Background and Objective: Ezrin links the cortical cytoskeleton to the plasma membrane and plays a role in regulating changes in cell shape. Recently, NSC668394 has been shown to inhibit a key step for its activity, i.e. phosphorylation at threonine 567. In neutrophils, another key regulatory step is the Ca2+-mediated cleavage of ezrin by calpain.
Methods: In this paper, we use NSC668394 as a pharmacological inhibitor to investigate the interplay between these two steps in regulating changes in neutrophil shape.
Results: NSC668394 reduced the amount of peripherally located ezrin in neutrophils, and increased Ca2+-dependent ezrin cleavage. Neutrophils with NSC668394-inhibited ezrin phosphorylation remained both phagocytic and chemotactically competent. However, phagocytosis was slightly impaired and chemotaxis could not be maintained over longer periods. The characteristic chemotactic morphology which neutrophils adopt was also aberrant. Although phosphorylation of ezrin plays a minor role in limiting the rapid changes in cell shape in neutrophils, inhibition of ezrin phosphorylation by NSC668394 prevented multiple and prolonged shape changes during extended chemotaxis.
Conclusion: The susceptibility of prolonged chemotaxis to inhibition by NSC668394 may point to a useful target for anti-inflammatory therapy. Inhibition of neutrophil chemotaxis towards chronically inflamed sites without compromising their ability to undergo phagocytosis is a much sought after the effect of anti-neutrophil therapy.