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28 LDLR and properdin cooperate in LPS-mediated cell signalling
  1. L Francescut1,
  2. S Byrne1,
  3. T Steiner2,
  4. S Francis2,
  5. C Stover1
  1. 1Department of Cardiovascular Science, University of Sheffield Medical School, Sheffield, UK

Abstract

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.

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