Distribution of IL-18 and IL-18 receptor in human skin: various forms of IL-18 are produced in keratinocytes

Abstract Human interleukin-18 (IL-18) enhances IL-12-mediated IFN-γ production by lymphocytes and Fas/ perforin-mediated cytolysis by NK cells. IL-18 is synthesized as a 24 kDa proform, and the proform is processed in the cytoplasm into an 18 kDa mature form. Active and precursor forms of IL-18 have been detected in immunocompetent cells, and active IL-18 exerts its functions through its receptor. We sought to determine which human skin cells are responsible for production of IL-18 and which express its receptor. Monoclonal antibodies against human IL-18 and polyclonal antibody against IL-18 receptor were provided for this analysis. Formalin-embedded and frozen sections of human epidermis were analyzed by immunoperoxidase and immunofluorescence staining. IL-18 was detected in all living cell layers of the epidermis, hair follicles, arrectores pilorum, eccrine ducts and endothelial cells. IL-18 was localized in the cytoplasm of cells in living epidermal cell layers. In contrast, IL-18 receptor was mainly detected in keratinocytes and expressed in the cell periphery in living cell layers. Since keratinocytes were the main source of IL-18 and its receptor, cultured human keratinocytes were further analyzed by immunoblotting. IL-18 receptor was identified as an 80 kDa single band. The mature 18 kDa and precursor 24 kDa forms of IL-18 were detected by our monoclonal antibody (mAb) 21 and mAb 132, respectively, while only the 18 kDa form was detected by a commercial mAb, 125-2H. Cultured keratinocytes showed positive granular staining for IL-18 in the cytoplasm and positive staining for IL-18 receptor mainly in the cell periphery. Our findings indicate that mature IL-18, precursor IL-18 and IL-18 receptor are simultaneously expressed with different localizations by human epidermal keratinocytes. Keratinocytes might be activated by their own IL-18 in an autocrine or paracrine fashion. Received: 14 August 2000 / Revised: 10 December 2000 / Accepted: 24 April 2001     

In vivo targeting of integrin receptors in human skin xenografts by intravenously applied antibodies

We examined whether systemically injected monoclonal antibodies (mAbs) directed to cell-surface glycoproteins of human keratinocytes reach their target antigens in xenograft transplants of normal human skin on SCID mice. The integrins _{6 4}, expressed in the basal cell layer of human epidermis, and glycoprotein T16 (gp 40/50), expressed in terminally differentiating keratinocytes of the stratum spinosum, were selected as targets. It was found that all injected mAbs selectively localized to their antigens and bound and saturated their targets even in the uppermost layers of the stratum malpighii. This could easily be monitored by direct immunofluorescence staining since SCID mice lack endogenous production of significant amounts of immunoglobulins. After a single injection, mAbs could still be detected at the target site after 14 days. Our results proved that heterologous immunoglobulins pass systemic capillary filters in this xenograft model and specifically bind to their target molecules. Thus, xenografted SCID mice provide a versatile model for studying cell-surface glycoprotein-mediated interactions by the use of functionally interfering antibodies under in vivo conditions in human skin.Part of this work was presented to the European Society of Dermatological Research, Amsterdam, 3–6 April 1993Supported by the Italian Association for Cancer Research (AIRC)Recipient of a grant from the Deutsche Forschungsgemeinschaft (KL 510/2-2)     

Absorption of boric acid through human skin depending on the type of vehicle

Summary The distribution of procollagen in normal hyperplastic, preneoplastic or neoplastic human epidermal lesions has been analysed in indirect immunofluoresence tests with the antibodies to procollagen raised in sheep to extracted procollagen, synthesised by newborn rat skin explants in culture. These antiprocollagen antibodies produced indirect immunofluorescence staining only of the papillary dermis of human skin. We reacted serial dilutions of the antibody with each skin lesion and recorded the maximum dilution at which a positive reaction was observed. All lesions examined displayed essentially the same procollagen immunofluorescence pattern. The fluorescence was localised as a fibrillar/diffuse area just under the epidermis. In conditions in which the epidermis is highly convoluted, the fluorescent band was found to follow the pattern of the epidermis and to surround epidermal islands in the deeper dermis. Our observations suggest that in the neoplastic lesions a new dermal topography, resembling the stratum papillare of normal human skin, is found in the deeper dermis surrounding the epidermal islands produced as the epidermal mass increases and invaginates further into the stroma. Malignant epidermal lesions were found to show a positive procollagen immunofluorescent staining at tenfold lower concentrations of the antibody than normal human skin in subepidermal regions.     

