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.

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.

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.

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.

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.

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.

Interferon-sensitive response element (ISRE) is mainly responsible for IFN-alpha-induced upregulation of programmed death-1 (PD-1) in macrophages.

Programmed death-1 (PD-1), an immunoinhibitory receptor, is upregulated in T cells, B cells, NKT cells, and monocytes upon activation. More specifically, T-cell-associated PD-1 is critically important for maintaining peripheral tolerance through the PD-1-B7-H1 pathway. However, the physiological role of macrophage-associated PD-1 remains unclear. We addressed the molecular mechanism underlying the regulation of PD-1 expression on macrophages in response to IFN-alpha. Based on a luciferase assay using promoter constructs, we found that the promoter region located between -1090 and -1105 nucleotides from the translational start site is essential for PD-1 expression. Electrophoretic mobility-shift assay and site-directed mutagenesis revealed that interferon-sensitive responsive element (ISRE) and STAT1 and STAT2 are primarily responsible for the constitutive expression of PD-1, as well as for the IFN-alpha-mediated upregulation of PD-1. In addition, AG490, a Janus-activated kinase/signal transducer and activator of transcription (JAK/STAT) inhibitor, markedly abolished the responsiveness of bone marrow-derived macrophages (BMM) to IFN-alpha. Our findings support the essential roles of ISRE, STAT1, and STAT2 in the regulation of constitutive and IFN-alpha-mediated PD-1 expression in macrophages.

Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation.

Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.