Background Mouse models are useful in analysing pathomechanisms of atherosclerosis. LPS (lipopolysaccharide) given systemically aggravates the development of atherosclerotic plaques in LDLR (Low-Density Lipoprotein Receptor)-deficient (LDLR−/−) mice, involving Factor B (an alternative complement pathway protease).1 LPS binds to lipoproteins, which are pathologically elevated in LDLR−/−, and this binding reduces LPS toxicity.2 We investigated the cellular and humoural contribution of complement properdin (prop) to LPS-mediated cell signalling in dependence of LDLR.
Materials and Methods LDLR−/−propKO were obtained by crossing LDLR−/− with UoL properdin-KO mice. For the humoural assay, RAW264.7 (mouse macrophages) were incubated with LPS (from cell wall of Escherichia coli, serotype O111:B4, Alexis; 1 ng/ml, 20 min, after optimisation). LPS and genotypically different mouse sera (LDLR−/−propWT, LDLR+/+propWT, LDLR−/−propKO, LDLR+/+propKO) were pre-incubated for 30 min before addition to RAW264.7 cells. For the cellular assay, splenocytes were prepared from different mouse genotypes and stimulated with LPS (100 ng/ml, 20 min). Cell lysates were analysed using GAPDH (Millipore), IkB (Santa Cruz) and phospho-p38 (Cell Signalling) antibodies. Specific bands were evaluated densitometrically.
Results LPS-mediated signalling is enhanced in the absence of properdin: preincubation with LPS of serum from LDLR−/−propKO leads to increased NF-κB signalling (approximately twofold) compared to preincubation with LPS of serum from LDLR+/+propWT. Stimulation with LPS of splenocytes from LDLR−/−propKO resulted in increased signalling (approximately fourfold) compared to stimulation of splenocytes from LDLR+/+propWT. LDLR−/− shows enhanced LPS-mediated stimulation compared with LDLR+/+ (consistent with3).
Conclusion We have discovered a properdin-dependent phenotype of LPS-mediated signalling. Properdin may modify the contribution of LPS to hyperlipoproteinemia-induced atherosclerosis.