Cilomilast, tacrolimus and rapamycin modulate dendritic cell function in the elicitation phase of allergic contact dermatitis

BACKGROUND: Cilomilast and tacrolimus as well as rapamycin are potential drugs for the treatment of allergic skin diseases like atopic dermatitis and allergic contact dermatitis. OBJECTIVES: To compare the in vitro and in vivo immunomodulatory effects of the phosphodiesterase 4 inhibitor cilomilast with those of tacrolimus and rapamycin. METHODS: The in vitro action of cilomilast, tacrolimus and rapamycin were tested in a mixed leucocyte reaction (MLR). In vivo, the inhibitory action of the immunomodulatory drugs was compared in the toluene-2,4-diisocyanate (TDI)-induced allergic inflammatory response with particular focus on dendritic cell (DC) function. RESULTS: Cilomilast, tacrolimus and rapamycin were all able to inhibit DC-mediated T-cell activation in a MLR. But it was demonstrated for cilomilast that the target cells are T cells rather than DC. In vivo, a combination of systemic and topical administration of each of these three substances significantly inhibited swelling in the murine ear 16 h after TDI challenge. There was also a reduction in the weight of the draining auricular lymph node, in lymphocyte cell count, and in the number of emigrated DC. The density of Langerhans cells in the epidermis was correspondingly higher in mice treated with cilomilast, tacrolimus and rapamycin than in those treated with vehicle. All three substances were found to inhibit DC migration ex vivo in a skin DC migration assay performed on ear tissue after TDI challenge. CONCLUSIONS: DC migration into the draining lymph node also takes place in the elicitation phase of allergic contact dermatitis and this migration can be influenced by tacrolimus and rapamycin, and, to a lesser extent, by cilomilast.     

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.     

特应性皮炎患者血清CXC、CC及C亚家族趋化因子的检测

目的 了解部分CXC、CC及C亚家族趋化因子在特应性皮炎患者血清中的水平和相互关系,及与Th1及Th2相关的细胞因子的关联。方法 用双抗体夹心ELISA方法检测51例特应性皮炎患者血清γ-干扰素诱导的单核因子（Mig）、胸腺和活化调控的趋化因子（TARC）、皮肤T细胞趋化因子（CTACK）及淋巴细胞趋化因子（Ltn）等水平。以SCORAD对特应性皮炎患者进行评分。结果 特应性皮炎患者血清Mig、TARC、CTACK及Ltn水平均显著高于正常人对照组（P < 0.05或 < 0.01）。患者血清CTACK水平与SCORAD呈显著正相关;血清Mig、TARC、CTACK及Ltn水平均与皮损体表面积百分比（BSA%）呈正相关;患者血清Ltn与TARC和CTACK显著相关。结论 CXC、CC及C亚家族的趋化因子在特应性皮炎的发病中可能起作用。血清CTACK水平可能是评估特应性皮炎严重度的一个较好的指标。     

Non-steroidal and steroidal anti-inflammatory drugs vary in their modulation of dendritic cell function in the elicitation phase of allergic contact dermatitis

The role of dendritic cells (DCs) in allergic contact dermatitis has been clearly demonstrated for the induction phase. However, the situation during the elicitation phase is very complex within a distinct inflammatory response. This study was performed to exploit DC migration in the elicitation phase in a mouse model of allergic contact dermatitis and to evaluate the effects of steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) on DC migration through skin in the elicitation phase of allergic contact dermatitis. Topically and systemically administered acetylsalicylic acid (ASA) did not reduce the inflammatory response. However, systemically administered ASA significantly reduced the DC migration to the draining lymph node. In contrast, topically administered indomethacin reduced the inflammatory response, but had only minor effects on DC migration, whereas diflorasone diacetate reduced both inflammatory reaction and DC migration. Thus, NSAIDs may differ in their inhibitory action in immunological inflammation.     

