TGF-beta/Smad signaling inhibits IFN-gamma and TNF-alpha-induced TARC (CCL17) production in HaCaT cells

BACKGROUND: A Th2 chemokine, thymus and activation regulated chemokine (TARC/CCL17), produced by keratinocytes, is implicated in the development of atopic dermatitis by recruiting CLA(+)CCR4(+) lymphocytes into lesional skin and its expression was induced by proinflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). However, it remains unknown how TARC expression is negatively regulated in keratinocytes. OBJECTIVE: We sought to determine whether transforming growth factor-beta 1 (TGF-beta 1) regulated TARC expression in keratinocytes. METHODS: The effect of TGF-beta 1 on mRNA and protein expression of IFN-gamma and TNF-alpha-induced TARC in a human keratinocyte cell line, HaCaT cells, was evaluated by using RT-PCR and ELISA. Adenovector-mediated gene transfer was used to determine the effect of Smad proteins on TARC expression in HaCaT cells. RESULTS: TGF-beta 1 inhibited mRNA and protein expression of IFN-gamma and TNF-alpha-induced TARC in HaCaT cells. The inhibitory effect of TGF-beta 1 on the TARC expression was suppressed by overexpression of Smad7, a major inhibitory regulator of Smad pathway for transforming growth factor-beta (TGF-beta) signaling, but not by PD98059, an inhibitor for ERK/mitogen-activated protein kinase (MAPK) pathway. In addition, overexpression of Smad2 or Smad3, major signal transducing Smads, was sufficient to inhibite the IFN-gamma and TNF-alpha-induced TARC production in HaCaT cells. CONCLUSION: TGF-beta1 inhibited IFN-gamma and TNF-alpha-induced TARC production in HaCaT cells via Smad2/3, suggesting that modulation of TGF-beta/Smad signaling pathway may be beneficial for the treatment of atopic dermatitis.     

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.     

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.     

Ultraviolet A irradiation inhibits thymus and activation-regulated chemokine (TARC/CCL17) production by a human keratinocyte HaCaT cell line

Ultraviolet A (UVA) irradiation modulates the immunological functions of skin. We examined the effect of UVA irradiation on the basal and the IFN-gamma-and TNF-alpha-stimulation-induced production of thymus-and activation-regulated chemokine (TARC/CCL17) using HaCaT cells. UVA irradiation inhibited the basal levels of both TARC mRNA expression and TARC protein production. UVA irradiation also significantly inhibited TARC mRNA expression and TARC protein secretion that were induced by co-stimulation with IFN-y and TNF-alpha. A time course study showed that: the significant suppression of TARC mRNA expression was detected 8 hours after irradiation and continued for 36 hours; the strongest inhibition of TARC protein secretion occurred in the first 8 hours after UVA irradiation and continued for 36 hours. Our data provide the first evidence that UVA inhibits TARC mRNA expression and TARC protein production by keratinocytes in a dose-dependent manner. These results may suggest an explanation for the UV-induced therapeutic effect.     

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.     

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.     

Reciprocal regulation of thymus and activation-regulated chemokine/macrophage-derived chemokine production by interleukin (IL)-4/IL-13 and interferon-gamma in HaCaT keratinocytes is mediated by alternations in E-cadherin distribution

Keratinocytes produce many cytochemokines that are involved in the pathogenesis of skin disorders. In particular, the CC chemokines thymus and activation-regulated chemokine (TARC)/macrophage-derived chemokine (MDC) play an important role in the infiltration of Th2 cells. This study was undertaken to examine the regulatory effects of interleukin (IL)-4, IL-13, and interferon (IFN)-gamma on TARC/MDC production in the human keratinocyte cell line HaCaT. HaCaT cells spontaneously secrete TARC and MDC. The production of TARC/MDC was downregulated by IL-4/IL-13, whereas it was upregulated by IFN-gamma. To explore these regulatory mechanisms, we investigated the capacity of cytokines to regulate expression of several adhesion molecules that may affect TARC/MDC production. Of the adhesion molecules examined, the constitutive surface expression of E-cadherin was downregulated by IL-4/IL-13, but was upregulated by IFN-gamma. Moreover, disruption of the homophilic adherence of E-cadherin by anti-E-cadherin antibody or calcium chelation abolished the production of TARC/MDC. We further examined the distribution of the adherens junction complex composed of E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin. IL-4/IL-13 decreased the levels of membrane staining for adherens junction proteins, whereas IFN-gamma increased membrane staining. Taken together, these results suggest that IL-4/IL-13 and IFN-gamma induce alternations in the distribution of adherens junctions in a different fashion and thereby contribute to the reciprocal regulation of TARC/MDC production.     

