Evaluation of the sensitizing potential of antibiotics in vitro using the human cell lines THP-1 and MUTZ-LC and primary monocyte-derived dendritic cells.

Since the 7th amendment to the EU cosmetics directive foresees a complete ban on animal testing, alternative in vitro methods have been established to evaluate the sensitizing potential of small molecular weight compounds. To find out whether these novel in vitro assays are also capable to predict the sensitizing potential of small molecular weight drugs, model compounds such as beta-lactams and sulfonamides - which are the most frequent cause of adverse drug reactions - were co-incubated with THP-1, MUTZ-LC, or primary monocyte-derived dendritic cells for 48 h and subsequent expression of selected marker genes (IL-8, IL-1ß, CES1, NQO1, GCLM, PIR and TRIM16) was studied by real time PCR. Benzylpenicillin and phenoxymethylpenicillin were recognized as sensitizing compounds because they are capable to induce the mRNA expression of these genes in moDCs and, except for IL-8, in THP-1 cells but not in MUTZ-LC. Ampicillin stimulated the expression of some marker genes in moDCs and THP-1 cells. SMX did not affect the expression of these genes in THP-1, however, in moDCs, at least PIR was enhanced and there was an increase of the release of IL-8. These data reveal that novel in vitro DC based assays might play a role in the evaluation of the allergenic potential of novel drug compounds, but these systems seem to lack the ability to detect the sensitizing potential of prohaptens that require metabolic activation prior to sensitization and moDCs seem to be superior with regard to the sensitivity compared with THP-1 and MUTZ-3 cell lines.

Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on Janus kinase function.

Thymic stromal lymphopoietin (TSLP) is an interleukin-(IL)-7-like cytokine with emerging pathological importance for the development of atopic diseases such as allergic asthma bronchiale. The TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor, shares the IL-7R alpha-subunit with the IL-7 receptor system. The specific TSLPR alpha-chain shows similarities with the gammac receptor chain, but has some unusual features within the receptor family in both its ligand-binding and cytoplasmic domain. The murine TSLPR signals via the signal transducers and activators of transcription STAT5 and STAT3, but is unique among cytokine receptors in that it activates STATs without the involvement of Janus (JAK) tyrosine kinases, but instead utilizes the Src type kinase Tec. Here, we show by Western blotting and reporter gene experiments in combination with the application of a specific JAK inhibitor that the human TSLP receptor, in contrast, requires the function of JAK1 and JAK2 for STAT activation. Moreover, we demonstrate that the human TSLPR mediates gene regulation not only through STAT5 and STAT3 but has also the potential to mediate transcription via STAT1. Our work should help to understand more thoroughly how TSLP triggers inflammatory responses in the course of atopic diseases.

Signal transduction around thymic stromal lymphopoietin (TSLP) in atopic asthma.

Thymic stromal lymphopoietin (TSLP), a novel interleukin-7-like cytokine, triggers dendritic cell-mediated inflammatory responses ultimately executed by T helper cells of the Th2 subtype. TSLP emerged as a central player in the development of allergic symptoms, especially in the airways, and is a prime regulatory cytokine at the interface of virus- or antigen-exposed epithelial cells and dendritic cells (DCs). DCs activated by epithelium-derived TSLP can promote naïve CD4+ T cells to adopt a Th2 phenotype, which in turn recruite eosinophilic and basophilic granulocytes as well as mast cells into the airway mucosa. These different cells secrete inflammatory cytokines and chemokines operative in inducing an allergic inflammation and atopic asthma. TSLP is, thus, involved in the control of both an innate and an adaptive immune response. Since TSLP links contact of allergen with the airway epithelium to the onset and maintainance of the asthmatic syndrome, defining the signal transduction underlying TSLP expression and function is of profound interest for a better understandimg of the disease and for the development of new therapeutics.

Characterization of SLCO5A1/OATP5A1, a solute carrier transport protein with non-classical function.

Organic anion transporting polypeptides (OATP/SLCO) have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold) and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20) and genes implicated in developmental processes (e.g. TGM2). A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F) revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.