Shinji Kagami

Significant elevation of serum levels of eotaxin-3/CCL26, but not of eotaxin-2/CCL24, in patients with atopic dermatitis: serum eotaxin-3/CCL26 levels reflect the disease activity of atopic dermatitis

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of T cells, eosinophils and macrophages in lesional skin. Recently, eotaxin‐2/CCL24 and eotaxin‐3/CCL26 were identified as CC chemokines that signal exclusively via the CCR3 receptor and have eosinophil‐selective chemoattractant activity, as does eotaxin/CCL11. We previously reported that serum levels of thymus and activation‐regulated chemokine (TARC)/CCL17 and macrophage‐derived chemokine (MDC)/CCL22 were correlated with the severity of AD. In this report, we investigated the participation of eotaxin‐2/CCL24 and eotaxin‐3/CCL26 in AD, first measuring the serum levels of eotaxin‐2/CCL24 and eotaxin‐3/CCL26 in 30 patients with AD, 20 patients with psoriasis vulgaris and 20 healthy controls. The serum levels of eotaxin‐3/CCL26 (but not eotaxin‐2/CCL24) were significantly higher in patients with AD than in either healthy controls or patients with psoriasis vulgaris; furthermore, the eotaxin‐3/CCL26 levels in patients with moderate and severe AD were significantly higher than eotaxin‐3/CCL26 levels in patients with mild AD. The serum eotaxin‐3/CCL26 levels tended to decrease after treatment, but there was no significant difference between groups. Moreover, the serum eotaxin‐3/CCL26 levels were significantly correlated with the serum TARC/CCL17 and MDC/CCL22 levels, eosinophil numbers in peripheral blood and the scoring AD (SCORAD) index. Our study strongly suggests that serum levels of eotaxin‐3/CCL26, but not of eotaxin‐2/CCL24, have a notable correlation with disease activity of AD and that eotaxin‐3/CCL26, as well as TARC/CCL17 and MDC/CCL22, may be involved in the pathogenesis of AD.

Interleukin-4 and interleukin-13 enhance CCL26 production in a human keratinocyte cell line, HaCaT cells

Eotaxin‐2/CCL24 and eotaxin‐3/CCL26 are CC chemokines and their receptor, CC chemokine receptor 3 is preferentially expressed on eosinophils. It was reported that vascular endothelial cells and dermal fibroblasts produced CCL26. However, the regulation of CCL24 and CCL26 production in keratinocytes has not been well documented. We investigated the expression and production of CCL24 and CCL26 in the human keratinocyte cell line, HaCaT cells. Reverse transcription and polymerase chain reaction was performed using these cells and Enzyme‐linked immunosorbent assay was carried out using supernatant of these cells. The production of CCL24 in HaCaT cells was slightly enhanced by IL‐4 and that of CCL26 was strongly enhanced by IL‐4 and IL‐13. Furthermore, TNF‐α generated a synergistic effect on IL‐4 enhanced CCL26 production. Dexamethasone, IFN‐γ and the p38 mitogen‐activated protein kinase inhibitor SB202190 inhibited IL‐4 enhanced CCL26 production. IL‐4 enhanced production of CCL26 was inhibited by leflunomide and JAK inhibitor 1, but not by JAK3 inhibitor, which indicates that it is mediated by JAK1‐STAT6‐dependent pathway. This result also strongly suggests the involvement of the type 2 IL‐4 receptor in IL‐4 enhanced production of CCL26. These results suggest that keratinocytes are involved in the migration of CC chemokine receptor 3 positive cells such as eosinophils in a Th2‐dominant situation like atopic dermatitis.

Prevalence of atopic dermatitis determined by clinical examination in Japanese adults

