Joanna Murray

Prostaglandin D2 and Its Metabolites Induce Caspase-Dependent Granulocyte Apoptosis That Is Mediated Via Inhibition of IκBα Degradation Using a Peroxisome Proliferator-Activated Receptor-γ-Independent Mechanism

Many inflammatory mediators retard granulocyte apoptosis. Most natural PGs studied herein (e.g., PGE2, PGA2, PGA1, PGF2α) either delayed apoptosis or had no effect, whereas PGD2 and its metabolite PGJ2 selectively induced eosinophil, but not neutrophil apoptosis. This novel proapoptotic effect does not appear to be mediated via classical PG receptor ligation or by elevation of intracellular cAMP or Ca2+. Intriguingly, the sequential metabolites Δ12PGJ2 and 15-deoxy-Δ12, Δ14-PGJ2 (15dPGJ2) induced caspase-dependent apoptosis in both granulocytes, an effect that did not involve de novo protein synthesis. Despite the fact that Δ12PGJ2 and 15dPGJ2 are peroxisome proliferator-activated receptor-γ (PPAR-γ) activators, apoptosis was not mimicked by synthetic PPAR-γ and PPAR-α ligands or blocked by an irreversible PPAR-γ antagonist. Furthermore, Δ12PGJ2 and 15dPGJ2 inhibited LPS-induced IκBα degradation and subsequent inhibition of neutrophil apoptosis, suggesting that apoptosis is mediated via PPAR-γ-independent inhibition of NF-κB activation. In addition, we show that TNF-α-mediated loss of cytoplasmic IκBα in eosinophils is inhibited by 15dPGJ2 in a concentration-dependent manner. The selective induction of eosinophil apoptosis by PGD2 and PGJ2 may help define novel therapeutic pathways in diseases in which it would be desirable to specifically remove eosinophils but retain neutrophils for antibacterial host defense. The powerful proapoptotic effects of Δ12PGJ2 and 15dPGJ2 in both granulocyte types suggest that these natural products control the longevity of key inflammatory cells and may be relevant to understanding the control and resolution of inflammation.

Interleukin-10 inhibits lipopolysaccharide-induced survival and extracellular signal-regulated kinase activation in human neutrophils

Lipopolysaccharide (LPS) induces a marked delay in human neutrophil apoptosis that is reversed by the anti‐inflammatory cytokine IL‐10. The effect of IL‐10 is specific since other agents that delay neutrophil apoptosis are not affected. To investigate mechanisms underlying the actions of IL‐10, we examined signaling pathways activated by LPS per se and in response to IL‐10. The MAPK kinase (MEK) 1 inhibitor PD098059, the protein kinase C (PKC) inhibitor Ro31,8220, and the phosphatidylinositol‐3 kinase (PI3‐K) inhibitor LY294002 all partially reversed LPS‐mediated retardation of neutrophil apoptosis, but the p38 MAPK inhibitor SB203850 did not. LPS activates the transcription factor NF‐κB, however, IL‐10 did not affect the ability of LPS to activate NF‐κB as assessed by IκB‐α proteolysis. Although IL‐10 did not alter activation of ERK by GM‐CSF or TNF‐α, it did inhibit activation induced by LPS. Thus our data illustrate that LPS‐induced neutrophil survival is regulated by the MAPK, PKC and PI3‐K pathways as well as NF‐κB, and can be reversed by IL‐10, through a mechanism involving inhibition of ERK activation. Because of the specific nature of this inhibition, we conclude that IL‐10 interferes with an ERK activation pathway, which is not involved in GM‐CSF or TNF‐α signaling.

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFα) stimulated apoptosis by a combination of high‐performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFα and the phosphatidylinositol‐3‐kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFα, compared to TNFα by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non‐invasive quantification of apoptosis.

