Epidermal Receptor Activator of NF-B Ligand Controls Langerhans Cells Numbers and Proliferation 1,2 Jean-Baptiste O. Barbaroux, 3 * Manfred Beleut, † Cathrin Brisken, †‡ Christopher G. Mueller, § and Richard W. Groves* ¶ Langerhans cells (LC) are the dendritic APC population of the epidermis, where they reside for long periods and are self- replicating. The molecular signals underlying these characteristics are unknown. The TNF superfamily member receptor activator of NF-B ligand (RANKL, TNFSF11) has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts. In this study, we have studied expression of the RANKL system in skin and have defined a key role for this molecule in LC homeostasis. In vitro and in vivo, human KC expressed RANKL and epidermal LC expressed cell surface RANK. In vitro, RANKL sustained CD34  progenitor-derived LC viability following 72-h cultures in cytokine-free medium (79.5  1% vs 55.2  5.7% live cells, respectively; n  4; p < 0.05). In vivo, RANKL-deficient mice displayed a marked reduction in epidermal LC density (507.1  77.2 vs 873.6  41.6 LC per mm 2 ; n  9; p < 0.05) and their proliferation was impaired without a detectable effect on apoptosis. These data indicate a key role for the RANKL system in the regulation of LC survival within the skin and suggest a regulatory role for KC in the maintenance of epidermal LC homeostasis. The Journal of Immunology, 2008, 181: 1103–1108. L angerhans cells (LC) 4 represent a subset of dendritic cells (DC) found in the epithelia of skin and mucosae. They express the common DC marker CD1a but can be distin- guished from other DC by the presence of intracellular Birbeck granules, containing the C-type lectin Langerin (CD207) (1), and by their expression of E-cadherin, enabling tight interaction with epithelial cells (2). LC are long-lived in comparison to other DC subtypes, and recent studies have highlighted the ability of LC- precursors to proliferate in situ (3). The molecular signals respon- sible for these characteristics are unclear. Recent data have suggested that the TNF superfamily molecule receptor activator of NF-B (RANK, TNFRSF11a) and its ligand, RANKL (TNFSF11), play a key role in the regulation of several DC functions including enhancement of their capacity to activate T cells (4), increased production of proinflammatory cytokines (5), and pro- longed cell survival (6, 7). Interestingly, it was found that though RANK/RANKL interactions are important factors in the regulation of DC functions, lack of either molecule did not affect the development and distribution of peripheral DC (8, 9). Normal ratios of various splenic DC subpopulations were observed as well as normal lympho- cyte, platelet, granulocyte, and monocyte counts in peripheral blood. This was despite the osteopetrotic phenotype and may be explained by the extramedullary hematopoiesis observed in the liver of RANKL -/- mice (8). In the skin, RANKL overexpression has been shown to promote LC activation and to control regulatory T cell ho- meostasis (10). RANK has been shown to be up-regulated upon DC maturation and RANKL signaling from activated T lymphocytes pro- vides a survival signal to some mature DC subtypes ensuring pro- longed cellular interaction and induction of a sufficient immune re- sponse (11). RANKL activity in vivo is regulated by a decoy receptor, osteoprotegerin (OPG, TNFRSF11b). Signaling through RANK en- gages TRAF-1, -2, -3, -5, and -6 and leads to the activation of several transcription factors such as NF-B, AP-1, ERK1/2, and NFAT. It has been associated with numerous physiological processes including or- ganogenesis of lymph nodes and mammary glands (8, 9, 12), bone homeostasis (13, 14), and regulation of DC function (15, 16). Both RANKL and RANK have recently been shown to be ex- pressed in breast (17) and thymic (18) epithelia and we, therefore, sought to determine their expression and function in human skin epidermis. We hypothesized that RANKL signaling might be of relevance to the extended longevity of LC in the skin and, being tightly intercalated between keratinocytes (KC), that KC may be a good source of such a LC sustaining signal. Materials and Methods Cell preparation Skin biopsies were obtained after informed consent from patients under- going breast reduction surgery or abdominoplasty. Epidermal sheets and epidermal single-cell suspensions were obtained as previously described (19, 20). Cord blood was obtained following informed consent. CD34 + *St. John’s Institute of Dermatology, King’s College London, London, United Kingdom; † Ecole Polytechnique Fe ´de ´rale de Lausanne, Institut Suisse de Recherches Experimen- tales sur le Cancer, Lausanne, Switzerland; ‡ National Center of Competence in Research Molecular Oncology, Lausanne, Switzerland; § Centre National de la Recherche Scienti- fique Laboratory of Therapeutic Immunology and Chemistry, Institut de Biologie Mo- le ´culaire et Cellulaire, Universite ´ Louis Pasteur, Strasbourg, France; and ¶ National Insti- tute for Health Research Biomedical Research Centre at Guy’s and St. Thomas’ National Health Service Foundation Trust and King’s College London, London, United Kingdom Received for publication June 7, 2007. Accepted for publication May 3, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Chelsea and Westminster Hospital Special Trustees and Department of Health via the National Institute for Health Research comprehensive Bio- medical Research Centre award to Guy’s and St. Thomas’ National Health Service Foun- dation Trust in partnership with King’s College London. Financial support was also pro- vided by Institut National de la Sante ´ et de la Recherche Me ´dicale, Centre National de la Recherche Scientifique, and Association pour la Recherche sur le Cancer. 2 J.-B.O.B. designed and performed the research and drafted the manuscript, M.B. performed the research, C.B. provided reagents, C.G.M. designed the research and drafted the manuscript, and R.W.G. designed the research and drafted the manuscript. 3 Address correspondence and reprint requests to Dr. Jean-Baptiste Barbaroux, St. John’s Institute of Dermatology, Guy’s Hospital, London SE1 9RT, U.K. E-mail address: jean-baptiste.barbaroux@kcl.ac.uk 4 Abbreviations used in this paper: LC, Langerhans cell; DC, dendritic cell; KC, keratinocyte; LCL-DC, LC-like DC; OPG, Osteoprotegerin; RANK, receptor activa- tor of NF-B; KO, knockout; PI, propidium iodide; CS, cytokine supplemented; WT, wild type. 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