THP-1 cells are widely applied to mimic monocytes in cell culture models. In this study, we compared the cytokine release from THP-1, peripheral blood mononuclear cells (PBMC), monocytes, or whole blood after stimulation with lipopolysaccharide (LPS) and investigated the consequences of different cytokine profiles on human umbilical vein endothelial cell (HUVEC) activation. While Pseudomonas aeruginosa-stimulated (10 ng/mL) THP-1 secreted similar amounts of tumor necrosis factor alpha (TNF-α) as monocytes and PBMC, they produced lower amounts of interleukin(IL)-8 and no IL-6 and IL-10. Whole blood required a higher concentration of Pseudomonas aeruginosa (1000 ng/mL) to induce cytokine release than isolated monocytes or PBMC (10 ng/mL). HUVEC secreted more IL-6 and IL-8 after stimulation with conditioned medium derived from whole blood than from THP-1, despite equal concentrations of TNF-α in both media. Specific adsorption of TNF-α or selective cytokine adsorption from the conditioned media prior to HUVEC stimulation significantly reduced HUVEC activation. Our findings show that THP-1 differ from monocytes, PBMC, and whole blood with respect to cytokine release after stimulation with LPS. Additionally, we could demonstrate that adsorption of inflammatory mediators results in reduced endothelial activation, which supports the concept of extracorporeal mediator modulation as supportive therapy for sepsis.
The extent to which bacterial products from contaminated dialysate enter a patient's blood depends upon the type and permeability of the hemodialysis membrane in use. This study was performed to assess the transfer of pyrogenic substances across both high- and low-flux membranes (DIAPES, Fresenius Polysulfone, Helixone, Polyamide S). All experiments were carried out in the saline-saline model. The dialysate pool was contaminated either with purified lipopolysaccharide (LPS) (250 and 500 EU/mL) or with sterile bacterial culture filtrates (20 EU/mL), and in vitro dialysis was performed under diffusive and convective conditions. A significant transfer of endotoxin was observed for both low- and high-flux DIAPES challenged with either LPS or with bacterial culture filtrates. Under identical conditions, no transfer of endotoxins was detectable across Fresenius Polysulfone and Helixone upon challenge with purified LPS. With bacterial culture filtrates, endotoxin concentrations for Polyamide S and Fresenius Polysulfone were about 10% and 1%, respectively, of those measured for DIAPES, whereas no transfer of endotoxin was detectable for Helixone. Using an alternative assay (induction of interleukin-1 receptor antagonist, IL-1Ra, in whole blood), only the DIAPES membrane showed the passage of cytokine-inducing substances. Thus, when saline is present in both the blood and dialysate compartments (i.e., the situation during predialysis priming procedures), dialysis membranes differ profoundly with respect to their permeability to endotoxins.
microRNAs (miRNAs) play an essential role in inflammation processes including sepsis. This study aimed to identify miRNAs as candidates for therapies that are involved in the innate immune response and to assess their potential functions in the activation of the endothelium. We stimulated THP-1 monocytes with 10 ng/ml LPS for 4 h and used the supernatant for the stimulation of human umbilical vein endothelial cells (HUVEC) or human pulmonary microvascular endothelial cells (HPMEC) for 16 h. miRNA array analysis (of 1,891 miRNAs) identified a 1.5-fold upregulation of miR-146a, miR-146b, and miR-155 in stimulated endothelial cells. HUVEC were transfected with miRNA inhibitors for miR-146a, miR-146b, and miR-155 to investigate the function of these miRNAs in endothelial inflammatory pathways. Inhibition of miR-146a resulted in a diminished release of interleukin (IL)-6 and IL-8 by respective 68% and 55% (P<0.001). Inhibition of miR-146b reduced the expression of IL-6 by 49% (P<0.001). Inhibition of miR-155 reduced the expression of IL-6 and IL-8 by respective 31% (P<0.001) and 14%. The inhibition of miR-146a, miR-146b, and miR-155 reduced the release of HSP10 by 50%, 35%, and 69% (P<0.05), respectively, but did not influence the expression of HSP27 or TXA2. In conclusion, miR-146a, miR-146b, and miR-155 are exerting anti-inflammatory properties by down-regulating IL-6 and IL-8, and influencing the expression of HSP10 in the activated endothelium. We provide evidence for the central role of selected miRNAs in sepsis and their use in the development of small interfering RNA therapeutics to target immune cells and sepsis pathways.
The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory. The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange. The system provides a reproducible, complex 3D in vitro culture system with large cell masses. During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates. The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory. The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities. We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl. 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm). HepG2 cells readily attached to the mcs while the vessel was rotating. Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs. After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built. HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel. We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs. The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.
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