VISTA Stimulation of VSIG4-Positive Macrophages Strongly Suppresses T Cell Proliferation via Excessive Nitric Oxide Production in Sepsis.

Organ damage and immune deficiency are important problems in sepsis caused by an excessive immune response. There is controversy about the cause of immune suppression. In this study, we investigated the roles of macrophages that exhibit excessive activity on T cell immunity. Peritoneal macrophages from mice with cecal ligation and puncture (CLP)-induced sepsis migrated to different organs. In particular, V-set immunoglobulin (Ig)-domain-containing 4 (VSIG4) positive macrophages appeared in the spleen 48 h after CLP induction. When cocultured with splenic T cells, VSIG4(+) cells inhibited the proliferation of activated T cells through the release of nitric oxide (NO) compared to VSIG4(-) cells. Stimulation of VSIG4(+) cells with V-domain Ig suppressor of T cell activation (VISTA) antibody increased the expression of several cytokine genes and the release of NO, but not phagocytosis, compared to those of hamster IgG-stimulated VSIG4(+) cells. When cocultured with splenic T cells, VISTA-stimulated VSIG4(+) cells induced excessive T cell suppression via more NO secretion compared to hamster IgG-stimulated VSIG4(+) cells. Taken together, the current study demonstrates that VSIG4(+) peritoneal macrophages play important roles in inducing immunosuppression and that VISTA acts as a costimulatory receptor in these cells. These data suggest that blocking the migration of VSIG4(+) cells might alleviate excessive immune activity and that blocking VISTA on VSIG4(+) macrophages might play a crucial role in the development of new therapies to prevent T cell suppression in sepsis.

VSIG4(+) peritoneal macrophages induce apoptosis of double-positive thymocyte via the secretion of TNF-α in a CLP-induced sepsis model resulting in thymic atrophy.

Thymic atrophy in sepsis is a critical disadvantage because it induces immunosuppression and increases the mortality rate as the disease progresses. However, the exact mechanism of thymic atrophy has not been fully elucidated. In this study, we discovered a novel role for VSIG4-positive peritoneal macrophages (V4(+) cells) as the principal cells that induce thymic atrophy and thymocyte apoptosis. In CLP-induced mice, V4(+) cells were activated after ingestion of invading microbes, and the majority of these cells migrated into the thymus. Furthermore, these cells underwent a phenotypic shift from V4(+) to V4(-) and from MHC II(low) to MHC II(+). In coculture with thymocytes, V4(+) cells mainly induced apoptosis in DP thymocytes via the secretion of TNF-α. However, there was little effect on CD4 or CD8 SP and DN thymocytes. V4(-) cells showed low levels of activity compared to V4(+) cells. Thymic atrophy in CLP-induced V4(KO) mice was much less severe than that in CLP-induced wild-type mice. In addition, V4(KO) peritoneal macrophages also showed similar activity to V4(-) cells. Taken together, the current study demonstrates that V4(+) cells play important roles in inducing immunosuppression via thymic atrophy in the context of severe infection. These data also suggest that controlling the function of V4(+) cells may play a crucial role in the development of new therapies to prevent thymocyte apoptosis in sepsis.

Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice.

CD44v6, a splice variant of the cell surface glycoprotein CD44, acts as a co-receptor for c-Met and is upregulated in tumors with high metastatic potential. Methods: We screened a phage-displayed peptide library for peptides that selectively bind to CD44v6-overexpressing cells and exploited them to block CD44v6 and deliver a pro-apoptotic peptide to tumors for cancer therapy. Results: CNLNTIDTC (NLN) and CNEWQLKSC (NEW) peptides bound preferentially to CD44v6-high cells than to CD44v6-low cells. The binding affinities of NLN and NEW to CD44v6 protein were 253 ± 79 and 85 ± 18 nM, respectively. Peptide binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases.

Blood lead concentration and exposure related factors in Korea from the National Environmental Health Survey (KoNEHS) II (2012-2014).

