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The Diadenosine Polyphosphates Ap,A and Ap,A and Adenosine
Triphosphate Interact With Granulocyte-Macrophage Colony-Stimulating
Factor to Delay Neutrophil Apoptosis: Implications for Neutrophi1:Platelet
Interactions During Inflammation
By Lakhdar Gasmi, Alexander G. McLennan, and Steven W. Edwards
Incubation of neutrophils with cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) delays
their loss of function and changes in cellular morphology
that are characteristic of apoptosis. Adenosine triphosphate
(ATP) and the diadenosine polyphosphates Ap,A and Ap,A
were almost as effective as GM-CSF in delaying neutrophil
apoptosis. The nucleotides could thus preserve cellular morphology, protect against chromatin fragmentation, and preserve functions such as NADPH oxidase activity and expression of CD16. Moreover, addition of ATP, Ap,A, and
Ap,A together with GM-CSF resulted in more pronounced
protection from apoptosisthan was observed during incubation with either the cytokine or the nucleotides alone. Because ATP, Ap,A. and Ap,A may be secreted from activated
platelets, these observations suggest that platelet-derived
products, perhaps acting in combination with endothelialderived or immune cell-derived cytokines, can regulate neutrophil function during certain types of inflammation.
0 1996 by The American Society of Hematology.
N
These effects include priming of the respiratory burst and
elevations in intracellular Ca2+
Because the dinucleoside polyphosphates are considerably more stable than
ATP in biologic s o l ~ t i o n sthey
, ~ ~may
~ ~ ~be of greater importance than ATP in inflammatory processes because they are
longer lived.
In this report, we have compared the effects of ATP,
Ap3A, and Ap4A with GM-CSF on neutrophil apoptosis. All
nucleotides could delay apoptosis, as assessed by morphologic and functional parameters and were almost as effective
as GM-CSF in this activity. Furthermore, their ability to
delay apoptosis was enhanced when added in combination
with GM-CSF. These data thus provide novel evidence that
platelet-derived products can regulate neutrophil function
during certain types of inflammation and that the effects of
cytokines on neutrophil function can be augmented by other
types of cell-derived components.
EUTROPHIL FUNCTION during acute inflammation
is regulated by a variety of pro- and anti-inflammatory
agents that are secreted by endothelial cells, immune cells,
or by neutrophils themselves.’ Within the circulation, neutrophils are only poorly responsive to patho-physiologic agonists and possess a very short half-life because they constitutively undergo apoptosis or programmed cell
During
inflammatory activation, neutrophil function becomes
primed, for example, by exposure to cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), so
that the primed cells respond much more aggressively to
stimulatory
Many neutrophil priming agents can
also delay apoptosis and so they serve the dual purpose of upregulation of function and extension of life span. Apoptotic
neutrophils are characterized by having condensed cytoplasm, highly fragmented chromatin, inability to generate
reactive oxidants, and decreased CD16 (FcyRIII) expression,5&13 and they are recognized and cleared by tissue macr ~ p h a g e s . ’ ~Clearance
,’~
of apoptotic neutrophils in this way
does not result in the leakage of cytotoxic neutrophil proteins
into tissues that would otherwise occur if the cells lysed via
necrosis.
There is now considerable evidence to show that plate1et:neutrophil interactions can occur in some inflammatory
conditions and that platelets can secrete a number of components that modulate neutrophil function.’“’’ The dense granules of platelets contain high concentrations of adenosine
triphosphate (ATP) and also of the dinucleoside polyphosphates, Ap3A and Ap4A,’y~20
and all of these compounds have
been shown to have many effects on neutrophil function.
From the Cellular and Metabolic Regulation Group, Department
of Biochemistry, University of Liverpool, Liverpool, UK.
Submitted July IO? 1995; accepted November 20, 1995.
Supported by the Medical Research Council (UK).
