title: Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.
abstract: Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Harald
authors.initials: H
authors.lastname: Schwarz
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Schmittner
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Albert
authors.initials: A
authors.lastname: Duschl
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Jutta
authors.initials: J
authors.lastname: Horejs-Hoeck
mesh_terms: D018949:Antigens, CD1 / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000737:chemistry / Q000276:immunology*; D004731:Endotoxins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*
chemical_list: D018949:Antigens, CD1; C437944:CD1C protein, human; D004731:Endotoxins; D006023:Glycoproteins; D008070:Lipopolysaccharides; D011994:Recombinant Proteins