The inflammatory and proliferative response of normal skin in a model for acute chemical injury: ornithine decarboxylase induction as a common feature in various models for acute skin injury.

Application of sodium dodecyl sulphate (SDS) on the skin of healthy volunteers was used as a model for acute chemical injury. The time course of the response with respect to cell proliferation was studied using ornithine decarboxylase (ODC) activity. Erythema, polymorphonuclear leucocyte (PMN) infiltration, and the induction of epidermal antiproteinase activity (SKALP/elafin) were used as markers for the inflammatory response. ODC induction was similar to that in other models of acute skin injury, such as tape-stripping and ultraviolet light radiation. The amount of PMN infiltration correlated with erythema, but not with ODC induction. In contrast with findings in the tape-stripping model, no induction of SKALP/elafin activity was found after SDS application. We conclude that cell proliferation as measured by ODC induction is a common feature in the various models for skin injury. Both the kinetics and the intensity of the inflammatory response, and the induction of epidermal antiproteinase activity, appear to vary, depending on the specific model.

Cell kinetic characterization of the epidermal growth factor dependent BALB/MK line using flow cytometric analysis of DNA content and iododeoxyuridine incorporation.

Epidermal hyperproliferation (psoriasis, wound repair) is the result of quiescent (G0) keratinocytes being recruited into the cell cycle. A detailed characterization of the cell cycle kinetic parameters of the mouse keratinocyte line (Balb/MK) has been carried out with the aid of bivariate iododeoxyuridine (IdUrd) and DNA analysis using flow cytometry, in order to establish whether it might provide a useful model for the study of the biochemical events controlling recruitment into the cell cycle. Balb/MK keratinocytes were cultured using low Ca2+ Dulbecco's modified Eagle's medium/F 12 in the presence of 10% dialysed fetal bovine serum and 4 ng/ml epidermal growth factor (EGF). IdUrd labelling followed by flow cytometric analysis of trypsinized cells showed that about 95% of the population were actively cycling, with a cell cycle time of around 14h and no significant contact inhibition. Omission of serum and EGF followed by IdUrd pulse-labelling indicated that the cells progressively withdrew from the cycle and, after 16h, less than 10% incorporated IdUrd. Subsequent restimulation with serum resulted in a synchronized cohort of cells being recruited. Entry into the S phase of the cell cycle (IdUrd incorporation) started at 8 h and was maximal between 12 h and 16h after stimulation. Restimulation with EGF alone reached a growth fraction of 87% after 24 h continuous labelling compared with 97% using serum together with EGF. Epidermal growth factor already showed a significant stimulation at 10 pg/ml compared with the controls. There is a broad plateau centred on 5 ng/ml, followed by a slight decline above this level. We conclude that the use of a cell line with defined cell cycle kinetic parameters which can be switched between the quiescent and cycling states in a fully defined medium will provide an ideal model for biochemical studies of the relevant signal transduction pathways in keratinocytes.

Effects of sphingosine, isoquinoline and tannic acid on the human tape-stripping model and the psoriatic lesion.

Published data (mainly from rodent skin) suggest a correlation between compounds which inhibit protein kinase C (PKC), have anti-inflammatory or antitumor characteristics and possess antipsoriatic potential. We have investigated the effects of topical application of sphingosine (a naturally occurring PKC inhibitor), isoquinoline (a component of coal tar which showed antipsoriatic capacities in the mouse tail model) and tannic acid (a plant phenol with antitumor activity) on human skin. In each case we have assessed (a) the level of induction of ornithine decarboxylase (ODC) following Sellotape stripping as an indicator for potential PKC inhibition in vivo, and (b) its effects on the lesions of chronic plaque psoriasis. The control group consisted of 18 healthy volunteers, used for the ODC induction experiments (0.0/0.1/0.2 M sphingosine in ethanol, 100% coal tar and 0/50 mM tannic acid in acetone) and 17 psoriatic patients used for double-blind scoring of two randomly selected lesions (0.0/0.1 M sphingosine in ethanol, 0.0/0.2% isoquinoline in white vaseline/lanette wax cream 50%/50% and 0/10% tannic acid in lanette wax cream) and also for some of the ODC induction experiments (0.0/0.2% isoquinoline and 0/10% tannic acid). Biopsies were taken 8 h after stripping and ODC activity was assessed by measurement of 14CO2 release. Lesions were scored with a modified psoriasis area and severity index on days 0, 7 (isoquinoline and tannic acid), 13 (sphingosine) and 21 (isoquinoline and tannic acid). Application of 0.1 or 0.2 M sphingosine resulted in a decrease of ODC activity of 52% and 66%, respectively (p < 0.01), but histologic sections showed intraepidermal necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclo-oxygenase products do not participate in the induction of ornithine decarboxylase in human epidermis.

