Interleukin 20 regulates dendritic cell migration and expression of co-stimulatory molecules.

BACKGROUND: Psoriasis is an inflammatory disease characterized by leukocyte skin infiltration. Interestingly, recent works suggest that the migration of dendritic cells (DCs) is abnormal in psoriatic skin. DCs have significant role in regulating the function of T lymphocytes, at least in part influenced by the local environment of cytokines. In psoriatic skin lesions the expression of IL-20 is highly up-regulated. It is unclear if this cytokine has any influence on DCs. METHODS: Here, we investigated the influence of IL-20 in monocyte-derived dendritic cell (MDDCs) in vitro. This work addressed IL-20 effects on DC maturation, receptor expression and signaling. By use of extra cellular matrix components mimicking the skin environment, we also studied the functional effects of IL-20 on the chemotactic migration of DCs. Based on the recent finding that CD18 integrin are shed during migration of myeloid leukocytes, the concentration of these adhesion molecules was measured in MDDCs culture supernatants post migration. RESULTS: Following stimulation with IL-20, immature human MDDCs enhanced the expression of the co-stimulatory molecule CD86, further enabling activation of the p38 MAPK, but not the STAT3, pathway. IL-20 increased the migration of MDDCs in a biphasic response narrowly controlled by the interleukin concentration. A concomitant change in the shedding of CD18 integrins suggested that these adhesion molecules play a role in the migration of the MDDCs through the extracellular matrix layer. CONCLUSION: Taken together, our findings points to a possible, yet subtle, role of IL-20 in DCs migration. The biphasic response suggests that the aberrant IL-20 expression in psoriasis impedes DC migration, which could be a part of the processes that precipitates the dysregulated inflammatory response associated with this disease.

Interleukin 20 protein locates to distinct mononuclear cells in psoriatic skin.

We previously demonstrated that mRNA for the pro-inflammatory cytokine interleukin 20 (IL-20) is expressed in suprapapillary keratinocytes of lesional psoriatic skin (LS). Here, we describe the distribution of IL-20 protein and the identity of the IL-20-positive cells in LS. We found that the main part of IL-20 immunoreactivity is present in mononuclear cells of the dermal papillae, and that the IL-20-positive cells located in the papillae were langerin+, CD1a+, CD4+ and CD303+. These cells might be immature dendritic cell. In situ hybridization for IL-20 mRNA on non-LS, ex vivo stimulated with IL-1ß revealed a colocalization between IL-20 mRNA and the keratinocyte marker CK14. No IL-20 mRNA was detected in the dermal mononuclear cells. Our results suggest that IL-20 is produced by keratinocytes, released into the epidermis and then possibly taken up by papillary mononuclear cells. Our study supports that IL-20 is involved in the pathogenesis of psoriasis.

CCL27 expression is regulated by both p38 MAPK and IKKß signalling pathways.

The skin-specific chemokine CCL27 is believed to play a pivotal role in establishing the inflammatory infiltrate characteristic for common inflammatory skin diseases. Through binding to the chemokine receptor 10 (CCR10), CCL27 mediates inflammation by promoting lymphocyte migration into the skin. Little is known about the regulation of CCL27 gene expression. The purpose of our study was to investigate the regulation of the IL-1ß-induced CCL27 gene expression in normal human keratinocytes (NHEK). Preincubation of NHEK with the inhibitory κB (IκB) kinase (IKK) inhibitor, SC-514, or the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, revealed a profound reduction in both CCL27 mRNA and CCL27 protein expression indicating the significance of these pathways in the regulation of CCL27 expression. Furthermore, the impact of inhibitors of mitogen- and stress-activated kinase 1 (MSK1) or the mitogen-activated protein kinase-interacting kinases (Mnk1+2), downstream kinases of p38 MAPK, on IL-1ß-induced CCL27 expression in NHEK were investigated. We identified seven NF-κB binding elements upstream from the CCL27 gene start codon using electrophoretic mobility shift assay (EMSA). Supershift analyses demonstrated the involvement of the p50/p65 NF-κB heterodimer. We conclude that IL-1ß-induced CCL27 gene expression in NHEK is regulated through the p38 MAPK/MSK1/Mnk1+2 as well as the IKKß/NF-κB signalling pathways.

