Sanju Iwamoto

TNF-α Drives Human CD14+ Monocytes to Differentiate into CD70+ Dendritic Cells Evoking Th1 and Th17 Responses

Many mechanisms involving TNF-α, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-α to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14+ monocytes cultured in the presence of TNF-α and GM-CSF converted to CD14+ CD1alow adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-α, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (Mφ). The mature DC (CD83+ CD70+ HLA-DR high CD14low) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-γ and TNF-α, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-α added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the Mφ (CD14highCD70+CD83−HLA-DR−) produced large amounts of MMP-9 and TNF-α without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated Mφ. Therefore, additional stimulation of monocytes with TNF-α may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated Mφ-mediated innate immunity.

Detection of circulating urothelial cancer cells in the blood using the CellSearch System

Circulating tumor cells (CTCs) have been shown to aid in the therapeutic management of patients. But, only a few attempts have been made at the detection of urothelial cancer cells in the blood. The purpose of this study was to test the hypothesis that CTCs are detected in patients with urothelial cancers using newly developed CellSearch Assay.

Bacillus Calmette-Guérin-pulsed dendritic cells stimulate natural killer T cells and γδT cells

Background:  Immunotherapy with bacillus Calmette‐Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG‐activated killer cells, but the roles of other T lymphocytes, such as NKT cells and γδT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and γδT cells. However, it is known that the patients prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG‐pulsed DCs stimulate NKT cells and γδT cells.

Lipopolysaccharide stimulation converts vigorously washed dendritic cells (DCs) to nonexhausted DCs expressing CD70 and evoking long-lasting type 1 T cell responses

A great variety of in vitro culture protocols for human monocyte‐derived dendritic cells (mo‐DCs) has been used to generate DCs suitable for use in immunotherapy. It is thought that activated DCs undergo one‐way differentiation into “exhausted” DCs. In the present study, we contrived an in vitro method for facilitating expression of CD70 by mature DCs. This was achieved by vigorous washing of mo‐DCs before exposure to lipopolysaccharide (LPS). Unexpectedly, these mature DCs retain expression of some interleukin (IL)‐12 family members after extended periods and maintain their ability to stimulate type 1 T cell responses. In contrast, DCs exposed to IL‐4 before LPS stimulation or LPS‐stimulated DCs not exposed to washing stress before activation failed to express CD70 and did differentiate into exhausted DCs. It is interesting that DCs expressing CD70 (CD70+ DCs) induced interferon‐γ production from purified, allogeneic CD8+ T cells through a direct CD27‐CD70 interaction. This is evidence for a pathway resulting in generation of CD8 T effectors by B7‐independent mechanisms. These data suggest that exposure of immature DCs to LPS stimulation contributes to their terminal differentiation into CD70+ DCs, which have potent ability to prolong type 1 T cell responses through alternative pathways.

Zoledronate Stimulates γδ T Cells in Prostate Cancer Patients

Androgen deprivation therapy is the mainstay of treatment for prostate cancer. Given its frequent failure, new therapy that reduces prostate cancer progression would be a breakthrough in treating this disease. Bisphosphonates are well-established agents for treating skeletal-related events (SREs) in prostate cancer patients with bone metastases. Exposure to bisphosphonates may not only reduce the incidence of SREs, but also have anticancer effects by modulating a patients immunity. The purpose of this study was to examine the effect of zoledronate (ZOL) on γδ T cells, serum prostate-specific antigen (PSA) levels, and velocities. The effect of ZOL, with and without IL-2, on γδ T cell activation was examined in vitro. Furthermore, the activated state and the number of γδ T cells and changes in serum PSA levels were examined for patients who received ZOL infusion for the prevention of SREs. We found that ZOL activated γδ T cells, and the number of γδ T cell was increased when IL-2 was administered with ZOL in vitro. Comparisons before and after the first ZOL infusion revealed that γδ T cells in peripheral blood were activated by ZOL. Moreover, after the first ZOL treatment, reduction in serum PSA was observed in 3 of 11 patients, and reduction in PSA velocity was observed in 5 of 10 patients. Our findings indicate that ZOL stimulates γδ T cells in vivo and in vitro. This study provides further insight into the ability of γδ T cells to induce an antitumor immune response.

