The relationship between clinical severity, the hydration state of the skin surface as assessed by a conductance with a 3.5 MHz high frequency impedance meter, and the amino acid content of the stratum corneum (SC) of six patients with ichthyosis vulgaris and 30 elderly persons with varying degrees of xerosis was investigated. With an increase in the severity of xerosis the SC showed a decrease in hydration and in its extractable amino acid content. There was a significant correlation between the hydration state and amino acid content of the SC. Although there was a significant correlation between the amino acid content of the lower leg SC and that of the forearm SC in the same subject, the former was generally lower, corresponding to the greater incidence of xerosis on the lower leg. These results suggest that a decreased amount of amino acids may be the result of low profilaggrin biosynthesis in the epidermis and that this is involved in the pathogenesis of these xerotic skin conditions. Clinical improvement of the xerosis following treatment with a urea-containing cream was not accompanied by any significant change in the amino acid content of the SC.

Dermatologists universally recognize that the unaffected skin of patients with atopic dermatitis tends to be dry and slightly scaly. To characterize the functional properties of the superficial stratum corneum in atopic xerosis, we studied the forearms of 28 patients with atopic dermatitis, aged 14 to 30 years, and 18 age-matched controls, with the use of mainly noninvasive methods. Patients with atopic xerosis showed markedly higher transepidermal water loss and markedly lower skin surface hydration levels than did the controls. The corneocytes in atopic xerosis tended to desquamate in clumps of cell aggregates instead of as individual cells. They contained a substantially lower amount of water-soluble amino acids, which play a role in the water-retaining capacity of stratum corneum, than did those of controls. Although the number of stratum corneum cell layers in atopic xerosis (21 +/- 4) was substantially larger than that in controls (15 +/- 1), its turnover time (7 +/- 2 days) was appreciably shorter than that for controls (14 +/- 2 days). Like those noted in the skin with increased epidermal proliferation, the size of superficial corneocytes in patients with atopic xerosis was substantially smaller than in controls. Histopathologic examination revealed acanthotic epidermis, mild perivascular mononuclear cell infiltrate, and pigment incontinence. Atopic xerosis, the dry skin of patients with atopic dermatitis, shows various stratum corneum functional impairments, probably reflecting increased epidermal proliferation due to a low-level ongoing dermatitis.

In order to identify the endogenous protease associated with stratum corneum (SC) desquamation, we examined properties of proteases in the stratum corneum of normal human skin. SC were obtained by tape stripping, washed in toluene and then dried. The proteolytic activity in SC was measured using peptidyl 4-methyl-coumaryl-7-amides (MCAs). The SC was dispersed uniformly in the reaction mixture with dimethylformamide and Triton X-100 and incubated with the peptidyl MCAs. The protease in the SC hydrolysed both Boc-Phe-Ser-Arg-MCA and Boc-Gln-Ala-Arg-MCA (substrates for trypsin) very effectively. The hydrolytic activity was inhibited by the serine protease inhibitors diisopropyl fluorophosphate (DFP), aprotinin, antipain and leupeptin, but not by chymostatin, a chymotrypsin inhibitor. These results show that one or more trypsin-like serine protease is present in the SC of normal human skin. Casein-acrylamide electrophoresis showed that the molecular weight of this serine protease was about 30 kDa. We have previously shown that cells dissociate from human SC sheets in a detergent mixture (N,N-dimethyldodecylamine oxide and sodium lauryl sulphate). This cell dissociation was inhibited by aprotinin and leupeptin. In addition, the proteolytic activity in the outer SC was higher than that in the inner SC, and the activity in the SC of scaly skin induced by SLS treatment was higher than that of untreated skin. These results strongly suggest that the trypsin-like serine protease described here is involved in SC desquamation.

We investigated the role of stratum corneum (SC) trypsin-like and chymotrypsin-like serine proteases in the degradation of desmoglein-1 (DSG-1) in the SC sheet. DSG-1, whose presence in the SC sheet was confirmed by Western blot analysis, was degraded completely during incubation of the SC sheet in Tris buffer. The degradation of DSG-1 was inhibited by the addition of protease inhibitors, such as aprotinin or a mixture of leupeptin and chymostatin. Either leupeptin or chymostatin alone did not inhibit its degradation. These results indicated that both trypsin-like and chymotrypsin-like serine proteases are involved in the degradation of DSG-1. We further examined the activities of the two proteases in the SC obtained from patients with ichthyosis vulgaris, in whom SC desquamation is abnormal. The enzymatic activities measured using synthetic substrates were significantly decreased in these ichthyotic SC samples. This result supports the idea that these proteases play an important role in normal SC desquamation.

The effects of protease inhibitors on cell dissociation were studied in vitro in order to examine the involvement of proteases in stratum corneum desquamation. Stratum corneum sheet (peeled from human backs after sunburn) was incubated in a detergent mixture containing 8 mM N,N-dimethyldodecylamine oxide, 2 mM sodium lauryl sulphate and 60 micrograms/ml kanamycin with or without protease inhibitors, and the number of released cells was counted after incubation for 48 h. Cell dissociation was inhibited strongly by antipain or aprotinin, but not at all by N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl]-agmatin, N-ethylmaleimide or pepstatin, which suggests that only serine proteases are associated with desquamation. Furthermore, leupeptin and chymostatin each reduced cell dissociation about half as effectively as aprotinin or antipain, while a mixture of leupeptin and chymostatin prevented stratum corneum dissociation as potently as antipain or aprotinin. In addition, the activity of chymotrypsin-like protease in scaly skin was higher than that in normal skin, as we have previously found for trypsin-like protease. These results suggest that both trypsin-like and chymotrypsin-like serine proteases are involved in stratum corneum desquamation.