Lecture title: Signaling via P13Ks in Neutrophils Dr Phillip Hawkins

Lecture title: Signalling via P13Ks in Neutrophils Dr Phillip Hawkins

Summary

PI3Ks play important roles in the signaling pathways used by a wide variety of cell surface receptors on neutrophils. Class IB PI3K plays a major role in the initial generation of PtdIns(3,4,5)P3 by Gi-coupled G-protein coupled receptors (GPCRs) (e.g., receptors for fMLP, C5a, LTB4). Class IA PI3Ks generate PtdIns(3,4,5)P3 downstream of receptors which directly or indirectly couple to protein tyrosine kinases such as integrins, Fc?Rs, cytokine receptors, and GPCRs. The PtdIns(3,4,5)P3 made by Class I PI3Ks regulates the activity of several different effector proteins, many of which are plasma membrane GEFs or GAPs for small GTPases (Segal 2009).

Class III PI3K generates PtdIns(3)P in the phagosome membrane and plays an important role in efficient assembly of the NADPH oxidase at this location. Much still remains to be discovered about the molecular details that govern activation of PI3Ks and the mechanisms by which these enzymes regulate complex cellular processes, such as neutrophil spreading, chemotaxis, phagocytosis, and killing of pathogens. However, it is clear from recent use of transgenic mouse models and isoform-selective PI3K inhibitors that these pathways are important in regulating neutrophil recruitment to sites of infection and damage in vivo. Thus, PI3K pathways may present novel opportunities for selective inhibition in some inflammatory pathologies (Nathan 2006).

Critical analysis

PI3K activity, resulting in the accumulation of PIP3 along the leading edge of a chemotaxing cell, has been proposed to be an indispensable signaling event that is required for cells to undergo chemotaxis to endogenous and exogenous chemoattractants. Some studies have suggested that this might be the case for chemoattractants such as IL8, whereas chemotaxis to other stimuli, such as the bacterial peptide Nformyl- methionyl-leucyl-phenylalanine (fMLP), might occur normally in the absence of PI3K activity. Herein, we systematically analyze the role of PI3K in mediating chemotaxis to fMLP, both in vitro and in vivo. Using short- and long-term in vitro assays, as well as an in vivo chemotaxis assay, we investigated the importance of PI3K in response to the prototypic chemoattractant fMLP. Exposure of neutrophils to fMLP induced an immediate polarization, which resulted in directional migration towards fMLP within 2-3 minutes (Alloy 2009).

PI3Kinhibited cells also polarized and migrated in a directional fashion towards fMLP; however, this process was delayed by ~15 minutes, demonstrating that PI3K accelerates the initial response to fMLP, but an alternative pathway replaces PI3K over time. By contrast, p38-MAPK-inhibited cells, or cells lacking MK2, were unable to polarize in response to fMLP. Long-term chemotaxis assays using a pan-PI3K inhibitor, a PI3K_-specific inhibitor or PI3K knockout neutrophils, demonstrated no role for PI3K in mediating chemotaxis to fMLP, regardless of the steepness of the fMLP gradient. Similar results were observed in vivo, with PI3K_-/- cells displaying a delayed, but otherwise normal, chemotactic response to gradients of fMLP. Together, these data demonstrate that, although PI3K can enhance early responses to the bacterial chemoattractant fMLP, it is not required for migration towards this chemoattractant (Hawkins ...