Extensive animal data suggest that prostaglandins are involved in the epidermal induction of ornithine decarboxylase (ODC). We undertook this study to investigate their role during induction of hyperproliferation in human skin. Topical indomethacin (Elmetacin) or vehicle only were applied under occlusion on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping and biopsies were carried out for the estimation of the levels of ODC. There was no significant difference in the level of ODC in the indomethacin-treated and control sites. Also, test sites were irradiated with 3 MEDs of UVB, and this was immediately followed by the application of indomethacin or vehicle only on the irradiated sites. After 8 h biopsies were taken and the levels of ODC were again similar in both sites. The data indicate that the cyclo-oxygenase products in human epidermis do not contribute to the induction of ODC.

Epidermal transglutaminase in the ichthyoses.

Membrane-bound transglutaminase (TGm) is responsible for the cross-linking of proteins to form the cornified envelope. Since abnormalities have been reported in the envelope in certain ichthyoses, we have carried out a survey of TGm concentrations in scales from these disorders. Surprisingly, a striking and specific increase in enzyme activity was found in patients with non-erythrodermic autosomal recessive lamellar ichthyosis. It is not clear how this increase is related to the underlying recessive mutation.

Induction of ornithine decarboxylase following sellotape stripping in normal and psoriatic skin.

Ornithine decarboxylase (ODC) was measured in the epidermis of healthy volunteers and the uninvolved skin of psoriatic patients at various times after sellotape stripping. Basal levels were less than 1 pmol/min/mg protein. Activity peaked to a maximum of 86 pmol/min/mg protein after 8 h; this was followed by an abrupt decline to a lower level which remained relatively constant for at least 36 h. No difference was seen between the response of controls and psoriatic patients. Pretreatment with topical corticosteroids reduced peak ODC levels to about one-half, but oral indomethacin had no effect.

Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.

Elastase inhibiting activity (EIA) was demonstrated in the epidermis from lesions and in psoriatic scale, whereas normal epidermis did not contain significant EIA. Two new elastase inhibitors were partially purified and characterized using psoriatic scale as a source. The two species (approximate molecular weights 10 and 20 kDa) were shown to be stable, and high-affinity inhibitors of human leucocyte elastase (Ki less than 10(-10) M). No activity against human cathepsin G could be demonstrated. Cultured human keratinocytes were shown to contain EIA activity similar to that found in psoriatic scale. EIA could also be demonstrated in human epidermis following the induction of an experimental inflammatory response by sellotape-stripping. We propose the acronym SKALP (skin-derived antileucoprotease) as a name for these new proteinase inhibitors.

Enzymatic distinction between two subgroups of autosomal recessive lamellar ichthyosis.

It has been proposed that the autosomal recessive lamellar ichthyoses may be divided into two subgroups, the erythrodermic (EARLI) and non-erythrodermic (NEARLI) forms. We report measurements of the enzymes beta-glucosidase, a recently described phosholipase, a short-chain carboxylesterase ("butyrase"), and a long-chain carboxylesterase ("palmitase") in aqueous extracts of scales from patients diagnosed according to clinical and micromorphologic criteria, and show that beta-glucosidase and phospholipase tend to be lower in the EARLI group, whereas butyrase is relatively low in the NEARLI group. The internal ratio of either butyrase/glucosidase or butyrase/phospholipase yields a clear separation of the two subgroups, supporting the concept of heterogeneity in this group of diseases.