The p38 MAPK regulates IL-24 expression by stabilization of the 3' UTR of IL-24 mRNA.

BACKGROUND: IL-24 (melanoma differentiation-associated gene-7 (mda-7)), a member of the IL-10 cytokine family, possesses the properties of a classical cytokine as well as tumor suppressor effects. The exact role of IL-24 in the immune system has not been defined but studies have indicated a role for IL-24 in inflammatory conditions such as psoriasis. The tumor suppressor effects of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis. Current knowledge on the regulation of IL-24 expression is sparse. Previous studies have suggested that mRNA stabilization is of major importance to IL-24 expression. Yet, the mechanisms responsible for the regulation of IL-24 mRNA stability remain unidentified. As p38 MAPK is known to regulate gene expression by interfering with mRNA degradation we examined the role of p38 MAPK in the regulation of IL-24 gene expression in cultured normal human keratinocytes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we show that anisomycin- and IL-1beta- induced IL-24 expression is strongly dependent on p38 MAPK activation. Studies of IL-24 mRNA stability in anisomycin-treated keratinocytes reveal that the p38 MAPK inhibitor SB 202190 accelerates IL-24 mRNA decay suggesting p38 MAPK to regulate IL-24 expression by mRNA-stabilizing mechanisms. The insertion of the 3' untranslated region (UTR) of IL-24 mRNA in a tet-off reporter construct induces degradation of the reporter mRNA. The observed mRNA degradation is markedly reduced when a constitutively active mutant of MAPK kinase 6 (MKK6), which selectively activates p38 MAPK, is co-expressed. CONCLUSIONS/SIGNIFICANCE: Taken together, we here report p38 MAPK as a regulator of IL-24 expression and determine interference with destabilization mediated by the 3' UTR of IL-24 mRNA as mode of action. As discussed in the present work these findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.

A Characterization of the expression of 14-3-3 isoforms in psoriasis, basal cell carcinoma, atopic dermatitis and contact dermatitis.

14-3-3 is a highly conserved protein involved in a number of cellular processes including cell signalling, cell cycle regulation and gene transcription. Seven isoforms of the protein have been identified; ß, γ, ε, ζ η σ and τ. The expression profile of the various isoforms in skin diseases is unknown. To investigate the expression of the seven 14-3-3 isoforms in involved and uninvolved skin from psoriasis, basal cell carcinoma (BCC), atopic dermatitis and nickel induced allergic contact dermatitis. Punch biopsies from involved and uninvolved skin were analyzed with quantitative reverse transcription-polymerase chain reaction to determine the mRNA expression of the 14-3-3 isoforms. The protein level of 14-3-3 isoforms was measured by Western blot technique in keratome biopsies from patients with psoriasis. Evaluation of dermal and epidermal protein expression was performed by immunofluorescence staining. Increased 14-3-3τ mRNA levels were detected in involved skin from patients with psoriasis, contact dermatitis and BCC. 14-3-3σ mRNA expression was increased in psoriasis and contact dermatitis, but not in BCC. In atopic dermatitis no significant difference between involved and uninvolved skin was found. The expression of the 14-3-3 isoforms was also studied at the protein level in psoriasis. Only 14-3-3τ expression was significantly increased in involved psoriatic skin compared with uninvolved skin. Immunofluorescence staining with 14-3-3τ- and 14-3-3σ-specific antibodies showed localization of both isoforms to the cytoplasm of the keratinocytes in the various skin sections. These results demonstrate a disease specific expression profile of the 14-3-3τ and 14-3-3σ iso-forms.

The expression of IL-20 and IL-24 and their shared receptors are increased in rheumatoid arthritis and spondyloarthropathy.

