Pivotal advance: The pattern recognition receptor ligands lipopolysaccharide and polyinosine-polycytidylic acid stimulate factor B synthesis by the macrophage through distinct but overlapping mechanisms.

Kaczorowski DJ, Afrazi A, Scott MJ, Kwak JH, Gill R, Edmonds RD, Liu Y, Fan J, Billiar TR.
Source: J Leukoc Biol
Publication Date: (2010)
Issue: 88(4): 609-18
Research Area:
Basic Research
Cells used in publication:
Endothelial, umbilical vein, human (HUVEC)
Species: human
Tissue Origin: vein
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-?B 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-?B. 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-?B 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.