Classification of chemotherapeutic agents based on their differential in vitro effects on dendritic cells.

Despite the crucial roles dendritic cells (DC) play in host immunity against cancer, the pharmacologic effects of many chemotherapeutic agents have remained mostly unknown. We recently developed a DC biosensor clone by engineering the stable murine DC line XS106 to express the yellow fluorescent protein (YFP) gene under the control of interleukin (IL)-1beta promoter. In this study, the resulting XS106 pIL1-YFP DC clone was used to screen 54 anticancer drugs. Each drug was tested at five concentrations (0.1-10 micromol/L) for its effects on YFP expression, cell viability, and granulocyte macrophage colony-stimulating factor-dependent growth. Our unbiased systematic screening unveiled a striking heterogeneity among the tested anticancer drugs in their effects on the three functional variables. Interestingly, 15 drugs induced significant YFP expression at subcytotoxic concentrations and were thus categorized as "DC-stimulatory" anticancer drugs. These drugs were subsequently found to induce at least one of the characteristic maturational changes in mouse bone marrow-derived DCs. For example, vinblastine, a prototypic drug of this class, induced the production of IL-1beta, IL-6, and IL-12, increased surface expression of CD40, CD80, CD86, and MHC class II, and augmented T cell-stimulatory capacity of DCs. Not only do these results illustrate the differential pharmacologic effects of commonly used chemotherapeutic agents on DCs, they may also provide a conceptual framework for rationale-based selection and combination of anticancer drugs for clinical application.

Identification of novel pharmacological activities of an antifungal agent, nystatin, to promote dendritic cell maturation.

As an unbiased functional screen to identify agents activating dendritic cells (DCs), we recently developed a DC-based biosensor system, in which a stable murine DC line XS106 was engineered to express the yellow fluorescent protein (YFP) gene under the control of the IL-1beta promoter. Here we report that nystatin (NYT), an antifungal drug of the family of polyene macrolide antibiotics, elevated YFP expression by the resulting XS106-pIL1-YFP DC biosensor clone in a dose-dependent fashion. With respect to the underlying mechanisms, NYT activated the NFkappaB p65 and c-Rel subunits in the parental XS106 DC line. Moreover, NYT dose-dependently increased the surface expression of major histocompatibility complex (MHC) class II (MHC II), CD40, CD54, CD80, and CD86 by murine bone marrow-derived DCs and triggered their robust production of IL-1beta, IL-6, IL-12, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha. Our results document previously unrecognized pharmacological activities of the most commonly used antifungal drug to promote DC maturation.

Discovery of novel immunostimulants by dendritic-cell-based functional screening.

Immunostimulants represent an emerging class of drugs for the treatment of infectious disorders and cancer. CpG oligonucleotides and imiquimod, prototypic drugs in this category, are now known to activate dendritic cells (DCs). Here we report the development of a highly sensitive, unbiased functional screen to detect DC-stimulatory signals. Because interleukin-1beta (IL-1beta) mRNA expression is closely associated with DC activation, we engineered DCs to stably express a fluorescent marker gene under the control of IL-1beta promoter. By screening about 3000 compounds with the resulting DC biosensor clone, we identified DC-stimulatory potentials of topoisomerase I inhibitors (camptothecin derivatives) and microtubule depolymerizing drugs (colchicine and podophyllotoxin). In response to treatment with each agent, bone marrow-derived DC preparations exhibited characteristic phenotypic and/or functional changes associated with DC activation. All of these agents also triggered nuclear factor-kappaB (NFkappaB) activation in DCs, suggesting a common pharmacologic mechanism of action. Furthermore, locally administered colchicine induced in situ maturation and migration of DCs and augmented both humoral and cellular immune responses. These results support the practical utility of the DC-based biosensor system to discover novel DC-targeted immunostimulants and unveil previously unrecognized (and totally unexpected) pharmacologic activities of several drugs that are commonly used for the treatment of various disorders.

Keratinocyte ATP release assay for testing skin-irritating potentials of structurally diverse chemicals.

Irritant dermatitis represents innate inflammatory responses to toxic chemicals. We have reported recently that ATP released from chemically injured keratinocytes may serve as a causative mediator for irritant dermatitis. In this study, we examined whether ATP release from keratinocytes would serve as a reliable readout for predicting skin irritating potentials of structurally diverse compounds. A vast majority (19/20) of the tested compounds, i.e., strong and weak irritant chemicals selected from the literature, induced rapid (<10 min) and significant (P<0.05) ATP release from Pam 212 keratinocytes. Two compounds caused no detectable skin inflammation in our standard mouse model, documenting relatively high sensitivity (false negative rate of 0/18) and specificity (false positive rate of 1/20) of our ATP release assay. Selected compounds, primarily those containing phenol residues or hydrophobic hydrocarbon chains, triggered rapid (<10 min) and robust leakage of a fluorescence probe from liposomes, suggesting that lipid bilayers serve as one, but not the only, target moiety on keratinocytes. Not only do our data support the pathogenic role for keratinocyte-derived ATP in irritant dermatitis, they also form the basis for a formal validation study to evaluate the utility of the keratinocyte-based in vitro assay in screening environmental and industrial chemicals.

CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness.

CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.