Suppression of IL-17A-induced CCL20 production by cytokine inducible SH2-containing protein 1 in epidermal keratinocytes

BackgroundLesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes.ObjectiveTo investigate whether CIS1 influences CCL20 production in epidermal keratinocytes.MethodsExpression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro.ResultsExpression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A.ConclusionCIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.     

Expression of CCR2 on monocytes and macrophages in chronically inflamed skin in atopic dermatitis and psoriasis

Monocytes form a significant component of the inflammatory reaction taking place in the skin of atopic dermatitis and psoriasis. Chemokines are pivotal in mediating the attraction of leucocytes to sites of inflammation. The CC-chemokine, monocyte chemotactic protein 1 (MCP-1/CCL2), is expressed by keratinocytes in both atopic dermatitis and psoriasis. MCP-1 binds to the chemokine receptor CCR2 which is known to be expressed on monocytes and macrophages. We examined the expression of CCR2 on peripheral blood monocytes from patients with psoriasis (n=8) and atopic dermatitis (n=7) and found it to be expressed on approximately 90% of the cells, whereas monocytes from healthy donors had a significantly lower CCR2 expression (p<0.05). Skin biopsies from patients suffering from atopic dermatitis and psoriasis revealed that CCR2-positive cells expressed CD163, a marker for monocytes/macrophages. However, not all CD163-positive cells expressed CCR2, which could be interpreted as a mechanism for retaining the macrophages in the skin. Furthermore, we found that keratinocytes are able to express MCP-1, when stimulated with tumour necrosis factor-alpha and/or interferon-gamma in a dose-dependent manner. Thus MCP-1 and CCR2 interaction is likely of importance for the monocyte/macrophage trafficking of inflammatory skin disorders.     

Expression of the C-C chemokine MIP-3 alpha/CCL20 in human epidermis with impaired permeability barrier function

External assault to the skin is followed by an epidermal response including synthesis of DNA, lipids, cytokines and migration of antigen presenting cells. MIP-3 alpha (CCL20, LARC, Exodus-1, Scya20) is a recently described C-C chemokine, predominantly expressed in extralymphoid tissue, which is known to direct migration of dendritic cell precursors and memory lymphocytes to sites of antigen invasion. We assessed the expression of MIP-3 alpha in human skin using semi-quantitative polymerase chain reaction. In vivo, MIP-3 alpha mRNA was constitutively expressed at low levels in untreated human epidermis. After acute disruption of the epidermal permeability barrier MIP-3 alpha mRNA was upregulated in the epidermal fraction, whereas dermal MIP-3 alpha mRNA levels remained unchanged. In vitro, MIP-3 alpha was increased in cultured keratinocytes treated with IL-1 alpha and TNF-alpha and was present in immature and mature dendritic cells, THP-1 monocytic cells and activated T cells. Finally, skin biopsies from patients with psoriasis, contact dermatitis and mycosis fungoides showed abundant expression. In biopsies from atopic dermatitis and graft vs. host disease a weak signal was present, whereas no expression was found in scleroderma and toxic epidermal necrolysis. We conclude that regulation of MIP-3 alpha mRNA is part of the epidermal response to external assault. Its upregulation may represent a danger signal for increased immunosurveillance in barrier disrupted skin and inflammatory skin conditions with impaired barrier function to counteract potential antigen invasion.     

CC chemokine receptor 4 expression on peripheral blood CD4+ T cells reflects disease activity of atopic dermatitis

