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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 291-301
Caspase-Mediated Proteolysis and Activation of Protein Kinase C
Plays a Central Role in Neutrophil Apoptosis
By
Asim Khwaja and
Louise Tatton
From the Department of Haematology, University College London Medical
School, London, UK.
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ABSTRACT |
Neutrophils undergo constitutive apoptosis when aged ex vivo. Recent
studies have indicated roles for Fas/CD95 and the nicotinamide adenine
dinucleotide phosphate (NADPH)-oxidase system in this process. We have investigated the role of protein kinase C (PKC) in
neutrophil death. We show that there is proteolysis and activation of
the novel isoform PKC in aged neutrophils and that this process is
accelerated by the addition of an agonistic Fas antibody. PKC proteolysis occurs before the onset of any detectable features of
apoptosis and pharmacologic inhibition of this enzyme inhibits neutrophil apoptosis. PKC cleavage and activation is dependent on
caspase-8/FADD-like interleukin-1 converting enzyme
(FLICE)-mediated processing of caspase-3/CPP32.
Neutrophil survival is prolonged by the addition of broad spectrum
(BD.fmk) or caspase-8 targeted (zIETD.fmk) peptide caspase inhibitors.
Inhibition of PKC does not prevent apoptosis triggered by factor
withdrawal in immature hematopoietic cells, including normal human
CD34+ progenitors indicating that within a given lineage,
the mechanisms of apoptosis may be differentiation-stage-specific. Ex
vivo aging of neutrophils leads to the increasing production of
reactive oxygen species and this is attenuated in cells treated with
either caspase or PKC inhibitors. Proteolytically activated PKC
acts as a molecular link between the Fas/CD95 receptor and the
NADPH-oxidase system and plays a central role in regulating the process
of neutrophil apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NEUTROPHILS PLAY a central role in host
defense against infectious microorganisms. The normal human neutrophil
has a circulatory half-life of 6 to 10 hours and the production rate is
about 1.5 × 109 cells/kg body weight per
day.1 After migration into the tissues, cells either
undergo spontaneous or activation-induced death by apoptosis and are
recognized and ingested by resident macrophages.2 This
serves to limit any damage to host tissues after their death. Neutrophils incubated ex vivo undergo constitutive apoptosis with about
half the cells dying in the first 24 hours.3 Survival of
neutrophils in culture can be prolonged by incubation with cytokines
such as granulocyte-macrophage colony-stimulating factor (GM-CSF),4 granulocyte CSF (G-CSF),5 and
interleukin-8 (IL-8)6 and by inflammatory mediators such as
bacterial lipopolysaccharide.7
Recent studies have given some insight into the mechanisms involved in
neutrophil apoptosis. Liles et al8 showed that neutrophils express significant basal levels of cell surface Fas/CD95, and when
cultured, produce Fas ligand, which leads to cell death in an autocrine
or paracrine manner. The molecular mechanism of Fas/CD95-induced cell
death has been characterized in the last few years.9 After receptor ligation by ligand or agonistic antibody, there is recruitment and stabilization of an adaptor protein known as FADD (Fas-associated death domain protein).10 This interacts with Fas/CD95 via a region known as the death domain, which binds to a homologous section
of the intracytoplasmic tail of the receptor. FADD binds via its death
effector domain to a member of the cysteinyl aspartate specific
protease (caspase) family, FADD-like interleukin-1 converting enzyme
(FLICE) or caspase-8.11 This process leads to
auto-processing of procaspase-812 to generate an active
molecule that is capable of cleaving and activating downstream
caspases.13 These enzymes are believed to mediate many of
the processes that culminate in programmed cell death by cleaving
specific target proteins.14 An increasing number of these
substrates have been identified. Some are involved in cell-cycle
regulation and DNA repair, such as Rb and DNA-PK, others are structural
proteins such as nuclear lamins, and some are proteins involved in
signal transduction.15,16 The cleavage of some proteins by
caspases can lead to their inactivation,16 but in other
instances may lead to increased enzymatic activity. For example,
proteolysis leads to activation of hPAK65 (a p21-activated kinase),17 MEKK-1 (an upstream regulator of the JNK
pathway),15 and of protein kinase C.18
Ectopic transfection of constructs encoding these truncated forms can
lead to some of the morphologic appearance of
apoptosis.15,17,19 However, their role in apoptosis in
primary cells is largely uncertain. In particular, it is unclear whether specific enzyme activation plays a causal role in certain forms
of apoptosis, acts to facilitate the process of cell death once
apoptosis is triggered, or is merely a consequence of the execution program.
There is evidence that the production of reactive oxygen species (ROS)
via the neutrophil nicotinamide adenine dinucleotide phosphate
(NADPH)-oxidase system also plays an important role in apoptosis.
Neutrophil apoptosis is inhibited by hypoxia and by the addition of
agents such as catalase, which tend to detoxify the products of the
neutrophil oxidative burst.20-23 Neutrophils isolated from
patients with chronic granulomatous disease (CGD) show a decreased rate
of spontaneous cell death, as well as a reduction in apoptosis induced
by the addition of agonistic Fas/CD95 antibodies.24
Phagocytosis-induced apoptosis in neutrophils, a process regulated by
the CD11b/CD18 integrin, is also dependent on the production of
ROS.25
Less is known about the intracellular signaling molecules involved in
regulating neutrophil survival and apoptosis. Several groups have shown
that elevation of cyclic adenosine monophosphate (cAMP)
inhibits neutrophil apoptosis.26-28 Agents that elevate cAMP also inhibit neutrophil respiratory burst activity, but it is not
clear whether the effects of cAMP on preventing apoptosis are mediated
by this mechanism.29,30 Frasch et al31 have shown that neutrophil apoptosis in reponse to stress stimuli (such as
ultraviolet [UV] irradiation and hyperosmolarity) can be inhibited by
blocking activation of the p38 stress-activated kinase. However, spontaneous or Fas/CD95-induced apoptosis was not affected by inhibiting p38 activity. The protective effects of cytokines such as
GM-CSF and G-CSF involve tyrosine kinase activity,32,33 but
the relevant downstream pathways have not been identified.
