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Exposure of Human Neutrophils to Exogenous Nucleotides Causes Elevation in
Intracellular Calcium, Transmembrane Calcium Fluxes, and an Alteration of a
Cytosolic Factor Resulting in Enhanced Superoxide Production in Response to
FMLP and Arachidonic Acid
By Richard A. Axtell, Rebecca R. Sandborg, James E. Smolen, Peter A. Ward, and Laurence A. Boxer
Exposure of human neutrophils to micromolar concentrations of both hydrolyzable and nonhydrolyzable purine
nucleotides caused the generation of transient rises in
intracellular calcium (Ca”), Ca2+ fluxes across the membrane, and primed the cells for enhanced production of
superoxide (O,-) when subsequently exposed to agonists
such as FMLP and arachidonic acid. The neutrophils were
most sensitive to adenosine triphosphate (ATP) and ATP-yS, which produced CaZ+ transients and enhanced 0,production at concentrations as low as 1 to 5 fimol/L, with
a doubling of 0,- generation at 25 to 50 fimol/L. Adenosine
diphosphate (ADP), guanosine triphosphate (GTP), and
5‘-adenylylimidodiphosphate(AMP-PNP) required approximately 10-fold higher concentrations to cause similar
effects. Adenosine did not cause Ca2+ fluxes or a Ca2+
transient and was inhibitory of 0,- production. There was
a strong correlation between a nucleotide‘s ability to
generate a Ca” response and its ability to enhance 0,generation. Nitrogen cavitation and subcellular fractionation of the neutrophils after a brief exposure to ATP,
ATP-7-S, and AMP-PNP revealed that the enhanced 0,generating capacity was stable and detectable in a cell-free
assay system. By combining variously treated cytosolic and
membrane fractions, it was found that the enhanced 0,production was attributable to a modification of a componentls) of the cytosol.
0 1990 by The American Society of Hematology.
N
exposure to ATP and ATP-7-S results in recruitment of
CDllb/CD18 to the cell membrane with a concomitant
increase in neutrophil aggregation and adhesion. Balazovich
and Boxer’ have reported that exogenous ATP is capable of
causing both the release of specific granule contents and the
activation and translocation of protein kinase C. Kuhns et
found that ATP, uridine triphosphate (UTP), and
inosine triphosphate (ITP), but not ADP (in concentrations
less than 200 pmol/L) were capable of generating a Ca2+
transient and enhancing FMLP-induced 0,- production. In
addition, Ward et a18 reported that with both rat and human
neutrophils, the non-hydrolyzable analogs S-adenylylimidodiphosphate (AMP-PNP) and &y-methyleneadenosine 5’triphosphate (AMP-PCP) were capable of enhancing 0,production in response to immune complexes. The nonhydrolyzable nucleotides were not tested with FMLP as the
stimulus. Further work by Ward et a13 has shown that both
ATP and ADP are capable of causing Ca2+ transients and
enhanced 0,- production in response to FMLP, but interpretation of these latter observations is somewhat confounded by
the inclusion of cytochalasin B in these assays.
In this report, we present data indicating that with several
purine nucleotides, both native and hydrolytically resistant,
there is a consistent correlation between a nucleotide’s ability
to raise intracellular Ca2+ and its ability to enhance 0,production in response to the agonists F M L P and arachidonic acid. We will also present data indicating that this
“priming” effect of exogenous nucleotides is mediated through
a modification of the cytosolic factor(s).
EUTROPHILS RESPOND to numerous extracellular
stimuli including bacterial components (formylated
peptides), products of complement activation (Cs), opsonized particles, and immune complexes. These factors act
through cell surface receptors to mediate chemotaxis, phagocytosis, degranulation, and oxygen burst activity. Recent
work in this and other laboratories has suggested the existence of a new class of exogenous mediators of neutrophil
activation. In vivo experiments have shown that there is
increased tissue damage and oxygen radical production when
neutrophils are stimulated in the presence of platelets.’.’
Subsequent in vitro experiments have confirmed that platelets will enhance superoxide (Oz-) production by neutrophils
in response to several stimuli, including immune complexes,
opsonized zymosan, C 3 and N-formyl-methionyl-leucylphenylalanine (FMLP) .
It has recently been shown that the platelet effect can be
mediated by either platelet lysates or secretory products from
thrombin-stimulated platelets, the apparent mediators being
adenosine triphosphate (ATP) and adenosine diphosphate
(ADP) contained in the dense granule^.^ Subsequent investigations have identified several effects of ADP, ATP, and
other purine nucleotides (and their non-hydrolyzable analogs) on human neutrophils. Freyer et a14 has reported that
From the Departments of Pediatrics and Pathology, The University of Michigan, Ann Arbor, MI.
Submitted March 17, 1989; accepted November 14.1989.
Supported in part by a National Institute of Health Grant No.
HL31963 and a grant from the Children’s Leukemia Foundation of
Michigan.
Address reprint requests to Richard A . Axtell, MD. Department
of Pediatrics. The University of Michigan. F6515 Mot1 Hospital.
Box 0238, Ann Arbor, MI 48109-0238.
