Human epidermis transforms exogenous leukotriene A4 into peptide leukotrienes: possible role in transcellular metabolism

Leukotriene B₄ formation can take place by cell interaction between keratinocytes and neutrophils. Thus, keratinocytes without proven 5-lipoxygenase activity can transform neutrophil-derived leukotriene A₄ into leukotriene B₄. The purpose of the present study was to investigate whether human epidermis is able to transform leukotriene A₄ sequentially into the peptide leukotrienes (LTC₄, LTD₄ and LTE₄). Epidermis isolated using the suction blister technique or keratomed skin specimens were incubated with either neutrophils or exogenously added leukotriene A₄. Peptide leukotrienes were determined by integrated optical density after RP-HPLC separation, and the identity of leukotrine C₄ was confirmed by (1) the retention time similarity with authentic leukotriene C₄; (2) the UV spectrum determined with an on-line diode array detector; and (3) conversion by -glutamyl transpeptidase of the peak coeluting with authentic leukotriene C₄ into a new peak coeluting with authentic leukotriene D₄. The results of this study showed that while human epidermis cannot form detectable amounts of peptide leukotrienes by itself, it can transform exogenous leukotriene A₄ into peptide leukotrienes. Furthermore, coincubation of human epidermis and neutrophils resulted in a marked increase (90%) in peptide leukotriene formation when compared with neutrophils alone, indicating that human epidermis can transform neutrophil-derived leukotriene A₄ into peptide leukotrienes. These results indicate that human skin contains leukotriene C₄ synthase activity capable of producing significant amounts of leukotriene C₄ from leukotriene A₄, and that the keratinocytes may play a more active role in peptide leukotriene formation in the skin than previously thought. Because neutrophil migration into the epidermis can provide the keratinocytes with leukotriene A₄, transcellular leukotriene biosynthesis may be important for peptide-leukotriene synthesis during skin inflammation.     

The effect of topical retinoids on the leukotriene-B4-induced migration of polymorphonuclear leukocytes into human skin

Summary Systemic retinoids are effective in a variety of inflammatory dermatoses. Disorders in which polymorphonuclear leukocytes (PMN) are involved, such as psoriasis and acne, respond particularly well to various retinoids. However, side-effects restrict the use of systemic retinoids to severe manifestations. Topical application might provide the possibility of avoiding the systemic side-effects of these compounds. In this communication we report on the modulation of transepidermal migration of PMN by topical application of all-trans-retinoic acid, 13-cis-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone. Test areas of healthy volunteers were pre-treated with these retinoids in a cream base and with corresponding placebo creams, and intraepidermal accumulation of PMN was quantified 24 h after epicutaneous challenge with leukotriene B₄ (LTB₄), using elastase as a marker enzyme. Topical treatment with 13-cis-retinoic acid resulted in a marked and statistically significant inhibition of the LTB₄-induced migration of PMN. All-trans-retinoic acid, arotinoid methyl sulphone and arotinoid ethyl sulphone reduced the accumulation of PMN slightly, but not statistically significantly. Topical treatment with arotinoid methyl sulphone had no effect.     

The chemokinetic response of psoriatic and normal polymorphonuclear leukocytes to arachidonic acid lipoxygenase products

Summary Polymorphonuclear leukocytes (PMN) from ten patients with chronic stable plaque psoriasis, five of whom had more than 40% skin involvement and five with less than 20% involvement, responded in a dose-related fashion to stimulation with the arachidonic acid lipoxygenase products 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B₄ (LTB₄) in an in vitro chemokinesis assay. There was no significant difference in either the random migration or the chemokinetic response of psoriatic PMN to LTB₄ when compared to the responses of PMN from a group of age- and sex-matched healthy controls. Psoriatic PMN migrated further in response to low doses of 5- and 12-HETE although the distance moved after maximal stimulation was no different to that observed in controls. No significant difference was observed in the responses of PMN obtained from patients with less than 20% skin involvement when compared to those with more extensive psoriasis. The small differences measured between the chemokinetic responses of psoriatic and control PMN to the lipoxygenase products tested are unlikely to be of pathogenetic significance.     

