Glucocorticoid in inflammatory proliferative skin disease reduces arachidonic and hydroxyeicosatetraenoic acids.

Psoriasis is a prototype of several common, glucocorticoid responsive, inflammatory proliferative skin diseases. Within 28 hours, glucocorticoid reduced the increased concentration of free arachidonic acid in diseased tissue. This reduction was observed prior to visible improvement of disease and may be an important molecular mechanism for the therapeutic efficacy of glucocorticoids in psoriasis and similar inflammatory diseases.

Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro.

Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha.

Unoccluded retinol penetrates human skin in vivo more effectively than unoccluded retinyl palmitate or retinoic acid.

The formation of all-trans retinoic acid is an oxidative process whereby retinol is converted to retinaldehyde and then to retinoic acid. Because retinol causes qualitative molecular changes similar to those produced by retinoic acid, we compared potency of retinol, retinaldehyde, and retinyl palmitate to retinoic acid and assessed the effects of occlusion. Retinoids were prepared in an experimental vehicle of 95% ethanol:propylene glycol (7:3) with anti-oxidant. Induction of retinoic acid 4-hydroxylase activity was the end point for comparison. Retinoic acid concentrations from 0.001% to 0.05% under occlusion produced a linear dose-response induction of 4-hydroxylase activity. The concentrations of the other retinoids under occlusion required to achieve significant induction of enzyme activity were 0.6% retinyl palmitate, 0.025% retinol, and 0.01% retinaldehyde. The linear dose-response was lost with retinoid concentrations in excess of 0.25% retinol or 0.5% retinaldehyde. Statistical analyses showed no difference in 4-hydroxylase activity between unoccluded and occluded retinol treated sites. By contrast, however, unoccluded sites treated with retinoic acid or retinyl palmitate had less induction of 4-hydroxylase activity than occluded sites. Retinol, retinaldehyde, and retinyl palmitate did not produce erythema but did increase epidermal thickness. Although retinol is a weaker retinoid than retinoic acid, the increased penetration of unoccluded retinol in comparison to unoccluded retinoic acid with this prototypic vehicle confers on retinol a more effective delivery of a retinoidal effect than unoccluded retinoic acid. Retinol at 0.25% may be a useful retinoid for application without occlusion because it does not irritate but does induce cellular and molecular changes similar to those observed with application of 0.025% retinoic acid.

Retinoic acid isomers applied to human skin in vivo each induce a 4-hydroxylase that inactivates only trans retinoic acid.

Application of all-trans retinoic acid to human skin for 4 d under occlusion produces a marked increase in retinoic acid 4-hydroxylase activity. In this study, the possible induction of other hydroxylase in response to 9-cis and 13-cis retinoic acid application to adult human skin in vivo was determined. Application of 0.1% all-trans, 0.1% 9-cis, and 0.1% 13-cis retinoic acid to human skin for 2 d resulted in induction of only all-trans retinoic acid 4-hydroxylase activity. The 4-hydroxylase activity in microsomes from the treated tissue ranged from 838 +/- 46 to 531 +/- 59 pg of 4- hydroxy all-trans retinoic acid formed/min/mg protein (n=6). These same preparations were unable to use 9-cis or 13-cis retinoic acid as substrate for the hydroxylation reaction. Extraction of the retinoic acid isomers from epidermis 48 h after application of 0.1% solution of each isomer yielded significant amounts of all-trans retinoic acid (36-72%) regardless of the isomer applied. The all-trans isomer produced by isomerization of both 9-cis and 13-cis retinoic acids is the likely inducer of the 4-hydroxylase. All-trans retinol and all-trans retinal were unable to compete with all-trans retinoic acid as substrate for 4-hydroxylase enzyme. The 4-hydroxylase induced in response to pharmacological doses of retinoic acids is specific for the all-trans isomer. The inability of 9-cis or 13-cis retinoic acid to induce their own hydroxylation and inactivation or act as substrate for the 4-hydroxylase in skin may have considerable implications in light of the clinical use of retinoids in the treatment of various diseases including cancers.

