Induction of eosinophil‐ and Th2‐attracting epidermal chemokines and cutaneous late‐phase reaction in tape‐stripped skin

Abstract: Skin barrier damage induces various harmful or even protective reactions in the skin, as represented by enhancement of keratinocyte cytokine production. To investigate whether acute removal of stratum corneum modulates the production of chemokines by epidermal cells, we treated ears of BALB/c and C57BL/6 mice by tape‐stripping, or acetone‐rubbing as a control of acute barrier disruption procedure. There was no difference between the tape‐stripped and acetone‐rubbed skin sites in the increased and recovered levels of transepidermal water loss. The mRNA expression levels of all the chemokines tested, including Th1 chemokines (CXCL10, CXCL9 and CXCL11), Th2 chemokines (CCL17 and CCL22) and eosinophil chemoattractant (CCL5), were higher in the epidermal cells from BALB/c than in those of C57BL/6 mice. In particular, CCL17, CCL22 and CCL5 were remarkably elevated in BALB/c mice and augmented by tape‐stripping more markedly than acetone‐rubbing, whereas Th1 chemokines were enhanced by acetone‐rubbing more remarkably. Tape‐stripping induced dermal infiltration of eosinophils in BALB/c but not C57BL/6 mice. In a contact hypersensitivity model, where BALB/c mice were sensitized on the abdomen and challenged on the ears with fluorescein isothiocyanate, mice exhibited higher ear swelling responses at the late‐phase as well as delayed‐type reactions, when challenged via the tape‐stripped skin. The challenge via tape‐stripped skin augmented the expression of IL‐4 and CCR4 in the skin homogenated samples, indicating infiltration of Th2 cells. These findings suggest that acute barrier removal induces the expression of Th2 and eosinophil chemokines by epidermal cells and easily evokes the late phase reaction upon challenge with antigen.     

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.     

TNF‐α and IL‐10 gene polymorphisms show a weak association with pemphigus vulgaris in the Slovak population

Backround Pemphigus vulgaris is a rare chronic autoimmune disease of skin and mucous membranes, with several cytokines participating in its development. The role of their gene polymorphisms in susceptibility to the disease is, however, not fully understood. Objective The aim of our case‐control study was to investigate whether some of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine genes (IL‐1α, IL‐1β, IL‐1RI, IL‐1Ra, IL‐4Rα, IL‐12, IFN‐γ, TGF‐β1, TNF‐α, IL‐2, IL‐4, IL‐6 and IL‐10) are associated with pemphigus vulgaris in the Slovak population. Methods DNA samples were obtained from 34 pemphigus vulgaris patients and 140 healthy controls of Slovak origin. Cytokine gene SNPs were determined using the polymerase chain reaction with sequence‐specific primers (PCR‐SSP) method. Results We found a weak association between pemphigus vulgaris and polymorphic variants in TNF‐α and IL‐10 genes only, with haplotypes TNF‐α–308G/–238G and IL‐10 –1082A/–819C/–592C being significantly overrepresented in pemphigus vulgaris patients (TNF‐α GG: 94.12% vs. 82.86%, P = 0.0216; IL‐10 ACC: 44.12% vs. 30.00%, P = 0.0309). Conclusions Our preliminary results suggest that certain TNF‐α and IL‐10 gene polymorphisms might contribute to genetic susceptibility to pemphigus vulgaris; however, their overall impact on disease development will be rather limited.     

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.     

The crucial role of IL‐22 and its receptor in thymus and activation regulated chemokine production and T‐cell migration by house dust mite extract

House dust mite (HDM) is known as one of the factors that causes atopic dermatitis (AD). Interleukin (IL)‐22 and thymus and activation regulated chemokine (TARC) are related to skin inflammatory disease and highly expressed in AD lesions. However, the effects of HDM on IL‐22 production in T cells and on TARC production and IL‐22Rα receptor expression in keratinocytes are unknown. To identify the role of HDM in keratinocytes and T cells, we investigated IL‐22Rα expression and TARC production in the human keratinocyte cell line HaCaT and IL‐22 production in T cells treated with HDM extract as well as their roles in HDM‐induced skin inflammation. HDM extract not only increased IL‐22Rα expression and TARC production in HaCaT but also enhanced IL‐22, tumor necrosis factor (TNF)‐α and interferon (IFN)‐γ production in T cells. The HDM extract‐induced IL‐22 from T cells significantly increased the production of IL‐1α, IL‐6 and TARC in HaCaT cells. In addition, we found that TARC produced in HDM extract‐treated HaCaT induced T‐cell recruitment. These results suggest that there is a direct involvement of HDM extract‐induced IL‐22 in TARC production and T‐cell migration. Taken together, TARC production in HaCaT through the interaction between IL‐22 and IL‐22Rα facilitates T‐cell migration. These data show one of the reasons for inflammation in the skin lesions of AD patients.     

