AhR mediates an anti-inflammatory feedback mechanism in human Langerhans cells involving FcεRI and IDO.

BACKGROUND: Aryl hydrocarbon receptor (AhR), an important regulator of immune responses, is activated by UVB irradiation in the skin. Langerhans cells (LC) in the epidermis of patients with atopic dermatitis (AD) carry the high-affinity receptor for IgE, FcεRI, and are crucially involved in the pathogenesis of AD by inducing inflammatory responses and regulating tolerogenic processes. OBJECTIVES: We investigated AhR and AhR repressor (AhRR) expression and functional consequences of AhR activation in human ex vivo skin cells and in in vitro-generated LC. METHODS: Epidermal cells from healthy skin were analyzed for their expression of AhR and AhRR. LC generated from CD34+ hematopoietic stem cells (CD34LC) were treated with the UV photoproduct and AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ). Cell surface receptors, transcription factors, and the tolerogenic tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) were analyzed using flow cytometry and quantitative PCR. RESULTS: Epidermal LC and CD34LC express AhR and AhRR. AhR was also found in keratinocytes, which lack AhRR. AhR activation of LC by FICZ caused downregulation of FcεRI in CD34LC without affecting their maturation. AhR-mediated regulation of FcεRI did not involve any known transcription factors related to this receptor. Furthermore, we could show upregulation of IDO mediated by AhR engagement. CONCLUSIONS: Our study shows that AhR activation by FICZ reduces FcεRI and upregulates IDO expression in LC. This AhR-mediated anti-inflammatory feedback mechanism may dampen the allergen-induced inflammation in AD.

Among the S100 proteins, S100A12 is the most significant marker for psoriasis disease activity.

BACKGROUND: Psoriasis is a chronic skin disease with deregulation of proteins in the immune system. These proteins include members of the heterogeneous S100 family, which have been discussed as potential biomarkers for disease severity. OBJECTIVE: The aim of this study was to evaluate the impact of S100A7, S100A8, S100A9 and S100A12 as possible markers for disease activity in patients with psoriasis skin disease. PATIENTS AND METHODS: S100A7, S100A8, S100A9 and S100A12 mRNA expression was determined in the skin of patients with psoriasis and controls (N = 341) by gene expression analyses. In addition, S100 serum levels were investigated by ELISA in an independent cohort of psoriasis patients (i) untreated, with different manifestations (skin/joints), (ii) under treatment (etanercept) and (iii) healthy controls, (N = 55). RESULTS: All S100-subtypes included are significantly upregulated in psoriasis skin lesions when compared with atopic dermatitis, lichen ruber and healthy donors. In untreated psoriasis patients, S100A12-serum levels showed the closest association with disease activity (PASI) (r = 0.542; P < 0.01). Serum levels decreased under treatment with etanercept (P < 0.05). CONCLUSION: Among the investigated S100-proteins, S100A12 showed the closest association with disease activity and therapeutic response and might therefore provide a valuable biomarker for psoriasis.

Generation and functional analysis of human TNF-α/iNOS-producing dendritic cells (Tip-DC).

BACKGROUND: A unique type of CD11c(pos) dendritic cells (DC) is abundant in inflamed tissue, for example, in chronic inflammatory skin diseases. Due to their remarkable production of tumor necrosis factor (TNF)-α and inducible NO synthase (iNOS), these cells have been referred to as TNF and iNOS-producing DC (Tip-DC). While Tip-DC have been mainly characterized in murine models of infection, functional data about their human counterpart are lacking. OBJECTIVES: We sought to generate human Tip-DC in vitro und thus provide a new model for the investigation of their phenotype and function. METHODS: We generated human Tip-DC from monocytic precursor cells of healthy individuals, atopic and psoriatic patients using human serum. Resting and stimulated cells were analyzed by flow cytometry, real-time PCR, and by ELISA. INOS activity was measured by fluorometric detection of NO. RESULTS: Tip-DC closely resembled their in vivo counterparts by expressing CD11c, CD86, and CD40 while lacking CD1a, CD1c, or CD207/Langerin. Bacterial stimulation of Tip-DC from healthy donors, atopic dermatitis, or psoriasis patients resulted in a similar increase in iNOS activity and TNF-α production. In kinetic experiments, TNF-α, a putative activator of Tip-DC, could not induce NOS2. Upon bacterial stimulation, TNFA, IL6, IL12B, and IL23A mRNA appeared in a first wave, while IL12A and NOS2 mRNA were up-regulated later on but not blocked by anti-TNF-α agents, implying a biphasic pro-inflammatory response. CONCLUSIONS: We developed a new model for the study of human Tip-DC and provide the first evidence of their pro-inflammatory capacity.

