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Prepublished online as a Blood First Edition Paper on June 28, 2002; DOI 10.1182/blood-2001-12-0266.
PHAGOCYTES
From the Department of Medicine, Pathology, and
Surgery, University of Washington, Seattle.
It is increasingly clear that there are caspase-dependent and
-independent mechanisms for the execution of cell death and that the
utilization of these mechanisms is stimulus- and cell type-dependent.
Intriguingly, broad-spectrum caspase inhibition enhances death receptor
agonist-induced cell death in a few transformed cell lines.
Endogenously produced oxidants are causally linked to
necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis
induced in human neutrophils by incubation with agonistic anti-Fas
antibody or by coincubation with tumor necrosis factor- Nearly 1 billion neutrophils per kilogram of body
mass are turned over daily in an average human adult.1
With a relatively short life span in vivo, mature human neutrophils
appear to be endowed with robust machineries that drive cell
death. Spontaneous neutrophil apoptosis is temporally associated with
an increase in caspase-3 activity2 and is attenuated
by caspase inhibitors.2,3 Agonistic activation of Fas, one
of the death receptor family members constitutively expressed in
circulating mature human neutrophils,4,5 also activates
caspase-3 activity2 and provokes neutrophil apoptosis that
is attenuated by caspase inhibitors.2,3 Thus, caspase
activation mediates spontaneous and Fas-induced neutrophil apoptosis.
There is increasing evidence for caspase-independent mechanisms of
apoptotic cell death.6 To distinguish these novel
caspase-independent forms of cell death from the classically described
apoptotic and necrotic cell death processes, terms such as
paraptosis7 and aponecrosis8 have been
proposed. The distinctions between apoptotic versus necrotic and
programmed versus nonprogrammed cell death are increasingly blurred
also,9,10 and neutrophils can undergo cell death in the
absence of classical apoptotic or necrotic
characteristics.2,11,12 Although it has been suggested
that human neutrophils are relatively deficient in the numbers of
mitochondria13 and caspases 2, 6, and 7,14,15
the execution of a death receptor agonist-induced caspase-independent
pathway has not previously been described.
In addition to the Fas ligand-Fas system, the tumor necrosis
factor- In the present study, we show that TNF- Reagents
Isolation of circulating mature human neutrophils
Cell death assessment by light and electron microscopy Unless indicated otherwise, 1 × 106 neutrophils at 5 × 106 cells per milliliter were used for each sample throughout our study. At predefined time points, total neutrophil viable cell counts that remained under various treatment conditions were determined by trypan blue dye exclusion with a hemacytometer. In parallel, cytospin slides were prepared, stained with Diff-Quick (Baxter, McGaw Park, IL), and examined by light microscopy for morphologic changes.For transmission electron microscopy (TEM), 5 × 106 freshly isolated neutrophils in 1 mL culture medium were incubated under a specified condition and processed as previously described.27 Specifically, at predetermined time points, cells were fixed in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde at 4°C, washed, and postfixed in distilled water containing 2% osmium tetroxide and a few drops of 2% aqueous potassium ferrocyanide. After block staining with 0.5% aqueous uranyl acetate for 15 minutes, the cells were embedded in 0.1 M sodium cacodylate buffer containing 2% agar. The cell pellet was then dehydrated in a graded series of ethanol and embedded in Eponate-12 resin (Ted Pella, Redding, CA). Finally, thin sections were cut on an LKB Nova ultramicrotome (LKB, Bromma, Sweden), stained with uranyl acetate and lead stain, and examined with a JEOL JEM 1200 EX (JEOL, Tokyo, Japan). Cell death assessment by flow cytometry Annexin V-FITC binds to exposed phosphatidylserine on early apoptotic (representing membrane-intact cells with externalized phosphatidylserine) and necrotic (representing "primary," nonapoptotic cells and "secondary,"28 late apoptotic cells with compromised membrane integrity) cells, and PI gains entry into necrotic cells.29 Utilizing these agents, early apoptotic and primary/secondary necrotic neutrophils were quantitatively determined by dual-parameter flow cytometry. Briefly, neutrophils were cultured under a specified condition in a humidified CO2 (5%) incubator at 37°C. At a predetermined time point, neutrophils were resuspended in 100 µL binding buffer (140 mM NaCl, 2.5 mM CaCl2, 1.5 mM MgCl2, and 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4) containing annexin V-FITC (20 µg/mL) and PI (2 µg/mL) for 15 minutes at room temperature. Samples were kept on ice and analyzed immediately with an EPICS XL-II flow cytometer (Beckman Coulter, Fullerton, CA).Cell death assessment by DNA fragmentation assays Internucleosomal DNA fragmentation in neutrophils was assessed qualitatively by agarose gel electrophoresis as described.4 Briefly, neutrophils (10 × 106 cells in 2 mL supplemented RPMI medium) at a predetermined time point after treatment were washed twice with phosphate-buffered saline (PBS), pelleted, and lysed (0.2% Triton X-100, 10 mM EDTA [ethylenediaminetetraacetic acid], and 10 mM Tris [tris(hydroxymethyl)aminomethane] HCl, pH 7.5) for 10 minutes on ice. After centrifugation (12 000g, 10 minutes), DNA-containing supernatant was extracted with phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol), precipitated overnight with 70% ethanol at 20°C, pelleted, and dissolved in TE
(10 mM Tris HCl, pH 7.5, and 1 mM EDTA). The samples were subsequently
digested with 1 mg/mL DNase-free RNase at 37°C for 3 hours; 5 µg
DNA from each sample was electrophoretically resolved in a
2% agarose gel containing 0.5 µg/mL ethidium bromide and visualized
under ultraviolet light.