Vitamin A uptake by human skin in vitro

Summary Results are presented establishing that epidermis accumulates vitamin A from serum retinolbinding protein (RBP). Strips of human breast skin (0.2–0.3-mm thick) were incubated in a serum-free medium. From the rate of glucose oxidation, the tissue was viable for at least 48 h at 32°C in 5% CO₂ air. [³H]-Retinol-RBP (10^-6 M) was added to the medium for 1–24 h, after which epidermis and dermis were split and separately extracted with hexane after saponification. [³H]-Retinol was isolated by high performance liquid chromatography (HPLC). Epidermis had 6–7 times higher affinity for [³H]-retinol than dermis. The uptake could be saturated by substrate and was inhibited with unlabelled retinol-RBP but not with serum albumin. Furthermore, although the uptake was temperature-dependent, it seemed independent of cellular energy production. The epidermal accumulation of [³H]-retinol was reduced by the filtering action of dermis. On the basis of these observations, an in vitro model for the delivery of vitamin A to human skin has been proposed.     

Accelerated acquisition of permeability barrier function in the skin of presenilin-1-deficient embryos

Presenilin-1 (PS1) is a transmembrane protein and is responsible for the development of early-onset familial Alzheimer’s disease. PS1 is essential for neurogenesis, somitogenesis, angiogenesis and cardiac morphogenesis. We report here that PS1 is involved in the development of skin barrier function. PS1-deficient embryos showed an accelerated acquisition of permeability barrier function at embryonic day 17.5 as manifested by the exclusion of a dye solution. While the expression of β-catenin and epidermal differentiation markers such as keratin 1 and loricrin was not substantially altered, an increased accumulation of E-cadherin was observed immunohistochemically in the mutant skin. These results suggest that PS1 regulates the acquisition of permeability barrier function in the skin.     

Inhibition of skin 11β-hydroxysteroid dehydrogenase activity in vivo potentiates the anti-inflammatory actions of glucocorticoids

Abstract Synthetic forms of glucocorticoids (GCs) with high potency are widely used to treat a number of dermatological conditions having an inflammatory or autoimmune etiology. While GCs are generally effective in their ability to suppress inflammatory processes, their chronic use can lead to detrimental systemic side effects. In this report, we describe a method by which the localized antiinflammatory potential of the natural GC cortisol can be significantly augmented without increasing the risk of negative systemic effects. 11β-Hydroxysteroid dehydrogenase (11β-HSD) is a naturally occurring enzyme in the skin. 11β-HSD functionally converts biologically active 11-hydroxy GCs to their biologically inactive 11-keto metabolites, thereby limiting the ability of GCs to mediate antiinflammatory activities. By topically applying specific inhibitors of 11β-HSD in conjunction with low doses of GCs, the antiinflammatory properties of cortisol can be significantly potentiated. It was observed that the generation of the effector phase of contact hypersensitivity (CH) responses could be suppressed by this combined treatment under conditions where the 11β-HSD inhibitor alone, or cortisol alone were only minimally effective. Only the combined treatment was effective at inhibiting the progression of an ongoing CH response. Received: 22 July 1997     

Bleomycin increases steady-state levels of type I collagen, fibronectin and decorin mRNAs in human skin fibroblasts

Abstract Bleomycin is a drug capable of inducing tissue fibrosis. In this study, the effects of bleomycin on gene expression of extracellular matrix encoding ·1(I) collagen, fibronectin and decorin were determined in vitro in human dermal fibroblasts. Northern blot analysis showed that bleomycin upregulated ·1(I) collagen, fibronectin and decorin gene expression dose-dependently between 1 nM and 1 μM. Bleomycin at 100 nM upregulated α1(I) collagen, fibronectin and decorin mRNA expression with a peak at 6 h following stimulation in normal skin fibroblast monolayers. Bleomycin enhanced mRNA expression encoding these extracellular matrix proteins in both normal dermal and scleroderma fibroblasts. Concomitant stimulation with bleomycin and interferon-γ (1000 U/ml), a representative antifibrotic cytokine, decreased ·1(I) collagen mRNA expression. Bleomycin also mildly upregulated mRNA expression of transforming growth factor-‚ (TGF-‚) and connective tissue growth factor (CTGF) coordinately in normal skin fibroblasts. Our results indicate that bleomycin modulates gene expression of extracellular matrix proteins in dermal fibroblasts, and this effect may be mediated by TGF-‚ and CTGF. Received: 20 April 2000 / Revised: 19 June 2000 / Accepted: 4 September 2000     