Serum levels of cutaneous T-cell attracting chemokine (CTACK) as a laboratory marker of the severity of atopic dermatitis in children

There are at least 13 scoring systems for the assessment of disease severity in atopic dermatitis (AD). Each system has its problems with interobserver and intraobserver variability. Cutaneous T-cell attracting chemokine (CTACK) is a skin-specific chemoattractant which may correlate with AD severity and obviate the issue of observer reliability. We evaluated whether serum CTACK concentrations were associated with the severity of AD in children according to the SCORing Atopic Dermatitis (SCORAD) index. Thirty-seven Chinese children with AD (23 boys, 14 girls; aged 1-11 years) and 13 controls were recruited. The median (interquartile range) overall SCORAD for AD patients was 29.7 (20.3-49.7). Serum concentrations of CTACK and two other atopy-related chemokines, macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), were measured by sandwich enzyme immunoassay. There were significant correlations between SCORAD (r = 0.394, P = 0.016), its area (r = 0.528, P = 0.001) and intensity components (r = 0.429, P = 0.008) with serum levels of CTACK. The serum concentrations of inflammatory markers MDC and TARC also correlated with the CTACK concentrations (r = 0.618, P < 0.001, and r = 0.587, P = 0.001, respectively). Serum CTACK concentration appears to be a skin-specific objective marker that correlates with various clinical and laboratory parameters of AD.     

TGF-beta1-mediated regulation of thymus and activation-regulated chemokine (TARC/CCL17) synthesis and secretion by HaCaT cells co-stimulated with TNF-alpha and IFN-gamma

Thymus and activation-regulated chemokine (TARC/CCL17) contributes not only to the recruitment of leukocytes, but is also involved in immune disorders, such as atopic dermatitis (AD) and bronchial asthma. We have previously reported that the levels of TARC were high in patients with AD and that lesional epidermis were strongly immunoreactive for TARC. In this paper, the effects of transforming growth factor (TGF)-beta(1) on the expression of TARC/CCL17 were examined in HaCaT cells, a human keratinocytes (KCs) cell line, co-stimulated with TNF-alpha and IFN-gamma. We found that TGF-beta(1) down-regulated the TARC synthesis and secretion of HaCaT cells co-stimulated with TNF-alpha and IFN-gamma in a dose-dependent manner. TGF-beta(1) at a concentration of 10ng/ml maximally inhibited this secretion. Northern blot analysis showed a similar inhibitory effect of TGF-beta(1) on TARC mRNA expression by HaCaT cells. The TGF-beta(1)-induced down-regulation of TARC/CCL17 in HaCaT cells suggests that TGF-beta(1) might regulate the TARC-related inflammatory processes, which may be important for understanding the pathogenesis of allergic diseases.     

CTACK /CCL27 expression in psoriatic skin and its modification after administration of etanercept

BACKGROUND: Tumour necrosis factor-alpha upregulates the expression of a cutaneous T cell-attracting chemokine (CTACK/CCL27), that promotes migration of cutaneous lymphocyte-associated antigen-positive lymphocytes into the skin. The role of CTACK/CCL27 in pathogenesis of psoriasis has recently been documented but no data are available at the present time on its modification in psoriatic cutaneous tissue after administration of etanercept. OBJECTIVES: To evaluate modifications of CTACK/CCL27 expression in skin of patients with psoriasis after administration of etanercept and their relation with disease activity. METHODS: Twenty-two patients with moderate to severe psoriasis underwent clinical, histological and immunohistochemical evaluations of disease activity at baseline and at 12 and 24 weeks after starting treatment with etanercept. RESULTS: All selected patients experienced an improvement of Psoriasis Area and Severity Index (PASI) score (P < 0.001) and Dermatology Life Quality Index score (P < 0.001) during the treatment. Skin histological abnormalities showed statistically significant modifications during treatment (P < 0.001). Immunohistochemical expression of CTACK/CCL27 decreased significantly (P < 0.001) and its relation with final PASI score was statistically significant (P < 0.05); the pattern of distribution of CTACK/CCL27 immunoreactivity significantly moved from diffuse and predominantly suprabasal to basal (P < 0.001) and the restoration of basal distribution of CTACK/CCL27 was also significantly related to clinical improvement of cutaneous disease (P < 0.001). CONCLUSIONS: Etanercept induces a clinical and histological improvement of psoriatic disease, promoting a reduction in CTACK/CCL27 cutaneous immunostaining and favouring the restoration of physiological CTACK/CCL27 epidermal expression. Moreover, CTACK/CCL27 reduction in cutaneous expression during administration of etanercept could be considered a favourable prognostic marker.     

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.     