15-Hydroxyeicosatetraenoic acid (15-HETE) specifically inhibits the LTB4-induced skin response

Summary 15-Hydroxyeicosatetraenoic acid (15-HETE), a 15-lipoxygenase product of arachidonic acid, inhibits leukotriene B₄ (LTB₄)-induced chemotaxis of polymorphonuclear leukocytes (PMNs) in vitro. In this study the effects of intradermal injections of LTB₄ were determined in the absence or presence of 15-HETE. For comparison intradermal injections of purified human complement split product C5a were performed in the absence or presence of 15-HETE. The skin response was evaluated by measuring the diameter of the wheal, the area of the flare and by intensity of the erythema (erythema index). LTB₄ and C5a were injected at the concentration of 200 ng/ml. At this concentration the maximal skin response of LTB₄ and C5a were equivalent. In contrast to C5a reaction, which resolved within 1 h, LTB₄-induced skin response lasted up to 18 h. In all subjects the skin response was significantly decreased when LTB₄ was injected together with 300 ng of 15-HETE. The decrease of wheal, flare, and erythema index averaged 81.9%, 56.6%, 53.6%, respectively, when all parameters were obtained at the maximal skin response. In contrast, the C5a-induced skin response was not affected by addition of 15-HETE, even when the final dose of 15-HETE was increased 10 times to 3 g. The LTB₄-induced reaction could last up to 18 h after injection. After the addition of 300 ng of 15-HETE the skin response resolved after 1 h. The present results demonstrate that 15-HETE is a specific inhibitor of the LTB₄-induced skin response and brings additional evidence in support of the ability of 15-HETE to regulate the proinflammatory effects of LTB₄ in vivo.     

Chemotactic lipoxygenase products in sera from patients with psoriasis

Summary Sera obtained from 12 patients with moderate to severe psoriasis were investigated for the presence of arachidonate lipoxygenase metabolites using reverse-phase high-performance liquid chromatography after extraction on silicic acid columns. Peaks which co-chromatographed with standards of synthetic leukotriene B₄ (LTB₄) were collected, and the material was tested for chemotactic activity. In the sera of 5 of the patients, chemotactic activity was demonstrable in these LTB₄ peaks. Although minor peaks cochromatographing with LTB₄ were found in control sera, none of them contained chemotactic material. Isolated monocytes from the psoriasis patients showed enhanced chemotactic activity as compared to monocytes obtained from healthy controls. The results of our study support the view that abnormal 5-lipoxygenase activity is present in psoriasis. Further investigation is required to determine whether LTB₄ is released from circulatory leukocytes or the skin.     

Lymphocytes and macrophages of the epidermis and dermis in lesional psoriatic skin,but not epidermal Langerhans cells,are depleted by treatment with cyclosporin A

Summary Since cyclosporin A (CsA) is an immuno-suppressive agent, its beneficial effect in psoriasis suggests that immune cells may play a role in the pathogenesis and resolution of psoriasis. To determine early effects of CsA in psoriasis, we quantitated immune cells using double immunofluorescence microscopy on biopsy specimens obtained prior to therapy and after 3,7, and 14 days of CsA therapy. CsA therapy resulted in significant reductions in the absolute number of immune cells (including T cells, monocytes/macrophages, and antigen presenting cells) contained within psoriatic skin. The effect was rapid, with over one-half of the reduction in the density of HLe1⁺ (human leukocyte antigen-1 positive or bone marrow derived) cells, including T cells, activated T cells, monocytes, and Langerhans cells (LCs), occurring within 3 days. Despite the overall reduction in the numbers of immunocytes in the skin, the proportion of T cells, Langerhans cells, and monocytes in relation to the total number of immune cells was unchanged with therapy, reflecting equally proportional losses of each subtype. Dermal CD1⁺DR⁺ cells (putative Langerhans cells), which are not found in normal skin but are present in lesional psoriasis skin, were virtually cleared from the papillary dermis after CsA therapy. Although absolute numbers of epidermal Langerhans cells, defined as cells expressing both CD1 (T6) and DR molecules (CD1⁺DR⁺), were also reduced after CsA, epidermal non-Langerhans CD1^-DR⁺ cells (macrophages, activated T cells, DR^- keratinocytes) demonstrated a proportionally greater decrease, with the ratio of CD1⁺DR⁺ Langerhans cells/non-Langerhans CD1^-DR⁺ epidermal cells changing from a mean of 0.82 at baseline to 1.92 at day 14. Thus, early in the course of therapy, CsA appears to be effective at clearing CD1^-DR⁺ cells while leaving LC relatively intact in the epidermis.This work was supported in part by the Babcock Foundation     

Effect of dithranol treatment on arachidonic acid and its lipoxygenase products in psoriasis

Summary The concentrations of arachidonic acid and its lipoxygenase metabolites were measured in exudates from lesional psoriatic skin during treatment with 0.25% dithranol (anthralin) applied topically for 10 days. Dithranol caused a reduction in the concentration of arachidonic acid at 10 days (167±42 and 358±55 ng ml^-1 treated and control sites respectively, p<0.05) concurrent with clinical improvement of the lesions. Neither 12-hydroxyeicosatetraenoic acid nor leukotriene B₄ concentrations were significantly affected. These results do not support the view that lipoxygenase products are of major importance in the pathogenesis of psoriasis.     