Recent studies indicate that Th1 and Th2 cells differ in their chemokine receptor expression and their responsiveness to various chemokines. Therefore, selective Th2 cell recruitment in Th2-predominant inflammatory diseases such as atopic dermatitis may be under the influence of some chemokines. It is reported that CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells whereas CXC chemokine receptor (CXCR) 3 is selectively expressed on Th1 cells. In this study we examined CCR4 and CXCR3 expression on peripheral blood CD4+ and CD8+ T cells obtained from adult atopic dermatitis subjects, and compared the results with those from patients with psoriasis vulgaris and healthy controls. CCR4 was preferentially expressed on CD4+ T cells from atopic dermatitis subjects and CXCR3 was preferentially expressed on CD4+ T cells from psoriasis vulgaris subjects. This CCR4 expression was prominent especially in severe atopic dermatitis subjects. CCR4 expression on CD4+ T cells in severe atopic dermatitis subjects decreased on improvement of disease activity. CD25 was preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. Cutaneous lymphocyte-associated antigen was also preferentially expressed on CCR4+CD4+ T cells but not on CXCR3+CD4+ T cells in atopic dermatitis subjects. CD4+ T cells in atopic dermatitis skin lesions were predominantly CCR4+ cells. Taken together, this study strongly indicates that CCR4+CD4+ T cells reflect disease activity and suggests that CCR4 expression is important for T cell infiltration into atopic dermatitis lesions. Thus, CCR4 may be a possible target for therapy of atopic dermatitis in the future.     

Statins inhibit chemotactic interaction between CCL20 and CCR6 in vitro: possible relevance to psoriasis treatment

Psoriasis is a chronic IL-23/Th17 pathway-associated skin disease. An increased expression of lesional CCL20 can recruit CCR6+ Th17, and lesional cytokine milieu persistently activates keratinocytes to produce CCL20. Lipid-lowering drugs, statins, are known to possess immune-modulating functions. In this study, we explored an inhibitory effect of statins on CCL20/CCR6 interaction. We demonstrated that IL-1β, TNF-α, and IL-17A significantly increased CCL20 production from HaCaT cells. However, these increments were markedly inhibited by fluvastatin and simvastatin, but not by pravastatin. In the chemotaxis migration assay, pretreatment with fluvastatin and simvastatin inhibited the migration of human CD4+ T cells towards CCL20. However, the level of CCR6 surface expression in memory CD4+ T cells was not affected. Our results suggest that not all, but specific types of statins may be of benefit in alleviating psoriasis partially via interrupting CCL20/CCR6 chemotactic interaction, the mechanism which may eventually lessen the infiltration of Th17 cells.     

A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin

Atopic dermatitis is an inflammatory skin disease in which the inflammation is characterized by the influx of lymphocytes into the dermis. It is generally believed that atopic dermatitis is a Th2-type disease, i.e., the T lymphocytes produce interleukin-4, interleukin-5, interleukin-10, and interleukin-13, although it has become evident in recent years that the cytokine profile in the skin changes during the course of the disease towards a Th1-Th2 mixed cytokine profile (interferon-gamma, tumor necrosis factor alpha, and interleukin-2). The lymphocytes that home into the skin express cutaneous lymphocyte-associated antigen, and it has recently been shown that most of the lymphocytes in this population express the chemokine receptor CCR4. CCR4 is the receptor for the CC chemokine TARC (thymus and activation regulated chemokine), and this chemokine is expressed predominantly by keratinocytes in the basal layer of the epidermis of lesional atopic dermatitis skin in mice. In humans, however, it was shown to be expressed in the endothelial cells of the dermis. We have examined the peripheral blood mononuclear cells of atopic dermatitis patients for the expression of cutaneous lymphocyte-associated antigen and CCR4 and compared them with peripheral blood mononuclear cells from normal controls. We found that the proportion of CLA+CCR4+ lymphocytes is upregulated in atopic dermatitis patients. In addition we have examined skin biopsies of lesional and non-lesional skin from atopic dermatitis patients and found that the keratinocytes, but not the endothelial cells, produce TARC in the lesional but not in the nonlesional skin. To gain insight in the stimulatory mechanisms for TARC production in keratinocytes, as previously observed in mice, we cultured HaCaT cells and found that interferon-gamma and tumor necrosis factor alpha work synergistically to induce TARC production. These observations suggest that the induction of TARC production in keratinocytes plays an important role in the late phase skin invasion by CCR4+CLA+ Th2-type lymphocytes in atopic dermatitis.     

