Epitopes for CD1a, CD1b, and CD1c antigens are differentially mapped on Langerhans cells, dermal dendritic cells, keratinocytes, and basement membrane zone in human skin

Epitopes for CDla, CDlb, and CDlc antigens are differentially mapped on Langerhans cells, dermal dendritic cells, keratinocytes, and basement membrane zone in human skin Masutaka Furue, MD, PhD,a Margit Nindl, MD, PhD,a Katsura Kawabe, AR,a Koichiro Nakamura, MD,a Yasumasa Ishibashi, MD, PhD,a and Kimitaka Sagawa, MD, PhDb Tokyo and Fukuoka, Japan Background: CDI antigens are classified serologically into at least three groups, CDla, CD Ib, and CD I c, and many kinds of monoclonal antibodies are available for each subgroup of CDI antigens. CDla, CDlb, and CDlc antigens have been shown to be selectively and differentially expressed on epidermal Langerhans cells and dermal dendritic cells in normal human skin. Objective: The objective was to further delineate the localization of epitopes of CDI antigens in human skin. Methods: We examined the immunoreactivity of 14 different CDI antibodies (seven CDla, five CD1b, and two CD 1c antibodies) with the immunoperoxidase technique. We also studied the reactivity of NU-T2 (CDlb) antibody by immunogold electron microscopy. Results: The epitopes for CDla, CDlb, and CDlc antigens were differentially mapped on epidermal Langerhans cells, dermal dendritic cells, keratinocytes, the luminal portion of eccrine gland ducts, and the basement membrane zone in human skin. Conclusion: These CD1 antibodies may be useful to analyze the phenotypic alteration of immune and nonimmune cells in various skin diseases. (J AM ACAD DERMATOL 1992;27:419-26.)

Human cluster of differentiation I (CDI) antigens are a family of structurally related glycoproteins that are noncovalently associated with /32-microglobulin, like MHC class I molecules, on the cell surface. I, 2 At least five different CDI genes have been identified and sequenced,3, 4 of which three express homologous proteins, CDla (49 kd), CDlb (45 kd), and CDlc (43 kd) molecules. 5,6 CDla, CD 1b, and CD 1c antigens can be detected serologically and many kinds of monoclonal antibodies are available for each subgroup of CDl antigens.?-9 These three CDl molecules are differentially expressed on cortical thymocytes, T-cell leukemias, epidermal Langerhans cells (ELC), dermal denFrom the Department of Dermatology, University of Tokyo"; and the Department of Immunology, Kumme University School of Medicine, Fukuoka. b Accepted for publication March 18, 1992. Reprint requests: Masutaka Fume, MD, Department of Dermatology, Yamanashi Medical College, 1110, Shimogatou, Tamaho-cho, Nakakoma-gun, Yamanashi-ken, 409-38, Japan.

16/1/37937

driticcells (DDC), and a subset ofB cells. I , 9-12 CDl molecules were originally described in humans and believed to be the homologues of the mouse thymus leukemia antigens (Tla).3, 4, 9However, this idea has been disputed by the recent finding that the CD 1 genes do not share any specific sequence homology with T Ia. 3, 4 Although the function of CD 1 molecules is not well understood, analysis of the predicted amino-acid sequences of CDl molecules reveals a low but significant level of homology to MHC class I and class II molecules, 3 suggesting a role of CD 1 molecules in antigen recognition by T cells. 13 Furthermore, Porcelli et al. 14 directly demonstrated the existence of a subset of CD4-CDS- T~cell lines (bearing either al/3 or 'Y15 T-cell receptors), which are not restricted to MHC molecules but specifically recognize CDla or CDlc molecules. In normal human skin CD I molecules have been shown to be selectively and differentially expressed on ELC and DDC.ll, 15, 16 However, from our routine immunohistologic studies, we had the impression that CD1a antibodies have a strong background