The purpose of this study was to analyze the expression of the two proinflammatory cytokines IL-20 and IL-24 and their shared receptors in rheumatoid arthritis and spondyloarthropathy. IL-20 was increased in plasma of rheumatoid arthritis patients compared with osteoarthritis patients and IL-24 was increased in synovial fluid and plasma of rheumatoid arthritis and spondyloarthropathy patients compared with osteoarthritis patients. IL-20 and IL-24 mRNA was only present at low levels in the synovium. In the synovial membrane, IL-20 protein was present in mononuclear cells and neutrophil granulocytes whereas IL-24 protein was observed in endothelial cells and mononuclear cells. IL-20 receptor type 1 and IL-22 receptor were expressed by granulocytes in the synovial fluid. In synovial fluid mononuclear cell cultures, stimulation with recombinant human IL-20 or recombinant human IL-24 induced monocyte chemoattractant protein 1 (CCL2/MCP-1) secretion, but not tumour necrosis factor alpha mRNA synthesis or IL-6 secretion. Both IL-20 and IL-24 showed correlations to CCL2/MCP-1 in plasma from rheumatoid arthritis and spondyloarthropathy patients. This study associates IL-20 and IL-24 to the synovium of rheumatoid arthritis and spondyloarthropathy and results indicate that the two cytokines contribute to disease pathogenesis through recruitment of neutrophil granulocytes and induction of CCL2/MCP-1.

HDAC activity is required for p65/RelA-dependent repression of PPARdelta-mediated transactivation in human keratinocytes.

Peroxisome proliferator-activated receptors (PPARs) play a key role in differentiation, inflammation, migration, and survival of epidermal keratinocytes. The NF-kappaB has long been known to play pivotal roles in immune and inflammatory responses, and furthermore NF-kappaB has been implicated in the regulation of epidermal homeostasis. Recent studies have established that p65/RelA is a potent repressor of PPARdelta-mediated transactivation in human keratinocytes. In this article we further investigate the molecular mechanisms dictating the NF-kappaB-dependent repression of PPARdelta in human keratinocytes. We demonstrate that repression is unique to p65/RelA, as no other member of the NF-kappaB family had an impact on PPARdelta-mediated transactivation. Interestingly, our results show that p65/RelA only represses PPARdelta-dependent transactivation when PPARdelta is bound to DNA via its DNA-binding domain. We show that repression is sensitive to inhibition of histone deacetylases (HDACs) by tricostatin A (TSA), suggesting that HDAC activity is indispensable for p65/RelA-mediated repression. Accordingly, we demonstrate that a ternary complex consisting of PPARdelta, p65/RelA, and HDAC1 is formed in vivo. Finally, we demonstrate that TSA relieves tumor necrosis factor-alpha (TNFalpha)-induced repression of PPARdelta-mediated transactivation of the PPARdelta target gene adipose differentiation-related protein (ADRP) indicating that cross-talk between PPARdelta and NF-kappaB is of biological significance in human keratinocytes.

Dimethylfumarate specifically inhibits the mitogen and stress-activated kinases 1 and 2 (MSK1/2): possible role for its anti-psoriatic effect.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway, which regulates the activity of different transcriptions factors including NF-kappaB, is activated in lesional psoriatic skin. The purpose of this study was to investigate the effect of fumaric acid esters (FAEs) on the p38 MAPK and the downstream kinases mitogen- and stress-activated protein kinase (MSK)1 and 2 in cultured human keratinocytes. Cell cultures were incubated with dimethylfumarate (DMF), methylhydrogenfumarate (MHF), or fumaric acid (FA) and then stimulated with IL-1beta before kinase activation was determined by Western blotting. A significant inhibition of both MSK1 and 2 activations was seen after preincubation with DMF and stimulation with IL-1beta, whereas MHF and FA had no effect. In addition, DMF decreased phosphorylation of NF-kappaB/p65 (Ser276), which is known to be transactivated by MSK1. Furthermore, incubation with DMF before stimulation with IL-1beta resulted in a significant decrease in NF-kappaB binding to the IL-8 kappaB and the IL-20 kappaB-binding sites as well as a subsequent decrease in IL-8 and IL-20 mRNA expression. Our results suggest that DMF specifically inhibits MSK1 and 2 activations and subsequently inhibits NF-kappaB-induced gene-transcriptions, which are believed to be important in the pathogenesis of psoriasis. These effects of DMF explain the anti-psoriatic effect of FAEs.

IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms.

IL-20 is a novel member of the IL-10 cytokine family with pleiotropic effects. Current knowledge of what triggers and regulates IL-20 gene expression is sparse. The aim of this study was to investigate the regulation of IL-20 expression in cultured normal human keratinocytes. The expression of IL-20 was rapidly induced by proinflammatory stimuli, in particular IL-1beta, IL-6, and UVB irradiation. Using kinase inhibitors and small-interfering RNA, we discovered that the p38 mitogen-activated protein kinase (MAPK) as well as inhibitory kappaB kinase-NF-kappaB signaling pathways are crucial for IL-20 expression. By electrophoretic mobility shift assay two kappaB-binding sites were identified upstream from the start codon in the IL-20 gene. Supershift analysis revealed binding of the p50/p65 heterodimer. Furthermore, the p38 MAPK was shown to exert its effects on IL-20 expression through activation of the downstream kinase mitogen- and stress-activated kinase 1 (MSK1), indicating transactivation of NF-kappaB driven IL-20 messenger RNA transcription as an important mechanism of action. IL-20 is assumed to be a key cytokine in the pathogenesis of psoriasis and possibly cancer, and therefore the p38 MAPK, MSK1, and NF-kappaB may be important new molecular targets for the modulation of IL-20 expression in these diseases.

Mitogen- and stress-activated protein kinase 1 is activated in lesional psoriatic epidermis and regulates the expression of pro-inflammatory cytokines.

Mitogen- and stress-activated protein kinase 1 (MSK1) is a downstream target of both the p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPKs). MSK1 stimulates transcription of different pro-inflammatory genes through activation of transcription factors. The purpose of this study was to investigate the expression and activation of MSK1 in lesional psoriatic skin and its role in cytokine production in cultured normal human keratinocytes. Western blotting revealed a consistent and significant increase in phosphorylated (activated) MSK1(Ser376) in lesional psoriatic skin. Immunofluorescence staining revealed the phosphorylated MSK1(Thr581) to be localized in the basal layers of the epidermis in lesional psoriatic skin. No staining was found in non-lesional psoriatic skin. Cultured human keratinocytes incubated with anisomycin or IL-1beta resulted in the phosphorylation of the p38 MAPK and MSK1(Ser376). MSK1(Ser376) phosphorylation was inhibited by pre-incubation with the p38 inhibitor SB 202190. Transfection of the keratinocytes with specific MSK1 small interfering RNA resulted in 80% reduction of MSK1 expression and 51, 40, and 31% decrease in IL-6, IL-8, and tumor necrosis factor-alpha protein production, respectively. This study demonstrates for the first time the expression of MSK1 in epidermal keratinocytes and increased activation focally in psoriatic epidermis. As MSK1 regulates the production of pro-inflammatory cytokines, it may play a role in the pathogenesis of psoriasis.

Surfactant protein D in atopic dermatitis and psoriasis.