IL‐24 is expressed during wound repair and inhibits TGFα‐induced migration and proliferation of keratinocytes

Please cite this paper as: IL‐24 is expressed during wound repair and inhibits TGFα ‐induced migration and proliferation of keratinocytes. Experimental Dermatology 2010; 19 : 714–722. Abstract: Interleukin (IL)‐24 is the protein product of melanoma differentiation‐associated gene 7 (MDA‐7). Originally identified as a tumor suppressor molecule, MDA‐7 was renamed IL‐24 and classified as a cytokine because of its chromosomal location in the IL‐10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL‐24 is expressed by cytokine‐activated monocytes and T lymphocytes. Here, we show that IL‐24 is expressed in keratinocytes during wound repair. Paraffin‐embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL‐24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor α (TGFα), TGFβ, IFNγ, and IFNβ, upregulated IL‐24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL‐24 in both in vitro wound repair and migration assays demonstrated that IL‐24 inhibits TGFα‐induced proliferation and migration of NHEKs. These data support the hypothesis that IL‐24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.     

Lipocalin‐2 exacerbates psoriasiform skin inflammation by augmenting T‐helper 17 response

Lipocalin‐2 (LCN2) is an antimicrobial protein and adipokine associated with insulin resistance, obesity and atherosclerotic disease. Psoriasis is a T‐helper (Th)1/Th17‐mediated, chronic inflammatory dermatosis related to metabolic syndromes and serum LCN2 levels are elevated in psoriatic patients. We examined the in vivo effects of LCN2 on topical imiquimod (IMQ)‐induced psoriasiform skin in BALB/c mice and in vitro on human keratinocytes (KC). Clinically, i.p. injected LCN2 exacerbated erythema and scaling in IMQ‐treated murine skin compared with phosphate‐buffered saline injection alone, and it augmented interleukin (IL)‐17A, IL‐17F, IL‐22, IL‐23p19, IL‐12p40, CCL20, tumor necrosis factor‐α, chemokine (C‐X‐C motif) ligand (CXCL)1, CXCL2, DEFB4, DEFB14, LCN2 and S100A7 mRNA levels of IMQ‐treated murine skin while it did not increase the mRNA levels of interferon‐γ, IL‐12p35 or CXCL10. LCN2 in synergy with IL‐17 increased mRNA levels of CCL20, LCN2 and DEFB4A but not of CXCL10 in human KC in vitro. These results suggest that LCN2 enhances the expression of Th17 cytokines/chemokines and antimicrobial peptides in murine IMQ‐treated psoriatic skin and KC. LCN2 may potentiate the development of psoriasis via the enhancement of Th17‐ and antimicrobial peptide‐mediated inflammation.     

Malassezia‐derived aryl hydrocarbon receptor ligands enhance the CCL20/Th17/soluble CD163 pathogenic axis in extra‐mammary Paget's disease

Malassezia yeast play a role in the pathogenesis of chronic dermatitis, especially in apocrine areas, by polarizing the local immunologic background to a Th2/Th17 state through aryl hydrocarbon receptor (AhR)‐dependent pathways. Extra‐mammary Paget's disease (EMPD) is an adenocarcinoma of apocrine origin, and except for cases associated with Malassezia yeast and their metabolites, the lesions typically develop in areas not exposed to environmental material. The purpose of this study was to investigate (a) the immunomodulatory effects of Malassezia metabolites on normal human keratinocytes (NHKCs), focusing on interleukin (IL)‐17 and related cytokines/chemokines (IL‐23, IL‐36γ, CCL20), (b) the expression of these factors in lesion‐affected skin in EMPD and (c) the activation of tumor‐associated macrophages (TAMs) by these factors. Malassezia metabolites augmented the expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), CCL20 and IL‐36γ mRNA in NHKCs in vitro. In lesion‐affected skin of patients with EMPD, epidermal keratinocytes expressed CYP1A1 and CCL20. In addition, Paget cells expressed CCL20 and IL‐23. IL‐17–producing cells were distributed adjacent to Paget cells. Compared to healthy donors, patients with EMPD exhibited significantly increased serum levels of soluble (s)CD163, CXCL5, CXCL10 and CCL20. In addition, serum levels of sCD163 decreased significantly following tumor resection. Our study demonstrates a possible mechanism for the development of EMPD involving AhR‐mediated signalling by epidermal keratinocytes and RANKL‐induced recruitment of Th17 cells and TAMs.     