419

Journal of the American Academy of Dermatology

420 Furue et af.

Table I. Monoclonal antibodies used in this study Antibody to

CDla

COlb

CDlc

Fig. 1. Differential staining of normal human skin by COl antibodies. A, OKT6 (COla) antibody stained both ELCs and DOCs. B, WM-25 (COl b) antibody stained a minor population of ODCs. No immunoreaction of ELCs was detected byCOlb antibody. C, M24l (COlc) antibody stained ODCs; however, no immunoreaction of ELCs was detected by CDlc antibody. D, In addition to ELCs and DOCs, BL-6 (COla) antibody reacted with keratinocytes in some areas of normal human skin. Areas of intercellular keratinocyte staining are marked by arrowheads. E, Intercellular keratinocyte staining by BL-6 (CD 1a) antibody was more prominent in follicular infundibulum. F, Acrosyringial cells of eccrine glands (arrowhead) were negative for BL-6 (CDla) antibody.

staining in the epidermis compared with CDlb or CDle antibodies. To further delineate the localization of epitopes of CDI antigens, we examined the immunoreactivity of 14 different CDl antibodies (seven CDla, fiveCDlb, and two CDlc antibodies) in human skin. MATERIAL AND METHODS Monoclonal antibodies. A large panel of murine monoclonal antibodies, including 14 CO 1 antibodies, was used in this study. We also used six control antibodies (antitype VII collagen, C02, C03, CD4, C08, and CD28 antibodies). The specificity and the source of these antibodies are listed in Table 1. Tissue specimens and immunoperoxidase staining. Skin samplesofnormalhumanskin(n = 10),psoriasis(n = 2), Bowen's disease (n = 2), Paget's disease (n = 2), sebaceous nevus (n = 1), and recessive-dystrophic epidermolysis bullosa hereditaria (n = 1) were examined in frozen sections. Frozen sections (6 j.tm) were first incubated with various concentrations (l :50 to 1:800) of primary monoclonal antibodies for 30 minutes at room temperature.

Gone

Source

OKT6 Leu 6 BL-6 (IOT6a) 66-IL-C7 M-Tl02 0-605 VfT6 NU-T2 4A76 (IOT6b) WM-25 FI1l-132 M-TlOI L161 M241

Ortho BD Immunotech Sanbio (Dr. Rieber)*9 (Dr. Boumsell)*9 (Dr. Knapp)*9 Nichirei Immunotech (Dr. Favaloro)*9 (Dr. Carriere)*9 (Dr. Rieber)*9 (Dr. Boumsell) *9 (Dr. Knowles)16 Chemicon Nichirei Nichirei Nichirei Nichirei Nichirei

Type VII collagen NU-Tl C02 C03 NU-T3 C04 NU-TH/I C08 NU-Ts/c C028 KOLT-2

BD, Becton-Dickinson Immunocytometry Systems, Mountain View, Calif.; Chemicon, Chemicon, Temecula, Calif. [mmunolech, Immunotech, Marseille, France; Nicherei, Nichirei Co., Tokyo, Japan; Ortho, Ortho Pharmaceuticals Corp., Raritan, N.J.; Sanbio, Sanbio U den, the Netherlands. *1:100 diluted ascites form.

After being washed in phosphate-buffered saline (PBS), sections were successively incubated with biotin-conjugated goat-anti-mouse IgG, and peroxidase-avidin complex (Vectastain kit, Vector Laboratory, Burlingame, Calif.), according to the manufacturer's recommendation. Peroxidase activity was visualized with 0.06% diaminobenzidine (Wako Co., Tokyo, Japan) and hydrogen peroxide. Counterstainingwas performed with methyl green. The density of ELC was enumerated by counting of immunoreactive ELC overlying 200 basal cells. The density of DDCs in the papillary dermis was enumerated by counting the immunoreactive DDCs underlying 200 basal cells. Immunogold electron microscopy. Frozen sections of sodium chloride (NaCI)-separated, unfixed normal human skin (6 ,urn) were incubated with a 1:50 dilution of primary monoclonal antibodies (NU-T2 antibody and anti-type VII collagen antibody) for 1 hour at 3r c. After being washed in PBS, sections were sequentially incubated with biotinylated goat anti-mouse IgG (Vectastain), Auro Probe One anti-biotin (1 nm gold-conjugated anti-biotin antibody, Amersham, England), and silver enhancement solution (Immuno Gold/Silver kit GOLDTAGS, Lipshaw Co., Detroit, Mich.), according to the manufacturer's recommendation. After being washed in

