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.
Granulocyte apoptosis is an important mechanism underlying the removal of redundant neutrophils from an inflammatory focus. The ability of many proinflammatory agents to impede this event suggests that such agents act not only in a priming or secretagogue capacity but also increase neutrophil longevity by delaying apoptosis. We have examined whether this hypothesis holds true for all neutrophil priming agents, in particular tumor necrosis factor-α (TNF-α), which has been variably reported to either induce, delay, or have no effect on neutrophil apoptosis. After 20 hours coincubation TNF-α inhibited neutrophil apoptosis; however, more detailed analysis demonstrated its ability to promote apoptosis in a subpopulation of cells at earlier (2 to 8 hours) times. Formyl-Met-Leu-Phe, platelet-activating factor, inositol hexakisphosphate, lipopolysaccharide, leukotriene B4 , and granulocyte-macrophage colony-stimulating factor all inhibited apoptosis at 6 and 20 hours. The early proapoptotic effect of TNF-α was concentration-dependent (EC50 2.8 ng/mL), abolished by TNF-α neutralizing antibody, and was not associated with any change in cell viability or recovery. Of relevance to the inflamed site, the ability of TNF-α to accelerate apoptosis was lost if neutrophils were primed with 1 μmol/L PAF or aged for 6 hours before TNF-α addition. The TNFR55-selective TNF-α mutants (E146K, R32W-S86T) induced neutrophil apoptosis but with a potency 14-fold lower than wild-type TNF-α. Although the TNFR75-selective mutant (D143F ) did not induce apoptosis, blocking antibodies to both receptor subtypes abolished TNF-α–stimulated apoptosis. Hence, TNF-α has the unique ability to induce apoptosis in human neutrophils via a mechanism where TNFR75 facilitates the dominant TNFR55 death effect. This may be an important mechanism controlling neutrophil longevity and clearance in vivo.
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