Address reprint requests to S. W. Edwards, PhD, Biochemistry
Department, University of Liverpool, PO Box 147, Liverpool L69
3BX, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 V.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8708-001$3.00/0
3442
MATERIALS AND METHODS
Materials. Ap4A, Ap3A, ATP, adenosine, adenosine deaminase,
met-Leu-Phe, PMA, diphenylamine, cytochrome c, lunrinol, perchloric acid, Triton X-100, RNAase, sarkosyl, and proteinase K were
from Sigma (Poole, UK); trichloroacetic acid (TCA) was from BDH
(Lutterworth, UK). Recombinant (r)GM-CSF (97% punty) was a
nonglycosylated peptide from Glaxo (Greenford, UK) and had an
activity of 1.5 mU/mg protein in the AML-193 proliferation assay.
Stock solutions of fMet-Leu-Phe and PMA were freshly made up
in DMSO and corresponding amounts of solvent were added to
controls.
Preparation of neutrophils. Neutrophils were isolated from heparinized human blood from healthy volunteers by one step centnfugation through neutrophil-isolating medium (NIM; Cardinal Associates, Sante Fe, NM) as described in the manufacturer’s instructions.
After hypotonic lysis to remove contaminating erythrocytes,?’ neutrophils were suspended in RPMI- 1640 medium (Flow Laboratories,
Rickmansworth, UK) supplemented with 2.5% fetal calf serum
(Sigma) and 2 mmollL L-glutamine (Flow Laboratories) and then
counted using a Fuchs-Rosenthal haemocytometer slide. Cell viability (>95%) and cell purity (>97%) after purification were routinely
determined by Trypan blue exclusion and Wright’s staining, respectively.
Culture of neutrophils. Neutrophils ( 5 X lO%nL) in RPMI 1640
supplemented with 2.5% fetal calf serum and 2 m m o l L L-glutamine
were incubated in polypropylene conical tubes at 37°C in the absence
(control) or presence of ATP, Ap4A, Ap,A, or adenosine (all at 50
pmol/L), added in the presence or absence of rGM-CSF (50 U/mL).
Blood, Vol 87,No 8 (April 15). 1996: pp 3442-3449
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3443
NUCLEOTIDES AND NEUTROPHIL APOPTOSIS
Some incubations also contained adenosine deaminase (1 U/mL) as
indicated. At various incubation times aliquots were removed and
processed as described below.
Survival and apoptosis. Aliquots of neutrophils were mixed with
0.1% Trypan blue, incubated for 3 minutes, and the number of viable
and nonviable neutrophils counted. Survival was expressed as the
percentage of neutrophils remaining viable (ie, those that excluded
Trypan blue) of the total number in the original suspension.
neutrophils were
For morphologic estimation of apoptosis,
cytocentrifuged, fixed and stained with May-Grlinwald-Giemsa
(Sigma), air dried, and then examined microscopically. A minimum
of 800 cells per cytospin were counted and the number of apoptotic
cells was expressed as percentage of the total cells on the slide.
NADPH oxidase acrivity. Chemiluminescence was assayed in a
reaction mixture containing equal numbers of viable (ie, Trypan
blue-excluding) neutrophils and 10 pmoVL luminol. After the addition of the stimuli (met-Leu-Phe at 1 pmoVL and PMA at 0.1 pgl
mL), photon emission was measured using an LKE! Wallac 1251
luminometer (LKB Wallac, Turku, Finland) in a final volume of
1 mL.” Superoxide secretion was monitored by determination of
superoxide dismutase-inhibitable reduction of cytochrome c29,30
in a
reaction mixture containing equal numbers of viable neutrophils and
75 pmoVL cytochrome c. After the addition of stimuli, absorbance
increases at 550 nm were measured using a Perkin Elmer Lambda
5 spectrophotometer in a final volume of 1 mL. Reference cuvettes
additionally contained 30 pg/mL superoxide dismutase.