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 U.S.C.section I734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7506-0003$3.00/0
1324
MATERIALS AND METHODS
Materials. Anhydrous dextrose was obtained from Columbus
Chemical Industries (Columbus, WI), fura-2 acetoxymethyl ester
was purchased from Molecular Probes (Junction City, OR), and 6%
dextran 75 in 0.9% sodium chloride for injection was obtained from
Abbott Laboratories (North Chicago, IL). Horse heart ferricytochrome C (grade VI), superoxide dismutase (SOD, from bovine
erythrocytes), FMLP, ethylene glycol tetra-acetic acid (EGTA),
Trizma base, Triton-X-100, adenosine 5’-triphosphate (disodium
salt), guanosine 5’4riphosphate (GTP; sodium salt type III), adenoBlood, Vol75, No 6 (March 151, 1990: pp 1324-1332
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PURINE NUCLEOTIDE PRIMING OF NEUTROPHILS
sine 5'-diphosphate (sodium salt), adenosine, citric acid trisodium
salt (dihydrate), citric acid (monohydrate), HEPES, PIPES, and
5'-adenylylimido-diphosphate (AMP-PNP, tetralithium salt), were
purchased from Sigma Chemical Company (St Louis, MO). Adenosine-5'-0-(3-thiotriphosphate)tetralithium salt (ATP-+) was obtained from Boehringer Manheim Biochemicals (Indianapolis, IN),
and Ficoll-Paque and Percoll were purchased from Pharmacia Fine
Chemicals (Piscataway, NJ). Arachidonic acid was obtained from
Nu-Chek-Prep, Inc (Elysian, MN), and sodium dcdeoxylsulfate
from Bio-Rad Laboratories (Richmond, CA). MAPTAM (bis-(2amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic
acid tetraacetoxymethyl ester) was purchased from Calbiochem (San Diego,
CA). All other reagents were laboratory grade.
Preparation of human neutrophils. Blood was collected from
healthy volunteers and anticoagulated with acid-citrate-dextrose
(ACD). The neutrophils were purified by dextran sedimentation
followed by hypotonic lysis to remove red blood cells and centrifugation through Ficoll-Paque to remove mononuclear cells? These
preparations contained greater than 95% neutrophils. For the whole
cell assays, the neutrophils were used immediately.
For the cell-free assays, the neutrophils were subcellularly fractionated following the Borregaard technique" with certain modifications. The cells were suspended in ice-cold relaxation buffer (100
mmol/L KCI, 3 mmol/L NaCl, 3.5 mmol/L MgC1, and 10 mmol/L
PIPES, pH 7.3) without 1 mmol/L ATP. For those experiments
involving exposure to nucleotides, the cells were suspended in
ice-cold relaxation buffer with the desired nucleotide concentration,
incubated for 5 minutes at 4"C, and then washed twice with
nucleotide-free relaxation buffer. Control cells were likewise washed
twice with nucleotide-free relaxation buffer. The cells were adjusted
to 7.5 x lo7 cells/mL and disrupted by nitrogen cavitation after
pressurization at 420 psi for 20 minutes. The cavitate was collected
into EGTA (pH 7.4) sufficient for a final concentration of 1.25
mmol/L and then centrifuged at 500g for 10 minutes at 4OC. The
supernatant was then loaded onto precooled discontinuous Percoll
gradients (also lacking in nucleotides) of densities 1.07 and 1.110
g/mL, and the gradients were centrifuged at 48,OOOg for 16 minutes
at 4OC. After centrifugation, the cytosolic and membrane fractions
were harvested with a Pasteur pipette. The cytosolic fraction (the
aqueous layer extending down to just above the membranes at the
aqueous-light Percoll interface) was then spun at 320,OOOg for 68
minutes to remove contaminating membranes. The membrane
fraction (extending from the aqueous-light Percoll interface down to
just above the specific granule bands) was centrifuged at 180,OOOg
for 95 minutes at 4°C to remove the Percoll. The membranes were
then collected from just above the Percoll pad and either used
immediately or stored at -70°C at a concentration of 5 x lo8 cell
equivalents/mL. Cytosol was also stored at -7OoC, and activity was
stable for at least 2 weeks.
For experiments using the intracellular CaZ+chelator MAPTAM,
the purified neutrophils (10' cells/mL) were suspended in a Ca2+free HEPES buffer (150 mmol/L NaCI, 5.6 mmol/L KCI, 1
mmol/L MgCI,, 10 mmol/L glucose, and 10 mmol/L HEPES, pH
7.3) to which either MAPTAM (final concentration 100 pmol/L) or
DMSO (the diluent for the MAPTAM, final concentration of
DMSO less than 0.5%) were added.".I2 The cells were then
incubated for 10 minutes at 37"C, diluted 10-fold with Ca2+-free
HEPES buffer, and incubated another 10 minutes at 37OC. The
neutrophils were then washed with nucleotide-free relaxation buffer
and processed as described above.
Continuous assays of superoxide production. Whole cell assays
were performed on a Uvikon 8 10 dual beam recording spectrophotometer (Kontron Instruments, Everett, MA) that monitored the SODinhibitable reduction of ferricytochrome C, following the technique
of Badwey et al.13 Production of 02-was monitored continuously at
1325
37°C for the duration of the respiratory burst, which was greater
than 95% complete by 5 minutes. Cells were kept at 4°C in
Dulbecco's phosphate-buffered saline (PBS) without glucose, CaZ+
or Mg2+at a concentration of lo7cells/mL until immediately before
their use. These cells were then diluted 1:lO with prewarmed PBS
with Ca2+, Mg2+ and glucose, and allowed to equilibrate for 5
minutes before the addition of the stimulus (FMLP or arachidonic
acid). When used, nucleotides or nucleosides were added 90 seconds
before the FMLP. FMLP was stored frozen in dimethyl sulfoxide
and was diluted with PBS immediately before its use. The nucleotides were dissolved in deionized water. 0,-production was
calculated using an extinction coefficient of 18.5 mmol/L-' cm-',
and results were expressed in nanomoles 0,produced per lo7cells.
For those assays examining the effects of extracellular Ca2+on
superoxide production, 10 pL of 300 mmol/L EGTA (pH 7.4 in
deionized water) was added 120 seconds before the FMLP, which
was 30 seconds before the nucleotide (if added).
