Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils

Abstract

It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-␣ (TNF-␣)-primed human neutrophils with N-formylmethionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3K␥, and the second phase is largely dependent on PI3K␦. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-␣ priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-␣-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3K␥ alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation. IntroductionNeutrophils are critical components of the immune system and have a vital role in combating bacterial and fungal infections. 1 A key weapon in the neutrophil armory is the so-called "respiratory burst," the generation of reactive oxygen species (ROS) by a multicomponent oxidase complex. 2,3 Patients with chronic granulomatous disease (CGD) caused by defective expression of active oxidase components experience recurrent, life-threatening infections. 4 The role of ROS in fighting infections is complex. ROS are involved in the killing process directly through the damaging actions of oxygen radicals and their halogenated derivatives and indirectly through the activation of phagosomal proteases. [5][6][7] It is also becoming apparent that ROS may regulate the neutrophil lifespan, modify the extracellular matrix through which the neutrophils migrate, and modulate the function of other cells participating in the inflammatory response. [8][9][10][11][12] Given the potential for self-damage, a key feature of the inflammatory response is to confine ROS generation in time and space to areas where it is required. One way in which this is thought to occur is through a form of signal integration in which prior exposure to local proinflammatory factors is necessary for maximal activation by subsequent oxidase-triggering signals. [13][14][15][16][17] One of best studied examples of this "priming" phenomenon is the ability of tumor necrosis factor-␣ (TNF-␣), a cytokine released primarily by macrophages, to dramatically augment the oxidase response to bacterially derived peptides (eg, N-formyl-methionyl-leucylphenylal...

Class IA Phosphatidylinositide 3-Kinases, rather than p110γ, Regulate Formyl-Methionyl-Leucyl-Phenylalanine-Stimulated Chemotaxis and Superoxide Production in Differentiated Neutrophil-Like PLB-985 Cells

“…These various results, which were apparently contradictory, suggest that both class I A and class I B PI3Ks are activated following fMLP stimulation. Moreover, a recent study using a series of PI3K isoform-specific inhibitors has provided evidence for a sequential activation of class I B and class I A in response to fMLP in TNF-␣-primed neutrophils (31). Although the latter study did not examine the direct effects of the chemotactic factor, the data provided also support the idea that multiple isoforms of PI3Ks are required for chemoattractant-stimulated PI(3,4,5)P 3 production.…”

“…Direct activation of p110␤ by G␤␥ has been described (32,33), and this mechanism could account in part for the activation of PI3K I A by fMLP. Different lines of evidence suggest that nonreceptor tyrosine kinases, such as the Src family tyrosine kinases, could be involved in the activation of PI3K by chemotactic factors (20,31). We also observed that PP2, an inhibitor of the Src family tyrosine kinases, significantly reduced the accumulation of PI(3,4,5)P 3 in fMLP-stimulated neutrophils (our unpublished data), suggesting that this family of enzymes is implicated in the regulation of the PI(3,4,5)P 3 response to the chemotactic agent.…”

Platelet Factor 4 (CXC Chemokine Ligand 4) Differentially Regulates Respiratory Burst, Survival, and Cytokine Expression of Human Monocytes by Using Distinct Signaling Pathways

“…In the latter cells, stimulation with the chemotactic peptide fMLP caused a rapid activation of p38 MAPK, Erk, and PI3K. Interestingly, inhibition of p38 MAPK and PI3K, but not Erk, resulted in a drastic reduction of ROS formation and neutrophil chemotaxis (42)(43)(44)(45)(46)(47). Noteworthy, PF4 activates in human neutrophils neither PI3K nor p38 MAPK and is indeed unable to induce ROS release or chemotaxis in these cells (19,21,48).…”

P2X1 Ion Channels Promote Neutrophil Chemotaxis through Rho Kinase Activation

“…Cells can indeed detect differences in the concentration of chemoattractant between points on their surface (41) and translate the extracellular gradient into an intracellular derivative, encoded by the signaling networks engaged by the chemoattractant. Localized accumulation of PI3Ps is a critical signal coordinating chemotaxis or movement and depends on PI3K␥ and SHIP-1 (31,40,42). The study of Chen et al (6) proposed that ATP released from developing leading edges can act to control chemotaxis.…”