Inhibitory effects of sulfonated shale oils (ammonium bituminosulphonates,Ichthyols) on enzymes of polyenoic fatty acid metabolism

The two commercial pharmaceutical preparations of ammonium bituminosulphonates, Leukichthol and Dark Ichthyol, were shown to inhibit the formation of 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) from external arachidonic acid by human polymorphonuclear leukocytes stimulated by ionophore A-23187 in a dose-dependent manner. Pure arachidonate 15-lipoxygenases from rabbit reticulocytes and soya beans, and the particulate prostaglandin endoperoxide synthase from sheep vesicular glands, were also inhibited. With the reticulocyte lipoxygenase, the Ichthyols suppressed the enzyme activity by two different mechanisms: (1) a prolongation of the lag period typical of lipoxygenase catalysis, and (2) by a lowering of the maximal enzymatic activity after the end of lag period. As expected, the first effect was reversed by the addition of the lipoxygenase product 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE). Ammonium bituminosulphonates are thus universal inhibitors of lipoxygenase activities, and the latter are of potential importance in inflammatory dermatoses.     

Prostaglandin E1 in normal human skin: Methodological evaluation,topographical distribution and data related to sex and age

Summary A methodological evaluation of a radioimmunoassay technique for PGE₁ measurement was applied to normal human skin. The detection limit of the assay was 15 pg and recovery (mean±SD) was 90±6%. The mean value of PGE₁ (±SEM) in nine punch biopsy specimens (4 mm in diameter) from a piece of skin surgically removed from one person was 117±14 pg/mg dry weight. The individual variation of PGE₁ activity in biopsy specimens from the same topographical area in ten persons belonging to the same sex and age group was 33±4 pg/mg dry weight. The influence has been elucidated of temperature and tissue processing, local anaesthesia, sex, age and topographical distribution on endogenous PGE₁ activity.Supported by grant 512–8125, 512–15539 and 12–1690 from the Danish Medical Research Council     

In situ depletion of CD4<Superscript>+</Superscript> T cells in human skin by Zanolimumab

CD4⁺ T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4⁺ T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, κ) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4⁺, but not CD8⁺ CD3⁺ T cells. The capacity of Zanolimumab to deplete the CD4⁺ T cells in the skin may be of importance in diseases where CD4⁺ T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.     

Effect of dihomogammalinolenic acid and its 15-lipoxygenase metabolite on eicosanoid metabolism by human mononuclear leukocytes in vitro: selective inhibition of the 5-lipoxygenase pathway

Summary The purpose of the present study was to determine the effect of the n-6 fatty acid, dihomogammalinolenic acid (DGLA, 203, n-6) on arachidonic acid (AA) (C204) metabolism by human peripheral mononuclear leukocytes (HPML). After incubation of HPML with A23187 (5 M) and DGLA, the cyclooxygenase (CO) and lipoxygenase (LO) products were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC) combined with radioimmunoassay. DGLA led to no change in PGE₂ formation, but at similar concentrations there was a dose-dependent decrease in LTB₄ formation (IC₅₀=45.0 M). The inhibition of LTB₄ formation by DGLA was associated with a dosedependent increase in its 15-LO metabolite 15-hydroxyeicosatrienoic acid (15-HETrE) and its CO metabolite prostaglandin E₁ (PGE₁). Incubation of HPLM with 15-HETrE (0–1.5 M) alone did not result in a change in PGE₂ formation, whereas 15-HETrE was a much more potent inhibitor of LTB₄ formation (IC₅₀=0.5 M) than DGLA. These results show that the addition of DGLA to HPML results in a selective inhibition of LTB₄ formation, presumably via its metabolite (15-HETrE).     

In vitro inhibition of leukotriene B4 formation by exogeneous 5-lipoxygenase inhibitors is associated with enhanced generation of 15-hydroxy-eicosatetraenoic acid (15-HETE) by human neutrophils

