Katharina Czaja

Rapid uptake of gold nanorods by primary human blood phagocytes and immunomodulatory effects of surface chemistry.

Nanoparticle-based in vivo applications should consider the omnipresence of the phagocytes in the bloodstream and tissue. We have studied the nanoparticle uptake capacities of the most important human primary leukocyte populations using a nanoparticle library encompassing both rod-shaped and spherical gold nanoparticles with diameters between 15 and 50 nm and a variety of surface chemistries. Cetyltrimethylammoniumbromide (CTAB)-stabilized nanoparticles were internalized rapidly within 15 min and in large amounts by macrophages and to a lower extent also by monocytes. Interestingly, we found that the uptake of nanorods by macrophages was more efficient than that of nanospheres. Blocking experiments and electron microscopic studies revealed macropinocytosis as the major uptake mechanism. Grafting of poly(ethylene oxide) (PEO) onto the nanorods was found to significantly delay their internalization for several hours. The long-term uptake of PEO-coated nanoparticles with positively or negatively charged end groups was almost identical. Particle surface chemistry strongly influenced the expression of inflammation-related genes within 1 day. Furthermore, the macrophage phenotype was significantly affected after 7 days of culture with nanorods depending on the surface chemistry. Thus, in vivo application of nanoparticles with certain surface functionalities may lead to inflammation upon particle accumulation. However, our data also suggest that chemical modifications of nanoparticles may be useful for immunomodulation.

IL-31 regulates differentiation and filaggrin expression in human organotypic skin models

BACKGROUND Atopic dermatitis (AD) is an inflammatory skin disease affecting 10% to 20% of children and 1% to 3% of adults in industrialized countries. Enhanced expression of IL-31 is detected in skin samples of patients with AD, but its physiological relevance is not known. OBJECTIVE We sought to determine the role of IL-31 in skin differentiation. METHODS We used human 3-dimensional organotypic skin models with either primary keratinocytes or HaCaT keratinocytes with inducible IL-31 receptor α to evaluate the effect of IL-31. The consequences were studied by using histology, the expression of markers analyzed by immunofluoresence and quantitative RT-PCR, and gene expression arrays. RESULTS We observed that IL-31 interferes with keratinocyte differentiation. Gene expression analysis revealed a limited set of genes deregulated in response to IL-31, including IL20 and IL24. In HaCaT keratinocytes with inducible IL-31 receptor α, IL-31 inhibited proliferation upon induction of IL-31 receptor α by inducing cell cycle arrest. As in primary cells, IL-31-treated HaCaT cells elicited a differentiation defect in organotypic skin models, associated with reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the stratum basale, and poor development of the stratum granulosum. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. The highly induced proinflammatory cytokines IL-20 and IL-24 were responsible for part of the effect on FLG expression and thus for terminal differentiation. CONCLUSION Our study suggests that IL-31 is an important regulator of keratinocyte differentiation and demonstrates a link between the presence of IL-31 in skin, as found in patients with AD, and filaggrin expression.

N-Cyano Sulfoximines: COX Inhibition, Anticancer Activity, Cellular Toxicity, and Mutagenicity

From insects to cancer: N-Cyano sulfoximines were evaluated for COX inhibition and antiproliferative activity against a panel of cancer cell lines. The most active compound exhibited potent COX-2 inhibition, some selectivity for COX-2 over COX-1, only slight cytotoxicity towards healthy cells (HaCaT skin cells), and no mutagenic potential (as determined by an Ames assay).

Dexpanthenol modulates gene expression in skin wound healing in vivo.

