Makoto Enomoto

TGF-β1 Reciprocally Controls Chemotaxis of Human Peripheral Blood Monocyte-Derived Dendritic Cells Via Chemokine Receptors

We examined the effect of TGF-β1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-β1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3α (MIP-3α) (for CCR-6), or stromal cell-derived growth factor-1α (for CXCR-4). Ligation by TNF-α resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3α, and stromal cell-derived growth factor-1α, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3β. Stimulation of mature DCs with TGF-β1 also enhanced TNF-α-induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF-α-induced expression of CCR-7 and chemotactic migratory ability to MIP-3β. Our findings suggest that TGF-β1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.

A putative chemoattractant receptor, C5L2, is expressed in granulocyte and immature dendritic cells, but not in mature dendritic cells.

Leukocyte chemoattractants are involved in the inflammatory response act via G protein-coupled receptors. We report the cloning of a novel human gene encoding the putative orphan receptor, C5L2, belonging to a subfamily of C3a, C5a and formyl Met-Leu-Ph receptors that are related to the chemokine receptor family. C5L2 transcript levels were abundant in granulocytes and immature dendritic cells but not in mature dendritic cells. We speculate that this receptor may regulate the activation of immature dendritic cells and play a role in the chemoattraction of leukocytes to inflammatory regions.

Fibromuscular cap composition is important for the stability of established atherosclerotic plaques in mature WHHL rabbits treated with statins

We examined the relationship between plaque vulnerability and fibromuscular cap composition using hydrophilic pravastatin and lipophilic fluvastatin. WHHL rabbits aged 10 months were given pravastatin (50 mg/kg) or fluvastatin (20 mg/kg) for 52 weeks. The atherosclerotic lesions were immunohistochemically or conventionally stained and the components were analyzed with a color image analyzer. Compared with the control group, the plasma cholesterol levels were decreased by about 25% in both statin groups. Pravastatin decreased the lipid components (macrophages+extracellular lipids) in whole aortic plaques by 34% and the fibrous caps of coronary plaques by 55%. Fluvastatin decreased the fibromuscular components (smooth muscle cells+collagen fibers) in whole aortic plaques and in the fibromuscular caps of the aortic and coronary plaques. In the pravastatin group, the vulnerability index, the ratio of (lipid components)/(fibromuscular components), was decreased in whole aortic plaques by 28% and in the fibromuscular caps of coronary lesions by 61%, while the indexes were increased in the fluvastatin group. The incidence of vulnerable plaques was decreased by 74% in the coronary plaques of the pravastatin group. Our results suggest that the stability of atheromatous plaques was improved due to a decrease of the lipid components and vulnerability index of the fibromuscular cap by pravastatin.

IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells

We analyzed the expression of IL-12Rβ1 and IL-12Rβ2 and the role of IL-12 in the activation of monocyte-derived dendritic cells (DCs) via IL-12Rβ1-mediated signaling events. Flow cytometric analysis revealed that IL-12Rβ1 was expressed in T cells, Con A blasts, and monocyte-derived DCs, but not in monocytes, while its transcript was detected in all of these cell types. Transcriptional expression of IL-12Rβ2 was observed in T cells, Con A blasts, and monocyte-derived DCs, but not monocytes. The ligation of DCs as well as Con A blasts by IL-12 induced the production of GM-CSF, IL-1β, IL-6, TNF-α, and IFN-γ at the transcription levels. Furthermore, stimulation of DCs with IL-12 induced IL-12p40 transcript, but not IL-12p35 transcript, whereas this stimulation caused the expressions of both transcripts in Con A blasts. Stimulation of DCs with IL-12 caused a tyrosine phosphorylation of several intracellular proteins, and the pattern of these events were distinct from those of IL-12-stimulated Con A blasts. IL-12 also induced tyrosine phosphorylation of IL-12Rβ1 as well as recruitment of several tyrosine-phosphorylated proteins to IL-12Rβ1 in DCs and Con A blasts. Receptor engagement of DCs as well as Con A blasts by IL-12 resulted in activation of Janus kinase 2 and Tyk2 kinases and Stat3 and Stat4 transcription factors and the association of these proteins to IL-12Rβ1. Stimulation with IL-12 caused a tyrosine phosphorylation and enzymatic activity of a family of mitogen-activated protein kinases, p38mapk. These results suggest that IL-12 acts directly on DCs to induce their functional activation via IL-12Rβ1-mediated signaling events.

