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C-C chemokine receptor (CCR)10 is a specific receptor for chemokine ligand (CCL)27, a selective chemoattractant for skin-associated memory T cells to cutaneous sites. In melanoma, CCR10 increases the ability of neoplastic cells to grow, invade tissues, disseminate to lymph nodes, and escape the host immune responses. In this study, we investigated the expression of CCR10 and its ligand CCL27 in squamous cell carcinoma (SCC). CCR10 and CCL27 were expressed in SCC, actinic keratosis (AK), Bowen's disease, and seborrheic keratosis (predominantly prickle cell type), but not in seborrheic keratosis (predominantly basal cell type) and basal cell carcinoma. Furthermore, CCR10 and CCL27 were overexpressed in SCC relative to Bowen's disease, an early stage of SCC. Consistently, a human SCC cell line, A253 cells, and HaCaT cells exhibited CCL27 production that was strongly induced by tumor necrosis factor-α and interleukin-1β. Finally, A253 cells expressed stronger intracellular CCR10 compared to HaCaT cells by flow cytometry. These results suggest that CCR10 and CCL27 overexpression in SCC is related to the progression of SCC and is useful for the diagnosis of SCC.

CC chemokine receptor 3 (CCR3), the sole receptor for eotaxins, is expressed on eosinophils and T helper type 2 (Th2) cells. In Hodgkin's disease, eotaxin-1 secreted by fibroblasts collects Th2 cells and eosinophils within the tissue. Similarly, many Th2 cells infiltrate the lesional skin of cutaneous T-cell lymphoma (CTCL). In this study, we investigated the role of eotaxins in the development of the Th2 environment of CTCL. We revealed that fibroblasts from lesional skin of CTCL expressed higher amounts of eotaxin-3 messenger RNA (mRNA) compared with those from normal skin. Lesional skin of CTCL at advanced stages contained significantly higher levels of eotaxin-3 and CCR3 mRNA, compared with early stages of CTCL. IL-4 mRNA was expressed in some cases at advanced stages. Immunohistochemistry revealed that keratinocytes, endothelial cells, and dermal fibroblasts in lesional skin of CTCL showed a stronger expression of eotaxin-3 than did normal skin. CCR3 + lymphocytes and IL-4 expression were observed in some cases of advanced CTCL. Furthermore, both serum eotaxin-3 and eotaxin-1 levels of CTCL patients at advanced stages were significantly higher than those of healthy individuals. The concentrations of these chemokines correlated with serum soluble IL-2 receptor levels. These results suggest that interaction of eotaxins and CCR3 regulates the Th2-dominant tumor environment, which is closely related to the development of CTCL.

CC chemokine receptor 10 and its ligand, CCL27, are important components of T cell-mediated cutaneous immunity, but whether they influence lymph node (LN) homing by T cells is unknown. In this study, CCL27 protein was detected in skin-draining LN by Western blotting and ELISA although CCL27 mRNA transcripts were low. CCL27 protein was present at higher levels in skin-draining LN compared with gut-draining LN and spleen. A single topical treatment of mouse skin with the contact sensitizer 2,4-dinitro-1-fluorobenzene (DNFB) resulted in a 13-fold increase in CCL27 protein accumulation in skin-draining LN within 1 h and a 5-fold elevation in CCR10 mRNA (normalized to the T cell marker CD2) within 6 h. DNFB treatment also resulted in rapid depletion of ∼75% of CCL27 from the epidermis. In summary, we describe a novel mechanism for the recruitment of CCR10-positive T cells to skin-draining LN following the rapid release of preformed CCL27 from the epidermis.

Luciferase-transduced B16 murine melanoma cells (luc-B16) inoculated in ear skin do not form tumors but prevent tumor formation by luc-B16 cells injected into the footpad. To determine the requirements for such immunity, we followed the fate of luc-B16 cells following ear injection. Surprisingly, small numbers of viable luc-B16 cells were detected in tumor-free mouse skin for up to 60 days post-inoculation. After 1 week, the number of Foxp3(+)CD4(+)CD25(+) T cells (along with foxp3 mRNA expression) increased rapidly in the injected ear skin. Residual tumor cells in ears were reduced in mice treated with anti-CD25 mAb and in CD4-deficient mice, but increased in CD8-deficient mice. Strikingly, the loss of luc-B16 cells in the ear skin, either spontaneously or following amputation of the injected ear, resulted in significantly enhanced tumor formation by parental and luciferase-expressing B16 cells after footpad injection. These studies suggest that small numbers of tumor cells (possibly regulated by CD4(+)CD25(+) regulatory T cells expressing Foxp3) are required for effective host anti-tumor responses at alternate inoculation sites.