As a complementary approach, internucleosomal DNA fragmentation was quantitatively assayed by antibody-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosome-associated histone-DNA complexes (Cell Death Detection ELISA plus kit; Roche Molecular Biochemicals, Mannheim, Germany) that accumulated in dying neutrophils with intact cell membrane.30 Briefly, neutrophils (1 × 104 cells in 200 µL supplemented RPMI medium) at a predetermined time point after treatment were washed, resuspended in 200 µL of the lysis buffer supplied by the manufacturer, and incubated for 30 minutes at room temperature. After pelleting nuclei (200g, 10 minutes), 20 µL of the supernatant (cytoplasmic fraction) was used in the enzyme-linked immunosorbent assay (ELISA) following the manufacturer's standard protocol. Finally, absorbance at 405 nm and 490 nm (reference wavelength), upon incubating with a peroxidase substrate for 5 minutes, was determined with a microplate reader (Bio-Tec Instruments, Winooski, VT). Signals in the wells containing the substrate only were subtracted as background. Caspase-3-like activity assay Neutrophils (2 × 106 cells in 400 µL supplemented RPMI medium) at a predetermined time point after treatment were washed with PBS and lysed in 100 µL buffer (10 mM potassium phosphate, 1 mM EDTA, 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL leupeptin, 10 µg/mL pepstatin A, and 10 mM dithiothreitol [DTT]) for 10 minutes on ice. After centrifugation (15 000g, 20 minutes, 4°C), protein concentration of the supernatant was determined with the BCA Protein Assay Reagent (Pierce, Rockford, IL) following the manufacturer's instructions. Subsequently, 40 µg of the sample was diluted to a final volume of 200 µL with the assay buffer (50 mM HEPES, 10% sucrose, 0.1% CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid], and 10 mM DTT) containing the fluorogenic caspase-3-preferred substrate Ac-DEVD-AMC (100 µM) and incubated for 2 hours at 30°C in a 96-well plate. Fluorescence was determined (excitation, 360 nm; emission, 460 nm) with a CytoFluor series 4000 plate reader (Applied Biosystems, Foster City, CA). Background fluorescence was determined in wells containing the assay buffer only.Subcellular fractionation and immunoblotting for cytochrome c release The procedure for the preparation of mitochondria-poor cytosol fraction was modified from that previously reported.31 Specifically, neutrophils (10 × 106 cells in 2 mL supplemented RPMI medium) were harvested at a predetermined time point after treatment, washed with PBS, and resuspended in 500 µL buffer (20 mM HEPES-potassium hydroxide [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA [ethyleneglycoltetraacetic acid], 1 mM DTT, 250 mM sucrose, 1 mM PMSF, 1% aprotinin, 1 mM leupeptin, 1 µg/mL pepstatin A, and 1 µg/mL chymostatin). Following homogenization with a glass Pyrex homogenizer and a type B pestle (40 strokes), unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifugation (1000g, 20 minutes, 4°C). The supernatant was recentrifuged (100 000g, 1 hour, 4°C) to generate the mitochondria-poor cytosolic fraction. Fifty micrograms of the cytosolic protein extracts were then resolved by gradient (4% to 20%) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose membrane. The membrane was blocked overnight with 5% milk in TBST (50 mM Tris HCl [pH 7.4], 150 mM NaCl, and 0.1% Tween 20) and then probed at room temperature for 1 hour with primary mouse monoclonal anticytochrome c antibody (1:1000 dilution) in TBS (50 mM Tris MCl [pH 7.4], 150 mM NaCl), supplemented with 0.05% Tween 20 and 1% bovine serum albumin. After 3 washes with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:5000 dilution) and the chemiluminescence of the expected protein bands (15 kDa) detected with the SuperSignal substrate, per the supplier's protocol.NF-  B activation in neutrophils, a nonradioactive
ELISA method (NF-B Transcription Factor Assay Kit; Active Motif,
Carlsbad, CA), which has been shown to be specific and quantitative as
well as to correlate well with the traditional electrophoretic mobility
shift assay (EMSA), was utilized.32 In brief,
5 × 106 neutrophils were cultured in 1 mL supplemented
RPMI medium in the absence or presence of TNF- (10 ng/mL) and
without or with zVAD (100 µM) before incubation. At 30, 60, 90, and
120 minutes, neutrophils were washed twice with ice-cold PBS and lysed
in 40 µL of the supplied lysis buffer supplemented with protease and phosphatase inhibitor cocktails. Subsequently, 20 µg of protein lysate per sample was incubated for 1 hour in wells containing immobilized NF-B consensus oligonucleotide in the absence or presence of competing, nonimmobilized, NF-B consensus
oligonucleotide. After extensive washing, total bound NF-B p65/p50
heterodimer and p50/p50 homodimer were detected with the supplied
anti-p50 antibody following the manufacturer's protocol. Wells
incubated with lysis buffer alone were developed in parallel and the
readout subtracted as background.