The retinoid X receptor agonist 9-cis-retinoic acid and the 24-hydroxylase inhibitor ketoconazole increase activity of 1,25-dihydroxyvitamin D3 in human skin in vivo.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] transactivates its target genes via the vitamin D receptor (VDR). VDR functions in physiology as a dimer complexed with retinoid X receptor (RXR), whose natural ligand is 9-cis-retinoic acid (9-c-RA). Inactivation of 1,25(OH)2D3 occurs through a cytochrome P-450 24-hydroxylase (OHase). The promoter of the human 24-OHase gene contains a 1,25(OH)2D3-responsive enhancer element (VDRE). We have used this VDRE containing gene as an endogenous reporter for vitamin D3-mediated gene activation in vivo. Normal adult human skin was keratomed after a 2-d exposure to 1,25(OH)2D3, 9-c-RA, all-trans-RA, and ketoconazole. 1,25(OH)2D3 caused a concentration-dependent increase in 24-OHase mRNA expression as determined by northern blot analysis. The activity of epidermal 24-OHase was also induced by 1,25(OH)2D3. Compared with vehicle, neither of the RA isomers nor ketoconazole alone induced 24-OHase mRNA. Addition of 9-c-RA or t-RA to 1,25(OH)2D3, however, caused a synergistic increase in 24-OHase mRNA. Similarly, 1,25(OH)2D3 plus ketoconazole increased 24-OHase mRNA synergistically. Ketoconazole inhibited ex vivo 1,25(OH)2D3-induced epidermal 24-OHase activity. Thus, 24-OHase mRNA induction is a sensitive reporter of 1,25(OH)2D3 activity in vivo; RXR bound to VDR is not a silent partner in vivo, because 9-c-RA enhances 1,25(OH)2D3-liganded RXR/VDR stimulation of the VDRE containing 24-OHase gene; ketoconazole inhibition of 24-OHase enhances 1,25(OH)2D3 activity by impeding its breakdown. Thus, the synergistic response of human skin to topical 1,25(OH)2D3 and/or 1,25(OH)2D3 analogs plus RXR retinoids and/or ketoconazole may be exploited to give a desired biologic/therapeutic response with less 1,25(OH)2D3, minimizing the potential calcemic risk from systemic absorption of 1,25(OH)2D3.

Determination of 5,12, and 15-lipoxygenase products in keratomed biopsies of normal and psoriatic skin.

Keratomed epidermal tissue from normal individuals and from the lesional and non-lesional skin of psoriasis patients served as source materials for the extraction, separation, and quantitation of eicosanoids that may be important to cutaneous function and pathophysiology. The eicosanoids were extracted in ethanol and buffer, partially purified on SEP-PAKs, separated by reverse phase microbore high-performance liquid chromatography, and quantitated by radioimmunoassay or integration of absorbency peaks obtained by high performance liquid chromatography. The involved areas from psoriasis patients contained a statistically significant seven- to 11-fold increase in the levels of leukotriene B4, 13-hydroxyoctadecadienoic acid-like compound, 15-hydroxyeicosatetraenoic acid compound and 12-hydroxyeicosatetraenoic acid in comparison to normal samples and a three- to sevenfold increase in comparison to uninvolved tissue. The uninvolved areas contained 40% to 100% increases in the levels of these compounds in comparison to normal tissue; these increases were statistically significant except for 13-hydroxyoctadecadienoic acid-like compound. From a single keratome biopsy, multiple eicosanoids can be separated and quantitated; in addition, levels before, during, and after therapy for psoriasis may be compared.

Liarozole inhibits human epidermal retinoic acid 4-hydroxylase activity and differentially augments human skin responses to retinoic acid and retinol in vivo.

