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IL-4Rα expression on airway smooth muscle cells is sufficient for the development of airway hyperresponsiveness., Production of the cytokines IL-4 and IL-13 is increased in both human asthma and mouse asthma models, and Stat6 activation by the common IL-4/IL-13R drives most mouse model pathophysiology, including airway hyperresponsiveness (AHR). However, the precise cellular mechanisms through which IL-4Rα induces AHR remain unclear. Overzealous bronchial smooth muscle constriction is thought to underlie AHR in human asthma, but the smooth muscle contribution to AHR has never been directly assessed. Furthermore, differences in mouse versus human airway anatomy and observations that selective IL-13 stimulation of Stat6 in airway epithelium induces murine AHR raise questions about the importance of direct IL-4R effects on smooth muscle in murine asthma models and the relevance of these models to human asthma. Using transgenic mice in which smooth muscle is the only cell type that expresses or fails to express IL-4Rα, we demonstrate that direct smooth muscle activation by IL-4, IL-13, or allergen is sufficient but not necessary to induce AHR. Five genes known to promote smooth muscle migration, proliferation, and contractility are activated by IL-13 in smooth muscle in vivo. These observations demonstrate that IL-4Rα promotes AHR through multiple mechanisms and provide a model for testing smooth muscle–directed asthma therapeutics.

Recent advances have been made in our understanding of the roles played by cutaneous dendritic cells (DCs) in the induction of contact allergy. A number of associated changes in epidermal Langerhans cell phenotype and function required for effective skin sensitization are providing the foundations for the development of cellular assays (using DC and DC-like cells) for skin sensitization hazard identification. These alternative approaches to the identification and characterization of skin sensitizing chemicals were the focus of a Workshop entitled "Dendritic Cells and Skin Sensitization: Biological Roles and Uses in Hazard Identification" that was given at the annual Society of Toxicology meeting held March 6-9, 2006 in San Diego, California. This paper reports information that was presented during the Workshop.

Cellular changes within resident skin dendritic cells (DCs) after allergen uptake and processing are critical events in the acquisition of skin sensitization. Here we describe the development of a set of selection criteria to derive a list of potential target genes from previous microarray analyses of human peripheral blood-derived (peripheral blood mononuclear cells (PBMCs)-DCs) treated with dinitrobenzene sulfonic acid for predicting skin-sensitizing chemicals. Based on those criteria, a probing evaluation of the target genes has been conducted using an extended chemical data set, comprising five skin irritants and 11 contact allergens. PBMCs-DCs were treated for 24 hours with various concentrations of chemicals and in each instance the expression of up to 60 genes was examined by real-time PCR analysis. Consistent allergen-induced changes in the expression of many genes were observed and further prioritization of the targets was conducted by analysis of the same genes in DCs treated with non-sensitizing chemicals to determine their specificity for skin sensitization. Real-time PCR analyses of multiple chemical allergens, irritants, and non-sensitizers have identified 10 genes that demonstrate reproducibly high levels of selectivity, specificity, and dynamic range consistent with providing the basis for robust and sensitive alternative approaches for the identification of skin-sensitizing chemicals.

Histoplasma capsulatumis a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytoseH. capsulatumyeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC killHistoplasmais via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mφ,Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin toHistoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation ofHistoplasma-infected DC at 18°C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degradeH. capsulatumyeasts. Further, culture ofHistoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasmaactivity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture ofHistoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasmaactivity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used byH. capsulatumyeasts to survive intracellularly within Mø.

The evaluation of the potential to induce allergic contact dermatitis is an important component of the overall safety assessment process of a new chemical entity that may be encountered through the skin. Currently, the murine local lymph node assay (LLNA) is the method of choice for assessing skin sensitization potential. However, despite the success of the LLNA as an alternative test method that results in a reduction in and refinement of animal usage, it does require the continued use of animals. Therefore, the development of an in vitro predictive test method for skin sensitization is still necessary. A number of different approaches have been taken to develop such a method including the examination of changes in cell surface marker expression in cultured human dendritic cells (DC). The purpose of this current study was to examine the effect of allergen exposure on peripheral blood mononuclear cell (PBMC)‐derived DC using a panel of cell surface markers known to be important in the development of aller...

The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water so...

