Figure 1.
Analysis of endotoxin contents by two commercially available endotoxin detection assays.
Endotoxin contamination levels of recombinant protein 1 from suppliers 1 and 2 were measured by EndoZyme and EndoLISA. Results show impurities in 1 µg of recombinant protein 1 from two different suppliers (rec.prot.1 suppl.1 and rec.prot.1 suppl.2). As controls, total amounts of 0.001–2 ng E.coli LPS, as well as solvent (PBS containing 0.1% BSA, undiluted), DC-medium without FBS (Med –FBS) and with FBS (Med + FBS; both diluted 1∶10 in endotoxin-free water) were used. Results show mean values of technical duplicates. n.d., not detectable; n.a., not applicable.
Table 1.
Measured impurities in protein preparations from different suppliers evaluated by LAL tests.
Figure 2.
Activation of NF-κB by endotoxin impurities in commercial preparations of a recombinant protein.
NF-κB activation in HEK293 cells transfected with an NF-κB luciferase reporter plasmid and plasmids encoding LPS-receptor components (TLR4, CD14, MD-2) is shown. Cells were exposed to recombinant protein 1 from supplier 1 or 2, LPS, or solvent, in the amounts stated. 20 h after induction, luciferase activity was measured. Results show mean and standard deviations of four independent experiments.
Figure 3.
Effects of low LPS concentrations on cytokine production in different human immune cells.
A 1×105 THP-1 cells, primary human monocytes, monocyte-derived DCs (moDCs), or CD1c+ DCs were either stimulated with different concentrations of LPS or solvent (PBS/BSA) as a control. After 16 h, supernatants were harvested and analysed by ELISA. Results show means and SD of at least four independent experiments per cell type. B Left panel: Cells were plated and stimulated as described above. After 24 h, supernatants were harvested and analysed for IL-1β expression by ELISA. Results show means and SD of at least five independent experiments per cell type. Right panel: 1×105 monocytes were stimulated as described above. After 6 h, cells were harvested and mRNA levels were measured by qRT-PCR. Results show means and SD of three independent experiments. One-way ANOVA with a Dunnett post-test was performed for each cell type individually. * p<0.05, ** p<0.01, *** p<0.001 compared to the solvent control of the respective cell type.
Figure 4.
Surface marker expression upon stimulation with low LPS concentrations.
Cells were plated and induced as described in Figure 3. After 16 h of induction, cells were harvested and stained for the expression of surface markers using appropriate amounts of fluorescent-labelled antibodies. The median of the fluorescence intensities of 1×104 cells each was recorded by flow cytometry. Fold change values were calculated to compare individual cell types with different compensation settings. Results show mean and SD of at least four independent experiments per cell type. Statistical analysis was performed as described in Figure 3.
Figure 5.
Endotoxin sensitivity correlates with CD14 expression.
A Expression of TLR4 and CD14 on different human immune cells. Cells were plated and induced as described in Figure 3. After 24 h, cells were stained for the expression of TLR4 and CD14 and analysed by flow cytometry. Results show the percentage of fluorescence-positive cells (% pos cells) compared to the respective isotype controls. Statistical analysis was performed as described in Figure 3. B Analysis of CD1c, CD14 and CD19 expression on primary human CD1c+ DCs. 1×105 CD1c+ DCs were stained for the expression of CD1c, CD14 or CD19 either directly after isolation (0 h), or after 24 h culture in 24-well plates containing 1 ml of DC-medium. Cells were then analysed by flow cytometry. One representative donor is shown. Light grey, isotype control; dark grey, respective surface marker staining.