Revisiting the Koebner phenomenon: role of NGF and its receptor system in the pathogenesis of psoriasis

Nerve growth factor (NGF) influences the key pathological events of psoriasis: keratinocyte proliferation, angiogenesis, and T-cell activation. We have systematically examined the kinetics of NGF expression, keratinocyte proliferation, and migration of T lymphocytes in the epidermis in Koebner-induced developing psoriatic plaques. In skin traumatized by the tape-stripping method (n = 12), a marked up-regulation of NGF in Koebner-positive lesions (n = 7) was observed 24 hours after trauma. Synthesis of NGF reached its maximum level in the 2nd week. Furthermore, cultured keratinocytes from nonlesional skin of psoriasis patients produced 10 times higher levels of NGF compared with keratinocytes from healthy individuals. To substantiate the in vivo effect of NGF secreted by keratinocytes in psoriatic plaques, we studied psoriatic plaques and normal human skin in a SCID-human skin xenograft model. The transplanted psoriatic plaques demonstrated marked proliferation of NGF-R (p75)-positive nerve fibers compared with only a few nerves in the transplanted normal human skin. Our results demonstrate that 1) in a developing psoriatic lesion, up-regulation of NGF together with keratinocyte proliferation are early events and precede epidermotropism of T lymphocytes; 2) keratinocytes in patients with psoriasis are primed to produce elevated levels of NGF; and 3) NGF synthesized by these keratinocytes is functionally active.     

IL-17 suppresses TNF-alpha-induced CCL27 production through induction of COX-2 in human keratinocytes

BACKGROUND: The chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on nuclear factor kappaB (NF-kappaB) activity and enhanced in lesions of patients with atopic dermatitis, psoriasis vulgaris, or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. OBJECTIVE: We examined the in vitro effects of IL-17 on TNF-alpha-induced CCL27 production in human keratinocytes. METHODS: Keratinocytes were incubated with TNF-alpha, IL-17, or both. CCL27 secretion and mRNA levels were analyzed by means of ELISA and RT-PCR, respectively. COX-2 promoter and NF-kappaB activities were analyzed by using luciferase assays. COX-2 protein levels were analyzed by means of Western blotting. RESULTS: IL-17 suppressed TNF-alpha-induced CCL27 secretion and mRNA expression and NF-kappaB activity in keratinocytes. The COX-2 inhibitor NS398 counteracted the effects of IL-17, and prostaglandin E(2) prevented counteraction by NS398. IL-17 alone or synergistically with TNF-alpha increased prostaglandin E(2) release from keratinocytes, and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-alpha increased COX-2 mRNA and protein levels, promoter activity, and mRNA stability. The stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-alpha induced dual phosphorylation of p38 MAPK and ERK. CONCLUSION: IL-17 might suppress TNF-alpha-induced CCL27 production by inhibiting NF-kappaB through induction of COX-2. The induction of COX-2 might be mediated by activation of p38 MAPK and ERK. T cell-derived IL-17 might alleviate T-cell skin infiltration through inhibition of CCL27 production.     

Distributions of melanoma growth stimulatory activity of growth-regulated gene and the interleukin-8 receptor B in human wound repair.

The alpha-chemokines have been implicated as regulators of proliferation and differentiation of normal keratinocytes and as mediators of keratinocyte maturation and migration in inflammatory processes that involve the skin. Using the cutaneous wound repair model, we examined the sites and temporal sequence of the appearance of melanoma growth stimulatory activity or growth-regulated gene (MGSA/GRO;ligand) and the type B interleukin (IL)-8 receptor (IL-8RB) to which MGSA/GRO binds. Human burn tissues (n = 44) representing days 2 to 12 after injury were obtained during surgical debridement, fixed in 4% paraformaldehyde, and embedded in paraffin. Immunolocalizations were performed with polyclonal antisera for both ligand and receptor, as well as a monoclonal antibody for the IL-8 RB. Western blot analysis confirmed the presence of the IL-8 RB in immunoprecipitates of epidermal keratinocyte lysates. In normal skin, MGSA/GRO protein was restricted to sites populated by differentiated keratinocytes (suprabasal compartments, inner root sheath cells, and dermal sweat ducts). MGSA/GRO protein was barely detectable within epithelial margins and islands of burn wounds where the migrating/proliferating keratinocyte populations reside, but staining intensities increased as cells matured into the outer layers. Weak diffuse staining was detected in areas of neutrophilic infiltration (granulation tissue and overlying exudates). By contrast, in normal skin the IL-8 RB was detected in specific locations within epidermal and dermal compartments of healing wounds. In the dermis, polyvalent antibodies detected receptor immunoreactivity most prominently in dermal sweat ducts and endothelium of capillaries, whereas this immunoreactivity was inconspicuous in sections stained with the monoclonal antibody. Receptor immunostaining was noted in migrating/proliferating keratinocytes in epithelial margins and islands but was in the outer layers or in hypertrophic epidermis adjacent to wounds. This same pattern was observed in epidermal appendages such as hair follicles and eccrine sweat ducts. In granulation tissues, IL-8 RB was noted in numerous fibroblasts and in subpopulations of macrophages and smooth muscle. The presence of both MGSA/GRO and its receptor in human burn wounds implicate this cytokine as an autocrine or paracrine mediator of epidermal regeneration in both the inflammatory and proliferative phases of cutaneous wound repair.     