Chromatin structure. Quantitation of low molecular weight
DNA was carried out as described previously.8 Briefly, 2.5 X IO6
neutrophils were centrifuged in microfuge tubes at 13,OOOg for 2
minutes, washed with cold phosphate-buffered saline (PBS) (10
mmoVL potassium phosphate, 0.9% NaCI, pH 7.4) and then lysed
with 10 mmoVL Tris, pH 7.5, 1 mmoVL EDTA, and 0.2% Triton
X-100. After 15 minutes of incubation on ice, low and high molecular weight DNA were separated by centrifugation at 13,OOOg at 4°C
for 20 minutes. Centrifugation-resistant low molecular weight DNA
in the supernatant was transferred to separate tubes and precipitated
overnight at 4°C with 12.5% trichloroacetic acid (TCA). Cold TCA
(12.5%) was also added to the pellets, which were then left overnight
at 4°C. Samples were then centrifuged at 13,000g at 4°C for 7
minutes, and DNA in the precipitates was extracted with 30 pL of
5 mmol/L NaOH and 30 pL of 1 m o m perchloric acid at 70°C for
20 minutes. Then, 120 pL diphenylamine reagent” was added to
each sample and incubated overnight at 37°C. One hundred twenty
microliters from each sample was then transferred to a well of a
flat-bottomed 96-well plate and the absorbance at 600 nm was measured using a Bio-Rad 3550 plate reader.
The extraction and electrophoresis of fragmented DNA was assessed using a previously described method* with some modifications. Briefly, 5 X IO6 neutrophils were washed, lysed, then centrifuged at 13,OOOg at 4°C for 20 minutes exactly as described above.
Low molecular weight DNA in the supematants was transferred to
separate microfuge tubes, mixed with 20 pg/mL RNase, and incubated for 1 hour at 37°C. Added to the pellets was 0.5 mL of 50
“OIL Tris, 10 mmol/L EDTA, 0.5% Sarcosyl, and 0.5 pg/mL
proteinase K; this was incubated overnight at 48°C. Low and high
molecular weight DNA were then extracted twice with 1 volume
phenolkhlorofodiso-amyl alcohol (25:24:1, respectively) and once
with chlorofordiso-amyl alcohol (24:l). DNA in the extracts was
then precipitated with 0.5 moln NaCl and 1 volume iso-propanol
for 18 hours at -20°C. The samples were then centrifuged for 10
minutes at 13,OOOg, and 200 p L of 70% ethanol was gently added
to the precipitate; the samples were recentrifuged for 2 minutes at
13,OOOg. air dried, and resuspended in water. Three microliters loading buffer (2.5% ficoll, 0.025% bromophenol blue, and 0.025% xylene cyanol) was added to each sample; this was heated at 75°C for
le
5 minutes, snap-cooled, and then electrophoresed along with DNA
markers on a 1% agarose gel containing 1 pg/mL ethidium bromide
at 30 V in Tris-acetate buffer. DNA was visualized under UV and
then photographed.
Receptor expression. Expression of CD16 (FcyRIII) was measured by FACS analysis using a standard indirect immunofluorescence technique as described p r e v i o u ~ l yCells
. ~ ~ ~were
~ suspended in
PBS/I% bovine serum albumin (BSA) (globulin free)/O.l% sodium
azide, pH 7.2, and incubated with the monoclonal antibody Leu1 la
(Becton Dickinson, Cowley, UK) as a first layer antibody. FITClabeled goat-antimouse immunoglobulin was used as a second layer
antibody. Both were used at saturating concentrations and nonimmune mouse IgG of the appropriate subclass was used as a specific
first layer control. Stained cells were fixed in 1% paraformaldehyde
in PBS and were analyzed using a Becton Dickinson Ortho Diagnostics Cytron analyzer. Fluorescence distributions represent a total of
5,000 gated events.
