Treatment with Caspase Inhibitor Does Not Prevent the Spontaneous Death of Neutrophils.
In this study, we used a well established in vitro
explore neutrophil spontaneous death. Human primary neutrophils
were prepared from freshly collected human-citrated whole blood
by using a Ficoll-Hypaque solution. After 21 h of culturing,
many neutrophils manifest clear morphological signs of apoptosis
such as cell shrinkage and nuclear condensation (Fig. 7, which
is published as supporting information
on the PNAS web site).
The number of neutrophils undergoing spontaneous death was quantified
by using FACS analysis. In the FACS assays, we use Annexin V,
an anticoagulant protein that has high affinity and selectivity
for phosphatidylserine, to detect phosphatidylserine exteriorization
and propidium iodide (PI), a membrane impermeable dye, to monitor
cell membrane integrity (Fig. 7). The phosphatidylserine exposure
was evident in neutrophils by 12 h, and the level increased
to 35 ± 4% at 24 h. We detected a concomitant increase
in the apoptotic (Fig. 1A
, R2) and necrotic (Fig. 1A
, R3 and
R4) populations at 48 h, suggesting that at later time points
many of the apoptotic cells have proceeded into secondary necrosis
(Fig. 1 A
Using this system, we investigated the role of caspases in neutrophilspontaneous death. As reported in ref. 20, the broad-spectrumcaspase inhibitor zVAD-fmk blocked caspase-dependent apoptosis,such as staurosporine-induced death of HEK293 cells and H2O2-eliciteddeath of HeLa cells, whereas the same drug did not suppressspontaneous neutrophil death at all (Fig. 1 C–E).
Akt Signaling Pathway Is Down-Regulated During Neutrophil Spontaneous Death. We recently showed that deactivation of PtdIns(3,4,5)P3/Aktsignal characterizes both caspase-dependent and -independentcell death. To investigate whether the PtdIns(3,4,5)P3/Akt survivalpathway is involved in neutrophil spontaneous death, we examinedthe activation of this signal in neutrophil spontaneous death.Akt is recruited onto the plasma membrane through its specificbinding to PtdIns(3,4,5)P3. Only the Akt molecules on the plasmamembrane can be phosphorylated and activated by two phosphatidylinositol-dependentprotein kinases and get activated, thus Akt phosphorylationhas been used widely as an indicator of Akt activation (12,14, 15, 19, 21–23). Our results show that during the courseof neutrophil death, levels of phospho-Akt decrease dramatically,whereas levels of total Akt do not change (Fig. 2A). This resultwas detected in all of the blood donors we examined (>10donors). The level of phospho-Akt declines by more than one-halfin only 15 h in culture (Fig. 2B). We next measured the Aktactivity directly. We used glycogen synthase kinase-3 (GSK-3),a substrate of Akt (24, 25), as a marker for in vivo Akt activation.Levels of phospho-GSK-3 decline during neutrophil death, witha time course similar to the decline in levels of phospho-Akt.By contrast, total GSK-3 levels do not change (Fig. 2C). Noticeably,the Akt activity started to decrease before any apoptotic morphologychanges were detected, indicating that Akt deactivation mightbe a causal mediator of neutrophil death. The decreases in phospho-Aktare selective. We measured activity of MAPK p44/42 (Erk1/2)pathway (Fig. 2D). During neutrophil spontaneous death, we detectno alteration in any of these phosphokinases 3–48 h afterblood was drawn from the donors.
Inhibition of PtdIns(3,4,5)P3/Akt Signaling Promotes Neutrophil Death. To test the hypothesis that deactivation of PtdIns(3,4,5)P3/Aktsignaling is a causal mediator of neutrophil spontaneous death,we examined whether inhibition of this pathway can promote furtherneutrophil death. We used a newly developed Akt inhibitor, Aktinhibitor I, to suppress the PtdIns(3,4,5)P3/Akt signaling inneutrophils. Moreover, because PI3K is upstream of Akt and inhibitionof PI3K is associated with deactivation of Akt, we also treatedneutrophil cultures with the PI3K inhibitor wortmannin. Treatmentwith these drugs markedly deactivates Akt without altering totalAkt levels (Fig. 3A and B). Both drugs promoted neutrophil deathas monitored by FACS analysis (Fig. 3C).
