" example in a newborn. Br J Haematol 30:499- see whether human MC also react wit h Pr h ab). Whether t his 505, 1975 6. Saurat JH, Didierjean L, Beucher F, Gluckman E: Immunofluores­ observation might have some relevance to t he fact that carbo· cent tracing of cytoplasmic components involved in keratinocyte hydrates are thought to be involved in neuronal recognition, differentiation. Br J Dermatol 98:155- 163, 1978 adhesion, and synaptogenesis [16,17] has to be explored. How­ 7. McLean IW, Nakane PK: Periodate-lysine-paraformaldehyde fix­ ever, it should be noted that the Pr h glycoconjugate was neither ative. A new fixative for immunoelectron microscopy. J Risto­ chern Cytochem 22:1077- 1083, 1974 concentrated at the basal pole of MC nor expressed in the 8. Farr AG, Nakane PK: Immuno-histochemistry with enzyme labeled endings of cutaneous nerves. ant ibodies: a brief review. J Immunol Methods 47:129- 144, 1981 The availability of a potent monoclonal antibody reacting 9. Doutre MS, Beylot C, Conte Ph, Bioulac P , Vezon G, Fizet D, with MC but not with neighboring epidermal cells in the rabbit Lassalle H: Les signes cutanes de Ia maladie des agglutinines froides. A propos d'un cas. Ann Derm Venereol 105:541- 545, lip, offers a new tool for the study of several aspects of MC 1978 biology; it makes it possible to study a large number of MC, 10. Tachibana T , Nawa T: Merkel cell differentiation in t he labial and to consider several important issues. (1) The antigenic mucous epithelium of t he rabbit. J Anat 131:145-155, 1980 properties of MC: this cell is considered to be of neuronal cell 11. Hartschuh W, Weihe E, Buchler M, Helmstaedter V, Feurle GE, line; however the epidermal origin is still discussed [10]. There­ Forssmann WG: Met-enkephalin-like immunoreactivity in Mer­ kel cells. Cell Tissue Res 201:343-348, 1979 fore, by using double staining with both Pr h ab and specific 12. Gu J , Polak JM, Tapia FJ, Marangos PJ, Pearse AGE: Neuron­ antibodies to common keratinocytic antigens it would be pos­ specific enolase in the Merkel cells of mammalian skin. The use sible to progress in this field [15]. (2) The kinetics of MC: no of specific antibody as a simple and reliable histologic marker. data are available regarding the possibility of division Am J Pathol 104:63-68, 1981 MC 13. Romer W, Seelig HP, Lenhard V, Roelcke D: The distribution of within the epidermis. In order to study this point it is necessary I/i, Pr and Gd antigens in mammalian tissues. Invest Cell Pathol to screen a large number of MC; the coupling of Pr h immu­ 2: 157-162, 1979 nostaining with autoradiography makes this possible [15] . 14. Feizi T : Carbohydrate differentiation antigens. Trends Biochem Antibody probes have been valuable in several fields of Sci (Amsterdam) 6:333-335, 1981 15 Saurat JH, Carraux P , Polla L, Didierjean L, Chavaz P: A mono­ dermatologic research. It might be that they bring the myste- · clonal ab to Merkel cell s (MC). Its use fo r t he study of MC rious MC on stage. kinetics and antigenic properties (abstr). J Invest Dermatol 80:317, 1983 16. Kelly P, Cotman CW, Gentry Ch, Nicolson GL: Distribution and mobility of lectin receptors on synapt ic membranes of identified The authors thank B. Habibi, M.D., who provided us with t he eluates neurons in t he central nervous system. J Cell Bioi 71:487- 496, a nd absorbates and established the Pr h specificity of the monoclonal 1976 antibody, and M. Spinelli and M. Pisteur who helped in the preparation 17. Sanes JR, Cheney JM: Lectin binding reveals a synapse-specific of the manuscript. carbohydrate in skeletal muscle. Nature 300:646-647, 1982