Psoriatic skin lesions contain biologically active amounts of an interleukin 1-like compound

The presence of neutrophil chemoattractant material in aqueous extracts of lesional psoriatic scale has been investigated by use of an agarose microdroplet chemokinesis method in combination with ultrafiltration and high-performance liquid chromatography (HPLC). Fractions were also assayed for murine thymocyte co-stimulating activity. Aqueous extracts of psoriatic scale contained significantly greater neutrophil chemokinetic activity than extracts of scale from normal skin. Successive ultrafiltration of extracts showed that the chemokinetic material was 10 to 30 kd. Heat lability and gel filtration HPLC characteristics suggested that the major chemokinetically active material in aqueous extracts of psoriatic scale is different from C5a des arg. Reversed-phase HPLC of 0.1% trifluoroacetic acid/acetonitrile extracts of psoriatic scale revealed two clearly resolved peaks of chemokinetic activity, the major peak consistently containing thymocyte co-stimulating activity. No significant neutrophil chemokinetic activity was seen in fractions after reversed-phase HPLC of scale from normal skin. These findings suggest that a major portion of the neutrophil chemoattractant activity in aqueous extracts of psoriatic scale is due to interleukin 1-like material, which may play a role in the pathogenesis of this disease.     

Lipid lymphocyte chemoattractants in psoriasis

In view of the evidence that lymphocyte infiltrates are a constant feature of the skin lesions of psoriasis and the demonstration that certain hydroxylated metabolites of arachidonic acid are present in lesional psoriatic skin and possess lymphocyte chemoattractant properties, lipid extracts of samples from lesional and normal skin were assayed to determine which are the predominant lipid lymphocyte chemoattractants in psoriasis. Dilution-related lymphocyte chemoattractant activity was found in lipid extracts of stratum corneum samples from psoriatic lesions, but not in similar extracts the samples from both sources contained equivalent amounts of this activity. Subsequent purification of lesional stratum corneum lipid extracts by straight and reversed phase high performance liquid chromatography (HPLC) revealed the presence of at least two different lipid chemoattractants, one major component being identified as 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12[R]-HETE) by its biological and chromatographic properties. These compounds may play a role in the pathogenesis of the lymphocyte infiltrates in psoriatic lesions.     

Cytokines in skin lesions of psoriasis

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.     

A roadmap from research to clinical testing of mesenchymal stromal cell exosomes in the treatment of psoriasis

The most clinically trialed cells, mesenchymal stromal cells (MSCs), are now known to mainly exert their therapeutic activity through paracrine secretions, which include exosomes. To mitigate potential regulatory concerns on the scalability and reproducibility in the preparations of MSC exosomes, MSC exosomes were produced using a highly characterized MYC-immortalized monoclonal cell line. These cells do not form tumors in athymic nude mice or exhibit anchorage-independent growth, and their exosomes do not carry MYC protein or promote tumor growth. Unlike intra-peritoneal injections, topical applications of MSC exosomes in a mouse model of IMQ-induced psoriasis alleviate interleukin (IL)-17, IL-23 and terminal complement complex, C5b9 in psoriatic skin. When applied on human skin explants, fluorescence from covalently labeled fluorescent MSC exosomes permeated and persisted in the stratum corneum for about 24 hours with negligible exit out of the stratum corneum into the underlying epidermis. As psoriatic stratum corneums are uniquely characterized by activated complements and Munro microabscesses, we postulated that topically applied exosomes permeate the psoriatic stratum corneum to inhibit C5b9 complement complex through CD59, and this inhibition attenuated neutrophil secretion of IL-17. Consistent with this, we demonstrated that assembly of C5b9 on purified human neutrophils induced IL-17 secretion and this induction was abrogated by MSC exosomes, which was in turn abrogated by a neutralizing anti-CD 59 antibody. We thus established the mechanism of action for the alleviation of psoriatic IL-17 by topically applied exosomes.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant for human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B4. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant fo human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy - 5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B₄. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

In vitro and in situ expression of IL-23 by keratinocytes in healthy skin and psoriasis lesions: enhanced expression in psoriatic skin