Propidium iodideKDI6 dual labeling. Dual labeling of neutrophils with propidium iodide (to measure chromatin structure) and
anti-CD16 antibodies (to detect FcyRIII expression) was carried
out as described p r e v i o u ~ l y , ’with
~ ~ ~some
~
modifications. Briefly,
neutrophils were labeled with first (Leulla) and second layer antibodies as described above. Cells were then washed twice with cold
PBS/I% BSA/O.I% azide and fixed for 15 minutes in ice-cold 70%
ethanol. The cells were then washed and the pellet suspended in
propidium iodide solution (2 pg/mL in PBS) and incubated for 1
hour in the dark at 4°C before. cytometric analysis.
Sraristical analysis. The paired Student’s r-test was used to evaluate the significance of differences between the sample means. Statistical significance was defined at P 5 .05.
RESULTS
Effects of ATP, ApA, and Ap& on neutrophil survival.
Neutrophil apoptosis can be assessed by various parameters,
including changes in cellular morphology. Thus, apoptotic
neutrophils have a condensed nucleus, condensed cytoplasm,
and decreased cell size. Neutrophil suspensions incubated
for 24 hours in the absence of any exogenous addition were
75% apoptotic (?lo%, n = 9) by these criteria. Addition of
Ap4A, Ap3A, or ATP (Fig 1) caused a slight but significant
(P > .05) protection against apoptosis, with values of 68%
( t l l % , n = 9), 68% (+14%, n = 7), and 71% (?5%, n =
5), respectively, when analyzed in a paired Student’s t-test.
Addition of GM-CSF alone likewise protected against
apoptosis (56% ? 13%, n = 21), but addition of nucleotide
2 GM-CSF together resulted in much better protection than
was observed with either nucleotide or cytokine alone.
Effects on DNA fragmentation. When neutrophils undergo apoptosis, their chromatin breaks down and becomes
highly fragmented. This can be detected as either an increased formation of low molecular weight DNA (which can
be quantified) or as a DNA “ladder” of nucleosome-sized
(180-200 bp) fragments after gel electrophoresis. High and
low molecular weight DNA was thus quantified after incubation of neutrophils for 24 hours in culture. In control (untreated) suspensions, 59% (t5%, n = 5) of the DNA was
fragmented into low molecular weight (Fig 2). In suspensions treated with either GM-CSF, Ap4A, or ATP, significantly less (P < .05) DNA was fragmented, with the three
agents possessing near equal potency in protecting against
DNA fragmentation. However, in suspensions incubated
with either ATP or Ap4A together with GM-CSF, signifi-
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GASMI,McLENNAN,AND
3444
Fig 1. Effects ofGM-CSF, Ap,A, A p d , and ATP on neutrophil
apoptosis. Neutrophils were incubated for 24 hours, as described in
Materials and Methods, and cellular morphology was assessed by
Trypan blue exclusion, nuclear condensation, cell size, and cell shape,
as described in reference 8. The number of apoptotic neutrophils
present after 24 hours is expressed as a percentage of the number
of cells counted. ( W ) control; ( 0 )nucleotide alone; ( S ) GM-CSF alone;
(U)nucleotide GM-CSF. Bars indicate mean values ? SD. Number
of separate experiments: Ap,A, n = 9; Ap,A, n = 5; ATP, n = 7.
+
cantly greater protection against chromatin breakdown was
observed compared with the effect of either compound alone
( P < .OS). Thus, the nucleotides and GM-CSF appeared to
have an additive effect on protection against DNA fragmentation. Similar results were observed when DNA fragmentation was analyzed by gel electrophoresis (data not shown).
Incubation with GM-CSF, ATP, and Ap4A resulted in decreased DNA fragmentation, but far lower levels of fragmentation were observed when either ATP or Ap4A were used
in combination with GM-CSF.
