Interactions between lipopolysaccharides and blood factors on the stimulation of equine polymorphonuclear neutrophils

In horses, the mechanisms of lipopolysaccharide (LPS) stimulation of isolated neutrophils to produce reactive oxygen species remain unknown. We re-investigated this problem by monitoring the luminol-enhanced chemiluminescence (CL) produced by LPS-stimulated equine neutrophils. The neutrophils were isolated from horse blood by discontinuous density gradient centrifugation (≥99% neutrophils; viability ≥98%). Increasing concentrations of Escherichia coli (E. coli) LPS (from 0.01–10 μg ml−1) were used to activate the neutrophils. When LPS was used directly, without another stimulator, the respiratory burst of neutrophils was not activated (N=12 horses; n=5 assays per horse). On the contrary, when LPS was added to whole blood, the neutrophils isolated from this blood were stimulated in a LPS dose-dependent manner, but polymyxin B added to whole blood suppressed this stimulation (N=2; n=6). LPS dissolved in autologous equine plasma stimulated the isolated neutrophils in a dose-dependent manner from 0.1–10 μg ml−1 (N=5; n=12). Heat inactivation of the plasma abolished this CL increase (N=2; n=5). LPS added to equine albumin did not stimulate the isolated neutrophils (N=2; n=5). On the contrary, the addition of γ-globulins (1 mg ml−1) to LPS (10 μg ml−1) led to the stimulation of neutrophils (N=2; n =5). We concluded that LPS did not directly stimulate the isolated equine neutrophils, but that plasmatic factors are needed for the stimulation of these cells by LPS.