Increased expression of interleukin-4 receptors on psoriatic epidermal cells.

Abnormal keratinocyte proliferation and differentiation, the main characteristics of psoriasis, may be induced and maintained by cytokines produced by activated resident and recruited inflammatory cells in lesional skin. As the epidermal cytokine profile is clearly altered in psoriasis and because increased expression of interleukin-4 receptor (IL-4R) has been reported in some epithelial proliferative diseases, we investigated the expression of IL-4R on psoriatic epidermal cells (ECs). The expression of IL-4R on freshly isolated ECs from healthy skin and untreated psoriatic lesions was studied by immunostaining using an IL-4R-specific antibody and by examining their capacity to bind biotinylated recombinant human IL-4 using flow cytometry. The number of IL-4R+ ECs and the number of binding sites per cell were significantly increased on psoriatic ECs as compared with healthy control ECs. Immunostaining confirmed these results, whereby staining was mainly observed in the lower epidermal layers. In addition, an increased IL-4R mRNA expression was also observed in psoriatic epidermis using a digoxigenin-labeled IL-4R RNA probe. In short-term in vitro cultures, lipopolysaccharide/phorbol-myristate-acetate-stimulated and unstimulated psoriatic ECs did not produce any immunoreactive IL-4. The results of this study together with the reported increased expression of IL-4R in epithelial neoplasias suggest an association between overexpression of IL-4R and abnormal keratinocyte activation and proliferation.     

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.     

The role of IL-22 in the pathogenesis of skin diseases

IL-22 is an IL-10 family cytokine that acts mainly on epithelial cells. It is produced by immune cell subsets, including CD4? T cells, natural killer cells, and natural killer T cells. In the skin, IL-22 mediates keratinocyte proliferation and epidermal hyperplasia, inhibits terminal differentiation of keratinocytes, and induces the production of antimicrobial proteins. Although IL-22 production was initially linked with IL-17 expression in Th17 cells, IL-22 production can also occur in an apparently unique subset of cells that lacks the production of IL-17 and IFN-γ (Th22). Interestingly, Th22 cells express skin homing chemokine receptors CCR4 and CCR10. Indeed, Th22 cells reside in the normal skin and are shown to be enriched in the lesional skin of inflammatory skin diseases, indicating the importance of IL-22 in skin homeostasis and pathogenesis of skin diseases. Although psoriasis is the first example of an organ-specific immune disorder for which the role of IL-22 has been comprehensively studied, a growing body of evidence indicates that this cytokine also plays a critical role in the pathogenesis of atopic dermatitis. In this review, we discuss the role of IL-22 in the pathogenesis of skin diseases, particularly focusing on psoriasis and atopic dermatitis. Targeting IL-22 may have promise as a potential therapeutic for various skin diseases.     

Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing

Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/glycerol-transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was approximately 80% complete in wild-type mice at 5 days vs approximately 50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation.     

Angiogenesis drives psoriasis pathogenesis

Psoriasis pathogenesis is closely associated with disease-inducing Th1 and Th17 cells. Yet, several studies suggest that aberrant keratinocyte or endothelial cell signalling significantly contributes to disease manifestation. Histological hallmarks of psoriatic skin include the infiltration of multiple immune cells, keratinocyte proliferation and increased dermal vascularity. Formation of new blood vessels starts with early psoriatic changes and disappears with disease clearance. Several angiogenic mediators like vascular endothelial growth factor, hypoxia-inducible factors, angiopoietins and pro-angiogenic cytokines, such as tumour necrosis factor (TNF), interleukin (IL)-8 and IL-17, are up-regulated in psoriasis development. Contact- and mediator-dependent factors derived from keratinocytes, mast cells and immune cells may contribute to the strong blood vessel formation of psoriasis. New technologies and experimental models provide new insights into the role of angiogenesis in psoriasis pathogenesis. Interestingly, many therapies target not only immune cells, but also protein structures of endothelial cells. Here we summarize the role of pro-angiogenic factors in psoriasis development and discuss angiogenesis as a potential target of novel therapies.     