Effects of NADPH oxidase activity. As neutrophils age
in culture, their ability to generate reactive oxidants via the
NADPH oxidase declines. GM-CSF treatment both primes
the respiratory burst and protects against this decline in oxidase activity. Incubation of neutrophils for2 hours with
GM-CSF primed luminol chemiluminescence generated in
response to stimulation by Met-Leu-Phe (Fig 3). Under
these conditions, the responses of primed neutrophils were
4.8-fold ( 21.6, n = 15) greater than in control cells. However, neither ATP, Ap4A, nor ApsA primed Met-Leu-Phe
stimulated oxidase activity after 2 hours of incubation. Instead, the responses of neutrophils incubated for 2 hours with
these nucleotides was decreased compared with controls. In
addition, in suspensions incubated with ATP plus GM-CSF
for 2 hours, Oxidase activity was decreased cornpared with
that observed in Cultures incubated with GM-CSF alone.
However, this decrease in the GM-CSF primed response was
not observed in cultures containing A ~ +
~ GM-CSF
A
or
"
ApsA GM-CSF at 2 hourS. All three nucleotides are unstable in bio1ogicsolutions,but the dhdenosine pobphosphates
are more stable than ATP. It has previously been shown that
EDWARDS
the nucleotide breakdown product, adenosine, can inhibit
some neutrophil response^.^^-^^ Thus, we tested the effects of
adenosine on oxidase activity in these experiments. Indeed,
incubation of cells for 2 hours with adenosine inhibited both
the control oxidase activity andthe GM-CSF primedresponse (Fig 3D). The effects of adenosine on oxidase activity
were very rapid. When adenosine was added to control suspensions 1 minute before the addition of Met-Leu-Phe, oxidase activity was inhibited by 60% ( 219%, n = 5). Similarly,
when added to primed suspensions (GM-CSF for 2 hours)
1 minute before Met-Leu-Phe stimulation, the response was
inhibited by 89% (+4%, n = 5). However, the addition
of adenosine deaminase (1 U/mL) before the addition of
adenosine prevented this inhibitory effect on oxidase activity
(Fig 4A). Similarly, when adenosine deaminase was added
to cultures containing ATP (Fig 4B), Ap4A, or Ap3A (data
not shown), the decreased oxidase activity that was observed
after 2 hours of incubation with the nucleotides was not
observed. These data thus indicate that the decreased oxidase
activity observed after 2 hours of incubation in nucleotidecontaining cultures is due to the inhibitory effects of adenosine that is released from the breakdown of the nucleotides.
Adenosine deaminase, by degrading the released adenosine,
prevents against this inhibition of oxidase activity. Adenosine did not affect PMA-stimulated NADPH oxidase activity
(data not shown).
When oxidase activity wasmeasured after 24 hours of
incubation, quite different results were obtained. In control
suspensions (Fig 3, which shows results from a series of
experiments where n = 5 ) , oxidase activity had decreased
Fig 2. Effects of GM-CSF, Apd, and ATp on DNA fragmentation.
Neutrophils were incubated with 50 U/mL GM-CSF, 50 pmol/L ATP,
or 50 prnol/L APJ for 24 hours and IOW molecular weight and high
molecular weight DNA were quantified as described in Materials and
Methods. Values quoted are means k SD (n = 5 experiments); an
asterisk P ) indicates values that are significantly different from controls and a dagger (t)indicates values that are significantly different
from values obtained in suspensions containing either GM-CSF, ATP,
or Ap,A alone.
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APOPTOSIS
NUCLEOTIDES AND NEUTROPHIL
3445
Fig 3. Effects
of
GM-CSF,
Ap,A, Ap3A, adenosine, and ATP
on NADPH oxidaseactivity. Neutrophils were incubated for 2
hours ( W or 24 hours ( 0 )as describedin Materials and Methods.
NADPH
oxidase
activity
was measured
by
luminol
chemiluminescence after addition of 10 pmol/L luminol and
stimulation by 1 pmol/LfMetLeu-Phe. Values presented are
means SD (n = 5 experiments);
an asterisk (*) represents values
significantly different from controls and a dagger (t) significantly different from values obtained in cultures containing
either GM-CSF alone or nucleotide alone. All suspensions contained equal numbers of Trypan
blue-excluding cells.
