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 ß-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 ß-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 αß 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.

Identification of a novel marker for dendritic cell maturation, mouse transmembrane protein 123.

Dendritic cells (DCs) are a group of professional antigen-presenting cells, and many genes are known to be associated with their maturation. We compared the transcriptional profiles of immature and mature mouse Langerhans cells using the suppressive, subtractive hybridization method and identified a novel gene of unknown function, termed herein transmembrane protein 123 (Tmem123), of which mRNA expression was enhanced in mature but not in immature Langerhans cells. Its expression was also enhanced in other mature DCs such as bone marrow-derived DCs (BMDCs) and splenic DCs. Interestingly, CD40 expression was up-regulated on mature BMDCs cultured with colchicine concurrently with the enhanced expression of Tmem123 compared with that of fresh BMDCs. Furthermore, the expression of CD40 was enhanced on Tmem123-transfected DC2.4 cells, a mouse BMDC-derived cell line, compared with that on mock-transfected DC2.4 cells. This enhancement of CD40 expression did not occur after deletion of lysosome/endosome targeting YXXϕ motifs (where X is any amino acid and ϕ is a bulky hydrophobic amino acid) in the Tmem123 cytoplasmic tail. By stimulation with anti-CD40 monoclonal antibody, these transfectants secreted an increased amount of IL-12/23 p40 compared with mock-transfected DC2.4 cells. Thus, our study demonstrates that Tmem123 may be used as a new maturation marker in DCs and that this molecule may be closely associated with the cell surface expression of CD40.

Significant attenuation of macrovascular involvement by bosentan in a patient with diffuse cutaneous systemic sclerosis with multiple digital ulcers and gangrene.

Systemic sclerosis (SSc) is characterized by vascular injuries, and bosentan has recently been proved to be efficacious for the prevention of new digital ulcers in SSc. We herein report a case of SSc in a patient with refractory digital ulcers and gangrene treated with bosentan. Stenosis of the ulnar artery, evaluated by magnetic resonance angiography, was attenuated by the bosentan treatment, suggesting that bosentan exerts a reverse remodeling effect against the pathological organic changes of arteries in SSc.

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.

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.

Successful use of intravenous cyclophosphamide pulse therapy for interstitial lung disease in a patient with systemic sclerosis on hemodialysis.

Interstitial lung disease and scleroderma renal crisis are major complications of systemic sclerosis, which occasionally coexist in patients with the diffuse cutaneous subtype. We herein report a case of diffuse cutaneous systemic sclerosis under hemodialysis due to a previous history of scleroderma renal crisis, whose interstitial lung disease was effectively and safely treated with a half dose of i.v. cyclophosphamide pulse. The dose of cyclophosphamide and the timing of hemodialysis leading to efficacy and low toxicity are discussed.

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

A subset of CC chemokine receptor-6(+) (CCR6(+)), γδ-low (GDL) T cells that express Th17 cytokines in mouse skin participates in IL-23-induced psoriasiform dermatitis. We use CCR6-deficient (knockout, KO) and wild-type (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 the total numbers of GDL T cells in the dermis of WT and CCR6 KO mice were equivalent. There was an ∼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 and epidermal recruitment of GDL T cells are impaired in CCR6-deficient mice. Thus, these data show that CCR6 regulates epidermal trafficking of γδ-T-cell subsets in the skin and suggest the potential of CCR6 as a therapeutic target for psoriasis.

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 CXCR4(flox/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 CXCR4(f/f) control mice in an IL-23-mediated psoriasiform dermatitis model and measured skin edema, and histologic and immunohistological changes. IL-23-treated K14-CXCR4KO mice showed a 1.3-fold increase in mean ear swelling, a 2-fold increase in epidermal thickness, and greater parakeratosis. IL-23-treated wild-type (WT) mice showed weak CXCR4 expression in areas of severe epidermal hyperplasia, but strong CXCR4 expression in nonhyperplastic regions, suggesting that CXCR4 may regulate KC proliferation. To test this hypothesis, we overexpressed CXCR4 in HaCaT KC cells and treated them with IL-22 and/or CXCL12 (chemokine (C-X-C motif) ligand 12). CXCL12 blocked IL-22-mediated HaCaT cell proliferation in vitro and synergized with IL-22 in upregulating SOCS3 (suppressor of cytokine signaling 3), a key regulator of STAT3 (signal transducer and activator of transcription 3). 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 has an unexpected role in inhibiting KC proliferation and mitigating the effects of proliferative T helper type 17 cytokines.

