Primary human keratinocytes efficiently induce IL‐1‐dependent IL‐17 in CCR6+ T cells

Abstract: Keratinocytes and activated T cells interact in skin inflammation by virtue of chemokines and cytokines. T cell‐derived IL‐17 has been described to play an important role in the course of psoriatic inflammation. In this study, we addressed how keratinocytes influence the secretion of IL‐17 in autologous T cell subsets. We found that a co‐culture of autologous keratinocytes and T cell‐receptor‐stimulated T cells markedly enhanced the production of IL‐17. Besides the importance of direct cell contact, this effect was mainly mediated by IL‐1 and could be blocked by the IL‐1 antagonist anakinra. An additional increase in IL‐17 production by IL‐23 was only seen in the presence of IL‐1, which thus plays a permissive role for the action of IL‐23. Importantly, co‐culture of keratinocytes with CCR6+ CD4+ T cells that are enriched for Th17 cells resulted in significantly higher IL‐17 production compared to co‐culture with CD4+ T cells.     

UVA‐1 exposure in vivo leads to an IL‐6 surge within the skin

UVA‐1 is a known promotor of skin ageing. Cytokines like IL‐1α, Il‐1β or TNF‐α, VEGF and IL‐6 orchestrate UV effects, and IL‐6 is furthermore an effector of UVA‐induced photoageing. We investigated how fractionated UVA‐1 doses influence the cytokine milieu and especially the IL‐6 levels in the skin in vivo. In a study with 35 participants, we exposed previously unirradiated human skin to three UVA‐1 irradiation regimes. Cytokine levels in interstitial skin fluid were measured up to 48 hours postexposure and compared to unirradiated control skin fluid. Our results show that IL‐6 levels increased significantly after UVA‐1 exposure at selected time points. The other candidates IL‐1α, Il‐1β or TNF‐α and VEGF show no significant response after UVA‐1 exposure in vivo. UVA‐1 thus raises selectively IL‐6 levels in vivo, a fact that underlines its role in photoageing and has potential implications for its modulatory effect on photoageing pathology.     

The balance between pro‐ and anti‐inflammatory cytokines is crucial in human allergic contact dermatitis pathogenesis: the role of IL‐1 family members

The interleukin (IL)‐1 family includes 11 members that are important in inflammatory processes. It includes various agonists and two antagonists, IL‐1Ra and IL‐36Ra. Our aim was to investigate whether the IL‐1 family is involved in allergic contact dermatitis (ACD). The expression of IL‐1 family members was evaluated by PCR and immunohistochemistry in the positive patch test reaction site (involved skin) and in the uninvolved skin of ACD patients. We also examined these cytokines in an ex vivo model of ACD. The antagonistic activity of IL‐36Ra was evaluated by injecting recombinant IL‐36Ra in uninvolved skin biopsies of ACD patients. IL‐1Ra and IL‐36Ra expression was quantified in mononuclear cells of nickel‐sensitized patients challenged in vitro with nickel. IL‐33 involvement in ACD was investigated by intra‐dermal injection of anti‐IL‐33 in the uninvolved skin of patients ex vivo. Results showed that IL‐1β, IL‐1Ra, IL‐36α, IL‐36β, IL‐36γ and IL‐33 expression, but not IL‐36Ra expression, was enhanced in ACD‐involved skin. Immunohistochemical analysis and ex vivo skin cultures confirmed these results. Injection of anti‐IL‐33 in ACD‐uninvolved skin inhibited IL‐8 expression, whereas IL‐36Ra inhibited IL‐36α, IL‐36β, IL‐36γ and IL‐8 expression. Nickel induced IL‐1Ra expression in lymphocytes of nickel‐sensitized patients. Hence, various IL‐1 agonists and antagonists may be involved in ACD pathogenesis.     

