Cyclosporin A induced augmentation of the beta-adrenergic adenylate cyclase response of pig epidermis.

It has been known that beta-adrenergic adenylate cyclase response is decreased in psoriatic-involved epidermis. Since the immunosuppressive agent, cyclosporin A, is reported to be effective on psoriasis clinically, the effect of cyclosporin A on beta-adrenergic adenylate cyclase response in pig skin was examined in vitro. Therapeutic serum levels of cyclosporin A (100-400 ng/ml) augmented the beta-adrenergic adenylate cyclase response of the epidermis. Highest levels of cyclosporin A (2-20 micrograms/ml) did not have any effect on its response. Both low Km and high Km cyclic AMP phosphodiesterases were not affected by cyclosporin A. Therefore, it is suggested that the clinical efficacy of cyclosporin A on psoriasis can be explained partially by its direct effect on the keratinocyte itself.

Application study of N-hexadecane on pig skin.

Pig skin was treated with N-hexadecane. Epidermal mitoses, epidermal and dermal histopathology, epidermal phospholipase A2 activity were investigated. The most prominent increase of mitoses was found 48 hours after three applications of n-hexadecane. Epidermal and dermal changes were observed mainly at the same time. Phospholipase A2 activity was decreased when increased mitoses and histological changes were seen. This paradoxical movements of the activity of this enzyme has also been reported in regard to psoriasis.

Cyclosporin A does not inhibit epidermal cell growth at therapeutic levels.

The direct effect of cyclosporin A on epidermal cells was examined by using a pig skin explant culture system. A dose-response study using cyclosporin concentration from one-tenth the expected serum level to 100 times the expected serum level was done, and epidermal outgrowth and the number of mitotic figures were measured. Cyclosporin inhibited epidermal cell outgrowth at concentrations of 60 and 100 times the expected serum level, but it did not inhibit epidermal cell outgrowth at the serum level or even at 30 times the serum level. Cyclosporin did not inhibit mitotic figures significantly. These results suggest that cyclosporin does not inhibit epidermal cells directly.

Immunohistochemical study of beta-adrenergic receptors in the psoriatic epidermis using an anti-alprenolol anti-idiotypic antibody.

In order to study the beta-adrenergic receptor-cyclic AMP system in the human skin, we have developed a monoclonal antibody against the receptor binding site in two fusion steps. We produced an anti-alprenolol monoclonal antibody using a hybridoma technique after the immunization of a mouse with alprenolol-BSA conjugate. This antibody was then used to immunize mice, whose splenocytes were used for the second fusion. Colonies were screened by enzyme linked immunosorbent assay (ELISA) using turkey erythrocyte membrane as an antigen. One of the positive colonies was grown in mice to obtain ascites fluids. This antibody showed the ability to compete with [125I]-iodocyanopindolol for the binding to beta-adrenergic receptors of turkey erythrocytes. The binding of the antibody to the turkey erythrocytes was prevented by the existence of the anti-alprenolol antibody and also L-alprenolol. This antibody showed a prominent inhibition of the adrenaline-stimulated adenylate cyclase activity of the turkey erythrocyte membrane. The data indicate that the anti-idiotypic monoclonal antibody against alprenolol binds to the beta-adrenergic receptors and acts like an antagonist. We further studied the localization of the beta-adrenergic receptors with this antibody in the normal human skin as well as in the psoriatic skin using immunohistochemical technique. A prominent decrease of the beta-receptors was observed in the psoriatic epidermis.

Ionophore A23187 stimulates arachidonic acid release and cyclic GMP accumulation in pig epidermis.

We studied the effect of Ionophore A23187 (A23187) on phospholipid degradation and the accumulation of cyclic GMP in pig epidermis. A23187 stimulated the release of AA, probably partially through the activation of phospholipase A2. A23187 also stimulated the accumulation of epidermal cyclic GMP, but the activity of cyclic GMP-dependent phosphodiesterases was not altered. Mepacrine, an inhibitor of phospholipase A2, inhibited the A23187-stimulated accumulation of cyclic GMP. The results suggest that A23187 stimulates the accumulation of epidermal cyclic GMP by a calcium-dependent process which also requires products of phospholipid degradation.