*
to approximately 40% of the value obtained after 2 hours of
incubation (2 hours response of controls from all experiments = I O mV ? 6.6 mV, n = 37; 24 hours response of
controls = 4 mV ? 1.8 mV, n = 22). The presence of GMCSF in the suspensions preserved oxidase activity to levels
two- to threefold greater than in control suspensions and
about 25% of the activity observed after 2 hours of incubation with the cytokine (2 hours response with GM-CSF from
all experiments = 42 mV ? 21 mV, n = 37; 24 hours
response with GM-CSF = I 1 mV ? 3.8 mV, n = 22). In
contrast to the results observed after 2 hours of incubation,
oxidase activity in suspensions treatedwith either ATP,
Ap,A, or Ap,A for 24 hours was significantly higher than
in control suspensions. Furthermore, the effects of either
ATP, Ap,A, or Ap4A plus GM-CSF appeared to be additive
and in all cases, the combined effects of nucleotide plus
cytokine were significantly greater ( P < .OS) than those
observed with cytokine alone or nucleotide alone. The protective effects of the nucleotides and cytokine were observed
when neutrophils were stimulated with either fMet-Leu-Phe
(Fig 3) or PMA (Fig S ) and also whenNADPH oxidase
activity was measured by cytochrome c reduction (data not
shown). The addition of adenosine deaminase to cultures did
not significantly affect the oxidase response obtained after
24 hours of incubation with either ATP,Ap,A,Ap.,A,
or
GM-CSF or combinations of cytokine and nucleotide (data
not shown).
l?fecrs on CD16 expression. Previous investigations
have shown that there is a link between neutrophil apoptosis
and expression ofCD16,'.''."
the low affinity receptor for
IgG-containing immune complexes. Functionally active neutrophils express high levels of this receptor, while apoptotic
neutrophils are CD 16-. Freshly isolated neutrophils express
high levels of CD16, but after 24 hours of incubation in
culture, only about 6% (?WO, n = 6) of the population
expressed this receptor (data not shown). The levels of fluorescence observed in the CD16- population were equivalent
to those observed when cells were stained with nonimmune
first layer antibody. When neutrophils were incubated for 24
hours with either GM-CSF, ATP, Ap,A, or Ap,A, there was
a significant increase in the population of CD16' cells (25%
? 8%; 16% ? 6%; 15% t 6%; IS% 5 S%, respectively).
Furthermore, there was a significant increase in the number
of CD16' cells observed after co-incubation with nucleotide
plus GM-CSF. In the presence of GM-CSF, the percentage
of CD16' cells in suspensions also containing ATP, Ap,A,
or Ap,A were 37% (54%). 47% (?7%), and 50% (?S%),
respectively.
We then simultaneously measured both chromatin structure
and CD16 expression in cultures incubated with GM-CSF and
nucleotides.After 2 hoursofincubationofcontrolcells
(no
additions) over 90% of the cells exhibited high expression of
CD16andhighpropidiumiodidefluorescence,indicatingthat
<IO% of the population showed signs
of apoptosis (data not
shown). However.by 24 hours incubation, >97% of the cells
exhibited low CD16 expression and low propidium iodidefluorescence (Fig 6A). In suspensions containing GM-CSF, 23% of
thecellshadhighCD16/propidiumiodidestaining(ie,were
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3446
McLENNAN,
GASMI,
AND EDWARDS
inflammatory response. Circulating cells constitutively undergo apoptosis and so are rapidly cleared from the circulation. However, in response to the generation of pro-inflammatory signals by endothelial cells or other immune cells,
apoptosis is delayed so that the functional life span of neutrophils is extended. Upon resolution of the inflammatory re-
Fig 4. Effect of adenosine deaminase on NADPH oxidase activity.