Continuous high‐dose antigen exposure preferentially induces IL‐10, but intermittent antigen exposure induces IL‐4

IL‐10 plays a critical role in the induction of specific T‐cell tolerance. To date, whether IL‐10 induction by antigen application is dose‐ or time‐dependent remains unclear. In this study, IL‐10 induction by allergen exposure was investigated in the several schedules. Oxazolone was repeatedly applied to mouse ear, and mRNA of inflammatory cytokines in lesional skins was measured. The results indicated that continuous high‐dose antigen exposure induces IL‐4 as well as abundant IL‐10 production. Monocytes/dendritic cells and T cells are major source of IL‐10. Allergen‐specific immunotherapy is resumed before antigen scattering: preseason. We evaluated safe‐loading dose of allergens in preseasonal therapy focusing Tr1 induction. Restarting immunotherapy with high dose effectively augmented IL‐10 expression accompanied with further induction of IL‐4 and inflammatory cytokines. Therefore, the protocol restarting with low‐dose antigen is preferential to obviate the risk of exacerbation or anaphylaxis.     

Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes

Please cite this paper as: Epidermal growth factor receptor tyrosine kinase inhibitors induce CCL2 and CCL5 via reduction in IL‐1R2 in keratinocytes. Experimental Dermatology 2010; 19 : 730–735. Abstract: Epidermal growth factor receptor tyrosine kinase (EGFR‐TK) is a transducer of mitogenic signals, and is involved in the pathogenesis and progression of a number of cancers, including non‐small cell lung cancer (NSCLC). Gefitinib is an EGFR‐TK inhibitor that is clinically used to treat NSCLC; however, this drug frequently causes adverse effects, including skin eruptions. The mechanism underlying these skin reactions is elusive, although it is assumed that they are caused by the inhibition of EGFR‐TK signalling in epidermal and adnexal cells. In this article, we demonstrate by immunocytochemistry that the skin lesions of patients treated with oral gefitinib had higher expression of CCL2 and CCL5 compared to normal human epidermis. Further, PD153035, a gefitinib prototype, induced CCL2 and CCL5 mRNA and protein expression in HaCaT and HSC‐1 keratinocyte cell lines with or without interleukin‐1 (IL‐1) treatment in vitro. PD153035 also reduced the levels of interleukin‐1 receptor 2 (IL‐1R2), an IL‐1 decoy receptor. Moreover, we demonstrate that reduction in IL‐1R2 by RNA interference increased IL‐1‐mediated CCL2 and CCL5 mRNA and protein expression. Taken together, our data strongly suggest that IL‐1‐mediated signalling is activated to induce the high expression of CCL2 and CCL5 via reduction in IL‐1R2 in the skin lesions caused by gefitinib.     

Long‐term maintenance of skin immune system in a NOD‐Scid IL2rγnull mouse model transplanted with human skin

We developed a NOD‐Scid IL2rγ^null mouse model transplanted with human skin that brings fundamental insight on in vivo cellular mechanisms of intradermal immunization and antigen presentation by dermal dendritic and epidermal Langerhans cells for skin T‐cell immunity. Indeed, T‐cell immunity is a crucial checkpoint for the induction of in vivo rapid control of skin infection. With the long‐term preservation of a complete human skin immune system, this model offers the unique opportunity not only to better understand mechanisms of skin immune response but also to test new compounds and devices for cutaneous routes of vaccination, as well as new therapeutics approach for skin diseases, allergies or infections.     

Granzyme A potentiates chemokine production in IL‐17‐stimulated keratinocytes

Plaque psoriasis presents with focal skin inflammation, partially maintained by IL‐17‐mediated interactions between infiltrating epidermal T cells and activated keratinocytes. Here we show that the majority of lesional epidermal CD8 T cells express granzyme A, alone or in combination with IL‐17. To assess proinflammatory properties of granzyme A in psoriasis, primary human keratinocytes were stimulated with granzyme A in the presence or absence of IL‐17. Out of 33 analysed keratinocyte‐derived inflammatory mediators, granzyme A potentiated IL‐17‐induced secretion of CXCL 1, CXCL 12 and CCL 4. Intriguingly, all three chemokines are implicated in psoriasis pathogenesis and are involved in recruitment of T cells, neutrophils and pDCs into inflamed tissues. Our results indicate that granzyme A produced by lesional CD8 T cells specifically increase the chemokine production from inflamed keratinocytes, thereby amplifying a chemotactic inflammatory loop that sustains psoriasis lesions.     

