Lipopolysaccharide-induced change of ADP-ribosylation of a cytosolic protein in bone-marrow-derived macrophages.
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ABSTRACT:
SUBMITTER: Hauschildt S
PROVIDER: S-EPMC1137782 | biostudies-other | 1994 Jan
REPOSITORIES: biostudies-other
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Project description:A limit to the clinical benefit of radiotherapy is not an incapacity to eliminate tumor cells but rather a limit on its capacity to do so without destroying normal tissue and inducing inflammation. Recent evidence reveals that the inflammasome is essential for mediating radiation-induced cell and tissue damage. In this study, using primary cultured bone marrow-derived macrophages (BMDM) and a mouse radiation model, we explored the role of NLRP3 inflammasome activation and the secondary pyroptosis underlying radiation-induced immune cell death. We observed an increasing proportion of pyroptosis and elevating Caspase-1 activation in 10 and 20?Gy radiation groups. Nlrp3 knock out significantly diminished the quantity of cleaved-Caspase-1 (p10) and IL-1? as well as the proportion of pyroptosis. Additionally, in vivo research shows that 9.5?Gy of radiation promotes Caspase-1 activation in marginal zone cells and induces death in mice, both of which can be significantly inhibited by knocking out Nlrp3. Thus, based on these findings, we conclude that the NLRP3 inflammasome activation mediates radiation-induced pyroptosis in BMDMs. Targeting NLRP3 inflammasome and pyroptosis may serve as effective strategies to diminish injury caused by radiation.
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Project description:Macrophages are phenotypically diverse cells performing a number of functions involved in immunity, inflammation, wound healing, tissue homeostasis and the foreign body reaction. In the latter, the type of biomaterial and the surrounding environment likely have an impact on macrophage phenotype and, subsequently, the severity of the reaction. The objectives for this study were to characterize the phenotype of bone marrow-derived murine macrophages in response to poly(ethylene glycol) (PEG)-based hydrogels, a promising class of materials for cell delivery. Gene expression was used as a measure of phenotype and characterized by IL-1?, TNF-?, iNOS, IL-12?, arginase, VEGF-A, and IL-10. Macrophages were cultured on PEG hydrogels, PEG hydrogels with RGD tethers, and medical grade silicone rubber, a well-characterized biomaterial, up to 96 h in the absence and presence of lipopolysaccharide (LPS) to simulate an inflammatory environment. Macrophage interrogation led to immediate up-regulation (10×) of IL-1? and TNF-? within 4h, followed by an increase in IL-10/IL-12? and a subsequent concomitant decrease in the pro-inflammatory genes by 96 h, suggesting a shift from classically activated to a regulatory phenotype. LPS stimulation led to a stronger early up-regulation of pro-inflammatory genes (e.g. 20-30× for IL-1? and TNF-?), followed by upregulation (4-6×) of arginase, suggesting a shift from an elevated classically activated to a wound healing phenotype. Material type played a significant role in regulating pro-inflammatory genes, which was most pronounced with PEG alone. Overall, our findings indicate that macrophages undergo similar phenotypic changes for the materials tested, but the magnitudes of these responses are highly material dependent.
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Project description:Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the alphaLbeta2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated. Cytohesin-1 enhanced binding of 35S-labeled guanosine 5'-[gamma-thio]triphosphate [35S]GTP[gammaS] or [3H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [35S]GTP[gammaS] bound, accelerated [35S]GTP[gammaS] release to a similar degree in the presence of unlabeled GDP or GTP[gammaS] and to a lesser degree with GDP[betaS]; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.
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Project description:"Experiment looks to identify metabolites in the major metabolic pathways, i.e. glycolysis, tca, urea cycle and ppp along with amino acids."
2018-08-02 | MSV000082689 | GNPS