Sphingosine‐1‐phosphate (S1P) enhances Fcg receptor‐mediated neutrophil (PMN) recruitment under flow and reactive oxygen species (ROS) generation

Abstract

Background S1P is a bioactive phospholipid released by platelets and endothelial cells that has been implicated in diverse biological functions. We hypothesized that S1P may influence immune-complex-mediated PMN activation. Methods and Results SIP (10μM) significantly augmented Fcγ receptor IIIb-dependent PMN adhesion at 1.5 dynes/cm2 to a substrate coated with sub-optimal immune complexes (without S1P 22.78 ±1.5 cells/field of view, with S1P 33.25 ±3.2, p<0.03). Using fluorescence spectrometry, we found that exogenous addition of S1P led to an enhanced neutrophil Fcγ receptor-mediated rise in intracellular Ca2+ levels in a pertussis toxin-independent manner, while inducing only a small Ca2+ influx by itself. Reactive oxygen species (ROS) generation was also synergistically enhanced by S1P and Fcγ receptor co-stimulation, as shown by flow cytometry with 123-DHR. Inhibition of ROS generation abolished calcium flux and vice-versa, as shown using n-acetyl cysteine and EGTA respectively. Conclusions These data indicate that S1P augments neutrophil activation in response to Fc receptor ligation, with an interdependent signaling loop involving increased intracellular Ca2+ levels and ROS generation. Taken together, S1P from activated platelets or endothelial cells may serve to amplify leukocyte recruitment and tissue injury at sites of immune complex deposition in vasculitis.