Volume 27 Number 3 September 1992

DijJerentiallocalization of CD} a, CD1 b, and CD] c 421

Fig. 2. Leu-6 (CDla) and M-Tl02 (CDla) staining in normal human skin. Leu-6 antibody reacted with ELCs and DDCs (A); however, M-Tl02 stained only a certain population of ELCs and DDCs (B). Luminal portion of eccrine gland ducts was weakly stained by Leu-6 (C) and was strongly stained by M-Tl02 (D). distilled water, sections were fixed with 1% osmium tetroxide for I hour at room temperature, dehydrated, and embedded in Epon. Unstained ultrathin sections were observed with a JEOL JEM 1OO-SX electron microscope. After the reaction products were confirmed, a counterstain with uranyl acetate was performed. Separation of dermoepidermal junction by NaO. The epidermis was separated from the dermis after immersinga piece of normal human skin in IN NaCI for 4 days. 15 After this 4-day-long procedure the epidermis was easily separated from the dermis, and the binding specificity of bullous pemphigoid antibody or epidermolysis bullosa acquisita antibody was not altered. In some experiments this NaCl-separated skin was used as a substrate for immunoperoxidase staining or for immunogold electron microscopy. RESULTS

Staining pattern of ELC and DDC As shown in Fig. I, A and D, ELCs were clearly stained with CDla antibodies. The staining pattern was intracytoplasmic and membranous, and the dendritic pattern of ELC was apparently demonstrable by CDla antibodies. However, the number of ELC stained by one (M-Tl02) of the seven different CDla antibodies was decreased, when compared with the other CDla antibodies (Fig. 2, B, Tables II and III). The staining pattern of ELCs by M-TI02 was mainly cytoplasmic, and the dendritic processes of ELC were difficult to recognize (Fig. 2, B). No immunoreaction of ELCs was observed by CD 1b and CD 1c antibodies (Fig. 1, Band C; Tables

II and III) nor by control antibodies (data not shown). In contrast, DDCs were strongly positive for CDlaorCDlc antibodies (Fig. 1, A, C, and D). The density of CDla+ DDCs was similar to that of CDlc+ DDCs (Table II). Again, an epitope for M-TI02 (CDla) was expressed on a certain population of DDCs (Fig. 2, B, Tables II and III). CDlb antibodies only stained a minor population of D DCs (Fig. 1, B, Tables II and III). In some sections, any CDlb+ DDC was not recognized at all (Table II).

Staining pattern of keratinocytes Besides ELCs, CDla antibodies stained keratinocytes intercellularly in some areas of normal human skin (Fig. 1, D and E). Among the CDla antibodies, BL-6 had the strongest reactivity with keratinocytes (Table III). M-TI02 showed very weak staining for keratinocytes (Table III). The intercellular keratinocytc staining by BL-6 was more prominent in the follicular infundibulum (Fig. 1, E), which was in sharp contrast to the negative staining of the acrosyringium of eccrine glands (Fig. I, F). In contrast to CD Ia antibodies, all CD 1b and CD 1cor other control antibodies exhibited no immunoreaction with the epidermis (Fig. 1, Band C; Tables II and III). M-T] 02 and Leu-6 strongly reacted with the luminal edge of eccrine gland ducts (Fig. 2, C and D; Table III), Other CDla antibodies also weakly

Journal of the American Academy of Dermatology

422 Furue et al. Table II. Numbers of cells immunoreactive with CDI antibodies* ELct

CDla

CDlb

CDlc

BL-6 OKT6 66-IL-C7 0-605 VIT6 Leu-6 M-T102 4A76 NU-T2 Fl11-132 M-TlOl WM-25 Ll61 M241

DDe:!:

N8-1

N8-2

NS-3

N8-1

NS-2

13.5 14.9 12.3 13.9 12.9 13.5 8.5 0 0 0 0 0

8.2 7.0 7.5 9.0 8.5 8.8 4.6

10.8 (13.2)§ 11.2 (12.2) 12.9 (11.8) 10.1 (11.5) 11.8 (12.1) 12.0 (12.3) 7.0 ( 7.5) 0 (0) 0 (0)