Early inflammatory markers in elicitation of allergic contact dermatitis

Background  

Allergic Contact Dermatitis (ACD) is regarded as a T-cell-mediated delayed-type hypersensitivity reaction. We studied the kinetics of the expression of CS-1 fibronectin, thymus and activation-regulated chemokine (CCL17/ TARC) and different chemokine receptors (CR) in skin biopsies from individuals suffering from back problems, with the antigen responsible of their contact dermatitis and an irrelevant antigen.     

The role of chemokines in allergic contact dermatitis

Chemokines are important mediators of immune-mediated skin diseases. Allergic contact dermatitis (ACD) is the most thoroughly investigated T cell-mediated disorder because of the ability to easily reproduce the lesions in humans and the availability of an excellent mouse model. Migration of dendritic cells from the skin to lymph nodes is absolutely required for induction of hapten sensitization, and depends upon expression of CCR7 by mature dendritic cells and SLC in the lymph nodes. During expression of ACD, recruitment of T lymphocytes is driven by chemokines exposed on the surface of endothelial cells or released by activated resident skin cells such as mast cells, fibroblasts and keratinocytes. Chemokines are produced in a coordinated and sequential manner, with IL-8 and RANTES induced by TNF-alpha during early stages, and MCP-1, IP-10, Mig, I-TAC, I-309 and MDC induced by IFN-gamma during later stages. Infiltrating monocytes, dendritic cells and T cells are additional sources of chemokines for further leukocyte accumulation. Distinct T cell subsets express different chemokine receptors, with type 2 cells mostly attracted by eotaxin, MDC, TARC and I-309, and type 1 cells sensitive to IP-10, Mig, I-TAC, RANTES and MIP-1beta. MCP-1 is effective on both subsets. T regulatory cells, which inhibit dendritic cell function and are probably involved in the termination of ACD, are sensitive to MCP-1, MIPs and TARC, but express high levels of CCR8 and are more specifically attracted by I-309. Targeting chemokines and chemokine receptors may offer new opportunities for therapeutic interventions in ACD and other chronic inflammatory skin diseases.     

Increased expression of chemokines in the skin of chronic proliferative dermatitis mutant mice

Chemokines direct the migration of leukocytes to sites of inflammation and are potential targets for anti-inflammatory therapy. Chronic proliferative dermatitis (cpdm/cpdm) mutant mice develop a persistent eosinophilic dermatitis associated with increased T(H)2 cytokines in the skin. Expression patterns of chemokines in the skin of cpdm/cpdm mice were evaluated to define the mechanisms driving cutaneous infiltration by leukocytes. RNA isolated from the skin of mutant and littermate control mice revealed a significant increase in Ccl1 (TCA-3), Ccl2 (MCP-1), Ccl11 (eotaxin), Ccl17 (TARC), Cxcl10 (IP-10), and the chemokine receptor Ccr3. The concentration of CCL11 protein was increased two- to threefold in the skin of cpdm/cpdm mice by enzyme-linked immunosorbent assay. In vitro culture of primary dermal fibroblasts from cpdm/cpdm and control mice with tumor necrosis factor, IL-4, and IL-13 stimulation did not reveal differences in their ability to secrete CCL11, suggesting that the increased chemokine expression observed in the skin of cpdm/cpdm mice is most likely caused by the increased T(H)2 cytokines in the dermis of this mouse model. Treatment of cpdm/cpdm mice with CCL11-neutralizing polyclonal antibodies did not affect the number of eosinophils in the skin or the severity of the dermatitis. Neutralizing multiple chemokines or chemokine receptors may be necessary to decrease eosinophil accumulation. The cpdm/cpdm mutant mouse is a potentially useful model to determine the role of various chemokines in eosinophil accumulation in chronic inflammation.     