To try and understand further the intracellular signalling pathways
that regulate neutrophil survival, we have investigated the role of
protein kinase C (PKC) in neutrophil apoptosis. The PKC family consists
of at least three groups of enzymes: the classical PKCs ( , , and
 ) that are dependent on diacylglycerol (DAG) and Ca2+
for activation; the novel group (,  ,  , and  ) dependent on DAG alone and the atypical PKCs ( and  / ) that do not require either DAG or Ca2+ for activation, but may be regulated by
3-phosphorylated inositides generated by the activity of
phosphoinositide 3-kinase(s).34-36 We show that in
neutrophils incubated ex vivo, there is caspase-dependent cleavage and
activation of the nPKC that precedes the onset of the earliest
features of apoptosis. Prevention of PKC proteolysis by the use of
peptide caspase inhibitors or pharmacologic inhibition of PKC
strongly inhibits spontaneous and Fas/CD95-induced apoptosis by
decreasing the production of reactive oxygen species. These effects are
not seen in myeloid precursor cells before terminal differentiation.
The results suggest an important role for cleavage and activation of
PKC in neutrophil apoptosis and provide a molecular link between the
Fas/CD95 and NADPH-oxidase pathways that are recognized as key
participants in neutrophil programmed cell death.
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MATERIALS AND METHODS |
Materials.
The PKC inhibitors bisindolylmaleimide (GF109203X) and Gö6976
were obtained from Calbiochem (San Diego, CA). Caspase
inhibitors BD-fmk, zIETD-fmk, and zVDVAD-fmk were from Enzyme Systems
Products (Dublin, CA) The phosphoinositide (PI) 3-kinase inhibitor
LY294002 was from Biomol (Plymouth Meeting, PA). Recombinant human (rh) G-CSF was donated by Chugai Pharmaceuticals (London, UK), rhGM-CSF by
Behringwerke/Hoechst (Marburg, Germany) and rhIL-3 by Sandoz (Frimley
Park, UK). rhIL-6 and stem cell factor were from Peprotech (Rocky Hill,
NJ). PKC antibodies were as follows: polyclonal anti-PKC (C-terminal) from Sigma (St Louis, MO); monoclonal PKC
(recognizes both I and II), , , , and monoclonal
antibodies (MoAbs) were from Transduction Labs (Santa Cruz, CA) and
polyclonal antibodies (C-terminal) to PKCII, PKC, and PKC
were from Santa Cruz (Santa Cruz, CA). A polyclonal ERK2 antibody was
from Santa Cruz. Agonistic anti-Fas/CD95 (clone CH11) was from UBI
(Lake Placid, NY).
Neutrophil isolation, culture, and annexin V binding assay for
apoptosis.
Neutrophils from normal volunteers were isolated by dextran
sedimentation, Ficoll hypaque density gradient (Amersham Pharmacia, Uppsala, Sweden) and hypotonic lysis of contaminating red
blood cells as described previously.37 Cells
were finally resuspended in RPMI 1640 supplemented with 1%
heat-inactivated fetal calf serum (FCS) and incubated at a density of
106 cells per mL in 24-well tissue-culture plates. At
stated time points, cells were washed once with phosphate-buffered
saline (PBS) and used in apoptosis quantitation assays. Fluorescein
isothiocyanate (FITC)-conjugated annexin V (Boehringer Mannheim,
Mannheim, Germany) was used according to the manufacturer's
instructions. Analysis of annexin V binding was performed by flow
cytometry (Epics; Coulter, Hialeah, FL)38 and peak
fluorescence of positive cells was at least 2 logs greater than the
negative fraction.
Cell lysis, gel electrophoresis, and immunoblotting.
Before cell lysis, neutrophils or HL-60 cells were washed once in PBS
and incubated in 1 mmol/L diisopropylfluorophosphate (DIFP) for 5 minutes on ice. Cells were pelleted by centrifugation and resuspended
in lysis buffer (50 mmol/L HEPES pH 7.5, 100 mmol/L NaCl, 1% Triton
X-100, 1 mmol/L EDTA, 1 mmol/L EGTA, 20 mmol/L NaF, 1 mmol/L Na
orthovanadate, 1 mmol/L Pefabloc, 10 µg/mL each of aprotinin,
pepstatin, and leupeptin). After 5 minutes on ice, cells were
centrifuged at 13,000g at 4°C for 10 minutes, assayed for
protein concentration (Bio-Rad, Richmond, CA) and the
supernatant added to boiling sample buffer and boiled for 10 minutes.
Equivalent protein amounts were loaded for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10%
acrylamide gels followed by transfer to nitrocellulose membranes
(Hybond C; Amersham, Arlington Heights, IL). Primary
antibodies were incubated with the nitrocellulose membrane for 60 minutes at room temperature at the following dilutions: anti-PKC
(C-terminal) 1:5,000; monoclonal PKC (recognizes both I and
II) 1:1,000; polyclonal (C-terminal) PKCII 1:1,000;
anti-PKC 1:500; anti-PKC (C-terminal) 1:2,000; anti-PKC
1:1,000; anti-ERK2 1:500.