Factors Preventing Neutrophil Spontaneous Death Restore Akt Kinase Activity. If Akt deactivation mediates neutrophil spontaneous cell death,then treatments that prevent neutrophil death should inhibitthe deactivation. Many extracellular factors, such as granulocyte–macrophagecolony-stimulating factor (GM-CSF), granulocyte colony-stimulatingfactor (G-CSF), and IFN- (LPS), have been shown to block neutrophilspontaneous death (4–7, 26, 27). We have confirmed theantineutrophil death effect of these reagents in our experimentalsystem (Fig. 4A). In addition, we showed that these factorsalso can prevent deactivation of Akt during neutrophil deathwith no alterations in total Akt levels (Fig. 4 B and C). Theseresults are consistent with our hypothesis that Akt deactivationis an essential causal mediator in neutrophil spontaneous death.
Augmentation of PtdIns(3,4,5)P3/Akt Signal by PTEN Depletion Prevents Neutrophil Spontaneous Death. If Akt deactivation mediates neutrophil spontaneous death, thenelevating this pathway should diminish neutrophil death. Totest this idea, we tried to augment PtdIns(3,4,5)P3/Akt signalingby disrupting PTEN gene. The tumor suppressor PTEN is a phosphatidylinositol3'-phosphatase that converts PtdIns(3,4,5)P3 to PtdIns(4,5)P2.Depletion of this lipid phosphatase leads to accumulation ofPtdIns(3,4,5)P3 on the plasma membrane and, thus, elevationof PtdIns(3,4,5)P3/Akt signaling. To examine the effect of elevatedPtdIns(3,4,5)P3/Akt signaling on neutrophil death, we isolatedneutrophils from PTEN knockout mice. Because of the early embryoniclethality of conventional Pten–/– mice (28), weused a conditional Pten knockout mouse, in which two loxP sequenceswere inserted on either side of the exon 5 of PTEN encodingthe phosphatase domain (Fig. 8, which is published as supporting informationon the PNAS web site). We then crossed this mouse with a myeloid-specificCre line, in which the Cre recombinase gene was inserted intothe endogenous M lysozyme locus (Fig. 8). Mice that are homozygousfor this allele are viable, fertile, normal in size, and donot display any gross physical or behavioral abnormalities (datanot shown). The expression of WT PTEN protein is completelyabolished in neutrophils isolated from either Cre+/–;PTENloxP/loxP or Cre+/+;PTEN loxP/loxP mice (Fig. 5A and data notshown). Thus, Cre-mediated deletion of the loxP-flanked PTENgene in myeloid cells is highly efficient. The amount of Crerecombinase expressed from only one copy of the Cre gene isenough to initiate PTEN deletion (Fig. 5A).
In neutrophils depleted of the PTEN gene, PtdIns(3,4,5)P3/Aktsignaling, monitored by Akt phosphorylation, is dramaticallyenhanced (Fig. 5A). This result is consistent with the roleof PTEN as a PtdIns(3,4,5)P3 phosphatase. PTEN-null neutrophilsdifferentiate normally, and neutrophil count in peripheral bloodis the same between PTEN knockout and WT mice (Fig. 5B and datanot shown). We measured the cell death rate of neutrophils directlyisolated from the bone marrow. We found that Pten-null neutrophilslive much longer than WT neutrophils, and this effect was observedat all time points examined. Only 5% of WT neutrophils couldlive>72 h in the culture, whereas 40% PTEN-null neutrophilswere detected healthy under the same condition (Fig. 5C). Delayeddeath of PTEN-null neutrophils can be reversed by treatmentwith the PI3K inhibitor wortmannin, suggesting this delayedneutrophil death is directly mediated by PtdIns(3,4,5)P3 signaling(Fig. 5D). The same result was obtained when in vitro-generatedneutrophils were used (Fig. 6). More than 85% of cells in WTgranulocyte colonies died at day 22, whereas only <10% ofPTEN-null neutrophils went to apoptosis.
In the myeloid-specific Cre line, the Cre recombinase gene wasinserted into the lysozyme locus and, therefore, the endogenouslysozyme gene is disrupted. To ascertain that the phenotypewe observed is caused by PTEN disruption instead of lysozymedeletion, we examined the neutrophils isolated from Cre+/+;PTEN wt/wt mice, which express WT PTEN but contain two mutatedlysozyme alleles. These cells die at a similar rate as WT neutrophils,suggesting that depletion of lysozyme does not affect neutrophilspontaneous death. Furthermore, the diminished cell death ofPTEN-null neutrophils could be reversed by treatment with PI3Kor Akt inhibitors, suggesting that this effect is directly causedby elevated Akt activity in PTEN-null neutrophils (data notshown). In fact, we routinely use only Cre heterozygous micein our assay (Fig. 5 and 6). Thus, effects caused by lysozymedepletion will be minimized.