Reactive oxygen species as signaling molecules in neutrophil chemotaxis

Abstract

Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPHoxidase- dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.

Keywords: NADPH oxidase; cell migration; chemokines; chronic granulomatous disease; innate immunity; signal transduction.

Figure 1

Multiple pseudopodia formation induced by nadPH inhibitor diphenyleneiodonium chloride (dPI) was more prominent in relatively shallow chemoattractant gradient. (a) relatively shallow chemoattractant gradient was generated in the upper section in the eZ-taxiscan device. (B) dependent on rOS in neutrophil chemotaxis relies on the feature of the gradient. neutrophils were treated with 50 µM dPI for 30 min and chemotaxis was induced by 100 nM fMLP. neutrophil purification, eZ-taxiscan chemotaxis assay, and analysis of cell tracks and morphology were conducted as previously described.14,18,19 Percentage of cells that display multiple pseudopodia (n = 20 cells, Fisher’s 2 × 2 test, *p < 0.05 versus untreated) during the course of the eZ-taxiscan chemotaxis assay was quantified as described by Hattori et al.14

Figure 2

Comparison of various chemotaxis assays. (a) needle assay. Chemoattractant gradient was formed by continuous passive diffusion of chemoattractant from a micropipette tip. equations describe the concentration gradient C(r,t) generated in the radial direction (neglecting convection). D denotes the diffusion constant for the chemoattractant (cm2/sec), q denotes the rate at which the chemoattractant is released (mols/sec), r is the radius from the needle tip (cm). (B) eZ-taxis scan chemotaxis device. Gradient is set up by addition of 1 µl chemoattractant to the chemoattractant reservoir, and allowing diffusion towards the cell reservoir. a linear gradient is setup across a 260 µm channel within the time scale of the experiment (30 mins) (as per manufacturer’s description). (C) Comparison of gradients generated by needle assay and eZ-taxiscan device. Percentage change in chemoattractant concentration across 10 µm sections are plotted for the steady state gradient in the needle assay (C ∼1/r) and a linear gradient in a eZ-taxiscan device. the chemoattractant gradient is most shallow near the source for the eZ-taxiscan assay and steepest near the source for the needle assay.

Figure 3

neutrophil chemotaxis in shallow chemoattractant gradient. In shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. their location and direction are random and are not oriented by chemoattractants. directional sensing is mediated by maintaining the most accurate existing pseudopod, while the ones facing wrong direction need to be quickly destroyed via actin depolymerization. Inhibition of actin depolymerization in misoriented pseudopods should lead to multiple pseudopod formation and reduced chemotaxis efficiency.