It is clear from large clinical studies that selected chemokine receptors are often up-regulated in a large number of common human cancers, including those of the breast, lung, prostate, colon, and melanoma. Chemokine receptors and their corresponding chemokine ligands have been demonstrated to play a number of nonredundant roles in cancer metastasis to vital organs as well as regional lymph nodes, the most frequent site of cancer metastasis. Chemokine receptors may potentially facilitate tumor dissemination at several key steps of metastasis, including adherence of tumor cells to endothelium, extravasation from blood vessels, metastatic colonization, angiogenesis, proliferation, and protection from the host response via activation of key survival pathways such as phosphatidylinositol-3 kinase and Akt. It is interesting that many of these roles are reminiscent of their functions in leukocyte and stem cell trafficking. Lastly, we discuss therapeutic applications for chemokine receptor antagonists in cancer therapy.

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.

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.

SummaryEotaxin-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.

CCR4, a member of the CC chemokine receptor family, is believed to play an important role in the pathogenesis of atopic dermatitis. To examine whether CCR4 single nucleotide polymorphism (SNP) is associated with susceptibility to atopic dermatitis, we investigated the allele and genotype frequencies of C1014T SNP of CCR4 in 198 Japanese patients with atopic dermatitis and controls by a PCR-restriction fragment length polymorphism method. There was no significant difference in allele or genotype frequencies between patients with atopic dermatitis and controls. Serum IgE levels and peripheral blood eosinophil counts were not significantly different among genotypes. There was also no significant difference in allele or genotype frequencies between the patient subgroup with and without asthma, with mild or moderate disease, with and without family history of atopic dermatitis, or with and without family history of atopic disorders. C1014T SNP of CCR4 does not appear to be associated with susceptibility to atopic dermatitis in Japanese patients.

CCR4, a member of the CC chemokine receptor family, is believed to play an important role in the pathogenesis of atopic dermatitis. To examine whether CCR4 single nucleotide polymorphism (SNP) is associated with susceptibility to atopic dermatitis, we investigated the allele and genotype frequencies of C1014T SNP of CCR4 in 198 Japanese patients with atopic dermatitis and controls by a PCR-restriction fragment length polymorphism method. There was no significant difference in allele or genotype frequencies between patients with atopic dermatitis and controls. Serum IgE levels and peripheral blood eosinophil counts were not significantly different among genotypes. There was also no significant difference in allele or genotype frequencies between the patient subgroup with and without asthma, with mild or moderate disease, with and without family history of atopic dermatitis, or with and without family history of atopic disorders. C1014T SNP of CCR4 does not appear to be associated with susceptibility to atopic dermatitis in Japanese patients. Key words: atopic dermatitis; CCR4; single nucleotide polymorphism.

SUMMARY 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.

Thymus and activation-regulated chemokine (TARC/CCL17) is a Th2-type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Langerhans cells (LC) are immature dendritic cells (DC) in the epidermis of the skin and play vital roles in immune response. In this study, we investigated TARC expression by murine freshly isolated LC and 48 h cultured (mature) LC, and the regulation of TARC production in cultured LC by various cytokines. Murine LC was prepared using a panning method from BALB/c mice. RT-PCR was performed using fresh and cultured LC to evaluate TARC mRNA levels. ELISA was carried out using supernatant of cultured LC to calculate secreted TARC protein levels. TARC mRNA was strongly upregulated during maturation of murine LC. TARC production by murine LC was upregulated by TNF-alpha and IL-4 and downregulated by IFN-gamma, dose-dependently. Th1 and Th2 cytokines reciprocally regulate the production of Th2-type chemokine TARC by murine LC. Th2 cytokine microenvironments in skin may increase TARC production by mature LC, providing attraction of Th2 cells in skin. This may be an amplification circuit in Th2-dominant inflammatory skin disease like atopic dermatitis.

