SummaryWe have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR. and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels ofbioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 • 106 DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison oflL-12 production, and blocking experiments employing an anti-CD40 ligand n'LAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Thl type cell-mediated immune responses. IL-12, a 70-kD heterodimeric cytokine composed of co-.valentty linked p35 and p40 chains has emerged as a central cytokine in the immune response (1). IL-12 stimulates NK cells, mediates Thl development, and fosters CTL development. It can be produced by monocytes and macrophages in response to intracellular pathogens, bacteria (e.g., staphylococci) and bacterial products. Recent reports indicate that dendritic cells (DC) also release bioactive IL-12. One report described that anti-IL-12 blocks the capacity of murine DC to skew the response of naive transgenic T cells to the Thl phenotype (2), and another shows induction of IL-12 p40/p35 mRNA in bone-marrow derived murine DC upon uptake ofmicroparticle-absorbed protein antigen (3). Human epidermal Langerhans cells are also a source of IL-12 (4). We have recently used several criteria for demonstration of IL-12 p40 and p35 mRNA as well as IL-12 p40 and bioactive p70 proteins, to show that murine and human DC release IL-12 upon conventional stimuli such as staphylococcus aureus (5). We also found that DC produced bioactive IL-12 upon interaction with T cells without standard stir " ~-,~h as bacterial products. Here, we describe the regulation oflL-12 in DC.

Migration from sites of antigen encounter to lymphoid organs is essential to the strong immunogenic function of dendritic cells (DC). In the skin, migration proceeds through dermal lymphatic vessels and is regulated in an incompletely understood way by inflammatory mediators. We studied the effects of tumor necrosis factor ␣ (TNF-␣) and interleukin-1␤ (IL-1␤) in mouse skin organ cultures by direct enumeration of migrating DC and by immunohistochemistry.

Little is known about the immigration of bone marrow-derived progenitors of Langerhans cells (LC) into the epidermis. We developed an in vivo system based on the tape stripping method that allowed us to study the immigration of LC into the epidermis after intradermal injection of bone marrow-derived dendritic cells (DC). Tape stripping induced a mechanical disruption of the epidermal barrier that led to skin inflammation and subsequent emigration of LC and dermal DC from the skin. Emigrating LC and dermal DC were observed in lymphatic vessels, and the numbers of LC and dermal DC in the draining lymph node increased. Up to 500 times more injected precursors migrated into tape-stripped epidermis as compared with unstripped epidermis. Newly immigrated cells were slender with one or two dendrites and acquired a more dendritic morphology after 2-4 days. They were both MHC II-positive and negative and they did not express Langerin/CD207, nor macrophage-mannose receptor/CD206 and Fc-epsilon receptor I. In contrast, all cells that had entered the epidermis expressed CD11c and CCR6, suggesting that they were LC. We conclude that this experimental system may serve as a valuable tool for the further characterization of LC-precursors and the conditions necessary for LC-immigration into the epidermis.

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