Stratum corneum thickness was estimated from water concentration profiles of the skin measured by a confocal Raman spectrometer. Stratum corneum apparent thickness (SCAT) was defined as the depth where the water content reached an almost constant value. Site variations were determined using 15 healthy Japanese subjects (6 males, 9 females), and age variations at the cheek and forearm were examined using 27 female Japanese subjects. There were marked site variations in mean SCAT; 16.8 microm for cheek, 22.6 microm for volar forearm, 29.3 microm for back of the hand, and 173.0 microm for palm. These variations were similar to reported stratum corneum thickness values obtained in biopsy tissues. The SCAT tended to become age-dependently thicker at the forearm, but not at the cheek. In addition, SCAT was increased up to two-fold by hydration for 90 min, while lesser increases were seen with shorter hydration periods.

Cornified envelopes (CEs), rigid and insoluble structures in the stratum corneum, which are assembled by crosslinking of several precursor proteins by transglutaminases, provide a hydrophobic foundation for barrier function; omega-hydroxyceramides are covalently attached to the outer surface of CE components, and onto this hydrophobic assembly, lamellar layers of intercellular lipids are organized. Morphologically irregular, fragile CEs are found in the deep layer of the stratum corneum or in certain disorders, such as psoriasis, whereas most CEs from healthy subjects are rigid and polygonal. We have established a staining method to characterize such fragile CEs as immature and less hydrophobic CEs, and employed it to examine regional differences in the properties of CEs, especially in relation to the barrier function of the skin. CEs from the outermost stratum corneum of the trunk and extremities of healthy subjects were relatively uniform in morphology with larger shape, and were homogeneous in hydrophobicity as judged from the use of an environment-sensitive fluorescent dye, Nile red. However, CEs from the face were strikingly heterogeneous, and consisted of both rigid and fragile CEs. Rigid CEs were Nile red-positive and little stained by anti-involucrin. In contrast, fragile CEs were Nile red-negative but strongly stained with anti-involucrin, as detected by indirect immunofluorescence. Thus, CEs from the face were stained with Nile red or involucrin in a mutually exclusive manner. Fragile CEs were stained with antibodies against other CE components, including loricrin, envoplakin, filaggrin, and isopeptides. Such fragile, involucrin-positive CEs were detected not only in the face, but also in the deep layer of the stratum corneum of the arm. In addition, experimental barrier disruption resulted in the appearance of involucrin-positive CEs in the outermost stratum corneum. These results suggest that involucrin-positive, fragile CEs are immature and less hydrophobic, and that their occurrence is closely related to impairment of the barrier function of the skin.

Keratinocytes produce not only interleukin 1 (IL-1) but also IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1. Because little is known about the presence of IL-1ra in the stratum corneum, we examined the content of IL-1ra in the stratum corneum, especially the balance between IL-1 and IL-1ra. IL-1 alpha and IL-1ra, but not IL-1 beta, were detected in the tape-stripped stratum corneum of healthy volunteers by enzyme-linked immunosorbent assays. IL-1 alpha and IL-1ra were bioactive as determined by thymocyte co-stimulation assay, and their molecular masses were 17 and 20 kDa, respectively, suggesting that the stratum corneum contains active forms of IL-1 alpha and IL-1ra produced by keratinocytes. The stratum corneum of an unexposed area, the inner side of the upper arm. contained more IL-1 alpha than a sun-exposed area, the face. In contrast, the stratum corneum of the sun-exposed area contained a markedly higher amount of IL-1ra than that of the unexposed area. The ratio of IL-1ra to IL-1 alpha was 8 in the unexposed area, and over 100 in the sun-exposed area. Therefore, IL-1 alpha activity was dominant in the unexposed area, and in contrast, IL-1ra activity was dominant in the sun-exposed area. An elevated level of IL-1ra was detected in the stratum corneum of the sun-exposed area independently of age. In the unexposed area, however, IL-1a increased, but IL-1ra decreased, with age, resulting in a significant decline of the ratio of IL-1ra to IL-1a with increasing age. Irradiation of 2 MED of ultraviolet B to the back skin, an unexposed area, resulted in striking elevation of IL-1ra in the stratum corneum in desquamating scales. These data suggest that IL-1ra in the epidermis may be inducible by chronic UV irradiation, although IL-1ra production in the epidermis may decrease with aging in the absence of any stimulus. IL-1ra in the epidermis may play a role in the regulation of IL-1-induced inflammatory responses, and an appropriate balance between IL-1 and IL-1ra may help to maintain homeostasis of the skin.

IL-1 receptor antagonist (IL-1ra) is a cytokine that competitively binds the IL-1 receptor to antagonize IL-1 activity without any agonist function. Previous experiments indicated that the ratio of IL-1ra to IL-1alpha in the normal stratum corneum (SC) was much higher in the sun-exposed face than in the sun-protected area, upper arms. It was also reported by another laboratory that IL-1ra is increased in the lesional skin of psoriatic patients. This study was designed to measure the contents of IL-1alpha and IL-1ra in non-lesional and pathological SC obtained from inflammatory skin diseases including psoriasis and non-psoriatic dermatoses such as atopic dermatitis. The SC materials were obtained with a non-invasive tape-stripping method. Their soluble fractions were prepared and assayed for IL-1alpha and IL-1ra by enzyme-linked immunosorbent assays. As a result we confirmed the previous findings that the ratio of IL-1ra to IL-1alpha in the normal SC was much higher in the face than in the sun-protected sites, the trunk as well as extremities. Next, we found that IL-1alpha contents were significantly reduced in the SC samples obtained from inflammatory skin regardless of whether their IL-1ra contents increased or unchanged. Moreover, we noted that an increased ratio of IL-1ra to IL-1alpha in the SC was not specific to psoriasis, but was also found in other inflammatory skin diseases including atopic dermatitis. This ratio was found to become lower after successful treatment of these skin lesions with topical glucocorticoids. We conclude from these observations that the increased ratio of IL-1ra to IL-1alpha in the SC is a non-specific phenomenon that can occur in any inflammatory skin diseases regardless of the inflammatory pattern, probably reflecting a skin regulation process against various kinds of inflammation.

The effects of four different magnesium salts on the cutaneous barrier recovery rate after barrier disruption were evaluated. We spread an aqueous solution of each salt on the flank skin of hairless mice, occluded the area with a plastic membrane for 20 min, and then left the skin surface to dry. All of the magnesium salts, except magnesium bis(dihydrogen phosphate), accelerated barrier repair. We next estimated the effects of magnesium chloride aqueous solutions which contained calcium chloride at different molar ratios. When the calcium to magnesium ratio was lower than 1, the mixture accelerated barrier repair. The application of an aqueous solution of 10 mM magnesium chloride and 10 mM calcium chloride was found to hasten the barrier recovery more effectively than a solution of 10 mM magnesium chloride. These results suggest that the effects of these metal ions are different depending on the counter ion and/or the method of application.