Monoclonal antibodies against epidermal cell surface and dermal-epidermal junction: antigens which may be epidermal cell spreading factors.

Two monoclonal antibodies which reacted with the epidermal cell surface (SF-1) and the dermal-epidermal-junction (SF-2), respectively, were obtained by immunizing mice with partially-purified human epibolin. The corresponding antigens were partially purified from fetal bovine serum by affinity chromatography using these antibodies. SDS-polyacrylamide gel electrophoresis showed that these antigens contained polypeptide components with molecular weights different from that of epibolin (mol. wt. 65,000 daltons); SF-1 antigen had a 68,000 dalton main component, and SF-2 antigen a broad 58,000-61,000 dalton main component. Both of these partially-purified antigens promoted the spreading of dissociated pig epidermal cells. SF-2 antigen also promoted the spreading of Pam cells (a murine keratinocyte line). The results suggest that proteins capable of promoting epidermal cell spreading may be present on the epidermal cell surface and at the dermal-epidermal junction. However, their physiological role in keratinization remains to be elucidated.

A cell-spreading inhibitor exists in serum and in epidermal basal cells.

A unique monoclonal antibody was obtained by immunizing mice with complement-inactivated fetal bovine serum (FBS). This antibody, named SI-1, stained epidermal basal cells of humans, pig, guinea pig, and rat by an indirect immunofluorescence technique after pretreatment of cryostat sections with alkali buffer (pH 9.6). After dissociating pig epidermal cells by trypsin, the SI-1 antibody stained exclusively and strongly one type of uniquely shaped cells. They were small and hanging-bell or columnar in shape with one convoluted side on the base, consisting of less than 2.8% of the dissociated epidermal cell population. The antigen contained in FBS was partially purified by affinity chromatography using the SI-1 antibody. The affinity-purified antigen inhibited the spreading of PAM cells, a spontaneously transformed murine keratinocyte line, in serum-free medium in a dose-dependent manner at concentrations of 10(-5) to 10 ng/ml. The antigen also inhibited the spreading of trypsinized pig epidermal cells in the range of 10(-2) to 10(3) ng/ml in the presence of 0.05% FBS. Although there have been a few reports indicating that serum inhibited both spreading and attachment, a specific factor in serum has not been purified before. This is, to our knowledge, the first presentation of a cell-spreading inhibitor contained in serum.

Monoclonal antibodies to the beta-adrenergic receptor: modulation of catecholamine-sensitive adenylate cyclase by the antibody.

We have developed two types of hybridomas producing monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor in order to study the beta-adrenergic-cAMP system of epidermis. Splenic cells from BALB/c mice immunized with partially purified turkey erythrocyte beta 1-receptors were fused with mouse myeloma cell line SP2/0-Ag14. Five hybridomas of 17 positive cells producing antibodies which could precipitate soluble turkey erythrocyte beta 1-receptors were cloned by the limiting dilution method. The antibodies cross-reacted with beta 1- and beta 2-adrenergic receptors and stained epidermal basal cells with immunocytochemical techniques. Neither type of antibody interfered with the antagonist binding, i.e., all antibodies bound to sites other than the ligand binding site on the surface. One type of antibody inhibited epinephrine-stimulated adenylate cyclase activity in our "leaky" epidermal cell system. The data suggest that the antibody interferes with the coupling of the receptor to the regulatory protein.

Stimulated mitotic counts in the non-lesional skin of patients with psoriasis and controls.

Mitotic counts have been determined in the symptomless skin of nine psoriatic patients and nine healthy controls 48 h after pressure-tape stripping. A significantly increased response was seen in the symptomless psoriatic epidermis compared with the controls.

Dexamethasone directly inhibits snake venom phospholipase A.

Dexamethasone was found to directly inhibit snake venom phospholipase A2 within 3 to 10 minutes. To detect this effect, the incubation time seems to be critical. Moreover, the inhibitory effect of dexamethasone and mepacrine were additive with each other. We speculate that this direct inhibitory effect on phospholipase A2 plays a part in its strong biological activity.

Predictive value of HLA antigen for methotrexate-induced liver damage in patients with psoriasis.