18.0 17.5 18.5 19.1 17.0 16.9 10.2 2.0 1.2 4.0

5.1 6.2 6.8 5.0 6.0 6.5 2.0 0 0 0 0 0 7.8 8.8

0 0 0 0

0 0

0 0

0

0

0

0

(0) (0) (0) (0)

1.5

2.5 19.5 17.9

I

NS-3

12.0 (15.6) 13.1 (14.1) 12.5 (13.0) 10.8 (14.0) 11.9 (13.8) 13.6 (15.2) 9.1 (8.6) l.l (2.2) 2.0 (2.3) 1.2 (2.7) 1.8 (1.9) 2.2 (2.4) 9.5 (12.5) 11.6 (13.6)

DDC, Dermal dendritic cells; ELC, epidermal Langerhans cells; NS, normal human skin. = LO). *Representative data obtained from normal human skin tMean numbers of CDI + ELCs overlying 200 basaL cells. At Least five different areas were examined in each section. :j:Mean numbers of CD I + DOCs in papillary dermis underlying 200 basal cells. At least five different areas were examined in each section. §Mean numbers of CDI + cells in normal human skin = L0).

en

en

Table III. Immunoreactivity of CDI antibodies

CDla

CDlb

CDlc

BL-6 OKT6 66-IL-C7 0-605 VIT6 Leu-6 M-TI02 4A76 NU-T2 F111-132 M-TlOI WM-25 Ll61 M241

-, Negative, ±, negative or very weak;

skin.

++ ++ ++ ++ ++ ++ +

++ ++ ++ ++ ++ ++ + ± ± ±

Kemtinocytes

Lumen of eccrine gland ducts

++ + + + + + ±

± ±/+ ±/+ ±/+ ±/+ +/++ ++

BMZ

++

± ±

++ ++ +, weak; ++, strong. The epitopes of COLa, CDI b, COle antigens were differentially localized in normal

stained the luminal portion of eccrine gland ducts in some sections, although the secretory portion of eccrine glands was always negative for CDla antibodies (Fig. 2, C and D). No immunoreaction was recognized in eccrine glands by CDlb and CDlc antibodies or by control antibodies (data not shown).

Staining pattern of basement membrane zone (BMZ) We observed linear BMZ staining by one of the CDlb antibodies (NU-T2) (Fig. 3, A). Other COl antibodies or control antibodies did not show this BMZ staining pattern (Table III). The BMZ staining by NU-T2 antibody was clearly demonstrated in

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Differential localization of CDla. CD] b. and CDlc

all normal human skin samples. The NU-T2 antibody also weakly reacted with the BMZ of the follicles and the eccrine glands. In addition to the skin, NU-T2 antibody reacted with the BMZ of nonnal human esophagus and stomach (Fig. 3, B and C). The reactivity of NU-T2 antibody was not seen in murine and guinea pig skin (data not shown). Formalin-embedded sections were also not stained by NU-T2 antibody like other CDI antibodies (data not shown). We next examined the immunoreactivity of NU-T2 antibody on NaCl-separated normal human skin. As shown in Fig. 3,D, NU-T2antibody reacted with the dermal edge of the separated skin like anti-type VII collagen antibody (Fig. 3, E). To delineate further the localization of the NU-T2 BMZ antigen, we performed immunogold electron microscopy (particle size 1 nm, with subsequent silver enhancement) on NaCI-separated unfixed normal human skin, with a preembedding immunostaining technique. Surprisingly, the NU-T2 BMZ antigen localized in the lowermost lamina lucida (Fig. 4, B), which was in sharp contrast to the subbasal lamina localization (on the anchoring fibrils) of type VII collagen, as has been demonstrated previously, 16 and in our control experiment (Fig. 4, C). No reaction product was found in the epidermis (Fig. 4, A). We finally examined the expression ofthe NU-T2 BMZ antigen in the various skin disorders. Although the NU-T2 BMZ antigen was intact in psoriasis and in sebaceous nevus, it was absent in malignant skin diseases such as Bowen's disease and genital Paget's disease (Fig. 3, F), which was in sharp contrast to the presence of type VII collagen in the BMZ underlying these skin tumors (Fig. 3, G). The normal-appearing skin of a patient with recessive-dystrophic epidermolysis bullosa hereditaria was also strongly positive for the NU~T2 BMZ antigen (data not shown). DISCUSSION We have examined the localization of epitopes of CD1a, CD 1b, and CD 1c antigens with 14 different CD1 antibodies. We found that ELCs were stained only by CD 1a antibodies; M -T 102 (CD 1a) antibody selectively reacted with a certain subpopulation of ELCs; and DDCs were strongly positive for CDla and CD 1c antibodies; however, CD I b antibodies only stained a minor population of DDCs; CDla antibodies (especially BL-6) also showed intercellu-

