The Human Hair Follicle: A Reservoir of CD40+ B7-Deficient Langerhans Cells that Repopulate Epidermis After UVB Exposure
1998; Elsevier BV; Volume: 110; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.1998.00162.x
ISSN1523-1747
AutoresAnita C. Gilliam, Inger B. Kremer, Yuichi Yoshida, Seth R. Stevens, Elena Tootell, Marcel B. M. Teunissen, Craig Hammerberg, Kevin D. Cooper,
Tópico(s)Immune Cell Function and Interaction
ResumoThe 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 migrating 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. 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 migrating 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. Epidermal Langerhans cells are specialized members of the human cutaneous bone marrow derived dendritic cell population (Silberberg-sinakin et al., 1976Silberberg-sinakin I. Thorbecke G.J. Baer R.L. Rosenthal S.A. Berezowsky V. Antigen-bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes.Cell Immunol. 1976; 25: 137-151Crossref PubMed Scopus (354) Google Scholar; Katz et al., 1979Katz S.I. Tamaki K. Sachs D.H. Epidermal Langerhans cells are derived from cells originating in bone marrow.Nature. 1979; 282: 324-326Crossref PubMed Scopus (725) Google Scholar; Volc-platzer et al., 1984Volc-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. 1984; 310: 1123-1124PubMed Google Scholar; Breathnach, 1991Breathnach S.M. Origin cell lineage ontogeny tissue distribution, kinetics of Langerhans cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 23-48Google Scholar), 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., 1977Rowden G. Lewis M.G. Sullivan A.K. Ia antigen expression on human epidermal Langerhans cells.Nature. 1977; 268: 247-248Crossref PubMed Scopus (452) Google Scholar; Toews et al., 1980Toews G.B. Bergstresser P.R. Streilein J.W. Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.J Immunol. 1980; 124: 445-453PubMed Google Scholar; Romani et al., 1991Romani N. Schuler G. Fritsch P. Identification and phenotype of epidermal Langerhans cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 49-72Google Scholar; Stingl and Shevach, 1991Stingl G. Shevach E.M. Langerhans cells as antigen-presenting cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 159-180Google Scholar; Teunissen et al., 1997Teunissen M.B.M. Kapsenberg M.L. Bos J.D. Langerhans cells and related skin dendritic cells.in: Bos J.D. 2nd edn. Skin Immune System. CRC Press, Boca Raton1997: 59-83Google Scholar). 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., 1977Rowden G. Lewis M.G. Sullivan A.K. Ia antigen expression on human epidermal Langerhans cells.Nature. 1977; 268: 247-248Crossref PubMed Scopus (452) Google Scholar), as well as in follicular epithelium (Breathnach, 1963Breathnach A.S. The distribution of Langerhans cells within the human hair follicle and some observations on its staining properties with gold chloride.J Anat. 1963; 97: 73-80PubMed Google Scholar), 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., 1986Murphy G.F. Messadi D. Fonferko E. Hancock W.W. Phenotypic transformation of macrophages to Langerhans cells in the skin.Am J Pathol. 1986; 123: 401-406PubMed Google Scholar; Breathnach, 1991Breathnach S.M. Origin cell lineage ontogeny tissue distribution, kinetics of Langerhans cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 23-48Google Scholar), some of which are of the dendritic antigen presenting cell lineage (Meunier et al., 1993Meunier L. Gonzalez-ramos A. Cooper K.D. 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. 1993; 151: 4067-4080PubMed Google Scholar) 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 presents antigen to naïve T cells (Schuler and Steinman, 1985Schuler G. Steinman R.M. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-546Crossref PubMed Scopus (847) Google Scholar; Teunissen et al., 1997Teunissen M.B.M. Kapsenberg M.L. Bos J.D. Langerhans cells and related skin dendritic cells.in: Bos J.D. 2nd edn. Skin Immune System. CRC Press, Boca Raton1997: 59-83Google Scholar). 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., 1992Caux C. Dezutter-dambuyant C. Schmitt D. Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells.Nature. 1992; 360: 258-261Crossref PubMed Scopus (1406) Google Scholar) and from peripheral blood precursors (Sallusto and Lanzavecchia, 1994Sallusto 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. 1994; 179: 1109-1118Crossref PubMed Scopus (4392) Google Scholar) 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, 1991Cruz Jr, P.D. Bergstresser P.R. The influence of ultraviolet radiation and other physical and chemical agents on epidermal Langerhans cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 253-267Google Scholar). After UVB exposure, epidermal Langerhans cells are decreased in density and altered in morphology, becoming less dendritic (Toews et al., 1980Toews G.B. Bergstresser P.R. Streilein J.W. Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.J Immunol. 