Dear Editor, Atopic dermatitis (AD) is an inflammatory skin disease that is characterized by pruritic and eczematous lesions persisting chronically. Studies on the prevalence of AD have produced widely varying figures which may be due to several factors such as subjects’ age, their community and the investigative methodology. There have been numerous studies on the prevalence of AD in children and adolescents, however, there have been few studies on AD in adults. Furthermore, most of these studies were based on hospital patient records or questionnaires. To the best of our knowledge, there has been no study of the prevalence of AD conducted by clinical examination in the general adult Japanese population. The objective of this study was to evaluate the prevalence of AD based on regular health check-ups by dermatologists in Japanese adults. The target population was government officials visiting the Health Service Center of Tokyo University for annual health check-ups in September 2004. Permission was obtained from the Board of the Health Service Center of Tokyo University. The government officials were told the purpose of the study, and those who granted consent participated in this study. AD was diagnosed by experienced dermatologists based on the Japanese Dermatological Association criteria for the disease. The severity of AD was graded as mild, moderate, severe or very severe according to the following criteria: (i) mild, skin involvement of mild eruption only; (ii) moderate, <10% surface area involvement of eruption with severe inflammation (severe eruption); (iii) severe, 710% but <30% skin involvement of severe eruption; and (iv) very severe, >70% of body involvement of severe eruption. The χ test was used to analyze the results, and P < 0.05 was considered statistically significant. A total of 2123 (1220 men and 903 women) officials were examined in this study. The average age was 38.8 ± 10.4 years (men: 39.6 ± 10.5; women: 37.7 ± 10.4) ranging 20–69 years. The prevalence of AD was 6.9% overall, and 9.8%, 8.7%, 4.4% and 2.6%, respectively, for those in their 20s, 30s, 40s and 50s/60s (Table 1). The prevalence of 30s was significantly higher than that of 40s (P < 0.0001). The prevalence of women was higher than that of men overall (9.3% vs 5.1%, P < 0.001), especially in 20s (13.1% vs 5.7%, P < 0.05) and 30s (11.5% 6.9%, P < 0.05). Table 2 depicts the severity of AD. Overall, 76.7%, 18.5%, 3.4% and 1.4% of those afflicted were in the mild, moderate, severe and very severe groups, respectively. There was no severe or very severe AD in those beyond their 40s, nor was there any very severe AD in men. This is the first study of the prevalence of AD determined by clinical examination in the general adult Japanese population. In 1999, Plunkett et al. reported the prevalence of AD based on clinical examination by dermatologists in adults in central Victoria, Australia. A total of 1457 people (670 men and 787 women) whose ages ranged 20–94 years were examined in their study. The prevalence of AD was 6.9% overall, and 5.7% and 8.1%, respectively, for men and women. There was a clear age-related variation: the prevalence was approximately 10%, 8%, 7%, 3%, 2%, respectively, for men in their 20s, 30s, 40s, 50s and 60s, and 22%, 13%, 7%, 7%, 2%, respectively, for women in their 20s, 30s, 40s, 50s and 60s. They classified AD into three categories: mild, moderate and severe. Of those with the disease, 82.8% were classified as being mild, 14.6% moderate, and 2.6% severe. Interestingly, their results and ours suggested the same tendency: (i) the overall prevalence of adult AD was approximately 7%; (ii) the prevalence of women was higher than that of men; (iii) the prevalence decreased with age; and (iv) approximately 80% of AD cases were in the mild group.

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

Abstract 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-α or IFN-γ, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-α or IFN-γ were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-α and IFN-γ. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-α and IFN-γ 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.

CCL17 transgenic mice show an enhanced Th2-type response to both allergic and non-allergic stimuli.

CC chemokine ligand (CCL)17 is implicated in the pathogenesis of atopic dermatitis (AD). To study the effect of CCL17 produced by keratinocytes (KC) during inflammation, we created transgenic (Tg) mice in which CCL17 is overexpressed in KC. Th2‐type contact hypersensitivity (CHS) was enhanced and Th1‐type CHS was suppressed in these mice. Increased numbers of CC chemokine receptor (CCR)4+ cells and mast cells infiltrated in Tg mice. Levels of IL‐4 mRNA were higher and those of IFN‐γ mRNA were lower in both acute and chronic CHS. Higher levels of serum IgE were observed after CHS. Numbers of CCR4+ cells among PBMC were increased in Tg mice challenged acutely on the trunk. Chronic irritation with croton oil induced dermatitis and an elevation of serum IgE levels. Tg mice showed enhanced ear swelling after tape stripping. CCL17 was thought to modify the inflammation caused by sensitizing reagents as well as irritant reagents by attracting CCR4+ cells into the lesional skin and creating a Th2‐dominant condition. AD‐like conditions such as increased number of mast cells and elevated levels of serum IgE were observed. Thus, CCL17 may participate in the pathogenesis of skin diseases such as AD by regulating both allergic and irritant inflammation.