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.

Inflammation and tumor microenvironment in lymph node metastasis.

In nearly all human cancers, the presence of lymph node (LN) metastasis increases clinical staging and portends worse prognosis (compared to patients without LN metastasis). Herein, principally reviewing experimental and clinical data related to malignant melanoma, we discuss diverse factors that are mechanistically involved in LN metastasis. We highlight recent data that link tumor microenvironment, including inflammation (at the cellular and cytokine levels) and tumor-induced lymphangiogenesis, with nodal metastasis. Many of the newly identified genes that appear to influence LN metastasis facilitate general motility, chemotactic, or invasive properties that also increase the ability of cancer cells to disseminate and survive at distant organ sites. These new biomarkers will help predict clinical outcome and point to novel future therapies in metastatic melanoma as well as other cancers.

Duration of remission period of narrowband ultraviolet B therapy on psoriasis vulgaris.

The remission period of psoriasis vulgaris following narrowband ultraviolet B (NB-UVB) light therapy with topical vitamin D(3) application was evaluated retrospectively to investigate the therapeutic efficacy. Fifty-two patients (60 cases) were treated with a 5-day/week protocol of NB-UVB light irradiation plus topical vitamin D ointment application for 1 month and followed up for at least 12 months. We considered re-exacerbation as the time when the patients needed treatment other than topical therapy. The remission period was defined as the duration from the end of treatment until re-exacerbation. Twenty-seven cases (56%) of psoriasis showed a remission period longer than 12 months. The patients with a past history of systemic therapy or phototherapy had a significantly shorter remission period than those without such a history. No statistically significant differences were observed in sex, age, period before treatment, Psoriasis Area and Severity Index score and total irradiation dose. A previous history of systemic therapy or phototherapy may mean that the disease is severe and sufficiently active to form multiple new lesions requiring these treatments. Our results suggest that the 5-day/week NB-UVB light protocol for 4 weeks is an effective and safe treatment for psoriasis vulgaris and can induce long-term remission.

A case of dermatomyositis with "liver disease associated with rheumatoid diseases" positive for anti-liver-kidney microsome-1 antibody.

We reported a case of dermatomyositis (DM) with liver disturbance in a 50-year-old Japanese female. She presented with fever, muscle weakness, and typical DM rashes. On clinical and serological examinations, the liver impairment was initially diagnosed as probable autoimmune hepatitis, which was denied by a histological study despite positive anti-liver-kidney microsome-1 antibody. Finally, she was diagnosed as having DM with "liver disease associated with rheumatoid diseases", and treatment with oral prednisone (40 mg/day) achieved normalization of liver and muscle enzyme levels as well as improvement of symptoms associated with DM. Liver involvement in patients with polymyositis (PM)/DM has not been well described and is considered to be uncommon. Full clarification of the etiology of liver impairment with a histological examination in collagen diseases including PM/DM is useful to determine the proper dose of corticosteroids for the treatment of collagen diseases and their liver complications.

Early inflammatory changes in the "perilesional skin" of psoriatic plaques: is there interaction between dendritic cells and keratinocytes?

Early inflammatory changes in psoriatic plaques were investigated immunohistochemically by studying the normal-appearing skin adjacent to the plaques (perilesional skin), lesional skin, and distant uninvolved skin from psoriasis patients. Perilesional epidermis contained numerous CD1a-positive Langerhans cells, some of which expressed HLA-DR, CD83, CD80, and CD86, at the same time expressing Langerin. There were also numerous CD83-positive, CD11c-positive, Langerin-negative dendritic cells (DCs) in the epidermal-dermal junction of perilesional skin. CD3-positive T lymphocytes were sparse in the perilesional skin. Perilesional epidermis expressed keratin K6 and K16, inflammatory keratins, and C/EBPbeta, a transcription factor related to inflammatory cytokines. Our results demonstrated the abundant distribution of activated DCs in the perilesional skin of psoriatic plaques, where early inflammatory changes occur in the epidermal keratinocytes, which suggests their involvement in the provocation of epidermal inflammation in the perilesional epidermis and further pathogenic roles in the formation of psoriatic plaques.