Summary Leukotrienes, products of the 5-lipoxygenase pathway of arachidonic acid metabolism, have been suggested to play a pathogenic role in psoriasis, because of their ability to induce skin inflammation and to stimulate epidermal proliferation. The 15-lipoxygenase product 15-hydroxy-eicosatetraenoic acid (15-HETE) has no proinflammatory capacity. In contrast, it can inhibit the activity of the 5-lipoxygenase. The purpose of the present study was to study the effect of 5-lipoxygenase inhibitors on the formation of 15-HETE by human neutrophils in vitro. Purified neutrophils were incubated with A 23187 (5 M) and arachidonic acid (25M) with and without different inhibitors of 5-lipoxygenase activity (RS 43179, benoxaprofen, NDGA, and CP 66248). Methods for identifying eicosanoids included RP-HPLC and radioimmunoassay. Formation of leukotriene B₄ was inhibited in a dose-dependent way, which was strongly correlated with a concomitant increase in the formation of 15-HETE (r=0.97, p<0.01). The cyclooxygenase inhibitor indomethacin did not change 15-HETE formation. The stimulation of 15-HETE formation was not associated with cell damage as assessed by LDH release. Furthermore, identical incubations of T lymphocytes, characterized by a low 5-lipoxygenase activity, did not result in increased 15-HETE formation. These results show that inhibition of 5-lipoxygenase activity can lead to increased formation of 15-HETE. Because 15-HETE inhibits formation of 5-LO products, it may amplify the effect of 5-lipoxygenase inhibitors.Part of this work has been presented at the 17th annual meeting of the European Society for Dermatological Research (ESDR), Amsterdam, The Netherlands, 29 March–1 April, 1987     

Alpha1-proteinase inhibitor in psoriasis: reduced activity in symptom-free patients and during flare

Summary The aim of this study was to quantitate the active fraction of the ₁-proteinase inhibitor (₁-PI) in psoriasis. Serum proteinase inhibitory capacity was measured vs porcine pancreatic elastase of a known active fraction against its specific substrate (Suc-Ala₃-pNA). The inhibitory capacity was determined in 21 symptom-free patients, 134 patients with skin lesions, and 23 healthy volunteers. Alpha₁-PI was found to be significantly decreased in symptom-free patients and in those with stationary lesions, in a manner similar to the reduced activity of neutrophil proteinases, elastase, and cathepsin G. The synthesis of ₁-PI was stimulated during the appearance of active psoriatic lesions, but to a much lesser degree in patients with early onset (21 years) than in patients with late onset of psoriasis (>21 years). The early onset subgroup differed by a more frequent familial occurrence of psoriasis and a more severe course of the disease. The data indicate that the regulation of the proteinase-₁-PI system in psoriasis is abnormal and this may contribute to the pathogenesis of the disease. The decreased ₁-PI during flare may be responsible for the disease activity, at least in patient with early onset of psoriasis.Presented in part at the 4th International Psoriasis Symposium, 6–11 July 1986, Stanford, USA     

Leukotriene B4 enhances adherence of human polymorphonuclear leukocytes to dermal microvascular endothelial cells in vitro

Summary Adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells (ECs) is a crucial step in the diapedesis of inflammatory cells to the site of inflammation. We have demonstrated that leukotriene B₄ (LTB₄), a metabolite of the arachidonic acid cascade, and N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) significantly enhance the binding of human PMNs to selected populations of human dermal microvascular endothelial cells (MECs) in vitro. MECs were isolated from the vascular-rich portion of foreskins of newborns. MECs were grown in Iscove's modified Dulbecco's media with 2% prepartum serum and 8% newborn calf serum on 1% gelatin-coated plastic dishes. PMNs isolated from five human donors were added to the culture dishes for varying time intervals (usually 30 min) in the presence and absence of the chemotactic stimuli LTB₄ and FMLP. Addition of PMNs to MECs in the absence of chemotactic stimuli results in baseline binding to the MEC monolayer. About one in every 150 ECs binds more than five PMNs. These selected ECs are randomly distributed throughout the monolayer. LTB₄ from 10^-10 to 10^-7 M increases the number of MECs which selectively bind PMNs by 260% at 10^-7 M. FMLP also increases adherence in qualitatively and quantitatively similar fashion. These data support a role for LTB₄ in the mediation of adherence of neutrophils to dermal MECs. In contrast to other endothelial cells from the large blood vessels, such as from umbilical veins or calf thoracic aortae, PMNs bind only to selected MECs in culture, even when stimulated with LTB₄ or FMLP. These findings suggest a specific subpopulation of MECs which can be induced to express specific finding sites by LTB₄.Part of this work has been presented at the Meeting of the Western Society of Investigative Dermatology in Carmel, February 3–6, 1986     

Prostaglandin I1 analogues,SM-10902 and SM-10906, affect human keratinocytes and fibroblasts in vitro in a manner similar to PGE1: therapeutic potential for wound healing