1980; 124: 445-453PubMed Google Scholar). 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., 1980Toews G.B. Bergstresser P.R. Streilein J.W. Sullivan S. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB.J Immunol. 1980; 124: 445-453PubMed Google Scholar; Aberer et al., 1981Aberer G. Schuler G. Stingl G. Hönigsmann H. Wolff K. Ultraviolet light depletes surface markers of Langerhans cells.J Invest Dermatol. 1981; 76: 202-210Abstract Full Text PDF PubMed Scopus (362) Google Scholar); their disappearance is closely related in time to the development of an immunosuppressive environment and antigen specific tolerance to epicutaneous hapten (Streilein et al., 1980Streilein J.W. Toews G.T. Gilliam J.N. Bergstresser P.R. Tolerance or hypersensitivity to 2,4-dinitro-1-fluorobenzene: the role of Langerhans cell density within epidermis.J Invest Dermatol. 1980; 74: 319-322Abstract Full Text PDF PubMed Scopus (70) Google Scholar; Cooper et al., 1992Cooper K.D. Oberhelman L. Hamilton T.A. 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. 1992; 89: 8497-8501Crossref PubMed Scopus (334) Google Scholar). 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., 1985Cooper K.D. Fox P. Neises G. Katz S.I. 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. 1985; 134: 129-137PubMed Google Scholar). At the same time that Langerhans cells disappear from the epidermis, infiltration of first the dermis and later the epidermis by IL-10 producing CD11b+ monocyte/macrophages (Kang et al., 1994Kang K. Hammerberg C. Meunier L. Cooper K.D. 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. 1994; 153: 5256-5264PubMed Google Scholar) 1Gilliam 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.)1Gilliam 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.) that are deficient in CD40, B7, and IL-12 2Kremer 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.)2Kremer 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.) 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, 1976Kripke M.L. Fisher M.S. Immunologic parameters for ultraviolet carcinogenesis.J Natl Cancer Inst. 1976; 57: 211-215Crossref PubMed Scopus (265) Google Scholar) and humans (Yoshikawa et al., 1990Yoshikawa T. Rae V. Bruins-slot W. Van den Berg J.-W. Taylor J.R. Streilein J.W. Susceptibility to effects of UVB radiation on induction of contact hypersensitivity as a risk factor for skin cancer in humans.J Invest Dermatol. 1990; 95: 530-536Abstract Full Text PDF PubMed Google Scholar) 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. 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., 1992Cooper K.D. Oberhelman L. Hamilton T.A. 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. 1992; 89: 8497-8501Crossref PubMed Scopus (334) Google Scholar), 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. AntibodiesImmunostaining was performed at least three times on each specimen with the following primary mouse monoclonal antibodies: CD1a (B17.20.9, 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 anti-mouse IgG2a for CD1a followed by Rhodamine Red-x conjugated avidin (Neutr-Avidin, Molecular Probes, Leiden, The Netherlands). Immunohistochemical proceduresSix 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., 1995Chadwick C.A. Potten C.S. Nikaido O. Matsunaga T. Proby C. Young A.R. 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. 1995; 28: 163-170Crossref PubMed Scopus (101) Google Scholar). Microscopy and photographyImages 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+ cellsCD1a+ cells in the upper area of the 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 0.0625 mm2 (Figure 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. AntibodiesFor 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-Blue-streptavidin was purchased from Molecular Probes (Eugene, OR). Flow cytometry protocolKeratome biopsies were obtained 72 h after four MED of UVB as previously described (Meunier et al., 1993Meunier L. Gonzalez-ramos A. Cooper K.D. 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. 1993; 151: 4067-4080PubMed Google Scholar). 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). Human hair follicle is a reservoir for Langerhans cells in skin depleted of epidermal Langerhans cells by UVBWhen human skin is exposed to four MED of UVB, interfollicular epidermal Langerhans cells (Figure 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 (Figure 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 (Figure 2a, arrows). At later time points after UVB (48, 72, 96 h, Figure 2b–d, respectively), CD1a+ cells are absent in interfollicular epidermis and dermis, but appear in and adjacent to follicles. At 96 h (Figure 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, Figure 3a). The immunostaining results presented here are representative of those in all biopsies from all patients.Figure 2UVB-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) 48 h, (c) 72 h, (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.View