The single‐chain anti‐TNF‐α antibody DLX105 induces clinical and biomarker responses upon local administration in patients with chronic plaque‐type psoriasis

It is not clear whether TNF‐α antagonists used in the treatment of psoriasis need to act systemically, or whether local inhibition of skin‐produced TNF‐α would be sufficient to silence skin inflammation. To answer this question, we conducted two multicentre, double‐blinded, randomized, placebo‐controlled clinical trials with the novel single‐chain anti‐TNF‐α‐PENTRA^®‐antibody DLX105. Upon intra‐dermal injection, DLX105 induced a mean local PASI decrease of 33% over baseline after 2 weeks of treatment, while the placebo response was only 12% (P = 0.001). The clinical response was accompanied by changes in biomarkers such as reductions in K16, Ki67 and epidermal thickness as well as decreased mRNA levels of IL‐17, TNF‐α, IL‐23p19, IL‐12p40 and IFN‐γ. Next, we applied the drug topically twice daily in a 0.5% hydrogel formulation. While the local PASI did not change, topical DLX105 mediated significant reductions of mRNA levels of key proinflammatory cytokines when compared to placebo, and this effect was further enhanced after weekly tape stripping of plaques to increase drug penetration. These results suggest that longer treatment periods and/or increased local drug concentrations might result in better therapeutic efficacy of topically applied DLX105. In sum, we can show for the first time that local inhibition of TNF‐α is sufficient to mediate a biological response in psoriasis that translates into clinical efficacy.     

Decreased interleukin‐21 expression in skin and blood in advanced mycosis fungoides

Interleukin (IL)‐21 is regarded as a potent antitumor agent, which increases the cytotoxicity of both natural killer (NK) and CD8⁺ T cells. In this study, we investigated the role of IL‐21 in mycosis fungoides (MF). IL‐21 mRNA expression levels in patch and plaque MF were significantly higher than those in normal skin. IL‐21 mRNA expression levels in tumor MF were significantly decreased compared with those in patch and plaque MF. Interestingly, mRNA expression levels of IL‐21 in MF lesional skin significantly correlated with those of T‐helper type‐1 cytokines/chemokines such as CXCL10, CXCL11 and γ‐interferon. Immunohistochemistry showed that IL‐21 was expressed by keratinocytes in patch and plaque MF. Furthermore, serum IL‐21 levels in patients with tumor MF were significantly lower than those of healthy controls and plaque MF. Thus, IL‐21 expression was significantly downregulated in skin and blood of patients with tumor MF, which may contribute to progression of MF. Our study suggests that recombinant IL‐21 would be a promising therapy for MF.     

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL‐6 confers a protective effect to lesional skin during ICD. How IL‐6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid‐specific knockout of the IL‐6Rα (IL‐6Rα^Δmyeloid) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant‐8 (JP8) fuel, two well‐characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL‐6Rα^Δmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro‐inflammatory cytokines IL‐1α and TNF‐α, but reduced expression of chemokine proteins including CCL2‐5, CCL7, CCL11, CXCL1 and CXCL10 in IL‐6Rα^Δmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL‐6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.     

CCL7 contributes to the TNF‐alpha‐dependent inflammation of lesional psoriatic skin

Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT‐PCR, we found CCL7, a chemokine ligand known to interact with multiple C‐C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non‐lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod‐induced psoriasis‐like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro‐psoriatic cytokines such as CCL20, IL‐12p40 and IL‐17C, while its blockade led to an increase in the antipsoriatic cytokine IL‐4. In humans receiving the TNF‐α‐blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF‐α‐dependent Th1/Th17‐mediated inflammation in lesional psoriatic skin.     