This study evaluated lead concentrations in blood and related factors among the South Korean population based on data from the Korea National Environmental Health Survey (KoNEHS) II (2012-2014) conducted by the National Institute of Environmental Research and the Ministry of Environment. Personal data were obtained from non-institutionalized civilian Korean individuals in an interview with trained community surveyors using a structured questionnaire (n = 6,455, aged 19 or older, mean age 49.7 years). The lead concentrations in whole blood were analyzed by atomic absorption spectrophotometry (AAS) with a Zeeman-effect-based background corrector. The precision and accuracy of the analytical methods were verified by internal and external quality controls (G-EQUAS, Germany). Statistical analysis was performed using weighted KoNEHS II data separated by sex, and the lead concentration was expressed as a geometric mean (GM). Multiple linear regression was performed using the SPSS 23.0 software package (SPSS Inc., Chicago, IL, USA). The total GM of lead concentrations was 19.5 µg/L. Lead concentrations increased with age and were higher in males (22.8 µg/L) than in females (16.6 µg/L). Smokers and drinkers had higher concentrations than non-smokers and non-drinkers of both sexes. People who used herbal medications had higher concentrations than those who did not among females. People of both sexes living in rural areas had higher lead concentrations than those in urban areas. Lead concentrations also varied with educational level, total family income, the type of water regularly consumed, and occupation. The average lead concentration of the general adult population in Korea has rapidly decreased over time from 45.8 µg/L (1999) to 19.45 µg/L (2012-2014); however, it remains higher than that of the United States, Germany, and Canada. The factors significantly related to lead concentration in South Korea were age, sex, smoking and alcohol drinking status, herbal medication usage by females, residential area, drinking water at home, and occupation. These factors could be used to improve occupational and environmental hygiene among the Korean population.

Ultravist Induces the Expression of MCP-1 and VCAM-1 in IL-4-Stimulated HUVECs.

The goal of the present study focused on the adverse reaction of contrast medium (CM) via the induction of inflammatory molecules in human umbilical vein endothelial cells (HUVECs). Ultravist-induced monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression was markedly increased in interleukin-4 (IL-4)-pretreated HUVECs in a time- and dose-dependent manner and was paralleled by concomitant production of MCP-1 and VCAM-1 proteins. MCP-1 and VCAM-1 gene expression by Ultravist in combination with IL-4 was mediated by the c-Jun N-terminal kinases (JNK1/2) signaling pathway. IL-4-pretreated Ultravist-stimulated HUVECs showed greatly increased migration and adhesion of THP-1 cells. Cell migration was decreased by treatment of CCR2 antagonist, and cell adhesion was also decreased by VCAM-1 blocking antibody. Furthermore, when tested in vivo under similar conditions, MCP-1 protein was significantly increased in Ultravist combined with IL-4-injected mice. Taken together, our findings suggest that MCP-1 blocking may be crucial in preventing the endothelial dysfunction induced by contrast medium in patients with inflammatory disease and atherosclerosis.

Decrease in the urine cotinine concentrations of Korean non-smokers between 2009 and 2011 following implementation of stricter smoking regulations.

This study aimed to determine if there was an association between the implementation of smoking regulation policies and the urine cotinine concentrations of Korean non-smokers. The subjects of this study were 4612 non-smoking Korean citizens (aged 19 or older) selected from the first stage of the Korean National Environmental Health Survey conducted by the National Institute of Environmental Research from 2009 to 2011. Cotinine concentrations in urine were measured by GC-MS (limit of detection: 0.05 ng/mL). Changes in the urine cotinine concentration were analyzed using a weighted general linear model and linear regression and values were shown as geometric mean (GM). The GM urine cotinine concentration decreased over time (2.92 ng/mL in 2009, 1.93 ng/mL in 2010, and 1.25 ng/mL in 2011). The total decrease in the subjects' urine cotinine concentration between 2009 and 2011 was 2.79 ng/mL, representing a relative decrease of 54.7%. The decrease in GM urine cotinine concentration in each subgroup ranged from 2.17 ng/mL to 3.29 ng/mL (relative decreases of 46.4% and 62.8%, respectively), with the largest absolute reductions in subjects in the following groups: females, aged 40-49 years, detached residence type, no alcohol consumption, employed, secondhand smoke exposure. All groups had negative regression coefficients, all of which were significant (p < 0.001). Our results provide indirect indicators of the effectiveness of smoking regulation policies including the revision of the National Health Promotion Act in Korea.

Cancer cell-associated cytoplasmic B7-H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation.

Aberrant B7-H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7-H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7-H4 transcription in primary CD138(+) multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7-H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7-H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7-H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7-H4 expression. Furthermore, knockdown of cytoplasmic B7-H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7-H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7-H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment.