The cell-free assays of NADPH oxidase activity were performed
according to the technique of Curnutte et all4 with the following
modifications: (1) 2 x lo6 cell equivalents of membrane were
combined with lo7cell equivalents of cytosol in each assay; (2) either
108 pmol/L sodium dodecyl sulfate (SDS)I5 or 61 pmol/L arachidonic acid-the optimal concentrations of each agent-were used as
stimuli; and (3) cytosol, membrane, and stimulus were incubated for
240 seconds before the reaction was initiated by the addition of
NADPH. 0,-generation was maximal within 30 seconds of the
addition of the NADPH. No 0,-was generated during the preincubation or if either cytosol or membrane were omitted from the
assay mixture.
Fura-2 assays of intracellular calcium concentrations. After
preparation at 4OC, cells were suspended in HEPES buffer (150
mmol/L NaCl, 5.6 mmol/L KCl, 2 mmol/L CaCl,, 1 mmol/L
MgCI,, 10 mmol/L glucose, and 10 mmol/L HEPES, pH 7.3) and
allowed to equilibrate at room temperature for 15 minutes. The
neutrophils were then loaded with fura-2/AM (10 pmol/L) at a cell
concentration of 5 x lo7mL. The cells were incubated at 37°C for 15
minutes and were then diluted 1:lO with HEPES buffer containing
0.5% bovine serum albumin (BSA) and incubated another 15
minutes. After incubation, the neutrophils were washed twice with
HEPES buffer and stored at room temperature at a concentration of
2 x io7ceh/mL.
Assays were performed at 37°C on a Perkin-Elmer 650-10s
fluorescence spectrophotometer. For each assay, 100 pL of the cell
suspension was added to 890 pL of prewarmed HEPES buffer in a
cuvette with a magnetic stir bar, and the cells were allowed to
equilibrate for 3 minutes. The stimuli were added as 10 pL aliquots,
and the change in fura-2 fluorescencewas monitored continuously at
an emission wavelength of 500 nm (10 nm slit width) and a single
excitation wavelength of 342 nm (5 nm slit width).I6 The intracellular Ca2+ levels were calculated using the formula: [Ca2+]i = kd
(F - Fmin)/(Fmax - F). The dissociation constant (kd) was taken
as 224 nm,I7 Fmax was determined by lysing the cells with 0.2%
Triton, and Fmin was determined by first adding 5 pL of 1 mol/L
Tris base to the cuvette to raise the pH to 8.5 and then adding 7.5
mmol/L EGTA (pH 8.5).
45caZ+ uptake and eflux. Assays of Ca2+fluxes were performed
in a manner similar to those methods described by Korchak et al.I8*l9
For uptake assays, purified neutrophils were resuspended in a buffer
consisting of 150 mmol/L NaCl,, 5 mmol/L KCl, 1.2 mmol/L
MgCI,, 0.4 mmol/L CaCI,, and 10 mmol/L HEPES, pH 7.45 and
incubated at 37°C for 5 minutes. 45Ca2+uptake was initiated by the
addition of stimulus or vehicle containing 150 pCi/mL 45CaC1,.
Assays were carried out for 2 minutes and then terminated by the
addition of 10 mmol/L EGTA, pH 7.4 to the incubation medium.
Aliquots of cell suspensions were immediately layered over silicon oil
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1326
AXTELL ET AL
and centrifuged in a microfuge for 20 seconds. The amount of
radioactivity in each cell pellet was measured using liquid scintillation counting. The amount of radioactivitytaken up by cells over two
minutes was calculated as the difference between that measured at 2
minutes and at a zero time control.
For efflux assays, neutrophil suspensions were incubated with 150
pCi/mL 4SCaC12for 45 minutes. Preliminary experiments indicated
that isotopic equilibrium was achieved within 45 minutes. After the
loading procedure, cells were washed three times at room temperature in unlabeled buffer, resuspended, and then incubated at 37OC
for 5 minutes. Efflux was initiated by the addition of an aliquot of the
cell suspension to the stimulus or vehicle and terminated by
centrifugation through silicon oil. Aliquots of cell supernatantswere
analyzed for radioactivity.
All results are expressed as counts per minute per lo6 cells and
were analyzed for statistical significance using a Student’s t test.
150-
-z
--
120-
I
+
90I
f
W
2
a
60-
30-
0.1
RESULTS
1
180-
1
10
100
NUCLEOTIDE [uMl
Nucleotide-induced Ca2+ transients in neutrophils.
Exposure of human neutrophils to 25 pmol/L ATP, ATP--/S, AMP-PNP, or G T P in the absence of FMLP produced
changes in intracellular Ca2+,as shown in Fig 1. ATP and
ATP-y-S caused transient rises in intracellular Ca2+ that
were initiated within seconds of the addition of nucleotide
and rose to a maximum within 20 seconds. The intracellular
Ca2+ concentration then diminished to the “resting” level
over the subsequent several minutes. At similar concentrations (25 pmol/L), AMP-PNP caused somewhat lower
elevations, and G T P caused markedly smaller Ca2+ transients. As can be seen in Fig 1, the patterns of Ca2+transients
induced by ATP and ATP-7-S were generally similar to that
produced by 0.1 pmol/L FMLP, although the duration of the
elevation of the intracellular Ca2+concentration was shorter
after the nucleotide stimulation than after exposure to the
chemotactic peptide.
The magnitude of the induced rise in the intracellular
Ca2+ concentration was found to be dependent on both the
O !
0
I
I
I
i
1
2
3
4
TIME ( m i d
Fig 1. Change in intracellular Ca” levels in human ne:ttrophils
in response to various nucleotides or adenosine as measured by
fura-2 fluorescence. Final concentrations of ATP (0-0).ATP-7-S
GTP (A-A),
and adenosine ( 0 - 0 )were
(X-X), AMP-PNP (0-0).
has been added for
25 pmol/L. Response to 0.1 pmol/L FMLP (0-0)
comparison.
