ABSTRACT
Amyloid-ß (Aß) oligomer toxicity is a crucial factor in the development of Alzheimer's disease. Therefore, the aim of therapeutic research is to target the modification of secretase activity, increase Aß degradation, reduce Aß formation, and modulate Aß-induced neuroinflammation. Recently, the p38 MAP kinase inhibitor CNI-1493 has been shown to reduce plaque load and has led to an improvement in memory performance in a transgenic mouse model. We examined the role of CNI-1493 in the microglial inflammatory response to Aß using both a microglia cell line as well as primary microglia isolated from mesocortices. MTT assays were performed to quantify cell viability. FACS analysis was used to measure phagocytosis. We used ELISA to analyse cytokine concentrations in response to CNI-1493 treatment. Western-blot/Dot-blot techniques were used to show the interaction of CNI-1493 with Aß-oligomers as well as to measure apoptosis in microglia cells. RT-PCR was used to analyze secretase expression, and secretase function was determined using fluorimetric assays. CNI-1493 is able to prevent oligomer formation as well as apoptosis in microglia. A significant reduction was found in the Aß-induced release of IL-6 and TNF-α in the presence of CNI-1493. Phagocytosis is an essential Aß removal mechanism and was enhanced by CNI-1493 in primary microglia. CNI-1493 also influenced the α-secretase product C83 with an increase in the treated cells, while a simultaneous reduction in Aß secretion was also observed. We hypothesize that CNI-1493 not only reduces neuroinflammation and consequent neurodegeneration, but also leads to a shift in AßPP-processing towards the non-amyloidogenic pathway. Therefore, CNI-1493 is a promising candidate for the treatment of AD.
Subject(s)
Amyloid beta-Peptides/toxicity , Hydrazones/pharmacology , Immunosuppressive Agents/pharmacology , Microglia/drug effects , Peptide Fragments/toxicity , Animals , Apoptosis/drug effects , Caspase 12/metabolism , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Embryo, Mammalian , Mice , Phagocytosis/drug effects , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Subcellular Fractions/drug effects , Subcellular Fractions/metabolismABSTRACT
BACKGROUND: Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. RESULTS: Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. CONCLUSION: Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition.
Subject(s)
Contact Inhibition/genetics , Glioblastoma/pathology , Intramolecular Oxidoreductases/genetics , Macrophage Migration-Inhibitory Factors/genetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Contact Inhibition/drug effects , Disease Progression , Gene Knockdown Techniques , Gene Targeting , Glioblastoma/genetics , Glioblastoma/metabolism , Humans , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/immunology , Intramolecular Oxidoreductases/metabolism , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/immunology , Macrophage Migration-Inhibitory Factors/metabolism , RNA, Small Interfering/pharmacology , TransfectionABSTRACT
Antiviral or antitumor immunity requires activation of cytotoxic CD8+ T cells by dendritic cells, which present viral or tumor antigens on major histocompatibility complex (MHC) class I molecules. The intracellular mechanisms facilitating MHC class I-restricted presentation of extracellular antigens ('cross-presentation') are unclear. Here we demonstrate that cross-presentation of soluble antigen occurred in an early endosomal compartment distinct from the endoplasmic reticulum where endogenous antigen is loaded onto MHC class I. Efficient cross-presentation required endotoxin-induced, Toll-like receptor 4- and signaling molecule MyD88-dependent relocation of the transporter associated with antigen processing, essential for loading of MHC class I, to early endosomes. Transport of cross-presented antigen from endosomes to the cell surface was inhibited by primaquine, which blocks endosomal trafficking. Thus, cross-presentation is spatially and mechanistically separated from endogenous MHC class I-restricted antigen presentation and is biased toward antigens containing microbial molecular patterns.
Subject(s)
Dendritic Cells/immunology , Histocompatibility Antigens Class I/immunology , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/immunology , ATP-Binding Cassette Transporters/metabolism , Animals , Antigen Presentation , Antigens/immunology , Antigens/metabolism , Cross-Priming , Dendritic Cells/metabolism , Endosomes/metabolism , Histocompatibility Antigens Class I/metabolism , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Mannose Receptor , Mannose-Binding Lectins/deficiency , Mannose-Binding Lectins/genetics , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/immunology , Ovalbumin/immunology , Ovalbumin/metabolism , Primaquine/pharmacology , Protein Subunits/immunology , Protein Transport/drug effects , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Solubility , T-Lymphocytes, Cytotoxic/immunology , Toll-Like Receptor 4/immunologyABSTRACT
The mechanisms that allow antigen-presenting cells (APCs) to selectively present extracellular antigen to CD8+ effector T cells (cross-presentation) or to CD4+ T helper cells are not fully resolved. We demonstrated that APCs use distinct endocytosis mechanisms to simultaneously introduce soluble antigen into separate intracellular compartments, which were dedicated to presentation to CD8+ or CD4+ T cells. Specifically, the mannose receptor supplied an early endosomal compartment distinct from lysosomes, which was committed to cross-presentation. These findings imply that antigen does not require intracellular diversion to access the cross-presentation pathway, because it can enter the pathway already during endocytosis.
