Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

We performed a comparative investigation of the immunomorphological characteristics of lymphatic and blood microvascular endothelial cells in normal human skin, cutaneous lymphangiomas, and hemangiomas, employing a pre-embedding immunogold electron microscopic technique. We stained for cell membrane proteins that are commonly used for light microscopic characterization of blood endothelial cells. With blood microvascular endothelial cells, we observed uniform labeling of the luminal cell membranes with monoclonal antibodies (MAbs) JC70 (CD31), EN-4 (CD31), BMA120, PAL-E, and QBEND-10 (CD34), and strong staining of the vascular basal lamina for Type IV collagen under normal and pathological conditions. In contrast, lymphatic microvascular endothelial cells in normal human skin and in lymphangiomas displayed, in addition to a luminal labeling, pronounced expression of CD31 and CD34 along the abluminal cell membranes. Moreover, CD31 was preferentially detected within intercellular junctions. The expression of CD34 was mostly confined to abluminal endothelial microprocesses and was upregulated in lymphangiomas and hemangiomas. Type IV collagen partially formed the luminal lining of initial lymphatics and occasionally formed bridges over interendothelial gaps. Our findings suggest a function of transmigration protein CD31 in recruitment of dendritic cells into the lymphatic vasculature. CD34 labeling may indicate early endothelial cell sprouting. The distribution of Type IV collagen also supports its role as a signal for migration and tube formation for lymphatic endothelial cells.

The demonstration of circulating autoantibodies directed against the constitutive desmosomal plaque proteins desmoplakin (dp) I and II in mucocutaneous lesions in a subset of patients with erythema multiforme major, suggests that humoral immune mechanisms may play a role in the pathogenesis of this severe skin disease. In this study we identified a specific peptide sequence--YSYSYS--representing an antigenic binding site for the human autoantibodies. This epitope is localized at the extreme carboxy terminal domain of dp thought to be responsible for the assembly of keratin filaments with desmosomes. To test the possibility whether these antibodies may exert any pathologic effects in vivo, human autoantibodies were affinity purified on a corresponding synthetic peptide matrix and peptide-specific antibodies were raised in rabbits. After repeated subcutaneous injections into newborn mice, affinity-purified human autoantibodies and anti-peptide rabbit IgG were detected on desmosomal plaques of keratinocytes overlying the injection site. Histologic and electron microscopic examinations showed hydropic degeneration of basal and suprabasal keratinocytes, dyskeratosis, signs of suprabasal acantholysis, and keratin filaments detached from the desmosomal plaques clumping around the nucleus. We demonstrate that autoantibodies are directed to an epitope within a dp domain crucial for the interaction of keratin filaments with desmosomes, and, when injected subcutaneously into newborn mice, produce pathologic changes. These findings imply that autoantibodies to dp could impair the function of desmosome-keratin filament complexes suggesting a pathogenic role in vivo.

In a previous report, we described autoantibodies against the desmosomal plaque proteins desmoplakin I and II (dp I and II) in patients with erythema multiforme (EM) major. In the present study we investigated ten EM major and eight EM minor patients for circulating autoantibodies and performed clinical and immunomorphological evaluations. Seven out of ten EM major patients revealed anti-dp I and II autoantibodies. Antigens were biochemically characterized by Western blotting and immunoprecipitation of epithelial-cell-derived protein extracts. These autoantibodies bind in vivo to lesional skin/mucosa in a pemphigus-type dotted pattern along the cytoplasmic membranes of keratinocytes. Ultrastructural immunolocalization studies confine in vivo bound autoantibodies to the cytoplasmic desmosomal plaque. Autoantibody binding studies with the sera of such patients demonstrate that the target antigens are not restricted to squamous epithelia but are also expressed in simple and transitional epithelia, on hepatocytes, and on cells of mesenchymal origin, e.g., myocardial cells. Comparing the clinicopathological features of ten patients with EM major, we could not define any discriminating clinical symptoms among patients with or without autoantibodies. Histopathological examination, however, revealed that only patients with EM major and autoantibodies against dp I and II show suprabasal acantholysis in lesional skin and mucous membranes, suggesting a potential role of the humoral immune response in the pathogenesis of this disease. These findings suggest that these autoantibodies define a subset of patients within the clinical spectrum of EM.

Accumulating evidence suggests that psoriasis may be a genetically determined immunogenic, inflammatory disorder based on an ongoing autoreactive Th-1 response. Systemic immunosuppressive therapy is highly effective but fraught with longterm side effects. Our research therefore focuses on therapeutic strategies that induce local immunosuppression in the skin by topical, transepidermal delivery of immunosuppressive drugs. SDZ 281-240 is a newly developed macrolide of the ascomycin type. It is immunosuppressive by mechanism of action similar to that of FK506 but has no antiproliferative activity against keratinocytes in vitro. To evaluate whether SDZ 281-240 exhibits antipsoriatic activity when applied topically, we tested 15 patients with severe, recalcitrant psoriasis, using a microplaque assay in randomized, double-blind, placebo-controlled study, comparing the therapeutic efficacy of the macrolide with a potent halogenated corticosteroid and vehicle. All patients showed a significant improvement of psoriatic lesions treated with two concentrations of the macrolide and, as expected, with the corticosteroid but not with placebo. Both concentrations of the macrolide led to clearing of psoriasis after 10 days of treatment and biopsies confirmed a reversal of the histopathological and immunopathological phenotype of psoriasis to that of normal skin. Thus, an immunosuppressive agent that interferes with early T cell activation can be designed to penetrate into psoriatic lesions when applied topically and to be functionally active within the skin to suppress the ongoing psoriatic process.