Fig 2. Maximal rise in intracellular Ca’+ occurring in response
to varying concentrations of four nucleotides and adenosine: ATP
(0-0).ATP-7-S (X-XI. GTP (A-A), ADP (0-0)
and adenosine
( 0 - 0 ) .No FMLP was present. Values are means k SD for three
experiments.
identity of the nucleotide and its concentration. In general,
there was an increase in the magnitude of the Ca2+transient
with increasing nucleotide concentration (Fig 2). There was
a consistent hierarchy of sensitivity to the various purine
nucleotides. ATP produced detectable rises in intracellular
Ca2+at 0.5 pmol/L and near maximal rises (130 nmol/L) at
10 pmol/L concentrations. The neutrophils were slightly less
sensitive to ATP-7-S with detectable Ca2+transients occurring at 1 pmol/L concentrations and a maximal response
occurring a t 50 pmol/L.
The neutrophils were approximately an order of magnitude less sensitive to G T P and ADP, exhibiting a threshold of
5 to 10 pmol/L, with a maximal Ca2+ rise occurring with
exposure to nucleotide concentrations in the 100 pmol/L
range. As reported by other investigators using fluorescent
probes such as f~ra-2/AM,”.~’
there was a wide variation in
the maximal amplitude of the Ca2+ transient seen from
experiment to experiment. In these experiments, the maximal rises in Ca2+ concentration ranged from 120 to 750
nmol/L, although mean values were in the range of 150 to
185 nmol/L. The near equivalent efficacy of ATP and
ATP-7-S in producing a Ca2+transient suggests that hydrolysis of the nucleotide is not required for this effect.
Nucleotide-induced Ca2+ fluxes in neutrophils in the
presence and absence of FMLP. FMLP-induced changes
in the intracellular Ca2+concentrations of neutrophils represent the net effect of three sequential events. Within 1 second
of exposure to the chemotactic peptide, there is a release of
Ca2+ from internal store^,'^.^' which is followed within 3
seconds by the initiation of an influx of Ca2+from extracellular
After 10 to 12 seconds, an efflux of Ca2+begins,
which eventually returns the intracellular Ca2+ concentration to resting
To determine whether the changes
in intracellular Ca2+observed in response to exposure to the
nucleotides were related to changes in Ca2+movement across
the cell membrane, we investigated the flux of 45Ca2+in
intact human neutrophils in response to ATP, A T P - y S , and
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1327
PURINE NUCLEOTIDE PRIMING OF NEUTROPHILS
Table 1. "CaZ+ Uptake in Response to FMLP and Various
Adenine Compounds
Calcium Uptake
cpmflo' cells
Stimulant
Control (n = 8)
FMLP (n = 8)
ATP (n = 6)
ATP-y-S (n = 6)
ATP
FMLP (n = 6)
Adenosine (n = 3)
+
1,267 t
4,210 +
2,980 +
4,052 f
5.470 i
1.1 14 +
176
348'
645'
543"
691'
208
% Control
100
375 t 50'
205 t 25"
357 56"
424 68"
102 t 6
*
*
Neutrophils were exposed to 0.1 pmol/L FMLP and/or 100 pmol/L
concentrations of ATP, ATP-7-S, or adenosine for 2 minutes, and the
uptake of 45Ca2+from the medium was measured as described in
"Materials and Methods."
'Differs significantly from control group (P< ,051.
FMLP. All measurements were performed after 2 minutes of
exposure to each stimulus. As seen in Table 1, the influx of
45Ca2+ from the medium was markedly increased over
control by the presence of 0.1 pmol/L FMLP. Exposure of
the neutrophils to 100 pmol/L concentrations of ATP and
ATP-y-S produced similar increases in 45Ca2+uptake. In
addition, treatment of the cells with ATP and FMLP
simultaneously resulted in a greater 45Ca2' uptake than that
produced in response to either stimulus alone, the net effect
being almost additive in nature.
W e then examined Caz+ efflux from cells that had been
preloaded with 4sCa2+.As seen in Table 2, exposure of the
neutrophils to 0.1 pmol/L FMLP resulted in the release of
34%of the 4sCa2+.Both ATP and ATP-y-S (at 100 pmol/L
concentrations) also produced a significant 4sCa2+efflux,
although the magnitude was less than that seen with the
chemotactic peptide alone. Once again, the greatest 45Ca2+
flux (44% of the preloaded total) occurred after simultaneous
exposure of neutrophils to ATP and FMLP. In marked
contrast, exposure to adenosine did not produce significant
45Ca2+influx or efflux in neutrophils. This is consistent with
our observation that adenosine fails to produce a Ca2+
transient (Figs 1 and 2).
Effects of nucleotides and adenosine on FMLP-induced
0,-production. It is commonly assumed that the FMLPinduced changes in intracellular Ca2+ concentration are a
prerequisite for several neutrophil functional responses, including adhesion, degranulation, phagocytosis and 0,p r o d u c t i ~ n . ' ~As
. ' ~previously illustrated, exposure of neutrophils to exogenous purine nucleotides causes similar elevations in intracellular Ca2+and generates Ca2+ fluxes across
0
0
20
a
n
b"
0 4
lom9
10-8
lo-'
lo-a
FMLP CONCENTRATION [MI
Fig 3. Effect of purine nucleotides and adenosine on FMLPinduced 0,- generation. Human neutrophils were incubated with
50 fimol/L concentrations of ATP (0-0).ATP-7-S (X--X), GTP
(A--A), or adenosine (0-0). before being stimulated by varying
concentrations of FMLP. The initial (maximal) rate of 0,- production was then measured. All points represent the mean value
obtained from three separate experiments. (FMLP alone.0 -0).
the membrane. These nucleotides do not, however, by themselves cause the generation of any O,-. They are, however,
capable of enhancing 0,production in response to agonists
such as FMLP and arachidonic acid. Pre-incubation of
neutrophils with these nucleotides caused an increase in both
the maximal rate of superoxide production and the total
amount of superoxide generated. As can be seen in Fig 3, 50
pmol/L concentrations of ATP, ATP-y-S, and G T P enhanced 0,-production over the full range of effective
chemotactic peptide concentrations. The most pronounced
enhancement induced by the nucleotides was seen a t 5 x
lo-' to 5 x
mol/L FMLP, where the rate of 0,production was more than doubled. In contrast, 50 pmol/L
concentrations of adenosine profoundly inhibited FMLPinduced 0,production.
