Epidermal Langerhans cells are specialized members of the

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1 The Human Hair Follicle: A Reservoir of CD40 B7-Deficient Langerhans Cells that Repopulate Epidermis After UVB Exposure Anita C. Gilliam,* Inger B. Kremer,* Yuichi Yoshida,* Seth R. Stevens,* Elena Tootell,* Marcel B.M. Teunissen, Craig Hammerberg,* and Kevin D. Cooper* *Department of Dermatology, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, U.S.A.; Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; School of Medicine, Kyushu University, Japan; VA Medical Center, Cleveland, Ohio, U.S.A. The ability of skin to maintain its protective structural and functional integrity depends on both resident and circulating cells. Until now, it was thought that dendritic antigen presenting cells of epidermis (Langerhans cells) were replaced by circulating bone marrow derived precursors. Here we show by immunostaining studies of timed biopsies taken from human skin after ultraviolet exposure, that hair follicle is a critical reservoir of Langerhans cells that repopulate epidermis depleted of Langerhans cells by a single four minimal erythema dose of ultraviolet B. Immunostaining with antibodies to thymidine dimers showed that ultraviolet B only penetrated the superficial hair follicle opening, whereas deeper follicle was relatively protected. Langerhans cells migrat- ing from hair follicle into epidermis 72 h after ultraviolet exposure have a partial deficiency of molecules important to T cell costimulation. We used four color flow cytometry to show that Langerhans cells isolated from epidermis 72 h after ultraviolet B can upregulate CD40 but not B7 1 or B7 2 expression in culture, suggesting a different phenotype of hair follicle Langerhans cells. Therefore, the hair follicle is a specialized immune compartment of the skin that serves as an intermediate reservoir of Langerhans cells between bone marrow and epidermis, and that may play a critical role in immune surveillance. Key words: antigen presenting cells/costimulatory molecules/dendritic cells. J Invest Dermatol 110: , 1998 Epidermal Langerhans cells are specialized members of the human cutaneous bone marrow derived dendritic cell population (Silberberg-Sinakin et al, 1976; Katz et al, 1979; Volc-Platzer et al, 1984; Breathnach, 1991), and are critical antigen presenting cells for initiating primary T cell mediated immune processes (i.e., delayed type hypersensitivity and contact hypersensitivity) (Rowden et al, 1977; Toews et al, 1980; Romani et al, 1991; Stingl and Shevach, 1991; Teunissen et al, 1997). They normally are present as highly dendritic cells with processes interdigitating between epidermal keratinocytes, with a cell density of about 2%. Langerhans cells are found in many epithelia, where contact with the external environment occurs (Rowden et al, 1977), as well as in follicular epithelium (Breathnach, 1963), although follicular Langerhans cells are poorly characterized. Other human leukocyte antigen (HLA) class II positive dendritic cells are found in the dermis (Murphy et al, 1986; Breathnach, 1991), some of which are of the dendritic antigen presenting cell lineage (Meunier et al, 1993) that may represent resident perivascular Langerhans cell variants, Langerhans cell precursors migrating into skin, or activated Langerhans cells migrating from skin. Upon activation, the Langerhans cell phenotype changes from a cell that efficiently takes up and processes antigen into one that efficiently Manuscript received August 29, 1997; revised December 4, 1997; accepted for publication December 23, Reprint requests to: Dr. Anita C. Gilliam, Department of Dermatology, Lakeside Hospital, Case Western Reserve University/University Hospitals of Cleveland, Euclid Avenue, Cleveland, OH presents antigen to naïve T cells (Schuler and Steinman, 1985; Teunissen et al, 1997). This maturation is accompanied by the inducible expression of several costimulatory molecules and the enhanced expression of HLA class II molecules. Recently, dendritic cells with features of Langerhans cells have been generated in vitro from human neonatal cord blood stem cell precursors (Caux et al, 1992) and from peripheral blood precursors (Sallusto and Lanzavecchia, 1994) using specialized culture conditions [granulocyte-macrophage colony stimulating factor, interleukin (IL)-4, and tunor necrosis factor-α]; however, the source (resident dermal precursors or blood