Neutrophil chemorepulsion in defined interleukin-8 gradients in vitro and in vivo

Abstract

We report for the first time that primary human neutrophils can undergo persistent, directionally biased movement away from a chemokine in vitro and in vivo, termed chemorepulsion or fugetaxis. Robust neutrophil chemorepulsion in microfluidic gradients of interleukin-8 (IL-8; CXC chemokine ligand 8) was dependent on the absolute concentration of chemokine, CXC chemokine receptor 2 (CXCR2), and was associated with polarization of cytoskeletal elements and signaling molecules involved in chemotaxis and leading edge formation. Like chemoattraction, chemorepulsion was pertussis toxin-sensitive and dependent on phosphoinositide-3 kinase, RhoGTPases, and associated proteins. Perturbation of neutrophil intracytoplasmic cyclic adenosine monophosphate concentrations and the activity of protein kinase C isoforms modulated directional bias and persistence of motility and could convert a chemorepellent to a chemoattractant response. Neutrophil chemorepulsion to an IL-8 ortholog was also demonstrated and quantified in a rat model of inflammation. The finding that neutrophils undergo chemorepulsion in response to continuous chemokine gradients expands the paradigm by which neutrophil migration is understood and may reveal a novel approach to our understanding of the homeostatic regulation of inflammation.

Neutrophils Induce Astroglial Differentiation and Migration of Human Neural Stem Cells via C1q and C3a Synthesis

“…6D), suggesting a repulsive effect. Interestingly, concentration-dependent chemoattraction versus chemorepulsion (fugetaxis) is consistent with previous reports for PMN and T cell responses to IL-8 and SDF1a gradients in vitro and in vivo (63,64). In particular, higher concentrations of IL-8 and greater CXCR2 occupancy in human PMN were associated with conversion from chemoattraction to chemorepulsion (63); moreover, both chemoattraction and chemorepulsion were sensitive to PI3K/AKT inhibitors.…”

Universal Microfluidic Gradient Generator

“…For this, we first demonstrate that any set of n + 2 concentrations at one level n, can be produced from the mixing of n + 1 solutions from the previous level, n − 1. In mathematical form, this requires us to prove that for any given set of concentrations and positions of the dividers at level n, C n,i and u n,i , with the following restrictions (1) and (2) there exists at least one set of and that simultaneously verifies the constrain for the position of dividers at two consecutive levels: (3) and the relation between concentrations at the two consecutive levels (4) To demonstrate this, we assume flow rates proportional to the width of the spacing between dividers and use the mass conservation equations for fraction mixing between any two dividers: (5) which we could also rewrite it in the following form: (6) By plugging u n−1,m−1 into the compatibility condition u n,m − 1 < u n − 1,m − 1 < u n,m we obtain after a couple of reductions (7) which implies, considering u n,m−1 < u n,m that (8) It can be seen immediately that the argument is reversible, and therefore, we obtain the equivalence between eqs 3 and 4. By using mathematical induction principles, we could then further prove that for any series of concentrations at the output, a complete set of intermediate concentrations and divider positions can be found for generating these concentrations, starting from two distinct input concentrations.…”

“…Such distributions usually occur in nonhomogeneous mediums, under various production, transport, and degradation conditions, and gradients of nonlinear, more or less complex shapes are usually formed. 6,7 Although linear gradients represent a good first-order approximation for in vivo gradients, and are useful in gaining insights into the biology of cellular responses, growing evidence suggests that a lot of complexity arises and many cellular responses are specific to spatial gradients that are not linear. For example, in bacteria migrating in chemoattractant gradients, the interplay between sensory adaptation and concentration changes due to displacement can lead to variations in chemotactic responses.…”

Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients

“…Assays using the Boyden chamber, 10 agarose or collagen gels, or shallow layer chambers, like the Dunn 11 or Zigmond 12 chambers, require several minutes for gradient stabilization and consequently are not appropriate for fast gradient changes. Previous attempts to gradient changes using microfluidic chemotaxis devices 13,14 also resulted in long switching times that limited their use to the generation of stable gradients only. 15 Micropipettes and photoactivated release of caged chemicals can generate new gradients in seconds, but these gradients can be easily perturbed by external factors and reproducibility of experimental conditions is generally a concern.…”