Large Image Figure ViewerDownload (PPT)Figure 3Langerhans 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.View Large Image Figure ViewerDownload (PPT) Immunostaining of sections containing follicles from the same specimen with CD11b/CD18 (Mac-1) (Figure 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 (Figure 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 source of Langerhans cells migrating to the epidermis (papillary dermis) contains few if any CD1a+ cells at 48, 72, or 96 h after UVB (Figure 2b – dFigure 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, Figure 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 Figure 4 shows, the numbers of Langerhans cells decrease with increasing distance from the hair follicle, with 100% representing control unirradiated skin. It has been shown that UVB only reaches the epidermis and superficial dermis (Bruls et al., 1984Bruls W.A.G. Slaper H. Van Der Leun J.C. Berrens L. Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths.Photochem Photobiol. 1984; 40: 485-494Crossref PubMed Scopus (390) Google Scholar). 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., 1994Qin 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. 1994; 83: 291-298Abstract Full Text PDF PubMed Scopus (24) Google Scholar; Vink et al., 1996Vink A.A. Strickland F.M. Bucana C. Cox P.A. Roza L. Yarosh D.B. Kripke M.L. Localization of DNA damage and its role in altered antigen-presenting cell function in ultraviolet-irradiated mice.J Exp Med. 1996; 183: 1491-1500Crossref PubMed Scopus (127) Google Scholar). 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 Figure 5b 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. Immunostaining with Ki-67, which stains nuclei of cells in cycle (Gerdes et al., 1984Gerdes 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. 1984; 133: 1710-1715PubMed Google Scholar), 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) (Figure 6) show that admixed with the green nuclei of Ki-67+ proliferating keratinocytes are occasional cells with yellow 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. We next asked if the Langerhans cells present in UVB exposed epidermis 72 h after UVB, which are predominantly associated with the hair follicle (Figure 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., 1996Foy T.M. Aruffo A. Bajorath J. Buhlmann J.E. Noelle R.J. Immune regulation by CD40 and its ligand gp39.Annu Rev Immunol. 1996; 14: 591-617Crossref PubMed Scopus (563) Google Scholar; Lenschow et al., 1996Lenschow D.J. Walunas T.L. Bluestone J.A. CD28/B7 system of T cell costimulation.Annu Rev Immunol. 1996; 14: 233-258Crossref PubMed Scopus (2275) Google Scholar). 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, 1985Schuler G. Steinman R.M. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-546Crossref PubMed Scopus (847) Google Scholar). After 1 d of culture, as expected, Langerhans cells from control skin strongly expressed B7–1, B7–2, and CD40 (Figure 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 (Figure 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. 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, 1987Miyauchi S. Hashimoto K. Epidermal Langerhans cells undergo mitosis during the early recovery phase after ultraviolet-B irradiation.J Invest Dermatol. 1987; 88: 703-708Abstract Full Text PDF PubMed Google Scholar); 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., 1979Katz S.I. Tamaki K. Sachs D.H. Epidermal Langerhans cells are derived from cells originating in bone marrow.Nature. 1979; 282: 324-326Crossref PubMed Scopus (725) Google Scholar; Volc-platzer et al., 1984Volc-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. 1984; 310: 1123-1124PubMed Google Scholar), 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., 1993Gimmi C.D. Freeman G.J. Gribben J.G. Gray G. Nadler L.M. Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation.Proc Natl Acad Sci USA. 1993; 90: 6586-6590Crossref PubMed Scopus (536) Google Scholar), 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., 1993Ding L. Linsley P.S. Huang L.-Y. Germain R.N. Shevach E.M. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression.J Immunol. 1993; 151: 1224-1234PubMed Google Scholar; Ozawa et al., 1996Ozawa 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. 1996; 26: 648-652Crossref PubMed Scopus (83) Google Scholar). 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., 1998Murphy G.F. Katz S. Kligman A.M. Topical tretinoin replenishes CD1a-positive epidermal Langerhans cells in chronically photo-damaged human skin.J Cutan Pathol. 1998; 25: 30-34Crossref PubMed Scopus (21) Google Scholar). 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 Foundation, and Christine Buisman Foundation (IBK); NIAID #AI-41766–04 (KDC); Case Western Reserve University Skin Disease Research Center, NIAMS (ACG and SRS), and the Veterans Administration Hospital (KDC).
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