Thymus- and activation-regulated chemokine (TARC/CCL17) induces a Th2-dominated inflammatory reaction on intradermal injection in mice

TARC/CCL17 (thymus- and activation-regulated chemokine) is a CC chemokine, which binds to the CC chemokine receptor-4 (CCR4) known to be distinctively expressed on Th2 lymphocytes. In atopic dermatitis (AD), the skin is invaded by Th2 lymphocytes in the acute phase. TARC/CCL17 is produced by the keratinocytes in AD lesions, and CCR4 is overexpressed on CLA+ (cutaneous lymphocyte-associated antigen) lymphocytes in the skin and blood. We, therefore, hypothesized that TARC/CCL17 is pivotal in mediating a Th2-dominated inflammation in the skin. To examine this, we injected BALB/c mice with murine TARC/CCL17 in concentrations ranging from 0.1 microg/ml to 10 microg/ml and examined the skin after 48 h. This revealed that TARC/CCL17 induces lymphocytic infiltration of the skin by CD4+ lymphocytes in a dose-dependent manner with a maximum response at 1 microg/ml. Additionally, TARC/CCL17 induced interleukin-4 mRNA but not interferon-gamma mRNA expression in the skin, suggesting that the lymphocytes invading the skin are Th2 cells. Additionally, TARC/CCL17 induced its own production in the keratinocytes along with cutaneous T-cell-attracting chemokine (CTACK/CCL27) mRNA. We, therefore, conclude that TARC/CCL17 induces a Th2-dominated inflammatory reaction when injected into the skin.     

Chemokine receptors in the pathogenesis and therapy of psoriasis

Chemokine receptors are G-protein-coupled, seven-transmembrane-spanning surface receptors that play key roles in cell trafficking, cell motility, and survival. These receptors are activated by small molecular weight chemotactic cytokines called chemokines. Chemokine receptors and their corresponding chemokine ligands play roles in the migration and localization of normal T cells (and other cells) during physiological responses in inflamed or infected skin. In psoriasis, the chemokine receptor CCR6 is expressed on the Th17 cells and γδ T cells, which produce a variety of cytokines (IL17 and IL22 among others), that play a role in the immunological activation. CCR6 and its ligand, CCL20, are highly expressed in psoriatic skin lesion and CCR6 is essential for the development of the psoriasiform phenotype following IL23 injection in mouse skin. In this review, we focus on the roles of chemokine receptors, particularly of CCR6, in the pathogenesis of psoriasis and discuss chemokine receptors as novel therapeutic targets for psoriasis.     

Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases

Thymus and activation-regulated chemokine (TARC)/CCL17 is constitutively expressed in the thymus and is produced by dendritic cells (DC), endothelial cells, keratinocytes (KC) and fibroblasts. TARC is designated a Th2 type chemokine since it binds to CCR4. We review the pathogenic role of TARC in skin diseases such as atopic dermatitis (AD), bullous pemphigoid (BP) and mycosis fungoides (MF) focusing on epidermal KC and Langerhans cells (LC), which are epidermal DC. We have determined that serum TARC levels sharply reflect the disease activity of AD, which is thought to be a Th2-dominant inflammatory skin disease especially in the acute phase. Serum TARC levels are also related to the disease activity of BP, which is a blistering autoimmune skin disease, and MF, which is a cutaneous T-cell lymphoma, but very high serum TARC levels are only seen in a limited number of various other skin diseases. TARC may be a useful laboratory marker for the diagnosis of AD, especially cases which are moderate to severe, and for the evaluation of disease activity of AD. IL-4 and TGF-beta1 downregulate TNF-alpha and IFN-gamma induced TARC production in the human KC cell line, HaCaT cells, while IL-4 upregulates, and IFN-gamma downregulates TARC production by mouse LC. Because TARC and its receptor CCR4 are believed to play important roles in the pathogenesis of AD, BP and MF, TARC and CCR4 may be possible future targets for therapy of these diseases.     

