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 crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells.

By screening 720 natural compounds in a standard 2-way allogeneic mixed leukocyte reaction assay, we identified a potent immunosuppressive capacity of crassin acetate (CRA), a coral-derived cembrane diterpenoid. CRA efficiently inhibited allogeneic mixed leukocyte reaction as well as antigen-specific activation of CD4 T cells by bone marrow-derived dendritic cells (DCs). With regard to cellular targets, CRA suppressed not only mitogen-triggered T-cell activation, but also lipopolysaccharide-induced DC maturation, indicating dual functionality. Treatment with CRA at nontoxic doses induced heme oxygenase-1 (HO-1) mRNA/protein expression and HO-1 enzymatic activity in DCs, suggesting a unique mechanism of action. In fact, lipopolysaccharide-induced DC maturation was also inhibited by structurally unrelated compounds known to induce HO-1 expression or carbon monoxide (CO) release. Allergic contact hypersensitivity response to oxazolone and oxazolone-induced Langerhans cell migration from epidermis were both prevented almost completely by systemic administration of CRA. Not only do our results support the recent concept that HO-1/CO system negatively regulates immune responses, they also form both conceptual and technical frameworks for a more systematic, large-scale drug discovery effort to identify HO-1/CO-targeted immunosuppressants with dual target specificity.

Development of intravital intermittent confocal imaging system for studying Langerhans cell turnover.

Although several studies have suggested relatively slow turnover of Langerhans cells (LCs), their actual lifespan remains elusive. Here we report the development of a new intravital imaging system for studying LC efflux and influx. Epidermal LCs expressing enhanced green fluorescent protein (EGFP) were visualized in anesthetized I-Abeta-EGFP knock-in mice by confocal microscopy. By overlaying two sets of EGFP+ LC images recorded in the same microscopic fields at time 0 and 24 hours later, we identified LC subpopulations that had disappeared from or newly emerged in the epidermis during that period. Of >10,000 LCs analyzed in this manner, an overwhelming majority (97.8+/-0.2%) of LCs showed no significant changes in the x-y locations, whereas 1.3+/-0.1% of the LCs that were found at time 0 became undetectable 24 hours later, representing LC efflux. Conversely, 0.9+/-0.1% of the LCs that were found at time 24 hours were not detectable at time 0, representing LC influx. From these frequencies, we estimated the half-life of epidermal LCs to range from 53 to 78 days, providing new insights into the immunobiology of LCs. Our intermittent imaging approach may be regarded as a technical breakthrough enabling direct visual assessment of LC turnover in living animals.

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.

Differential activation profiles of multiple transcription factors during dendritic cell maturation.

Immature dendritic cells (DC) at the environmental interfaces, such as the skin, constantly survey the tissue for the emergence of microbial products and pro-inflammatory mediators. Upon recognition of such "danger" signals, they undergo dynamic reprogramming of gene expression and functions, the process known as DC maturation, which plays critical roles in both innate and adaptive immune responses. Although DC have been shown to discriminate different maturation stimuli by expressing stimulus-specific signature genes and unique phenotypic and functional properties, underlying mechanisms for this extraordinary plasticity remain relatively unclear. We hypothesized that DC might activate unique sets of transcription factors (TF) upon sensing different stimuli. To test this hypothesis, we transduced a mouse epidermal-derived DC line XS106 to express the luciferase reporter gene under the control of each of 15 different cis-enhancer elements. The resulting DC panels were then exposed to 14 different microbial, endogenous, environmental, and pharmacological agents that produced unique maturational changes. This approach allowed systematic determination of TF activation profiles in DC. Our results revealed striking diversity, with different classes of stimuli triggering preferential activation of distinct sets of TF. We propose that differential TF usage represents a previously unrecognized mechanism regulating the direction of DC maturation.

CD1a and langerin: acting as more than Langerhans cell markers.

Langerhans cells (LCs) represent a unique DC subset populating the outermost body surface, i.e., the epidermis. Although CD1a and langerin (CD207) are used as specific markers to distinguish LCs from other DC subsets, their immunological functions have remained mostly unknown. A new paper (see the related article beginning on page 701) demonstrates that LCs utilize these markers to induce cellular immune responses to Mycobacterium leprae: CD1a mediates the presentation of nonpeptide antigens to T cells, while langerin facilitates uptake of microbial fragments and perhaps their delivery to a specialized subcellular compartment.

Generation and function of reactive oxygen species in dendritic cells during antigen presentation.

Although reactive oxygen species (ROS) have long been considered to play pathogenic roles in various disorders, this classic view is now being challenged by the recent discovery of their physiological roles in cellular signaling. To determine the immunological consequence of pharmacological disruption of endogenous redox regulation, we used a selenium-containing antioxidant compound ebselen known to modulate both thioredoxin and glutaredoxin pathways. Ebselen at 5-20 micro M inhibited Con A-induced proliferation and cytokine production by the HDK-1 T cell line as well as the LPS-triggered cytokine production by XS52 dendritic cell (DC) line. Working with the in vitro-reconstituted Ag presentation system composed of bone marrow-derived DC, CD4(+) T cells purified from DO11.10 TCR-transgenic mice and OVA peptide (serving as Ag), we observed that 1) both T cells and DC elevate intracellular oxidation states upon Ag-specific interaction; 2) ebselen significantly inhibits ROS production in both populations; and 3) ebselen at 5-20 micro M inhibits DC-induced proliferation and cytokine production by T cells as well as T cell-induced cytokine production by DC. Thus, Ag-specific, bidirectional DC-T cell communication can be blocked by interfering with the redox regulation pathways. Allergic contact hypersensitivity responses in BALB/c mice to oxazolone, but not irritant contact hypersensitivity responses to croton oil, were suppressed significantly by postchallenge treatment with oral administrations of ebselen (100 mg/kg per day). These results provide both conceptual and technical frameworks for studying ROS-dependent regulation of DC-T cell communication during Ag presentation and for testing the potential utility of antioxidants for the treatment of immunological disease.

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.