Serum Gastrin-Releasing Peptide Levels Correlate with Pruritus in Patients with Atopic Dermatitis

effectors of the Wnt pathway, i.e., c-myc and Skp2 were not expressed in KSCs by reverse transcription–PCR (Figure 1b). Further, probing cell lysates of WIF1-arrested keratinocytes in western blots revealed that Wnt3A/ WIF1 treatment resulted in increased p21 protein levels (Figure 2o) demonstrable quantitatively (Figure 2p). Thus, WIF1 may achieve its cell cycle arrest in keratinocytes at least in part through derepression of p21 transcription. In conclusion, we report that WIF1 is, to our knowledge, previously unreported as a marker of interfollicular KSCs, and that it inhibits cell cycle progression in human keratinocytes even in the presence of activating Wnt signals (Wnt3A). Although canonical Wnt signaling appears to be dispensable during development in the interfollicular epidermis (Huelsken et al., 2001; Nguyen et al., 2009), our data suggest that inhibition of Wnt signaling may be required for keeping interfollicular stem cells quiescent and differentiating cells from proliferating during homeostasis.

Serum IL-31 levels are increased in patients with cutaneous T-cell lymphoma.

RESULTS As previously reported (4), serum IL-31 levels were significantly higher in patients with AD (2.13 ± 0.26 pg/ ml) than those of healthy controls (0.94 ± 0.28 pg/ml; p < 0.01; Fig. 1a). In addition, serum IL-31 levels were significantly higher in patients with CTCL (3.19 ± 0.74 pg/ml) than those of healthy controls (p < 0.05; Fig. 1a). We subsequently examined serum IL-31 levels in CTCL patients with different types of skin lesions. Serum IL31 levels in patients with patch and plaque, tumour and erythroderma were 1.05 ± 0.30 pg/ml, 6.25 ± 2.11 pg/ml and 5.00 ± 1.46 pg/ml, respectively (Fig. 1b). Serum IL-31 levels in patients with tumour were extremely high, which were significantly higher than those with healthy controls (p < 0.01) and patients with patch and plaque (p < 0.05). Serum IL-31 levels in patients with erythroderma were also significantly higher than those in healthy controls (p < 0.01). Serum IL-31 levels in patients with stage I, stage II, stage III and stage IV were 0.95 ± 29 pg/ml, 3.97 ± 1.43 pg/ml, 2.07 ± 0.00 pg/ ml and 7.98 ± 2.56 pg/ml, respectively (Fig. 1c). Serum IL-31 levels in patients with stage IV were significantly higher than those with healthy controls and patients with stage I (p < 0.05). Serum IL-31 levels in patients with stage II were significantly higher than those with healthy controls (p < 0.05). We also found that serum IL-31 levels correlated significantly with serum sIL-2R and LDH levels (r = 0.43, p < 0.05 and r = 0.34, p < 0.05, respectively; Fig. 1d, e), which have been reported to reflect disease activity of CTCL (8, 9). Thus, serum IL31 levels correlate with disease activity of CTCL.

CCR4 and CCR10 are expressed on epidermal keratinocytes and are involved in cutaneous immune reaction.

CC chemokine ligand (CCL)17 and CCL27 produced by epidermal keratinocytes (KCs) recruit CC chemokine receptor (CCR)4 and CCR10 expressing T cells into the skin, respectively, resulting in enhanced skin inflammation. However, CCR4/CCL17 and CCR10/CCL27 interactions in epidermal KCs have not been investigated. The purpose of this study was to evaluate the role of the CCR4/CCL17 and CCR10/CCL27 loops in cutaneous immune reaction. Normal human KCs (NHKs) and HaCaT KCs expressed both CCR4 and CCR10 at mRNA and protein levels. CCR4 ligand CCL17 but not CCR10 ligand CCL27 induced production of IL-12 p40, granulocyte/monocyte colony-stimulating factor (GM-CSF) and nerve growth factor (NGF) by KCs. Both CCL17 and CCL27 induced migration of KCs in Boyden chamber assay and wound scratch assay. This study revealed that CCR4 and CCR10 are expressed on epidermal KCs and that both are functional in terms of skin cytokine production and/or migration to their ligand CCL17 and CCL27, respectively. Thus this study provided new insight into chemokine/chemokine receptors of KCs.

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

Abstract:  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‐α and interleukin‐1β. CCL27 production was downregulated by inhibitors of p38 mitogen‐activated protein kinase and nuclear factor‐kappa B (NF‐κB). By contrast, CCL28 production was downregulated by inhibitors of extracellular signal‐regulated kinase and NF‐κB. 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.

The -431C>T polymorphism of thymus and activation-regulated chemokine increases the promoter activity but is not associated with susceptibility to atopic dermatitis in Japanese patients.

Background:  Thymus and activation‐regulated chemokine (TARC) plays an important role in the pathogenesis of atopic dermatitis (AD). We recently detected the single nucleotide polymorphism (SNP) (−431C>T) in the 5′‐flanking region of TARC gene.