For EMSA, nuclear lysates were prepared as previously
described33 from neutrophils (5 × 106 cells
in 1 mL supplemented RPMI medium) incubated for 1 hour without or with
TNF- Measurement of intracellular oxidative activity Neutrophil intracellular oxidative metabolism was assessed using the cell-permeable, fluorogenic DCFH-DA as described.34 Briefly, the neutrophil sample (1 × 106 cells in 200 µL supplemented RPMI medium) was supplemented with DCFH-DA (5 µM) 30 minutes before the completion of a predetermined duration of incubation under a specified condition. Upon intracellular hydrolysis and subsequent oxidization, fluorescent DCF was generated. Cells were washed with ice-cold PBS and resuspended in 0.5 mL PBS supplemented with 1% FBS. Accumulation of intracellular fluorescence in live cells was determined by flow cytometry.Statistical analysis Data are expressed as means ± SEM. For normally distributed data, a t test or paired t test was used to evaluate the differences between sets. For nonnormally distributed data, the Mann-Whitney U test was used. GraphPad Prism (version 2.01; GraphPad Software, San Diego, CA) was used for all statistical analyses. Statistical significance was defined as P < .05.
Broad-spectrum caspase inhibitors augment TNF- -stimulated neutrophils in a dose-dependent manner, with maximal inhibition at 100 µM.14 At this concentration,
zVAD also was reported to inhibit agonistic anti-Fas antibody-induced neutrophil cell death2,3 and to attenuate TNF--induced
neutrophil cell death in the absence (assessed by
morphology)17 or presence (assessed by DNA hypodiploidy and
phosphatidylserine accessibility)14 of
cycloheximide. In time-course experiments, we confirmed
that zVAD effectively attenuated spontaneous and anti-Fas
antibody-induced neutrophil cell death, measured both by total viable
cell counts (Figure 1) and by
dual-parameter flow cytometric analysis of cellular annexin V-FITC
binding and PI accessibility (data not shown). Unexpectedly, zVAD
failed to inhibit but instead augmented TNF--induced neutrophil
cell death in the same experiments (Figure 1). To characterize further
this surprising observation, a series of experiments were performed. In
these analyses, pretreatment with zVAD sensitized neutrophils to
TNF--induced cell death over 6 hours in a dose-dependent manner
(Figure 2A), an effect that was minimal
but appeared to be present at 5 µM and clearly discernible at zVAD
concentrations at and above 25 µM. This sensitizing effect also
occurred when zVAD was added up to 2 hours after TNF- stimulation
(data not shown). Similarly, with zVAD pretreatment, TNF- at and
above 1 ng/mL still induced neutrophil cell death in a dose-dependent manner (Figure 2B). Corroborating these results, TNF--induced cytosolic cytochrome c accumulation was enhanced with zVAD pretreatment (Figure 2C). In confirmatory experiments, the cleavage of a fluorogenic substrate for caspase-3-like proteases, Ac-DEVD-AMC, was abrogated in
cell lysates prepared from neutrophils incubated with TNF- and zVAD
(Figure 3A). Importantly, zVAD in and of
itself was not cytotoxic, because it clearly prolonged cell survival in
spontaneously aging and anti-Fas antibody-stimulated neutrophils
(Figure 1). Furthermore, the sensitization of neutrophils to
TNF--induced cell death was not limited to zVAD, because another
broad-spectrum, cell-permeable caspase inhibitor, Boc-D, produced a
similar effect (Figure 3B). Some specificity for broad-spectrum caspase
inhibition was suggested by the fact that zFF, a cell-permeable
cathepsin L-selective inhibitor, did not significantly alter
TNF--induced neutrophil cell death (Figure 3B). Taken together,
although it is possible that zVAD exerts its effect nonspecifically in
TNF--stimulated neutrophils, these experiments indicate that
broad-spectrum caspase inhibitors are not cytotoxic per se, and their
paradoxic effects in sensitizing to TNF--induced cell death is in
contrast to Fas agonist stimulation.
zVAD-sensitized, TNF- -stimulated neutrophils in vitro. Interestingly, under light
microscopy, whereas neutrophils incubated with zVAD alone appeared
normal morphologically and TNF--stimulated neutrophils exhibited
classic features of apoptosis, significant numbers of neutrophils
concurrently incubated with both of these reagents showed apoptoticlike
and necroticlike changes6 (Figure 4B). Examination by TEM
confirmed that zVAD-sensitized, TNF--stimulated neutrophils
developed atypical but apoptoticlike ultrastructural alterations
(Figure 4C). These included the loss of normal membrane ruffles and
"smoothing out" of the cell surface, extensive cytoplasmic
fragmentations, and formation of many membrane-bound bodies.