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.     

Absence of CD4 or CD8 lymphocytes changes infiltration of inflammatory cells and profiles of cytokine expression in skin wounds,but does not impair healing

The involvement of lymphocytes in skin wound healing has not been studied extensively. This study shows that CD4 and CD8 cells are present in significant numbers in skin wounds with peak levels at days 5–10 and 7–10, respectively. Both subsets expressed inflammatory and/or regulatory cytokines. To examine the function of CD4 and CD8 lymphocytes in tissue repair, wound healing was examined in mice deficient for either CD4 or CD8 cells. Wounds in CD4 deficient mice exhibited an initial delayed infiltration of CD8 cells followed by a relative increase in CD8 cells at day 10 and thereafter. Wounds in CD4 deficient mice also displayed up‐regulated expression of IL1β, IL‐6, IL‐17, IFN‐γ, CXCL‐1 and down‐regulated expression of IL‐4 as compared to wild‐type mice. In contrast, wounds in CD8 deficient mice showed significantly decreased infiltration of CD4+ cells, neutrophils, and macrophages along with down‐regulated expression of IL1β, IL‐6, TNF‐α, CXCL‐1, CCL‐2 and up‐regulated expression of IL‐4 as compared to wild‐type mice. Despite these significant changes in cytokine expression and inflammatory cell infiltrate, the rate of wound closure, wound breaking strength, collagen content and angiogenesis in either CD4 or CD8 deficiency showed no significant difference from that of wild‐type mice. The results suggest that, despite being present and involved in wound inflammation, neither CD4+ nor CD8+ cells play critical roles in the healing process of skin wounds. Further studies are needed to investigate whether these cells might play critical roles in wounds that experience stress such as ischemia or infection.     

IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes

Please cite this paper as: IL‐1 signalling determines the fate of skin grafts expressing non‐self protein in keratinocytes. Experimental Dermatology 2010; 19 : 723–729. Abstract: Although IL‐1 is a known inflammatory cytokine during pathogen infection, the role of IL‐1 in skin graft rejection, particularly where foreign antigen is expressed exclusively in keratinocytes, is less understood. Here, we use a syngeneic skin graft system, where antigens are expressed in epithelial cells via either a keratin 14 or keratin 5 promoter, to explore the role of IL‐1 in graft rejection and induction of epithelial antigen‐specific effector CD8⁺ T‐cell function. Keratin 5 ovalbumin (K5mOVA) transgenic skin grafts destined for rejection demonstrated increased expression of IL‐1β and its receptors compared to K14 HPV16 E7 transgenic grafts that do not reject spontaneously. Rejection of OVA grafts lacking the IL‐1 receptor (IL‐1R1) was delayed and associated with decreased numbers of antigen‐specific CD8 T cells. In contrast, K14E7 grafts survived on immunocompetent, syngeneic recipients with decreased graft levels of IL‐1β and IL‐1R1 and 2. However, in the absence of the IL‐1 receptor antagonist, IL‐1Ra, skin grafts were spontaneously rejected and an E7‐specific CD8 T‐cell response was primed. Thus, expression of the HPV16E7 oncoprotein in epithelial cells prevents IL‐1β‐associated skin graft rejection and induction of antigen‐specific CD8 T‐cell responses. Enhancing IL‐1β signalling, via blocking of the IL‐1 receptor antagonist, may represent an alternative strategy for treatment of HPV16E7‐associated cancers.     

Proteomic analysis of stratum corneum in Cutaneous T‐Cell Lymphomas and psoriasis

Stratum corneum collected by tape stripping from 10 and 24 subjects with cutaneous T‐cell lymphomas (CTCL) or psoriasis, respectively, were compared using quantitative label‐free mass spectrometry analysis. A non‐supervised statistical analysis (Posneg NMF) based on 352 differentially expressed proteins in both CTCL and psoriasis samples was able to separate the two disease groups and finally able to identify a set of 112 proteins that contributed most and significantly to the separation when compared to non‐lesional samples. In addition, Luminex assay revealed that the increase in the amount of chemokines related to the inflammatory response, and immune cell infiltration and recruitment in lesional stratum corneum in CTCL, including CXCL8, CXCL9, CXCL10, CCL27, TNF and sICAM‐1 was in agreement with published data on entire skin biopsies. Proteome analysis using quantitative methods including mass spectrometry and Luminex technology offered the possibility to investigate the relevant protein signature in CTCL and may be helpful to diagnose and investigate the efficacy of treatments in clinical investigations using non‐invasive methods in future.     