derived dendritic cells) of Langerhans cells in epidermis in vivo and their maturation steps are not known. A useful model for the study of Langerhans cell entry into skin and phenotype after functional challenge is ultraviolet B (UVB) exposed skin (Cruz and Bergstresser, 1991). After UVB exposure, epidermal Langerhans cells are decreased in density and altered in morphology, becoming less dendritic (Toews et al, 1980). At 72 h, CD1a Langerhans cells are markedly decreased in numbers in UVB exposed epidermis and do not begin to reappear in large numbers until days 4 6 (Toews et al, 1980; Aberer et al, 1981); their disappearance is closely related in time to the development of an immunosuppressive environment and antigen specific tolerance to epicutaneous hapten (Streilein et al, 1980; Cooper et al, 1992). By 72 h, depletion of cells bearing HLA-DR but not OKT6 from epidermal suspensions reduces T cell proliferation in allogeneic epidermal cell-lymphocyte reactions, showing indirectly that at that time, Langerhans cells are not significantly contributing to induction of T cell proliferation by UV exposed skin (Cooper et al, 1985). At the same time that Langerhans cells disappear from the epidermis, infiltration of first the dermis and later the epidermis by IL X/98/$10.50 Copyright 1998 by The Society for Investigative Dermatology, Inc. 422

2 VOL. 110, NO. 4 APRIL 1998 HUMAN HAIR FOLLICLE producing CD11b monocyte/macrophages (Kang et al, 1994) 1 that are deficient in CD40, B7, and IL-12 2 and that induce T cell proliferation well is seen. We report here that the hair follicle is a reservoir for epidermal Langerhans cells after UVB injury to human skin. (This follicular reservoir paradigm is also seen in cutaneous thermal injury and in the autoimmune disease vitiligo, in which follicular keratinocytes and melanocytes replenish epidermal cell populations.) After UVB, Langerhans cells in follicular epithelium divide and appear to migrate from follicular infundibulum into surrounding epidermis. In addition, the early repopulating epidermal Langerhans cells present 72 h after UVB show an altered phenotype compared with Langerhans cells from unirradiated epidermis. These Langerhans cells have a selectively reduced capacity to express the costimulatory markers B7 1 and B7 2, but not CD40, in culture compared with Langerhans cells from unexposed control skin, suggesting an intermediate maturation step in the transition from hair follicle to epidermis with possible implications for T cell activation. Because of the demonstrated linkage of susceptibility in animals (Kripke and Fisher, 1976) and humans (Yoshikawa et al, 1990) to UVB induced skin tumors with development of an immunosuppressive state in UVB exposed skin, our data suggest the importance of a mechanism for a more rapid recovery of the protective cutaneous immune system after UVB than may be possible from blood precursors alone. MATERIALS AND METHODS Subjects Healthy adult volunteers (n 8) participated in the study after Institutional Review Board approval of the protocol and informed consent. As described previously (Cooper et al, 1992), four minimal erythema dose (MED) was delivered to buttock skin from Westinghouse FS20 bulbs at different time points. At the final time point, punch biopsies or keratomes were obtained from all UVB exposed and nonirradiated control sites. Immunohistochemical studies Antibodies Immunostaining was performed at least three times on each specimen with the following primary mouse monoclonal antibodies: CD1a (B , IgG2a; Immunotech, Westbrook, ME), CD11b (anti-mac-1/bear 1, IgG1; Immunotech), Ki-67 (Ki-S5, IgG1; Boehringer, Indianapolis, IN), and antibodies to cyclobutane pyrimidine dimers (TDM-2, IgG2a; gift of Dr. Osamu Nikaido, Kanazawa University, Japan). Secondary antibodies used for immunoperoxidase studies were biotin conjugated goat anti-mouse IgG1 or IgG2a (Caltag, San Francisco, CA) followed by peroxidase labeled streptavidin (Kirkegaard & Perry, Gaithersburg, MD). Double immunofluorescence staining for Ki-67 and CD1a was performed using fluorescein isothiocyanate conjugated goat anti-mouse IgG1 for Ki-67 (Boehringer), and biotin conjugated goat antimouse IgG2a for CD1a followed by Rhodamine Red-x conjugated avidin (Neutr-Avidin, Molecular Probes, Leiden, The Netherlands). Immunohistochemical procedures Six micrometer frozen sections were blocked with 10% goat serum, treated with primary antibody or its isotype