Neutrophil fractionation into apoptotic/nonapoptotic.
Neutrophils were incubated ex vivo for 8 hours as described above.
Cells were washed once in annexin V binding buffer (150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L CaCl2,
10 mmol/L HEPES, pH 7.4) and resuspended in 90 µL of binding buffer plus 10 µL of annexin V microbeads (kind gift of Miltenyi Biotech, Bergisch Gladbach, Germany) per 107 cells. Cells were
incubated for 15 minutes at 12°C and then washed once in a large
volume of binding buffer. Cells were loaded onto a mini-MACS column,
washed with binding buffer, and both positively selected (by magnet)
and flow-through cells collected. Cell lysates were made as described
above, protein concentrations quantitated, and equal amounts of protein
loaded for Western blotting. The purity of cells was verified by using
annexin V-FITC and in the magnetically selected fraction 94% ± 3% of cells were annexin V positive and in the flow-through
fraction 4% ± 2% were positive.
PKC kinase assay.
At appropriate time points, 5 × 106 neutrophils were
processed and Triton X-100 lysates prepared as described above.
Immunoprecipitation of PKC isoforms was performed by the addition of 1 µg of a polyclonal anti-C-terminal antibody for 45 minutes and
protein A-agarose for 45 minutes. Immunoprecipitates were washed twice
with lysis buffer and once with kinase buffer (20 mmol/L Tris.Hcl, pH
7.5, 10 mmol/L MgCl2). Samples were resuspended in a kinase
mixture of 50 µL containing kinase buffer plus 20 µmol/L adenosine
triphosphate (ATP), 5 µg H1 histone, and 2 µCi
[-32P]ATP (Amersham). Incubation was for 5 minutes at
30°C and was terminated by the addition of sample buffer and
boiling for 10 minutes. Phosphorylated proteins were separated by
SDS-PAGE, analyzed by autoradiography, and phosphorylation of substrate
histone H1 quantitated using NIH Image software (NIH, Bethesda, MD). In
experiments evaluating the effects of caspase inhibitors or agonistic
Fas antibody on PKC activity, inhibitors were present throughout the
cell culture period, but were not added further to the
immunoprecipitated PKC. In experiments examining the effects of PKC
inhibitors, GF109203X and Gö6976, these were present throughout
the culture period at a concentration of 1 µmol/L; due to the
competitive, reversible nature of these inhibitors, further compound
was added to a final concentration of 500 nmol/L to the kinase mixture
before the addition of ATP.
In vitro cleavage of PKC by caspases.
Purified recombinant caspases 3 and 8 and PKC were from Pharmingen
(San Diego, CA). A total of 100 ng of PKC was incubated with 10 ng
of caspase-3 or 150 ng of caspase-8 in a final volume of 50 µL of
assay buffer (20 mmol/L 1,4 piperazinediethanesulfonic acid [PIPES],
pH7.2, 100 mmol/L NaCl, 10 mmol/L dithiothreitol [DTT],
1 mmol/L EDTA, 0.1% 3-[(chlolamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS), 10% sucrose) for 2 hours at 37°C. Parallel tubes were incubated with 1 µmol/L AcDEVD-CHO (Bachem, Saffron Walden, UK) as a caspase inhibitor. The reaction was terminated by the
addition of sample buffer and proteins were analyzed by SDS-PAGE and
immunoblotting using the anti-PKC antibody.
CD34 isolation/culture and culture of HL-60 and Jurkat cell lines.
Peripheral blood CD34+ cells were collected and purified
from individuals with relapsed/resistant lymphomas undergoing stem cell
mobilization for subsequent use after high-dose cytotoxic therapy.
Cells were isolated and purified by immunoaffinity techniques as
described. CD34+ cells surplus to defined clinical
requirements were used for experimental purposes using a protocol with
local ethics committee approval. A second round of selection using a
mini-MACS column as per the manufacturer's instructions was performed
and yielded cells with 93% purity. Factor withdrawal-induced apoptosis
was measured in freshly purified cells by incubation in a defined serum
free medium (BIT; Stem Cell Technologies, Vancouver, Canada). To
differentiate CD34+ cells, they were cultured in Iscove's
modified Dulbecco's medium (IMDM)/20% FCS plus recombinant IL-3,
IL-6, and stem cell factor (all at 20 ng/mL) for 7 days and then the
same mixture supplemented with G-CSF (50 ng/mL) for a further 7 days.
Factor withdrawal-induced apoptosis was measured after extensive
washing and resuspension in serum-free medium. HL-60 and Jurkat cells
were maintained in RPMI plus 10% fetal bovine serum.
Measurement of intracellular hydrogen peroxide.
Neutrophils were incubated in culture conditions as described above. At
stated time points, cells were incubated in 5 µmol/L dichlorofluorescein diacetate (DCF-DA) for 60 minutes and then analyzed
by flow cytometry. To control for variations in background fluorescence
or dye uptake with time, parallel samples were incubated with the
flavoprotein inhibitor diphenylene iodonium (DPI; Sigma) at 20 µmol/L
for 15 minutes before DCF-DA addition. DPI inhibits oxidase activity,
and in pilot experiments, inhibited the f-met-leu-phe-induced activation of the respiratory burst by over 94%. For each experimental condition, the DPI/DCF-DA sample was used as the control to which the
DCF-DA alone sample was corrected. Hydrogen peroxide activity was
expressed as the ratio of the mean channel fluorescence DCF-DA alone:DPI/DCF-DA. To verify that the addition of caspase inhibitors (BD.fmk, VDVAD.fmk, and IETD.fmk) did not directly affect the DCF-DA
assay, they were incubated with neutrophils subsequently stimulated
with phorbol ester to activate the respiratory burst. None of the
caspase inhibitors reduced the response to phorbol ester in the DCF-DA assay.
