Tomonori Takekoshi

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and β-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

Epidermal CCR6+ γδ T Cells Are Major Producers of IL-22 and IL-17 in a Murine Model of Psoriasiform Dermatitis

Cytokine components of Th17 pathway play vital roles in human psoriasis. Although much is known about TCR αβ T cells in psoriasis, the role of unconventional T cells, including γδ T cells, is unclear. In this study, using an IL-23 skin injection model of psoriasiform dermatitis in mice, we demonstrate that IL-22, IL-17A, and the IL-23R were highly enriched in a population of CCR6+, TCR γδ-low expressing (GDL) T cells that accumulated in the epidermis after IL-23 injections. GDL cells were distinct from resident TCR γδ-high, Vγ3+,CCR6− T cells in the epidermis that did not change appreciably in numbers following IL-23 injection. Large numbers of CCR6+ cells were detected at or above the level of the epidermal basement membrane by confocal microscopy 5 d after repeated IL-23 injections at the same time GDL cells increased in numbers in the epidermis. TCR δ-deficient mice (lacking γδ T cells) exhibited decreased ear swelling and downregulated expression of IL-22 and IL-17A in the epidermis following IL-23 injection. Our data suggest that a subset of γδ T cells play a critical role in IL-23–mediated psoriasiform dermatitis.

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.

CCR6 is required for epidermal trafficking of γδ T cells in an IL-23-induced model of psoriasiform dermatitis

A subset of CCR6+, γδ-low (GDL) T cells that express Th17 cytokines in mouse skin participates in IL-23-induced psoriasisform dermatitis. We use CCR6-deficient (KO) and wildtype (WT) mice to analyze skin trafficking patterns of GDL T cells and function-blocking mAbs to determine the role of CCR6 in IL-23-mediated dermatitis. Herein, CCL20 was highly upregulated in IL-23-injected WT mouse ear skin as early as 24 hours after initial treatment, and large numbers of CCR6+ cells were observed in the epidermis of IL-23-injected WT mice. Anti-CCL20 mAbs reduced psoriasiform dermatitis and blocked recruitment of GDL T cells to the epidermis. In CCR6 KO mice, GDL T cells failed to accumulate in the epidermis after IL-23 treatment, but total numbers of GDL T cells in the dermis of WT and CCR6 KO mice were equivalent. There was a ~70% reduction in the proportion of IL-22+ GDL T cells in the dermis of CCR6 KO mice (vs. WT mice), suggesting that effector function as well as epidermal recruitment of GDL T cells are impaired in CCR6-deficient mice. Thus, these data show CCR6 regulates epidermal trafficking of γδ T cell subsets in skin and suggest the potential of CCR6 as a therapeutic target for psoriasis.

Polymorphisms of vitamin D receptor gene in Japanese patients with psoriasis vulgaris

We examined polymorphisms of vitamin D receptor (VDR) gene in Japanese patients with psoriasis vulgaris (PsV). We also studied the association between VDR gene polymorphisms and the response to vitamin D (VD) topical treatment in psoriatic patients. FokI, BsmI, ApaI and TaqI genotypes were determined by restriction fragment patterns in patients (n = 115) and controls (n = 69). In addition, 54 psoriatic patients were divided into two groups in terms of their response to VD (tacalcitol) topical treatment: non-responsive (n = 30) and responsive (n = 24) patients. The frequencies of B allele and t allele were lower in patients than in controls (9 vs. 19%: p < 0.01, 7 vs. 14%: p < 0.05, respectively). In regard to response to VD treatment, F allele was lower in non-responsive patients than in controls (47 vs. 64%, p < 0.05). We show that polymorphisms of VDR gene are associated with Japanese patients with PsV. Allelic variance in the VDR gene or other genes in linkage disequilibrium with this gene might predispose to the development of PsV.

A Locked, Dimeric CXCL12 Variant Effectively Inhibits Pulmonary Metastasis of CXCR4-Expressing Melanoma Cells Due to Enhanced Serum Stability

The CXC chemokine receptor-4 (CXCR4) plays a critical role in cancer by positively regulating cancer cell metastasis and survival. We previously showed that high concentrations of the CXCR4 ligand, wild-type CXCL12 (wtCXCL12), could inhibit colorectal cancer metastasis in vivo, and we have hypothesized that wtCXCL12 dimerizes at high concentration to become a potent antagonist of CXCR4. To address this hypothesis, we engineered a covalently locked, dimeric variant of CXCL12 (CXCL122). Herein, we show that CXCL122 can not only inhibit implantation of lung metastasis of CXCR4-B16-F10 melanoma cells more effectively than AMD3100, but that CXCL122 also blocks the growth of established pulmonary tumors. To identify a basis for the in vivo efficacy of CXCL122, we conducted Western blot analysis and ELISA analyses, which revealed that CXCL122 was stable for at least 12 hours in serum, whereas wtCXCL12 was quickly degraded. CXCL122 also maintained its antagonist properties in in vitro chemotaxis assays for up to 24 hours in serum, whereas wtCXCL12 was ineffective after 6 hours. Heat-inactivation of serum prolonged the stability and function of wtCXCL12 by more than 6 hours, suggesting enzymatic degradation as a possible mechanism for wtCXCL12 inactivation. In vitro analysis of amino-terminal cleavage by enzymes dipeptidylpeptidase IV (DPPIV/CD26) and matrix metalloproteinase-2 (MMP-2) resulted in 25-fold and 2-fold slower degradation rates, respectively, of CXCL122 compared with wtCXCL12. In summary, our results suggest CXCL122 possesses greater potential as an antimetastatic drug as compared with AMD3100 or wtCXCL12, potentially due to enhanced serum stability in the presence of N-terminal degrading enzymes. Mol Cancer Ther; 11(11); 2516–25. ©2012 AACR.