IL‐17A synergistically enhances TLR3‐mediated IL‐36γ production by keratinocytes: A potential role in injury‐amplified psoriatic inflammation

Skin injury can trigger formation of new lesions in psoriasis (Koebner phenomenon). The mechanisms through which injury exacerbates psoriasis are unclear. During wound repair, epidermal keratinocytes are activated and produce abundant IL‐36γ, further promoting the skin inflammation. IL‐17A is the cornerstone cytokine in the pathogenesis of psoriasis. We sought to investigate the effects of IL‐17A on injury‐induced keratinocyte activation and IL‐36γ production. Here, we demonstrated that dsRNA released from necrotic keratinocytes induced the expression of IL‐36γ. Silencing of TLR3 by siRNA decreased the IL‐36γ induction by necrotic keratinocyte supernatant. Co‐stimulation with dsRNA and IL‐17A synergistically increased the expression of IL‐36γ and other proinflammatory mediators (CCL20, CXCL8, DEFB4 and LCN2) in keratinocytes. The synergistic effects were not dependent on TLR3 upregulation, TNF receptor signalling and mRNA stabilization. Co‐stimulation with dsRNA and IL‐17A resulted in an accumulation of IκBζ. The synergistic upregulation of IL‐36γ and proinflammatory mediators were inhibited by IκBζ siRNA. Co‐stimulation with IL‐17A and poly(I:C) markedly activated the p38 MAPK and NF‐κB pathway, compared with poly(I:C). Blockade of p38 MAPK and NF‐κB suppressed dsRNA/IL‐17A–mediated IκBζ and IL‐36γ induction. These findings demonstrated that IL‐17A synergistically enhanced the dsRNA‐mediated IL‐36γ production through a p38 MAPK‐, NF‐κB–, and IκBζ‐dependent mechanism.     

IL‐33 is secreted by psoriatic keratinocytes and induces pro‐inflammatory cytokines via keratinocyte and mast cell activation

IL‐33 is a novel pro‐inflammatory cytokine and ligand for the orphan receptor ST2. Although originally defined as an inducer of Th2‐mediated responses, IL‐33 was recently found to be involved in arthritis, a Th1/Th17‐mediated disease. Here, we assessed the ability of IL‐33 to promote inflammation via mast cells (MCs) and keratinocytes (KCs) activation in psoriasis. IL‐33 resulted elevated in the skin but not in the serum of psoriasis patients. IL‐33 was secreted by psoriasis KCs and HaCaT cells after TNF‐α stimulation. In HMC‐1, TNF‐α, but not IL‐17, could induce a robust increase in IL‐33 expression. In HaCaT cells, TNF‐α was able to induce IL‐6, MCP‐1 and VEGF, and the addition of IL‐33 reinforced these increases. TNF‐α + IL‐33 combination showed similar results in primary KCs and ex vivo skin organ culture. In conclusion, our study suggests that IL‐33 may be involved in psoriasis biology via MCs and KCs.     

Increased levels of serum IL‐31 in chronic spontaneous urticaria*

Please cite this paper as: Increased levels of serum IL‐31 in chronic spontaneous urticaria. Experimental Dermatology 2010; 19: 464–466. Abstract: IL‐31 represents a novel cytokine involved in pruritic skin diseases including atopic dermatitis (AD). We, therefore, aimed at investigating IL‐31 levels in chronic spontaneous urticaria (CU). We included 46 patients with CU, 26 non‐atopic skin healthy subjects as negative and 28 patients with AD as positive controls. IL‐31 serum levels were analysed using commercial ELISA kit. IL‐31 serum levels were higher in patients with CU compared to healthy controls (P < 0.001), but lower compared to patients with AD (P < 0.001). There was no difference in IL‐31 serum levels in autologous serum skin test positive or negative CU patients and patients with infectious trigger factors including helicobacter pylori infection. IL‐31 serum levels may play a role in the pathophysiology of CU. This is supported by the finding that not all patients with CU respond to antihistamine treatment but to the treatment with immunosuppressive drugs.     

