Regulation of anti-inflammatory cytokines IL-10 and TGF-β in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-β production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-β significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-β. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase 1/2.     

Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: involvement of protein kinase C-alpha

The major house-dust mite allergen, Der f 2, stimulates the phospholipase D (PLD) in T lymphocytes from Dermatophagoides farinae specific allergic individuals. PLD activity increased more than two-fold in T cells from allergic patients compared with those cells from normal controls with maximal responses within 30 min after exposure of Der f 2. A well-known PLD activator PKC-alpha was found to be translocated to membrane from cytosol in Der f 2-treated T cells from Dermatophagoides farinae specific allergic individuals. Down-regulation of PKC-alpha with phorbol myristate acetate pretreatment for 24 h abolished Der f 2-induced PLD activation. Ro 320432, PKC inhibitor also reduced the effects of Der f 2-induced PLD activation suggesting that PKC-alpha acts as upstream activator of PLD in Der f 2-treated T cells. Taken together, the present data suggest that Der f 2 can stimulate PLD activity through the PKC-alpha activation in T cells from Dermatophagoides farinae allergic individuals.     

Enhancement of antitumor effect using dendritic cells activated with natural killer cells in the presence of Toll-like receptor agonist

Dendritic cells (DCs) play a role in natural killer (NK) cell activation, while NK cells are also able to activate and mature DCs. Toll-like receptors (TLRs) on the surface of DCs and NK cells induce the maturation and activation of these cells when engaged with their cognate ligand. We investigated to generate potent DCs by maturation with NK cells in the presence of TLR agonist in vitro and tested the efficacy of these DC vaccinations in mouse colon cancer model. The optimal ratios of DCs versus NK cells were 1:1 to 1:2. Immature DCs were mature with NK cells in the presence of lipopolysaccharide, which is TLR4 agonist, and further addition of IL-2 induced phenotypically and functionally mature bone marrow-derived DCs. These potent DCs exhibited not only high expression of several costimulatory molecules and high production of IL-12p40 and IL-12p70, but also high allogeneic T cells stimulatory capacity, and the induction of the high activities to generate tumor-specific CTLs. Consistently, vaccination with these DCs efficiently inhibited CT-26 tumor growth in mouse colon cancer model when compared to other vaccination strategies. Interestingly, combination therapy of these DC-based vaccines and with low-dose cyclophosphamide showed dramatic inhibition effects of tumor growth. These results suggest that the DCs maturated with NK cells in the presence of TLR agonist are potent inducer of antitumor immune responses in mouse model and may provide a new source of DC-based vaccines for the development of immunotherapy against colon cancer.     

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.     

Identification of novel peptides that stimulate human neutrophils

Neutrophils play a key role in innate immunity, and the identification of new stimuli that stimulate neutrophil activity is a very important issue. In this study, we identified three novel peptides by screening a synthetic hexapeptide combinatorial library. The identified peptides GMMWAI, MMHWAM, and MMHWFM caused an increase in intracellular Ca2+ in a concentration-dependent manner via phospholipase C activity in human neutrophils. The three peptides acted specifically on neutrophils and monocytes and not on other non-leukocytic cells. As a physiological characteristic of the peptides, we observed that the three peptides induced chemotactic migration of neutrophils as well as stimulated superoxide anion production. Studying receptor specificity, we observed that two of the peptides (GMMWAI and MMHWFM) acted on formyl peptide receptor (FPR)1 while the other peptide (MMHWAM) acted on FPR2. Since the three novel peptides were specific agonists for FPR1 or FPR2, they might be useful tools to study FPR1- or FPR2-mediated immune response and signaling.     