control immunoglobulin, treated with biotin conjugated secondary antibody, peroxidase conjugated streptavidin (Kirkegaard & Perry), then detected with Diaminobenzidine Reagent Set (DAB) (Kirkegaard & Perry) and counterstained with hematoxylin. Before staining frozen tissue sections with antibodies to cyclobutane thymidine dimers (TDM-2), nuclear DNA was denatured with 70 mm NaOH in 70% ethanol for 2 min, followed by neutralization for 1 min in 100 mm Tris-HCL, ph 7.5 in 70% ethanol, three washes of 70% ethanol, and two washes of phosphate buffered saline (Chadwick et al, 1995). Microscopy and photography Images from immunostaining experiments were obtained using a Zeiss (Thornwood, NY) Axiophot microscope and Kodak (Rochester, NJ) Ektachrome 160T film (bright field studies) or P1600 film (immunofluorescence studies). These were scanned (SprintScan, Polaroid, Cambridge, MA) and formatted as tiff images in Adobe Photoshop and Illustrator. Analysis: quantitation of CD1a cells CD1a cells in the upper area of the 1 Gilliam AC, Moser A, Tootell E, Kang K, Cooper KD: Distinct physical and temporal compartments of antigen-presenting cells and IL-10 and TNFα expression in human UV-exposed skin by in situ hybridization and immunostaining. J Invest Dermatol 108:638, 1997 (abstr.) 2 Kremer IB, Cooper KD, Teunissen MBM, Stevens SR: Diminished CD40 expression on macrophages infiltrating UV-exposed skin is responsible for IL- 2Rα T cell activation. J Invest Dermatol 108:538, 1997 (abstr.) follicle and in the epidermis adjacent to the follicle were counted in unirradiated control skin and in skin 72 h after UVB using an ocular micrometer grid with 200 magnification. The compartments are delineated as A E with the areas of A, B1 B2, C1 C2, D1 D2, and E1 E2, each corresponding to mm 2 (Fig 4). After counting the number of CD1a cells in three sections per individual specimen (n 3), CD1a cells in UVB irradiated skin were expressed as a percentage ( SEM) of the mean count in the equivalent compartment in unirradiated control skin. Flow cytometry Antibodies For flow cytometry, the following mouse monoclonal antibodies were used: biotin conjugated anti-hla-dr (IgG2a; Becton Dickinson, Mountain View, CA), fluorescein isothiocyanate conjugated anti-cd1a (IgG1; Ortho, Raritan, NJ), and anti-cd40 (IgG1; Pharmingen, San Diego, CA), phycoerythrin conjugated anti-cd1a (OKT6; IgG1; Ortho), anti-b7 1 (CD80; IgG1; Becton Dickinson), anti-b7 2 (CD86; IgG1; Ancell, Bayport, MN), or isotype matched control antibodies from the same companies. Cascade-Bluestreptavidin was purchased from Molecular Probes (Eugene, OR). Flow cytometry protocol Keratome biopsies were obtained 72 h after four MED of UVB as previously described (Meunier et al, 1993). Epidermis was separated from dermis by incubation in dispase, digested in trypsin, and dissociated in 0.1% DNase (Sigma, St Louis, MO) in Hanks balanced salt solution containing 10% fetal bovine serum (Hyclone, Logan, UT). The suspension was filtered through 100 µm nylon mesh and cultured for 24 h in vitro in RPMI with 10% heat inactivated human AB serum (North American Biology, Miami, FL) to allow costimulatory molecule expression. Cells were incubated with human IgG (Sigma) to prevent nonspecific Fc receptor binding. Dead cells were identified by addition of ethidium monoazide (Molecular Probes) for 10 min under visible light for cross-linkage to DNA. Flow cytometry was performed using an Epics Elite (Coulter Cytometry). RESULTS Human hair follicle is a reservoir for Langerhans cells in skin depleted of epidermal Langerhans cells by UVB When human skin is exposed to four MED of UVB, interfollicular epidermal Langerhans cells (Fig 1a d, arrows) become less dendritic and are decreased in number after 24 h, with almost complete disappearance of CD1a cells at 72 h. Note the occasional CD1a dermal Langerhans cell-like cells in control unirradiated skin (Fig 1a) but not in irradiated skin. Lower power views of skin specimens from a single subject show numerous CD1a Langerhans cells in both follicular epithelium and epidermis in control tissue (Fig 2a, arrows). At later time points after UVB (48, 72, 96 h, Fig 2b d, respectively), CD1a cells are absent in interfollicular epidermis and dermis, but appear in and adjacent to follicles. At 96 h (Fig 2d), there are clearly increased numbers of CD1a cells in the follicular orifice and in the adjacent epidermis. At the same time, the epidermis and dermis at some