Modulation of mediators derived from whole blood or monocytic cells stimulated with lipopolysaccharide reduces endothelial cell activation

Introduction

Modulation of inflammatory mediators with specific or selective adsorbents may represent a promising supportive therapy for septic patients. The aims of this study were to modulate mediator concentrations from lipopolysaccharide (LPS)-stimulated whole blood or monocytic THP-1 cells with specific or selective adsorbents and to compare the influence on endothelial cell activation.

Methods

Whole blood or THP-1 cells (1 × 10⁶ cells per ml medium containing 10% human plasma) [1] were stimulated with 10 ng/ml LPS from Escherichia coli for 4 hours. Mediator modulation was performed with either a specific adsorbent for TNFα which was based on sepharose particles functionalized with anti-TNFα antibodies, or with a selective albumin-coated polystyrene divinylbenzene copolymer (PS-DVB) [2]. Human umbilical vein endothelial cell (HUVEC) activation was monitored for 15 hours by measuring secretion of IL-6 and IL-8, as well as surface expression of the adhesion molecules ICAM-1 and E-selectin.

Results

Conditioned media derived from whole blood (CMB) or THP-1 cells (CMT) both contained approximately 1,300 pg/ml TNFα which is known to be an important stimulator for HUVEC [1, 2]. However, CMB led to a significantly higher HUVEC activation as compared to CMT, as indicated by increased secretion of IL-6 and IL-8 (IL-6: 52,000 vs. 2,000 pg/ml; IL-8: 295,000 vs. 43,000 pg/ml), as well as significantly increased E-selectin surface expression (50 vs. 12 mean fluorescence intensity for CMP and CMT, respectively). Adsorption of inflammatory mediators from the conditioned medium of whole blood or THP-1 cells either with the specific TNFα adsorbent or with the selective PS-DVB beads resulted in decreased endothelial cell activation, as shown by statistically significant reduction of IL-6 and IL-8 secretion from HUVEC, as well as statistically significant reduction of surface expression of the adhesion molecules ICAM-1 and E-selectin. The reduction of HUVEC activation was more pronounced when applying the selective adsorbent showing that the modulation of more than one cytokine is more effective than removing TNFα alone.

Conclusion

Inflammatory mediator modulation with specific or selective adsorbents reduces endothelial cell activation and thus may support the development of new therapies for sepsis.