Metabolic inactivation of all-trans retinoic acid to 4-hydroxy retinoic acid occurs via a cytochrome P-450 enzyme. We investigated the effects of liarozole on the retinoic acid 4-hydroxylase activity of human epidermis and its ability to modify in vivo human skin responses to retinoic acid and all-trans retinol. Retinoic acid 4-hydroxylase activity induced in vivo by 4 d treatment with retinoic acid (0.1%) was inhibited in vitro by liarozole in a concentration-dependent manner. Comparable micromolar concentrations of liarozole were extracted from stratum corneum-free epidermis treated with 3% liarozole. Retinoic acid levels in liarozole-treated skin increased to 19 +/- 5 ng/g wet wt (mean +/- SEM, p < 0.002, n = 17) at 18 h and to 6 +/- 2 ng/g wet wt (p = 0.38, n = 17) at 48 h as compared to vehicle (not detectable). At 48 h, retinoic acid 4-hydroxylase activity was induced 9-fold over vehicle (p < 0.03, n = 8). At 96 h, no significant erythema or increased epidermal thickness was found when either retinoic acid (0.001%), all-trans retinol (0.0250%), or liarozole (3%) was applied individually, but when 0.001% retinoic acid and 3% liarozole were applied together, both erythema and increased epidermal thickness occurred. In contrast, 0.025% all-trans retinol and 3% liarozole together caused increased epidermal thickness but no erythema. These data demonstrate that, at doses used here, liarozole, although an effective inhibitor of retinoic acid 4-hydroxylase, cannot function alone like a retinoid in vivo, probably because of retinoic acid 4-hydroxylase induction. In the presence of a low dose retinoic acid or all-trans retinol, however, liarozole can amplify human skin responses to each retinoid in a manner characteristic of the retinoid at a higher dose (erythema and hyperplasia with retinoic acid; no erythema but hyperplasia with all-trans retinol).

Papaverine and Ro 20-1724 inhibit cyclic nucleotide phosphodiesterase activity and increase cyclic AMP levels in psoriatic epidermis in vitro.

The comparative inhibitory potency of papaverine and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) on cyclic AMP-phosphodiesterase (cAMP-PDE) and cyclic GMP-phosphodiesterase (cGMP-PDE) activities and their effect on the levels of cAMP and cGMP were examined in psoriatic epidermis. At concentrations of 5 X 10(-4) M, papaverine inhibited the hydrolysis of both cAMP and cGMP by either the low or high Km psoriatic epidermal PDE nearly 100% (p less than .0001) while Ro 20-1724 selectively inhibited the hydrolysis of cAMP 94% (p less than .0001) but had no significant effect on cGMP hydrolysis. When keratomed psoriatic epidermal slices were incubated in 5 X 10(-4) M papaverine or Ro 20-1724 the tissue levels of cAMP were increased 343% or 1395% respectively (p less than .001) with no concomitant change in the levels of cGMP. Selective inhibition of cAMP hydrolysis by Ro 20-1724 and its greater effectiveness in elevating cAMP levels in slices of psoriatic epidermis is one explanation for its clinical superiority in treating psoriatic lesions.

In vitro synthesis of 12-hydroxy-eicosatetraenoic acid is increased in uninvolved psoriatic epidermis.

Certain arachidonic acid (AA) metabolites have been detected in psoriatic skin lesions. In this study the capacity of normal epidermis and clinically uninvolved psoriatic epidermis to transform AA into lipoxygenase products was determined in vitro. After incubating homogenized epidermis with exogenous AA, the extracted lipids were isolated by reverse-phase high-performance liquid chromatography. Each chromatographic peak was characterized by its UV absorption spectrum and identified by its coelution with the appropriate authentic standard and by radioimmunoassay of its eluate fraction. Identified compounds were quantitated by integrated UV absorbance. Leukotriene B4 (LTB4) was also identified by neutrophil chemokinesis. Normal epidermis generated 15-hydroxy-eicosatetraenoic acid (15-HETE) and 12-HETE, the latter being more abundant. 5-Lipoxygenase products (LTB4, LTC4, and 5-HETE) were not detected. However, an unknown compound exhibiting a triplet UV absorbtion spectrum with maximum at 274 mm was formed. Its formation was inhibited by 5,8,11,14-eicosatetraynoic acid, but not by indomethacin or a specific 5-lipoxygenase inhibitor (REV 5901). These data suggest that a di-HETE with a triene structure is one possible candidate for the unknown compound. Compared with normal epidermis, the formation of 12-HETE and the unknown di-HETE by uninvolved psoriatic epidermis was increased by 54% and 63%, respectively. The formation of 12-HETE and the unknown di-HETE in uninvolved psoriatic epidermis was stimulated to the same degree in the presence of the phospholipase inhibitor quinacrine. These results indicate that uninvolved psoriatic epidermis has an increased capacity to metabolize free AA into 12-lipoxygenase products.