423

Fig. 3. BMZ staining by NU-T2 (CDlb) antibody. NU-T2 antibody reacted with BMZ of normal human skin (A), esophagus (B), and stomach (c, arrowheads). NU-T2 antibody reacted with dermal edge of NaCI-separated normal human skin (D), as did anti-type VII collagen antibody (E). NU-T2 BMZ antigen was absent in BMZ underlying clusters of Paget's cells in genital Paget's disease (F, arrowheads). In contrast to NU-T2 BMZ antigen, type VII collagen was present in BMZ of Paget's disease (G).

lar keratinocyte staining even in the normal skin; M-TI02 (CDla) and Leu-6 (CDla) antibodies had considerably strong reactivity with the luminal portion of eccrine gland ducts; NU-T2 (CD1b) antibody clearly reacted with the BMZ of normal human skin, esophagus, and stomach; the NU-T2 BMZ antigen localized in the lowermost lamina lucida by immunogold electron microscopy; and the NU-T2 BMZ antigen was lost in the BMZ underlying malignant epidermal tumors such as Paget's disease and Bowen's disease. Although ELCs in situ are known to be CDla+ and CDlc-, 17 Teunissen et a1. 18 have reported that ELCs in epidermal cell suspension after trypsinization are positive for both CDla (OKT6) and CDlc (M241). Their data may suggest that an epitope for M24l on ELCs is unmasked by trypsinization. An epitope for M-T102 (CDla) was expressed on a certain population of ELCs in the present study (approximately 40% to 70% of all ELCs), suggesting that this antibody may be useful to study the phenotypic characteristics of histiocytosis X and other ELC-increasing diseases such as atopic dermatitis. 19

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Journal of the American Academy of Dermatology

Furue et al.

.,

."- . ., ........ r

Fig. 4. Immunogold electron microscopy (particle size 1 nm with subsequent silver enhancement) was performed on NaCl-separated, unfixed normal human skin, with a preembedding immunostaining technique. A, No reaction product was found in epidermis. Arrows, Hemidesmosomes. (X20,OOO.) B, In specimens incubated with NU-T2 antibody, reacted gold particles (arrowheads) were found in lowermost lamina lucida. BL, Basal lamina; arrows, anchoring fibrils. (X25,OOO.) C, In specimens incubated with anti-type VII collagen antibody, reacted gold particles (arrowheads) were localized below basal lamina, connected with anchoring fibrils (arrow). (X25,OOO.)

We used the term DDC as a purely descriptive one that was not meant to be synonymous with either "indeterminate cells" or "dermal dendrocytes." Almost similar numbers of DDCs were stained by CD la or CD Ic antibodies, which is in sharp contrast to the extremely limited staining of DDCs by CD 1b (approximately up to 20% of CDla+ or CDlc+ DDCs). We have recently observed a massive increase of CDI b+ DDCs in skin lesions in a patient with refractory anemia with an excess of blasts in transformation (unpublished data), indicating that CDlb+ DDCs may participate in the pathogenesis of some of the skin lesions. We must wait for the results of double-staining studies to confirm whether CDla+, CDlb+, or CDlc+ DDCs represent mutually distinct subpopulations of DDCs. It also would be worthwhile to investigate the relation between CDI + DDCs and other recently identified skinresiding DDCs, such as factor XlIIa+,20 CD36+,21 or CD34+22 DDCs. With dual and triple color analysis, Cooper et al. 23 have recently reported that the subset of human dermal class II MHC+ antigenpresenting cells most closely related to ELCs were CDlb+, CDlc+, and CDllc+. They also found that ELCs did have the potential to express CDlb, CD36, and CDllc because ELCs in atopic dermatitis or mycosis fungoides abnormally expressed CDl band CD36, and ELCs in histiocytosis X could be observed to coexpress CD 11 c.