Additionally, features resembling cellular degranulation were
occasionally seen. Apoptoticlike nuclear changes were also noted
and included rounding up of the nuclear contour, dense condensation of
the nuclear chromatin, as well as fragmentation of the nuclear lobes.
Consistent with the results obtained by flow cytometry, most cells
appeared to maintain membrane integrity at early time points in spite
of the ultrastructural changes. While significant numbers of
necroticlike cells were also observed, they were less frequent and were
noted only later. Intriguingly, as demonstrated by DNA ladder formation
(Figure 5A) and cytoplasmic
oligonucleosome-associated histone assay (Figure 5B), we observed that
zVAD not only failed to attenuate DNA fragmentation but appeared to
augment it in several experiments. Similar enhancement of
TNF--induced DNA ladder formation by zVAD was previously reported
in the mouse fibroblast cell line NIH3T3.22 Furthermore,
Li et al reported that endonuclease G was released from mitochondria in
murine embryonic fibroblasts and directly resulted in nuclear DNA
ladder formation during TNF--induced cell death in the presence of
zVAD.35 Thus, these studies indicate that apoptoticlike
cell death processes can proceed despite blockade of caspases by
broad-spectrum inhibitors. Taken together, it appears that, during
broad-spectrum caspase inhibition, TNF--induced neutrophil cell
death occurred through processes that exhibited both apoptoticlike and
necroticlike features.
Protein synthesis inhibition alters cell death response in
TNF- can also extend neutrophil survival when
utilized at low concentrations (0.1 to 1 ng/mL)19 and/or when incubated with neutrophils for long periods (12 hours or more).16 These apparently diverse responses to TNF-
suggest certain functional heterogeneity in circulating neutrophils.
Alternatively, TNF- induces intracellular competing kinetics of
neutrophil death and survival signals that, on temporal balance,
determine cell fate.16,17,19 In an attempt to reconcile
some of the differences between the findings presented above and those
reported by others,17 time-course studies were compiled
over a 4-month period. They revealed that the net magnitude of
TNF--attributable decrease in cell survival relative to control,
measured by dual-parameter flow cytometry, occurred at 2 hours after
stimulation and remained statistically significant at 4 and 6 hours but
waned and became more variable (Figure
6A). In contrast, zVAD not only failed to significantly protect neutrophils from TNF--induced cell death at 2 hours after stimulation but highly significantly augmented it in a
sustained manner at 4 and 6 hours (Figure 6A). Of note, in some
experiments, zVAD appeared to confer cytoprotection in TNF--stimulated neutrophils. Nonetheless, paradoxic enhancement of
cell death still occurred at other time points in the same experiments.
Moreover, parallel assays invariably showed significant reduction in
total viable cell counts (data not shown), suggesting that a
significant proportion of cells underwent necroticlike cell
death in these experiments. However, because TNF- induces both cell
death and survival signals that are potentially complex, the effect of
zVAD could not be extrapolated outside of the time points and the in
vitro system examined. Thus, depending upon the time points assessed
and the particular cell death assays performed, divergent
interpretations could be reached.
Similar to other TNF- NF- B activation plays a critical
cytoprotective role during constitutive and TNF--stimulated
neutrophil cell death.17 To determine if zVAD exerted its
cell death-enhancing effect in TNF--stimulated neutrophils by
preventing NF-B activation, time-course studies of NF-B
activation were performed (Figure 7). As
observed in L929 and NIH3T3 cells in which broad-spectrum caspase
inhibitors also sensitized to TNF-induced cell
death,20,22,36 zVAD did not appreciably suppress NF-B
activation by TNF-.