The newly synthesized prostaglandin (PG) I₁ analogues, SM-10902 and SM-10906, were compared with PGE₁ in terms of their biological effects on cultured normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) in order to evaluate their therapeutic potential for cutaneous wound healing. The PGI₁ analogues had a direct effect on cell proliferation of HDFs as did PGE₁, but inibited cell growth of NHKs in contrast to the stimulatory effect observed with PGE₁. In contrast to NHKs stimulated with PGI₁ analogues, which exhibited low levels of adenosine 3,5-cyclic monophosphate (cAMP). HDFs stimulated with these analogues responded in a dose-dependent manner with extremely high levels of cAMP. Conditioned media (CM) derived from media in which HDFs had been incubated with both the PGI₁ analogues promoted NHK proliferation. HDF production of interleukin (IL)-6 increased in response to the PGI₁ analogues. Since IL-6 was shown to promote cell growth of NHKs, enhancement of NHK proliferation by CM was thought to be due to IL-6 derived from HDFs stimulated with the PGI₁ analogues.     

Metabolism of topical retinaldehyde and retinol by mouse skin in vivo: predominant formation of retinyl esters and identification of 14-hydroxy-4, 14-retro-retinol

Abstract We have previously shown that retinaldehyde (RAL), a natural metabolite of (β-carotene and retinol (ROL), can be used topically in human skin and exerts biological activity: it may be a convenient way to deliver multipotential vitamin A activity in epidermis. RAL can be converted enzymatically into 2 pathways: one leads to ROL (and then retinyl esters), the other to retin-oic acid (RA). The aim of the present study was 2-fold: (i) to see if RAL is metabolised in vivo when topically applied on mouse skin, and (ii) if so. to an-alyse the occurrence and relative importance of the 2 metabolic pathways as compared to ROL. We studied by HPLC the metabolites detectable in mouse tail skin upon topical application of RAL and ROL. As compared to vehicle-treated controls, RAL-treated mouse skin contained low amounts of all-trans RA and 13-cis-RA, whereas ROL content increased 10-fold and retinyl esters 30-fold after RAL application. As compared to RAL. ROL-treated mouse skin showed no detectable RA, slightly less retinyl esters but a significant amount of 14-hydroxy-4, 14-retro-ROL (14-HRR). a metabolite not previously reported in the skin. 14-HRR was the predominant polar metabolite of ROL. These data indicate that keratinocytes metabolise topical RAL. thus confirming the concept of using RAL as a precursor. Both pathways are used but in significantly different proportions. Thus, only a low proportion of RAL is metabolised into all-trans-RA, which may explain the low irritancy profile of topical RAL and supports the concept of a controlled delivery of ligands. That keratinocytes predominantly channel RAL into storage forms indicates that RAL, should also be considered as a convenient way to load the epidermis with vitamin A. The detection of 14-HRR. a metabolite not previously reported in skin, that promotes growth of B lymphocytes and activation of T lymphocytes, suggests distinct potentials of topical ROL and RAL.     

Origin Use of CD4, CD8, and CD1a Immunostains in Distinguishing Mycosis Fungoides from its Inflammatory Mimics: A Pilot Study

Patch-stage/early mycosis fungoides (MF) is difficult to differentiate from benign dermatoses, despite several robust histologic criteria. Most studies include advanced lesions and data about early disease is limited.

Objectives:

(1) To compare the CD4:CD8 ratio in patch-stage MF versus inflammatory mimics. (2) To study patterns of CD1a expression in the epidermis and dermis in the two groups.

Materials and Methods:

Twenty cases each of early MF and inflammatory dermatoses were selected. The diagnoses were established after clinicopathologic correlation, repeat biopsies, and follow-up. The inflammatory group included pityriasis lichenoides chronica, actinic reticuloid, lichenoid purpura, and various psoriasiform dermatoses. Immunohistochemistry was done for CD4, CD8, and CD1a. Epidermal CD4, CD8 cells were quantified and CD1a was graded semi-quantitatively in the epidermis and dermis.

Results:

The average CD4:CD8 ratio was 4.2 in MF (range: 1-16.8), and 0.9 in inflammatory diseases (range: 0.43-5), which was statistically significant (P < 0.0001). None of the MF cases had a ratio <1. Four cases of pityriasis lichenoides chronica had a ratio >1. CD1a cells had a continuous or confluent epidermal pattern in almost all cases of MF, while they occurred as small or large groups in the dermis. In inflammatory dermatoses, there were either isolated or scattered CD1a+ cells in both epidermis and dermis.

Conclusions:

Elevated CD4:CD8 ratio favors MF. But there is an overlap in the lower range with pityriasis lichenoides chronica. These cases require good clinicopathologic correlation and follow-up. Patterns of CD1a expression are more reliable. Immunostains buttress morphology and are a valuable addition.