Iopromide in combination with IFN-γ induces the activation of HMC-1 cells via IL-4 and MCP-1 expression.

In this study, we investigated whether IFN-γ has a role in contrast-medium-induced adverse reactions. Iopromide, a nonionic iodinated contrast agent, slightly induced mast cell proliferation and significantly increased the expression of IL-4 and MCP-1 at low doses. The pretreatment of cells with IFN-γ dramatically increased the expression of iopromide-induced IL-4 and MCP-1. An evaluation of mast cell activator secretion revealed that IFN-γ- or IL-4-pretreated HMC-1 cells released dramatically increased levels of ß-hexosaminidase and histamine when stimulated with iopromide. We also found that the migration of EoL-1 and THP-1 cells was significantly increased in culture conditions with iopromide-stimulated IL-4-pretreated HMC-1 cells. Taken together, our findings suggest that measuring IFN-γ or IL-4 levels in serum would be helpful as a potential biomarker of adverse patient reactions and that blocking IFN-γ or IL-4 may be crucial in preventing the delayed allergy-like reaction induced by contrast medium in patients with various diseases.

Effects of perfluorooctane sulfuric acid on placental PRL-family hormone production and fetal growth retardation in mice.

Perfluorooctane sulfuric acid (PFOS) is a persistent organic pollutant, causes fetal growth retardation but the mechanism is still unclear. This study focused on PFOS-induced toxicity such as placental trophoblast cell histopathological changes, endocrine function (i.e., prolactin (PRL)-family hormone production) and subsequent fetal growth retardation in mice. Maternal body weight gain, placental and fetal weights were significantly decreased in proportion to PFOS dosage. Placental efficiency (fetal weight/placental weight) was significantly reduced dose-dependently. Necrotic changes were observed in PFOS-treated placental tissues, and the area of injury increased dose-dependently. Finally, mRNA levels and maternal serum concentrations of the PRL-family hormones (mPL-II, mPLP-Cα, mPLP-K) were significantly reduced dose-dependently. In addition, the changing pattern between PRL-family hormone concentrations and fetal body weight was positively correlated. These results suggest that gestational PFOS treatment induces placental histopathological changes and disruption of endocrine function, finally may lead to fetal growth retardation in mice.

Blood lead concentration and related factors in Korea from the 2008 National Survey for Environmental Pollutants in the Human Body.

This study evaluated blood lead concentrations in the Korean general population and the correlation between various exposure sources using data from the 2008 Korea National Survey for Environmental Pollutants in the Human Body (National Institute of Environmental Research, Korea). The general and occupational characteristics were gathered from 5136 participants who were 20 years of age and older using a structured questionnaire. Blood lead concentrations were analyzed using an atomic absorption spectrophotometer. Statistical analysis was performed using multiple linear regressions of the log lead concentrations to the independent variables such as age, gender, smoke, herbal medication and drug consumption, drinking water, and living area. Geometric mean (GM) blood lead concentrations in Korean adults were 19.7 µg/l. The blood lead concentrations increased with age; the highest concentrations were found in the 50-69-year age group (p<0.001). Males were higher than in females (p<0.001). Current smokers and drinkers had higher concentrations than nonsmokers (p<0.001) and nondrinkers (p<0.001), respectively. People who took herbal medication and drug consumption were higher than those who did not (p<0.001). Education level was negatively associated with blood lead concentration (p<0.001). People living in or around industrial areas had elevated blood lead concentration (p<0.001). Family income was also negatively associated with lead concentration, but not significantly. For drinking water, the underground water (spring or well water) drinking group had higher concentrations than other types of water drinking groups, but not significantly (p=0.063). The blood lead concentrations by occupation were significant (p<0.034): the highest was in laborer and Agricultural-Fishery-Forestry and the lowest in office workers. In women, blood lead concentrations tended to decrease with increasing delivery times, but not significantly. The blood lead concentration (GM) of the general adult population in Korea has decreased over time from 45.8 µg/l (1999) to 19.7 µg/l (2008). Although it is still higher than in other countries such as the United States and Canada, it is rapidly decreasing. Gender, age, smoking and alcohol drinking status, herbal medication and drug consumption, education level, living area and occupation were significantly related to the blood lead concentrations in Korea.