Despite the similar responses of neutrophils to 50 pmol/L
concentrations of ATP, ATP-y-S, and GTP, there were
substantial differences in the sensitivity of FMLP-stimulated
neutrophils to lower concentrations of the nucleotides. In the
experiments shown in Fig 4, the concentration of the chemotactic peptide was held constant at
mol/L and the
nucleotide concentrations were varied between 0.1 pmol/L
and 100 pmol/L. The neutrophils were most sensitive to
ATP, with a doubling of the rate of 0,production occurring
Table 2. "Ca*+ Efflux from Neutrophils in Response to FMLP and Various Adenine Compounds
Calcium Efflux
Treatment
cpmflo' Cells
% Control
% Release
Control (n = 7)
FMLP (n = 7)
ATP (n = 6)
ATP-y-S (n = 4)
ATP
FMLP (n = 4)
Adenosine (n = 3)
284 + 47
2,434 + 330
991 t 164"
1,013 t 217'
2,908 + 299'
265 t 93
100
884 t 67'
331 r 15"
318 + 42"
1,136 i 97"
83 + 4
4 + 0.3
34 + 2'
1 4 2 1'
1 3 + 1'
44 t 3'
3 + 1
+
Results are expressed as the percent of internalized 45Ca2+released after 2 minutes of stimulation with the indicated agonists. Final concentrations
were 0.1 pmol/L for FMLP and 100 pmol/L for ATP, ATP-y-S, and adenosine.
'Differs significantlyfrom control group (P< .05).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1328
AXTELL ET AL
at a concentration of 5 pmol/L. They were slightly less
sensitive to ATP-7-S (the rate of 0,production doubling at
10 pmol/L), and approximately an order of magnitude less
sensitive to GTP and ADP. As before, adenosine profoundly
inhibited FMLP-induced 0,-production, with 76% inhibition at 0.1 pmol/L and 93% inhibition at 100 pmol/L.
When the ability of these four nucleotides to generate
Ca2+transients and to augment FMLP-induced 0,production in human neutrophils were compared, a close correlation
between the two phenomena was evident (Figs 2 and 4).The
lowest concentration of each nucleotide at which a Ca2+
transient was detected was essentially the same concentration at which enhancement of 0,-production by that
nucleotide was first seen. The subsequent dose-response
curves for the two phenomena were then nearly superimposable for each nucleotide. Thus, the nucleotide concentration
that produced the maximal Ca2+transient was the concentration that produced maximal enhancement of 0,production.
Effect of EGTA on superoxide production and Ca2+
transients. The addition of EGTA to the extracellular
CI
SO01
400
CI
5
300
u
.-
n
2
U
200
100
I
0
I
1
I
2
TIME (min)
1
1
4
3
Fig 5. Effect of chelation of external Ca2+ on the changes in
intracellular CaZ+ as monitored by fura-2 fluorescence. Neutrophils were exposed to 0.1 pmol/L FMLP in the absence (X-X) or
presence (X---XI of EGTA, or 28 pmol/L ATP in the absence
(0-0) or presence (O----OI of EGTA. Where indicated, 10
mmol/L EGTA (pH 7.4) was added 30 seconds before the stimulus.
Q
U
T
T
1 1
6
5
lBO1
b"
1601
/
P
w
0
2
n
EI
n.
l o o + - -
1
2
ew
fw
u)
4
m
1
""1
8o
40
medium (to chelate-free Ca2+)immediately before stimulation of neutrophils by FMLP has been shown to decrease the
magnitude of the rise in intracellular Ca2+and to hasten the
return of the intracellular Ca2+ concentration to resting
levels.18-21A similar effect of EGTA on human neutrophils
occurred with the Ca2+transient evoked by ATP. As can be
seen in Fig 5, when 10 mmol/L EGTA (adequate to reduce
extracellular Ca2+to less than 0.1 pmol/L)22 was added 30
seconds before the stimulus, the usual initial rapid rise (0 to 5
seconds) was essentially intact. However, later sustained
elevations ( 5 to 20 seconds) were reduced, as was the
maximal increment in Ca2+concentration. The intracellular
Ca2+ concentration subsequently returned more quickly to
the baseline value. Presumably, these changes reflect an
abrogation of the influx of extracellular Ca2+ and are
consistent with the data (Table 1) that indicated that both
ATP and ATP-7-S induced uptake of Ca2+from the extracellular medium.
Extracellular EGTA also affected FMLP-induced 0,production. As seen in Table 3, the initial rate of 0,production was not significantly diminished by the addition
6
c
Table 3. Effect of EGTA on FMLP-Induced 0,- Production by
Human Neutrophils in the Presence and Absence of ATP
z
w
0
% Reference Control
a
W
n.
t
1
I
1
0.1
1
10
100
NUCLEOTIDE CONCENTRATION [uMI
Fig 4. Enhancement of FMLP-induced 0,- production is a
function of nucleotide/nucleoside identity and concentration.
Human neutrophilswere incubated with increasing concentrations
of ATP (O--O). ATP-y-S (X--X). GTP. (A--A), ADP
and
adenosine (O--O), and then stimulated with 0.1 pmol/L FMLP. The
effect on the maximal 0,- production rate was then compared for
the different nucleotides and adenosine. The nucleotide or adenosine were added after 210 seconds of incubation and the FMLP at
300 seconds. Values are means f SD for three experiments.