Epidermal chalone--past to present concept.

How the constantly renewing epidermis maintains its homeostatic state remains a mystery. A "negative feedback" mechanism of control with chalone, a tissue-specific, species-nonspecific inhibitor of mitosis, has been suggested by Bullough and Laurence. Progress in isolating, purifying, and characterizing epidermal chalone has been slow and difficult because of the lack of a fast, assurate, and meaningful assay system. A G2 assay (depending upon mitotic counts) was used to obtain the data which served as the basis for postulating the existence of chalone. Since a control point with the G1 phase of the cell cycle is considered to be the one with physiologic implications, in vivo and in vitro assays with pure primary epidermal cell cultures have been used to investigate a G1 block. The incorporation of tritiated thymidine into DNA is often used as a measure of the rate of prokiferation. However, inhibition of thymidine transport and phosphorylation have been encountered in several systems and negate this method as an accurate measure of cell proliferation.

Cyclic AMP, cyclic GMP, and glucocorticoids as potential metabolic regulators of epidermal proliferation and differentiation.

The two cyclic nucleotides, cyclic AMP and cyclic GMP, appear to be central to the metabolic regulation of cell proliferation and differentiation in various cells. Moreover, in many systems glucocorticoids appear to act in concert with or parallel to cyclic AMP. The available evidence suggests that these three molecular species--cyclic AMP, cyclic GMP, and glucocorticoids--may be essential to the normal regulation of epidermal proliferation and differentiation. In 1970, we suggested that perturbed epidermal homeostasis, exemplified by psoriasis, might be associated with low cellular levels of cyclic AMP and, in 1972, with high levels of cyclic GMP as well. Subsequent measurements of these two cyclic nucleotides in our laboratory showed a probable reduction in the cyclic AMP/cyclic GMP ratio in lesional psoriatic tissue. This led to the hypothesis that the cardinal features of psoriatic epidermis--glycogen accumulation, excessive proliferation, and reduced cell specialization--are the results of this reduced ratio. A corollary of this hypothesis was that a psoriatic lesion could not begin or exist without this altered cyclic nucleotide ratio. Recently, four different agents--lithium, a beta adrenergic blocking agent, antimalarials, and iodide--have been found to exacerbate psoriasis and to reduce the formation of cyclic AMP in various tissues. Consequently we believe that cyclic nucleotides are of central importance in the pathogenesis of the epidermal component of psoriasis.

Extraction of human epidermis treated with retinol yields retro-retinoids in addition to free retinol and retinyl esters.

Vitamin A, all-trans-retinol, is metabolized to retinoic acid in vivo by a tightly controlled two-step conversion. Retinoic acid then binds to nuclear receptors and modulates cellular proliferation and differentiation. Because only a small fraction of retinol applied topically can be metabolized to retinoic acid, alternative pathways of retinol metabolism in skin were investigated. Retinol (0.4%) was applied to adult human skin under occlusion for 6 h to 4 d. The conversion of retinol into various metabolites such as 14-hydroxy-4,14-retro-retinol, anhydroretinol, 4-oxo-retinol, retinyl esters, and retinyl glucuronides was investigated. The level of 14-hydroxy-retro-retinol was increased from undetectable at time 0 to 326 ng/g wet weight of tissue at 6 h (6% of the retinol level) and maintained approximately the same concentration at 24 h to 409 ng/g wet weight (1.9% of the retinol level); it decreased to 48 ng/g wet weight of tissue (12% of its maximum level) by 4 d. Anhydroretinol was undetectable at time 0, increased only slightly at 6 h, and remained at the same level. We did not detect 4-oxo-retinol. Because 14-hydroxy-retro-retinol was found in the retinol-treated areas, its effects on epidermis were compared with those of retinol. Topical application of trans-retinol (0.3%) significantly increased both epidermal thickness and cellular retinoic acid binding protein II mRNA, whereas 14-hydroxy-4,14-retro-retinol (0.3%) did not increase either of these well-characterized cutaneous retinoid responses. Retinol, when applied topically in pharmacologic doses to human epidermis, remained as free retinol, was metabolized primarily to retinol ester, and was metabolized to a lesser extent to retro-retinoids and didehydroretinol.