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RESULTS |
The PKC inhibitor bisindolylmaleimide inhibits neutrophil apoptosis.
Neutrophils undergo spontaneous apoptosis when incubated ex vivo
(Fig 1) with survival of 57% ± 4% at
24 and 25% ± 5% at 48 hours. As has been reported previously,
neutrophil survival is promoted by incubation with G-CSF.5
Addition of the bisindolylmaleimide PKC inhibitor,
GF109203X,39,40 also maintains viability with survival of
78% ± 3% and 65% ± 5% at 24 and 48 hours,
respectively (Fig 1), suggesting that activation of PKC is causally
involved in neutrophil apoptosis. To investigate which member(s) of the PKC family could be involved in this process, we examined the expression of PKC isoforms by immunoblotting with specific antibodies. Comparison of PKC expression in neutrophils was made with the myeloid
cell line HL60, which can be induced to differentiate along the
granulocytic pathway (Fig 2).41
Of the classical PKCs, neutrophils expressed significant levels of
PKC (using an antibody that recognizes both I and II
isoforms), a low level of PKC and PKC was not expressed in either
HL60s or neutrophils. PKC was the only novel PKC isoform detected.
Although HL60 cells express both and / atypical PKCs,
neutrophils express a low level of PKC only. These results are
consistent with those published by other groups.42-44
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| Fig 1.
Effect of the PKC inhibitor, GF109203X, on neutrophil
apoptosis. (A) Neutrophils were incubated in the absence (control) or
presence of GF109203X (1 µmol/L), G-CSF (50 ng/mL), or GM-CSF (10 ng/mL) and apoptosis quantified by annexin V binding/flow cytometry at
24 and 48 hours. Mean ± standard error (SE) of 11, 10, and 8 separate
experiments for GF109203X, G-CSF, and GM-CSF, respectively. (B)
Concentration response curve for effects of GF109203X on neutrophil
survival. Cell death was measured at 24 hours by annexin V binding and
flow cytometry. Mean ± SE of three separate experiments.
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| Fig 2.
PKC isoforms expressed in neutrophils and HL-60 cells.
Cell lysates from unstimulated freshly isolated neutrophils (PMN) or
HL60 cells (20 µg of total protein were loaded per lane) were
immunoblotted with various antibodies specific for PKC isoforms.
Isoforms that were probed for, but not detected, in either cell type
were PKC,  , . Neutrophil/HL60 expression of the ERK2 MAPkinase
is shown for comparison. Lysates from one of two separate experiments
analyzed are shown.
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Inhibition of classical PKCs does not affect neutrophil survival.
GF109203X has been shown to inhibit classical and novel PKCs, but to
have no effect on the kinase activity of the atypical PKC.40,45 PKC activity is known to be regulated by
3-phosphorylated inositides generated by the action of phosphoinositide
3-kinase.35 Incubation with the PI 3-kinase inhibitor,
LY294002,46 at 20 µmol/L did not attenuate neutrophil
apoptosis (control 57% ± 6%, LY294002 60% ± 6% survival at
24 hours, n = 6), further suggesting that PKC is unlikely to play a
significant role in neutrophil apoptosis. In control experiments, this
concentration of LY294002 abolished the activation of the PI 3-kinase
target Akt/protein kinase B in response to GM-CSF, and this inhibitory
effect was maintained for at least 24 hours (data not shown).
Therefore, the antiapoptotic effects of GF109203X in neutrophils could
be attributed to inhibition of the cPKCs and/or or of the
nPKC. The PKC inhibitor, Gö6976, has been shown to inhibit
classical PKCs, but to have a limited effect on PKC
activity.45,47 Incubation with Gö6976 at
concentrations up to 10 µmol/L did not inhibit neutrophil apoptosis
(control 78% ± 2%, Gö6976 78% ± 3% survival at 24 hours, n = 3). Gö6976 (5 µmol/L) was functional under these
conditions as shown by a 68% inhibition of phorbol ester-stimulated
neutrophil hydrogen peroxide production as detected by the flow
cytometric DCF assay. These results suggest that the effects of
GF109203X on neutrophil survival are likely due to inhibition of PKC activity.
PKC undergoes proteolytic cleavage and activation in neutrophils
before the development of features of apoptosis.
Induction of apoptosis in certain cell lines has been shown to result
in the proteolytic cleavage of PKC leading to the generation of an
active catalytic (C-terminal) fragment.18 Immunoblotting with an antibody raised to the C-terminal portion of PKC showed that
a significant proportion of neutrophil PKC is proteolytically cleaved by 8 hours of incubation and that only the C-terminal fragment
(and no full-length protein) can be detected at 20 hours (Fig 3). In contrast, using an
anti-C-terminal antibody to PKCII, which is the major neutrophil
isoform,43,48 there was no detectable proteolysis of
PKCII between 0 and 20 hours of neutrophil incubation. Immunoblotting with an MoAb that recognizes both PKC isoforms showed
no loss of full-length protein for up to 20 hours (data not shown).
Immunoblotting with an anti-C-terminal PKC antibody showed the
presence of a lower molecular weight fragment, but this was present in
freshly isolated neutrophils and did not alter with aging in culture.
The degree of PKC proteolysis was not significantly affected by
incubation with GF109203X.
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| Fig 3.