(o---o),
Parameter Analyzed
Initial 0,-Rate
Total 0,-Produced
Burst Duration
FMLP Alone
9 5 & 5.2
71.6 f 11'
70.3 f 7.2'
ATP
+ FMLP
79.3 & 5.5'
45.7 & 1.5'
60 f 5.0'
Results are expressed as the percent of values obtained in the absence
of EGTA & SD. Baseline 0,-rates were 5 1 nmol 0,-/min/107 cells for
0.1 pmol/L FMLP alone and 9 2 nmol O,-/min/lO'
for FMLP
10pmol/L ATP. Baseline total 0,-production was 122 nmol/107 cells
for O.lpmol/L FMLP alone and 2 4 2 nmol/107 cells for FMLP
10pmol/L ATP. Baseline burst duration was 4.85 minutes for FMLP
alone and 5.0 minutes for ATP plus FMLP.
'Differs significantly from non-chelated results ( P < .05).
+
+
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PURINE NUCLEOTIDE PRIMING OF NEUTROPHILS
of 3 mmol/L EGTA (adequate to reduce extracellular CaZ+
to less than 0.1 pmol/L).22 However, the burst duration and,
hence, the total 0,-produced was significantly decreased,
consistent with the hypothesis that a Ca2+ influx was necessary for the maintenance but not the initiation of the
respiratory burst.
The chelation of extracellular Ca2+ had a more pronounced effect on the ability of ATP to augment FMLPinduced superoxide production. There was a greater than
20% reduction in the nucleotide’s enhancement of the initial
rate of 0,-production, although the rate was still higher
than with FMLP alone. The burst duration was also shortened with the result that the total 0,-generated was
decreased by more than 50%. It appears that the mechanism
by which ATP enhances 0,-production is substantially
dependent on the presence of extracellular Ca2+.
Eflect of exogenous nucleotides on 0,-production in a
cell-free assay. To attempt to elucidate the mechanism by
which nucleotides are able to enhance 0,-production, we
examined the effect of a brief exposure to exogenous nucleotides on NADPH oxidase activity when the cells were
subsequently fractionated and then reconstituted in a cellfree assay. This approach allows one to assess the stability of
any modification and to identify the site of that modification
(ie, cytosol, membrane, or both). As elaborated in “Materials and Methods,” purified neutrophils were exposed to
various concentrations (0, 1, 5, or 50 pmol/L) of ATP,
ATP-y-S, or AMP-PNP for 5 minutes at 4OC, washed,
cavitated, and fractionated. The cytosolic and membrane
fractions from the variously exposed cells were then combined in the indicated combinations, and 0,production was
assayed. Both SDS and arachidonic acid produced qualitatively identical responses, but SDS was used in the majority
of the assays because it was more stable than arachidonic
acid and gave more consistent results.
In the experiments shown in Fig 6, the cytosol and
membranes from cells exposed to 0, 1, 5, or 50 pmol/L
concentrations of either ATP, ATP-y-S, or AMP-PNP were
15’i
1004
NUCLEOTIDE CONCENTRATION (UM)
Fig 6. Effect of nucleotide exposure on 0,- production in a
cell-free assay. SDS (108 pmol/L) was used as the stimulus for
membrane and cytosol from cells exposed various concentrations
of ATP (0-0).ATP-7-S (X-X), and AMP-PNP (IT-0).Results are
plotted as the percent enhancement compared with subcellular
fractions from cells not exposed to nucleotides. Values are
means f SD of at least three experiments.
1329
ARACHIDONIC ACID CONCENTRATION (UM)
Fig 7. Effect of nucleotides on arachidonic acid-induced 0,production in intact neutrophils. Cells were exposed to 25 pmol/L
concentrations of ATP (0-0);ATP-7-S [X---X), AMP-PNP (El---O),
or no nucleotide (0-0) and then stimulated with the indicated
concentration of arachidonic acid. Results are plotted as nmol
0,-/min/107 cells and each point is the mean f SD for three
experiments.
recombined, and 0,generation in response to 108 pmol/L
SDS was measured. As can be seen, all three nucleotides
caused significant enhancement of 0,-generation when
compared with cells not exposed to nucleotide. The effect was
evident at 1 pmol/L concentrations of the nucleotides and
increased with increasing nucleotide concentration up to 50
pmol/L. Nucleotide concentrations greater than 50 pmol/L
did not consistently produce further enhancement of cell-free
0,-generation (data not shown). Brief exposure to 5 to 50
pmol/L concentration of ATP or AMP-PNP resulted in
more than a doubling of the rate of 0,production, while 50
pmol/L ATP-y-S was somewhat less effective a t enhancing
0,-generation. Comparison of Figs 4 and 6 reveals that the
degree of 0,enhancement, the range of effective concentrations, and the efficacy of both native and non-hydrolyzable
nucleotides are quite similar for the whole cell and the
cell-free systems.
Direct comparison of the nucleotide effects on 0,production are possible only for amphiphiles such as arachidonic
acid, which are capable of eliciting 0,-generation in both
systems. Figure 7 shows the effect of 25 pmol/L concentrations of three nucleotides (ATP, A T P - y S , and AMP-PNP)
on arachidonic acid-induced 0,production in intact neutrophils. As with FMLP, the nucleotide was added 90 seconds
before the agonist, and as with FMLP, micromolar concentrations of all three nucleotides enhanced 0,-production. At
intermediate concentrations of the fatty acid (30 to 60
pmol/L), these nucleotides produced a 60% to 160% increase
in 0,production, although at maximally stimulatory concentrations (80 pmol/L) there was little or no enhancement.
Once again, the effects of the nucleotides were first seen a t
submicromolar (0.5 pmol/L) concentrations and reached a
maxima at approximately 100 pmol/L (data not shown).