distance from follicles showing prominent UVB injury (necrosis of epidermal keratinocytes) still lack CD1a cells. The CD1a cells in perifollicular epidermis adjacent to follicles at 48, 72, and 96 h may represent either Langerhans cells that are migrating from follicle to epidermis, or Langerhans cells left in the epidermis after UVB injury; the immunostaining results of this time course showing CD1a cells at progressive distances from the follicular orifice, suggest that Langerhans cells are migrating from follicle to epidermis. These changes can be appreciated in the low power view of a biopsy taken 72 h after UVB exposure from another subject (n 3, Fig 3a). The immunostaining results presented here are representative of those in all biopsies from all patients. Immunostaining of sections containing follicles from the same specimen with CD11b/CD18 (Mac-1) (Fig 3b) highlights the unique character of follicular epithelium and of epidermis adjacent to follicles after UVB; numerous infiltrating Mac-1 macrophages are seen in epidermis and dermis at some distance from but not adjacent to or within follicles, presenting the reverse image of CD1a staining (Fig 3a). Therefore, the hair follicle appears to be a relatively protected environment for and serves as a source of Langerhans cells that begin to replenish the epidermis 48 h after four MED UVB. Meanwhile, the epidermis at some distance from the follicle is injured, without CD1a cells, and is a site for migration of and infiltration by Mac-1 macrophages. Further support of follicular Langerhans cells as a reservoir for epidermal Langerhans cells is the observation that the other possible

3 424 GILLIAM ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 1. UVB-induced changes in epidermal Langerhans cells. Interfollicular CD1a epidermal Langerhans cells in human skin irradiated with four MED UVB change in morphology and numbers, becoming less dendritic at 6 h (b), depleted in number at 24h(c), and completely absent by 72 h (d), compared with control unirradiated skin (a). Immunoperoxidase staining with CD1a (brown staining cells, arrows) of tissue collected at time points after UVB. Note occasional CD1a dermal cells in control skin (a) but not in irradiated skin. Scale bar, 43 µm. Figure 2. UVB-induced changes in hair follicle Langerhans cells. CD1a Langerhans cells (brown staining cells, arrows) at time points after four MED UVB [(a) control unirradiated skin, (b) 48h,(c)72h,(d) 96 h] are present in progressively increased numbers in the follicular opening, whereas interfollicular epidermis distant from the hair follicle becomes devoid of CD1a cells. Sclae bar, 138 µm. Figure 3. Langerhans cells and macrophages occupy different compartments in UV-exposed skin. Lower power views of a biopsy specimen of skin 72 h after four MED UVB stained with CD1a (a) or CD11b (b) show that CD1a Langerhans cells and CD11b macrophages are present in distinctly different compartments in the skin after UVB. Scale bar, 344 µm. source of Langerhans cells migrating to the epidermis (papillary dermis) contains few if any CD1a cells at 48, 72, or 96 h after UVB (Fig 2b d, 3a). To quantitatively document that follicular epithelium contains increased numbers of Langerhans cells 72 h after UVB, we counted CD1a cells in epidermal compartments (n 3, inset, Fig 4). Epithelium at the follicular orifice (inset: A, B1 B2) contains significantly more CD1a cells than the immediately adjacent epidermis (C1 C2, D1 D2, and E1 E2). As Fig 4 shows, the numbers of Langerhans cells decrease with increasing distance from the hair follicle, with 100% representing control unirradiated skin. Deep follicular epithelium is protected from UVB It has been shown that UVB only reaches the epidermis and superficial dermis (Bruls et al, 1984). To evaluate the depth of direct UVB exposure in our skin samples and to show that follicular epithelium is relatively photoprotected, we performed immunostaining of biopsy tissue taken from buttock skin 10 min after UVB with antibodies to cyclobutane pyrimidine dimers (Qin et al, 1994; Vink et al, 1996). We confirmed that fewer thymidine dimers are found at increasing depth in the epidermis. Figure 5(a) shows no staining in control unirradiated skin, whereas Fig 5(b) shows numerous positive cells in the epidermis and superficial papillary dermis but not in the deeper follicle, with a sharp line of demarcation in the superficial portion of the hair follicle, showing that infundibulum and deep follicular epithelium are indeed protected from UVB radiation. Proliferating Langerhans cells are found in the hair follicle Immunostaining with Ki-67, which stains nuclei of cells in cycle (Gerdes et al, 1984), identifies numerous proliferating cells in follicular epithelium 72 h after UVB. Double-staining immunofluorescence studies using anti-ki-67 (fluorescein isothiocyanate) and anti- CD1a (rhodamine) (Fig 6) show that admixed with the green nuclei of Ki-67 proliferating keratinocytes are occasional cells with yellow