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

Lysophosphatidylcholine (LPC) is a bioactive lipid generated by phospholipase A2-mediated hydrolysis of phosphatidylcholine. In the present study, we demonstrate that LPC stimulates phospholipase D2 (PLD2) activity in rat pheochromocytoma PC12 cells. Serum deprivation induced cell death of PC12 cells, as demonstrated by decreased viability, DNA fragmentation, and increased sub-G1 fraction of cell cycle. LPC treatment protected PC12 cells partially from the cell death and induced neurite outgrowth of the cells. Overexpression of PLD2 drastically enhanced the LPC-induced inhibition of apoptosis and neuritogenesis. Pretreatment of the cells with 1-butanol, a PLD inhibitor, completely abrogated the LPC-induced inhibition of apoptosis and neurite outgrowth in PC12 cells overexpressing PLD2. These results indicate that LPC possesses the neurotrophic effects, such as anti-apoptosis and neurite outgrowth, through activation of PLD2.     

HPMA‐Based Nanocarriers for Effective Immune System Stimulation

The selective activation of the immune system using nanoparticles as a drug delivery system is a promising field in cancer therapy. Block copolymers from HPMA and laurylmethacrylate‐co‐hymecromone‐methacrylate allow the preparation of multifunctionalized core‐crosslinked micelles of variable size. To activate dendritic cells (DCs) as antigen presenting cells, the carbohydrates mannose and trimannose are introduced into the hydrophilic corona as DC targeting units. To activate DCs, a lipophilic adjuvant (L18‐MDP) is incorporated into the core of the micelles. To elicit an immune response, a model antigen peptide (SIINFEKL) is attached to the polymeric nanoparticle—in addition—via a click reaction with the terminal azide. Thereafter, the differently functionalized micelles are chemically and biologically characterized. While the core‐crosslinked micelles without carbohydrate units are hardly bound by DCs, mannose and trimannose functionalization lead to a strong binding. Flow cytometric analysis and blocking studies employing mannan suggest the requirement of the mannose receptor and DC‐SIGN for effective micelle binding. It could be suppressed by blocking with mannan. Adjuvant‐loaded micelles functionalized with mannose and trimannose activate DCs, and DCs preincubated with antigen‐conjugated micelles induce proliferation of antigen‐specific CD8+ T cells.     

Sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.     

Activated natural killer cell-mediated immunity is required for the inhibition of tumor metastasis by dendritic cell vaccination

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-gamma production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8(+) T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.     

Phospholipase D activity is elevated in hepatitis C virus core protein-transformed NIH3T3 mouse fibroblast cells

Hepatitis C Virus (HCV) is associated with a severe liver disease and increased frequency in the development of hepatocellular carcinoma. Overexpression of HCV core protein is known to transform fibroblast cells. Phospholipase D (PLD) activity is commonly elevated in response to mitogenic signals, and has also been overexpressed and hyperactivated in some human cancer cells. The aim of this study was to understand how PLD was regulated in the HCV core protein-transformed NIH3T3 mouse fibroblast cells. We observed that PLD activity was elevated in the NIH3T3 cells overexpressing HCV core protein over the vector alone-transfected control cells, however, expression levels of PLD protein and protein kinase C (PKC) in the HCV core protein-transformed cells was similar to the control cells. Phorbol 12-myristate 13-acetate (PMA), which is known to activate PKC, stimulated PLD activity significantly more in the core protein-transformed cells, in comparison with that of the control cells. PLD activity assay using PKC isozyme-specific inhibitor and PKC translocation experiment showed that PKC-delta was mainly involved in the PMA- induced PLD activation in the core-transformed cells. Moreover, in cells overexpressing HCV core protein, PMA also stimulated p38 kinase more potently than that of the control cells, and an inhibitor of p38 kinase abolished PMA-induced PLD activation in cells overexpressing HCV core protein. Taken together, these results suggest that PLD might be implicated in core protein-induced transformation.     

