Interleukin-10 inhibits human lymphocyte IFN- production by suppressing natural killer cell stimulatory factor/interleukin 12 synthesis in accessory cells. DCs, and dengue computer virus contamination induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue computer virus in natural contamination and the vigor of CMI is usually modulated by the relative presence or absence of IFN- in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies. Dendritic cells (DCs) are bone marrow-derived cells that form a system of professional antigen-presenting cells and are an important component of the innate immune response. They are comprised of at least three unique subpopulations, one in the lymphoid/plasmacytoid lineage and two in the myeloid lineage (1, 20, 26). Myeloid DCs are found in most nonlymphoid organs including the epidermis (Langerhans cells), dermis, gastrointestinal and respiratory mucosa, and the Zinc Protoporphyrin interstitia of vascular organs (37). Following the uptake and processing of antigen in the periphery, immature myeloid DCs differentiate to an activated/mature state and migrate to the T-cell-rich areas of lymphoid organs. Activated DCs are the unique stimulators of main T-cell responses and potent stimulators of memory responses, and they produce an array of cytokines and chemokines (26, 44, 50, 55). Thus, DCs are crucial in the initiation of antimicrobial immunity, and they provide a crucial step in the development of adaptive immunity. Dengue is an emerging arboviral disease where the adaptive immune response plays a significant role in determining the severity of clinical illness. The dengue viruses are a group of four antigenically related mosquito-borne flaviviruses that produce a spectrum of clinical illness and significant morbidity throughout the tropics (30, 35). Dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) represent the most severe and potentially life-threatening manifestations of a dengue viral contamination. DHF/DSS is usually characterized by the rapid onset of plasma leakage and coagulopathy near the time of defervescence and viremia resolution. The most significant risk factor for the development of DHF/DSS is usually acquisition of a second, heterotypic dengue computer virus contamination (3, 11, Zinc Protoporphyrin 13). During this second dengue computer virus infection, it is postulated that this preexisting, cross-reactive, adaptive immune response prospects to excessive cytokine production, match activation, and the release of other phlogistic factors which produce DHF/DSS. Both the humoral and cellular components of adaptive immunity have been implicated in this process (12, 40). The principal target of dengue computer virus infection has been presumed to be blood monocytes and tissue macrophages (14, 46). However, myeloid DCs residing in the epidermis (Langerhans cells) and dermis are the predominant cells of the innate immune system that dengue computer virus encounters following the bite of an infected mosquito. A recent study exhibited the permissiveness of immature myeloid DCs to dengue computer virus infection but did not address the effect of viral contamination around the DCs (53). In this study, we further investigated the conversation between dengue computer virus and myeloid DCs. Immature myeloid DCs were generated from plastic-adherent peripheral blood mononuclear cells (PBMC) and were considered representative of myeloid interstitial DCs (1). Viral replication, DC maturation and activation, and cytokine production were examined in the hope of understanding the factors that guide formation of antiviral adaptive immunity, and, under certain conditions, increase the risk of developing severe disease. MATERIALS AND METHODS Generation of DCs. Immature myeloid DCs were generated from PBMC using previously explained techniques (38, 39, 44). Peripheral blood was collected in heparinized tubes from Zinc Protoporphyrin healthy adult volunteers. PBMC were isolated on Histopaque gradients (Sigma Chemical Co., St. Louis, Mo.), washed two times with RPMI 1640 medium (Gibco BRL, Gaithersburg, Md.), and incubated with neuraminidase-treated sheep reddish blood cells for 1 h on ice. Erythrocyte rosette-negative cells were collected and isolated using Histopaque gradient centrifugation. The T-cell-depleted, erythrocyte rosette-negative cells were cultured (3 106 cells/well) for 1 h Zinc Protoporphyrin in 24-well plates at 37C in a CO2 incubator with RPMI 1640 and 10% heat-inactivated fetal calf serum (FCS; Gibco BRL). Nonadherent cells were removed, and medium was replaced with the addition of human recombinant interleukin-4 (rIL-4; Rabbit Polyclonal to TFE3 500 U/ml; Endogen Inc., Woburn, Zinc Protoporphyrin Mass.) and human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF; 800 U/ml; Endogen). New medium and cytokines (rIL-4 plus rGM-CSF) were replaced every 2 to 3 3 days. After 7 days, the loosely adherent DCs.