TLR2 down-regulates FcεRI and its transcription factor PU.1 in human Langerhans cells.

BACKGROUND: Epidermal Langerhans cells (LC) expressing the high-affinity receptor for IgE (FcεRI) play a key role in atopic dermatitis (AD). AD skin is highly colonized with Staphylococcus aureus (S.a.), which are sensed by Toll-like receptor 2 (TLR2). We hypothesized that TLR2 may impact on the expression of FcεRI on LC. OBJECTIVES: To study a putative impact of TLR2 signaling on FcεRI, we analyzed FcεRI and known transcription factors of the receptor after ligand binding to TLR2. METHODS: We generated LC from CD34(+) progenitors in vitro (CD34LC) expressing FcεRI and TLR2 as well as its partners TLR1 and TLR6. The expression of FcεRI and known transcription factors of the receptor was analyzed on the protein and RNA level by flow cytometry, Western blotting, and real-time PCR. RESULTS: For CD34LC from 123 donors, we observed a high heterogeneity in FcεRI surface expression correlating with mRNA level of its α-chain. Stimulation of TLR1/2 or TLR2/6 dramatically down-regulated FcεRI on protein and mRNA level of both α- and γ-chain. Further analysis of putative transcription factors for FCER1A revealed the lack of GATA1 in CD34LC, weak expression of ELF1 and YY1, and high expression of PU.1. While ELF1 and YY1 appeared to be little affected by TLR2 engagement, PU.1 was significantly down-regulated. CONCLUSIONS: Taken together, our findings show that in human, LC ligation of TLR2 by S.a.-derived products down-regulates FcεRI and its transcription factor PU.1, thus suggesting that FcεRI is controlled by PU.1 in these cells.

The analysis of tyrosinase-specific mRNA in blood samples of melanoma patients by RT-PCR is not a useful test for metastatic tumor progression.

Reverse transcription (RT) of the tyrosinase mRNA and specific cDNA amplification by nested polymerase chain reactin (PCR) have been reported to facilitate the early detection of circulating tumor cells in melanoma patients. The significance and practical value of this procedure for the diagnosis of tumor dissemination in melanoma patients are unclear. In the current study we analyzed peripheral blood samples of 65 melanoma patients of different clinical stages for the presence of tyrosinase mRNA by RT-PCR using nested oligonucleotide primers specific for tyrosinase cDNA. Furthermore, blood samples were evaluated for tumor cell growth by cell culture assays in vitro. No tyrosinase mRNA was detectable in blood samples of 26 patients with primary melanoma and 16 patients with regional lymph node metastases. In five of 13 patients with visceral metastases we found at least one blood sample positive for tyrosinase mRNA during a 2-to 4-mo interval. Analyses of different blood samples of patients with visceral metastases taken in a 2-h interval, furthermore, indicate that tumor cells only transiently persist in the peripheral blood. We obtained in vitro proliferating melanoma cells from two blood samples derived from different patients with visceral melanoma metastases. This demonstrates that viable melanoma cells indeed circulate in the peripheral blood with retained proliferative capacity in vitro. The analysis of blood samples by RT-PCR for tyrosinase mRNA, however, is not suitable for the early detection of tumor progression in melanoma patients.

The CD7- T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA- T cells.

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7- T cells express the CD4CD45RO+CD45RA- memory phenotype. We analysed CD4+CD45RA- peripheral blood lymphocytes that were separated into CD7+ and CD7- for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7- subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7- subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-gamma) secretion was not significantly different in CD7+ and CD7- T cells upon stimulation in vitro. The data indicate that the CD4+CD7- T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA- memory T cells. Since CD4+CD7- T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7- T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.