Keratinocytes contribute to cutaneous immune responses through the expression of cytokines. We investigated whether human keratinocytes can express IL-23, a newly defined IFN-gamma-inducing cytokine composed of a unique p19 subunit and a p40 subunit shared with IL-12. Cultured keratinocytes from normal and lesional psoriatic skin were found to express constitutively mRNA for both subunits of IL-23. Low but significant levels of the heterodimeric IL-23 protein could be detected in cell lysates and supernatants from stimulated keratinocytes by immunoblotting and ELISA. Functional analysis showed that these low levels of keratinocyte-derived IL-23 were sufficient to enhance the IFN-gamma production by memory T cells. Immunostaining of skin sections confirmed expression of both subunits of IL-23 by keratinocytes in situ and also revealed expression of this cytokine in the dermal compartment. IL-23 expression was significantly higher in psoriatic lesional skin, compared with normal and psoriatic nonlesional skin. The immunostained preparations of cultured cells and IL-23 levels in culture supernatants did not show any difference between normal and psoriatic keratinocytes indicating no intrinsic aberration of IL-23 expression in keratinocytes from psoriatic skin. Double staining of cytospin preparations demonstrated that IL-23 p19 is also expressed by epidermal Langerhans cells, dermal dendritic cells, and macrophages. Psoriasis is a chronic inflammatory skin disease mediated by IFN-gamma-expressing type 1 memory T cells. As IL-23 is important to activate memory T cells to produce IFN-gamma, its augmented expression of IL-23 by keratinocytes and cutaneous APC may contribute to the perpetuation of the inflammation process in this disease.     

Physical characterization of the stratum corneum of an in vitro psoriatic skin model by ATR-FTIR and Raman spectroscopies

The stratum corneum is an important permeability barrier for the skin. The disorganization of the skin protective barrier characterizes some skin diseases such as psoriasis. Indeed, psoriatic skin is known to be more permeable than normal human skin. An in vitro human skin substitute may be obtained by the auto-assembly method. This method was adapted to produce psoriatic substitutes. FTIR spectroscopy is a well-established method to evaluate the order of hydrocarbon chains in terms of population of trans and gauche conformers. Using ATR-FTIR, we have compared the physicochemical properties of the stratum corneum in skin models derived from uninvolved and involved psoriatic cells with those derived from normal cells. Our results suggest that the stratum corneum of involved psoriatic skin substitutes is less organized than that of normal skin substitutes. Also, it seems that the properties of uninvolved psoriatic skin may vary with seriousness of the disease. The development of a new psoriatic skin model would be helpful in the design of new treatments and to increase the understanding of the mechanisms of this pathology.     

Noninvasive characterization of human stratum corneum of undiseased skin of patients with atopic dermatitis and psoriasis as studied by Fourier transform Raman spectroscopy

Etiopathogenetic regulatory disorders of epidermal metabolism and the subsequent changes in the molecular pattern of the stratum corneum play an important role in the clinical differentiation of particular dermatoses (e.g., psoriasis, atopic dermatitis). In this study we present in vitro Fourier transform Raman spectra of the stratum corneum from healthy skin, as well as from clinically undiseased skin of the right heel of atopic and psoriatic volunteers. Differences in the averaged spectra were detected, particularly in the spectral ranges of 1112-1142 (lipid band), 1185-1220, and 1394-1429 cm(-1). By using the first derivative of the averaged spectra and/or a statistical evaluation of the spectroscopic data it was possible to distinguish the skin types examined.     

Leukotrienes C4 and D4 in psoriatic skin lesions

Chemoattractant arachidonate lipoxygenase products have been recovered from the skin lesions of psoriasis, and may play a role in eliciting the intra-epidermal neutrophil infiltrate that characterises this disease. In view of evidence for lipoxygenase activity in psoriasis, the characteristic vasodilation in psoriatic lesions, and the vasodilator properties of leukotriene (LT) C4 and D4 in human skin, the presence of these LTs in psoriatic lesions has been investigated. Skin chamber fluid from abraded psoriatic lesions contained significantly greater amounts of immunoreactive material than that from clinically normal skin, as determined by a double antibody radioimmunoassay (RIA) that uses antiserum cross-reacting with both LTC4 and LTD4. Purification of lesional chamber fluid and scale extracts by high performance liquid chromatography (HPLC) and RIA of fractions showed immunoreactivity which co-eluted with standard LTC4 and LTD4. These findings suggest that LTC4 and LTD4 may play a role in mediating the vasodilation and increased blood flow that characterise psoriatic skin lesions.     

