Dermal accumulation of Langerhans cells in lichen planus is associated to abundant production of Activin A.

<p>Sections from normal skin (NS) (<i>a</i> and <i>d</i>) and lichen planus (LP) (<i>b, c, e, f</i>) biopsies were stained for Langerin (<i>a</i>–<i>c</i>) and Activin A (<i>d–f</i>). In normal skin, Langerin⁺ cells are regularly distributed in basal and suprabasal layers and show multiple fine dendrites; no positive cells are detectable in the dermis (panel <i>a</i>). In LP biopsies, in addition to intraepidermal LC, accumulation of Langerin⁺ cells is observed in the dermis within the dense monuclear cell infiltrate (panel <i>b</i>). At high power view, Langerin⁺ cells show an ovoidal/dendritic shape (panel <i>c</i>) and are found surrounding Factor VIII⁺ dermal blood vessels (arrow head, inset in <i>c</i>). Serial sections from the same tissue blocks were stained for Activin A. Normal skin (panel <i>d</i>) showed weak intraepithelial reactivity (red arrow head); in the dermis, mast cells and occasional spindle cells were positive for Activin A (black arrow heads). In LP, Activin A was strongly induced in the superficial layers of epidermis; in the dermis, a diffuse reactivity can be observed in numerous cells within the inflammatory infiltrate (panel <i>e</i>). This cell population includes endothelial cells and a mixture of non-lymphoid mononuclear cells (panel <i>f</i>). Magnification 100x (<i>a, b, d, e</i>; scale bar 200 micron) and 400x (<i>c, f</i>; scale bar 50 micron).</p

Activin A induces Langerhans cells differentiation in epidermis-depleted skin explants.

<p>Langerin expression was evaluated in the dermal layer, separated from skin explants by dispase digestion and subsequently treated for 72 hrs after i.d. injection with 100 ng Activin A (magnification 100X, inset 400X.).</p

Intradermal injection of Activin A induces the differentiation of dermal and epidermal Langerhans cells in human skin explants.

<p>Langerin expression was evaluated in the epidermis and dermis (full thickness skin explants) of skin explants, untreated and 72 hrs after i.d. injection of medium or 100 ng Activin A (magnification 100X, inset 400X) The number of Langerin⁺ cells were quantified in skin explants by evaluating six different skin sections (0.05 mm²/field; means±SD). * p<0.05 by Student's t test vs. medium (lower right panel).</p

Activin A promotes Langerhans cell differentiation from human CD14⁺ monocytes.

<p>(A) Phenotypic analysis of monocytes cultured for 6 days with GM-CSF and IL-4 in the presence of Activin A (Act A-LC) or TGFβ1 (TGFβ1-LC). Cells were stained with the indicated moAbs (filled histograms) or isotype-matched negative control moAbs (open histograms). Percentages of positive cells are shown in the upper right corner of each histogram. The figure shows one experiment representative of at least five independent cultures. (B) Electron microscopy analysis of Act A-LC. Act A-LC exhibited abundant dendritic membrane protrusions and lobulated or indented nuclei (left panel, 3,000X, bar 40 µm). Cytoplasm presented a rough endoplasmic reticulum, many multilamellar organelles and numerous electron-dense structures reminiscent of Birbeck granules (right panel, 12,000X, bar 1 µm). The inset shows rod-shaped Birbeck granules (200,000X, bar 20 µm). (C) TGFβ1 and Activin A mRNA expression in Act A-LC and TGFβ1-LC cultures. Monocytes were cultured in the presence of Act A or TGFβ1 for the indicated time and the expression of TGFβ1 and Activin A mRNA was determined by real-time PCR, relative to GAPDH mRNA used as internal control. The expression level in freshly isolated monocytes was assumed as the 1.0 value. Similar results were obtained in three different donors. (D) Effects of different TGF family members on LC differentiation. Monocytes were cultured for 6 days with GM-CSF in the presence of 10 ng/ml TGFβ1, 100 ng/ml Activin A, or 100 ng/ml BMP6 and analyzed for Langerin, E-caderin and CCR6 expression by flow cytometry analysis. Data are representative of at least four independent cultures.</p

Phenotypical and functional characterization of CD40L-activated Act A-LC

<p>(A) Expression of maturation markers by Act A-LC. Act A-LC were incubated with CD40L-transfected fibroblasts for 40 hrs and stained with anti-CD80, CD83, CCR7 and CXCR4 moAbs (filled histograms) or isotype-matched negative control Abs (open histograms). Results obtained with TGFβ1-LC are also shown for comparison. The percentage of positive cells is reported in each panel. Data shown are representative of three independent experiments. (B) Allostimulatory capacity of Act A-LC. Irradiated immature or CD40L-matured Act A-LC (or TGFβ1-LC) were cultured with 2×10⁵ allogeneic purified T cells. Proliferation was assayed as uptake of [H³]thymidine added in the last 16 hrs of a 6-day culture assay. Results are expressed as mean counts per minute (cpm)±SD of one representative experiment performed in triplicate. Values are at the net of T cell proliferation in the absence of DC (3250±250 cpm). (C) Act A-LC migrate in response to CCL20. Immature or CD40L-mature Act A-LC or TGFβ1-LC were applied to the upper wells of the chemotaxis chamber. CCL20 was added to the lower level of the chamber. The number of cells migrated to the lower chamber was counted. Each assay was performed in triplicate and the results are expressed as the mean±SD number of migrated cells (representative of three experiments). (D) Cytokine release by Act A-LC. Immature or CD40L-mature Act A-LC or TGFβ1-LC were assessed for their ability to release the indicated cytokines by ELISA. Results are the average determination (±SD) of four independent experiments.</p

DataSheet_1_Neutrophils inhibit γδ T cell functions in the imiquimod-induced mouse model of psoriasis.docx

BackgroundPsoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood.MethodsIn this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development.ResultsBy analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice.ConclusionsOverall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.</p

Multidisciplinary Management of Spondyloarthritis-related Immune-mediated Inflammatory Disease

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