The amounts of the epidermal proteins filaggrin, involucrin, cystatin A and Ted-H-1 antigen produced during the terminal differentiation of keratinocytes were immunohistochemically measured in lesional and nonlesional skin of atopic dermatitis (AD) patients. In addition, the amount of filaggrin in the skin of the inner surface of the upper arm of AD patients (nonlesional skin) and normal controls, obtained by punch biopsy, was measured by an enzyme-linked immunosorbent assay (ELISA) technique. The immunohistochemical study showed that all four proteins were decreased in lesional skin. By contrast, only filaggrin was decreased in nonlesional skin of AD patients. The ELISA showed that the amount of filaggrin in the skin of the inner surface of the upper arm was 2.48 +/- 0.45 microgram/7 mm2 (n = 8) in AD patients, which was 32% of that in the normal controls (7.7 +/- 0.55 microgram/7 mm2; n = 4). This decrease in filaggrin production in atopic skin may be one of the reasons why atopic skin can easily become dry, because filaggrin is thought to be the precursor protein of the emollient factors in the stratum corneum. The evidence that only the expression of filaggrin was suppressed in AD patients, though the genes of filaggrin and involucrin are localized to a very restricted portion of the same gene 1q21, indicates that the filaggrin gene does not share regulatory elements with the involucrin gene.

Because it has been suggested that the majority of the activity hydrolysing [N-methyl-14C] sphingomyelin is due to sphingomyelin acylase in the lesional skins of atopic dermatitis (AD), in this study we used immunologic techniques to localize and quantitate sphingomyelinase in AD lesional and normal skin. A polyclonal antibody raised against a synthetic polypeptide corresponding to a portion of the amino acid sequence deduced from the cDNA of human acid sphingomyelinase, cross-reacted with a 58 kDa, pI 5.8 human epidermal protein in an immunoblot analysis. The 58 kDa protein-rich fraction, partially purified by immunoprecipitation, converted [N-methyl-14C]-sphingomyelin to 14C-phosphorylcholine and ceramides. The reaction products were immunohistochemically observed in the intercellular domain from the upper spinous cell layer to the upper stratum corneum cell layers in the lesional skin of AD patients. Immunoelectron-microscopically, gold particles appeared to be concentrated in the intercellular domains of the granular-upper stratum corneum cells in the lesional skin of AD patients. The total amount of the 58 kDa protein in a 7 mm2 area of the skin was measured by quantitative immunoblot analysis; and was slightly increased in the lesional skin samples [3.5 +/- 0.3 microg per 7 mm2 (n = 7)], as compared with the nonlesional skin samples of AD patients [2.8 +/- 0.19 microg per 7 mm2 (n = 10)] and with the normal skin samples [2.7 +/- 0.22 microg per 7 mm2 (n = 10)]. This difference (between the lesional skin of AD and the nonlesional skin of AD or the normal control) was significant (nonpaired student's t test, p < 0.05).

In old age, the epidermis tends to become dry and flaky, especially on the lower legs. However, this does not occur on the face, although long-term ultraviolet light irradiation has important effects on the differentiation of facial keratinocytes. Therefore, the differentiation of the epidermal cells of the facial skin in the young and the aged was immunohistochemically examined using antibodies against four differentiation products: filaggrin, involucrin, cystatin A (CTA) and carbonic anhydrase-like protein. The results showed that there was no difference in the amounts of the above three proteins in facial skin between the young and the aged with the exception of CTA. The amount of CTA was much greater in the facial skin of the aged. However, there was a striking decrease of filaggrin content in the skin of the lower leg in the aged. One of the reasons why no scaly skin is found on the face in the aged may be the fact that especially filaggrin does not decrease with aging.

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In the skin of atopic dermatitis patients, the amount of ceramides in the stratum corneum is decreased. Although the cause of this decrease may be due to the higher activity of acylase, a decrease in the activity of sphingolipid activator proteins may also be the cause. A polyclonal antibody to saposin D, elicited by immunizing rabbits with the synthetic polypeptide from cDNA of saposin D, cross-reacted with a single 65-kDa epidermal protein of pI 5.6 in a 2-dimensional immunoblot study, suggesting that it was prosaposin, the precursor protein of saposin D, from its molecular weight and demonstrating its immunohistochemical localization in the innermost cell layers of the stratum corneum of the skin. The antigenic material was also observed in the epithelium of the esophagus, pneumocytes of the lungs, hepatocytes, and glandular cells of the stomach. Immunoelectron microscopy showed the antigenic material in the cytoplasm of the granular cells and the intercellular spaces, either between the stratum granulosum and the stratum corneum or on the stratum corneum cell envelope. By ELISA, the amount of the 65-kDa protein in the inner surface skin of the upper arm of atopic dermatitis patients (nonlesional skin) [4.1 +/- 2.0 microg per 7 mm2 (mean +/- SD), n = 10] was found to be significantly decreased (p < 0.05) to 66% of that in the normal control (6.2 +/- 1.5 microg per 7 mm2, n = 10). Therefore, the suppression of prosaposin synthesis may be related to the abnormal stratum corneum formation in atopic skin through lower activation of glucosylcerebrosidase or sphingomyelinase.