Both IL-4 and IL-13 inhibit the TNF-alpha and IFN-gamma enhanced MDC production in a human keratinocyte cell line,HaCaT cells

BACKGROUND: Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented. OBJECTIVE: In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells. METHODS: HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels. RESULTS: MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-alpha or IFN-gamma, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-alpha or IFN-gamma were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-alpha and IFN-gamma. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-alpha and IFN-gamma enhanced MDC production in HaCaT cells in a dose-dependent manner. CONCLUSION: Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.     

姜黄素对IL-17诱导的人表皮角质形成细胞株CCL20表达的影响

目的探讨姜黄素对IL-17诱导的人表皮角质形成细胞株(HaCaT细胞株)CCL20表达的影响。方法用IL-17刺激HaCaT细胞,并加入不同浓度的姜黄素(2.5μmol/L,5μmol/L,10μmol/L)共培养24h。提取总RNA并收集细胞上清液,分别进行荧光定量PCR和ELISA实验,从mRNA及蛋白表达水平两方面观察姜黄素对CCL20表达的影响。结果 IL-17能够有效地诱导HaCaT细胞CCL20的表达。加入姜黄素共培养后,CCL20在mRNA及蛋白表达水平均明显下降(P均<0.05)。结论姜黄素对IL-17诱导的HaCaT细胞CCL20的表达具有明显的抑制作用,可为其对银屑病的治疗提供理论依据。     

喜树碱对低氧诱导HaCaT细胞趋化因子配体20表达的影响

目的观察喜树碱(CPT)对低氧(2%O2)培养下人永生化角质形成细胞(HaCaT细胞)趋化因子配体(CCL20)表达的影响,探讨CPT治疗斑块状银屑病可能的作用机制。方法将HaCaT细胞分为常氧组(21%O2点)和低氧(2%O2点)组,培养12 h,实时荧光定量聚合酶链反应(RT-PCR)检测CCL20 mRNA 的相对表达,酶联免疫吸附试验(ELISA)Kit测定细胞培养上清中CCL20表达;将12.5、25.0、50.0、100.0、200.0 nmol/L的CPT作用低氧诱导12 h的HaCaT细胞,ELISA Kit测定细胞培养上清中CCL20表达。结果常氧组和低氧组培养12 h,HaCaT细胞CCL20 mRNA 表达(ΔCT值)分别为-15.19±0.13和-13.70±0.10,两组间表达差异有统计学意义(t=-14.430,P=0.001);低氧培养12 h后HaCaT细胞(1×105个细胞)较常氧组CCL20蛋白表达增多,分别为(112.18±28.66)pg/ml、(64.36±47.85)pg/ml,但两组间表达差异无统计学意义(t=-1.485,P=0.212);100.0、200.0 nmol/L CPT处理低氧诱导12 h的HaCaT细胞(1×105个细胞)CCL20的表达分别为(64.35±19.70)pg/ml、(74.35±23.85)pg/ml,溶媒对照组为(112.18±28.66)pg/ml,组间多重比较差异有统计学意义(P0.05)。结论低氧可诱导HaCaT细胞CCL20 mRNA 表达增加;100.0、200.0 nmol/LCPT可抑制HaCaT细胞CCL20蛋白的表达。     

Mechanism of thymus- and activation-regulated chemokine (TARC)/CCL17 production and its modulation by roxithromycin