(A) Neutrophils were incubatedin the absence (control) orpresence
of adenosine deaminase (ADA, 1 UlmL), adenosine (50 pmollL), or
both together for 2 minutes before stimulation by 1 p m o l l L fMetLeu-Phe and measurement of luminol chemiluminescence. Values
given are means (rSD, n = 3). (B) Neutrophils were incubated for2
hours in the absence (control) or presence of adenosine deaminase
(ADA, 1 UlmL), ATP 150 pmollL), or GM-CSF (50 UlmL) or combinations thereof, as indicated. NADPH oxidase activity was then stimulated by 1 pmollLfMet-Leu-Phe and measured by luminol chemiluminescence. Mean values are presented (-tSD, n = 3).
nonapoptotic,Fig 6B), while in culturescontainingAp4Athe
nonapoptotic cells represented 12% of the population (Fig W).
However, in cultures containing both ApA and GM-CSF, the
nonapoptotic cells comprised over 55% of the total population
(Fig 6D). Similar results were obtained in cultures containing
ATP + GM-CSF (35% nonapoptotic) or Ap3A + GM-CSF
(52% nonapoptotic).
DISCUSSION
Regulation of neutrophil function by apoptosis has clear
advantages for both the activation and resolution,of the acute
Fig 5. Effects of GM-CSF, ApA, Ap,A, and ATP on neutrophil
chemiluminescence. Neutrophils were incubated for 24 hours and
NADPH oxidase was stimulated by theaddition of 0.1 p g l m L PMA
and measured by luminolchemiluminescence. Suspensions were incubated in the absence (control) or presence of GM-CSF with: (A)
ATP, (B) APIA, and (C) Ap,A. All suspensions contained equal numbers of Trypan blue-excluding cells. (0)controls (no additions), (01
GM-CSF alone, (B) nucleotide alone, and (0)nucleotide plus GMCSF. Typical traces from at least five separate experiments.
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APOPTOSIS
NUCLEOTIDES AND NEUTROPHIL
Fig 6. Effects of GM-CSF and ApJ, on CD16 expression and chromatin structure. Neutrophils were incubated for 24 hours in the absence (AI or presence of GM-CSF (B), ApJ (Cl, or GM-CSF + ApJ
(Dl. After this incubation, chromatin structure was analyzed by propidium iodide staining in suspensions thatwere simultaneously
stained for expression ofCD16. Similar results were obtained in three
other experiments.
sponse, the functional capacity of apoptotic neutrophils is
lost and the cells are cleared by tissue macrophage^.^.'".^'
Clearance of nonfunctional neutrophils in this way does not
lead to lysis that would release degradative granule enzymes
into tissues. Many agents are now known to delay neutrophil
apoptosis and we show here, for the first time, that the platelet products Ap3A and Ap4A (andalso ATP) can themselves
exert this effect. There are many ways to detect neutrophil
apoptosis, such as morphologic and functional parameters,
and in many respects these nucleotides are as effective as
GM-CSF. However, the effects of these compounds on
apoptosis are additive when used in combination with GMCSF. This indicates that in vivo, dual control of neutrophil
apoptosis during inflammation is possible. For example, in
some cases of inflammation, endothelial cell signals or immune cell signals (cytokines) can combine with plateletderived signals (ATP or dinucleoside polyphosphates) to
control neutrophil apoptosis.
The mechanisms that result in the combined effects of
nucleotides and GM-CSF are notknown at present. This
may result from the combined stimulation of both the GMCSF receptor and the nucleotide receptor(s). The extracellular effects of mono-and di-adenosine polyphosphates on
various cells are mediated via P, type purino receptors, and
the rank order of potency of binding of various nucleotides
has allowed for the division of these P' receptors into several
sub-types." On neutrophils, ATP is believed to mediate its
effects via a P,-type purino r e c e p t ~ r , ' ' ~and
' ~ ~its
~ ~biochemical properties indicate that it may resemble the Pzyreceptor
identified on erythrocyte^."".^' These receptors are coupled
3447
to phospholipase C activation via a pertussis toxin sensitive
G-protein. Whether Ap4A and Ap3A bind to the ATP receptor on neutrophils is unknown, but theyalso activate a pertussis toxin sensitive rise in intracellular Ca" (unpublished
results). The mechanisms by which GM-CSF itself regulates
apoptosis are unknown, but they probably reside in the ability of this cytokine to stimulate gene expression.'."ATP,
ApIA, and Ap4A can all elevate intracellular Ca2+levels in
neutrophils,'" and elevations in the levels of this cation have
been implicated in the control of apoptosis?' Alternatively,
GM-CSF has been reported to possess a nucleotide-binding
site thatis capable of binding both ATP and AP,A.~'.~
Whether such a direct GM-CSF/nucleotide interaction alters
the properties of the cytokine to enhance or prolong its function on neutrophils remains to be tested.