Nocturnal wrist movements are correlated with objective clinical scores and plasma chemokine levels in children with atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is a distressing disease associated with pruritus and sleep disturbance. Scratching due to pruritus is an important mechanism in the exacerbation of AD but is difficult to document in the home environment. OBJECTIVES: To evaluate whether nocturnal wrist activities, defined as average acceleration in the early hours of sleep, were correlated with components of the SCORing Atopic Dermatitis (SCORAD) index and various AD-associated chemokine markers. METHODS: Patients with AD aged under 18 years were recruited and the severity of eczema was assessed with the SCORAD index. Concentrations of plasma AD-associated chemokines [cutaneous T-cell attracting cytokine (CTACK); macrophage-derived chemokine (MDC); thymus and activation regulated chemokine (TARC)], interleukin (IL)-18, serum total IgE, and eosinophil counts were measured in these patients. Healthy children with noninflammatory and nonitchy skin conditions as well as healthy children of staff volunteers were recruited as controls. All children were instructed to wear the DigiTrac monitor on their dominant wrist before sleeping. The monitor was programmed to record limb motion between 22.00 and 08.00 h the following morning. RESULTS: Twenty-four Chinese children with AD (mean +/- SD age 12.6 +/- 3.7 years) and 15 normal children (mean +/- SD age 11.9 +/- 3.4 years) were recruited. The median (interquartile range) SCORAD was 54.8 (32.8-70.2). Plasma concentrations in pg mL(-1) of CTACK, MDC, TARC and IL-18 in the patients were 105 (92-172), 1648 (973-4214), 258 (100-850) and 415 (304-539), respectively. When compared with controls, most wrist activities occurred at frequencies between 1 and 3 Hz. These activities were most consistent over the first 3 h of sleeping and correlated significantly with disease severity, extent, intensity, and AD-associated chemokine markers CTACK, MDC and TARC. However, there was no significant correlation between wrist activities and the subjective symptom of pruritus or sleep loss. CONCLUSIONS: This is the first study to demonstrate that wrist activities, nonintrusively measured by the DigiTrac monitor at home, are closely correlated with the objective clinical scores and levels of peripheral blood chemokine markers for AD but not with the reported symptoms of pruritus or sleep loss. We propose that wrist activities between 1 and 3 Hz for the first 3 h are a good indicator of AD severity in children and should substitute for the pruritus and sleep-loss components of the SCORAD.     

Wy14643, an agonist for PPARα, downregulates expression of TARC and RANTES in cultured human keratinocytes

Beneficial effects of Wy14643, an agonist for peroxisome proliferator‐activated receptor (PPAR)α, on permeability barrier homeostasis‐related functions of keratinocytes such as up‐regulation of epidermal differentiation‐related molecules and lipid synthesis, have been demonstrated. The present study demonstrated that Wy14643 reduced the expression of thymus and activation‐related chemokine (TARC) and regulated on activation normal T cell expressed (RANTES) in both single‐ and 3D‐cultured human keratinocytes. The combined data of the present and previous studies support the notion that Wy14643 could be a therapeutic agent that might simultaneously and directly modulate permeability barrier dysfunction and allergic inflammation in the pathogenesis of atopic dermatitis. As for the anti‐microbial barrier function, the present study demonstrated that Wy14643 up‐regulated expression of the anti‐microbial peptide, human β defensin 3, in cultured human keratinocytes only in mRNA levels but not in protein ones, suggesting that Wy14643 might not directly account for the up‐regulation of the anti‐microbial peptide which has been reported in vivo.     

Serum and tissue CTACK/CCL27 chemokine levels in early mycosis fungoides may be correlated with disease-free survival following treatment with interferon alfa and psoralen plus ultraviolet A therapy

Background Neoplastic T‐cell recruitment into the skin is a critical step in the pathogenesis of mycosis fungoides (MF), and the cutaneous T‐cell attracting chemokine, CTACK/CCL27, might be involved. Objectives To investigate the clinical and prognostic significance of CTACK/CCL27 levels in patients with early‐stage MF. Methods Serum samples and skin biopsy specimens were collected from 15 patients at the time of diagnosis and after the end of treatment with psoralen plus ultraviolet A/interferon alfa‐2b combination therapy. Serum samples were also collected from 20 healthy donors as controls. CTACK/CCL27 serum levels were analysed by enzyme‐linked immunosorbent assays. CTACK/CCL27 tissue expression was determined by immunohistochemistry on skin biopsy specimens taken at diagnosis and after therapy. Event‐free survival was taken as the primary clinical outcome. Results In patients with MF at diagnosis, CTACK/CCL27 serum levels were not significantly different from healthy controls, whereas CTACK/CCL27 expression in the skin was increased in 87% of cases compared with normal controls. After therapy, all patients obtained a clinical complete remission, serum levels did not change significantly and tissue expression remained abnormal in 80% of patients, even if complete histological remission was recorded. Serum levels were not significantly different in cases with different intensity of cutaneous immunostaining. Eight patients experienced a relapse: the combination of high CTACK/CCL27 levels both in sera and skin increased the probability of experiencing an event at 51 months from 36% to 83%. Conclusions Our data seem to indicate that CTACK/CCL27 levels in skin and sera after therapy might be correlated with risk of recurrence.     

