Lipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein–Induced IL-6 Secretion From Human Endothelial Cells In Vitr
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动脉硬化血栓血管生物学 2005年第9期
Departments of Investigative and Cardiac Biology (S.S.N., R.N.W., T.-L.Y) and Protein Chemistry (P.J.M., G.F.S., K.O.J.) GlaxoSmithKline Pharmaceuticals, King of Prussia, Penn
To the Editor,
In a recent article, Taylor et al1 have shown that C-Reactive Protein (CRP), per se, does not activate endothelial cells; rather, lipopolysacchride (LPS) and azide were responsible for the observed cellular activation. We would like to provide our data to further support the conclusion by Taylor and colleagues.
We have tested >10 batches of commercial human CRP preparations from 3 companies (Calbiochem, TriChem, and Sigma). Human saphenous vein endothelial cells (HUVECs, passages 3 to 5) were grown in MCDB-131 medium supplemented with 10% FBS. The cells were incubated with CRP for 24 hours, and the culture supernatants were assessed for IL-6 using the IL-6 ELISA kit (R&D). It was reported previously that CRP induced IL-6 production from HUVECs.2 Our main observations are summarized as follows.
All recombinant human CRP (rCRP, Escherichia coli–derived) that we tested stimulated HUVECs to secrete IL-6 significantly, and all of these CRP preparations contained endotoxin (10 to 100 EU/mL) as measured by the LAL gel clot assay (Associates of Cape Cod).3 The CRP-induced IL-6 secretion was correlated with the level of endotoxin contamination. Moreover, all these CRP preparations lost their ability to activate HUVECs after endotoxin was removed using a detoxi-gel column (Pierce). Furthermore, reintroducing LPS to CRP to a level equivalent to that presented before purification re-established the ability of CRP to stimulate HUVECs (Figure ). The CRP preparation purified from human plasma (Sigma) had low endotoxin levels (<0.31 EU/mL) and did not stimulate HUVECs (up to 100 μg/mL of CRP). However, the CRP purified from human plasma and purchased from TriChem contained high levels of endotoxin (160 EU/mL) and caused a robust production of IL-6 (up to 5 ng/mL). Analysis by size exclusion chromatography and SDS-PAGE suggest that removal of LPS by detoxi-gel column did not result in change in CRP structurally. Taken together, endotoxin contamination plays a confounding role in the interpretation of biologic actions previously attributed to CRP.
Role of LPS in CRP-induced IL-6 production from HUVECs in vitro. HUVECs were incubated for 24 hours with medium alone; rCRP (recombinant human CRP, 1 mg/mL, Calbiochem) containing endotoxin 80 EU/mL; p-rCRP (LPS-removed rCRP, LPS <0.31 EU/mL); or p-rCRP plus LPS. The final concentrations of CRP are indicated in the figure. Supernatants were harvested and analyzed for IL-6. Results are expressed as mean±SEM of experiments performed in triplicate samples. **P<0.01 vs basal (medium alone). EU indicates endotoxin unit.
References
Taylor KE, Giddings JC, van den Berg CW. C-reactive protein-induced in vitro endothelial cell activation is an artifact caused by azide and lipopolysacchride. Arterioscler Thromb Vasc Biol. 2005; 25: 1225–1230.
Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA. Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation. 2002; 105: 1890–1896.
Bacterial endotoxin test, p3349–3350. In: 8th Supplement to USP, XXII-NF-XVII, March 15, 1993. U.S. Phamacopeial Convention, Inc, Rockville, MD.(Sandhya S. Nerurkar; Patr)
To the Editor,
In a recent article, Taylor et al1 have shown that C-Reactive Protein (CRP), per se, does not activate endothelial cells; rather, lipopolysacchride (LPS) and azide were responsible for the observed cellular activation. We would like to provide our data to further support the conclusion by Taylor and colleagues.
We have tested >10 batches of commercial human CRP preparations from 3 companies (Calbiochem, TriChem, and Sigma). Human saphenous vein endothelial cells (HUVECs, passages 3 to 5) were grown in MCDB-131 medium supplemented with 10% FBS. The cells were incubated with CRP for 24 hours, and the culture supernatants were assessed for IL-6 using the IL-6 ELISA kit (R&D). It was reported previously that CRP induced IL-6 production from HUVECs.2 Our main observations are summarized as follows.
All recombinant human CRP (rCRP, Escherichia coli–derived) that we tested stimulated HUVECs to secrete IL-6 significantly, and all of these CRP preparations contained endotoxin (10 to 100 EU/mL) as measured by the LAL gel clot assay (Associates of Cape Cod).3 The CRP-induced IL-6 secretion was correlated with the level of endotoxin contamination. Moreover, all these CRP preparations lost their ability to activate HUVECs after endotoxin was removed using a detoxi-gel column (Pierce). Furthermore, reintroducing LPS to CRP to a level equivalent to that presented before purification re-established the ability of CRP to stimulate HUVECs (Figure ). The CRP preparation purified from human plasma (Sigma) had low endotoxin levels (<0.31 EU/mL) and did not stimulate HUVECs (up to 100 μg/mL of CRP). However, the CRP purified from human plasma and purchased from TriChem contained high levels of endotoxin (160 EU/mL) and caused a robust production of IL-6 (up to 5 ng/mL). Analysis by size exclusion chromatography and SDS-PAGE suggest that removal of LPS by detoxi-gel column did not result in change in CRP structurally. Taken together, endotoxin contamination plays a confounding role in the interpretation of biologic actions previously attributed to CRP.
Role of LPS in CRP-induced IL-6 production from HUVECs in vitro. HUVECs were incubated for 24 hours with medium alone; rCRP (recombinant human CRP, 1 mg/mL, Calbiochem) containing endotoxin 80 EU/mL; p-rCRP (LPS-removed rCRP, LPS <0.31 EU/mL); or p-rCRP plus LPS. The final concentrations of CRP are indicated in the figure. Supernatants were harvested and analyzed for IL-6. Results are expressed as mean±SEM of experiments performed in triplicate samples. **P<0.01 vs basal (medium alone). EU indicates endotoxin unit.
References
Taylor KE, Giddings JC, van den Berg CW. C-reactive protein-induced in vitro endothelial cell activation is an artifact caused by azide and lipopolysacchride. Arterioscler Thromb Vasc Biol. 2005; 25: 1225–1230.
Verma S, Li SH, Badiwala MV, Weisel RD, Fedak PW, Li RK, Dhillon B, Mickle DA. Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. Circulation. 2002; 105: 1890–1896.
Bacterial endotoxin test, p3349–3350. In: 8th Supplement to USP, XXII-NF-XVII, March 15, 1993. U.S. Phamacopeial Convention, Inc, Rockville, MD.(Sandhya S. Nerurkar; Patr)