Hypothesis: vascular twin naevi and somatic recombination in man.

Can the genetic concept of somatic recombination explain the phenomenon of vascular twin naevi in man? If so, this cutaneous disease would be a human counterpart of twin spots observed in animals and plants.

Membrane-bound phospholipase C activity in normal and psoriatic epidermis.

We report the quantification of a membrane-bound phospholipase C in human epidermis which is active against the physiologically relevant substrate, phosphatidylinositol 4,5-bisphosphate. The level of this enzyme is significantly increased in the psoriatic lesion, both on a weight and protein basis. Etiological implications of this observation are discussed.

Studies on the time course of dithranol-induced inflammation by quantification of alkaline phosphatase.

An inflammatory response of the skin to dithranol-induced free radicals seems to be essential for its clinical efficacy. In normal volunteers this response was evaluated at the level of the microvasculature following 30 min, 2 h and 24 h applications, using a functional parameter (erythema) and a biochemical parameter (alkaline phosphatase). The results of 'short contact' and 24 h applications were similar. In all schedules a maximum erythema was seen 2-3 days after the application which had resolved totally after 6-8 days. A marked discrepancy was established between the duration of functional and biochemical abnormalities; the alkaline phosphatase activity reached a maximum 1 day after the culmination of the erythema and persisted up to at least 7 days after disappearance of the erythema. These findings are discussed in the light of the day-to-day management of psoriasis with dithranol.

Phosphatidylinositol 4,5-bisphosphate phospholipase C activity in particulate preparations from rat brain.

We describe the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a particulate rat brain preparation in the presence of the cationic detergent, cetrimide. The optimum cetrimide concentration was in the range 0.2-0.4 mg/ml; at higher or lower concentrations, the reaction rate diminished abruptly, suggesting that the electrical charge density of the micelle is critical for enzymatic attack. In other respects, such as its partial requirement for Ca++ and its pH optimum of about 7.0, the particulate enzyme seems similar to soluble preparations which have been reported previously. Interestingly, the particulate preparation could be stimulated about fourfold by a soluble brain extract in the presence of 1 mM guanosine triphosphate, confirming that the enzyme is the catalytic subunit in a membrane-bound signal-transduction system.

Recruitment of quiescent (G0) cells following epidermal injury is initiated by activation of the phosphoinositol cycle.

Under normal circumstances, the rate of production of new cells by the epidermis is rather low, but injury results in a burst of mitotic activity that continues until repair is complete. It is now recognized that most cells of the germinative population are in a resting (G0) state, and "postinjury cell renewal" is the consequence of G0 cells entering the mitotic cycle. The biochemical events triggering this process, however, are unknown. Here we show that phorbol myristate acetate (PMA) is able to induce G0 mobilization in the epidermis of the nude mouse. Further, we demonstrate that amiloride (an inhibitor of the membrane Na+-H+ pump), applied topically to human skin, abolishes almost completely the regenerative response after experimental injury. We suggest that activation of the phosphoinositol cycle may initiate recruitment of G0 cells in the epidermis.

A unique phospholipase A2 in human epidermis: its physiologic function and its level in certain dermatoses.

It is now well established that epidermis, like many other tissues, contains a phospholipase A2 that is responsible for the initiation of the arachidonic acid cascade. Here we report that human epidermis also contains a second, quite distinct enzyme of the phospholipase A2 group, which is unique in its extreme activity against phospholipids in true solution. It also differs from the classic cutaneous enzyme in that (a) its activity is not reduced by pretreatment of the skin with corticosteroids in vivo nor by treatment of the epidermal homogenate with alkaline phosphatase in vitro, and (b) its activity is reduced, rather than increased, in the lesions of inflammatory diseases such as psoriasis. The enzyme seems to occur mainly in fully differentiated keratinocytes, its level being low in the basal cell layer of epidermis and in keratinocytes cultured in vitro. On the basis of these observations, we suggest that this new phospholipase A2 is responsible for the degradation of phospholipids that accompanies the terminal keratinization process.