Contribution of CARD9‐mediated signalling to wound healing in skin

The inflammatory response after skin injury involves the secretion of a variety of cytokines and growth factors that are necessary for tissue repair. Caspase recruitment domain‐containing protein 9 (CARD9) is an essential signalling adaptor molecule for NF‐κB activation upon triggering through C‐type lectin receptors (CLRs), which are expressed in macrophages and dendritic cells. However, the role of CARD9 in inflammatory responses at the wound site has not been elucidated. In this study, we analysed the role of CARD9 in the healing process of skin wounds. Wounds were created on the backs of wild‐type (WT) C57BL/6 mice and CARD9 gene‐disrupted (knockout [KO]) mice. We analysed per cent wound closure, and the wound tissues were harvested for analysis of leucocyte accumulation and cytokine and chemokine expressions. CARD9KO mice exhibited significant attenuation of wound closure compared with WT mice on days 5, 7 and 10 postwounding, which was associated with decreased macrophage accumulation and reduced TNF‐α, IL‐1β, CCL3 and CCL4 expressions. These results suggest that CARD9 may be involved in the wound‐healing process through the regulation of macrophage‐mediated inflammatory responses.     

Role of IL‐10 and TGF‐β in melanoma

IL‐10 and TGF‐β are immunosuppressive cytokines expressed in tumors including melanoma and, therefore, deemed major cause for failing antitumor immune responses. Re‐evaluating their role, we compared their expression by quantitative RT‐PCR in melanoma and skin of healthy individuals, tested their induction in dendritic cells and T cells co‐cultured with tumor cells, and their effects on the immune cells. Both cytokines as well as their receptors were expressed in melanoma at significantly lower levels than in healthy skin. Consequently, the expressions of IL‐10‐responsive SOCS‐3 and TGF‐β‐responsive Smad‐7 were low in tumors but high in healthy skin. T cells co‐cultured with tumor cells developed an anergic state without increased IL‐10 or TGF‐β expression. In vitro tumor‐induced immature dendritic cells produced high IL‐10 levels and less efficiently induced T‐cell proliferation. Nonetheless, they could be induced to mature, and blocking IL‐10 did not alter the capacity of the resulting mature dendritic cells to stimulate T cells. Mature dendritic cells co‐cultured with tumor cells produced increased IL‐10 but decreased TGF‐β and more efficiently induced T‐cell proliferation. The lack of correlation of IL‐10 and TGF‐β with immune deficits in situ and in vitro suggests re‐evaluating their roles in cancer.     

CD4+ T cells producing interleukin (IL)‐17, IL‐22 and interferon‐γ are major effector T cells in nickel allergy

Background It has been suggested that interleukin (IL)‐17 and IL‐22 play important roles in the elicitation of human allergic contact dermatitis; however, the frequencies of T cell subtypes producing IL‐17 and IL‐22 in human allergic contact dermatitis are unknown. Objectives To determine the frequencies of CD4⁺, CD8⁺ and γδ T cells producing IL‐17, IL‐22 and interferon (IFN)‐γ in the blood and skin from nickel‐allergic patients. Patients/materials/methods Blood samples were collected from 14 patients and 17 controls, and analysed by flow cytometry. Biopsies were taken from 5 patients and 6 controls, and analysed by immunohistochemistry and flow cytometry of skin lymphocytes. Results We found an increased frequency of γδ T cells in the blood, but no differences in the distribution of cytokine‐producing CLA⁺ T cell subtypes in nickel‐allergic patients as compared with controls. In nickel‐allergic patients, there was massive cellular infiltration dominated by CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ in nickel‐challenged skin but not in vehicle‐challenged skin. Conclusion CD4⁺ T cells producing IL‐17, IL‐22 and IFN‐γ are important effector cells in the eczematous reactions of nickel‐induced allergic contact dermatitis in humans.