NADPH oxidase is not required for zVAD enhancement of cell death in
TNF- In activated human neutrophils, the NADPH oxidase system assumes a
critical role as the generator of superoxide and hydrogen peroxide
during the respiratory burst.40 Patients with defects in
the components of this system manifest clinically with impairment in
host defense and develop chronic granulomatous disease.41 Importantly, several studies suggest that oxidants produced by this
system participate in constitutive and Fas and TNF receptor agonist-induced neutrophil cell death.3,19,42-44 However,
one study proposes that the role of intracellular oxidants as effectors during neutrophil cell death is context-dependent and that NADPH oxidase is not required for spontaneous and Fas activation-induced cell death.2 Two additional studies suggest that oxidants
do not play significant roles during TNF-
An increasing number of studies have described caspase-independent
cell death upon agonistic death receptor stimulation. However, few have
documented death receptor-mediated, caspase-independent cell death in
nontransformed primary mammalian cells. Thus far, human peripheral
blood T48,49 and B50 lymphocytes are the only
primary human cell types in which caspase-independent cell death has
been reported. Several observations suggest that the mechanisms for
executing caspase-independent cell death must also exist in circulating
mature human peripheral blood neutrophils. The most apparent is that,
although there is modest prolongation of survival afforded by
broad-spectrum, cell-permeable, irreversible caspase inhibitors,
neutrophils eventually undergo constitutive or Fas agonist-induced cell
death.3 Also, PMA induces rapid neutrophil cell death that
is characterized by minimal induction of caspase-3-like activities and
insensitivity to broad-spectrum caspase inhibition.2
Furthermore, PMA-induced neutrophil cell death is characterized by
morphologic features distinct from that classically described for
apoptosis and necrosis.2,11 Thus, perhaps, it was not
surprising that caspase-independent cell death mechanisms were also
involved in TNF- Several investigators have observed sensitization of death receptor
agonist-induced cell death by broad-spectrum caspase inhibitors in
transformed cell lines.20-24,37-39 Nevertheless, it is
important to emphasize that several distinct differences are noted in
our study. First, the sensitizing effect imposed by broad-spectrum caspase inhibition was observed for TNF- Second, the constellation of specific features of cell death occurring
in TNF- The third distinctive feature of this paradoxical cell death in
TNF- Our results regarding the paradoxical enhancement of cell death in
TNF-
Available data from nonprimate animal models of regional and systemic
inflammation suggest great promise for the caspases as therapeutic
targets in human diseases.60,61 Our studies show that
neutrophils undergo exaggerated apoptoticlike and necroticlike cell
death when stimulated by TNF- In conclusion, our data suggest that activation of the TNF receptor but
not the Fas receptor induces interacting, caspase-dependent and
-independent cell death mechanisms in human neutrophils. Although the
molecular basis remains to be elucidated, the paradoxical augmentation
of cell death in TNF-
We acknowledge Dr Douglas Bannerman, Dr Raymond Doty, and Dr Xianwu Li for their thoughtful inputs in the preparation of this manuscript.
Submitted December 18, 2001; accepted June 5, 2002.
Prepublished online as Blood First Edition Paper, June 28, 2002; DOI 10.1182/blood-2001-12-0266.
Supported by National Institutes of Health (NIH) SCOR HL30542 (J.M.H.), American Lung Association RG and American Heart Association 96004550 (P.I.C.), NIH R01 HL62995 (W.C.L.), and NIH R01 AI25606 (H.R.)
C.-Y.L. and A.T. contributed equally to the preparation of this manuscript.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Peter I. Chuang, Division of Pulmonary and Critical Care Medicine, Harborview Medical Center, Mail-stop 359762, 325 9th Ave, Seattle, WA 98104; e-mail: ichuang{at}u.washington.edu.
1. Dancey JT, Deubelbeiss KA, Harker LA, Finch CA. Neutrophil kinetics in man. J Clin Invest. 1976;58:705-715[Medline] [Order article via Infotrieve].
2.
Fadeel B, Ahlin A, Henter JI, Orrenius S, Hampton MB.
Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species.
Blood.
1998;92:4808-4818
3.
Khwaja A, Tatton L.
Caspase-mediated proteolysis and activation of protein kinase C
4.
Iwai K, Miyawaki T, Takizawa T, et al.
Differential expression of bcl-2 and susceptibility to anti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes, and neutrophils.
Blood.
1994;84:1201-1208
5.
Liles WC, Kiener PA, Ledbetter JA, Aruffo A, Klebanoff SJ.
Differential expression of Fas (CD95) and Fas ligand on normal human phagocytes: implications for the regulation of apoptosis in neutrophils.
J Exp Med.
1996;184:429-440 6. Leist M, Jaattela M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol. 2001;2:589-598[CrossRef][Medline] [Order article via Infotrieve].
7.
Sperandio S, de Belle I, Bredesen DE.
An alternative, nonapoptotic form of programmed cell death.
Proc Natl Acad Sci U S A.
2000;97:14376-14381 8. Formigli L, Papucci L, Tani A, et al. Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol. 2000;182:41-49[CrossRef][Medline] [Order article via Infotrieve]. 9. Kitanaka C, Kuchino Y. Caspase-independent programmed cell death with necrotic morphology. Cell Death Differ. 1999;6:508-515[CrossRef][Medline] [Order article via Infotrieve].
10.
Ma F, Zhang C, Prasad KV, Freeman GJ, Schlossman SF.
Molecular cloning of porimin, a novel cell surface receptor mediating oncotic cell death.