The collectin surfactant protein-D (SP-D) shows antimicrobial and immuno-regulatory properties and has recently been detected in the basal layers of normal human skin. This molecule potentially plays an important role in inflammatory skin diseases and therefore SP-D content and location was examined using immunohistochemistry on skin biopsies from patients with the two major dermatologic diseases, psoriasis and atopic dermatitis. SP-D was located in the stratum basale of all biopsies with similar intense staining in both diseased and normal skin. Differences were detected in stratum spinosum where involved psoriatic skin showed intense staining through the entire region significantly different from uninvolved and normal skin. Lesional atopic skin showed moderate staining extending through the basal three-fourths of stratum spinosum. Using real time polymerase chain reaction analysis, no substantial up-regulation of SP-D mRNA was detected in lesional psoriatic skin, and a comparison of serum levels of SP-D between patients with atopic dermatitis or psoriasis and a group of age matched healthy controls did not show significant differences. In conclusion SP-D was significantly more abundant in the stratum spinosum of lesional psoriatic and atopic skin due to more cells producing the molecule rather than up-regulation of production in single cells of diseased skin. Further studies are needed to show if SP-D plays a role in the protection against skin infections or modulation of the inflammatory process in these common skin diseases.

Protein expression of TNF-alpha in psoriatic skin is regulated at a posttranscriptional level by MAPK-activated protein kinase 2.

Alterations in specific signal transduction pathways may explain the increased expression of proinflammatory cytokines seen in inflammatory diseases such as psoriasis. We reveal increased TNF-alpha protein expression, but similar TNF-alpha mRNA levels, in lesional compared with nonlesional psoriatic skin, demonstrating for the first time that TNF-alpha expression in lesional psoriatic skin is regulated posttranscriptionally. Increased levels of activated MAPK-activated protein kinase 2 (MK2) together with increased MK2 kinase activity were found in lesional compared with nonlesional psoriatic skin. Immunohistochemical analysis showed that activated MK2 was located in the basal layers of the psoriatic epidermis, whereas no positive staining was seen in nonlesional psoriatic skin. In vitro experiments demonstrated that both anisomycin and IL-1beta caused a significant activation of p38 MAPK and MK2 in cultured normal human keratinocytes. In addition, TNF-alpha protein levels were significantly up-regulated in keratinocytes stimulated with anisomycin or IL-1beta. This increase in TNF-alpha protein expression was completely blocked by the p38 inhibitor, SB202190. Transfection of cultured keratinocytes with MK2-specific small interfering RNA led to a significant decrease in MK2 expression and a subsequent significant reduction in the protein expression of the proinflammatory cytokines TNF-alpha, IL-6, and IL-8, whereas no change in the expression of the anti-inflammatory cytokine IL-10 was seen. This is the first time that MK2 expression and activity have been investigated in an inflammatory disease such as psoriasis. The results strongly suggest that increased activation of MK2 is responsible for the elevated and posttranscriptionally regulated TNF-alpha protein expression in psoriatic skin, making MK2 a potential target in the treatment of psoriasis.

Tumor necrosis factor-alpha-induced CTACK/CCL27 (cutaneous T-cell-attracting chemokine) production in keratinocytes is controlled by nuclear factor kappaB.

CTACK/CCL27 is pivotal in mediating the migration of lymphocytes into the skin, through the binding to the chemokine receptor CCR10. CCL27 is continuously expressed by keratinocytes, but highly upregulated in inflammatory skin diseases such as atopic dermatitis and psoriasis. CCL27 can be induced in cultured keratinocytes by tumor necrosis factor (TNF)-alpha, which is also known to induce activity of the transcription factor NF-kappaB. NF-kappaB plays a vital role in controlling inflammation through its regulation of transcription of chemokines and pro-inflammatory cytokines. We show here that inhibition of NF-kappaB with the non-specific NF-kappaB inhibitors SSC (sodium salicylate), DCIC (3,4-dichloroisocoumarin) and PAO (phenylarsine oxide) results in a downregulation of TNF-alpha-induced CCL27 production. To substantiate the result and to investigate the role of NF-kappaB we investigated if specific antisense oligonucleotides against the p50 and p65 subunits of NF-kappaB had the same effect. Inhibition of either p50 or p65 production with antisense oligonucleotides resulted in a significant downregulation of TNF-alpha-induced CCL27 production. These results indicate that CCL27 expression is under the control of NF-kappaB, and that NF-kappaB, as indicated by others, may be an attractive target for therapy in inflammatory skin diseases.