Regulation of Involucrin in Psoriatic Epidermal Keratinocytes: The Roles of ERK1/2 and GSK-3β

Psoriasis, a common inflammatory skin disease, is characterized by epidermal hyperplasia, abnormal differentiation, angiogenesis, immune activation, and inflammation. Involucrin is an early terminal differentiation marker of epidermal keratinocytes. In this study, we determined the immunolocalization of involucrin in psoriatic lesions and normal skin of individuals without psoriasis by means of immunofluorescence (IF) assay. Furthermore, the regulation of involucrin by interleukin (IL)-13, IL-17A, endothelin (ET)-1, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ was investigated by Western blot. Extracellular regulate protein kinases 1/2 (ERK1/2) and glycogen syntheses kinase-3β (GSK-3β) inhibitors were also included to define the roles of these signals in the production of involucrin in both psoriatic and normal keratinocytes. In psoriatic lesional skin, involucrin was detected in the stratum spinosum, but not in the basal or the cornified layer. In normal skin, involucrin was restricted to the granular layer and the upper stratum spinosum. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ up-regulate expression of involucrin in both psoriatic and normal keratinocytes. However, this effect was abolished by ERK1/2 and GSK-3β inhibitors. In conclusion, involucrin is up-regulated in psoriatic keratinocytes. IL-13, IL-17A, ET-1, TNF-α, and IFN-γ could increase involucrin protein levels in psoriatic and normal keratinocytes. The ERK1/2 and GSK-3β signaling pathways may play positive roles in regulating epidermal differentiation as observed in psoriasis.     

Leukotrienes C4 and D4 psoriatic skin lesions

Chemoattractant arachidonate lipoxygenase products have been recovered from the skin lesions of psoriasis, and may play a role in eliciting the intra-epidermal neutrophil infiltrate that characterises this disease. In view of evidence for lipoxygenase activity in psoriasis, the characteristic vasolidation in psoriatic lesions, and the vasodilator properties of leukotriene (LT) C₄ and D₄ in human skin, the presence of these LTs in psoriatic lesions has been investigated. Skin chamber fluid from abraded psoriatic lesions contained significantly greater amounts of immunoreactive material than that from clinically normal skin, as determined by a double antibody radioimmunoassay (RIA) that uses antiserum cross-reacting with both LTC₄ and LTD₄. Purification of lesional chamber fluid and scale extracts by high performance liquid chromatography (HPLC) and RIA of fractions showed immunoreactivity which co-eluted with standard LTC₄ and LTD₄. These findings suggest that LTC₄ and LTD₄ may play a role in mediating the vasodilation and increased blood flow that characterise psoriatic skin lesions.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.     

Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin

Interferon α-producing plasmacytoid dendritic cells (pDC) are crucial contributors to pro-inflammatory or tolerogenic immune responses and are important in autoimmune diseases such as psoriasis. pDC accumulate in the lesional skin of psoriasis patients, but are rarely found in the affected skin of patients with atopic dermatitis (AD). While homeostatic chemokine CXCL12 and inducible pro-inflammatory CXCR3 chemokine ligands may regulate pDC influx to psoriatic skin, the mechanism responsible for selective pDC recruitment in psoriasis vs. AD remains unknown. Circulating pDC from normal donors express a limited number of chemoattractant receptors, including CXCR3 and CMKLR1 (chemokine-like receptor 1). In this work, we demonstrate that circulating pDC from normal donors as well as psoriasis and AD patients express similar levels of CXCR3 and responded similarly in functional migration assays to CXCL10. We next found that blood pDC from normal, AD, and psoriasis patients express functional CMKLR1. In contrast to normal skin, however, lesional skin from psoriasis patients contains the active form of the CMKLR1 ligand chemerin. Furthermore, in affected skin from psoriatic patients the level of active chemerin was generally higher than in AD skin. Taken together, these results indicate that local generation of active chemerin may contribute to pDC recruitment to psoriatic skin.     