Incubation of neutrophils ex vivo leads to the generation
of a C-terminal proteolytic fragment of PKC. Lysates prepared from
neutrophils aged for the indicated times. Cells (20 µg of total
protein loaded per lane) were analyzed by immunoblotting using an
antibody that recognizes the C-terminal end of PKC (A) or antibodies
to the C-terminal portion of PKCs  and II (B).
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Annexin V binding (a marker of apoptosis) indicated that only 6% ± 3% and 45% ± 5% of neutrophils were apoptotic at 8 and 20 hours,
respectively, suggesting that PKC proteolysis precedes the onset of
cell death. To verify this, annexin V-coated magnetic microspheres
were used to separate neutrophils into annexin V negative and positive
fractions and whole-cell lysates prepared for immunoblotting.
Figure 4 shows that there is a significant proportion of cleaved PKC in the annexin V-negative cells and only
the C-terminal fragment can be found in annexin V-positive cells.
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| Fig 4.
Separation of neutrophils using annexin V-coated
magnetic microspheres shows that PKC proteolysis precedes any
detectable features of apoptosis. (A) Neutrophils were incubated for 12 hours ex vivo and then separated by using annexin V-coated
microspheres, which bind to cells with external exposure of membrane
phosphatidylserine, an early marker of apoptosis. Subsequent staining
with FITC-conjugated annexin V and flow cytometry shows negative cells
in the upper panel and positively selected cells in the lower panel.
(B) Lysates prepared from the two fractions were analyzed by
immunoblotting with anti-PKC antibody. Equal amounts of protein were
loaded: lane 1, annexin V negative fraction; lane 2, annexin V
positive. This experiment was performed twice with similar results.
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There is increased PKC catalytic activity in neutrophils incubated
ex vivo.
In view of the detection of a C-terminal PKC fragment, which could
have altered activity, kinase assays were performed on immunoprecipitated PKC. Figure 5 shows
that there is spontaneous activation of PKC detectable at 8 hours of
incubation. This activation can be blocked by incubation with the
cPKC/nPKC inhibitor, GF109203X, but not with the cPKC inhibitor,
Gö6976. In contrast, there was no increase in the activity of
PKC or PKC at 8 hours. The basal level of activity of PKC and
PKC was inhibited by Gö6976 confirming the activity of this
inhibitor.
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| Fig 5.
Measurement of PKC kinase activity in aged neutrophils.
(A) Neutrophils were incubated in the absence (t8h) or presence of the
caspase-8 inhibitor, IETD.fmk (25 µmol/L), agonistic anti-Fas
antibody (500 ng/mL), PKC inhibitor, GF109203X (1 µmol/L), or PKC
inhibitor, Gö6976(1 µmol/L) for 8 hours. PKC was then
immunoprecipitated using an anti-C-terminal antibody from neutrophil
lysates and subjected to a kinase assay using histone H1 as a
substrate. Due to the competitive, reversible nature of the PKC
inhibitors (which would wash out during the immunoprecipitation
protocol), further compound was added to a final concentration of 500 nmol/L to the kinase mixture before the addition of ATP.
Phosphorylation of substrate was quantified after autoradiography and
normalized to the time 0 value (100%). Results are mean ± SE of
three separate experiments. (B) Representative autoradiograph from a
PKC kinase assay. (C) PKCs and II were immunoprecipitated
using anti-C-terminal antibodies after neutrophil aging for 8 hours in
the absence or presence of the PKC inhibitor, Gö6976, and
subjected to a kinase assay using histone H1 as a substrate as detailed
above. Mean ± SE of three separate experiments.
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Spontaneous and agonistic Fas antibody-induced proteolysis and
activation of PKC is inhibited by peptide caspase inhibitors.
Previous studies have indicated a role for Fas/CD95 in neutrophil
apoptosis. Incubation of neutrophils with an agonistic Fas antibody led
to more rapid death compared with control cells
(Fig 6) with over 30% of neutrophils
undergoing apoptosis within 8 hours. Proteolysis of PKC was
detectable by 2 to 4 hours of incubation with the anti-Fas antibody
(Fig 6B), and there was a slight increase in PKC kinase activity at
8 hours compared with control cells (Fig 5).
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| Fig 6.
Effects of an agonistic anti-Fas/CD95 antibody on PKC
proteolysis and neutrophil apoptosis. (A) Neutrophils were incubated in
the absence (control) or presence of agonistic Fas/CD95 antibody (500 ng/mL), PKC inhibitor, GF109203X (1 µmol/L), and caspase-8 inhibitor,
IETD.fmk (25 µmol/L) and cell survival measured by annexin V binding
using a flow cytometric assay. Mean ± SE of three separate
experiments. (B) Neutrophils were incubated with agonistic Fas/CD95
antibody (500 ng/mL) with or without preincubation with the caspase-8
inhibitor, IETD.fmk (25 µmol/L) for the indicated times. Lysates were
prepared, equal amounts of protein (20 µg) loaded per lane, and
immunoblotted with an anti-C-terminal PKC antibody. (C) Neutrophils
were incubated with agonistic Fas/CD95 antibody (500 ng/mL) for the
indicated times. Lysates were prepared, equal amounts of protein (20 µg) loaded per lane, and immunoblotted with anti-C-terminal
antibodies to PKC or PKC.