4 VOL. 110, NO. 4 APRIL 1998 HUMAN HAIR FOLLICLE 425 Figure 4. The numbers of CD1a cells 72 h after four MED UVB are high in the hair follicle and in the adjacent epidermis and form a gradient inversely related to distance from the follicle. The numbers of CD1a cells per square grid ( mm 2 ) in the upper area of the follicle and in the epidermis near the follicle were counted in unirradiated control skin and in skin 72 h after UVB using an ocular micrometer grid under the microscope. The image in the right corner shows the square compartments (A E) defined for counting. The percentage of CD1a cells SEM in each compartment (mean of three experiments) at 72 h versus control unirradiated skin (100%) is presented. Figure 6. Proliferating Langerhans cells are found in the hair follicle. Occasional proliferating Langerhans cells are seen in the follicular epithelium when double stained for CD1a (red staining dendritic cell bodies) and for Ki- 67 (yellow nuclei, white arrows) at 72 h after UVB. The remainder of Ki-67 cell nuclei staining green represent keratinocytes in the cell cycle. Scale bar,26 µm. Figure 5. Deep follicular epithelium is protected from UVB. The depth of penetration ( ) of UV in skin collected 10 min after a single dose of four MED UVB is shown by the presence of thymidine dimers (brown staining keratinocyte nuclei) in the epidermis and the upper follicular epithelium. Protected follicular epithelium deep to the infundibulum shows no thymidine dimers. Scale bar, 138 µm; inset, 72 µm. double-staining nuclei (white arrow) and red dendritic cell bodies that are positive for both CD1a and Ki-67, representing proliferating Langerhans cells. Therefore, although the majority of proliferating cells in the follicular epithelium of UVB irradiated skin are keratinocytes, there are also proliferating Langerhans cells. Hair follicles in control skin also showed a few CD1a Ki-67 cells (not shown); the numbers in both UV and control conditions are small. Newly migrating follicular Langerhans cells are deficient in upregulation of B7 but not of CD40 costimulatory molecules We next asked if the Langerhans cells present in UVB exposed epidermis 72 h after UVB, which are predominantly associated with the hair follicle (Figs 2b d, 3a, 4), are normal Langerhans cells. The ability of Langerhans cells to stimulate T cells depends on the presence of surface costimulatory molecules such as B7 and CD40 (Foy et al, 1996; Lenschow et al, 1996). Therefore, we investigated the expression of these costimulatory molecules using flow cytometry of Langerhans cells purified from control or UVB irradiated epidermis 72 h after four MED UVB, when Langerhans cells present in cell suspensions are likely to be those that have migrated from the hair follicle. [We verified that in routine histology of intact keratome and in dispase-split epidermal and dermal components of the keratome, the upper region of the hair follicle stays with the epidermis (not shown).] Epidermal cell suspensions were cultured in vitro for 24 h to mimic the maturation process observed when Langerhans cells migrate from the skin to the lymph nodes (Schuler and Steinman, 1985). After 1 d of culture, as expected, Langerhans cells from control skin strongly expressed B7 1, B7 2, and CD40 (Fig 7a, left panels, b). In contrast, Langerhans cells from UVB irradiated skin essentially failed to upregulate B7 1 or B7 2 during culture, but were clearly positive for newly expressed CD40 removed by trypsinization (Fig 7a, right panels, b), evidence for a partial deficit in costimulatory molecule expression. Although migration in this ex vivo model cannot be directly studied, together these data suggest that follicular Langerhans cells are sources for replenishment of epidermal Langerhans cells after a single dose of UVB, and that additional not yet characterized maturation steps occur after their migration into epidermis.