The identification of potential skin sensitizing chemicals is a key step in the overall skin safety risk assessment process. Traditionally, predictive testing has been conducted in guinea pigs. More recently, the murine local lymph node assay (LLNA) has become the preferred test method for assessing skin sensitization potential. However, even with the significant animal welfare benefits provided by the LLNA, there is a need to develop non-animal test methods for skin sensitization. Mechanistic understanding of allergic contact dermatitis has increased substantially in recent years. For example, a number of changes are known to occur in epidermal Langerhans cells, the principal antigen-presenting dendritic cell in the skin, as a result of exposure to chemical allergens, including the internalization of surface major histocompatibility complex (MHC) class II molecules via endocytosis, the induction of tyrosine phosphorylation, the modulation of cell surface markers, and cytokine expression. The application of this knowledge to the design of predictive in vitro alternative tests provides both unique opportunities and challenges. In this review, we have focused specifically on the impact of chemical exposure on dendritic cells and the potential use of that information in the development of cell-based assays for assessing skin sensitization potential of chemicals in vitro.

A critical step in the induction of allergic contact allergy is the activation and subsequent migration of Langerhans cells (LC), an important antigen presenting dendritic cell (DC) of the skin. As the Langerhans cells migrate, they undergo a maturation process. It has been proposed that contact allergen exposure can induce DC maturation. While changes in DC gene expression profiles induced by various maturation stimuli have been explored, there are no published reports describing genomic-scale analysis of the changes induced by chemical allergen exposure. Therefore, to explore the concept of chemical allergen-induced DC maturation and to identify genes that are regulated by exposure to allergens we examined, at the transcriptional level, the effects of exposure to a contact allergen on DC. Peripheral blood-derived DC were exposed for 24 h to either 1mM or 5 mM dinitrobenzenesulfonic acid (DNBS). Changes in gene expression were analyzed using Affymetrix U95Av2 GeneChip. Comparison of mean signal values from replicate cultures revealed 173 genes that were significantly different (P < or = 0.001) between 1 mM DNBS treated and untreated control DC and 1249 significant gene changes between 5 mM DNBS treated and control DC. Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was used to evaluate the observed transcript changes for selected genes in DC derived from a second donor. Comparison of the fold-changes in transcript levels between the two platforms and donors revealed a good correlation in both direction and magnitude. RT-PCR analysis was also used to assess the allergen specificity of a selected number of genes in DC derived from a third donor. Many of the gene expression changes were found to be induced only by exposure to the allergen, DNBS, and not by exposure to a structurally similar non-allergen, benzenesulfonic acid. A number of gene expression changes induced by allergen exposure were found to be consistent with what is known of the DC maturation process, and thus provide support for the theory of contact allergen-induced DC maturation. Additionally, it is hoped that some of the transcript changes identified through this approach will be shown to be suitable for use in the development of an in vitro predictive assay for contact sensitization.

Mice lacking NHE3, the major absorptive Na(+)/H(+) exchanger in the intestine, are the only animal model of congenital diarrhea. To identify molecular changes underlying compensatory mechanisms activated in chronic diarrheas, cDNA microarrays and Northern blot analyses were used to compare global mRNA expression patterns in small intestine of NHE3-deficient and wild-type mice. Among the genes identified were members of the RegIII family of growth factors, which may contribute to the increased absorptive area, and a large number of interferon-gamma-responsive genes. The latter finding is of particular interest, since interferon-gamma has been shown to regulate ion transporter activities in intestinal epithelial cells. Serum interferon-gamma was elevated 5-fold in NHE3-deficient mice; however, there was no evidence of inflammation, and unlike conditions such as inflammatory bowel disease, levels of other cytokines were unchanged. In addition, quantitative PCR analysis showed that up-regulation of interferon-gamma mRNA was localized to the small intestine and did not occur in the colon, spleen, or kidney. These in vivo data suggest that elevated interferon-gamma, produced by gut-associated lymphoid tissue in the small intestine, is part of a homeostatic mechanism that is activated in response to the intestinal absorptive defect in order to regulate the fluidity of the intestinal tract.

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As the induction of cell-mediated immunity to Hc is of critical importance in host defense, we sought to determine whether dendritic cells (DC) could function as a primary APC for this pathogenic fungus. DC obtained by culture of human monocytes in the presence of GM-CSF and IL-4 phagocytosed Hc yeasts in a time-dependent manner. Upon ingestion, the intracellular growth of yeasts within DC was completely inhibited compared with rapid growth within human macrophages. Electron microscopy of DC with ingested Hc revealed that many of the yeasts were degraded as early as 2 h postingestion. In contrast to macrophages, human DC recognized Hc yeasts via the fibronectin receptor, very late Ag-5, and not via CD18 receptors. DC stimulated Hc-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of viable and heat-killed Hc yeasts, but greater proliferation was achieved after ingestion of viable yeasts. These data demonstrate that human DC can phagocytose and degrade a fungal pathogen and subsequently process the appropriate Ags for stimulation of lymphocyte proliferation. In vivo, such interactions between DC and Hc may facilitate the induction of cell-mediated immunity.