Platelets can secrete sufficient amounts of dinucleoside
polyphosphates so that local concentrations as high as 100
pmol/L may be produced in certain circumstances." Thus, if
such concentrations are generated under conditions in which
neutrophils and platelets are involved in the inflammatory
response, then these compounds may regulate neutrophil
apoptosis in vivo. It is noteworthy that the dinucleotides are
considerably more stable in biologic solutions than ATP,"."
but both are hydrolyzed to adenosine. Indeed, after 2 hours
of incubation, suspensions containing ATP and the dinucleotides had decreased oxidase activity compared with control
suspensions. Furthermore, incubation of cells with ATP (but
not Ap4A or Ap'A) for 2 hours partially abrogated the priming effects of GM-CSF. Indeed, addition of adenosine to
the cultures could mimic these effects, and adenosine was
inhibitory when added to suspensions for less than 1 minute
before stimulation. Furthermore, addition of adenosine deaminase to degrade adenosine, resulting either from exogenous addition or via release from degraded nucleotides, restored NADPH oxidase activity. Thus, we conclude that the
inhibitory effects on oxidase activity observed after 2 hours
of incubation are due to degradation of the nucleotides to
adenosine. This mechanism thus represents an additional
negative control of neutrophil function. However, by 24
hours incubation, cultures containing these nucleotides all
showed signs of delayed apoptosis, even though by this time
the nucleotides have largely been degraded. Thus, the signal
for delayed apoptosis must have triggered the cells before
the nucleotides had broken down. Indeed, addition of these
nucleotides to neutrophils can result in elevations in intracellular Ca" within 1 minute.'" Therefore, itis possible that
such rapid receptor occupancy/signaling may commit neutrophils to delayed apoptosis before the extracellular nucleotides are degraded.
In contrast to these short term effects, ATP, Ap,A. and
Ap4A all preservedoxidase activity after 24 hours of incubation, while adenosine and adenosine deaminase had no effect
at this time. These observations indicate that the inhibitory
effects of adenosine are transient and reversible, but the
protective effects of the nucleotides are more long term.
Although ATP and the dinucleotides possessed near equal
potency in delaying neutrophil apoptosis and preserving
function, it is possible that the dinucleotides are more potent
than ATP in vivo because of their greater stability.
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3448
GASMI, McLENNAN, AND EDWARDS
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1. Edwards SW: Biochemistry and Physiology of the Neutrophil.
New York, NY, Cambridge University Press, 1994
2. Haslett C: Resolution of acute inflammation and the role of
apoptosis in the tissue fate of granulocytes. Clin Sci 83:639, 1992
3. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM,
Haslett C: Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its
recognition by macrophages. J Clin Invest 83:865, 1989
4. Wyllie AH, Kerr JFR, Cume AR: Cell death: The significance
of apoptosis. Int Rev Cytol 68:25 1, 1980
5. Edwards SW, Watson F, MacLeod R, Davies JM: Receptor
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From www.bloodjournal.org by guest on October 1, 2016. For personal use only.
1996 87: 3442-3449
The diadenosine polyphosphates Ap3A and Ap4A and adenosine
triphosphate interact with granulocyte-macrophage
colony-stimulating factor to delay neutrophil apoptosis: implications
for neutrophil: platelet interactions during inflammation
L Gasmi, AG McLennan and SW Edwards
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