Dermis-derived 15-hydroxy-eicosatetraenoic acid inhibits epidermal 12-lipoxygenase activity.

The purpose of the present study was to analyze the metabolism of arachidonic acid (AA) in normal human dermis. After incubating homogenized dermis with exogenous AA, the extracted lipids were isolated by reversed-phase high-performance liquid chromatography. Each chromatographic peak was characterized by its UV absorption spectrum and identified by its coelution with the appropriate authentic standard and by radioimmunoassay of its eluate fraction. Identified compounds were quantitated by integrated optical density. Homogenized human dermis transformed AA into both cyclooxygenase and lipoxygenase products, but predominantly 15-hydroxy-eicosatetraenoic acid (15-HETE). Cultured fibroblasts from normal human dermis also mainly metabolized AA into 15-HETE. To determine whether dermis-derived 15-HETE might modify the AA metabolism of epidermis, normal human epidermis was incubated with dermis. Increasing amounts of dermis resulted in an increasing inhibition of epidermal 12-HETE formation. Similarly, 15-HETE alone induced a dose-dependent decrease of epidermal 12-HETE formation, while the formation of prostaglandin E2 was unchanged. Since 12-HETE possess the ability to elicit skin inflammation and to stimulate epidermal DNA synthesis, 15-HETE formation may be a way whereby dermis regulates important epidermal activities.

In vitro model of essential fatty acid deficiency.

The polyunsaturated fatty acids linoleic acid (18:2, n-6) and arachidonic acid (20:4, n-6) are essential for normal skin function and structure, both as eicosanoid precursors and as components of lipids forming cell membranes. Adult human keratinocytes grow optimally in serum-free medium (MCDB 153) that contains no fatty acids. These keratinocytes expand rapidly and produce normal epidermis upon in vivo grafting. Analysis of lipid extracts of epidermis and of cultured keratinocytes was done to determine the fatty acid composition of cells grown in essential fatty acid (EFA)-deficient medium. Gas chromatography and high-performance liquid chromatography analyses were done of the fatty acids in the entire cell and in a thin-layer chromatography separated fraction containing those lipids that form cellular membranes. Comparison of snap-frozen epidermis and epidermal basal cell suspensions to passage 1 to 4 cultures shows that the cells are in an extreme essential fatty acid-deficient state by the first passage. The amount of the saturated fatty acids 16:0, 18:0, and 14:0 is unchanged by culture. The polyunsaturated fatty acids are found to be significantly decreased, the cells balancing their lack with a significant increase in the relative abundance of the monounsaturated fatty acids, 18:1 and 16:1. Greater than 85-90% of the fatty acids was found in lipids associated with membranes and no unusual fatty acids were detected. Because the serum-free medium is fatty acid free and the cells cannot synthesize essential fatty acids, the rapid division of the cells results in the predominance of an extreme EFA-deficient cell type. The essential fatty acid-deficient keratinocyte is an excellent adult, normal epidermal cell model that can be used to study EFA deficiency and the effect of the eicosanoid and fatty acids on cell function and structure.

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells.

Tissue levels of all-trans retinoic acid (RA) are maintained through coordinated regulation of biosynthesis and breakdown. The major pathway for all-trans RA inactivation is initiated by 4-hydroxylation. A novel cytochrome P-450 (CYP26) that catalyzes 4-hydroxylation of all-trans RA has recently been cloned. We have investigated regulation and properties of RA 4-hydroxylase in immortalized human keratinocyte HaCaT cells. In the absence of added retinoid, RA 4-hydroxylase (CYP26) mRNA and protein were minimally detected. Addition of all-trans RA rapidly induced RA 4-hydroxylase mRNA (within 2 h) and activity (within 6 h). Induction of both mRNA and activity was transient, returning to baseline within 48 h, and completely dependent on mRNA synthesis (i.e., blocked by actinomycin D). The synthetic retinoid CD367, which specifically activates nuclear RA receptors, also rapidly induced RA 4-hydroxylase activity. This induction, however, unlike that of all-trans RA, was long-lived (>48 h). This difference was attributable to lack of metabolic inactivation of CD367 in HaCaT cells. CD2665, which inhibits RA receptor-dependent gene transcription, blocked retinoid induction of RA 4-hydroxylase, indicating that it is mediated by RA receptors. Addition of excess unlabeled substrates specific for 10 distinct mammalian P-450 subfamilies did not compete with all-trans RA for RA 4-hydroxylase activity. RA 4-hydroxylase did not hydroxylate 9-cis RA or 13-cis RA. Inhibition of RA 4-hydroxylase activity by ketoconazole potentiated activation of RA receptors by all-trans RA. In summary, RA 4-hydroxylase is a unique, highly specific cytochrome P-450 isoenzyme, whose expression is regulated by its natural substrate, all-trans RA, through activation of RA receptors. RA 4-hydroxylase functions to limit the levels, and thereby the biologic activity of all-trans RA in HaCaT cells.