Topical application of dexpanthenol is widely used in clinical practice for the improvement of wound healing. Previous in vitro experiments identified a stimulatory effect of pantothenate on migration, proliferation and gene regulation in cultured human dermal fibroblasts. To correlate these in vitro findings with the more complex in vivo situation of wound healing, a clinical trial was performed in which the dexpanthenol-induced gene expression profile in punch biopsies of previously injured and dexpanthenol-treated skin in comparison to placebo-treated skin was analyzed at the molecular level by Affymetrix® GeneChip analysis. Upregulation of IL-6, IL-1β, CYP1B1, CXCL1, CCL18 and KAP 4–2 gene expression and downregulation of psorasin mRNA and protein expression were identified in samples treated topically with dexpanthenol. This in vivo study might provide new insight into the molecular mechanisms responsible for the effect of dexpanthenol in wound healing and shows strong correlations to previous in vitro data using cultured dermal fibroblasts.

High-Resolution Transcriptional Profiling of Chemical-Stimulated Dendritic Cells Identifies Immunogenic Contact Allergens, but Not Prohaptens

Allergic contact dermatitis is a complex syndrome and knowledge about the in vitro detection of small-molecular-weight compounds, particularly prohaptens, is limited. Therefore, we investigated chemical-induced gene expression changes in human antigen-presenting cells upon stimulation with immunogenic contact allergens, prohaptens and irritants. Monocyte-derived dendritic cells (moDCs) and THP-1 cells were stimulated with the prohapten cinnamic alcohol (CAlc), the hapten cinnamic aldehyde (CAld), an irritant and an obligatory sensitizer in vitro. Whole-genome screening and consecutive PCR analysis of differential gene expression in moDCs stimulated with either CAld or the obligatory sensitizer revealed coregulation of 11 marker genes which were related to immunological reactions (IL-8, CD1e, CD200R1, PLA2G5, TNFRSF11A), oxidative or metabolic stress responses (AKR1C3, SLC7A11, GCLM) or other processes (DPYLS3, TFPI, TRIM16). In contrast, the prohapten CAlc and the irritant did not change marker gene expression. In THP-1 cells, CAld and the positive control elicited similar expression changes in only 4 of the previously identified genes (IL-8, TRIM16, CD200R1, GCLM). In conclusion, we provide important insights into the pathophysiological basis of allergic contact dermatitis, identify marker genes suitable for skin hazard assessment and demonstrate that contact-allergenic prohaptens escape in in vitro detection if their skin metabolism is not taken into account.

Control of the Physical and Antimicrobial Skin Barrier by an IL-31–IL-1 Signaling Network

Atopic dermatitis, a chronic inflammatory skin disease with increasing prevalence, is closely associated with skin barrier defects. A cytokine related to disease severity and inhibition of keratinocyte differentiation is IL-31. To identify its molecular targets, IL-31–dependent gene expression was determined in three-dimensional organotypic skin models. IL-31–regulated genes are involved in the formation of an intact physical skin barrier. Many of these genes were poorly induced during differentiation as a consequence of IL-31 treatment, resulting in increased penetrability to allergens and irritants. Furthermore, studies employing cell-sorted skin equivalents in SCID/NOD mice demonstrated enhanced transepidermal water loss following s.c. administration of IL-31. We identified the IL-1 cytokine network as a downstream effector of IL-31 signaling. Anakinra, an IL-1R antagonist, blocked the IL-31 effects on skin differentiation. In addition to the effects on the physical barrier, IL-31 stimulated the expression of antimicrobial peptides, thereby inhibiting bacterial growth on the three-dimensional organotypic skin models. This was evident already at low doses of IL-31, insufficient to interfere with the physical barrier. Together, these findings demonstrate that IL-31 affects keratinocyte differentiation in multiple ways and that the IL-1 cytokine network is a major downstream effector of IL-31 signaling in deregulating the physical skin barrier. Moreover, by interfering with IL-31, a currently evaluated drug target, we will have to consider that low doses of IL-31 promote the antimicrobial barrier, and thus a complete inhibition of IL-31 signaling may be undesirable.

