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• W2002256497 abstract "HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 25, No. 9Lipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein–Induced IL-6 Secretion From Human Endothelial Cells In Vitro Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBLipopolysaccharide (LPS) Contamination Plays the Real Role in C-Reactive Protein–Induced IL-6 Secretion From Human Endothelial Cells In Vitro Sandhya S. Nerurkar, Patrick J. McDevitt, Gilbert F. Scott, Kyung O. Johanson, Robert N. Willette and Tian-Li Yue Sandhya S. NerurkarSandhya S. Nerurkar 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 Search for more papers by this author , Patrick J. McDevittPatrick J. McDevitt 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 Search for more papers by this author , Gilbert F. ScottGilbert F. Scott 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 Search for more papers by this author , Kyung O. JohansonKyung O. Johanson 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 Search for more papers by this author , Robert N. WilletteRobert N. Willette 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 Search for more papers by this author and Tian-Li YueTian-Li Yue 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 Search for more papers by this author Originally published1 Sep 2005https://doi.org/10.1161/01.ATV.0000175753.82842.ccArteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:e136To 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. Download figureDownload PowerPointRole 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.1 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.LinkGoogle Scholar2 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.LinkGoogle Scholar3 Bacterial endotoxin test, p3349–3350. In: 8th Supplement to USP, XXII-NF-XVII, March 15, 1993. U.S. Phamacopeial Convention, Inc, Rockville, MD.Google Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Jia Z, Li H, Liang Y, Potempa L, Ji S and Wu Y (2018) Monomeric C-Reactive Protein Binds and Neutralizes Receptor Activator of NF-κB Ligand-Induced Osteoclast Differentiation, Frontiers in Immunology, 10.3389/fimmu.2018.00234, 9 Kapetanovic R, Bokil N and Sweet M (2015) Innate immune perturbations, accumulating DAMPs and inflammasome dysregulation: A ticking time bomb in ageing, Ageing Research Reviews, 10.1016/j.arr.2015.02.005, 24, (40-53), Online publication date: 1-Nov-2015. Vilahur G and Badimon L (2015) Biological actions of pentraxins, Vascular Pharmacology, 10.1016/j.vph.2015.05.001, 73, (38-44), Online publication date: 1-Oct-2015. Halford M, He Y and Stacker S (2014) Expression and purification of bioactive, low-endotoxin recombinant human vitronectin, BioTechniques, 10.2144/000114181, 56:6, (331-333), Online publication date: 1-Jun-2014. Montecucco F, Noble S and Mach F (2012) Inflammatory Biomarkers Inflammation and Atherosclerosis, 10.1007/978-3-7091-0338-8_24, (481-495), . Grammer T, Marz W, Renner W, Bohm B and Hoffmann M (2008) C-reactive protein genotypes associated with circulating C-reactive protein but not with angiographic coronary artery disease: the LURIC study, European Heart Journal, 10.1093/eurheartj/ehn191, 30:2, (170-182) Khuseyinova N and Koenig W (2007) C-Reactive Protein and Other Inflammatory Markers in Cardiovascular Disease Therapeutic Lipidology, 10.1007/978-1-59745-533-6_5, (69-112), . Khuseyinova N and Koenig W (2006) Biomarkers of outcome from cardiovascular disease, Current Opinion in Critical Care, 10.1097/01.ccx.0000244119.16377.75, 12:5, (412-419), Online publication date: 1-Oct-2006. Casas J, Shah T, Cooper J, Hawe E, McMahon A, Gaffney D, Packard C, O'Reilly D, Juhan-Vague I, Yudkin J, Tremoli E, Margaglione M, Di Minno G, Hamsten A, Kooistra T, Stephens J, Hurel S, Livingstone S, Colhoun H, Miller G, Bautista L, Meade T, Sattar N, Humphries S and Hingorani A (2006) Insight into the nature of the CRP–coronary event association using Mendelian randomization, International Journal of Epidemiology, 10.1093/ije/dyl041, 35:4, (922-931), Online publication date: 1-Aug-2006. September 2005Vol 25, Issue 9 Advertisement Article InformationMetrics https://doi.org/10.1161/01.ATV.0000175753.82842.ccPMID: 16127022 Originally publishedSeptember 1, 2005 PDF download Advertisement" @default.
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