Regulation of cell cycles.

Of the major achievements in cell biology during the last 25 years, none is more important than the understanding of regulation of cell cycles. In 1953 two fundamental observations concerning DNA were made. Watson and Crick suggested that the three-dimensional structure of DNA exists as a double helix with specific base pairings, and Howard and Pelc observed that DNA is replicated during a specific phase in the mitotic cycle. Thus developed the theory of cell cycles. Next, investigators explored which events occur during each phase of the cycle and what controls the readout of the genes of proliferation or differentiation. In 1961, Jacob and Monod proposed that for prokaryotic cells the operon is the mechanism which controls the readout of the genes; and by the end of the 1960s, several investigators had defined the role of cyclic AMP and its mechanism of action at the gene level. The control mechanisms of eukaryotic cells are less well defined. Basically there are two types of regulatory molecules: those that arrive at the cell surface and send messages inside the cell; and those that enter the cell, bind to receptors, and then enter the nucleus to interact with the genes. During the past five to ten years, the cell surface and its receptors have received considerable attention as the recognition and control areas for cell proliferation and differentiation, and currently the role of the cyclic nucleotides and prostaglandins is being investigated. Various model systems are now available for detailed studies of these control mechanisms.

Topical cyclosporine A inhibits the phorbol ester induced hyperplastic inflammatory response but not protein kinase C activation in mouse epidermis.

Cyclosporine A (CsA) is efficacious in the treatment of psoriasis. Although CsA is known to inhibit T-lymphocyte proliferation in vitro, whether this is its mode of action in psoriasis is uncertain. 12-0-tetradecanoylphorbol-13-acetate (TPA) induces an inflammatory, hyperplastic response in mouse skin, with many of the biochemical and histologic aberrations that occur in psoriatic epidermis. Protein kinase C is the major cellular phorbol ester receptor, and most responses of cells to TPA are mediated by PK-C, which is directly activated by TPA. We therefore have investigated the effects of CsA on pleiotypic responses induced by TPA and whether CsA acts in vivo as a direct inhibitor of PK-C. Simultaneous application of CsA (1.7 mumol) and TPA (10 nmol) to mouse skin significantly reduced inflammatory cell infiltration and increased epidermal thickness induced by TPA treatment alone. CsA had to be applied within 30 min of TPA application in order to have a significant inhibitory effect. Optimal doses of CsA inhibited TPA-induced ODC activity, TGase activity, arachidonic acid release, and interleukin-1 beta (IL-1 beta) mRNA to the same degree (approximately 80%), despite measurement at widely different times (30 min-12 h) required to obtain maximal induction by TPA. CsA did not, however, directly inhibit activation of PK-C by TPA. These data demonstrate that CsA blocks the pleiotypic responses of mouse skin to TPA treatment involving biochemical events, inflammatory cell infiltration, and epidermal hyperplasia. The molecular site(s) of action of CsA appears to be distal to the initial activation of PK-C by TPA and clearly inhibits PK-C inducible events. Furthermore, the above data suggest that CsA may directly affect keratinocytes in vivo.

Levels of cyclosporin in epidermis of treated psoriasis patients differentially inhibit growth of keratinocytes cultured in serum free versus serum containing media.