Click-Chemistry-Mediated Cell Membrane Glycopolymer Engineering to Potentiate Dendritic Cell Vaccines

Dendritic cell vaccine (DCV) holds great potential in tumor immunotherapy owing to its potent ability in eliciting tumor-specific immune responses. Aiming at engineering enhanced DCV, we report the first effort to construct a glycopolymer-engineered DC vaccine (G-DCV) via metabolicglycoengineering and copper-free click-chemistry. Model G-DCV was prepared by firstly delivering tumor antigens, ovalbumin (OVA) into dendritic cells (DC) with fluoroalkane-grafted polyethyleneimines, followed by conjugating glycopolymers with a terminal group of dibenzocyclooctyne (DBCO) onto dendritic cells. Compared to unmodified DCV, our G-DCV could induce stronger T cell activation due to the enhanced adhesion between DCs and T cells. Notably, such G-DCV could more effectively inhibit the growth of the mouse B16-OVA (expressing OVA antigen) tumor model after adoptive transfer. Moreover, by combination with an immune checkpoint inhibitor, G-DCV showed further increased anti-tumor effects in treating different tumor models. Thus, our work provides a novel strategy to enhance the therapeutic effectiveness of DC vaccines.     

Major house dust mite allergen, Der p I, activates phospholipase D in human peripheral blood mononuclear cells from allergic patients: involvement of protein kinase C

The major house-dust-mite allergen, Der p I, stimulates the phospholipase D (PLD) in peripheral blood mononuclear cells (PBMC) from allergic patients with maximal responses after 30 min exposure. At 30 min, Der p I stimulated PLD activity by 1.4-fold in mild, 1.6-fold in moderate and 2-fold in severe allergic patients over control values (p < 0.05). When the cells were pretreated for 24 h with phorbol myristate acetate to down-regulate protein kinase C (PKC), PLD stimulation by Der p I was largely abolished. These results indicate that in PBMC from allergic patients, Der p I can stimulate PLD activity, and that PKC activation is involved in this stimulation.     

Dexamethasone enhances phospholipase D activity in M-1 cells

Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.     

ATP-induced focal adhesion kinase activity is negatively modulated by phospholipase D2 in PC12 cells

Extracellular ATP has been known to modulate various cellular responses including mitogenesis, secretion and morphogenic activity in neuronal cells. In the ATP-induced morphogenic activity, focal adhesion kinase(s) such as Fak have been suggested to play a critical role. Binding of ATP to its specific cell surface receptor in PC12 cells induces phospholipase D (PLD) activity. However, the role of PLD on ATP-induced Fak activation in PC12 cells remains unclear. In this study, we investigated the role of PLD on the ATP-induced Fak activation and paxillin phosphorylation using two established cell lines: wild type PLD2- and lipase-inactive mutant PLD2-inducible PC12 cells. Stimulation of cells with ATP caused PLD2 activation via classical protein kinase C activation. ATP also induced Fak activation, and paxillin phosphorylation, and were dramatically reduced by wild type PLD2 overexpression but not by lipase-inactive mutant PLD2 overexpression. When the PC12 cells were pretreated with propranolol, a specific inhibitor for phosphatidic acid phosphohydrolase resulting in the accumulation of PA, ATP-induced Fak activation and paxillin phosphorylation were also reduced. We found that inhibition of tyrosine phosphatases by pervanadate completely blocked PLD2-dependent Fak and paxillin dephosphorylation. Taken together, we suggest that PLD2 activity might play a negative role in ATP-induced Fak and paxillin phosphorylation possibly through tyrosine phosphatases.     

Receptor activator of NF-kappaB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.     

