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Derived Macrophages (derived + macrophage)
Selected AbstractsRole of the leucine-rich repeat domain of cryopyrin/NALP3 in monosodium urate crystal,induced inflammation in miceARTHRITIS & RHEUMATISM, Issue 7 2010Hal M. Hoffman Objective The mechanism by which monosodium urate monohydrate (MSU) crystals intracellularly activate the cryopyrin inflammasome is unknown. The aim of this study was to use a mouse molecular genetics,based approach to test whether the leucine-rich repeat (LRR) domain of cryopyrin is required for MSU crystal,induced inflammation. Methods Cryopyrin-knockout lacZ (Cryo,Z/,Z) mice and mice with the cryopyrin LRR domain deleted and fused to the lacZ reporter (Cryo,LRR Z/,LRR Z) were generated using bacterial artificial chromosome,based targeting vectors, which allow for large genomic deletions. Bone marrow,derived macrophages from Cryo,LRR Z/,LRR Z mice, Cryo,Z/,Z mice, and congenic wild-type (WT) mice were challenged with endotoxin-free MSU crystals under serum-free conditions. Phagocytosis and cytokine expression were assessed by flow cytometry and enzyme-linked immunosorbent assay. MSU crystals also were injected into mouse synovial-like subcutaneous air pouches. The in vivo inflammatory responses were examined. Results Release of interleukin-1, (IL-1,), but not CXCL1 and tumor necrosis factor ,, was impaired in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mouse bone marrow,derived macrophages compared with WT mouse bone marrow,derived macrophages in response to not only MSU crystals but also other known stimuli that activate the cryopyrin inflammasome. In addition, a comparable percentage of MSU crystals taken up by each type of bone marrow,derived macrophage was observed. Moreover, total leukocyte infiltration in the air pouch and IL-1, production were attenuated in Cryo,Z/,Z and Cryo,LRR Z/,LRR Z mice at 6 hours postinjection of MSU crystals compared with WT mice. Conclusion MSU crystal,induced inflammatory responses were comparably attenuated both in vitro and in vivo in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal,induced inflammation in this model. [source] Alteration of RANKL-Induced Osteoclastogenesis in Primary Cultured Osteoclasts From SERCA2+/, Mice,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2009Yu-Mi Yang Abstract RANKL is essential for the terminal differentiation of monocytes/marcrophages into osteoclasts. RANKL induces long-lasting oscillations in the intracellular concentration of Ca2+ ([Ca2+]i) only after 24 h of stimulation. These Ca2+ oscillations play a switch-on role in NFATc1 expression and osteoclast differentiation. Which Ca2+ transporting pathway is induced by RANKL to evoke the Ca2+ oscillations and its specific role in RANKL-mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca2+ ATPase type2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2+/,). The BMD in the tibias of SERCA2+/, mice increased >1.5-fold compared with wildtype mice (WT). RANKL-induced [Ca2+]i oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2+/, bone marrow,derived macrophages (BMMs). Forty-eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2+/, BMMs. In addition, RANKL treatment of SERCA2+/, BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL-mediated induction of SERCA2 plays a critical role in the RANKL-induced [Ca2+]i oscillations that are essential for osteoclastogenesis. [source] Role of the leucine-rich repeat domain of cryopyrin/NALP3 in monosodium urate crystal,induced inflammation in miceARTHRITIS & RHEUMATISM, Issue 7 2010Hal M. Hoffman Objective The mechanism by which monosodium urate monohydrate (MSU) crystals intracellularly activate the cryopyrin inflammasome is unknown. The aim of this study was to use a mouse molecular genetics,based approach to test whether the leucine-rich repeat (LRR) domain of cryopyrin is required for MSU crystal,induced inflammation. Methods Cryopyrin-knockout lacZ (Cryo,Z/,Z) mice and mice with the cryopyrin LRR domain deleted and fused to the lacZ reporter (Cryo,LRR Z/,LRR Z) were generated using bacterial artificial chromosome,based targeting vectors, which allow for large genomic deletions. Bone marrow,derived macrophages from Cryo,LRR Z/,LRR Z mice, Cryo,Z/,Z mice, and congenic wild-type (WT) mice were challenged with endotoxin-free MSU crystals under serum-free conditions. Phagocytosis and cytokine expression were assessed by flow cytometry and enzyme-linked immunosorbent assay. MSU crystals also were injected into mouse synovial-like subcutaneous air pouches. The in vivo inflammatory responses were examined. Results Release of interleukin-1, (IL-1,), but not CXCL1 and tumor necrosis factor ,, was impaired in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mouse bone marrow,derived macrophages compared with WT mouse bone marrow,derived macrophages in response to not only MSU crystals but also other known stimuli that activate the cryopyrin inflammasome. In addition, a comparable percentage of MSU crystals taken up by each type of bone marrow,derived macrophage was observed. Moreover, total leukocyte infiltration in the air pouch and IL-1, production were attenuated in Cryo,Z/,Z and Cryo,LRR Z/,LRR Z mice at 6 hours postinjection of MSU crystals compared with WT mice. Conclusion MSU crystal,induced inflammatory responses were comparably attenuated both in vitro and in vivo in Cryo,LRR Z/,LRR Z and Cryo,Z/,Z mice. Hence, the LRR domain of cryopyrin plays a role in mediating MSU crystal,induced inflammation in this model. [source] Osteopontin deficiency impairs wear debris,induced osteolysis via regulation of cytokine secretion from murine macrophagesARTHRITIS & RHEUMATISM, Issue 5 2010Sadanori Shimizu Objective To investigate the molecular mechanisms underlying particle-induced osteolysis, we focused on osteopontin (OPN), a cytokine and cell-attachment protein that is associated with macrophage chemoattractant and osteoclast activation. Methods We compared OPN protein levels in human periprosthetic osteolysis tissues with those in osteoarthritis (OA) synovial tissues. To investigate the functions of OPN during particle-induced osteolysis in vivo, titanium particles were implanted onto the calvaria of OPN-deficient mice and their wild-type (WT) littermates. Mice were killed on day 10 and evaluated immunohistologically. The effects of OPN deficiency on the secretion of inflammatory cytokines were examined using cultured bone marrow,derived macrophages (BMMs). BMMs from OPN-deficient and WT mice were cultured with titanium particles for 12 hours, and the concentrations of inflammatory cytokines in the conditioned media were measured by enzyme-linked immunosorbent assay. Results Expression of OPN protein was enhanced in human periprosthetic osteolysis tissues as compared with OA synovial tissues. In the particle-induced model of osteolysis of the calvaria, bone resorption was significantly suppressed by OPN deficiency via inhibition of osteoclastogenesis, whereas an inflammatory reaction was observed regardless of the genotype. Results of immunostaining indicated that OPN protein was highly expressed in the membrane and bone surface at the area of bone resorption in WT mice. When BMMs were exposed to titanium particles, the concentration of proinflammatory cytokines, such as tumor necrosis factor ,, interleukin-1, (IL-1,), IL-1,, and IL-6, as well as chemotactic factors, such as monocyte chemoattractant protein 1 and macrophage inflammatory protein 1,, in the conditioned medium were significantly reduced by OPN deficiency. Whereas phagocytic activity of BMMs was not attenuated by OPN deficiency, phagocytosis-mediated NF-,B activation was impaired in OPN-deficient BMMs. These data indicated that OPN was implicated in the development of particle-induced osteolysis via the orchestration of pro-/antiinflammatory cytokines secreted from macrophages. Conclusion OPN plays critical roles in wear debris,induced osteolysis, suggesting that OPN is a candidate therapeutic target for periprosthetic osteolysis. 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