Repeated administration of IL‐31 upregulates IL‐31 receptor A (IL‐31RA) in dorsal root ganglia and causes severe itch‐associated scratching behaviour in mice

We investigated the effects of repeated administration of interleukin‐31 (IL‐31) on itch‐associated scratching counts (long‐lasting scratching, LLS) and IL‐31‐related receptor mRNA expression in mice. Intra‐dermal (i.d.) injection of IL‐31 (100 and 300 ng/site) every 12 h for 3 days significantly increased LLS. Repeated administration of IL‐31 also increased the expression of IL‐31 receptor A (IL‐31RA) and oncostatin M receptor beta (OSMRβ) in dorsal root ganglia (DRG). After the repeated administration of IL‐31 was discontinued, IL‐31RA expression decreased and reached the baseline level 2 days after the last dose of IL‐31. LLS changed along with DRG IL‐31RA expression. Moreover, IL‐31‐induced IL‐31RA protein expression was confirmed by Western blotting analysis. These data suggest that IL‐31 upregulates IL‐31RA expression in DRG neuron cell bodies, and cutaneous‐injected IL‐31‐induced itching is enhanced by DRG IL‐31RA expression in mice.     

Heparinoid suppresses Der p‐induced IL‐1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.     

Evidence on the direct correlation between miR‐31 and IL‐22 axis in IMQ‐induced psoriasis

Psoriatic skin is characterized by a deregulated profile of miRNAs that are contributing in disease development. In this study, we focus on miR‐31, one of the upregulated miRNAs known to promote keratinocytes proliferation and migration. Moreover, miR‐31 expression induction was dependent on a large panel of cytokines including IL‐22. Here, we aimed at investigating the relationship between miR‐31‐5p and IL‐22 axis; and by searching novel molecular target for miR‐31‐5p in keratinocytes. Our data identify a direct correlation between miR‐31‐5p and IL‐22 in psoriasis with Pwp1 as new potential target. These findings confirm the important role of miR‐31 in psoriasis onset and provide a basis for further investigations in miRNAs field in context of skin diseases.     

IL‐36γ has proinflammatory effects on human endothelial cells

Interleukin‐36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL‐36 is now a recognised characteristic of psoriatic skin inflammation. IL‐36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL‐36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL‐36 receptor. Adhesion molecules VCAM‐1 and ICAM‐1 are upregulated by IL‐36γ stimulation, and this is reversed by the presence of the endogenous IL‐36 receptor antagonist. IL‐36γ‐stimulated endothelial cells secrete the proinflammatory chemokines IL‐8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T‐cells following IL‐36γ stimulation of endothelial cells. These results suggest a role for IL‐36γ in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.     

Allergic contact dermatitis beyond IL‐1β role of additional family members

Contact dermatitis is one of the most frequent pathological manifestations of the skin and plays a central role in clinical dermatology. The IL‐1 family consists a large group of cytokines, which currently contains 11 members with different pro‐ and anti‐inflammatory properties. Among the more pro‐inflammatory‐acting cytokines from the IL‐1 family, IL‐1β, IL‐18, IL‐33 and IL‐36 have been shown to be upregulated in different inflammatory mouse experimental models or skin diseases. The article by Mattii et al. represents a thorough analysis of the expression of IL‐1 family members including IL‐33 in skin samples from patients with allergic contact dermatitis. Although a lot of research is performed in this area, data from human samples are rather scarce. Therefore, Mattii et al. support the development of novel therapeutic concepts, which might include the use of antagonistic molecules targeting the IL‐1 family network.     

IL-1 signalling is dispensable for protective immunity in Leishmania-resistant mice

Leishmaniasis is a parasitic disease affecting ～12 million people. Control of infection (e.g. in C57BL/6 mice) results from IL-12-dependent production of IFNγ by Th1/Tc1 cells. In contrast, BALB/c mice succumb to infection because of preferential Th2-type cytokine induction. Infected dendritic cells (DC) represent important sources of IL-12. Genetically determined differences in DC IL-1α/β production contribute to disease outcome. Whereas the course of disease was not dramatically altered in IL-1RI(-/-) mice, local administration of IL-1α to infected C57BL/6 mice improved disease outcome. To definitively elucidate the involvement of IL-1 in immunity against leishmaniasis, we now utilized IL-1α/β-double-deficient C57BL/6 mice. C57BL/6 mice are believed to be a good surrogate model for human, self limited cutaneous leishmaniasis (CL). Leishmania major-infected IL-1α/β(-/-) mice were resistant to experimental CL comparable to controls. In addition, DC-based vaccination against leishmaniasis in C57BL/6 mice was independent of IL-1. Thus, in Leishmania-resistant C57BL/6 mice, IL-1 signalling is dispensable for protection.