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.

Background: Both atopic dermatitis (AD) and psoriasis vulgaris (PsV) are characterized as chronic and relapsing inflammatory skin diseases associated with various immunologic abnormalities. Cutaneous T cell-attracting chemokine (CTACK; CCL27) is a member of the CC chemokine family and a functional ligand for CC chemokine receptor 10. It is selectively expressed in skin and attracts CC chemokine receptor 10-expressing skin-homing memory T cells. The epidermal keratinocyte is a main source of CTACK, suggesting the involvement of various inflammatory skin diseases. Objective: The purpose of this investigation was to clarify whether CTACK produced by keratinocytes is detected in the sera of patients with AD and PsV and to examine the correlation between the serum CTACK levels and disease activity of patients with AD and PsV. Methods: We measured the serum CTACK levels in 50 patients with AD, 30 patients with PsV, and 22 healthy control subjects. We also divided 50 patients with AD into 3 groups (ie, those with mild, moderate, and severe disease) and compared them among 3 categories. Moreover, we compared the serum CTACK levels of patients with AD and PsV with clinical or laboratory data. Immunohistochemical staining of CTACK and IFN-induced protein of 10 kd (IP-10; CXCL10) was performed on the lesional skin of patients with AD and PsV. Results: The serum CTACK levels in patients with AD and PsV were significantly higher than those in healthy control subjects. The serum CTACK levels in patients with AD significantly correlated with scoring atopic dermatitis (SCORAD) scores, serum soluble IL-2 receptor levels, serum soluble E-selectin levels, serum thymus and activation-regulated chemokine levels, and serum macrophage-derived chemokine levels. Serum CTACK levels in patients with PsV significantly correlated with the serum IP-10 levels but not with the Psoriasis Area and Severity Index score. Immunohistochemical staining showed CTACK was strongly expressed in lesional ke-ratinocytes of patients with AD and PsV, whereas IP-10 was strongly expressed in lesional keratinocytes of patients with PsV and focally in those with AD. Conclusion: These results suggest that CTACK might be one of the important chemokines for the pathogenesis of AD and PsV. (J Allergy Clin Immunol 2003;111:592-7.)

Background: Mycosis fungoides (MF) belongs to cutaneous T-cell lymphoma and is clinically divided into 3 stages: patch, plaque, and tumor stage. Thymus and activation-regulated chemokine (TARC/CCL17) is a member of the CC chemokines and is a chemoattractant for CC chemokine receptor 4 (CCR4)- and CC chemokine receptor 8 (CCR8)-expressing cells. Objective: In this study, we examined the involvement of TARC among patients with each stage of MF. Methods: We investigated the expression of TARC, CCR4, and CXC chemokine receptor 3 in patients with each stage of MF by immunohistochemistry. We measured serum TARC levels in 20 patients with MF in varying degrees and compared them with 10 patients with psoriasis vulgaris and 10 healthy controls. In addition, we compared serum TARC levels in patients with MF with other laboratory data. Results: Immunohistochemical staining revealed that TARC was expressed in the lesional keratinocytes in the patch, plaque, and tumor stages. CCR4 was expressed on the epidermotropic cells in both patch and plaque stages and on the large cell-transformed cells in the tumor stage, whereas CXC chemokine receptor 3 was constantly expressed on the small cells in the lesional dermis. Serum TARC levels in patients with MF were significantly higher than those in patients with psoriasis vulgaris or healthy controls. Moreover, serum TARC levels in patients with the tumor stage of MF (n = 5) were remarkably higher than those with patch stage (n = 8) or plaque stage (n = 7). Serum TARC levels significantly correlated with serum lactate dehydrogenase levels ( r = 0.62), serum immunoglobulin E levels ( r = 0.60), serum soluble interleukin 2 receptor levels ( r = 0.72), and serum macrophage-derived chemokine levels ( r = 0.70). Conclusion: These data strongly indicate that serum TARC levels are useful for assessing the disease activity of patients with MF and that TARC and CCR4 may be involved in the pathogenesis of MF. (J Am Acad Dermatol 2003;48:23-30.)

