title: Pivotal advance: The pattern recognition receptor ligands lipopolysaccharide and polyinosine-polycytidylic acid stimulate factor B synthesis by the macrophage through distinct but overlapping mechanisms.
abstract: TLRs and complement are critical to the host response in sepsis, trauma, and ischemia/reperfusion. We hypothesize that TLR stimulation leads to synthesis and release of complement components by macrophages, an important source of extrahepatic complement. RAW264.7 macrophages or peritoneal macrophages from WT and TLR4-, TLR3-, TRIF-, or MyD88-deficient mice were cultured under standard conditions. In some experiments, cells were pretreated with inhibitors of MAPKs or a NF-κB inhibitor. Cells were stimulated with TLR ligands at known stimulatory concentrations. Intratracheal and i.p. injections were also performed in mice. RT-PCR, Western blotting, and immunocytochemistry were used for analysis. Using a RT-PCR-based panel, we demonstrate that of 18 complement components tested, factor B of the alternative pathway is the most robustly up-regulated complement component in macrophages in response to LPS. This up-regulation results in release of factor B into the media. Up-regulation of factor B by LPS is dependent on TLR4, TRIF, JNK, and NF-κB. A screen of other TLR ligands demonstrated that stimulation with poly I:C (dsRNA analog) also results in up-regulation of factor B, which is dependent on JNK and NF-κB but independent of TLR3 and TRIF. Up-regulation of factor B is also observed after intratracheal and i.p. injection of LPS or poly I:C in vivo. PRR stimulation profoundly influences production and release of factor B by macrophages. Understanding the mechanisms of PRR-mediated complement production may lead to strategies aimed at preventing tissue damage in diverse settings, including sepsis, trauma, and ischemia/reperfusion.
authors.affiliation: Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kaczorowski
authors.forename: Amin
authors.initials: A
authors.lastname: Afrazi
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Scott
authors.forename: Joon H
authors.initials: JH
authors.lastname: Kwak
authors.forename: Roop
authors.initials: R
authors.lastname: Gill
authors.forename: Rebecca D
authors.initials: RD
authors.lastname: Edmonds
authors.forename: Yujian
authors.initials: Y
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Fan
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
mesh_terms: D000818:Animals; D015153:Blotting, Western; D011415:Complement Factor B / Q000096:biosynthesis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D007369:Interferon Inducers / Q000276:immunology; D008024:Ligands; D008070:Lipopolysaccharides / Q000276:immunology*; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D011070:Poly I-C / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D051193:Toll-Like Receptors / Q000276:immunology* / Q000378:metabolism
chemical_list: D007369:Interferon Inducers; D008024:Ligands; D008070:Lipopolysaccharides; D051193:Toll-Like Receptors; D011415:Complement Factor B; D011070:Poly I-C