Coexpression of CCR6 and CD146 (MCAM) is a marker of effector memory T-helper 17 cells

Effector memory T (TEM) cells are a subpopulation of memory T cells that express receptors mediating migration to inflamed tissues and produce various cytokines. Effector memory T‐helper (Th)17 (Th17EM) cells are thought to be essential for inflammation in Th17‐mediated diseases, but have not been studied in detail. To identify superior surface markers to isolate a homogeneous population of Th17EM cells from peripheral blood, CD4+ T cells were isolated from the peripheral blood of healthy donors based on the expression of CCR7, CCR6 and CD146 using six‐color flow cytometry. After 4 days of culture in the presence of anti‐CD3/28 beads, intracellular cytokines were determined by flow cytometric analysis. To investigate the relevance of Th17EM cells in Th17‐mediated disease, the frequencies of TEM‐cell subsets in psoriasis were quantified using six‐color flow cytometry. An enzyme‐linked immunosorbent assay was performed to confirm the interleukin (IL)‐17‐producing capacity of TEM‐cell subsets from the peripheral blood of a patient with psoriasis. CCR6+CD146+ TEM (CD4+CD45RA−CCR7−) cells had a greater capacity to produce IL‐17 than CCR6+CD146− or CCR6−CD146+ TEM cells. Although the percentage of CCR6+CD146+ cells in TEM cells was not significantly different between patients with psoriasis and controls, three of eight patients had a higher percentage of CCR6+CD146+ TEM cells than the mean +5 standard deviations of the controls. Coexpression of CCR6 and CD146 is a useful marker for Th17EM cells. Increasing the number of CCR6+CD146+ Th17EM cells in peripheral blood may facilitate estimation of systemic Th17‐cell activity in Th17‐mediated diseases.

Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies

BACKGROUND AIMS This study developed a new method to expand CD3(-)CD56(+) natural killer (NK) cells from human peripheral blood mononuclear cells (PBMCs) without feeder cells for clinical trials. METHODS PBMCs from healthy subjects were co-stimulated with anti-CD3 and anti-CD52 monoclonal antibodies and cultured for 14 days in newly developed NKGM-1 medium containing autologous plasma and interleukin-2. Expanded NK cells were examined for cell number, phenotype, in vitro and in vivo cytotoxicity and interferon (IFN)-γ secretion. We also evaluated the proliferative ability of NK cells after cryopreservation. A patient with advanced pancreatic cancer was treated with autologous-expanded NK cells through the use of this method in combination with chemotherapy. RESULTS Expanded NK cells expressed higher levels of activating molecules compared with resting NK cells and exhibited potent cytotoxicity against K562 cells and IFN-γ secretion by cytokine stimulation. Significant anti-tumor activity was observed in immunodeficient mice injected with the human pancreatic cancer cell line BxPC-3. Large-scale cultures generated a median 5.7 × 10(9) NK cells from 20 mL of peripheral blood (n = 38) after 14 days of culture and 8.4 × 10(9) NK cells after 18 days of culture through the use of a cryopreservation procedure. The number of NK cells and cytotoxic activity in the peripheral blood of the patient with pancreatic cancer greatly increased, and successful clinical responses were observed after multiple infusions of expanded NK cells. CONCLUSIONS These data demonstrate that this simple and safe methodology for the ex vivo expansion of NK cells can be used for cancer immunotherapy.

Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells.

Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E(2) (PGE(2)) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE(2)-producing enzymes, cyclooxygenase-2 and microsomal PGE(2) synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-κB) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-κB pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

Time course-changes in phosphatidylcholine profile during oxidative modification of low-density lipoprotein

BackgroundOxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.MethodsHuman and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry.ResultsWhen human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL.ConclusionsThese results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.

Tumor necrosis factor alpha and lymphotoxin stimulate human myeloblastic leukemia cell (ML-1) invasion through a reconstituted basement membrane (Matrigel) with concomitant induction of 92 kDa gelatinase secretion.

We have examined the effects of tumor necrosis factor alpha (TNF) and lymphotoxin (LT) on gelatinase (72 kDa and 92 kDa) and tissue inhibitor of metalloprotease 1 (TIMP1) secretion by human myeloblastic leukemia cells (ML-1) in vitro. TNF (0.1-30 ng/ml) significantly stimulated 92 kDa gelatinase secretion in a dose-dependent manner, but did not significantly stimulate 72 kDa gelatinase secretion. LT also significantly stimulated 92 kDa gelatinase secretion, but the stimulation was less effective compared to TNF. TNF, but not LT, concentrations at 30 ng/ml slightly stimulated TIMP1 secretion. Because 92 kDa gelatinase is thought to play a pivotal role in tumor invasion, we examined the effect of TNF or LT on ML-1 cell invasion through a reconstituted basement membrane (Matrigel). Exposure of ML-1 cells to TNF (3, 10, and 30 ng/ml) or LT (3, 10, and 30 ng/ml) stimulated ML-1 cell invasion through Matrigel in a dose-dependent manner in vitro. The data suggest that TNF- and LT-stimulated 92 kDa gelatinase secretion could play an important role in TNF- or LT-stimulated ML-1 cell invasion.