Plasmacytoid dendritic cells have a cytokine-producing capacity to enhance ICOS ligand-mediated IL-10 production during T-cell priming.

Plasmacytoid dendritic cells (pDCs) have the potential to prime CD4(+) T-cells to differentiate into IL-10-producing T regulatory cells through preferential expression of inducible co-stimulatory ligand (ICOS-L). Although pDCs produce cytokines such as type-I IFNs, TNF-α, or IL-6 accompanying up-regulation of ICOS-L expression during activation in response to toll-like receptor (TLR)-ligands or IL-3, the roles of the pDC-derived cytokines in T-cell priming remain largely elusive. Therefore, we investigated the functional involvement of these cytokines in generating IL-10-producing T regulatory cells. We found that either IFN-α or IL-6 enhanced the pDC- or ICOS-L-driven generation of IL-10-producing T-cells from naive CD4(+) T-cells and their regulatory functions. However, IFN-α stimulation in the absence of ICOS-L showed only a marginal tendency to increase the T-cell production of IL-10 and thus pDC-derived type-I IFNs in response to CpG could function together with ICOS-L. In addition, IL-6 functioned to generate IL-10-producing T-cells only on T-cell priming by pDCs activated by IL-3 or under IL-4-mediated T(h)2 conditions. Thus, type-I IFNs and IL-6 act as supplementary factors for the ICOS-L-dependent IL-10-producing T-cell differentiation in pDCs activated along the TLR-dependent and IL-3-dependent pathways, respectively. We also showed that pDC-derived TNF-α induced ICOS-L expression on pDCs in an autocrine manner and that IL-6 promoted ICOS expression on T-cells, contributing to the ICOS/ICOS-L-mediated T-cell response. Our results suggest that the ICOS-L-mediated tolerogenic pDC function in adaptive immunity is backed up by the elaborate cytokine-producing ability of pDCs.

Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells.

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio-defence systems through the production of cytokines such as interferon-α (IFN-α) and interleukin-12 (IL-12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti-microbial innate immune responses but also in the pathogenesis of IFN-related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA-mediated innate immunity by possessing DNA-sensing toll-like receptor 9 (TLR9). We here found that double-stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte-derived DCs (moDCs), leading to the preferential production of IFN-α but not IL-12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA-mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co-localization of endosomal LAMP1 staining, and the dsDNA-mediated IFN-α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single-stranded RNA (ssRNA) stimulated moDCs to secrete IL-12 but not IFN-α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA-mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN-α production and acquired immunity via IL-12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA-mediated activation of protective or pathogenic immunity.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, function as inhibitors of cellular and molecular components involved in type I interferon production.

OBJECTIVE: Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs. METHODS: We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNalpha production, and intracellular signaling. RESULTS: Statins inhibited IFNalpha production profoundly and tumor necrosis factor alpha production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFNalpha production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNalpha production by PDCs from SLE patients and SLE serum-induced IFNalpha production. CONCLUSION: Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE.

Thymic stromal lymphopoietin plays an adjuvant role in BCG-mediated CD8(+) cytotoxic T cell responses through dendritic cell activation.

Although Bacillus Calmette-Guérin (BCG) has historically emerged as a potent adjuvant in cancer immunization through dendritic cell (DC) activation, the efficacy of its antitumor effect has been limited. Therefore, the strategy of adjuvant therapy using BCG needs to be improved by adding enhancers. Here we found that thymic stromal lymphopoietin (TSLP) acts as an enhancer for the BCG-mediated antitumor effect. While BCG-stimulated DCs induced CD8(+) T cell production of IFN-gamma without strong cell expansion, TSLP-stimulated DCs induced robust CD8(+) T cell expansion without high quantities of IFN-gamma production. Notably, DCs stimulated with both BCG and TSLP induced robust expansion of CD8(+) T cells that produced a large amount of IFN-gamma with a potent cytolytic activity related to granzyme B expression. Our data suggest that TSLP is a good adjuvant to enhance the BCG-mediated cytotoxic T cell effect through DC activation, and provide a functional basis for a novel strategy for antitumor immune-based therapy.

Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice.

Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vaccine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine XCR1 was the only chemokine receptor selectively expressed in CD8alpha(+) conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8(+) T cells increased XCL1 production upon activation. These expression patterns were conserved in human blood cells, including the BDCA3(+) DC subset. Thus, in human and mice, certain DC subsets should be chemotactic towards NK or activated CD8(+) T cells through XCR1.

Expression and characterization of vanilloid receptor subtype 1 in human dental pulp cell cultures.

The expression of the vanilloid receptor subtype 1 (VR1, TRPV1) was detected in human dental pulp fibroblasts (PF-10) using RT-PCR, Western blotting, and immunocytochemical analysis. As revealed by ELISA, capsaicin induced IL-6 expression in PF-10 cells, and the VR1 antagonist capsazepine dose-dependently inhibited capsaicin-induced IL-6 production, indicating that capsaicin-induced IL-6 expression is related to VR1 activation. The interaction between capsaicin and mitogen-activated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) were detected after capsaicin stimulation. p38 MAPK is involved in capsaicin-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. The result of EMSA showed that capsaicin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappa B (NF-kappaB) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.