There was a significant consistency in the effects of these
nucleotides on 0,-production, whether the cells are stimulated with amphiphile immediately as intact cells (Fig 7) or if
they are disrupted, reassembled, and then stimulated with
arachidonic acid or SDS (Fig 6). Although the mechanisms
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AXTELL ET AL
1330
by which amphiphiles elicit 0,generation in the whole cell
and cell-free systems may be different, the similarities with
respect to the range of effective nucleotide concentrations (1
to 50 pmol/L), the degree of enhancement (50% to 200%)
and the efficacy of both hydrolyzable and non-hydrolyzable
analogs suggest that the mechanisms by which the resultant
0,production is enhanced may be the same in both systems.
In the preceding cell-free experiments, isolated membranes were recombined with their corresponding cytosols,
and the 0,-generated was quantitated. With the cell-free
system, one can independently assay the membranes and
cytosol from neutrophils treated with varying concentrations
of various nucleotides. In the experiments shown in Table 4,
neutrophils were exposed to 50 pmol/L ATP-y-S, 50 pmol/L
AMP-PNP, or no nucleotide, washed, fractionated and then
assayed for superoxide production in response to SDS. As
expected, reconstituting the cytosol and membranes from
neutrophils exposed to ATP-7-S resulted in 0,production
a t nearly twice the rate of components from nonexposed
neutrophils, and recombination of membrane and cytosol
from neutrophils treated with AMP-PNP resulted in 0,production that was almost three times the basal rate. By
combining the three different membrane preparations with
standard cytosol (not exposed to any nucleotide) it could be
seen that the different membrane preparations were nearly
identical with respect to their ability to support 0,production. The converse combination of standard (non-nucleotide
exposed) membrane with the three cytosols, revealed that the
cytosol accounted for virtually all the observed enhancement
of 0,-production. ATP-7-S cytosol doubled the rate and
AMP-PNP cytosol tripled the rate of 0,-production, as
compared with cytosol from cells not exposed to exogenous
nucleotide.
The cell-free system can also be used to further define the
role of Ca2+ in nucleotide-induced priming. Extracellular
Ca2+is required by intact cells for maximal 0,production
in response to agonists such as FMLP, arachidonic acid,
immune complexes, and C s , which complicates the assessment of the requirement for Ca2+ as an intermediary in
nucleotide-induced priming. The cell-free 0,-generating
system does not require Ca2+ for optimal 0,-production,
and excess Ca2+is actually inhibit~ry.,~
Thus, this is an ideal
system for evaluating the role of Ca2+.Intact neutrophils at
4OC in Ca2+-freerelaxation buffer are capable of generating
5
-C
Y
+
+
m
0
Y
1
0
2
1
3
TIME ( m l n )
Fig 8. Effect of temperature on nucleotide and FMLP-induced
Caz+ transients as measured by fura-2 fluorescence. Neutrophils
were suspended in &+-free relaxation buffer at either 4°C (------I
or 37°C (-1
and then stimulated with either 25 pmol/L ATP-7-S
(X) or 0.1 pM FMLP (e).Tracings are representative curves.
calcium transients in response to FMLP and micromolar
concentrations of ATP, A T P - 7 3 , and AMP-PNP, although
the amplitude and rate of rise are less than those generated at
37OC in the presence of extracellular Ca2+(Fig 8). When the
cells are loaded with 100 pmol/L MAPTAM (an intracellular Ca2+chelator) this transient is no longer detectable with
fura-2/AM (data not shown), consistent with results reported by Korchak et a1.12*24
We therefore evaluated the
effect that MAPTAM loading of neutrophils had upon the
ability of exogenous nucleotides to enhance 0,production
when the cells were subsequently fractionated and assayed in
a cell-free system. As can be seen in Fig 9, MAPTAM alone
generation in the cell-free system, but its
had no effect on 0,presence completely abrogated the usual twofold enhance-
,,i
1
25
Table 4. Cell-Free 0,- Production by Subcellular Fractions of
Neutrophils Exposed to ATP-y-S, AMP-PNP, or No Nucleotide
Cytosol
Membranes
No Nucleotide
ATP-y-S
AMP-PNP
No nucleotide
ATP-7-S
AMP-PNP
100
98 f 6
99 6
209 t 9.5
180 t 4.6
306 t 9.2
*
-
298
f
MAPTAM
ATP-7-5
20
The rate of 0,-production by cytosol and membrane from neutrophils
not exposed to any exogenous nucleotide was arbitrarily assigned a value
of 100%. and the other combinations' 0,- production are expressed as a
percentage of this standard mixture. Actual rate of 0,-production by
non-nucleotide cytosol and membrane was 15.6 nmol/min/107 cell
equivalents of membrane. Stimulus was 108 pmol/L.SDS. Values are
means t SD for at least three experiments (cavitations). ATP--(-S: 5 0
pmol/L; AMP-PNP, 50 pmol/L.
-
+
-
+
+
-
+
Fig 9. Effect of chelation of intracellular Ca2+ on nucleotideinduced enhancement of 0,- production assayed in the cell-free
system. Neutrophils loaded with 100 pmol/L MAPTAM were
compared with control cells (sham-loaded with DMSO) with
respect to the ability of 50 pmol/L ATP-y-S to enhance SDSinduced 0,- production. Values are expressed as 0,- nmol/min
generated by lo7 cell equivalents of membrane f SD, and
represent data obtained from 12 cavitations of three separate cell
preparations.