Thymus- and activation-regulated chemokine (TARC/CCL17) and its receptor, CC chemokine receptor 4 (CCR4), have been proven to be involved in a number of allergic diseases, especially atopic dermatitis (AD). The purpose of this study was to examine the expression and distribution of TARC and CCR4 mRNAs in samples of AD (n=15, acute lesions 8, chronic lesions 7) and normal skin (n=6). The expression and distribution of TARC and CCR4 mRNAs were detected with the in situ reverse transcription (RT) -polymerase chain reaction (PCR) technique. TARC mRNA was expressed in epidermal keratinocytes, dermal endothelial cells and infiltrating cells. CCR4 mRNA was expressed in dermal endothelial cells and infiltrating cells. In acute AD lesional skin, there were more positive cells, and the staining intensity was stronger than in chronic lesions (p

Interleukin (IL)-13 plays an important role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD). We investigated the allele and genotype frequencies of three IL-13 single nucleotide polymorphisms (SNPs) (A704C and C1103T in the promoter region and G4257A in exon 4) in Japanese patients with AD. For A704C and C1103T SNPs, there were no significant differences in allele or genotype frequencies between AD patients and controls. For G4257A SNP, A allele was significantly increased in AD patients (39.5%) compared with controls (29.4%) (P = 0.016). The same proportion of each genotype and allele was observed in the patient subgroup with and without asthma. Serum IgE levels and peripheral eosinophil counts were not significantly different among genotypes in G4257A SNP. There was also no significant difference in allele or genotype frequencies between AD patients with mild disease and those with severe disease, between those with family history of AD and those without it, or between those with family history of atopic disorders and those without it. This result suggests that 4257A allele is associated with susceptibility to AD and that it may function in the pathogenesis of AD itself, presumably by other mechanisms than inducing IgE production.

Interleukin-12 (IL-12) is believed to play an important role in inducing Th1-type cytokine profiles. Atopic dermatitis (AD) and psoriasis vulgaris (PsV) are considered to be Th2 and Th1 type disease, respectively. The IL-12 p40 subunit gene (IL12B) is located at chromosome 5q31-33 and linkage findings of AD on 5q31 were reported. Recently single nucleotide polymorphism (SNP) (1188A/C) of IL12B has been reported. In function, it has been reported that this SNP is associated with IL12B mRNA expression levels. To learn whether this SNP is associated with susceptibility to AD or PsV, we investigated the genotype and allele frequencies of the SNP in AD patients, in PsV patients and in controls, examining 164 AD patients, 143 PsV patients and 100 healthy individuals in Japanese population. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism method. The A allele was decreased in AD patients (40.9%, p=0.031) and increased in PsV patients (60.1%, p=0.035) compared with controls (50.5%). This suggests that IL12B SNP is associated with susceptibility to AD and PsV, presumably by affecting the Th1/Th2 balance.

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.

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT1 and mCysLT2. mCysLT1 and mCysLT2 were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT2. Pranlukast, a specific inhibitor for human CysLT1, antagonized mCysLT2responses as determined by Ca2+ elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT1 mRNA was expressed mainly in skin, lung, and small intestine. mCysLT2 was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT1 and mCysLT2 were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT2 between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.

SummaryAtopic 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, macrophage-derived chemokine (MDC)/CCL22, a CC chemokine, was identified as a selective chemoattractant for CC chemokine receptor 4 (CCR4)-expressing cells, in addition to thymus and activation-regulated chemokine (TARC). We have previously reported that serum TARC levels correlate with the severity of AD. In this report, we investigated the participation of MDC in AD. First, we measured serum MDC levels in 45 patients with AD, 25 patients with psoriasis vulgaris and 25 healthy controls. Serum MDC levels in AD patients were significantly higher than those in healthy controls and psoriasis patients. Furthermore, the increases in serum MDC levels in AD patients were greater in the severely affected group than in the moderate or mild groups. We compared serum MDC levels in 11 AD patients, before and after treatment, and observed a significant decrease after treatment. Moreover, the serum MDC levels significantly correlated with the Scoring AD (SCORAD) index, serum soluble (s) E-selectin levels, serum soluble interleukin-2 receptor (sIL-2R) levels, serum TARC levels and eosinophil numbers in peripheral blood. Our study strongly suggests that serum MDC levels have a notable correlation with disease activity and that MDC, as well as the CC chemokine TARC, may be involved in the pathogenesis of AD.

