Proinflammatory effects of bacterial recombinant human C-reactive protein are caused by contamination with bacterial products, not by C-reactive protein itself

Abstract

Intravenous administration to human volunteers of a commercial preparation of recombinant human C-reactive protein (CRP) produced in Escherichia coli was recently reported in this journal to induce an acute phase response of serum amyloid A protein (SAA) and of CRP itself, and to activate the coagulation system. The authors concluded that CRP is probably a mediator of atherothrombotic disease. Here we confirm that this recombinant CRP preparation was proinflammatory both for mouse macrophages in vitro and for mice in vivo, but show that pure natural human CRP had no such activity. Furthermore mice transgenic for human CRP, and expressing it throughout their lives, maintained normal concentrations of their most sensitive endogenous acute phase reactants, SAA and serum amyloid P component (SAP). The patterns of in vitro cytokine induction and of in vivo acute phase stimulation by the recombinant CRP preparation were consistent with contamination by bacterial products, and there was 46.6 EU of apparent endotoxin activity per mg of CRP in the bacterial product, compared with 0.9 EU per mg of our isolated natural human CRP preparation. The absence of any proinflammatory activity in natural CRP for macrophages or healthy mice strongly suggests that the in vivo effects of the recombinant preparation observed in humans were attributable to proinflammatory bacterial products and not human CRP.

Figure 1.

Activation by TNF-α production by wild type and different knockout mouse macrophages. A, Stimulation with Sh. flexneri LPS at 100 ng/ml (solid bars) or synthetic BLP at 20 ng/ml (open bars), showing the dependence on TLR4 and TLR2 respectively and the requirement for MyD88 for both, compared with baseline production in medium alone (hatched bars). B, Effect of increasing concentrations of the different human CRP preparations showing complete absence of activity in the natural material (open bars), compared to marked dose dependent stimulation by the recombinant product that required TLR4, consistent with LPS activity. Similar results were obtained regardless of whether or not the recombinant CRP material was dialyzed before testing.

Figure 2.

Activation of NF-κB in THP-1 cells. Sh. flexneri LPS at 50 ng/ml (solid bars) activated significantly only in the presence of both soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP). Natural human CRP at 100 μg/ml (open bars) had no activity alone or in the presence of both soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP). Recombinant bacterial CRP at 100 μg/ml (hatched bars) produced substantial activation in the presence of sCD14 and LBP, consistent with the presence of LPS. Similar results were obtained regardless of whether or not the recombinant CRP material was dialyzed before testing.

Figure 3.

Activation of NF-κB in TLR2 transfected HEK 293 cells. Expression of TLR2 (solid bars) enables dose dependent NF-κB activation by bacterial lipopeptides compared to absence of any effects in control cells transfected with vector alone (open bars). Neither natural nor recombinant CRP preparations produced any activation at all in either TLR transfected or control cells, suggesting absence of lipopeptide. Similar results were obtained regardless of whether or not the recombinant CRP material was dialyzed before testing.