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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.

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.

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.

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.

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).

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.

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]

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

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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