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PURINE NUCLEOTIDE PRIMING OF NEUTROPHILS
1331
nucleotide’s ability to generate a Ca2+ transient and its
ability to enhance 0,-production, strongly support such a
hypothesis. In addition, the observations in the cell-free
system that the abolition of the nucleotide-induced Ca2+
transient with MAPTAM resulted in the absence of any
DISCUSSION
enhancement of 0,-production further strengthens this
conclusion. However necessary an elevation in intracellular
Exogenous nucleotides are capable of exerting numerous
Ca2+may be for the manifestation of the nucleotide response,
effects on human neutrophils. In contrast to many other cell
it is only an intermediary since the nucleotide effect on 0,types, such as 5774 murine macrophage^:^.^^ thymocyte^,^'
production
persists long after intracellular Ca2+levels have
and Friend erythroleukemia cells2’ that require high (0.3 to 5
returned to baseline. In addition, in the cell-free 0,assay
mmol/L) concentrations of the nucleotides for an effect to be
system the enhanced activity of the cytosol persists, despite
seen, neutrophils respond to more physiologic (1 to 50
the presence of EGTA.
pmol/L) concentrations. Meyer et a12’ calculated that plateRecently published work with transformed myeloid and
lets contain 1.9 pmol of secretable ATP and 2.6 pmol of
monocytic cell lines has provided further insight into the
secretable ADP per 10” platelets. Thus, assuming a normal
effects of purine nucleotides on hematopoietic cells. Dubyak
platelet count of 300,00O/pL, a serum concentration of 22
et a134 and Cowen et al,35 working with “granulocytes”
pmol/L ATP and 32 pmol/L ADP could be achieved.
differentiated from HL-60 cells, have shown that exposure to
Although such a massive systemic release of platelet granules
micromolar concentrations of ATP, ATP--&
and ADP
would never occur, it is reasonable to assume that nucleotide
resulted in the generation of Ca2+ transients and phosphoconcentrations in the 5 to 30 pmol/L range are achieved in
lipid remodeling as evidenced by inositol triphosphate accuthe vicinity of a fibrin clot or wherever platelet aggregation
mulation. These investigators found the same hierarchy of
and degranulation have occurred. This makes it highly likely
nucleotide sensitivity-ATP > ATP-y-S > ADP > A M P >
that the effects discussed in this report are physiologically
adenosine-as has been reported for P2 purinergic receptors
relevant.
in other system^.^'.^^ Greenberg et al,37 working with 5774
These nucleotides appear to be exerting their effects
macrophages, found a similar hierarchy of response to the
through a surface receptor, a conclusion based on the
various nucleotides and determined that the rises in intracelmicromolar sensitivity and the near-equivalent efficacy of the
M a r Ca2+ occurring in response to micromolar concentranon-hydrolyzable analogs (indicating that the nucleotides
tions (less than 50 pmol/L) of purine nucleotides represented
are not serving as a source of high energy phosphate). In
both a release from internal stores and an influx across the
addition, our cell-free experiments demonstrated that the
cell membrane. Finally, Seifert et a13’ have reported that
priming effects of these nucleotides persisted after brief
purine nucleotides are capable of enhancing the 0,producexposure and subsequent washing (less than 0.05% remaintion by DMSO and dibutyryl cyclic A M P (CAMP) differening after two washes) (data not shown), indicating that their
tiated HL-60 cells occurring in response to FMLP, as is seen
continued presence is not required for the persistence of the
in normal mature neutrophils. The dibutyryl-CAMP differenpriming phenomenon. All of these observations support the
tiated cells, however, were capable of generating 0,-in
hypothesis that these purine nucleotides are modifying neuresponse to treatment with cytochalasin B (1 pg/mL) and
trophil function by acting upon a “purinergic” receptor
ATP (1 to 100 pmol/L) alone (without FMLP), which is a t
mechanism such as that described by Burnstock3’ and Su3’in
neural tissue. Agonists such as ATP, ADP, ATP-y-S, and
variance with what is seen in normal mature neutrophils.’
AMP-PNP could be binding to the “P2” receptor, while the
The actual mechanism by which the neutrophil is modified
inhibitory adenine compounds A M P and adenosine act by
to respond more vigorously to stimuli such as FMLP,
binding to the “Pl” receptor. The greater efficacy of the
arachidonic acid, and immune complexes is yet to be determined. From the information obtained from our cell-free
nucleotide triphosphates ATP and ATP-y-S as compared
studies, one can exclude membrane changes such as inwith ADP is also consistent with such a hypothesis.
creased receptor number, altered receptor affinity, or a
One of the first events that occurs after ligand binding in
several cell types26s2’*32s33
is a rise in intracellular Ca2+.This
modification of the membrane bound portion of the oxidase
from consideration. Likewise, although granule fusion occurs
cation then acts as a second messenger producing alterations
in response to these nucleotide^:^ it does not appear to have a
in cell function that are distinct for each cell type. This signal
transduction mechanism has been well described in neutrorole in this priming process. It is clear that the nucleotides are
phil for ligands such as FMLP.12,’8-20
Based on the present
able to effect a stable (for several hours a t 4OC) modification
data, it would appear that Ca2+performs a similar role for
of a cytosolic constituent(s), but how this interacts with the
neutrophil responses to the nucleotides. In intact cells, the
components of the NADPH oxidase remains to be discovobservation that exogenous EGTA blunts both the Ca2+
ered. Potential mechanisms include alteration of cytosolic
response and the ability of the nucleotides to enhance 0,- components by the action of proteases, phosphatases, or
production, as well as the tight correlation between a
kinases.
ment that is normally seen after exposure to 50 pmol/L
ATP+ before cavitation (Fig 6). This confirms the requirement for a transient elevation of intracellular Ca2+ for
nucleotide-mediated priming of neutrophils to occur.
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1990 75: 1324-1332
Exposure of human neutrophils to exogenous nucleotides causes
elevation in intracellular calcium, transmembrane calcium fluxes, and
an alteration of a cytosolic factor resulting in enhanced superoxide
production in response to FMLP and arachidonic acid
RA Axtell, RR Sandborg, JE Smolen, PA Ward and LA Boxer
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