Stimulation with tumor necrosis factor (TNF)alpha and interferon (IFN)gamma synergistically induced thymus- and activation-regulated chemokine (TARC)/CCL17 production from HaCaT keratinocytes (KC). Inhibitors for nuclear factor kappa B (NFkappaB), parthenolide, and Bay 11-7085, and an inhibitor of p38, SB202190, inhibited TNFalpha- and IFNgamma-induced production of CCL17 by HaCaT KC. Surprisingly, an inhibitor of epidermal growth factor receptor tyrosine kinase, PD153035, enhanced the production of CCL17 in HaCaT KC. Roxithromycin (RXM), a 14-membered ring macrolide, suppressed CCL17 production by HaCaT KC induced by IFNgamma and TNFalpha. RXM partially suppressed p38 phosphorylation and NFkappaB-driven luciferase activity induced by TNFalpha and IFNgamma. Degradation of inhibitor of nuclear factor kappa B (IkappaB) alpha upon stimulation with IFNgamma and TNFalpha was not affected by the addition of RXM. Through elucidating the mechanism of CCL17 production, our study indicates that RXM suppresses the production through the inhibition of p38 and NFkappaB, independent of the inhibition of IkappaB degradation.     

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.     

Effect of an antiallergic drug (Olopatadine hydrochloride) on TARC/CCL17 and MDC/CCL22 production by PBMCs from patients with atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by the predominant infiltration of Th2-type cells in lesional skin. Thymus and activation-regulated chemokine (TARC/CCL17) and monocyte-derived chemokine (MDC/CCL22) are Th2-type cytokines, and it has been reported that serum CCL17 and CCL22 levels are associated with AD disease activity. Olopatadine hydrochloride (Olopatadine) is an antiallergic drug with selective histamine H(1) receptor antagonist activity. The effect of Olopatadine on chemokine production by peripheral blood mononuclear cells (PBMCs) in AD patients has not been completely elucidated. OBJECTIVES: This study was undertaken to clarify the effects of Olopatadine on CCL17 and CCL22 production by PBMCs from patients with AD during the treatment. METHODS: We measured plasma levels of CCL17, CCL22, IFNgamma, IL-12 and IL-18 in 15 patients with AD before and after treatment with oral Olopatadine (10 mg/day) for 4 weeks. We also examined disease activity using SCORAD index, eosinophil numbers in peripheral blood and serum levels of LDH. PBMCs from the patients were taken before and after the treatment and cultured with or without dust mite allergen extract (DME) for 3 or 5 days. CCL17, CCL22, IFNgamma, IL-12 and IL-18 levels in the supernatants of cultured PBMCs were measured. RESULTS: SCORAD index and eosinophil numbers in peripheral blood significantly decreased during treatment of AD patients with oral Olopatadine and topical corticosteroids for 4 weeks. The plasma levels of CCL17 and CCL22 significantly decreased after the treatment compared with before the treatment (p<0.05) and were significantly correlated with SCORAD index. PBMCs from AD patients taken after the treatment and cultured with DME for 5 days, showed significantly lower levels of CCL17 production than those taken before the treatment (p=0.018). PBMCs from AD patients taken after the treatment and cultured with DME for 5 days, also showed significantly lower levels of IFNgamma production than those taken before the treatment (p=0.012). CONCLUSION: Our data demonstrate that Olopatadine inhibits CCL17 and CCL22 production by PBMCs from AD patients, which are important regulators of Th2 recruitment in the skin.     

Interferon (IFN)-gamma is a main mediator of keratinocyte (HaCaT) apoptosis and contributes to autocrine IFN-gamma and tumour necrosis factor-alpha production