5 426 GILLIAM ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY DISCUSSION This study demonstrates the critical and unique role of hair follicles in the immune system of the skin. We show that Langerhans cells proliferate in and migrate from the hair follicle into UVB exposed epidermis, beginning to repopulate the epidermis as early as 48 h after a single dose of four MED UVB. The ability of Langerhans cells to divide and migrate is known (Miyauchi and Hashimoto, 1987); the factors that induce Langerhans cell division, maintain Langerhans cell/ keratinocyte ratios in epidermis, and initiate migration are not well characterized. Based on bone marrow transplantation studies of lethally irradiated mice and humans, in which repopulation of epidermis by Langerhans cells was shown to be entirely by cells with donor phenotype (Katz et al, 1979; Volc-Platzer et al, 1984), the route of entry into skin appeared to be via blood borne precursors originating in bone marrow. Whether these precursors take up residence in the perivascular dermis, enter directly into the epidermis, or enter the epidermis via the hair follicle is unknown. The present data using a physiologic skin challenge of a single four MED of UVB, which results in almost complete depletion of interfollicular epidermal CD1a cells, indicate that one pathway for repopulation of epidermal Langerhans cells is a local one via the hair follicle. This study suggests that newly emigrated follicular Langerhans cells have a different phenotype than interfollicular epidermal Langerhans cells, in that they are able to induce CD40 but not B7 expression in culture; the properties acquired in culture are thought to model the functional changes that occur during stimulated Langerhans cell migration from the epidermis to regional lymph nodes. The afferent pathway of Langerhans cell trafficking from blood and tissue into epidermis is less well understood; our data identifying follicular Langerhans cells as a resource for epidermal Langerhans cells will be useful for dissecting out that differentiation pathway. Because T cell activation in the absence of B7 costimulation can result in antigen specific unresponsiveness (Gimmi et al, 1993), the presence of B7 deficient Langerhans cells in UV exposed skin is likely to have consequences for immune activation and tolerance induction. Changes in the cytokine environment in UVB irradiated skin, such as the presence of IL-10 producing UV macrophages, will also influence the phenotype of Langerhans cells repopulating the epidermis from the hair follicle, because IL-10 can directly downregulate B7 expression (Ding et al, 1993; Ozawa et al, 1996). Therefore, Langerhans cells in the hair follicle may be exposed to different microenvironments of cytokines and growth factors as they migrate to the epidermis following UVB. Further studies of the follicular and epidermal microenvironments in which Langerhans cells reside will provide important information about possible mechanisms of Langerhans cell maturation and function, and roles in immune surveillance. Note added in proof: Follicular infundibular Langerhans cells may also replenish epidermis in chronically UV-exposed human skin (Murphy et al, 1998). We thank the many volunteers who donated skin biopsies and keratomes for these studies. We appreciate also the excellent secretarial assistance of Virginia Ehrbar with this manuscript. This work was supported in part by grants from the Dermatology Foundation (ACG, Mary Kay Cosmetics Grant; SRS, Procter and Gamble Fellowship); Ohio Regents Pilot Study Grant (ACG); National Alopecia Areata Foundation (ACG; 44); Netherlands Organization for Scientific Research Fellowship, Dutch Cancer Figure 