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Epitope Mapping of CDla, CDlb, and CDlc Antigens in Human Skin: Differential Localization on Langerhans Cells, Keratinocytes, and Basement Membrane Zone

Masutaka Furue, Margit Nindl, Katsura Kawabe, Koichiro Nakamura, Yasumasa Ishibashi, Kimitaka Sagawa Department of Dermatology, University of Tokyo, Tokyo; and Department of Immunology (KS), Kurume University School of

Medicine, Kurume, Japan

CD 1 antigens are classified into at least three groups, CDla, CD1h, and CDlc. In order to delineate the localization of epitopes of CDl antigens in human skin, we examined the immunoreactivity of fourteen different CDl antibodies (seven CDla, five CDlb, and two CDlc antibodies). The

Human CDl antigens are a family of structurally re­lated glycoproteins that are non-covalently asso­ciated with P2-microglobulin on the cell surface like MH C class I molecules [1]. At least five differ­ent CD 1 genes have been identified and sequenced

[2] of which three express homologous proteins, CDla (49 kd), CD1b (45 kd), and CD1c (43 kd) molecules, respectively [3,4]. These antigens can be detected serologically, and many different monoclonal antibodies are available for each subgroup [5 - 7]. The three CDl molecules are differentially expressed on cortical thy­mocytes, T-cell leukemias, epidermal Langerhans cells (ELC), der­mal dendritic cells (DDC), and a subset ofB cells [7 -12]. In order to further clarify the localization of epitopes of CDl antigens, we examined the immunoreactivity of 14 different CD1 antibodies (seven CD1a, five CD1b, and two CD1c antibodies) in human skin (Table I).

STAINING PATTERNS

ELC and DDC ELC were clearly stained by all CD1a antibodies (Fig lA). However, the number of ELC reacting to M-T102 (CDla) was only 4.0-7.0% of that of EL~ that wer~ I?ositive for other six CD1a antibodies (Table II). No Immunosta111l11g of ELC was observed with CD1b and CD1c antibodies or with control antibodies (Fig 1, Tables II and III).

On the other hand, DDC were strongly positive for CDla or CDlc antibodies, and the density of CD1a+ DDC was similar to that of CDlc+ DDC. Again, an epitope for M-TI02 (CDla) was expressed on a smaller population of DDC. CDlb antibodies only stained a very minor population ofDDC (0 - 20% ofDDC) (Fig 1, Table II).

Keratinocytes Besides ELC, CDla antibodies stained keratino­cytes interceUularly in some areas of normal human skin (Fig lA).

Reprint reque~ts to: Dr. Masutaka Fume, Department of Dermatology, Yamanashi Medical College, Tamaho, Nakakoma, Yamanaslu, 409-38,

Japan. Abbreviations

BMZ: basement membrane zone DDC: dermal dendritic cells ELC: epidermal Langerhans cells

epitopes for CDla, CDlb, and CDlc are differentially local­ized on epidermal Langerhans cells, dermal dendritic cells, keratinocytes, the luminal portion of eccrine gland ducts, and the basement membrane zone in normal human skin.] Invest DermatoI99:23S-26S, 1992

Among the CD1a antibodies, BL-6 had the strongest reactivity to keratinocytes. Intercellular keratinocyte staining by BL-6 was more prominent in the follicular infundibulum, which was in sharp con­trast to the negative staining of the acrosyringium of eccrine glands. In contrast to CDla antibodies, all CDlb, CD1c, and control anti­bodies exhibited no immunoreactivity in the epidermis.

M-TI02 and Leu6 reacted to the luminal edge of ecrrine gland ducts (Fig 1D). Other CD1a antibodies also weakly stained the luminal portion of eccrine gland ducts in some sections, although the secretory portion of eccrine glands was always negative for CDla antibodies. No immunoreactivity was recognized in eccrine glands with CD1b and CDlc antibodies or with control antibodies.

Basement Membrane Zone (BMZ) Interestingly, we observed linear BMZ staining with one of the CD1b antibodies (NU-T2) (Fig 2A). AU other CDl antibodies and control antibodies did not show this BMZ staining pattern (Table III). BMZ staining by NU­T2 antibody was clearly demonstrated in all normal human skin samples and in the BMZ of normal esophagus and stomach (Fig 2B). This reactivity of NU-T2 antibody was not seen in murine or guinea pig skin. Formalin-embedded sections were also not stained with NU-T2 antibody, as is the case with other CD1 antibodies. As shown in Fig 2C, NU-T2 antibody reacted to the dermal edge of the NaCI-separated normal human skin.