Proc Natl Acad Sci U S A.
2001;98:9778-9783 11. Takei H, Araki A, Watanabe H, Ichinose A, Sendo F. Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis or necrosis. J Leukoc Biol. 1996;59:229-240[Abstract].
12.
Liu J, Akahoshi T, Jiang S, et al.
Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3.
J Leukoc Biol.
2000;68:187-193
13.
Moulding DA, Quayle JA, Hart CA, Edwards SW.
Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival.
Blood.
1998;92:2495-2502
14.
Yamashita K, Takahashi A, Kobayashi S, et al.
Caspases mediate tumor necrosis factor- 15. Santos-Beneit AM, Mollinedo F. Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells. J Leukoc Biol. 2000;67:712-724[Abstract].
16.
Murray J, Barbara JA, Dunkley SA, et al.
Regulation of neutrophil apoptosis by tumor necrosis factor-
17.
Ward C, Chilvers ER, Lawson MF, et al.
NF-
18.
Weinmann P, Gaehtgens P, Walzog B.
Bcl-xl- and Bax-
19.
van den Berg JM, Weyer S, Weening JJ, Roos D, Kuijpers TW.
Divergent effects of tumor necrosis factor
20.
Vercammen D, Beyaert R, Denecker G, et al.
Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.
J Exp Med.
1998;187:1477-1485
21.
Khwaja A, Tatton L.
Resistance to the cytotoxic effects of tumor necrosis factor
22.
Luschen S, Ussat S, Scherer G, Kabelitz D, Adam-Klages S.
Sensitization to death receptor cytotoxicity by inhibition of Fas-associated death domain protein (FADD)/caspase signaling. Requirement of cell cycle progression.
J Biol Chem.
2000;275:24670-24678
23.
Foghsgaard L, Wissing D, Mauch D, et al.
Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor.
J Cell Biol.
2001;153:999-1010
24.
Vercammen D, Brouckaert G, Denecker G, et al.
Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.
J Exp Med.
1998;188:919-930 25. Grisham MB, Engerson TD, McCord JM, Jones HP. A comparative study of neutrophil purification and function. J Immunol Methods. 1985;82:315-320[CrossRef][Medline] [Order article via Infotrieve]. 26. Vuorte J, Jansson SE, Repo H. Evaluation of red blood cell lysing solutions in the study of neutrophil oxidative burst by the DCFH assay. Cytometry. 2001;43:290-296[CrossRef][Medline] [Order article via Infotrieve].
27.
Aprikyan AA, Liles WC, Park JR, Jonas M, Chi EY, Dale DC.
Myelokathexis, a congenital disorder of severe neutropenia characterized by accelerated apoptosis and defective expression of bcl-x in neutrophil precursors.
Blood.
2000;95:320-327 28. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980;68:251-306[Medline] [Order article via Infotrieve].
29.
Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger CP, Roos D.
Human neutrophils lose their surface Fc
30.
Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA.
Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.
Proc Natl Acad Sci U S A.
1995;92:7162-7166
31.
Yang J, Liu X, Bhalla K, et al.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science.
1997;275:1129-1132
32.
Renard P, Ernest I, Houbion A, et al.
Development of a sensitive multi-well colorimetric assay for active NF
33.
Zen K, Karsan A, Stempien-Otero A, et al.
NF- 34. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983;130:1910-1917[Abstract]. 35. Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature. 2001;412:95-99[CrossRef][Medline] [Order article via Infotrieve].
36.
Liu Y, Tergaonkar V, Krishna S, Androphy EJ.
Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor
37.
Li M, Beg AA.
Induction of necrotic-like cell death by tumor necrosis factor
38.
Boone E, Vanden Berghe T, Van Loo G, et al.
Structure/function analysis of p55 tumor necrosis factor receptor and Fas-associated death domain. Effect on necrosis in L929sA cells.
J Biol Chem.
2000;275:37596-37603
39.
Kim SO, Ono K, Han J.
Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages.
Am J Physiol Lung Cell Mol Physiol.
2001;281:L1095-L1105
40.
Babior BM.
NADPH oxidase: an update.
Blood.
1999;93:1464-1476
41.
Roos D, de Boer M, Kuribayashi F, et al.
Mutations in the X-linked and autosomal recessive forms of chronic granulomatous disease.
Blood.
1996;87:1663-1681
42.
Kasahara Y, Iwai K, Yachie A, et al.
Involvement of reactive oxygen intermediates in spontaneous and CD95 (Fas/APO-1)-mediated apoptosis of neutrophils.
Blood.
1997;89:1748-1753
43.
Watson RW, Rotstein OD, Jimenez M, Parodo J, Marshall JC.
Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils.
Blood.
1997;89:4175-4181 44. Lundqvist-Gustafsson H, Bengtsson T. Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils. J Leukoc Biol. 1999;65:196-204[Abstract].
45.