7. Langerhans cells in skin 72 h after four MED UVB are deficient in their ability to upregulate B7 1 and B7 2, but not CD40. Epidermal cells were isolated from UV exposed and nonirradiated control keratomes obtained 72 h after UV exposure and analyzed by four color flow cytometry. (a) Representative flow diagrams of B7 1, B7 2, and CD40 expression on CD1a HLA-DR ethidium monoazide (viable) Langerhans cells. The left panels show Langerhans cells from control skin (C-Langerhans cells), the right panels show Langerhans cells from UV-exposed skin (UV-Langerhans cells). Percentages of positive cells are given in the upper right boxes in each panel. Only 2% of UV-Langerhans cells express B7 1, compared with 50% of C-Langerhans cells. Similarly, only 19% of UV-Langerhans cells express B7 2, compared with 86% of C-Langerhans cells. In contrast, the percentage of UV- Langerhans cells expressing CD40 is almost the same as that of C-Langerhans cells (71 and 76%, respectively). (b) Mean values of B7 1, B7 2, and CD40 expression (delta mean fluorescence) for four experiments. Delta mean fluorescence for B7 1 and B7 2 was significantly lower for Langerhans cells from UV irradiated skin (y) as compared with nonirradiated control skin (j); * indicated p 0.05.

6 VOL. 110, NO. 4 APRIL 1998 HUMAN HAIR FOLLICLE 427 Foundation, and Christine Buisman Foundation (IBK); NIAID #AI (KDC); Case Western Reserve University Skin Disease Research Center, NIAMS (ACG and SRS), and the Veterans Administration Hospital (KDC). REFERENCES Aberer G, Schuler G, Stingl G, Hönigsmann H, Wolff K: Ultraviolet light depletes surface markers of Langerhans cells. J Invest Dermatol 76: , 1981 Breathnach AS: The distribution of Langerhans cells within the human hair follicle and some observations on its staining properties with gold chloride. J Anat 97:73 80, 1963 Breathnach SM: Origin cell lineage ontogeny tissue distribution, kinetics of Langerhans cells. In: Schuler G (ed.). Epidermal Langerhans Cells, CRC Press, Boca Raton, 1991, pp Bruls WAG, Slaper H, Van Der Leun JC, Berrens L: Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40: , 1984 Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J: GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 360: , 1992 Chadwick CA, Potten CS, Nikaido O, Matsunaga T, Proby C, Young AR: The detection of cyclobutane thymine dimers (6 4) photolesions and the Dewar photoisomers in sections of UV-irradiated human skin using specific antibodies, and the demonstration of depth penetration effects. J Photochem Photobiol 28: , 1995 Cooper KD, Fox P, Neises G, Katz SI: Effects of ultraviolet radiation on human epidermal cell alloantigen presentation: Initial depression of Langerhans cell-dependent function is followed by the appearance of T6- Dr cells that enhance epidermal alloantigen presentation. J Immunol 134: , 1985 Cooper KD, Oberhelman L, Hamilton TA, et al: UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: Relationship to dose, CD1a-DR epidermal macrophage induction, and Langerhans cell depletion. Proc Natl Acad Sci USA 89: , 1992 Cruz PD Jr, Bergstresser PR: The influence of ultraviolet radiation and other physical and chemical agents on epidermal Langerhans cells. In: Schuler G (ed.). Epidermal Langerhans Cells, CRC Press, Boca Raton, 1991, pp Ding L, Linsley PS, Huang L-Y, Germain RN, Shevach EM: IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J Immunol 151: , 1993 Foy TM, Aruffo A, Bajorath J, Buhlmann JE, Noelle RJ: Immune regulation by CD40 and its ligand gp39. Annu Rev Immunol 14: , 1996 Gerdes J, Lemke H, Baisch H, Wacker H-H, Schwab U, Stein H: Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 133: , 1984 Gimmi CD, Freeman GJ, Gribben JG, Gray