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.

Background: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of T H 2-type cells in lesional skin. Thymus and activation-regulated chemokine (TARC/CCL17) is a chemokine that attracts CC chemokine receptor 4–positive (CCR4 + ) or CCR8 + cells. Objective: The purpose of this study was to investigate the participation of TARC in AD. Methods: We measured serum TARC levels in 40 patients with AD, 20 healthy control subjects, and 20 patients with psoriasis. We also examined disease activity by using SCORAD score; serum soluble E-selectin, soluble IL-2 receptor, IgE, and GM-CSF levels; and eosinophil numbers in peripheral blood, as well as correlations between TARC levels and these factors. The positivity of CCR4 of CD4 + CD45RO + cells in PBMCs was examined by using FACS analysis. Immunohistochemical staining of TARC and GM-CSF was performed in the lesional skin of patients with AD. Results: The serum TARC levels of patients with AD were significantly higher than those of healthy control subjects and patients with psoriasis. The serum TARC levels significantly correlated with eosinophil number ( r = 0.61), SCORAD score ( r = 0.60), and serum soluble E-selectin levels ( r = 0.58) and weakly correlated with serum soluble IL-2 receptor levels ( r = 0.34) in patients with AD. The TARC levels of patients with AD decreased after the treatment in accordance with the improvement of clinical symptoms. The CCR4 positivity of CD4 + CD45RO + cells in PBMCs of patients with AD was also higher than that of healthy control subjects. Immunohistochemical staining revealed that TARC was positive in ke-ratinocytes in the epidermis and in vascular endothelial cells, T cells, and dendritic cells in the dermis. Conclusion: Serum TARC levels are associated with disease activity of AD, and TARC may play an important role in the pathogenesis of AD. (J Allergy Clin Immunol 2001;107:535-41.)

Skin fibrotic disorders are understood to develop under the influence of some growth factors, such as transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), or connective tissue growth factor (CTGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, we investigated the in vivo effects of growth factors by injecting TGF-beta, CTGF, and bFGF into the subcutaneous tissue of newborn mice. A single application of TGF-beta or bFGF resulted in the formation of transient granulated tissue that disappeared despite 7 days of consecutive injections. A single CTGF injection also caused slight granulation. However, injecting TGF-beta plus CTGF produced long-term fibrotic tissue, which persisted for at least 14 days. Also, fibrotic tissue was observed when CTGF was injected from 4 to 7 days after TGF-beta injections for the first 1-3 days. In situ hybridization analysis revealed the expression of CTGF mRNA in the fibroblasts at least in a few fibrotic conditions. These findings suggest that either CTGF mRNA or an application of exogenous CTGF protein is required for the development of persistent fibrosis. From our study, it appears that interaction of several growth factors is required for persistent fibrotic tissue formation, with TGF-beta causing the induction and CTGF needed for maintenance of skin fibrosis. The animal model on skin fibrosis by exogenous application of growth factors developed in this study may prove useful for future studies on fibrotic disorders.

A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG. In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp[A*]pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ([A*]), the latter of which is found at position 64 in the yeast initiator tRNA(Met).

Desmoplastic malignant melanoma (DMM) consists of amelanotic spindle-shaped melanoma cells and is accompanied by desmoplasia with fibrous stromata. It has a strong tendency for local infiltrative growth and recurrence and a propensity for neurotropism. It is not yet known which cytokine is responsible for the desmoplasia in DMM. In the present study, we investigated the roles of several fibrogenic cytokines and cytokine receptors in DMM: basic fibroblast growth factor (bFGF), connective tissue growth factor (CTGF), transforming growth factor-beta, platelet-derived growth factor (PDGF) and PDGF receptors. Immunostaining and in situ hybridization were conducted in four cases of DMM and four cases of amelanotic malignant melanoma (AMM) as negative controls for desmoplasia. PDGF-beta receptor, bFGF and CTGF were intensely expressed in the DMM specimens in comparison with the AMM specimens. The reaction of PDGF-B ligand and CTGF to PDGF-beta receptor, in addition to the expression of bFGF, may contribute to the desmoplasia in DMM.