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The effect of cell permeable peptide inhibitors of caspase family
members14 on PKC proteolysis and neutrophil survival was
measured. Figure 7A shows that PKC
proteolysis is markedly inhibited by the broad spectrum caspase
inhibitor, boc.aspartyl.fmk (BD.fmk),14,49 and by the
caspase-8 inhibitor, IETD.fmk,50 but not by another
inhibitor designed to target caspase-2, zVDVAD.fmk.51 Caspase-8 is recruited to the intracytoplasmic domain of oligomerized Fas/CD95 via its association with the adaptor protein
FADD.11 Previous work has shown that PKC can be cleaved
by the action of caspase-319; the caspase-8 inhibitor
IETD.fmk could potentially block PKC proteolysis in intact cells by
preventing the processing of caspase-3 by caspase-8. Alternatively,
caspase-8 could also cleave PKC directly. We incubated purified
active caspases 3 and 8 with purified recombinant PKC and found that
only caspase-3 was capable of cleaving PKC, indicating that PKC
is not a direct target of caspase-8 (Fig 7B). Neutrophil survival was
enhanced in the presence of both BD.fmk and IETD.fmk (Figs 6 and 7C).
VDVAD.fmk had no effect. IETD.fmk also inhibited anti-Fas-induced
PKC proteolysis and apoptosis (Figs 6B and 7D).
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| Fig 7.
Role of caspases in PKC proteolysis and neutrophil
survival. (A) Effect of caspase inhibitors on PKC proteolysis in
neutrophils. Neutrophils were either lysed immediately after
preparation (t0 con) or after 8 hours incubation in the absence (cont)
or presence of broad spectrum caspase inhibitor, BD.fmk, the caspase-8
inhibitor, IETD.fmk (both at 25 µmol/L), the caspase-2 inhibitor,
VDVAD.fmk (50 µmol/L), or with agonistic Fas antibody (500 ng/mL) as
indicated and immunoblotted with anti-PKC antibody. (B) Effect of
recombinant caspases on PKC proteolysis. Purified recombinant
caspase-3 or caspase-8 was incubated with recombinant PKC ± caspase inhibitor DEVD-CHO and proteolysis estimated by immunoblotting
with an anti-PKC antibody. For comparison, a lysate from 8-hour aged
neutrophils is shown (PMN). (C) Effect of caspase inhibitors on
neutrophil survival. Neutrophils were incubated in the absence (cont)
or presence of the broad spectrum caspase inhibitor, BD.fmk, the
caspase-8 inhibitor, IETD.fmk (both at 25 µmol/L), or the caspase-2
inhibitor, VDVAD.fmk (50 µmol/L) for 20 hours and survival measured
by annexin V binding and flow cytometry. Mean ± SE of four separate
experiments. (D) Effect of the caspase-8 inhibitor, IETD.fmk, and the
PKC inhibitor, GF109203X, on apoptosis induced by an agonistic Fas
antibody. Neutrophils were preincubated with IETD.fmk (25 µmol/L) or
GF109203X (1 µmol/L) for 15 minutes and then anti-Fas antibody added
(500 ng/mL). Cell survival was measured by annexin V binding in a flow
cytometric assay. Mean ± SE of three separate experiments.
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GF109203X does not inhibit apoptosis in immature hematopoietic cells.
Preincubation with GF109203X decreased the proapoptotic effects of
anti-Fas antibody on neutrophils (Fig 7D). In contrast, incubation of
Jurkat T-cells with GF109203X before the addition of anti-Fas antibody
did not prevent apoptosis with survival of 24% in anti-Fas-treated
cells and 11% in GF109203X+anti-Fas cells (measured at 24 hours). In
Jurkat cells, PKC proteolysis was readily detectable by
immunoblotting within 4 hours of Fas stimulation (data not shown). We
further investigated the effect of GF109203X on apoptosis triggered by
growth factor withdrawal in more primitive hematopoietic cells.
GF109203X had no protective effect on factor-withdrawal-induced apoptosis in primary human CD34+ cells (survival at 24 hours in control cells 60%; +GF109203X, 58%). In addition,
CD34+ cells were differentiated ex vivo in a growth factor
mixture including G-CSF.52 After 14 days, when over 80% of
cells were myelocytes and metamyelocytes, growth factors were removed.
Under these conditions, PKC inhibition did not ameliorate apoptosis (control, 47%; GF109203X, 50%). These results indicate that the effect of GF109203X on cell death is restricted to terminally differentiated neutrophils.
GF109203X and the caspase inhibitor IETD.fmk inhibit spontaneous
production of reactive oxygen species in neutrophils incubated ex vivo.
There is increasing evidence that the production of ROS plays a
critical role in neutrophil apoptosis; in particular,24 neutrophils from patients with chronic granulomatous disease show prolonged neutrophil survival ex vivo. We used the DCF-DA fluorescent probe coupled with flow cytometry37 to look at the effects
of GF109203X on neutrophil production of ROS.
Figure 8 shows that there was a sustained
increase in the spontaneous production of ROS in neutrophils incubated
ex vivo and that this was inhibited by incubation with the PKC
inhibitor, GF109203X. Incubation with the inhibitor, Gö6976, did
not significantly affect ROS production (control, 2.1 ± 0.2-fold
increase; Gö6976, 2.43 ± 0.1-fold; duplicate measurements in
two separate experiments; mean ± standard deviation [SD]).
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| Fig 8.
The effect of various agents on neutrophil production of
ROS. Freshly isolated neutrophils were incubated in the absence
(control) or presence of the caspase-8 inhibitor, IETD.fmk (25 µmol/L), the caspase-2 inhibitor, VDVAD.fmk (50 µmol/L), the PKC
inhibitor, GF109203X, or an agonistic Fas antibody (500 ng/mL).
Relative production of ROS was measured by a flow cytometric assay
(DCF-DA) at the indicated time points. Mean ± SE of three separate
experiments for VDVAD.fmk and four separate experiments for the rest.