Role of leukotrienes in the regulation of human granulocyte behaviour: dissociation between agonist-induced activation and retardation of apoptosis

Since most inflammatory mediators that stimulate granulocyte responsiveness also delay apoptosis, it is often assumed that activation and longevity are causally related. Using isolated human peripheral blood neutrophils and eosinophils, we examined this association by exploiting the proinflammatory lipid mediators, the leukotrienes (LTs), and investigated granulocyte function and apoptosis. LTB4 induced elevation of intracellular free Ca2+ concentration ([Ca2+]i), cell polarisation and retardation of neutrophil apoptosis, although the antiapoptotic effect occurred only at concentrations 300 nM. LTB4‐induced activation was attenuated by CP‐105,696, a BLT1‐specific antagonist suggesting classical LTB4 receptor BLT1 involvement. Despite demonstrating the presence of the neutrophil intracellular LTB4 receptor peroxisome‐proliferator activator receptor‐α (PPARα) in neutrophils, the selective PPARα agonist WY‐14,643 did not mimic LTB4‐induced prosurvival effects. LTB4‐induced survival, however, also appeared to be mediated by BLT1 since CP‐105,696 inhibited the LTB4‐mediated antiapoptotic effect. Furthermore, based on studies with CP‐105,696 and 5‐lipoxygenase inhibitors, lipopolysaccharide (LPS)‐, granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐, dexamethasone‐ and dibutyryl‐cAMP (db‐cAMP)‐induced delay of neutrophil apoptosis did not involve autocrine production of LTB4. Although LTB4 and LTD4 induced human eosinophil [Ca2+]i elevation and polarization, these LTs did not influence eosinophil apoptosis. Furthermore, LTB4‐ and LTD4‐induced eosinophil activation was attenuated by CP‐105,696 and the Cys‐LT1 receptor antagonist montelukast, respectively, highlighting specific receptor dependency. Thus, mediator‐triggered granulocyte activation and antiapoptotic pathways are distinct events that can be differentially regulated.

Characterization of the survival effect of tumour necrosis factor-alpha in human neutrophils

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFalpha (tumour necrosis factor-alpha) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-kappaB (nuclear factor-kappaB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFalpha increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFalpha addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFalpha, implying a TNFR1-dependent process. Finally, I-kappaBalpha (inhibitory kappaB-alpha) and NF-kappaB time-course studies demonstrated that the survival effects of both GM-CSF and TNFalpha could be explained by maintenance of functional NF-kappaB.

Oxygen levels determine the ability of glucocorticoids to influence neutrophil survival in inflammatory environments

GCs are highly effective in treating a wide range of inflammatory diseases but are limited in their ability to control neutrophilic lung inflammation in conditions such as COPD. Neutrophil apoptosis, a central feature of inflammation resolution, is delayed in response to microenvironmental cues, such as hypoxia and inflammatory cytokines, present at inflamed sites. GCs delay neutrophil apoptosis in vitro, and this may therefore limit the ability of GCs to control neutrophilic inflammation. This study assesses the effect GCs have on hypoxia‐ and inflammatory cytokine‐induced neutrophil survival. Human neutrophils were treated with GCs in the presence or absence of GM‐CSF or inflammatory macrophage‐CM at a range of oxygen concentrations (21–1% oxygen). Neutrophil apoptosis and survival were assessed by flow cytometry and morphological analysis and neutrophil function, by stimulus‐induced shape change and respiratory burst. Dexamethasone promoted neutrophil survival at 21%, 10%, and 5% oxygen but not at 1% oxygen. Interestingly, GM‐CSF and inflammatory CM increased neutrophil survival significantly, even at 1% oxygen, with cells remaining functionally active at 96 h. Dexamethasone was able to reduce the prosurvival effect of GM‐CSF and inflammatory CM in a hypoxic environment. In conclusion, we found that GCs do not augment neutrophil survival in the presence of severe hypoxia or proinflammatory mediators. This suggests that GCs would not promote neutrophil survival at sites of inflammation under these conditions.

Constitutive apoptosis in equine peripheral blood neutrophils in vitro

Highlights • Equine peripheral blood neutrophils underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h.• Equine neutrophils undergoing apoptosis develop the structural and functional features of apoptosis observed in many cell types, including human neutrophils.• Neutrophils undergoing apoptosis had diminished zymosan activated serum-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate.• The constitutive rate of neutrophil apoptosis was promoted by lipopolysaccharide, tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a).• Equine and human neutrophil apoptosis differed in terms of responses to lipopolysaccharide and in the time-dependence of the response to dexamethasone.