Active transport of contact allergens in human monocyte-derived dendritic cells is mediated by multidrug resistance related proteins

The multidrug resistance related proteins (MRPs) function as efflux transporters of a variety of large organic anions or their conjugates. In recent studies we demonstrated that antigen-presenting cells express a specific pattern of MRPs. MRP-mediated efflux activity of human monocyte-derived dendritic cells (moDCs) was analyzed using an in vitro transport assay. The efflux transport of radiolabeled contact allergens was inhibited using the specific MRP inhibitor indomethacin. Treatment with indomethacin increased intracellular concentration of [³H] eugenol and [³H] isoeugenol in moDCs. In addition by using MRP1 expressing inside-out membrane vesicles we revealed that the transport of eugenol is mediated by MRP1. Human DCs were employed to assess the sensitizing potential of contact allergens and alters their cytokine gene expression profile. Hence, to survey the functionality of indomethacin after stimulation with contact allergens IL-8 and TRIM16 regulation was measured by a DC-based in vitro assay. Incubation with isoeugenol after pre-treatment with indomethacin leads to increased IL-8 and TRIM16 gene expression. These results strongly support the functional role of MRPs in the active efflux of contact allergens also in antigen-presenting cells like moDCs, a novel mechanism which could possibly play a role in the pathogenesis of contact allergy.

Downregulation of STRA6 Expression in Epidermal Keratinocytes Leads to Hyperproliferation-Associated Differentiation in Both In Vitro and In Vivo Skin Models

Retinoids are known to affect skin cell proliferation and differentiation and are key molecules that target retinoid and retinoic acid receptors (RXRs and RARs), leading to physiological and pharmacologic effects. Our aim was to elucidate the role of the retinol-binding protein receptor STRA6, mediating cellular uptake of retinol, on skin structure and function. Our results indicate that STRA6 is constitutively expressed in human epidermal keratinocytes and dermal fibroblasts and is regulated via RAR/RXR-mediated pathways. HaCaT (Human adult low Calcium high Temperature) cells with stable STRA6 knockdown (STRA6KD) showed increased proliferation. Consistently, human organotypic 3D skin models using stable STRA6KD HaCaT cells showed a significantly thicker epidermis and enhanced expression of activation, differentiation, and proliferation markers. The effects were reversible after treatment with free retinol. Human skin reconstitution employing STRA6KD HaCaT cells leads to massive epithelial thickening under in vivo conditions in SCID mice. We propose that STRA6KD could lead to cellular vitamin A deficiency in keratinocytes. Consequently, STRA6 has a role for regulating retinoid homeostasis and in helping to program signaling that drives proliferation and differentiation of human skin cells. By its influence on hyperproliferation-associated differentiation, STRA6 could also have a role in skin regeneration and could be a target for pharmacological approaches to improve wound healing.

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-1β, 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.

Microarray-based detection of specific IgE against recombinant ω-5-gliadin in suspected wheat-dependent exercise-induced anaphylaxis.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a rare IgE-mediated food allergy. Component-resolved measurement of specific IgE (sIgE) against ω-5-gliadin by fluorescence enzyme immunoassay (FEIA) has been postulated as a good predictive decision criterion in the diagnosis of WDEIA. More recently, microarray technology has been introduced into component-resolved diagnostics. The aim of this study was to evaluate the performance of an allergen microarray in the detection of sIgE against ω-5-gliadin in 10 patients with suspected WDEIA and high levels of sIgE against ω-5-gliadin (mean: 9.31±7.53 kU/L, range: 4.24-25.8) as measured by FEIA. Using an old version of the microarray assay (ImmunoCAP ISAC™, Phadia), sIgE against ω-5-gliadin was detected in only 3 of the first 6 patients. The same samples and those of another 4 patients were then analysed with an improved version of the microarray system, yielding elevated levels of sIgE against ω-5-gliadin in all patients. In conclusion, the old version of the microarray was not reliable for the detection of sIgE against ω-5-gliadin in the examined individuals with suspected WDEIA. In contrast, the improved version of the microarray seems to be as adequate as FEIA in the detection of sIgE against ω-5-gliadin. However, further large-scale studies are warranted to confirm these results.