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Incubation with the caspase-8 inhibitor, IETD.fmk, was also associated
with a reduction in production of ROS, and this effect was not seen in
cells incubated with the caspase-2 inhibitor, VDVAD.fmk, mirroring the
effects of these inhibitors on neutrophil survival (Fig 7). In two
separate experiments, incubation of neutrophils with the broad spectrum
caspase inhibitor, BD.fmk, led to a decrease in ROS production compared
with control cells (mean increase at 8 hours in control cells 2.1 ± 0.2 and in BD.fmk-treated cells 1.51 ± 0.1). Production of ROS was
more pronounced in neutrophils incubated with an agonistic anti-Fas
antibody (Fig 8). These results suggest that in this context ROS
production is dependent on caspase activity, which is likely to be
triggered via activation of the Fas/CD95 pathway.
| |
DISCUSSION |
In this study we have evaluated the role of PKC in neutrophil apoptosis
and showed that the novel PKC isoform PKC plays an important part in
mediating cell death. Neutrophils isolated from normal individuals and
incubated ex vivo undergo constitutive apoptosis with about half of the
cells dying in the first 24 hours. Other investigators have shown that
this phenomenon can be inhibited by incubation with a variety of growth
factors and cytokines.4-7 We show that incubation with the
PKC inhibitor, GF109203X, also inhibits this form of cell death to
levels comparable to cytokines such as G-CSF with only about 20% of
cells undergoing apoptosis in the first 24 hours. Of the three major
PKC isoenzyme groups, we show that neutrophils contain the classical
PKCs and , the novel PKC, and the atypical PKC. This
distribution of PKCs is similar to that reported by other
groups.42-44
The early development of PKC inhibitors was based on compounds such as
staurosporine and H7, which act as catalytic domain inhibitors and are
competitive with respect to ATP binding.40 Although these
were effective, they lacked specificity. Staurosporine can, for
example, inhibit many other types of kinases such as ribosomal S6
kinase, c-AMP-dependent kinase, and the src tyrosine kinase.40 The bisindolylmaleimide inhibitors are synthetic
compounds based on staurosporine and have yielded potent and selective
PKC inhibitors.39 However, these compounds vary in their
potency against different PKC isoforms. The GF109203X compound used in this study has been shown to effectively inhibit the cPKCs and and the nPKCs and at concentrations in the nmol/L range; the
50% inhibitory concentration (IC50) for PKC is much higher at about
6 µmol/L.45 This specificity coupled with the
distribution of PKC isoforms in neutrophils suggested that the effects
of GF109203X on neutrophil survival could be a result of its effects on
either the novel isoform PKC or the classical isoforms PKC/.
We used the properties of another inhibitor,
Gö6976,45 to discriminate between these
possibilities. This inhibitor has been shown to inhibit the cPKC
isoforms in the low nmol/L range, but does not affect the activity of
PKC at concentrations up to 3 µmol/L.45 This failed to
have any effect on neutrophil survival, suggesting that PKC was the
most likely isoform responsible for the proapoptotic effects seen.
In 1995, Emoto et al showed that in U937 cells, induction of apoptosis
in response to ionizing radiation was associated with proteolytic
cleavage of PKC leading to the generation of an approximately 40 kD C-terminal fragment.18 This was
catalytically activated due to the loss of the N-terminal inhibitory
domain. Subsequent work from the same group showed that this cleavage
(at aspartate 330) could be reproduced in vitro using purified
caspase-3/CPP32.19 Transfection and transient
overexpression of the PKC catalytic fragment could induce an
apoptotic morphology in HeLa and NIH3T3 cells.19 These
results suggested that PKC activation by proteolysis could be a
participant in the apoptotic program and not just a consequence of the
execution phase. Proteolytic activation of PKC has been implicated
in several models of apoptosis. Caspase-3-dependent activation of
PKC and PKC in response to a variety of apoptotic stimuli was
reported by Shao et al.53 Mizuno et al54 have shown that in human leukemic cell lines triggered to undergo
Fas-mediated apoptosis, the nPKCs, including PKC, undergo limited
proteolytic cleavage, and that this parallels the activation of
caspase-3 and apoptosis. No data showing the effects of PKC inhibition
were reported in this report, but in certain cell types, phorbol ester addition alone could induce apoptosis.
More recently, Denning et al55 have reported that in human
keratinocytes UV radiation-induced apoptosis is associated with the
proteolytic activation of PKC and that cell death can be inhibited
by GF109203X preincubation. PKC proteolytic activation has also been
implicated in cerebellar granule cell death.56 We have
shown in this report that inhibition of PKC activity with GF109203X
does not inhibit all forms of apoptosis, or indeed even all forms of
apoptosis triggered by Fas ligation in different cell types. It has
recently been shown that induction of apoptosis by Fas can lead to the
cleavage of a number of signalling proteins.57 It is likely
that caspase-mediated cleavage of different proteins may be critical
for the apoptotic phenotype in various cell types and that this may be
influenced by their extracellular environment. In neutrophils, cleavage
and activation of PKC appears to play a crucial role in mediating
apoptosis, but this may not be a rate-limiting step in other cell types.