We report a case of squamous cell carcinoma of the left leg caused by previous irradiation to the giant pigmented nevus. In treating skin cancers originated from radiation dermatitis, we think it is important to resect surrounding radiation dermatitis lesions as well as tumor itself.

A novel 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting group for the 5′ position of nucleoside 3′-phosphoramidites was successfully applied to the solid-phase synthesis of both an oligodeoxyribonucleotide (TpTpT and TpTpTpT) and an octaribonucleotide in combination with a 2′- O -Thp protecting group (UpCpApGpUpUpGpG). The LMNBz group was simply unmasked due to its base-labile property by a two-step procedure, i.e. , treatments with 0.5 M hydrazine hydrate in 1:4 acetic acid - pyridine, and then with 0.5 M imidazole in acetonitrile.

A novel 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting group for the 5′ position of nucleoside 3′-phosphoramidites was successfully applied to the solid-phase synthesis of both an oligodeoxyribonucleotide (TpTpT and TpTpTpT) and an octaribonucleotide in combination with a 2′- O -Thp protecting group (UpCpApGpUpUpGpG). The LMNBz group was simply unmasked due to its base-labile property by a two-step procedure, i.e. , treatments with 0.5 M hydrazine hydrate in 1:4 acetic acid - pyridine, and then with 0.5 M imidazole in acetonitrile.

Expression of the chemokine receptor CXCR4 by tumor cells promotes metastasis, possibly by activating prosurvival signals that render cancer cells resistant to immune attack. Inhibition of CXCR4 with a peptide antagonist, T22, blocks metastatic implantation of CXCR4-transduced B16 (CXCR4-luc-B16) melanoma cells in lung, but not the outgrowth of established metastases, raising the question of how T22 can best be used in a clinical setting. Herein, whereas the treatment of CXCR4-luc-B16 cells in vitro with the CXCR4 ligand CXCL12 did not reduce killing induced by cisplatin or cyclophosphamide, CXCL12 markedly reduced Fas-dependent killing by gp100-specific (pmel-1) CD8+ T cells. T22 pretreatment restored sensitivity of CXCR4-luc-B16 cells to pmel-1 killing, even in the presence of CXCL12. Two immune-augmenting regimens were used in combination with T22 to treat experimental lung metastases. First, low-dose cyclophosphamide treatment (100 mg/kg) on day 5 in combination with T22 (days 4–7) yielded a ∼70% reduction of B16 metastatic tumor burden in the lungs compared with cyclophosphamide treatment alone (P < 0.001). Furthermore, whereas anti–CTL antigen 4 (CTLA4) monoclonal antibody (mAb; or T22 treatment) alone had little effect on established B16 metastases, pretreatment with T22 (in combination with anti-CTLA4 mAb) resulted in a 50% reduction in lung tumor burden (P = 0.02). Thus, in vitro, CXCR4 antagonism with T22 renders B16 cells susceptible to killing by antigen-specific T cells. In vivo, T22 synergizes with cyclophosphamide or anti-CTLA4 mAb in the treatment of established lung metastases, suggesting a novel strategy for augmenting the efficacy of immunotherapy. [Mol Cancer Ther 2006;5(10):2592–9]

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. Objectives: To examine whether the −431C>T SNP of the TARC gene is associated with susceptibility to AD and whether it affects the promoter activity of the TARC gene. Methods: We investigated the genotype and allele frequencies of the SNP in 193 AD patients and 158 healthy controls by polymerase chain reaction-restriction fragment length polymorphism method. We compared the promoter activities between TARC promoter carrying 431C and that carrying −431T by transient-transfection assay in DJM-1 cell line. Results: There were no significant differences in genotype or allele frequencies between AD patients and controls (genotype: P = 0.38, allele: P = 0.22). Luciferase activity was higher in −431T constructs than in −431C constructs (2.3-fold, P = 9.5 × 10−6). Conclusion: These results suggest that the −431C>T SNP of the TARC gene enhances the promoter activity of TARC gene but is not associated with susceptibility to AD in Japanese population.

