Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide

Abstract

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.

Figure 1

Scheme of the cell culture model. THP-1 cells, peripheral blood mononuclear cells (PBMC), monocytes, or whole blood are stimulated with lipopolysaccharide (LPS). The harvested conditioned medium containing LPS and cytokines is treated with adsorbents to modulate mediators of inflammation and is subsequently used to stimulate human umbilical vein endothelial cells (HUVEC).

Figure 2

Cytokine release upon stimulation of THP-1, PBMC, and monocytes with lipopolysaccharide. Cultures of one million cells per mL of medium containing 10 vol% plasma were stimulated with 10 ng per mL of LPS from P. aeruginosa (black circles). Unstimulated cultures served as control (white circles). Concentrations of TNF-α, IL-6, IL-8, IL-10, and IL-1beta are given as mean ± SD (n = 3).

Figure 3

Effect of conditioned media on HUVEC. Conditioned media (white bars) derived from an 8 h stimulation of THP-1, PBMC, or monocytes with LPS were applied onto HUVEC and the cytokine release was measured. Concentrations of IL-6, IL-8, IL-10 and TNF-α are expressed as mean ± SD (n = 3). Black bars indicate control media without LPS.

Figure 4

Effect of LPS on cytokine secretion from whole blood. Freshly drawn blood was stimulated with 10 or 1000 ng/mL LPS from P. aeruginosa or E. coli. Samples without LPS served as control. Concentrations of TNF-α, IL-1β, IL-6, IL-8, and IL-10 are expressed as mean ± SD (n = 3).

Figure 5

Effect of mediator modulation on HUVEC activation. Whole blood was stimulated with 100 ng/mL LPS from E. coli for 4 h and diluted tenfold with M199 to obtain conditioned medium derived from blood (CMB). THP-1 cells were stimulated with 10 ng/mL LPS from E. coli for 4 h in medium M199 supplemented with 10 vol% human plasma to yield conditioned medium derived from THP-1 cells (CMT). HUVEC were stimulated with CMB and CMT, respectively. Concentrations of TNF-α, IL-6 and IL-8 prior to mediator modulation (CM) and after specific TNF-α adsorption or selective cytokine adsorption (PS-DVB) are shown. Data are given as mean of three experiments ± standard deviation. Significance was accepted at P ≤ 0.05. n.s.: not significant.

Figure 6

Effect of mediator modulation on adhesion molecule expression. The experimental setup was identical to Figure 5. HUVEC surface expression of the adhesion molecules E-selectin (a) and ICAM-1 (b) is expressed as mean fluorescent intensity (mfi) minus basal expression of adhesion molecules on HUVEC. Data are given as mean of three experiments ± standard deviation. Significance was accepted at P ≤ 0.05. n.s.: not significant.