EFFECTIVENESS OF PHOTOFRIN II IN ACIWATION OF MACROPHAGES AND in vitro KILLING OF RETINOBLASTOMA CELLS

Abstract Administration of a small dose (300 ng/mouse) of photofrin II (PII) to mice, followed by 4 days of exposure to only ambient fluorescent light in animal quarters, induced Fc-receptor-mediated phagocytic and superoxide-generating capacities of peritoneal macrophages by five- and seven-fold, respectively. When these mice were kept in the dark for 4 days, no activation of macrophages was observed. These results suggest that macrophage activation is a consequence of photodynamic activation. Much higher doses (> 3000 ng/mouse) suppressed macrophage activity. However, 2 months after administration of 3000 ng PII/mouse, greatly enhanced phagocytic and superoxide-generating capacities of peritoneal macrophages were observed.
In vitro photodynamic activation of macrophages was analyzed after white or red fluorescent light exposure of mouse peritoneal cells (mixture of macrophages and B and T lymphocytes) in media containing PII. A short (10 s) white fluorescent light treatment of peritoneal cells in a medium containing 0.03 ng PII/mL produced the maximal level of phagocytic activity of macrophages. Illumination with the same total fluence of red fluorescent light requires a threefold higher concentration of PII to achieve the same extent of enhanced phagocytic activity of macrophages. Thus, photodynamic activation of macrophages with PII by white fluorescent light was more efficient than by red fluorescent light. Similarly, photodynamic killing of retinoblastoma cells was more efficient with white than red fluorescent light. The concentration of hematoporphyrin (HP) or PII required for direct photodynamic killing of retinoblastoma cells was roughly four orders of magnitude greater than that required for activation of macrophages. These results suggest that effective photodynamic therapy may be achieved with milder treatments that stimulate macrophage activity, an important component of immunopotentiation.     

Mucosal dendritic cells shape mucosal immunity

Dendritic cells (DCs) are key modulators that shape the immune system. In mucosal tissues, DCs act as surveillance systems to sense infection and also function as professional antigen-presenting cells that stimulate the differentiation of naive T and B cells. On the basis of their molecular expression, DCs can be divided into several subsets with unique functions. In this review, we focus on intestinal DC subsets and their function in bridging the innate signaling and adaptive immune systems to maintain the homeostasis of the intestinal immune environment. We also review the current strategies for manipulating mucosal DCs for the development of efficient mucosal vaccines to protect against infectious diseases.     

Role of phospholipase D1 in glucose-induced insulin secretion in pancreatic �� cells

As glucose is known to induce insulin secretion in pancreatic β cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic β-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.     

Oxygen-Deficient Molybdenum Oxide Nanosensitizers for Ultrasound-Enhanced Cancer Metalloimmunotherapy

Oxygen-deficient molybdenum oxide (MoO_X) nanomaterials are prepared as novel nanosensitizers and TME-stimulants for ultrasound (US)-enhanced cancer metalloimmunotherapy. After PEGylation, MoO_X-PEG exhibits efficient capability for US-triggered reactive oxygen species (ROS) generation and glutathione (GSH) depletion. Under US irradiation, MoO_X-PEG generates a massive amount of ROS to induce cancer cell damage and immunogenic cell death (ICD), which can effectively suppress tumor growth. More importantly, MoO_X-PEG itself further stimulates the maturation of dendritic cells (DCs) and triggeres the activation of the cGAS-STING pathway to enhance the immunological effect. Due to the robust ICD induced by SDT and efficient DC maturation stimulated by MoO_X-PEG, the combination treatment of MoO_X-triggered SDT and aCTLA-4 further amplifies antitumor therapy, inhibits cancer metastases, and elicits robust immune responses to effectively defeat abscopal tumors.     

In vitro induction of anterior gradient-2-specific cytotoxic T lymphocytes by dendritic cells transduced with recombinant adenoviruses as a potential therapy for colorectal cancer

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration, and cellular transformation, and is one of the specific mRNA markers for circulating tumor cells in patients with gastrointestinal cancer. We investigated the feasibility of AGR2 as a potent antigen for tumor immunotherapy against colorectal cancer (CRC) cells using dendritic cells (DCs) transduced with a recombinant adenovirus harboring the AGR2 gene (AdAGR2). DCs transduced with a recombinant adenovirus encoding the AGR2 gene (AdAGR2/DCs) were characterized. These genetically-modified DCs expressed AGR2 mRNA as well as AGR2 protein at a multiplicity of infection of 1,000 without any significant alterations in DC viability and cytokine secretion (IL-10 and IL-12p70) compared with unmodified DCs as a control. In addition, AdAGR2 transduction did not impair DC maturation, but enhanced expression of HLA-DR, CD80, and CD86. AdAGR2/DCs augmented the number of IFN-γ-secreting T-cells and elicited potent AGR2-specific cytotoxic T lymphocytes capable of lysing AGR2-expressing CRC cell lines. These results suggest that AGR2 act as a potentially important antigen for immunotherapy against CRC in clinical applications.