816 NATURE M EDICINE • VO LUM E 6 • N UM BER 7 • JULY 2 0 0 0 ARTICLES Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine 1,2 . M acrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes , we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-de- rived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/ interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukoctye antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60–80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-posi- tive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin. Although several groups of investigators have postulated that dengue virus (DV) replication occurs in human skin, the initial target cell has not been identified 3,4 . To determine if human DCs were permissive for growth of DV, we compared the ability of human DCs, macrophages, monocytes and T- and B-cell lym- phocytes to support DV growth. We monitored the expression of DV antigens using immunofluorescence, cytofluorometry and immunohistochemistry of cytospins with monoclonal antibod- ies specific for the DV envelope glycoprotein (antibodies 3H5, 2H2) and nonstructural protein, which is produced only during productive DV infection (antibody 7E11). After exposure of im- mature blood-derived DCs to the prototype New Guinea C (NGC) strain of DV, serotype 2, at a multiplicity of infection (MOI) of 0.2, 25–40% of the cells were infected (Figs. 1 c and d and 2); with a MOI of 2.0, the infection rate increased to 50–60% (data not shown). In contrast, exposure of macrophages from the same blood donor to DV(NGC) at an MOI of 0.2 resulted in in- fection of 1–2% of cells (Fig. 1 a and b). At an MOI of 2.0, a maxi- mum of 5% of macrophages were infected with DV(NGC) (data not shown). In similar experiments using CD14 + blood mono- cytes, B- and T-cell lymphocytes and unfractionated peripheral blood mononuclear cells (PBMCs), we detected dengue antigen in less than 1% of cells (data not shown). Expression of DV(NGC) in DCs was also demonstrated by flow immunocyto- chemistry (Figs. 1 g and h ) cytometry; DV envelope glycoprotein and nonstructural protein 1 could be detected by intracellular stain in g (Figs. 2 a and c and 3 b). To demonstrate infection of DCs with DV other than the NGC prototype strain, we also tested three primary patient isolates (serotypes 1, 2 and 4). DCs were similarly permissive for infection by primary DV strains (Fig. 2 c). By both immunofluorescence (not shown) and flow cytometry (Fig. 2 a), we found immature but not mature DCs to be permis- sive to infection with DV(NGC). Others have also found that im- mature DCs are more susceptible to other viral infections than mature DCs (ref. 5). Although expression of nonstructural protein 1 (detected by antibody 7E11) was indicative of active DV replication, we com- pleted several experiments to confirm this observation. Using immunofluorescence, we measured DV antigen expression in DCs at 2, 6, 12, 24 and 48 hours after the cells were pulsed with DV(NGC). We first found DV antigens (envelope glycoprotein and nonstructural protein 1) at 12 hours; peak expression occurred between 24 and 48 hours (data not shown). Human skin Langerhans cells are targets of dengue virus infection SHUENN-JUE L. W U 1,2 , G ERALDINE G ROUARD-VOGEL 3 , W ELLINGTON SUN 4 , JOHN R. MASC O LA 3 , ELEN A BRACHTEL 3,5 , RAVITHAT PUTVATANA 1 , MARK K. LOUDER 3 , LUIS FILGUEIRA 6 , MARY A. MAROVICH 3 , H EN RY K. W ONG 8 , ANDREW BLAUVELT 7 , G ERALD S. MURPHY 1,2 , MERLIN L. RO BB 3 , BRUCE L. INNES 4 , DEBO RAH L. BIRX 3 , C URTIS G. H AYES 1 & SARAH SCHLESINGER FRAN KEL 3,5 1 Viral and Rickettsial Diseases Department, Naval Medical Research Center, Bethesda, Maryland 20889-5607, USA 2 Uniformed Service University of Health Sciences and Departments of Preventative Medicine and Biometrics, Department of Medicine, Bethesda, Maryland 20889, USA 3 Division of Retrovirology, W alter Reed Army Institute of Research and the Henry M. Jackson Foundation, 13 Taft Court, Suite 200, Rockville, Maryland 20850, USA 4 Division of Viral Diseases, W alter Reed Army Institute of Research, Silver Spring, Maryland 20889-5607, USA 5 Department of Infectious and Parasitic Disease Pathology, Armed Forces Institute of Pathology, W ashington DC 20306-6000, USA 6 Institute of Anatomy, University of Zurich-Irchel, Zurich, Switzerland 7 Dermatology Branch, National Cancer Institute, Bethesda, Maryland 20892, USA 8 Department of Dermatology W alter Reed Army Medical Center, Silver Spring, Maryland 20889-5607, USA 9 B.L.I. present address: SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426-0989, USA S.-J.W . and G.G.-V. contributed equally to this study. Correspondence should be addressed to S.S.F.; email: sfrankel@hiv.hjf.org © 2000 Nature America Inc. • http://medicine.nature.com © 2000 Nature America Inc. • http://medicine.nature.com