Adhesion molecule mapping in normal human skin

Summary Adhesion molecules are a rapidly growing group of cell surface receptors providing cell-cell and cell-matrix interactions. Their physiological role in tissue homeostasis as well as cellular migration and differentiation is increasingly appreciated. In the present study we have analyzed the expression pattern of most adhesion molecules of the integrin family as well as of adhesion molecules belonging to the immunoglobulin superfamily in normal human skin. We provide evidence that expression of adhesion molecules in the various cutaneous cell systems follows a constant distribution. Moreover, the physiological mononuclear infiltrate of the skin also expresses a variety of adhesion molecules enabeling these cells to migrate or to reside within the skin. Furthermore, our results indicate that intercellular adhesion molecule-1 is not a prerequisite for lymphocyte epidermotropism as frequently stated. Our data provide a rational basis to analyze changing adhesion molecule expression in inflammatory and neoplastic skin diseases.     

Remittance spectroscopy mapping of human skin in vivo

Remittance spectroscopy of human skin may be influenced by probe application pressure and body site.

Methods:

We investigated remittance spectroscopy qualities of human skin in vivo in different areas: a) forearm, b) frontal, c) back, d) back of the hand, e) palms and f) cheek. Twenty volunteers of skin type 2–3 free of inflammatory skin diseases, were enrolled into the study. Spectroscopy readings were performed with a fiber optic spectrometer (Ocean Optics, USA). The readings were taken with standardized force (0 and 100 pont) by applying the probe vertically to the skin surface. The remittance in relation to wavelength was registered. White light with wavelengths from 420 to 750 nm were used. Individual remittance values and their standard deviations were obtained from 20 readings each.

Results/Conclusions:

Spectroscopic patterns of skin are influenced by external force and regional factors. Standardization remains critical for the use of this approach in bioengineering of skin.     

PTEN基因在皮肤肿瘤中的研究进展

PTEN基因是一个抑癌基因,其缺失、突变和表达减少可存在多种皮肤肿瘤中.PTEN基因主要通过PI3K/Akt、FAK/p130途径及Shc/ras/MARK途径影响细胞的分化、凋亡、移动及信号转导等方面,参与皮肤肿瘤的发生发展,与皮肤肿瘤的恶性转化有着密切的关系.因此,PTEN基因在保持皮肤内环境稳定和抑制皮肤肿瘤的发生方面起着重要的作用.概述紫外线辐射对PTEN功能的调节及PTEN基因在多种皮肤肿瘤发生发展中的作用.     

Presence of immunoreactive beta-endorphin in human skin

The production and its induction by ultraviolet radiation (UVR) of proopiomelanocortin (POMC)-derived peptides by keratinocytes has been reported, albeit not consistently. Recently we demonstrated that only under specific culturing conditions human keratinocytes are capable of producing a beta-endorphin (betaE)-like peptide with the characteristics of beta-lipotropin (betaLPH). Here the presence and UV-induction of betaE-immunoreactivity (betaE-IR) in keratinocytes in human skin in vivo was investigated. betaE-IR was detectable by immunohistochemistry in keratinocytes of the follicular matrix and to some extent in cells of sweat ducts, but was absent from epidermal keratinocytes. Absence of betaE-IR was confirmed by radioimmunoassay of HPLC-fractionated extracts of normal epidermis. Repeated exposure to solar-simulated UVR had no effect. This investigation is the first to demonstrate the presence of betaE-immunoreactive material in the follicular matrix of corporal hairs and in duct cells of sweat glands. The possible meaning of these results is discussed.     