Human keratinocytes express fractalkine/CX3CL1

BACKGROUND fractalkine/CX3CL1 is a unique chemokine that has properties of both chemoattractants and adhesion molecules. The major source of this chemokine in the skin is still controversial. OBJECTIVE studies were undertaken to determine the expression of fractalkine in human skin. METHODS RT-PCR, Western blotting, and immunostaining were performed with normal human epidermal keratinocytes (NHEK) and HaCaT cells, human keratinocyte cell line, for the presence of fractalkine. Biopsy specimens of normal and diseased skin were also investigated. RESULTS we identified that NHEK and HaCaT cells expressed fractalkine mRNA and protein. The combination of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma upregulated their expression by NHEK. Immunohistochemistry demonstrated fractalkine expression in keratinocytes in lichen planus and psoriasis vulgaris. RT-PCR also showed that lesional skin of psoriatic patients expressed higher levels of fractalkine mRNA than non-lesional skin from the same patients. CONCLUSION these results suggests that keratinocytes strongly express fractalkine in lichen planus and psoriasis vulgaris and that the fractalkine-CXC3CR1 system in the diseased skin can be a target for the treatment.

Interleukin 12 production by monocytes from patients with psoriasis and its inhibition by ciclosporin A.

Background  Psoriasis is a T‐helper (Th)1 cytokine‐mediated chronic skin disease and interleukin (IL)‐12 has been shown to play a major role in the development of Th1 responses.

Drug-induced hypersensitivity syndrome associated with transient hypogammaglobulinaemia and increase in serum IgE level.

Drug-induced hypersensitivity syndrome (DIHS) is a rare but severe disease with multiorgan failure. Recently, the association of the human herpesvirus (HHV) family, particularly of HHV-6, with DIHS has been reported. We report a 43-year-old female diagnosed as having DIHS based on the clinical course and laboratory examinations. The HHV-6 reactivation was demonstrated by significantly increased levels of the specific antibody in her paired sera and by polymerase chain reaction of HHV-6 DNA. Notably, transient hypogammaglobulinaemia was detected in the early stage of the disease, which was associated with the disease activity. By contrast, the serum IgE level and eosinophils were increased 2 or 3 weeks later. In addition, serum levels of interferon γ, interleukin (IL)-4 and soluble IL-2 receptor, which were increased in the early phase of the disease, decreased gradually after the corticosteroid therapy.

CXCR4 Negatively Regulates Keratinocyte Proliferation in IL-23-Mediated Psoriasiform Dermatitis

CXCR4 is expressed by basal keratinocytes (KCs), but little is known about its function in inflamed skin. We crossed K14-Cre and CXCR4flox/flox (f/f) transgenic mice, resulting in mice with specific loss of the CXCR4 gene in K14-expressing cells (K14-CXCR4KO), including basal KCs. K14-CXCR4KO pups had no obvious skin defects. We compared K14-CXCR4KO and CXCR4f/f control mice in an IL-23-mediated psoriasisform dermatitis model and measured skin edema, histologic, and immunohistological changes. IL-23-treated K14-CXCR4KO mice showed a 1.3-fold increase in mean ear swelling, 2-fold increase in epidermal thickness, and greater parakeratosis. IL-23-treated WT mice showed weak CXCR4 expression in areas of severe epidermal hyperplasia, but strong CXCR4 expression in non-hyperplastic regions, suggesting CXCR4 may regulate keratinocyte proliferation. To test this hypothesis, we overexpressed CXCR4 in HaCaT keratinocyte cells and treated them with IL-22 and/or CXCL12. CXCL12 blocked IL-22-mediated HaCaT cell proliferation in vitro and synergized with IL-22 in upregulating SOCS3, a key regulator of STAT3. SOCS3 was required for CXCR4-mediated growth inhibition. In human psoriatic skin, both CXCR4 and SOCS3 were upregulated in the junctional region at the border of psoriatic plaques. Thus, CXCR4 plays an unexpected role in inhibiting KC proliferation and mitigating the effects of proliferative Th17 cytokines.