Cyclosporin A, which is a specific immunosuppressive compound, has recently been demonstrated to be of significant benefit in the treatment of psoriasis. Because hyperplasia is a major feature of psoriasis, we have investigated whether this drug acts directly to inhibit keratinocyte growth. We have determined the concentration range of cyclosporin in the epidermis of psoriatic patients undergoing cyclosporin therapy and the effect of this concentration range on the growth of cultured keratinocytes. After 7 days of treatment, psoriatic involved epidermis contained 1.1 +/- 0.3 ng cyclosporin/micrograms DNA. Based on tissue wet weight this is approximately 2.8 micrograms cyclosporin/ml. This value was 10 times that of trough blood samples. On day 3 of treatment, involved epidermis contained 7 times more cyclosporin than scale, while on day 7 this ratio was reduced to 2. This suggests that cyclosporin was initially associated with the lower layers of the epidermis and distributed upward with time. Exposure of adult human keratinocytes, cultured on collagen in the presence of human serum, to cyclosporin (0.1-30 micrograms/ml, 0.4-13-fold higher than lesional cyclosporin) for 2-6 d had no effect on the rate of incorporation of thymidine into DNA. Cyclosporin (1-30 micrograms/ml) also had not effect on the reinitiation of DNA synthesis of quiescent cells subsequent to the reintroduction of serum. In contrast, cyclosporin (1-10 micrograms/ml) inhibited growth of keratinocytes cultured on plastic culture dishes in serum free media (MCDB 153). For a given dose of cyclosporin, cell associated drug was 2-3 times greater in serum free compared to serum containing media. This may contribute in part to the sensitivity of keratinocytes in serum free media to growth inhibition by cyclosporin. These data demonstrate that human epidermis contains a high concentration of cyclosporin after oral administration, and that, under certain conditions, concentrations of cyclosporin within the range found in vivo can inhibit growth of cultured keratinocytes. Because cyclosporin regulates lymphocyte function in vivo and in vitro, the demonstrated antiproliferative effects of cyclosporin on psoriatic keratinocytes in vivo may be due to inhibition of a mononuclear leukocyte-derived keratinocyte growth factor(s) in combination with direct inhibition of keratinocyte growth.

Papaverine: its effects on cyclic AMP in vitro and psoriasis in vivo.

Psoriatic epidermis shows excessive cell proliferation, incomplete morphogenesis, and glycogen accumulation. These three features may result from coordinate misregulation by the previously reported abnormal lesional ratio of cyclic AMP/cyclic GMP. If so, it is possible that psoriasis might improve by topical application of an agent known to alter cellular levels of cyclic nucleotides. In the present study papaverine (3.0 x 10-4 m) produced a 210percent increase (.02 smaller than p smaller than .05) in epidermal cyclic AMP levels in vitro. Therefore, 1 percent papaverine cream (approximately 2.7 x 10-2m papaverine) was chosen to establish whether a cyclic AMP elevating agent could improve a soriatic lesion. A double-blind comparison of 1 percent papaverine cream versus cream alone was conducted on matched lesions in 45 patients. Of those patients showing differential improvement (i.e., only one lesion of the pair improved), 81 percent (p=0.011) of those showing a response in the center of the lesion and 77 percent (p=0.046) of those improving at the edge had applied papaverine. Although no attempts were made to develop a papaverine formulation with immediate therapeutic utility, the numerically significant papaverine results suggest future therapeutic developments.

Decreased urinary polyamines in patients with psoriasis treated with etretinate.

Oral administration of the aromatic retinoid etretinate is effective therapy for psoriasis and other epidermal hyperproliferative disorders. Since polyamine metabolism is known to be important in cell growth and differentiation, we measured urinary levels of the polyamines putrescine, spermidine, and spermine as a reflection of cutaneous polyamine metabolism in 19 psoriatic patients treated with etretinate for 16 weeks. Using thin-layer chromatography, polyamine determinations were performed on urine collected pretherapy, during therapy, and 8 weeks after therapy was concluded. Good to excellent clearing of psoriasis occurred in 18 of 19 patients. All urinary polyamines showed a downward trend in the first week of therapy, prior to significant clinical improvement. At week 16 of therapy, the greatest reduction in mean urinary polyamine content occurred. Mean putrescine levels decreased from pretherapy to week 16 by 27% (p less than 0.001), mean spermidine values fell by 34% (p less than 0.001), and mean spermine levels declined by 37% (p = 0.005). These data are consistent with the hypothesis that etretinate inhibits polyamine biosynthesis.