BACKGROUND: Apoptosis of keratinocytes or intestinal epithelial cells is an important pathophysiological mechanism of organ damage during acute graft-versus-host disease. OBJECTIVES: To analyse in detail the mediators and their mutual interaction leading to keratinocyte apoptosis. METHODS: Experiments were performed using a keratinocyte cell line (HaCaT) and human skin explant cultures. RESULTS: Supernatants (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLCs) induced apoptosis in HaCaT cells and also in keratinocytes from skin biopsies. Although both interferon (IFN)-gamma and Fas ligand (FasL) were detected in MLC-SN by enzyme-linked immunosorbent assay, the apoptosis-inducing capacity could be fully abrogated by neutralization of IFN-gamma, but not by neutralization of FasL. Recombinant (r) IFN-gamma induced HaCaT keratinocyte apoptosis in a dose- and time-dependent manner. Induction of HaCaT apoptosis by rFasL alone was induced only at higher doses than present in MLC-SN, but apoptosis was dramatically enhanced in the presence of rIFN-gamma. Further synergistic effects with IFN-gamma in the induction of apoptosis were also observed with agonistic antitumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2 antibody, soluble TRAIL and TNF-alpha. However, in contrast to FasL and TRAIL, TNF-alpha alone did not induce HaCaT apoptosis. Interleukin-1beta and lipopolysaccharide did not enhance the apoptosis-inducing effect of IFN-gamma. Beside its apoptosis-inducing capacity in HaCaT cells, rIFN-gamma also induced autocrine IFN-gamma production, and combined treatment with IFN-gamma and TNF-alpha induced autocrine TNF-alpha production. Neutralization of autocrine IFN-gamma protected HaCaT cells from apoptosis. CONCLUSIONS: Taken together, our data suggest a central role for IFN-gamma in HaCaT keratinocyte apoptosis but also show the importance of co-acting mediators such as TNF-alpha, TRAIL and FasL, which potentiate the effect of paracrine and autocrine IFN-gamma and TNF-alpha release.     

IL-10 augments the IFN-gamma and TNF-alpha induced TARC production in HaCaT cells: a possible mechanism in the inflammatory reaction of atopic dermatitis

The CC-chemokine TARC is known to be a ligand for the CCR4 receptor which in turn is known to be expressed selectively on the Th(2)-subset of lymphocytes. Atopic dermatitis is generally believed to be a Th(2)-type disease, and TARC has been shown to be expressed in the skin lesions of a murine model of AD. IL-10 is an interleukine generally known for its ability to inhibit cytokine production, however it has been found to be highly expressed in the skin from AD patients. We show in this report that IL-10 is able to augment the TARC inducing effects of TNFalpha and IFNgamma in HaCaT cells, a property that may be important in the determination of the composition of the cells of the inflammation in the skin of AD patients. In addition, we show that the IL10 agonist IT 9302, a nona-peptide from the carboxylic end of IL-10, has the same effect on TARC production from HaCaT cells.     

Macrophage-derived chemokine (MDC)/CCL22 produced by monocyte derived dendritic cells reflects the disease activity in patients with atopic dermatitis

BACKGROUNDS: Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by high serum levels of IgE and Th2-type cytokines such as IL-4, IL-5 or IL-13. Chemokines attract leukocytes in inflamed tissues. We have previously found that thymus and activation regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are highly secreted in the plasma levels of AD patients. Dendritic cells (DCs) are antigen-presenting cells that are divided into two subgroups including monocyte derived DCs (MoDCs) and plasmacytoid DCs (pDCs). OBJECTIVES: The aim of the study was to elucidate CCL17 and CCL22 production by MoDCs in AD patients, psoriasis vulgaris (PsV) patients and healthy controls (HC). METHODS: MoDCs were obtained from AD patients, PsV patients or HC and were cultured. In addition, the chemokine levels were measured in the supernatants. RESULTS: We found that the CCL22 levels produced by MoDCs in AD patients to be significantly higher than those of PsV patients and HC. There was a significant correlation between the CCL22 levels produced by MoDCs and the SCORAD index. No significant difference in the CCL17 levels produced by MoDCs was detected among AD patients, PsV patients or HC. Immunosuppressive drugs such as dexamethasone (Dex), tacrolimus and cyclosporine (Cys) inhibited the CCL22 production by MoDCs in the AD patients. CONCLUSION: These data suggest that the CCL22 level produced by MoDCs thus reflects the disease activity of AD and it may also play an important role regarding the production of CCL22 in the pathogenesis of AD.