SPINK7对IL-22介导的角质细胞异常增殖及炎症应答的影响

目的:探讨SPINK7对IL-22刺激后角质形成细胞HaCaT的过度增殖及炎症应答的影响及其可能的机制.方法:采用IL-22刺激体外培养的HaCaT细胞.采用RT-PCR检测SPINK7和细胞炎症因子的mRNA表达;Western blot检测SPINK7,cyclin D1、survivin和Wnt/β-cateni...     

Neutrophil defensins enhance lung epithelial wound closure and mucin gene expression in vitro

Human airways are frequently exposed to potentially harmful agents that cause tissue injury. Upon such injury, a repair process is initiated that comprises cell migration, proliferation, and differentiation. We have previously shown that human neutrophil defensins (human neutrophil peptides 1-3 [HNP1-3]) induce airway epithelial cell proliferation. Because of the role of cell proliferation in epithelial wound repair, we investigated the effect of HNP1-3 on airway epithelial wound closure and mucin gene expression in vitro. Using NCI-H292 airway epithelial cell cultures, we demonstrated that HNP1-3 cause a dose- and time-dependent increase of wound closure as well as increased cell migration. Furthermore, HNP1-3 caused a biphasic activation of the mitogen-activated protein kinase extracellular-regulated kinase 1 and 2 (ERK1/2). Both the effects of HNP1-3 on wound closure and ERK1/2 activation were blocked by specific inhibitors of the mitogen-activated protein kinase kinase MEK, whereas inhibitors of epidermal growth factor receptor tyrosine kinase, phosphatidylinositol 3-kinase, and Src did block defensin-enhanced wound closure but not ERK1/2 activation. Finally, HNP1-3 increased mRNA encoding the mucins MUC5B and MUC5AC, suggesting a role for defensins in mucous cell differentiation. These results indicate that neutrophil defensins increase epithelial wound repair in vitro, which involves migration and proliferation, and mucin production. Neutrophil defensin-enhanced wound repair appears to require epidermal growth factor receptor activation and downstream signaling pathways.     

IL-22 and IL-20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-γ are not

Psoriasis is a common chronic skin disease with a largely unknown pathogenesis. We demonstrate here that transgenic over-expression of interleukin (IL)-22 in mice resulted in neonatal mortality and psoriasis-like skin alterations including acanthosis and hypogranularity. This cutaneous phenotype may be caused by the direct influence of IL-22 on keratinocytes, since this cytokine did not affect skin fibroblasts, endothelial cells, melanocytes, or adipocytes. The comparison of cytokines with hypothesized roles in psoriasis pathogenesis determined that neither interferon (IFN)-γ nor IL-17, but only IL-22 and, with lower potency, IL-20 caused psoriasis-like morphological changes in a three-dimensional human epidermis model. These changes were associated with inhibited keratinocyte terminal differentiation and with STAT3 upregulation. The IL-22 effect on differentiation-regulating genes was STAT3-dependent. In contrast to IL-22 and IL-20, IFN-γ and IL-17 strongly induced T-cell and neutrophilic granulocyte-attracting chemokines, respectively. Tumor necrosis factor-α potently induced diverse chemokines and additionally enhanced the expression of IL-22 receptor pathway elements and amplified some IL-22 effects. This study suggests that different cytokines are players in the psoriasis pathogenesis although only the IL-10 family members IL-22 and IL-20 directly cause the characteristic epidermal alterations. Kerstin Wolk and Harald S. Haugen equally contributed to this work.     

Inhibitory effect of Paeonia lactiflora Pallas extract (PE) on poly (I:C)-induced immune response of epidermal keratinocytes

Epidermal keratinocytes provide protective role against external stimuli by barrier formation. In addition, kertinocytes exerts their role as the defense cells via activation of innate immunity. Disturbance of keratinocyte functions is related with skin disorders. Psoriasis is a common skin disease related with inflammatory reaction in epidermal cells. We attempted to find therapeutics for psoriasis, and found that Paeonia lactiflora Pallas extract (PE) has an inhibitory potential on poly (I:C)-induced inflammation of keratinocytes. PE significantly inhibited poly (I:C)-induced expression of crucial psoriatic cytokines, such as IL-6, IL-8, CCL20 and TNF-α, via down-regulation of NF-κB signaling pathway in human keratinocytes. In addition, PE significantly inhibited poly (I:C)-induced inflammasome activation, in terms of IL-1β and caspase-1 secretion. Finally, PE markedly inhibited poly (I:C)-increased NLRP3, an important component of inflammasome. These results indicate that PE has an inhibitory effect on poly (I:C)-induced inflammatory reaction of keratinocytes, suggesting that PE can be developed for the treatment of psoriasis.     