Kettritz R, Falk RJ, Jennette JC, Gaido ML.
Neutrophil superoxide release is required for spontaneous and FMLP-mediated but not for TNF
46.
Salamone G, Giordano M, Trevani AS, et al.
Promotion of neutrophil apoptosis by TNF- 47. Olejnicka BT, Dalen H, Brunk UT. Minute oxidative stress is sufficient to induce apoptotic death of NIT-1 insulinoma cells. APMIS. 1999;107:747-761[Medline] [Order article via Infotrieve].
48.
Dumont C, Durrbach A, Bidere N, et al.
Caspase-independent commitment phase to apoptosis in activated blood T lymphocytes: reversibility at low apoptotic insult.
Blood.
2000;96:1030-1038 49. Holler N, Zaru R, Micheau O, et al. Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol. 2000;1:489-495[CrossRef][Medline] [Order article via Infotrieve].
50.
Drenou B, Blancheteau V, Burgess DH, Fauchet R, Charron DJ, Mooney NA.
A caspase-independent pathway of MHC class II antigen-mediated apoptosis of human B lymphocytes.
J Immunol.
1999;163:4115-4124 51. Wong GH, Goeddel DV. Fas antigen and p55 TNF receptor signal apoptosis through distinct pathways. J Immunol. 1994;152:1751-1755[Abstract]. 52. Schulze-Osthoff K, Krammer PH, Droge W. Divergent signalling via APO-1/Fas and the TNF receptor, two homologous molecules involved in physiological cell death. EMBO J. 1994;13:4587-4596[Medline] [Order article via Infotrieve]. 53. Estelles A, Charlton CA, Blau HM. The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1- mediated caspase-8 activity and apoptosis. Dev Biol. 1999;216:16-28[CrossRef][Medline] [Order article via Infotrieve].
54.
Schotte P, Van Loo G, Carpentier I, Vandenabeele P, Beyaert R.
Lithium sensitizes tumor cells in an NF- 55. Dusi S, Della Bianca V, Donini M, Nadalini KA, Rossi F. Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton. J Immunol. 1996;157:4615-4623[Abstract].
56.
Monney L, Otter I, Olivier R, et al.
Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2.
J Biol Chem.
1998;273:6121-6131
57.
Nakagawa T, Yuan J.
Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis.
J Cell Biol.
2000;150:887-894
58.
Jones BE, Lo CR, Liu H, et al.
Hepatocytes sensitized to tumor necrosis factor-
59.
Blomgren K, Zhu C, Wang X, et al.
Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia. A mechanism of "pathological apoptosis"?
J Biol Chem.
2001;276:10191-10198 60. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature. 2000;407:810-816[CrossRef][Medline] [Order article via Infotrieve].
61.
Oberholzer C, Oberholzer A, Clare-Salzler M, Moldawer LL.
Apoptosis in sepsis: a new target for therapeutic exploration.
FASEB J.
2001;15:879-892
62.
Wakasugi K, Schimmel P.
Two distinct cytokines released from a human aminoacyl-tRNA synthetase.
Science.
1999;284:147-151
63.
Devireddy LR, Teodoro JG, Richard FA, Green MR.
Induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by IL-3 deprivation.
Science.
2001;293:829-834
64.
Suzuki K, Hasegawa T, Sakamoto C, et al.
Cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis: role in decreased responsiveness to inflammatory cytokines.
J Immunol.
2001;166:1185-1192
65.
Hoffmann PR, deCathelineau AM, Ogden CA, et al.
Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.
J Cell Biol.
2001;155:649-660
66.
Hirt UA, Gantner F, Leist M.
Phagocytosis of nonapoptotic cells dying by caspase-independent mechanisms.
J Immunol.
2000;164:6520-6529
67.
Brown SB, Clarke MC, Magowan L, Sanderson H, Savill J.
Constitutive death of platelets leading to scavenger receptor-mediated phagocytosis. A caspase-independent cell clearance program.
J Biol Chem.
2000;275:5987-5996 68. Chang NS, Pratt N, Heath J, et al. Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity. J Biol Chem. 2000;276:3361-3370[Medline] [Order article via Infotrieve]. 69. Wang X, Ono K, Kim SO, Kravchenko V, Lin SC, Han J. Metaxin is required for tumor necrosis factor-induced cell death. EMBO Rep. 2001;2:628-633[CrossRef][Medline] [Order article via Infotrieve].
70.
Vancompernolle K, Boonefaes T, Mann M, Fiers W, Grooten J.
Tumor necrosis factor-induced microtubule stabilization mediated by hyperphosphorylated oncoprotein 18 promotes cell death.
J Biol Chem.
2000;275:33876-33882
© 2003 by The American Society of Hematology.