TLR5 activation by flagellin induces doxorubicin resistance via interleukin-6 (IL-6) expression in two multiple myeloma cells.

Multiple myeloma (MM) is an incurable B-cell hematologic malignancy characterized by the clonal expansion of malignant plasma cells in the bone marrow (BM). MM cells interact with various cells within the BM microenvironment, leading to skeletal destruction, angiogenesis, and drug resistance. Therefore, control of the cell-host interaction and growth factors is important to improve patient outcome with conventional treatments. In this study, we investigated flagellin-induced cell proliferation, cytokines expression, and the mechanisms of cancer drug resistance that lead to the failure of cytotoxic therapies for MM. The human MM line KMS28BM expresses the TLR5 gene as well as the protein at high levels. When treated with the specific TLR5 ligand flagellin, KMS28BM cells showed increased proliferation, increased IgG λ production, and high-level expression and secretion of the pro-inflammatory cytokine IL-6, via NF-κB activation through p38 and PI3K/AKT signaling. Furthermore, flagellin-stimulated KMS28BM cells were shown to have "increased doxorubicin and apoptosis resistance" through the inhibition of caspases and PARP activity, and this result was reversed by blocking IL-6. Thus, increased cell viability and the chemoresistance of flagellin-stimulated KMS28BM cells may result from autocrine or paracrine signaling mediated by secreted IL-6. These findings indicate that TLR5 activation by flagellin may elicit chemoresistance in MM patients who have suffered from recurrent bacterial infections.

Secondhand smoke exposure and urine cotinine concentrations by occupation among Korean workers: results from the 2008 Korea National Survey for Environmental Pollutants in the Human Body.

This study aimed to estimate the status of secondhand smoke (SHS) exposure through urine cotinine analysis among nonsmoking workers in Korea and to analyze factors affecting urine cotinine concentrations. Data were based on "The 2008 Korea National Survey for Environmental Pollutants in the Human Body," a cross-sectional study of the National Institute of Environmental Research of Korea. We selected 1448 nonsmoking adult workers from 200 localities to participate in this survey. Urine cotinine concentrations were analyzed using a gas chromatograph-mass selective detector. We calculated separate covariate-adjusted geometric means for socio-demographic variables for males, females, and total subjects by analysis of covariance (ANCOVA). Statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, Ill.). The prevalence of self-reported exposure to SHS was 36.9%. The geometric mean (95% confidence interval) of urine cotinine concentrations among all participants was 16.50 (14.48-18.80) µg/L. Gender, living area, education, and SHS exposure showed significant differences in urine cotinine concentrations. The urine cotinine concentrations of farmworkers and blue-collar workers such as skilled agricultural, forestry, and fishery workers, and elementary occupations were higher than those of white-collar workers such as clerical support workers, technicians, and associate professionals. Such a high proportion of the population having high urine cotinine levels indicates widespread exposure to SHS among nonsmoking workers in Korea. Furthermore, the urine cotinine levels among nonsmoking workers exposed to SHS varied by occupation. The measured urine cotinine concentration is suggested to be a valuable indication of SHS exposure in Korea.

Tetrahydropteridines possess antioxidant roles to guard against glucose-induced oxidative stress in Dictyostelium discoideum.

Glucose effects on the vegetative growth of Dictyostelium discoideum Ax2 were studied by examining oxidative stress and tetrahydropteridine synthesis in cells cultured with different concentrations (0.5X, 7.7 g L(-1); 1X, 15.4 g L(-1); 2X, 30.8 g L(-1)) of glucose. The growth rate was optimal in 1X cells (cells grown in 1X glucose) but was impaired drastically in 2X cells, below the level of 0.5X cells. There were glucose-dependent increases in reactive oxygen species (ROS) levels and mitochondrial dysfunction in parallel with the mRNA copy numbers of the enzymes catalyzing tetrahydropteridine synthesis and regeneration. On the other hand, both the specific activities of the enzymes and tetrahydropteridine levels in 2X cells were lower than those in 1X cells, but were higher than those in 0.5X cells. Given the antioxidant function of tetrahydropteridines and both the beneficial and harmful effects of ROS, the results suggest glucose-induced oxidative stress in Dictyostelium, a process that might originate from aerobic glycolysis, as well as a protective role of tetrahydropteridines against this stress.

Altered sugar donor specificity and catalytic activity of pteridine glycosyltransferases by domain swapping or site-directed mutagenesis.