With regard to keratinocyte staining, intercellular keratinocyte staining has already been demonstrated in lichen sclerosus et atrophicus, malignant melanoma, and lymphoma. 24-27 Focal intercellular keratinocyte staining and follicular epithelial staining by CD la has also been reported in normal skin by Willemze (reply in Pincelli et a1. 27 ), which is compatible with our results. This keratinocyte staining may account for the high background of immunolabeling of CD 1a in routine immunohistologic studies. The biologic significance of the CDla positivity on keratinocytes is unclear. However, recently Bleicher et al. 28 demonstrated that one ofthe murine CD 1 antigens is expressed only on gastrointestinal tract epithelium and in the cytoplasm of hepatocytes. The gastrointestinal distribution of murine CD 1 suggests that this molecule may be important in epithelial immunity. Because of the prominent keratinocyte expression of CDla molecules in various dermatoses, CDla+ keratinocytes may playa significant role in the cutaneous immunity. Because CDla expression by ELCs is upregulated by interleukin 129 and is down-regulated by granulocyte/ macrophage colony-stimulating factor,3o it would also be interesting to study the effects of various cytokines on the expression of CD Ia antigens by ELCs or by keratinocytes. With regard to the positive luminal staining of eccrine gland ducts by CDla (M-T102, Leu-6) in

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Differential localization of CDla, CDi b, and CDic 425

un-

our study, Gomes et al. 3! had already shown the munoreactivity of one of the CDla antibodies (D-47) to epithelial cells of the eccrine secretory portion. Although the immunoreactive sites of M-T102 and Leu-6 are different from thoseofD-47, these data indicate that epitopes of CD 1a molecules (perhaps cross-reactive epitopes) are also present on eccrine glands. It was surprising that one of the CD 1b antibodies (NU-T2) stained the BMZ of epidermis. This NU-T2 BMZ antigen presents not only in the BMZ of the stratified epithelium but also in the BMZ of the simple epithelium because the esophageal and gastric BMZ was immunolabeled by NU-T2. Biochemical and immunologic studies have demonstrated that the cutaneous BMZ includes many molecular components such as bullous pemphigoid antigen,32 cicatricial pemphigoid antigen,33 laminin,34 AA3 antigen,35 GB3 antigen,36 type IV collagen,3? KF-l antigen,38 nidogen,39 heparan sulfate proteoglycan,40 chondroitin sulfate proteoglycan,41 LDA-l antigen,42 LH7:2 antigen,43 AF-l and AF-2 antigen,44 type VII collagen (epidermolysis bullosa acquisita antigen),15, 17 cx!-microglobulin,45 thrombospondin,46 tenascin,4? and fibronectin. 48 Immunogold electron microscopy clearly showed that the NU-T2 BMZ antigen localizes in the lowermost lamina lucida. The NU-T2 BMZ antigen may be added to a list of defined molecules that are found in the lamina lucida such as bullous pemphigoid antigen, cicatricial pemphigoid antigen, laminin, AA3 antigen, GB3 antigen, and fibronectin. Our preliminary immunoprecipitation studies failed to identify the molecular weight of the NU-T2 BMZ antigen. It is of interest that the NU-T2 BMZ antigen was lost in the BMZ of the malignant epidermal tumors. de Moragas et al.49 previously reported that the bullous pemphigoid antigen was absent in anaplastic squamous cell carcinoma. The NU-T2 BMZ antigen may be another possible differentiation marker of skin tumors. REFERENCES

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