Lipopolysaccharide is required for leukocyte adhesion to Toraymyxin^® filters used in the treatment of sepsis

Introduction

Extracorporeal hemoperfusion with polymyxin B is a novel septic treatment, shown to improve hemodynamics, organ dysfunction, and mortality through the removal of circulating lipopolysaccharide (LPS). This therapy can also remove activated leukocytes, which likely contributes to reduced inflammation and improved patient outcome; however, the mechanistic role of LPS in the removal of leukocytes remains unclear.

Objective

To determine whether the presence of LPS and/or activation of leukocytes by LPS alters their ability to bind to polymyxin-bound filters used for extracorporeal hemoperfusion of septic patients.

Methods

Toraymyxin^® filters were opened under sterile conditions and 2 cm² sections were incubated for 2 hours under various conditions. Experiment 1: filters were exposed to (1) whole blood collected from a health volunteer, (2) blood with 700 ng/ml LPS (Escherichia coli 0127:B8), or (3) blood pre-incubated for 2 hours in 700 ng/ml LPS. Experiment 2: filters were pre-exposed to LPS then incubated with blood alone or blood with LPS. Experiment 3: filters were exposed to blood containing increasing LPS concentrations (1 pg/ml to 500 ng/ml) or TNFα (15 pg/ml to 10 ng/ml). In all experiments, following incubation, filters were washed, stained (methylene blue + eosin) and the number of adhered leukocytes were counted by light microscopy. Endotoxin activity of the collected whole blood in both the absence and presence of LPS was determined by an endotoxin activity assay (EAA™).

Results

The presence of LPS significantly increased (3.77 ± 0.54-fold, P = 0.005) the number of adhered leukocytes to Toraymyxin^® filters. Moreover, pre-incubation of the blood with LPS, to activate inflammatory cells, further increased leukocyte adhesion (7.59 ± 1.08-fold increase vs. control, P = 0.002, or vs. non-incubated LPS, P = 0.03). Pre-exposure of Toraymyxin^® filters to LPS versus vehicle control increased leukocyte adhesion, both for blood alone (7.60 ± 1.51-fold increase, P = 0.004) or blood incubated with LPS (24.43 ± 5.32-fols vs. 7.59 ± 1.08-fold increase, P = 0.019). Moreover, while the presence of TNFα or low levels of LPS did not induce leukocyte binding to Toraymyxin^® filters, increasing LPS concentrations induced a dose-dependent increase in adhesion (Figure 1). Sterile blood was confirmed by EAA to have low endotoxin activity (EAA™ <0.3), while blood containing 700 ng/ml LPS had high endotoxin activity (EAA™ = 0.8).

Figure 1

*, **, *** = versus all other groups, P < 0.05.

Conclusions

While leukocyte activation by LPS increases their adhesion to Toraymyxin^® filters, the activation of leukocytes by TNF did not alter binding, indicating the essential need for the presence of LPS possibly as a bridging molecule in the mechanism responsible for the removal of leukocytes during extracorporeal hemoperfusion with Toraymyxin^® filters.