The biological effects of CRP are not attributable to endotoxin contamination: evidence from TLR4 knockdown human aortic endothelial cells

C-reactive protein (CRP) is the prototypic marker of inflammation and a strong predictor of cardiovascular events in humans. There are questions regarding the validity of the biological effects reported for CRP, in spite of adherence to rigorous control measures minimizing endotoxin [lipopolysaccharide (LPS)] contamination in these in vitro studies. In this study, we addressed the key question of endotoxin contamination in CRP preparations using Toll-like receptor 4 (TLR4) knockdown endothelial cells. Human aortic endothelial cells (HAECs) transfected with prevalidated TLR4 small interfering RNA (siRNA) and scrambled siRNA controls were challenged with pleural fluid-derived CRP or LPS for 12-16 h. Secreted interleukin-6 (IL-6), IL-1 beta, IL-8, and plasminogen activator inhibitor-1 (PAI-1) levels and endothelial Nitric oxide synthase (eNOS) activity were determined. TLR4 knockdown in HAECs significantly decreased LPS-induced IL-1 beta, IL-6, and IL-8, whereas the stimulatory effects of CRP were similar in both scrambled control and TLR4 knockdown cells. Furthermore, CRP significantly stimulated PAI-1 levels in both control and TILR4-transfected cells and inhibited eNOS activity, whereas LPS effects were negated in TLR4-transfected cells. The data presented cogently demonstrate and further confirm that the biological effects of CRP on HAECs are independent of LPS and thus are attributable to native protein per se. This is the first study to positively authenticate the significance of earlier in vitro reports on CRP biological effects.