We next examined the expression of the NU-T2-BMZ antigen in various skin disorders. Although the NU-T2-BMZ antigen was intact in psoriasis or in sebaceous nevus, it was absent in malignant skin diseases such as Bowen's disease and genital Paget's disease (Fig 2D), which was in sharp contrast to the presence of type VII colla­gen in the BMZ underlying these malignant skin tumors (Fig 2E).

In order to further determine the localization of the NU -T2-BMZ antigen, we performed immunogold electron microscopy on NaCI-separated unfixed normal human skin. The NU-T2-BMZ antigen localized in the lowermost lamina lucida just above the basal lamina (Fig 3B). No reaction product was found in the epidermis (Fig 3A).

DISCUSSION

Although ELC in situ are known to be CDla+ and CD1c- [11], Teunissen et al have reported that ELC in epidermal cell suspension after trypsinization are positive for both CDla(OKT6) and

0022-202X/92/S05.00 Copyright © 1992 by The Society for Investigative Dermatology, Inc.

23S

24S FURUE ET AL

Table I. Monoclonal Antibodies Used in this Study

Antibody to Clone

COl a OKT6 Leu 6 BL-6 (IOT6a) 66-IL-C7 M-T102 0-605 VIT6

CD lb NU-T2 4A76(IOT6b) WM-25 Fl11-132 M-TI0l

CD l c L161 M241

Type VII collagen CD2 NU-T1 CD3 NU-T3 CD4 NU-TH / I CD8 NU-Ts/c CD28 KOLT-2

• Ortho, Raritan, NJ , USA. • Becton Dickinson, Mountain View, CA, USA. , Immunotech, Marseille, France. , Sanhio, Uden, Netherlands. , I : 100 diluted ascites form. I Nichirci Co., Tokyo, Japan. I Chemicon, Temecula, CA, USA.

Source

Ortho' B.D.' Immunotech' Sanbiod

(Dr. Rieber)' [7] (Dr. Boumsell)' [7] (Dr. Kn:rp)' [7] Nichire' Immunotech (Dr. Favaloro)' [7] (Dr. Carriere)' [7] (Dr. Rieber)' [7] (Dr. Boumsell)' [7] (Dr. Knowles) [11] ChemiconK Nichirei Nichirei Nichirei Nichirei Nichirei

CD l c(M241) [12]. Their data suggests that an epitope for CD lc on ELC is unmasked by trypsinization. The epitope for M-Tl02 (COla) was expressed on a subpopulation of ELC in the present study (approximately 40-70% of all ELe), suggesting that this antibody m ay be useful for studying the phenotypic characteristics of histiocytosis X or other diseases in which ELC increase in num­ber, such as atopic dermatitis [13]' Similar numbers of DOC were stained w ith CDlaor CDlc antibodies, which is in sharp contrast to the extremely limited staining of DOC by CDlb (approximately 0 -20% of CDla+ or CDlc+ ODe). Double-staining studies should determine whether CD 1 a+, CD 1 b+, and CD 1 c+ D DC repre­sent mutually distinct subpopulations. It would also be interesting to investigate the relationship between CD1+ DOC and other re­cently identified skin-residing DOC such as factor Xllla+ [14], CD36+ [15], or CD34+ [16] DOC.

Figure 1. Differential staining of normal human skin by CO l antibodies. A: BL-6 (COl a) antibody stained both ELC and DOC (arrow/leads) . BesidesELC and DOC, BL-6 reacted to keratinocytes in some areas of normal human skin (arrows). B: WM-25 (CDlb) antibody stained a very minor population of DOC. No immunoreaction of ELC was observed by CDl b antibody. C: M24 l (CDl c) anti­body stained DOC; however, no immunoreaction ofELC was observed by CDlc antibody. D: The luminal portion of eccrine gland ducts was stained by M-T102 (COla). Arrows, secretory portions of eccrine glands.

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Table U. Numbers of Immunoreactive Cells for CD1 Antibodies'

ELO DDC'

NS-ld NS-2 NS-3 NS-l NS-2 NS-3

COla BL-6 13.5 8.2 10.8 18.0 5.1 12.0 OKT6 14.9 7.0 11.2 17.5 6.2 13.1 66-IL-C7 12.3 7.5 12.9 18.5 6.8 12.5 0-605 13.9 9.0 10.1 19.1 5.0 10.8 VIT6 12.9 8.5 11.8 17.0 6.0 11.9 Leu6 13.5 8.8 12.0 16.9 6.5 13.6 M-TI02 8.5 4.6 7.0 10.2 2.0 9.1

CDlb 4A76 0 0 0 2.0 0 1.1 NU-T2 0 0 0 1.2 0 2.0 F111-132 0 0 0 4.0 0 1.2 M-TI0l 0 0 0 1.5 0 1.8 WM-25 0 0 0 2.5 0 2.2

CDlc L161 0 0 0 19.5 7.8 9.5 M241 0 0 0 17.9 8.8 11.6

• Representative data obtained from normal human skin (n = 10). b Mean numbers of CD1+ ELC overlying 200 basal cells. At least five different areas

were examined in each section. , Mean numbers of CD1+ DOC in the upper dermis underlying 200 basal cells. At

least five different areas were examined in each section. , NS, normal human skin.