For an investigation of a possible association between HLA antigens and postmethotrexate liver damage, the A, B, C, and Dr loci of the HLA antigens were determined in thirty-two patients with severe psoriasis who had been treated with methotrexate. There was no association between HLA antigens and increasing severity of liver disease in these patients. HLA-A3 was increased in frequency in the group of patients with the more severe liver damage but was not significant when corrected for the number of antigens tested. There appears to be no predictive value in HLA typing of psoriasis patients prior to starting methotrexate therapy.

Skin cancer in patients treated with 8-methoxypsoralen plus longwave ultraviolet radiation.

Although severe biochemical and cytologic changes occur in both melanocytes and keratinocytes during psoralen photochemotherapy, a review of the published literature does not reveal any increased incidence of skin cancers from this type of therapy. Perhaps not enough time has elapsed to show such effects.

Calcium-activated, phospholipid-dependent protein kinase in pig epidermis.

We investigated calcium-activated, phospholipid-dependent protein kinase in pig epidermis. Pig epidermal homogenates were centrifuged at 30,000 g for 30 min, and the supernatant was applied on a DEAE-cellulose column for purification. The partially purified enzyme was stimulated by simultaneous addition of Ca2+ and phospholipid. Successive addition of small amounts of diolein further activated the enzyme activity. The calcium-activated phospholipid-dependent protein kinase preferentially phosphorylated serine residues and its endogenous substrate protein in the pig epidermis has a molecular weight of about 97,000.

Control of epidermal cell proliferation in vitro.

Epidermis from explants of keratome slices of domestic pig skin can be induced to migrate, proliferate, and stratify if at least two serum protein factors and a source of nucleosides are present. The factor which is necessary for cell movement or spreading is destroyed by heating the serum to 100 degrees C and is resistant to 80 degrees C heating. This factor has a molecular weight of 65,000 and is identical to the 'epibolin' found by Stenn. Proliferation is dependent on a serum factor which is destroyed by heating to 80 degrees C. However, cells which are denied this factor can make their own proliferation factor(s). Cell migration and cell proliferation usually occur together but they can be dissociated and are therefore independent phenomena. Increased cell proliferation can be achieved by some retinoids at the proper concentration but steroids, epidermal growth factor, a phorbol ester (TPA), and the use of injured skin as a keratome source failed to show in vitro hyperproliferation. Mitotic inhibition, however, can be induced by a variety of metabolic poisons (methotrexate, 5-fluorouracil, hydroxyurea), steroids, retinoids at high concentrations, and agents which increase adenosine 3',5'-monophosphate (cAMP).

Agents that activate cyclic AMP-dependent protein kinase inhibit explant culture growth and mitotic activity.

Epidermal cells contain 4 separate surface receptors which are linked to adenylate cyclase. Activation of any one of these receptors leads to the accumulation of cAMP within the cell which in turn leads to the activation of cAMP-dependent protein kinase. The levels of cAMP accumulation within the cell caused by the 4 activators are not the same. Epinephrine, histamine, adenosine, and prostaglandins of the "E" series cause easily measurable concentrations of cAMP within 5 min of exposure. Prostaglandin F2 alpha causes only a small nonsignificant increase. Similarly, 2 phosphodiesterase inhibitors, which inhibit the breakdown of cAMP formed within the cell, differ in their ability to accumulate cAMP when cells are exposed to these agents alone. Isobutylmethylxanthine causes a measurable increase in cAMP, while theophylline, a weak inhibitor of phosphodiesterase, gives a nonsignificant increase in cAMP. Recently, experiments have shown that agents that give only slight increases in cAMP by biochemical measurements, that is, prostaglandins F2 alpha and theophylline, are equally able to activate protein kinase within the cell. Since activation of protein kinase is the only mechanism for an increase in cAMP to have a physiologic effect, all of these agents that do activate protein kinase should cause physiologic effects. Using an explant culture system, we show in this paper that this supposition is correct and that all agents that activate protein kinase do result in inhibition of mitotic activity regardless of whether or not they are able to raise cAMP to a level that can be biochemically measured as being significantly different from the baseline value.