Interleukin-11 production is increased in organ cultures of lesional skin of patients with active plaque-type psoriasis as compared with nonlesional and normal skin. Similarity to interleukin-1β, interleukin-6 and interleukin-8

Increased levels of several cytokines, mainly proinflammatory mediators, have been reported in psoriatic lesions. Little information, if any, is available concerning other cytokines, especially those initially studied as marrow differentiation agents. Using the experimental approach of measuring cytokines released by skin organ cultures. IL-11 and three other proinflammatory cytokines (IL-1β, IL-6 and IL-8) were determined using commercially available ELISA kits in supernatants of ten biopsies from lesional and nonlesional psoriatic skin areas and in supernatants of biopsies from ten normal volunteers. The results obtained showed that the amounts of IL-11 and the other three modulators were significantly increased in the material from the lesional areas ( P < 0.01). The amounts of IL-11, which is known to have functional activity similar to the proinflammatory cytokines and to share a receptor component with IL-6, were also correlated with the disease severity index ( R = 0.69, P = 0.04). In addition, a nearly significant correlation was noted between the amounts of IL-11 released by the lesional and the nonlesional skin biopsies ( R = 0.66, P = 0.05). More detailed studies are needed to clarify whether IL-11 plays a specific functional role in psoriasis, but this study emphasizes the complexity of the pathogenesis of psoriasis and the cytokine network, including activation of proinflammatory and haemopoietic biological response modifiers, in this disease. Received: 14 October 1996     

Calmodulin levels in psoriasis and other skin disorders

Summary Calmodulin was measured in a range of skin disorders characterized by some degree of epidermal hyperproliferation and/or dyskeratinization. In psoriasis the marginal zone of spreading plaques was also investigated. Within the spectrum of diseases we studied, calmodulin increase proved to be rather specific for lesional skin of psoriasis and seborrheic dermatitis; high levels were also found in psoriatic scales. Calmodulin increase in the marginal zone of the psoriatic lesion parallelled the sharp morphological demarcation.Although calmodulin may play a role in the maintenance of epidermal hyperproliferation in psoriasis, it seems unlikely to be of significance in its initiation.     

Identification of chemical compositions of skin calcified deposit by vibrational microspectroscopies

Calcinosis cutis is characterized by the deposition of calcium salts in the subcutaneous tissues. Both Fourier transform infrared (FTIR) and Raman microspectroscopic analysis have been applied to easily get the chemical compositions of the skin calcified deposit (SCD), which was surgically excised from a female patient. This SCD was cut into two parts for histopathological (H&E stain) examination and vibrational microspectroscopic study. The result indicates that the whole SCD in the skin lesion was found to be a well-developed, mature and hard mass. Several FTIR absorption bands at 873, 961 and 1,031 cm⁻¹ [the stretching modes of carbonate and phosphate of hydroxyapatite (HA)], 1,547 and 1,658 cm⁻¹ (the amide I and II bands of collagen) were detected in the IR spectrum of SCD. The Raman spectral bands at 1,665 and 1,450 cm⁻¹ (collagen); 1,519 and 1,156 cm⁻¹ (β-carotene); and 1,072 and 958 cm⁻¹ (HA) were also obtained. To our knowledge, this is the first report using FTIR and Raman microspectroscopies to quickly identify and quantify three predominant components, collagen, β-carotene and type B carbonated HA, in the SCD of a patient.     

Cytochemical expression of epidermal peroxidase and cytochrome oxidase activities in pathological skin conditions of man

Summary The cytochemical expression of epidermal peroxidase and cytochrome oxidase activity was recently well documented in normal human skin.We report here its expression in basal and squamous cell carcinomas, actinic keratoses, psoriasis, allergic contact dermatitis, seborrheic keratoses, and autosomal dominant ichthyosis vulgaris. The two enzyme activities were evaluated using the diaminobenzidine method. If present, the two enzymes were always localized in the same organelles as in normal epidermis endogenous peroxidase in the nuclear envelope and endoplasmic reticulum, and cytochrome oxidase in mitochondria. In basal and squamous carcinomas, actinic keratoses and psoriasis, the keratinocytes lost their peroxidase activity, but maintained their cytochrome oxidase activity. In seborrheic keratoses, allergic contact dermatitis and ichthyosis vulgaris, the cytochrome oxidase activity was greatly reduced or abolished in keratinocytes, Langerhans' cells, and melanocytes, whereas the peroxidase activity was present as in normal epidermis. These results indicate that the two peroxidatic enzymes studied are not interrelated and alternatively suppressed by different cellular dysfunctions.A part of this work was presented at the combined 12th SCUR Annual Meeting and 6th International Dermatopathology Colloqium, April 1985, Florence, Italy