Table III. Immunoreactivity of CDl Antibodies·

Lumen of Keratin- Eccrine

ELC DOC ocytes Gland Ducts BMZ

CDla BL-6 ++ ++ ++ ± OKT6 ++ ++ + ±/+ 66-IL-C7 ++ ++ + ±/+ 0-605 ++ ++ + ±j+ VIT6 ++ ++ + ±/+ Leu6 ++ ++ + +/++ M-TI02 + + ± ++

CD1b 4A76 ± NU-T2 ± ++ Fll1-l32 ± M-T101 ± WM-25 ±

CD1c L161 ++ M241 ++

• -, negative; ~, negative ~r weak; +, weak; ++, strong. The epitopes of COla, CDlb, CO le antIgens were dIfferentially localized III normal skin.

VOL. 99, NO.5, SUPPLEMENT, NOVEMBER t992

.J

A B

c E' '

With respect to keratinocytes, intercellular staining has already been demonstrated in several skin diseases, such as lichen sclerosus

t atrophicus, malignant melanoma, and lymphoma [17,18J. Focal ~ ntercellular keratinocyte staining and follicul ar epithelial staining ~y CD1a has be~n also .reported in normal skin .by Willemze (r~ply in [18]), which IS consistent with o~r observations. Tlll.s keratll1o­eyte staining may a~count .for ~he high backgro.und fO.r unmunola­beling with CDla III routl11e Immunohistologic studies. The blO-

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, .'Y

" ~ .. " ,

~B

. ' .

EPITOPE MAPPING OF COt 25S

Figure 2, Thc BMZ staining by NU-T2 (CDlb) anti­body. NU-T2 antibody reacted to the BMZ of normal human skin (A) and esophagus (B) . NU-T2 antibody reacted to the dermal edge (arrow) of the NaCI-separated normal human skin (C) (arrowheads, melanin pigments). The NU-T2-BMZ antigen was absent in the BMZ under­lying the clusters of Paget's cells in genital Paget's disease (D, arrows). In contrast to NU-T2-BMZ antigen, type VII collagen was consistently present (but weakly in some areas) in the BMZ of Paget's disease (E).

logic significance of the CD la positivity on keratinocytes is unclear. However, very recently, Bleicher et al have demonstrated that one of the murine CD1 antigens is expressed on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes [19J . Concerning the luminal staining of eccrine gland ducts with CDla (M-TI02, Leu-6) observed in our study, Gomes et al have already shown immunoreactivity with a different CD 1a antibody (D-47) to epithe­lial cells of the eccrine secretory region [20J. Although the immuno-

, .

, , *

BL *

Figure 3,. Immunogold electron microscopy (particle size.1 nm with subse9uent si lver enhancement) was performed on NaCI-separated, unfixed norma] ~uman skm, uSlI1g a pre-cmbeddlllg IlTImunostall1111g techlllque. A: no reactIOn product was found 111 the epidermis. Arrows, hemidesmosomes. (Magnifica­tIOn X 20,000.) B: 111 specimens Il1cubated with the NU-T2 antibody the reacted gold particles (arrowheads) were found in thc lowermost lamina lucida just above the basal lamina (BL) . Asterisks, anchoring fibrils . (Magnification X 25,000.)

26S FURUE ET AL

reactive sites by M-Tl02 and Leu6 to eccrine glands are quite dif­ferent from those of 0-47 ,these data indicate that epitopes of COla molecules do exist in eccrine glands.

It was of interest that one CD 1 b antibody (NU -T2) stained the BMZ of epidermis. This NU-T2-BMZ antigen was present not only in the BMZ of the skin but also in the BMZ of the esophagus and stomach. The immunogold electron microscopy showed that the NU-T2-BMZ antigen localized in the lowermost lamina lucida just above the basal lamina. However, our preliminary immunopre­cipitation studies have failed to identify its molecular weight. This NU-T2-BMZ antigen was absent from the BMZ of the malignant epidermal tumors, and based on the report of de Moragas et ai, that the bullous pemphigoid antigen is absent in anaplastic squamous cell carcinomas [21] , the NU-T2-BMZ antigen may be an addi­tional differentiation marker for skin tumors.

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