Previous studies have shown that barrier disruption increases epidermal mRNA levels of interleukin-1 alpha (IL-1 alpha). We used immunohistochemistry to examine IL-1 alpha expression in hairless mouse skin under basal conditions and following barrier abrogation. In untreated mice, IL-1 alpha was present in the dermis and nucleated epidermal layers in a diffuse, generalized pattern. In essential fatty acid deficient mice IL-1 alpha was present in all epidermal layers and the dermis, with prominent staining in the stratum corneum. After acute barrier disruption with tape-stripping, IL-1 alpha increased in the epidermis and dermis within 10 min, remained elevated at 2 and 4 h, and decreased to near basal levels by 24 h. Moreover, intense, perinuclear, basal cell staining appeared at 10 min, persisting until 4 h after barrier disruption. Since the increase in IL-1 alpha immunostaining after acute barrier abrogation precedes the increase in mRNA, we hypothesized that the IL-1 alpha might derive from a pre-formed pool. Prolonged occlusion of normal skin, a treatment that specifically reduces epidermal mRNA levels of IL-1 alpha, decreased basal immunostaining for IL-1 alpha and blunted the increase in IL-1 alpha usually seen following barrier disruption. Moreover, tape-stripping of skin, maintained ex vivo at 4 degrees C, resulted in increased IL-1 alpha immunostaining within the upper nucleated epidermal layers, as well as release of mature IL-1 alpha into the medium, as measured by Western blotting and enzyme-linked immunosorbent assay. In addition, the stratum corneum attached to the tape contained IL-1 alpha. These studies show that acute barrier disruption induces both the immediate release and dispersion of IL-1 alpha from a pre-formed, epidermal pool, as well as increased IL-1 alpha synthesis; both mechanisms are consistent with a role for IL-1 alpha in the regulation of proinflammatory and homeostatic processes in the skin.
Acute disruption of the cutaneous permeability barrier with either solvents or tape-stripping stimulates a homeostatic metabolic response in the subjacent nucleated layers of the epidermis that results in a rapid restoration of normal permeability barrier function. When the aged epidermal permeability barrier is stressed, it reveals a diminished capacity for recovery, in comparison to young epidermis, analogous to other organs in the aged when stressed. Although the signals that regulate this homeostatic response by the epidermis have not yet been resolved, acute permeability barrier disruption stimulates release of prestored IL-1alpha, and increased production of potentially regulatory cytokines, including IL-1alpha and TNFalpha in the epidermis. In these studies, we addressed the hypothesis that cytokine dysregulation explains the permeability barrier abnormality in aged epidermis, assessing the regulation of IL-1 and TNF signaling in aged vs young mice. To determine whether the IL-1 family of cytokines plays a key role in the permeability barrier abnormality of the aged, permeability barrier recovery rates were compared in transgenic mice lacking the functional IL-1 type 1 receptor vs wild-type mice at various ages. Knockout of the IL-1 type 1 receptor exacerbates the defect in permeability barrier homeostasis that is seen in age-matched, wild-type counterparts. Furthermore, the sluggish permeability barrier recovery in aged epidermis is associated with, and at least in part attributable to, altered expression of the IL-1 family of cytokines and receptors both under basal conditions and after acute barrier perturbations. Whereas modulations in cytokine expression with epidermal permeability barrier perturbation are qualitatively similar in aged epidermis, they greatly differ quantitatively. In contrast, examination of TNFalpha mRNA and protein basally, and following barrier perturbation revealed no alterations in aged epidermis. Together, these results show that selective alterations in the IL-1 family of cytokines occur with aging and that defects in IL-1 signaling may contribute to the epidermal permeability barrier abnormality of aged skin.
Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgGs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Preor nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or singlestranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.Alternating purine-pyrimidine sequences having a strong potential to adopt stable leftghanded Z-conformations (e.g., in negatively supercoiled DNAs) are repetitive elements in prokaryotic and eukaryotic DNA genomes. Some of these sequences are transcribed into RNA. The in vivo conformational states of these nucleic acids and their functions are not currently known (for reviews, see refs. 1 and 2).Z-DNA sequences exist in deproteinized negatively supercoiled plasmids, viral DNAs, and fixed (protein-deplet- A detailed report on the extent of bromine modification used to elicit the antibodies used here and its effect on their immunochemical specificities will be published elsewhere (C.C.H., D.A.Z., S. K. Wolk, W. Ross, and I. Tinoco, Jr., unpublished data). Polynucleotides were extensively dialyzed against TE buffer (10 mM Tris HCl, pH 7.4/0.1 mM EDTA). Polynucleotide kinase labeling was by the procedure of Silberklang et al. (6).Radioimmunoassay (RIA) and Nitrocellulose Filter Binding Assays. Direct RIA or competition assays with unlabeled polymers or drugs were as described (3-5). The standard RIA buffer was 200 mM NaCl/40 mM Tris HCl, pH 7.5/4 mM EDTA. Reaction mixtures (20 ,ul) containing 20 ng of polynucleotide substrate were equilibrated for 5 min at 37°C before adding 5 ,ul of the first antibody (either rabbit or mouse). Primary reaction mixtures were incubated for 1 hr at 37°C; this was followed by addition of 1 ,ul of the appropriate second antibody, either affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG (2.4 mg/ml). After an additional hour at 37°C, immune complexes were washed (three times) with RIA buffer, centrifuged at 15,000 x g (10°C), resuspended, and then assayed for radioactivity in 5 ml of Aquasol-2 (New England Nuclear) in a liquid scintillation counter (Beckman). A machine background of 15-35 cpm was subtracted. Filter-binding assays were performed using Millititer nitrocellulose filters on a vacuum manifold (Millipore). Reactions were as described for RIA, except for the omission of the second antibody, and were terminated by the...
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