Neutrophils undergoing spontaneous apoptosis show a rapid cleavage of
PKC such that a significant proportion is cleaved at 8 hours and
almost all by 20 hours. In contrast, there is no evidence of
proteolysis of PKC or PKC over this time course. The proteolytic changes in PKC are accompanied by increased enzyme activity and precede detectable features of apoptosis. At the 8-hour time point, only about 5% of cells display any features of apoptosis. One of the
earliest markers of apoptosis is the externalization of plasma membrane
phosphatidylserine (PS) detectable by the binding of annexin
V.58-60 This PS exposure precedes the morphologic
appearances of apoptosis, changes in membrane permeability to agents
such as trypan blue and propidium iodide, or the characteristic DNA fragmentation. Functionally PS exposure is one of the mechanisms by
which apoptotic cells are recognized by macrophages and targeted for
ingestion.61 Separation of annexin V negative and positive neutrophil fractions using magnetic microspheres showed that annexin V
negative cells contain a significant proportion of the catalytic PKC
fragment. These results indicate that proteolytic activation of PKC
takes place before the onset of apoptosis and that inhibition of PKC
by GF109203X attenuates the apoptotic process.
Previous work has suggested a role for the Fas/CD95 pathway in
neutrophil apoptosis.8,24 This pathway, which was initially thought to have a major role in T-cell regulation alone,9
has been shown to be involved in apoptosis in several cell types in response to various inducers of cell death.62-64 Liles et
al8 showed that spontaneous neutrophil death in vitro could
be inhibited by blocking the Fas/CD95 ligand-receptor interaction and
that neutrophils produced FasL suggesting an autocrine/paracrine
mechanism of death. Neutrophil apoptosis can be accelerated by
incubation with agonistic Fas/CD95 antibodies and this is associated
with more rapid PKC proteolysis. After oligomerization of Fas/CD95 by ligand or MoAb, there is activation of caspase-8/FLICE, which is
recruited to the receptor complex by
FADD/MORT-1.11,12,50,65 Caspase-8 is capable of activating
other effector caspases, such as caspase-3, by proteolytic removal of a
prodomain.13 Thus caspase-8 sits at the apex of the
Fas/CD95 pathway and its inhibition has been shown to prevent apoptosis
in response to Fas/CD95 ligation.11,50,66
We show that PKC proteolysis and activation is inhibited by
preincubation with certain peptide caspase inhibitors. These are
designed to mimic the preferred substrates of individual family members
or to act as general inhibitors.14 The broad spectrum inhibitor, BD-fmk, decreases PKC proteolysis and apoptosis as might
be expected. The inhibitor, IETD-fmk, targeted to
caspase-8,50 also prevents both spontaneous and
Fas/CD95-induced PKC proteolysis and apoptosis. Using purified
recombinant enzymes, we show that caspase-3, but not caspase-8, can
cleave PKC indicating that the effects of IETD-fmk are due to
indirect inhibition of caspase-3 by preventing its activation by
caspase-8 in the intact cell. These results confirm and extend those of
Ghayur et al19 showing a major role of Fas/CD95 in
spontaneous neutrophil apoptosis and indicate that PKC proteolysis
plays a significant part in this process. The cleavage of PKC by
caspase-3 in neutrophils where there are no other detectable features
of apoptosis suggests that mechanisms to compartmentalize caspase-3
activation are likely to exist in the early stages of programmed cell death.
In addition to Fas/CD95, the neutrophil NADPH-oxidase system has been
shown to play an important role in cell death. The production of ROS in
other cell types can also predispose to apoptotic death.67 Coxon et al25 showed that phagocytosis of opsonized
particles by human neutrophils induced apoptosis in a CD11b/CD18
integrin and ROS-dependent manner. Kasahara et al24 showed
that neutrophils from patients with CGD had much slower rates of
constitutive or Fas/CD95-induced apoptosis compared with normals.
Apoptosis of normal neutrophils can be inhibited by incubation with
catalase, which leads to the rapid intracellular breakdown of hydrogen
peroxide.22,24 We show that neutrophils incubated ex vivo
produce increasing amounts of ROS with time and that this increase is
inhibited by coincubation with either the PKC inhibitor, GF109203X,
with the caspase-8 inhibitor, IETD-fmk, or the broad spectrum caspase
inhibitor, BD.fmk. ROS production is not affected by incubation with
Gö6976 or with the caspase-2 inhibitor, VDVAD.fmk. This
suggests that activation of Fas/CD95 by endogenously produced Fas
ligand promotes the formation of ROS in a caspase-8/PKC-dependent
manner. Acceleration of the apoptotic process with activating anti-Fas
antibodies leads to an increase in ROS production. Triggering the
caspase cascade may also lead to the activation and/or dysregulation of
other proteins involved in formation of ROS such as the Rac guanine nucleotide dissociation inhibitor D4-GDI68 and the
p21-activated kinase PAK65.17 The latter may regulate the
NADPH-oxidase complex by phosphorylating p47phox.69 The
results shown here suggest that activation of PKC is crucial to the
production of ROS in this system.
The precise role of PKC in the activation of the neutrophil oxidase
system is not clear. The respiratory burst can be efficiently activated
by PKC-activating phorbol esters or by synthetic analogues of
diacylglycerol.70 These effects can be inhibited by
competitive PKC inhibitors such as GF109203X.71 PKC may be
involved in the phosphorylation and translocation of the p47 and p67
phox proteins, but the isoforms involved have not been
defined.72-76 The data on the effects of PKC inhibitors on
the respiratory burst induced by the bacterial peptide, fMLP, are
contradictory, but this may relate to the use of nonselective
inhibitors or to alternative pathways of oxidase
activation.71,74,77-79 One group who used more selective
inhibitors has suggested that chemotactic peptide-induced activation of
the respiratory burst may depend on the activity of
nPKCs,47 of which neutrophils only appear to express the isoform.
We have also shown that the effect of GF109203X on cell survival is
dependent on the stage of differentiation. Primary peripheral blood-derived CD34+ cells and progeny induced to
differentiat |
TOP
ABSTRACT
INTRODUCTION