Chronic toxicity and carcinogenicity in mice of the purified mycotoxins, luteoskyrin and cyclochlorotine*

Abstract Long-term feeding studies in mice, initiated in 1960, have been carried out on luteoskyrin and cyclochlorotine, obtained in purified form from Penicillium islandicum Sopp, an infectant of yellowed rice. In the case of luteoskyrin, five different experiments were conducted to investigate the importance of strain, sex, dose level, frequency of dosage and type of basal grain diet on the development of acute, subacute and chronic toxicity, and especially on hepatoma induction. The results provide evidence that these two mycotoxins are responsible for the hepatotoxic and hepatocarcinogenic effects of yellowed rice. The effects of the mycotoxins differed in that necrosis was observed in the early stages with luteoskyrin, and fibrosis and cirrhosis with cyclochlorotine, but the chronic effects on the liver were similar. The tumorigenic effect of luteoskyrin on the liver was more marked in males than in females.

Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2. Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte–macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-α to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 107 mDCs for 10 weeks and given 350–700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0–1). Median survival from the first vaccination was 240 days (range 31–735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocyte-derived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.

Chemokine Receptor Expressions and Responsiveness of Cord Blood T Cells

Chemokines and their receptors play a critical role in the selective attraction of various subsets of leukocytes. We examined the chemokine receptor expressions and responsiveness of cord blood (CB) T cells. Flow-cytometric analysis revealed that peripheral blood (PB) T cells expressed CCR-1, CCR-2, CCR-5, CCR-6, CXC chemokine receptor-3 (CXCR-3), and CXCR-4, while CB T cells expressed only CXCR-4 on their surface. Chemotactic migratory response of CB T cells to macrophage-inflammatory protein (MIP)-1α, monocyte chemoattractant protein-1, RANTES, MIP-3α, monokine induced by IFN-γ, and IFN-γ-inducible protein-10 was significantly impaired compared with those of PB T cells. In contrast, the ability of CB T cells to migrate to MIP-3β, 6Ckine, and stromal cell-derived factor-1α was greater than that of PB T cells, and these events were correlated with the expression levels of CCR-7 and CXCR-4, respectively. Engagement of CD3 and CD28 specifically up-regulated CXCR-3 expression and chemotaxis to monokine induced by IFN-γ and IFN-γ-inducible protein-10, whereas this stimulation down-regulated CCR-7 expression and chemotaxis to MIP-3β and 6Ckine in PB T cells, but not in CB T cells. These results suggest that PB T cells and CB T cells exhibit distinct chemokine responsiveness via different chemokine receptor repertoire.

Stability of atheromatous plaque affected by lesional composition: study of WHHL rabbits treated with statins.

Abstract: We examined how lesional composition is related to the stability of coronary plaques. WHHL rabbits (12 months old) were given pravastatin and fluvastatin orally for 52 weeks. Both statins decreased plasma cholesterol levels by about 25%, but the suppressive effects on the degree of coronary plaques were mild. Macrophage (Mφ) contents in fibromuscular cap regions were decreased by pravastatin, and smooth muscle cell (SMC) contents in those were decreased by fluvastatin. The plaque vulnerability index was low in Mφ‐poor plaques, but high in SMC‐poor plaques. Our results suggest that reduction in Mφs in the fibromuscular cap is related to plaque stabilization and that reduction in SMCs in the fibromuscular cap is related to plaque destabilization.

Cytotoxicity and carcinogenicity of pterosins and pterosides, 1-indanone derivatives from bracken (Pteridium aquilinum)

Über 30 Chemikalien aus getrockneten jungen Blättern und Wurzelstöcken des japanischen FarnkrautesPteridium aquilinum wurden isoliert und als Sesquiterpen-Derivate mit 1-Indanonkern, Pterosine und deren Glukoside, Pteroside, identifiziert. Eine nur geringe Toxizität für HeLa-Zellen ohne Chromosomenschädigung wurde festgestellt. Langzeitfütterung von Pterosin B und Pterosid B ergab keine Tumorbildung in Wistar-Ratten, obwohl die rohen Blätter und Wurzelstöcke kanzerogen waren.

N-hydroxylation of the carcinogen 2-acetylaminofluorene by human liver tissue invitro

Abstract N-Hydroxylation and 7-hydroxylation of 2-acetylaminofluorene were demonstrated in vitro with microsomes or the 9,000 × g supernatant fraction obtained from human livers of 5 autopsy cases. Two livers with severe fatty changes or marked jaundice showed no ability to hydroxylate 2-acetylaminofluorene. N-Hydroxy-2-acetylaminofluorene was also metabolized by the 9,000 × g supernatant fraction of human liver under hydroxylating conditions; 7-hydroxy-2-acetylaminofluorene was one of the metabolites.