Schemes to extend the operational region of gasoline compression ignition were explored using single (optial) and 4-cylinder 4-stroke engines equipped with an electromagnetic valve train. This report focuses mainly on the use of direct fuel injection devices (multi-hole and pintle types),exhaust gas recirculation (EGR) through valve timing, and their effects on the compression ignition operating ranges, and emissions. Also considered is charge boost HCCI using a mechanical supercharger. The results indicated that use of either direct fuel injection or charge boost increased (relative to homogeneous charge operation using port injection) the upper load range from an IMEP peak of about 400 kPa to 650 kPa, but the use of direct fuel injection deteriorated both the co-variation in IMEP (up to about 6%) and the NO x emission levels (up to about 8 g/kWh). In contrast, charge boost retained the very low NOx emission levels of port injection HCCI. At the lower load range, a small amount of fuel injection during negative valve overlap expanded the operational range at the lower load range. It is presumed that the EGR with negative valve overlap has a similar effect as injection during negative overlap namely the oxidation of residual unburned hydrocarbons from the prior cycle during this period. This facilitates compression ignition.

The CC chemokine receptor CCR4 is selectively expressed on Th2-type CD4(+) T cells. Therefore, its ligands, thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine, can facilitate the recruitment, activation and development of Th2 polarized cells. The allergic inflammation of atopic dermatitis is characterized by a predominance of Th2-type cells. Some reports suggest that CCR4 and TARC are important for the pathogenesis of atopic dermatitis. The future aspects of atopic dermatitis therapy that specifically targets CCR4 are discussed. (c) 2002 Prous Science. All rights reserved.

It is known that both interleukin-4 (IL-4) and IL-13 are produced by Th2-type cells and share similar biological functions with each other. However, recently accumulated evidences have revealed that IL-4 may be involved in the Th1-type response. Both thymus and activation-regulated chemokine (TARC/CCL17), a ligand for CC chemokine receptor 4 that is mainly expressed on Th2-type cells, and interferon-induced protein of 10kDa (IP-10/CXCL10), a ligand for CXC chemokine receptor 3 that is mainly expressed on Th1-type cells, are produced by keratinocytes after the stimulation with the primary cytokines such as tumor necrotic factor-alpha (TNF-alpha) and/or interferon-gamma (IFN-gamma). In this study, we investigated the regulation of TARC or IP-10 production from HaCaT cells, an immortalized human keratinocyte cell line, after stimulation with TNF-alpha, IFN-gamma, IL-4 and/or IL-13. Without stimulation, HaCaT cells did not produce TARC. When both TNF-alpha and IFN-gamma were added, they increased synergistically (P

Eotaxin is believed to play an important role in atopic dermatitis (AD) as a potent chemoattractant and activator of eosinophils and Th2 lymphocytes. The eotaxin gene is located at chromosome 17q21.1–q21.2, and linkage findings of AD on chromosome 17 were reported. Recently we have identified single nucleotide polymorphisms (SNPs) of eotaxin gene (−426C>T, −384A>G, 67G>A). To learn whether eotaxin gene SNPs are associated with susceptibility to AD or phenotypes of AD, we investigated the genotype frequencies at each SNP of the gene in AD patients and in controls. We examined 140 Japanese AD patients and 140 healthy Japanese individuals. Genotyping was performed using the polymerase chain reaction–restriction fragment length polymorphism method. No significant difference was observed in allele or genotype frequencies of any SNP between AD patients and controls. Serum immunoglobulin E (IgE) levels were significantly lower in CT and TT genotype than in CC (P=0.038) in −426C>T SNP, and lower in GG than in AA and AG with borderline significance (P=0.053) in −384A>G SNP in AD patients. Eotaxin gene SNPs in the promoter and exon regions are not associated with susceptibility to AD, but two of them in the promoter region are associated with phenotype of AD.

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.

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