G, Nadler LM: Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. Proc Natl Acad Sci USA 90: , 1993 Kang K, Hammerberg C, Meunier L, Cooper KD: CD11b macrophages that infiltrate human epidermis after in vivo ultraviolet exposure potently produce IL-10 and represent the major secretory source of epidermal IL-10 protein. J Immunol 153: , 1994 Katz SI, Tamaki K, Sachs DH: Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature 282: , 1979 Kripke ML, Fisher MS: Immunologic parameters for ultraviolet carcinogenesis. J Natl Cancer Inst 57: , 1976 Lenschow DJ, Walunas TL, Bluestone JA: CD28/B7 system of T cell costimulation. Annu Rev Immunol 14: , 1996 Meunier L, Gonzalez-Ramos A, Cooper KD: Heterogeneous populations of class II MHC cells in human dermal cell suspensions. Identification of a small subset responsible for potent dermal antigen-presenting cell activity with features analogous to Langerhans cells. J Immunol 151: , 1993 Miyauchi S, Hashimoto K: Epidermal Langerhans cells undergo mitosis during the early recovery phase after ultraviolet-b irradiation. J Invest Dermatol 88: , 1987 Murphy GF, Messadi D, Fonferko E, Hancock WW: Phenotypic transformation of macrophages to Langerhans cells in the skin. Am J Pathol 123: , 1986 Murphy GF, Katz S, Kligman AM: Topical tretinoin replenishes CD1a-positive epidermal Langerhans cells in chronically photo-damaged human skin. J Cutan Pathol 25:30 34, 1998 Ozawa H, Aiba S, Nakagawa S, Tagami H: Interferon-gamma and interleukin-10 inhibit antigen presentation by Langerhans cells for T helper type 1 cells by suppressing their CD80 (B7 1) expression. Eur J Immunol 26: , 1996 Qin X, Zhang S, Nakatsuru Y, Oda H, Yamazaki Y, Suzuki T, Nikaido O, Ishikawa T: Detection of active UV-photoproduct repair in monkey skin in vivo by quantitative immunohistochemistry. Cancer Lett 83: , 1994 Romani N, Schuler G, Fritsch P: Identification and phenotype of epidermal Langerhans cells. In: Schuler G (ed.). Epidermal Langerhans Cells, CRC Press, Boca Raton, 1991, pp Rowden G, Lewis MG, Sullivan AK: Ia antigen expression on human epidermal Langerhans cells. Nature 268: , 1977 Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony -stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179: , 1994 Schuler G, Steinman RM: Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J Exp Med 161: , 1985 Silberberg-Sinakin I, Thorbecke GJ, Baer RL, Rosenthal SA, Berezowsky V: Antigenbearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell Immunol 25: , 1976 Stingl G, Shevach EM: Langerhans cells as antigen-presenting cells. In: Schuler G (ed.). Epidermal Langerhans Cells, CRC Press, Boca Raton, 1991, pp Streilein JW, Toews GT, Gilliam JN, Bergstresser PR: Tolerance or hypersensitivity to 2,4-dinitro-1-fluorobenzene: the role of Langerhans cell density within epidermis. J Invest Dermatol 74: , 1980 Teunissen MBM, Kapsenberg ML, Bos JD: Langerhans cells and related skin dendritic cells. In: Bos JD (ed.). Skin Immune System, 2nd edn, CRC Press, Boca Raton, 1997, pp Toews GB, Bergstresser PR, Streilein JW, Sullivan S: Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 124: , 1980 Vink AA, Strickland FM, Bucana C, Cox PA, Roza L, Yarosh DB, Kripke ML: Localization of DNA damage and its role in altered antigen-presenting cell function in ultravioletirradiated mice. J Exp Med 183: , 1996 Volc-Platzer B, Stingl G, Wolff K, Hinterberger W, Schnedl W: Cytogenetic identification of allogeneic epidermal Langerhans cells in a bone-marrow-graft recipient. [letter]. N Engl J Med 310: , 1984 Yoshikawa T, Rae V, Bruins-Slot W, Van den Berg J-W, Taylor JR, Streilein JW: Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in humans. J Invest Dermatol 95: , 1990