Reference ranges of T lymphocyte subsets by single-platform among healthy population in southwest China

The anti-inflammatory effect of glycyrrhizin has been widely recognized, while the specific mechanism of glycyrrhizin in psoriasis remains poorly understood. In the imiquimod-induced mouse model of psoriasis (IMD), we found that glycyrrhizin can substantially improve the adverse symptoms in mice. The hematoxylin-eosin staining results showed that glycyrrhizin can also improve the pathological state of skin cells in IMD mice. Using enzyme-linked immunosorbent assay (ELISA), we found that glycyrrhizin substantially inhibited the expression of IL-17A and IFN-γ in the serum of IMD mice. In order to simulate the effect of IL-17A on keratinocytes in psoriasis, we treated HaCaT cells with 100 ng/mL IL-17A (IL-17A-HaCaT cells) for 48 h. Then, using cell-counting kit-8 (CCK-8) and ELISA assays, we found that glycyrrhizin inhibited the proliferation of IL-17A-HaCaT cells and reversed the promotion of IL-6, CCL20, and TNF-α induced by IL-17A. Further, western blotting (WB) results indicated that glycyrrhizin promoted the expression of SIRT1 and inhibited the expression of STAT3 and phosphorylated STAT3 (p-STAT3). By treating IL-17A-HaCaT cells with EX-527 (a potent and selective inhibitor of SIRT1), combined with CCK-8 and WB experiments, we initially found that EX-527 inhibited the proliferation of IL-17A-HaCaT cells and promoted the expression of STAT3, p-STAT3, and acetylated STAT3 (a-STAT3). However, when glycyrrhizin was added at the same time, the proliferation of IL-17A-HaCaT cells increased, and the expression of STAT3, p-STAT3, and a-STAT3 reduced. We then knocked down the expression of SIRT1 via small interfering RNA in IL-17A-HaCaT cells, and the results were consistent with those of EX-527. Together, these results indicated that glycyrrhizin improved psoriasis by inhibiting the expression of IL-17A and IFN-γ in vivo and suppressed the proliferation of IL-17A-HaCaT cells and the expression of STAT3, p-STAT3, and a-STAT3 by upregulating SIRT1 in vitro.     

Bovine colostrum promotes growth and migration of the human keratinocyte HaCaT cell line

Bovine colostrum represents a rich source of growth factors, which are known to play a central role in wound healing. The aim of our study was to investigate the possible mitogenic and motogenic effects induced by colostrum on human keratinocytes. Cell proliferation evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test and 5-Bromo-2′-deoxyuridine incorporation revealed that colostrum exerts a growth promoting activity. Scratch assay and immunofluorescence of actin cytoskeleton showed its effectiveness also in inducing cell migration. Furthermore, colostrum treatment increases the levels of tyrosine phosphorylated proteins and the activated forms of the extracellular signal-regulated kinases 1 and 2 and such effects appear to be repressed by the tyrosine kinase inhibitor genistein. Our results indicate that the biological activities of colostrum are specifically mediated by the growth factor-induced activation of tyrosine kinase receptors and underline the relevance of the synergistic action exerted by the growth factors in stimulating keratinocyte proliferation and migration essential for tissue repair.     

Modulation of keratinocyte-derived interleukin-8 which is chemotactic for neutrophils and T lymphocytes.

Interactions between T lymphocytes, neutrophils, and epidermal cells are believed to play a central role in the pathophysiology of psoriasis and other inflammatory cutaneous disorders. Although there is strong evidence that lymphocyte-function-associated antigen-1 (LFA-1) positive T cells are retained in the epidermis via intercellular adhesion molecule-1 (ICAM-1) expression induced on keratinocytes, the molecular basis for the directed migration of T cells or neutrophils towards the epidermis is not known. To investigate whether epidermal keratinocyte-derived products may be important in the migration of T cells and neutrophils into the epidermis, human keratinocytes were cultured in the presence of various cytokines and chemotactic activity of the supernatants were assessed. TNF-alpha stimulation produced directed migrational responses for both neutrophils and T-lymphocytes (both CD4 and CD8), but not B lymphocytes; 69% of T-cell movement and 80% of neutrophil migration induced by the TNF-alpha treated keratinocyte cell supernatants could be inhibited by anti-interleukin-8 (IL-8) serum. Using the same antibody, IL-8 was immunoprecipitated from the supernatants of TNF-stimulated 35S-labelled keratinocytes, and a single 7-kd band product detected by SDS-PAGE. In keeping with these biological activities and protein data, Northern blot analysis of total cellular RNA extracted from keratinocyte monolayers hybridized with a 32P-labelled 1-kb cDNA to IL-8 mRNA, revealed induction of the IL-8 gene in the presence of TNF-alpha and IL-1 beta, but not IFN-gamma. The protein kinase C agonist, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a known stimulator of psoriasiform cutaneous inflammation when applied directly to murine epidermis, strongly induced keratinocyte elaboration of IL-8 mRNA. These studies demonstrate that activated human keratinocytes are capable of producing biologically active IL-8, and provide evidence that keratinocytes can play a key role in mediating the influx of T cells and neutrophils into the epidermis.