ABSTRACT
Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.
Subject(s)
Glioma/pathology , Matrix Metalloproteinase 14/metabolism , Microglia/pathology , Animals , Blotting, Western , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Enzyme Precursors/metabolism , Female , Gelatinases/metabolism , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , Male , Matrix Metalloproteinase 14/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/metabolism , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Toll-Like Receptors/metabolism , Tumor Burden , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Inflammatory bowel disease (IBD) results from a breakdown of tolerance towards the indigenous flora in genetically susceptible hosts. Failure of dendritic cells (DC) to interpret molecular microbial patterns appropriately when directing innate and adaptive immune responses is conceivable. Primary (conventional, non-monocyte generated) CD1c(+)CD11c(+)CD14(-)CD16(-)CD19(-) myeloid blood or mucosal dendritic cells (mDC) from 76 patients with Crohn's disease (CD) or ulcerative colitis (UC) in remission, during flare-ups (FU) and 76 healthy or non-IBD controls were analysed by fluorescence activated cell sorter (FACS) flow cytometry and real-time polymerase chain reaction. Cytokine secretion of freshly isolated, cultured and lipopolysaccharide (LPS)-stimulated highly purified mDC (purity >95%) was assessed using cytometric bead arrays (CBA). More cultured and stimulated circulating mDC express CD40 in IBD patients. Stimulated circulating mDC from IBD patients secrete significantly more tumour necrosis factor (TNF)-alpha and interleukin (IL)-8. Toll-like receptor (TLR)-4 expression by mDC was higher in remission and increased significantly in flaring UC and CD patients compared with remission (P < 0.05) and controls (P < 0.001). Fluorochrome-labelled LPS uptake by mDC was evaluated at different time-points over 24 h by measuring mean fluorescence intensity (MFI). Circulating mDC from IBD patients take up more LPS and the uptake begins earlier compared with controls (P < 0.05 in CD-FU and UC-FU at 24 h). The frequency of mucosal mDC (P < 0.05) and the number of CD40 expressing mucosal mDC is significantly greater in UC and CD compared with non-IBD controls (P < 0.001 versus P < 0.01, respectively). Our data suggest an aberrant LPS response of mDC in IBD patients, resulting in an inflammatory phenotype and possibly intestinal homing in acute flares.
Subject(s)
Dendritic Cells/immunology , Inflammatory Bowel Diseases/immunology , Intestinal Mucosa/immunology , Acute Disease , Adult , Antigen Presentation , CD40 Antigens/analysis , Case-Control Studies , Cells, Cultured , Colitis, Ulcerative/immunology , Crohn Disease/immunology , Female , Flow Cytometry , Humans , Immunization , Immunophenotyping , Inflammation , Lipopolysaccharides , Lymphocyte Activation , Male , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Toll-Like Receptor 2/genetics , Toll-Like Receptor 4/genetics , Tumor Necrosis Factor-alpha/analysisABSTRACT
SNARE proteins - rab3A - parietal cells - H+/K+-ATPase When stimulated by histamine, acetylcholine, or gastrin the luminal compartments of oxyntic parietal cells display conspicuous morphological changes. The luminal plasma membrane surface becomes greatly expanded, while the cytoplasmic tubulovesicles are decreased in parallel. Due to these membrane rearrangements the H+/K(+)-ATPase obtains access to the luminal surface, where proton secretion occurs. The stimulation-induced translocation of H+/K(+)-ATPase involves a fusion process. Exocytotic membrane fusion in neurons is achieved by the highly regulated interaction of mainly three proteins, the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 (synaptosomal-associated protein of 25 kDa), also referred to as SNARE proteins. Using immunofluorescence microscopy we analysed the subcellular distribution of neuronal synaptic proteins and rab3A in resting and stimulated parietal cells from pig and rat. In resting cells all synaptic proteins colocalized with the H+/ K(+)-ATPase trapped in the tubulovesicular compartment. After stimulation, translocated H+/K(+)-ATPase showed a typical canalicular distribution. Syntaxin, synaptobrevin, SNAP25 and rab3A underwent a similar redistribution in stimulated cells and consequently localized to the canalicular compartment. Using immunoprecipitation we found that the SNARE complex consisting of synaptobrevin, syntaxin and SNAP25, which is a prerequisite for membrane fusion in neurons, is also assembled in parietal cells. In addition the parietal cell-derived synaptobrevin could be proteolytically cleaved by tetanus toxin light chain. These data may provide evidence that SNARE proteins and rab3A are functionally involved in the stimulation-induced translocation of the H+/K(+)-ATPase.
Subject(s)
Carrier Proteins/metabolism , H(+)-K(+)-Exchanging ATPase/metabolism , Membrane Proteins/metabolism , Multigene Family , Parietal Cells, Gastric/metabolism , rab3A GTP-Binding Protein/metabolism , Animals , Antibodies, Monoclonal , Gastric Mucosa/cytology , Gastric Mucosa/metabolism , Gastrins/pharmacology , Microscopy, Fluorescence , Nerve Tissue Proteins/metabolism , Precipitin Tests , R-SNARE Proteins , Rats , Rats, Wistar , Swine , Synaptosomal-Associated Protein 25ABSTRACT
Stimulation of parietal cells causes fusion of intracellular tubulovesicles with the canalicular plasma membrane thereby increasing the apical membrane area up to tenfold. The presence of the SNARE proteins synaptobrevin, syntaxin1, and SNAP25 in parietal cells and their intracellular redistribution after stimulation suggest a SNARE-mediated mechanism. Here we show that NSF and alpha, beta-SNAPs which are involved in the dissociation of the SNARE complex in neurons also occur in parietal cells exhibiting subcellular distributions similar to the ones obtained for SNARE proteins and for the H+, K(+)-ATPase. More importantly proteolytic cleavage of synaptobrevin by tetanus neurotoxin completely inhibits the cAMP-dependent increase of acid secretion further supporting the crucial role SNARE proteins play in parietal cells.