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ro,G CSF and CCL2MCP 1 relative to saline injected wild variety mice.A similar boost in serum levels of IL 6,CXCL10IP 10,CXCL1Gro and CCL2MCP 1 was evident in doxorubi cin treated IL 1R mice relative to control mice.In contrast,serum levels of IL AZD2858 1B,TNF and G CSF had been not considerably elevated in doxorubicin treated IL 1R deficient mice relative to their sham injected counterparts.To determine whether there was a statistically considerable interaction amongst drug and genotype,the effect of IL 1R deficiency on the doxo rubicin induced inflammatory response was determined by 2 x 2 ANOVA.A considerable drug x genotype interaction on serum levels of IL 6,and G CSF was observed following Bonferroni correction for numerous comparisons but not for the other serum analytes examined.
Although doxorubicin improved serum levels of IL 6 in both wild variety and IL 1R deficient mice,the magnitude on the response was roughly 2 fold greater in magnitude AZD2858 in wild variety mice than in IL 1R deficient IU1 mice.The effect of IL 1 defi ciency on doxorubicin induced GCSF levels was especially striking.Although serum levels of G CSF had been 10 fold higher in wild variety doxorubicin treated mice relative to controls,there was no observable effect of doxorubicin on levels of GCSF in IL 1R deficient mice.Doxorubicin induced IL 1B release from primed bone mar row derived macrophages.Many different agents such as asbestos,silicates and uric acid crystals are unable to induce expression of IL 1B in cultured macrophages.
However,when these agents are added to macrophages that Neuroblastoma have been previously primed by LPS to express pro IL 1B,the pro IL 1B is processed by caspase 1 and secreted as active IL 1B through activation on the inflammasome.25,29,30 To determine whether doxorubicin would induce the expres sion and release of IL 1B,LPS primed and unprimed BMDM had been exposed to doxorubicin for 8 h.Cell lysates and media had been analyzed for both pro IL 1B and mature IL 1B,respectively.In unprimed BMDM,doxo rubicin failed to induce expression of pro IL 1B or secretion of IL 1B into the medium.In contrast,LPS primed BMDM exposed to doxorubicin exhibited a dose dependent boost in expression of pro IL IU1 1B.LPS primed macrophages exposed to doxorubicin also showed improved release of IL 1B.Measured by ELISA,the release of IL 1B from LPS primed macrophages was maximal following exposure to AZD2858 10 uM of doxorubicin and was decreased slightly in cells exposed to 25 and 100 uM doxorubicin,respectively.
These data demonstrate that doxorubicin was unable to induce the expression of pro IL 1B in naive BMDM,but that doxorubicin was powerful in mediating both improved expression of pro IL 1B and also the release of IL 1B when added to IU1 LPS primed BMDM.Daunorubicin induced IL 1B release from primed BMDM.Daunorubicin is an anthracycline that's believed to act by similar mechanisms as doxorubicin.31 To determine whether daunorubi cin would act like doxorubicin in mediating the release of IL 1B,LPS primed or unprimed BMDM had been exposed to daunorubicin for 8 h.As with doxorubicin,daunoru bicin failed to induce pro IL 1B in unprimed BMDM or to release IL 1B into the medium.Exposure of LPS primed BMDM to doses of daunoru bicin greater than 0.
25 uM resulted in improved expression of pro IL 1B.Release of IL 1B from LPS primed macrophages was maximal following exposure to 1 or 2.5 uM daunorubicin.These data demon strate that daunorubicin,like doxorubicin,mediated the release of AZD2858 IL 1B LPS primed BMDM.Doxorubicin induced IL 1B release demands ASC,cas pase 1 and NLRP3.To determine if BMDM respond to doxorubicin through formation on the NLRP3 inflamma some,we tested whether the doxorubicin induced release of IL 1B from wild variety BMDM would differ quantitatively from BMDM deficient in ASC,caspase 1 or NLRP3.LPS primed or unprimed BMDM had been exposed to doxorubicin for 8 h,at which time cell lysates and media had been analyzed for pro IL 1B and mature IL 1B,respectively.
Doxorubicin failed to induce expression of pro IL 1B or release of IL 1B from all four strains of unprimed BMDM.In all four strains of BMDM that had previ ously been primed by LPS,exposure to doxorubicin improved the expression of pro IL 1B.As expected,doxorubi cin induced the release of IL 1B from LPS primed wild variety BMDM.On the other hand,the release of IL 1B from LPS primed IU1 BMDM exposed to doxorubicin was considerably suppressed in each on the mutant strains of BMDM.These data suggest that ASC,caspase 1 and NLRP3 are each necessary for doxorubicin to mediate the processing and release of IL 1B from BMDM.Activation on the NLRP3 inflammasome leads to the pro cessing and release of caspase 1 from cells.32,33 To further verify that NLRP3 was necessary for inflammasome activation,primed or unprimed WT and NLRP3 BMDM had been exposed to 10 uM doxorubicin for 8 h and also the release of p10 caspase 1 within the medium was measured.Doxorubicin induced the release of p10 caspase 1 in wt cells,but not in cells deficient in NLRP3,demonstrating that NLRP3 was necessary for