The experiments presented here were performed with primary human OBs only, whereas Chhanas studies were carried out mostly with murine MC3T3 E1 cells, and the only viability data with human primary OBs of the published Mdm2 report used the MTT assay, which is, at best, an assay evalu ating cell proliferation and that requires controlling several important parameters, to be an indirect test of cell viability. Moreover, in the present study, we evaluate only PI incorporation by OBs, which repre sents a useful quantification of necrotic and late apoptotic cells. Interestingly, although OB prolif eration is reduced by MSU, their catabolic functions are activated because they are always alive after 7 days of culture.

The absence of degradation of MSU by these nonprofessional phagocytes was corroborated Inhibitors,Modulators,Libraries with the findings of MSU directly encrusted in the ir regular matrix of gouty lesions of bone. Although visualization Inhibitors,Modulators,Libraries of MSU inside vacuoles was de layed for up to 24 hours, and NLRP3, which precedes the cleavage of LC3 I into LC3 II, appeared within 3 hours in MSU stimulated OB, intracellular signaling indicated a rapid activation of both autophagy and phagocytosis. Moreover, the process of phagocytosis ap peared an absolute necessity for subsequent autophagy of MSU, as shown by the absence of MSU autophagy secondarily to phagocytosis blockade. These sequences of phagocytosis followed by autophagy seem logical, be cause autophagy is aimed at destroying intracellular par ticles, whereas phagocytosis, also aimed at degrading foreign particles, is the process that will internalize extracellular particles.

However, phagocytosis could have been sufficient to destroy MSU. Inhibitors,Modulators,Libraries Interestingly, MSU in the presence of OBs, nonprofessional phagocytes, can act as a danger signal and trigger the autophagy process through the rapid induction of NLRP3 to complete the degradation of MSU. It has been reported that autoph agy participates in degrading extracellular microorgan isms linking autophagy to phagocytosis. It is also important to stress that NLRP3 activated by MSU in OBs does not engage the inflammasome signaling path ways, as it does in professional phagocytes, because expression of the adaptor ASC was not increased, and no activation of caspase 1 was detected in MSU stimulated OBs. Our results demonstrate that NLRP3 has an inflammasome independent, cell intrinsic effect in OBs ingesting MSU microcrystals.

MSU interaction with OBs also seems particularly ori ginal at the level of kinetics and regulation of phagocyt osis. First, engulfment of MSU by OBs is related to a process of phagocytosis, because cytochalasin D blocked entirely MSU internalization, whereas colchicine, an in hibitor of microtubule polymerization, had no effect. OBs can ingest various foreign particles like MSU, titan ium, Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries latex beads, or microorganisms like Escherichia www.selleckchem.com/products/Tipifarnib(R115777).html coli or Candida albicans.