CCL20、CCR6及IL-17A在寻常型银屑病患者血清中的表达及意义

目的观察趋化因子CCL20及其受体CCR6、白细胞介素(IL)-17A在银屑病患者血清中的变化,并探讨其在银屑病发病中的作用。方法采用酶联免疫吸附法测定26例斑块型、7例点滴型银屑病患者及8名健康人血清中CCL20、CCR6及IL-17A的浓度,并对银屑病皮损面积和严重程度指数(PASI)评分、病程进行相关性分析。结果斑块型银屑病患者血清CCL20、CCR6及IL-17A浓度(748.74±268.72,10.20±3.75,39.22±13.21)均显著高于健康对照组(578.45±204.93,6.79±4.61,25.54±13.04)(P分别0.05,0.01,0.01)。点滴型银屑病与对照组差异无统计学意义。CCL20、CCR6与PASI评分呈正相关(r=0.416,r=0.350)(P0.05)。CCL20、CCR6及IL-17A三者之间均存在显著正相关(r=0.852,r=0.801,r=0.825)(P0.001),与病程无明显相关性。结论 CCL20、CCR6及IL-17A参与斑块型银屑病的病理过程。     

趋化因子CCL27在常见炎陛皮肤病中的作用

临床常见的炎性皮肤病,如,银屑病、特应性皮炎和接触性皮炎等,其典型特征是T淋巴细胞浸润为主的皮肤炎症。趋化因子CCL27主要由皮肤角质形成细胞产生,其惟一受体是CCRl0。CCL27和CCRl0的相互作用,诱导皮肤记忆T细胞向局部皮肤的聚集,形成并维持了各种炎性皮损,因而已成为药物研究的新靶点。尽管目前尚无真正的临床药物可用,但体外研究和动物实验的良好效果为炎性皮肤病的治疗开启了一扇新的大门。     

Kinetics and differential expression of the skin-related chemokines CCL27 and CCL17 in psoriasis, atopic dermatitis and allergic contact dermatitis

CCL27 and CCL17 are chemokines believed to be involved in the process of establishing the inflammatory infiltrate, characteristic for the various inflammatory skin diseases. The skin-specific CCL27 binds the chemokine receptor-10 (CCR10), and CCL17 is a chemokine receptor-4 (CCR4) ligand. The purpose of our study was to characterize the expression of CCL27 and CCL17 in the inflammatory skin diseases: psoriasis, atopic dermatitis (AD) and acute allergic contact dermatitis (ACD) induced in nickel-sensitive individuals. Surprisingly, our studies revealed a markedly decreased CCL27 mRNA and protein expression in psoriatic lesions compared with non-lesional psoriatic skin. A minor CCL17 mRNA increase was measured in lesional psoriatic skin. No alterations were found in AD. In ACD, we found a pronounced (90-fold) raise in CCL17 mRNA and a 50-fold increase in CCL17 protein compared with normal skin. A kinetic ACD study of CCL17 expression showed the highest mean value 24 h after hapten application. Furthermore, we found the mRNA levels of CCR10 and CCR4 paralleling the results of their corresponding ligands. Overall, our principal findings were a distinct decrease in CCL27 in lesional psoriatic skin and a marked upregulation of CCL17 in ACD. These findings underscore the differential cutaneous T-cell recruitment in different inflammatory diseases.     

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.     

Lack of evidence for TARC/CCL17 production by normal human keratinocytes in vitro

BACKGROUND: thymus and activation-regulated chemokine (TARC)/CCL17 is a CC chemokine that selectively attracts Th2-type lymphocytes. Immunohistochemical analyses have revealed that TARC is expressed in the epidermal keratinocytes of atopic dermatitis (AD), suggesting TARC involvement in the pathogenesis of the disease. However, keratinocyte TARC production has been described only in the transformed keratinocyte cell line HaCaT. OBJECTIVE: to examine TARC production in normal human epidermal keratinocytes (NHEK) in vitro. METHODS: the expression of TARC mRNA and protein were examined in NHEK and HaCaT cells stimulated with various cytokines. RESULTS: stimulation with inflammatory cytokines, including interleukin (IL)-1, IL-4, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and tumor necrosis factor (TNF)-alpha failed to induce TARC mRNA expression in NHEK. However, stimulation with IFN-gamma and TNF-alpha together enhanced expression slightly. ELISA analysis failed to detect TARC protein in NHEK culture supernatant, even following stimulation with IFN-gamma and TNF-alpha. In contrast, HaCaT cells produced TARC protein even without stimulation of cytokines. CONCLUSION: these results indicate that production of TARC by HaCaT cells is a phenomenon specific to the cell line and the observation on TARC in HaCaT cells can not be generalized. NHEK do not produce TARC protein in vitro.     