Up-regulation of macrophage inflammatory protein-3 alpha/CCL20 and CC chemokine receptor 6 in psoriasis

Autoimmunity plays a key role in the immunopathogenesis of psoriasis; however, little is known about the recruitment of pathogenic cells to skin lesions. We report here that the CC chemokine, macrophage inflammatory protein-3 alpha, recently renamed CCL20, and its receptor CCR6 are markedly up-regulated in psoriasis. CCL20-expressing keratinocytes colocalize with skin-infiltrating T cells in lesional psoriatic skin. PBMCs derived from psoriatic patients show significantly increased CCR6 mRNA levels. Moreover, skin-homing CLA+ memory T cells express high levels of surface CCR6. Furthermore, the expression of CCR6 mRNA is 100- to 1000-fold higher on sorted CLA+ memory T cells than other chemokine receptors, including CXCR1, CXCR2, CXCR3, CCR2, CCR3, and CCR5. In vitro, CCL20 attracted skin-homing CLA+ T cells of both normal and psoriatic donors; however, psoriatic lymphocytes responded to lower concentrations of chemokine and showed higher chemotactic responses. Using ELISA as well as real-time quantitative PCR, we show that cultured primary keratinocytes, dermal fibroblasts, and dermal microvascular endothelial and dendritic cells are major sources of CCL20, and that the expression of this chemokine can be induced by proinflammatory mediators such as TNF-alpha/IL-1 beta, CD40 ligand, IFN-gamma, or IL-17. Taken together, these findings strongly suggest that CCL20/CCR6 may play a role in the recruitment of T cells to lesional psoriatic skin.     

Sphingosylphosphorylcholine is upregulated in the stratum corneum of patients with atopic dermatitis

To clarify the functional relevance of sphingomyelin (SM) deacylase to the ceramide deficiency seen in atopic dermatitis (AD), we developed a new highly sensitive method and measured the metabolic intermediate sphingosylphosphorylcholine (SPC) that accumulates in the stratum corneum. SPC in intercellular lipids extracted from stratum corneum was reacted with [(14)C]acetic anhydride to yield [(14)C-C(2)]SM, which was then analyzed by TLC. In both the lesional and non-lesional stratum corneum obtained from patients with AD, there was a significant increase in the content of SPC over that of healthy control subjects. There was a reciprocal relationship between increases in SPC and decreases in ceramide levels of stratum corneum obtained from healthy controls, and from lesional and non-lesional skin from patients with AD. Comparison with other sphingolipids present in the stratum corneum demonstrated that there is a significant positive correlation between SPC and glucosylsphingosine, another lysosphingolipid derived from glucosylceramide by another novel epidermal enzyme, termed glucosylceramide deacylase. In contrast, there was no correlation between SPC and sphingosine, a degradative product generated from ceramide by ceramidase. These findings strongly suggest the physiological relevance of SM deacylase function in vivo to the ceramide deficiency found in the skin of patients with AD.     

Interleukin 6 and 8 levels in plasma and fibroblast cultures in psoriasis

Fibroblasts have been implicated in psoriatic inflammatory processes. The aim of the study was to evaluate soluble interleukin 2 receptor (sIL-2R), interleukin 6 (IL-6), and interleukin 8 (IL-8) plasma levels in psoriatic patients and IL-6 and IL-8 levels in fibroblast culture supernatants. Cytokines levels in plasma and supernatants were measured by ELISA. Plasma sIL-2R, IL-6, and IL-8 levels were higher before the treatment in comparison to healthy controls (P < 0.001) and decreased after treatment. Fibroblasts from healthy controls, psoriatic lesional skin, and noninvolved psoriatic skin, when stimulated with tumor necrosis factor alpha, released considerable amounts of IL-6 and IL-8. No significant difference between healthy controls and psoriatic fibroblasts was observed. Monitoring plasma sIL-2R levels could be employed as a reliable method of psoriasis activity. IL-8 and IL-6 plasma levels seem to reflect psoriasis activity, and treatment response, respectively. Fibroblasts are not a major source of increased IL-6 and IL-8 production in psoriasis.