Demonstration of reversible priming of human neutrophils using platelet- activating factor

Abstract

Exposure of neutrophils to agents such as lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major upregulation of subsequent agonist-induced NADPH oxidase activation. This priming effect is a prerequisite for neutrophil-mediated tissue damage and has been widely considered to be an irreversible process. We have investigated the potential for neutrophils to recover from a priming stimulus by studying the effects of platelet-activating factor (PAF). PAF did not stimulate respiratory burst activity directly, but caused a rapid (maximal at 10 minutes) and concentration-dependent (EC50 50.2 nmol/L) increase in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide anion release. At time-points > 10 minutes, this priming effect spontaneously declined, with return to basal levels of fMLP- stimulated superoxide anion generation by 120 minutes. An identical priming time-course was observed with N-methyl carbamyl PAF, a nonmetabolizable analogue of PAF, indicating that the transient nature of PAF-induced priming was not secondary to PAF metabolism. Two structurally diverse PAF receptor antagonists (UK-74,505 and WEB 2086), added 10 minutes after PAF addition, increased the rate of decay of the priming effect. In contrast, TNF-alpha-induced priming, which was of a similar magnitude to that observed for PAF, was slower to evolve (maximal at 30 minutes) and remained constant for at least 120 minutes. The reversible nature of PAF-induced priming was confirmed by demonstrating that PAF-, but not TNF-alpha-, induced cell polarization (shape change) and CD11b-dependent neutrophil binding of albumin-coated latex beads was also transient, with return to basal, unstimulated levels by 120 minutes. Furthermore, cells that had spontaneously deprimed following PAF exposure retained their capacity to be fully reprimed by a subsequent addition of either PAF or TNF-alpha. These data imply that neutrophil priming is not an irreversible event: the demonstration of a cycle of complete priming, depriming, and repriming offers the potential for functional recycling of neutrophils at sites of inflammation.

Priming and de‐priming of neutrophil responses in vitro and in vivo

“…With PAF this was shown to be a spontaneous event, with a resetting of fMLF-triggered NADPH oxidase response and near-complete recovery from a polarised to a rounded morphology. 7 Our group has recently shown the potential for this process to occur in vivo in humans, with immediate (first pass) trapping of injected exogenously (GM-CSF)primed neutrophils within the very narrow pulmonary capillaries, followed by their subsequent gradual release back into the systemic circulation, a finding that we propose to reflect in situ de-priming. 8 Furthermore, the ability of repeated mechanical deformation of neutrophils (achieved by optical lasers or by passage through a series of multiple 5-μm micro-channels) highlights the mechano-sensitive nature of neutrophils and the potential for active and rapid depriming of these cells.…”

“…Primed neutrophils ingest and kill bacteria, with the risk of collateral tissue damage (6). Primed neutrophils are also "primed to die" in response to cues at the inflammatory site (7) or may de-prime spontaneously (8) mechanisms occurs. For example, GM-CSF and TNF both promote p47 phox phosphorylation to prime the respiratory burst, but rely on ERK and p38 respectively 12 ; RNAseq confirms that they activate both cytokine-specific and shared pathways.…”

Respiratory Barrier as a Safeguard and Regulator of Defense Against Influenza A Virus and Streptococcus pneumoniae

“…In addition, S. pneumoniae causes cell damage and loss of planar epithelial architecture at the mucosal surface (117,176). Pneumococci are able to evade neutrophils by expressing a polysaccharide capsule that also physically reduces deposition of complement and antibodies (177,178), and by molecular mimicry wherein bacterial phosphorylcholine moieties bind PAFr, preventing PAF from initiating neutrophil phagocytosis and bactericidal activities (135,(179)(180)(181). In this respect, IAV is a perfect partner for S. pneumoniae, providing it with a compromised mucosal epithelial barrier that is permissive for it to establish infection, while at the same time dampening antibacterial host responses.…”

Potential mechanisms regulating pulmonary pathology in inflammatory bowel disease

“…Neutrophil priming is a reversible process, and the equilibrium between unstimulated and primed neutrophils is dynamic [45]. Studies by Kitchen et al [45] have demonstrated the ability of platelet-activating factor to de-prime neutrophils in vitro, and evidence to support in vivo de-priming has been supplied by a mathematical model of neutrophil kinetics in IBD [70]. The lung has been proposed as the key anatomic site of neutrophil de-priming [48].…”

“…Primed neutrophils are not fully activated but are hyperresponsive to further agonist-induced activation. In addition to increased oxidative potential, primed neutrophils exhibit elevated CD11b expression and a conformational shape change [44,45], contributing to increased pulmonary retention times, as the diameter of a neutrophil is greater than the diameter of the lung microvascular capillaries [46,47]. An elegant study by Summers et al [48] demonstrated that primed neutrophils are retained in the lung of healthy subjects for up to 40 min longer than unprimed neutrophils, which passed through the lungs in under 15 s. Thus, inflammatory-driven changes to neutrophil priming status can lead to neutrophil retention in the lungs [48,49].…”