Role of the chemokine receptor CCR4 and its ligand thymus- and activation-regulated chemokine/CCL17 for lymphocyte recruitment in cutaneous lupus erythematosus

Skin-infiltrating T lymphocytes are thought to play a major role in the pathogenesis of cutaneous lupus erythematosus (CLE). In this study, we investigated the role of the chemokine receptor 4 (CCR4) and its ligand thymus- and activation-regulated chemokine (TARC/CCL17) for the recruitment of T cells in inflamed skin of patients with CLE. We found significant numbers of CCR4+ T lymphocytes in the skin of all patients with CLE. Interestingly, a subset of patients with disseminated scarring skin involvement were characterized by both lesional and circulating CD8+ T cells expressing CCR4. Destruction of epidermal and adnexal structures was histomorphologically associated with CCR4+ cytotoxic T cells invading basal layers of the epidermis where keratinocytes showed apoptotic death. The CCR4 ligand TARC/CCL17 was strongly expressed in skin lesions and elevated in the serum of CLE patients. The functional relevance of lymphocytic CCR4 expression could be confirmed by TARC/CCL17-specific in vitro migration assays. Our investigations suggest that CCR4 and TARC/CCL17 play a role in the pathophysiology of CLE. In particular, cytotoxic CD8+ T cells expressing CCR4 appear to be involved in scarring subtypes of CLE.     

CCL28 production in HaCaT cells was mediated by different signal pathways from CCL27

Both CCL27 and CCL28 are ligands for CCR10 and attract CCR10(+) lymphocytes. We previously demonstrated that CCL27 and CCL28 were strongly expressed in sera and lesional keratinocytes of patients with atopic dermatitis and psoriasis vulgaris. However, the regulation of CCL27 and CCL28 production in keratinocytes has not been well documented. In this study, we showed that CCL27 and CCL28 expression and production by a human keratinocyte cell line, HaCaT cells, were strongly induced by inflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta. CCL27 production was downregulated by inhibitors of p38 mitogen-activated protein kinase and nuclear factor-kappa B (NF-kappaB). By contrast, CCL28 production was downregulated by inhibitors of extracellular signal-regulated kinase and NF-kappaB. Our study results suggest that CCL28 produced by keratinocytes is mediated by different signal pathways from CCL27 and that both CCL27 and CCL28 are involved in the pathogenesis of inflammatory skin diseases.     

Changes of epidermal mu-opiate receptor expression and nerve endings in chronic atopic dermatitis

There is increasing evidence that neuropeptides such as a substance P, neurotrophins or beta-endorphin, an endogenous agonist for mu-opioid receptor, are involved in the pathogenesis of atopic dermatitis in which mental stress and scratching deteriorate the disease. mu-Opioid receptor, a G-protein-coupled receptor, can be downregulated and internalized by agonists and other factors in vitro. In this study, we investigated the regulation of mu-opioid receptor and nerve endings in atopic dermatitis patients. Skin biopsies from atopic dermatitis patients revealed a significant downregulation of mu-opiate receptor expression in epidermis of atopic dermatitis. Permeabilization of the skin showed that the receptor in keratinocytes from atopic dermatitis is internalized. The mRNA expression pattern of the mu-opiate receptor is different in epidermis taken from patients with chronic atopic dermatitis compared to normal skin. In atopic dermatitis, the mRNA is concentrated in the subcorneal layers of the epidermis and in normal skin in the suprabasal layers. Staining of the nerve endings using protein gene product 9.5 shows a different pattern of epidermal nerve endings in normal skin compared to atopic dermatitis. In normal skin, the epidermal nerve endings are rather thick. However, in atopic dermatitis, the epidermal nerve endings are thin and run straight through the epidermis. Based on these observations and combining the 'intensity' and 'pattern' hypothesis, we propose a new theory especially for histamine-unrelated, peripheral induction of chronic pruritus. We suggest that 'itch' is elicited in the epidermal unmyelinated nerve C-fibers and 'pain' in the dermal unmyelinated nerve fibers. The downregulation of the opioid receptor in the epidermis contributes to the chronic itching. We call this new hypothesis the 'layer hypothesis'.