Dose effects of LPS on neutrophils‐ in a whole blood flow cytometric assay of phagocytosis and oxidative burst

Abstract

Whole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2',7' dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial lipopolysaccharide (LPS) on phagocytosis.Blood from healthy donors was preincubated with log doses of bacterial LPS B (0.1-1,000 ng/ml) or sterile pyrogen-free saline at 37°C from 0-120 minutes. LPS increased both P and OB in a dose-dependent manner (up to 62 and 121%, respectively) at all time points tested, and this effect on P and OB could be detected even with no preincubation. This LPSinduced phagocytic activity could be blocked by the addition of polymyxin B (10 pglml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA at doses > 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re-emphasize the necessity of using pyrogen-free reagents in any study of neutrophil function. 0 1992 Wiley-Liss, Inc.

Neutrophil dysfunction in alcoholic hepatitis superimposed on cirrhosis is reversible and predicts the outcome

“…As endotoxin is known to be elevated in patients with alcoholic liver disease, we explored whether this transmissible factor in the patients' plasma imparting the functional disturbance in neutrophils was endotoxin. Endotoxin not only is a priming agent 6 but has been reported to fully activate neutrophils 12,13 by up-regulating the NADPH oxidase assembly. 32 The highest levels of endotoxin reported in patients with liver disease are found in portal venous blood, 33,34 highlighting the importance of the bowel and altered gut permeability as a possible source of endotoxin.…”

“…9 Endotoxin is known to be elevated in patients with AH 10 and has the ability to both prime 6,11 and activate neutrophils. 12,13 The clarification of this controversy has important clinical implications. Current strategies for treating AH include the administration of immunosuppressive agents such as corticosteroids and, more recently, anti-tumor necrosis factor (TNF)␣ strategies, which may further potentiate susceptibility to infection.…”

Role of MyD88 in Diminished Tumor Necrosis Factor Alpha Production by Newborn Mononuclear Cells in Response to Lipopolysaccharide

“…In addition, LPS can interact with cells via at least two pathways that are either dependent or independent of plasma proteins (5,11,12,30,47,52,53). The two pathways of LPS activation may serve different roles in gram-negative bacterial infection.…”

ICAM-1–targeted thrombomodulin mitigates tissue factor–driven inflammatory thrombosis in a human endothelialized microfluidic model

“…The latter was induced by 90-minute preincubation of the WB with 50 ng/mL LPS, conditions previously used to induce neutrophil oxidative burst and monocyte TF expression in human WB. 37,38 LPS was chosen to better simulate human disease states, such as sepsis, in which activated leukocytes and other blood components may contribute to inflammatory thrombosis. 4,5 While both agents demonstrated antithrombotic effect in this setting, hTM/ R6.5 was significantly more effective in inhibiting fibrin deposition than shTM (P 5 .05; Figure 6B; supplemental Table 8 …”