Are age‐specific high serum IgE levels associated with worse symptomatology in children with atopic dermatitis?

BACKGROUND: Atopic dermatitis (AD) is a distressing disease associated with excoriations, pruritus, sleep disturbance, and elevation of serum total immunoglobulin E (IgE) levels. OBJECTIVE: To evaluate whether serum IgE levels correlate with the symptomatology and plasma chemokine levels in children with AD. METHODS: AD patients aged younger than 18 years were recruited from the pediatric dermatology clinic of a university teaching hospital, and the AD severity was evaluated using the SCORing Atopic Dermatitis (SCORAD) index. Concentrations of serum total IgE, eosinophil count, and plasma AD-associated chemokines [cutaneous T-cell-attracting cytokine (CTACK), thymus and activation-regulated chemokine (TARC)] were measured. RESULTS: One hundred and seventeen Chinese children with AD (64 boys and 53 girls), with an age (mean +/- standard deviation) of 10.7 +/- 4.4 years, were recruited. Their overall SCORAD index (mean +/- standard deviation) was 51.1 +/- 22.8. The total serum IgE level divided by the age-specific upper limit (AE) correlated well with the extent and intensity of AD, except for oozing/crusting, which was significant only in males. There was a significant correlation between AE and pruritus or sleep loss only in females. Levels of IgE, CTACK, and TARC, and eosinophil count, differed significantly between patients with mild, moderate, and severe disease. AE correlated well with TARC (r = 0.50, P < 0.001) and eosinophil count (r = 0.41, P < 0.001), but not with CTACK (r = 0.11, P = 0.270). The prediction of moderate to severe eczema by AE gave an area under the receiver-operating characteristic curve of 0.76 (95% confidence interval, 0.65-0.86; P = 0.004). An optimum positive predictive value of 94.2% was achieved with a cut-off point of AE of 2.95, sensitivity of 75.0%, and specificity of 66.7%. CONCLUSION: AE correlates significantly with various objective clinical scores and chemokine markers of AD, and is a useful indicator for predicting moderate to severe AD in children.     

Enhanced TARC production by dust-mite allergens and its modulation by immunosuppressive drugs in PBMCs from patients with atopic dermatitis

BACKGROUND: Thymus and activation regulated chemokine (TARC) is a CC chemokine that attracts CCR4+ T cells. We reported previously that TARC is an important chemokine that defines Th2 imbalance in the pathogenesis of atopic dermatitis (AD). OBJECTIVES: This study was undertaken to clarify TARC producing cells in peripheral blood mononuclear cells (PBMCs), the regulation of dust mite-allergen clude extract (DME) and different immunosuppressive drugs (Tacrolimus (FK506), cyclosporine (CsA), dexamethasone (Dex)) on TARC production by peripheral PBMCs from AD patients in vitro. METHODS: Monocyte derived dendritic cells (MoDCs) were generated from and TARC mRNA levels were examined and comapared with those from T cells in PBMCs from AD patients. PBMCs were cultured with or without DME and/or immunosuppressive drugs (Tacrolimus, CsA, Dex) for 7 days and TARC levels were measured. RESULTS: PBMCs from AD patients which were cultured with DME stimulation for 7 days showed significantly higher levels of TARC production than those from healthy controls. RT-PCR demonstrated that TARC mRNA was expressed in CD4+ T cells, CD8+ T cells and MoDCs. Tacrolimus, CsA and Dex individually suppressed TARC production by PBMCs from AD patients which were co-cultured with DME for 7 days. Gel shift analysis revealed differential inhibitory effects of these immunosuppressive drugs on NFkappaB activity in PBMCs from AD patients. CONCLUSION: Our data demonstrate that TARC producing cells are MoDCs, T cells as well as epidermal keratinocytes in AD. We suggest that MoDCs might regulate the immune responses by attracting T cells and CD25+ T cells in the pathogenesis of AD. We also showed the important role of DME on TARC production and the inhibitory effect of the immunosuppressive drugs on TARC production by PBMCs from AD patients, that can regulate ongoing immune responses in the pathogenesis of AD.