Horseradish peroxidase degrades lipid hydroperoxides and suppresses lipid peroxidation of polyunsaturated fatty acids in the presence of phenolic antioxidants.

Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and alpha-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction.

Substance P enhances expression of lipopolysaccharide-induced inflammatory factors in dental pulp cells.

To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.

A classic temporal optic disc pit showing progression in the corresponding optic nerve fiber and visual field defects.

PURPOSE: We report a rare case with a classic temporal optic disc pit (ODP) showing the progression of an associated nerve fiber layer defect (NFLD) with the corresponding visual field defect (VFD). METHODS: We describe the patient's medical records and review the pertinent literature. RESULTS: A 54-year-old woman had a temporal ODP, which was considered to be congenital, associated with both NFLD expanded to both the upper and lower sides of the horizontal line and corresponding VFD and a small ODP-like excavation at the 6.5 o'clock position on the disc edge with a narrow NFLD OD. During the 5-year follow-up period, both the NFLD and VFD associated with the temporal pit progressed without serous retinal detachment. The small ODP-like excavation located at the 6.5 o'clock position also showed progressive NFLD in the temporal lower quadrant with advanced VFD, which suggested that the excavation might be associated with glaucoma. CONCLUSION: Based on the observation that the VFD occupied the two temporal quadrants with no step, an NFLD with corresponding VFD associated with a classic temporal ODP, although not considered to be related to glaucoma, can progress.

A case of thrombotic thrombocytopenic purpura induced by acute pancreatitis.

Thrombotic thrombocytopenic purpura (TTP) is a multisystemic microvascular disorder that may be caused by an imbalance between unusually large von Willebrand factor multimers and the cleaving protease ADAMTS13. In acquired TTP, especially in secondary TTP with various underlying diseases, the diagnosis is difficult because there are many cases that do not exhibit severe deficiency of ADAMTS13 or raised levels of ADAMST13 inhibitors. It is well known that collagen disease, malignancy, and hematopoietic stem cell transplantation can be underlying conditions that induce TTP. However, TTP induced by acute pancreatitis, as experienced by our patient, has rarely been reported. Our patient completely recovered with treatments using steroids and plasma exchange (PE) only. In cases where patients develop acute pancreatitis with no apparent causes for hemolytic anemia and thrombocytopenia, the possibility of TTP should be considered. Treatments for TTP including PE should be evaluated as soon as a diagnosis is made.

Quercetin induces the expression of peroxiredoxins 3 and 5 via the Nrf2/NRF1 transcription pathway.

PURPOSE: The flavonoids have potent antioxidant and free-radical scavenging properties and are beneficial in the prevention and treatment of ocular diseases including glaucoma. The authors have previously reported that antiglaucoma agents could transcriptionally activate the antioxidant protein peroxiredoxin (PRDX)2. The purpose of this study was to investigate whether quercetin can activate transcription factors and induce the expression of the PRDX family. METHODS: To demonstrate whether quercetin can transcriptionally induce the expression of the PRDX family, trabecular meshwork cells were treated with quercetin, and PRDX expression and transcription factors were both investigated by Western blot analysis, reporter assays, and siRNA strategies. Subsequently, cellular sensitivity to oxidative stress was determined. RESULTS: Expression of the PRDX3 and PRDX5 genes was induced by quercetin in a time- and dose-dependent manner. NRF1 transactivates the promoter activity of both PRDX3 and PRDX5 but not PRDX2 and PRDX4. Quercetin can also induce the expression of Nrf2 and NRF1 but not of Ets1, Ets2, or Foxo3a. Knockdown of NRF1 expression significantly reduced the expression of both PRDX3 and PRDX5. Reporter assays showed that NRF1 transactivated the promoter activity of both PRDX3 and PRDX5 and that the downregulation of NRF1 with siRNA repressed the promoter activity of both PRDX3 and PRDX5. Furthermore, the downregulation of NRF1, PRDX3, and PRDX5 renders trabecular meshwork cells sensitive to hydrogen peroxide. Finally, NRF1 activation by quercetin was completely abolished by the knockdown of Nrf2. CONCLUSIONS: Quercetin upregulates the antioxidant peroxiredoxins through the activation of the Nrf2/NRF1 transcription pathway and protects against oxidative stress-induced ocular disease.

Transcriptional regulation of activating transcription factor 4 under oxidative stress in retinal pigment epithelial ARPE-19/HPV-16 cells.