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  A. Biragyn, M. Coscia, K. Nagashima, M. Sanford, H. A. Young, and P. Olkhanud Murine {beta}-defensin 2 promotes TLR-4/MyD88-mediated and NF-{kappa}B-dependent atypical death of APCs via activation of TNFR2 J. Leukoc. Biol., April 1, 2008; 83(4): 998 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Shan, R. B. Marchase, and J. C. Chatham Overexpression of TRPC3 increases apoptosis but not necrosis in response to ischemia-reperfusion in adult mouse cardiomyocytes Am J Physiol Cell Physiol, March 1, 2008; 294(3): C833 - C841. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Cross, R. J. Moots, and S. W. Edwards The dual effects of TNF{alpha} on neutrophil apoptosis are mediated via differential effects on expression of Mcl-1 and Bfl-1 Blood, January 15, 2008; 111(2): 878 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Wu, S. Chiocca, W. T. Beck, and Y.-Y. Mo Gam1-associated alterations of drug responsiveness through activation of apoptosis Mol. Cancer Ther., June 1, 2007; 6(6): 1823 - 1830. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. E. Steinberg and S. Grinstein Unconventional Roles of the NADPH Oxidase: Signaling, Ion Homeostasis, and Cell Death Sci. Signal., March 27, 2007; 2007(379): pe11 - pe11. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Daryadel, R. F. Grifone, H.-U. Simon, and S. Yousefi Apoptotic Neutrophils Release Macrophage Migration Inhibitory Factor upon Stimulation with Tumor Necrosis Factor-{alpha} J. Biol. Chem., September 15, 2006; 281(37): 27653 - 27661. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Martinet, G. R. Y. De Meyer, J.-P. Timmermans, A. G. Herman, and M. M. Kockx Macrophages but Not Smooth Muscle Cells Undergo Benzyloxycarbonyl-Val-Ala-DL-Asp(O-Methyl)-Fluoromethylketone-Induced Nonapoptotic Cell Death Depending on Receptor-Interacting Protein 1 Expression: Implications for the Stabilization of Macrophage-Rich Atherosclerotic Plaques J. Pharmacol. Exp. Ther., June 1, 2006; 317(3): 1356 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Kim and M.-S. Lee Essential Role of STAT1 in Caspase-Independent Cell Death of Activated Macrophages through the p38 Mitogen-Activated Protein Kinase/STAT1/Reactive Oxygen Species Pathway Mol. Cell. Biol., August 1, 2005; 25(15): 6821 - 6833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Parker, M. K. B. Whyte, S. K. Dower, and I. Sabroe The expression and roles of Toll-like receptors in the biology of the human neutrophil J. Leukoc. Biol., June 1, 2005; 77(6): 886 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Cowburn, J. F. White, J. Deighton, S. R. Walmsley, and E. R. Chilvers z-VAD-fmk augmentation of TNF{alpha}-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species Blood, April 1, 2005; 105(7): 2970 - 2972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Maianski, D. Roos, and T. W. Kuijpers Bid Truncation, Bid/Bax Targeting to the Mitochondria, and Caspase Activation Associated with Neutrophil Apoptosis Are Inhibited by Granulocyte Colony-Stimulating Factor J. Immunol., June 1, 2004; 172(11): 7024 - 7030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wang, X. Li, L. Wang, P. Ding, Y. Zhang, W. Han, and D. Ma An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression J. Cell Sci., March 15, 2004; 117(8): 1525 - 1532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Okada, S. Adachi, T. Imai, K.-i. Watanabe, S.-y. Toyokuni, M. Ueno, A. S. Zervos, G. Kroemer, and T. Nakahata A novel mechanism for imatinib mesylate-induced cell death of BCR-ABL-positive human leukemic cells: caspase-independent, necrosis-like programmed cell death mediated by serine protease activity Blood, March 15, 2004; 103(6): 2299 - 2307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. A. Holtz and K. L. O'Malley Parkinsonian Mimetics Induce Aspects of Unfolded Protein Response in Death of Dopaminergic Neurons J. Biol. Chem., May 23, 2003; 278(21): 19367 - 19377. [Abstract] [Full Text] [PDF]
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-stimulated neutrophils
Top
Abstract
Introduction
) under protocols
approved by the Human Subjects Review Committee, University of
Washington. Informed consent was provided according to the Declaration
of Helsinki. Following erythrocyte sedimentation with hetastarch
(Abbott Laboratories, Abbott Park, IL) or dextran T-500 (Amersham
Pharmacia Biotech, Piscataway, NJ), neutrophils were separated from the
leukocyte fraction over a discontinuous gradient (450g,
25°C, 30 minutes) utilizing dextran Ficoll-Paque Plus
(Amersham Pharmacia Biotech) as previously described.
Survival = TNFLive, the percentage of
TNF-
plays a central role in neutrophil apoptosis.
Blood.
1999;94:291-301
RIII and acquire annexin V binding sites during apoptosis in vitro.
Blood.
1995;85:532-540
or lipopolysaccharide.
J Biol Chem.
1999;274:28808-28815