CY-007 and CY-049 pteridine glycosyltransferases (PGTs) that differ in sugar donor specificity to catalyze either glucose or xylose transfer to tetrahydrobiopterin were studied here to uncover the structural determinants necessary for the specificity. The importance of the C-terminal domain and its residues 218 and 258 that are different between the two PGTs was assessed via structure-guided domain swapping or single and dual amino acid substitutions. Catalytic activity and selectivity were altered in all the mutants (2 chimeric and 6 substitution) to accept both UDP-glucose and UDP-xylose. In addition, the wild type activities were improved 1.6-4.2 fold in 4 substitution mutants and activity was observed towards another substrate UDP-N-acetylglucosamine in all the substitution mutants from CY-007 PGT. The results strongly support essential role of the C-terminal domain and the two residues for catalysis as well as sugar donor specificity, bringing insight into the structural features of the PGTs.

Dictyostelium phenylalanine hydroxylase is activated by its substrate phenylalanine.

We have studied the regulatory function of Dictyostelium discoideum Ax2 phenylalanine hydroxylase (dicPAH) via characterization of domain structures. Including the full-length protein, partial proteins truncated in regulatory, tetramerization, or both, were prepared from Escherichia coli as his-tag proteins and examined for oligomeric status and catalytic parameters for phenylalanine. The proteins were also expressed extrachromosomally in the dicPAH knockout strain to examine their in vivo compatibility. The results suggest that phenylalanine activates dicPAH, which is functional in vivo as a tetramer, although cooperativity was not observed. In addition, the results of kinetic study suggest that the regulatory domain of dicPAH may play a role different from that of the domain in mammalian PAH.

CD40 stimulation induces vincristine resistance via AKT activation and MRP1 expression in a human multiple myeloma cell line.

Various co-stimulatory receptors are expressed in multiple myeloma (MM) both in immune microenvironment and in the tumor microenvironment in vivo. In relapsed human MM, these receptors are known to increase cell proliferation and induce conventional drug resistance. However, the mechanism of drug resistance induced via co-stimulatory receptors is poorly understood. In this study, we examined the role of CD40 expressed on MM cell lines. Out of all of the KMS MM cell lines, the KMS28BM cells expressed high levels of the CD40 receptor. When stimulated with anti-CD40 antibody or recombinant human CD40L, the proliferation of KMS28BM cells was increased 1.7 fold. In CD40-stimulated KMS28BM cells, signaling via the AKT pathway caused an increase in the expression of multidrug resistance-associated gene 1 (MRP1) and IL-6 by 2.2 fold and 30 fold, respectively, but not the MDR1 gene. Furthermore, CD40-stimulated KMS28BM cells were observed to be substantially resistant to the anticancer drug vincristine, and when cells were treated with the MRP1 specific inhibitor, MK-571, drug resistance was decreased. We also found that CD40-stimulated, MRP1-expressing KMS28BM cells significantly increased calcein efflux, and calcein efflux was inhibited through treatment with MK-571. Therefore, blocking CD40 and inhibiting MRP1 are potential targets to treat CD40-induced drug resistance in multiple myeloma.

All-trans retinoic acid induces TLR-5 expression and cell differentiation and promotes flagellin-mediated cell functions in human THP-1 cells.

Toll-like receptor 5 (TLR-5), which is expressed on macrophages and dendritic cells (DCs), is a crucial cell surface molecule that senses microbial-associated molecular patterns and initiates host innate immune responses upon infection with invaders that express flagellin. Little information is known about the induction factors and mechanisms of TLR-5 expression. In this study, we demonstrate that all-trans retinoic acid (ATRA) significantly up-regulated TLR-5 expression in human macrophage THP-1 cells by co-activating NF-κB and the RARα receptor and inducing the differentiation of CD11b(-)CD11c(-) THP-1 cells to CD11b(+)CD11c(low) cells. Furthermore, when stimulated with flagellin, ATRA-induced THP-1 cells expressed multiple cytokines, including TNF-α, IL-1beta, and IL-12p40, and several co-stimulatory molecules, such as CD40, CD80, CD86, and MHC class I and II. We also showed that when ATRA-induced THP-1 cells were stimulated with flagellin, the cells displayed an allostimulatory capacity rather than phagocytic activity. Taken together, our findings suggest that ATRA is a crucial immunostimulatory cofactor that induces the activation of macrophages and their subsequent differentiation into dendritic-like cells.

Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases.

BACKGROUND AND PURPOSE: Cilostazol is a specific inhibitor of 3'-5'-cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti-inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti-inflammatory effect of cilostazol on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. EXPERIMENTAL APPROACH: We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs). KEY RESULTS: Cilostazol suppressed production of nitric oxide (NO), prostaglandin E(2) (PGE(2)) and the proinflammatory cytokines, interleukin-1 (IL-1), tumour necrosis factor-alpha, and monocyte chemoattractant protein-1 (MCP-1), in a concentration-dependent manner. Inhibitory effects of cilostazol were not affected by treatment with an adenylate cyclase inhibitor, SQ 22536, indicating that these actions of cilostazol were cAMP-independent. Cilostazol significantly inhibited the DNA binding and transcriptional activity of NF-kappaB. Moreover, cilostazol blocked signalling upstream of NF-kappaB activation by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but without affecting the activity of p38 MAPK. CONCLUSION AND IMPLICATIONS: Our results demonstrate that suppression of the NF-kappaB, ERK, JNK signalling pathways may inhibit LPS-induced NO and PGE(2) production. Therefore, cilostazol may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators and cytokine production in activated microglia.

Programmed death-1 receptor negatively regulates LPS-mediated IL-12 production and differentiation of murine macrophage RAW264.7 cells.

While programmed death-1 (PD-1), a co-inhibitory member of CD28 immunoglobulin superfamily plays negative roles in effector functions of T cells and B cells, little is known about the function of PD-1 expressed on innate immune cells. In this study, we demonstrate that IL-12 production was greatly suppressed in LPS-stimulated RAW264.7 cells upon PD-1 engagement with B7-H1.Fc fusion protein, and was restored in the presence of antagonistic anti-PD-1 mAb. PD-1-mediated suppression of IL-12 production in LPS-stimulated RAW264.7 cells was mediated by inhibition of Janus N-terminal-linked kinase (JNK) signaling pathway, and to a lesser extent, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway through the recruitment of SHP-2 to PD-1 cytoplasmic tail. B7-H1.Fc-mediated PD-1 engagement also downregulates the expression of co-stimulatory molecules such as CD80, CD86, MHC class I and II proteins in LPS-stimulated RAW264.7 cells. Furthermore, the endocytic activity is enhanced but the allostimulatory capacity is suppressed in LPS-treated RAW264.7 cells upon PD-1 engagement. Taken together, our results reveal a novel function of macrophage PD-1 in the negative regulation of IL-12 synthesis and differentiation into dendritic cell-like cells.

Granulocyte colony-stimulating factor-induced immature myeloid cells inhibit acute graft-versus-host disease lethality through an indoleamine dioxygenase-independent mechanism.

Granulocyte colony-stimulating factor (G-CSF)-mobilized donor graft tissue used for peripheral blood stem cell transplantation contains a large number of immature myeloid cells that suppress alloreactive donor T cells, resulting in an inhibition of acute graft-versus-host disease (GVHD). However, the molecular mechanism underlying the suppressive function of immature myeloid cells is not fully understood. Here, we investigated whether indoleamine 2,3-dioxygenase (IDO) is related to the suppressive mechanism of G-CSF-induced immature myeloid cells (gMCs). We found that Gr-1(+) CD11b(+) cells were highly induced in G-CSF-injected donor graft tissue, which is a phenotype of immature myeloid cells, resulting in an inhibition of acute GVHD lethality by suppressing alloreactive donor T-cell expansion. IDO was not detected in primary isolated gMCs; however, this enzyme was markedly induced after treatment with interferon-gamma (IFN-gamma). This level was significantly higher in IFN-gamma-treated gMCs than in bone marrow myeloid cells, which promote alloreactive T-cell responses. We next investigated the functional role of IDO in gMC-mediated inhibition of acute GVHD lethality. We found no changes in gMC-mediated survival or alloreactive donor T-cell suppression when IDO activity was blocked using 1-methyl tryptophan. In addition, there was no difference in gMC-mediated survival rates between recipients transferred with either wild-type gMCs or IDO(-/-) gMCs. Taken together, our data suggest that gMC-mediated inhibition of lethal acute GVHD is through an IDO-independent mechanism.