PURPOSE: Oxidative stress plays an important role in the pathogenesis of various ocular diseases such as retinopathy, glaucoma, and age-related macular degeneration. Activating transcription factor 4 (ATF4) is induced by various stressors, including endoplasmic reticulum (ER) and oxidative stress, and ATF4 expression is regulated translationally through the PERK pathway of eIF2α phosphorylation. Transcriptional regulation of the ATF4 gene under oxidative stress was investigated in human papillomavirus 16 (HPV-16)-transformed retinal pigment epithelial ARPE-19/HPV-16 cells. METHODS: Retinal pigment epithelial cells, trabecular meshwork cells, and corneal endothelial cells were treated with anoxia and thapsigargin (TG). Gene expression of ATF4 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and transcription factors was investigated by Western blot analysis, reporter assays, chromatin immunoprecipitation (ChIP) assays, and small interfering (si)RNA strategies. Cellular sensitivity to oxidative stress was determined. RESULTS: The expression of two transcriptional factors, ATF4 and Nrf2, was significantly induced by anoxia and TG. The Nrf2 regulator Keap1 was downregulated by anoxia. Downregulation of Nrf2 abolished ATF4 expression. On the other hand, downregulation of Keap1 enhanced the expression of both Nrf2 and ATF4. The promoter activity of ATF4 was transactivated by the co-transfection of Nrf2 expression plasmids and reduced by the transfection of Nrf2-specific siRNA. The ChIP assays demonstrated that Nrf2 bound to the promoter of the ATF4 gene. Nrf2 downregulation nearly abolished the ATF4 induction by anoxia and TG. Consistent with these findings, the promoter activity of ATF4 was augmented by treatment with TG, HCA, H(2)O(2), and anoxia. However, stress induction of ATF4 promoter activity was observed, even when a mutation was introduced into the antioxidant-responsive elements site. Furthermore, stress induction of the ATF4 promoter was completely abolished when the 5' untranslated region of the ATF4 gene was deleted. Downregulation of ATF4 rendered ARPE-19/HPV-16 cells sensitive to oxidative stress. CONCLUSIONS: These results suggest that the stress induction of ATF4 is significantly regulated transcriptionally through a Nrf2-dependent mechanism and may be a double-edged sword in the pathogenesis of various retinopathies.

Inhibitor of IkappaB kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells.

INTRODUCTION: Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IkappaB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs. METHODS: We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-alpha production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-alpha production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U. RESULTS: Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-alpha production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-alpha production, more strongly inhibited IFN-alpha production than TNF-alpha production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-alpha production by human PBMCs induced by the SLE serum and the in vivo serum IFN-alpha level induced by injecting mice with poly U. CONCLUSIONS: These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.

Nipradilol and timolol induce Foxo3a and peroxiredoxin 2 expression and protect trabecular meshwork cells from oxidative stress.

PURPOSE: Oxidative stress plays an important role in pathogenesis of glaucoma. The purpose of this study is to investigate the novel effect of antiglaucoma drugs on the expression of antioxidant peroxiredoxins of trabecular meshwork (TM) cells. METHODS: The expression of the peroxiredoxin family was investigated using immortalized TM cell lines. Cells were treated with antiglaucoma drugs and analyzed for the expression of peroxiredoxin, and cellular sensitivity to oxidative stress. Furthermore, the effect of antiglaucoma drugs on the molecular regulation of the expression of peroxiredoxin was examined using a reporter assay and siRNA strategy. RESULTS: Glaucomatous TM cells highly express peroxiredoxin 2 when compared with normal TM cells. Nipradilol and timolol, but not latanoprost, induce the expression of peroxiredoxin 2 through the activation of the Foxo3a transcription factor. TM cells showed reduced sensitivity to H(2)O(2) when cells were treated with either nipradilol or timolol, but not with latanoprost. In addition, both Foxo3a and PRDX2 expression were enhanced by drug-induced signal transduction through its receptor. CONCLUSIONS: These results indicate that both nipradilol and timolol possess a novel mechanism of action and function as potent protective agents against oxidative stress.

Substance P activates p38 mitogen-activated protein kinase to promote IL-6 induction in human dental pulp fibroblasts.

Substance P (SP) induces the expression of proinflammatory cytokines, such as interleukin (IL)-6, which are implicated in pulp inflammation. To determine the signal pathway of SP-induced IL-6, we examined the activities of the mitogen-activated protein kinases (MAPKs) in human dental pulp cell (PF-10) cultures. SP induced the phosphorylation of p38 MAPK within 5 min; this activation persisted for up to 40 min and was independent of the activation of extracellular signal-related kinases (ERK-1 and ERK-2) that were induced after SP stimulation of PF-10 cells. As shown by electrophoretic mobility shift assay p38 MAPK was not involved in SP-induced activation of nuclear factor-kappa B (NF-kappaB). However, p38 MAPK mediated SP-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. Our results suggest that the activation of p38 MAPK is important for NF-kappaB-independent regulator of neurogenic inflammation in dental pulp tissues.