Endotoxin Activation of Macrophages Does
Not Induce ATP Release and Autocrine
Stimulation of P2 Nucleotide Receptors
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J Immunol 2000; 165:7189-7198; ;
doi: 10.4049/jimmunol.165.12.7189
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References
Reza D. Beigi and George R. Dubyak
Endotoxin Activation of Macrophages Does Not Induce ATP
Release and Autocrine Stimulation of P2 Nucleotide Receptors1
Reza D. Beigi and George R. Dubyak2
W
ithin minutes after binding to Toll family receptors on
macrophages, bacterial endotoxin (LPS) triggers activation of the three major mitogen-activated protein
kinase pathways (p38, c-Jun N-terminal kinase, and extracellular
regulated kinase; ERK)3 as well as the kinase cascade that culminates in nuclear translocation of NF-␬B (1– 4). These rapidly
evoked signals modulate the expression of multiple inflammatory
response genes via transcriptional and/or translational regulation.
As a result, macrophages synthesize and release of a variety of
inflammatory mediators for several hours following the initial exposure to endotoxin. These include TNF-␣, IL-1␤, IL-6, plateletactivating factor (5), PGE2, and NO (1, 3). Many of these mediators act in an autocrine fashion via cell surface receptors to
provide positive or negative feedback to the macrophage signaling
cascades initiated by endotoxin (1, 3).
It has recently been proposed that endotoxin-activated macrophages also release nucleotides, such as ATP, to provide an additional pathway for autocrine or paracrine modulation of endotoxindependent responses. This hypothesis is based on observations that
treatment of macrophages with ATP receptor antagonists, includDepartment of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106
Received for publication August 15, 2000. Accepted for publication September
13, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant GM36387, National
Institutes of Health Training Grant HL07415, and American Heart Association (National) Grant-in-Aid 9950305N.
2
Address correspondence and reprint requests to Dr. George R. Dubyak, Department
of Physiology and Biophysics, Room E565, Case Western Reserve University School
of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4970. E-mail address:
[email protected]
3
Abbreviations used in this paper: ERK, extracellular regulated kinase; oATP, periodate-oxidized ATP; ␣,␤-meATP, ␣,␤-methylene-ATP; FL-AAM, firefly luciferase
ATP assay mix; PEP, phosphoenolpyruvate; PK, pyruvate kinase.
Copyright © 2000 by The American Association of Immunologists
ing periodate-oxidized ATP (oATP) and pyridoxal-phosphate-6azophenyl-2⬘,4⬘-disulfonic acid (6), represses several of the short
and long term macrophage responses to endotoxin, including ERK
activation, NF-␬B translocation, and the release of TNF-␣, IL-1␤,
NO, arachidonic acid, and cyclooxygenase-dependent products of
arachidonic acid metabolism (5–11). In further support of this
model, macrophages are known to express both G protein-coupled
(P2Y) and ionotropic (P2X) nucleotide receptors, including the
P2X7 pore-forming receptor (12–14). Additionally, a subset of
macrophage responses to endotoxin is modulated by costimulation
with exogenous nucleotides (5, 15–21).
Experimental measurements of ATP release from macrophages
following endotoxin activation have yielded conflicting results (5,
6, 22). This may be due in part to the fact that routine experimental
procedures, such as solution exchange and cell washing, have been
shown to elicit nucleotide release from a variety of cell types (23–
26). During prolonged endotoxin activation, macrophages produce
and secrete a variety of cytotoxic mediators, including reactive
oxygen and nitrogen species (1, 3). Under these conditions macrophages may be more fragile and thus more likely to release nucleotides as a result of the fluid shear or mechanical trauma that
occurs during experimental manipulation.
We have examined whether short or long term exposure to endotoxin can cause macrophages to release ATP using on-line assays designed to minimize cellular trauma while providing a continuous readout of extracellular ATP concentrations. Although
murine macrophages constitutively release and hydrolyze ATP to
maintain a basal extracellular ATP concentration in the 0.1–1 nM
range, there are no increases in the extracellular concentrations of
ATP or ATP metabolites following either acute or prolonged treatment with endotoxin. This result argues strongly against the hypothesis of autocrine signaling by released nucleotides during
macrophage activation by endotoxin. We suggest that the ability of
nucleotide receptor antagonists to interfere with endotoxin signaling in macrophages is due to inhibitory effects on molecular species other than ATP receptors.
0022-1767/00/$02.00
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Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of
endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation
of NF-␬B and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine
macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP,
producing steady-state extracellular concentrations of ⬃1 nM when assayed as monolayers of 106 adherent cells bathed in 1 ml
of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In
addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be
significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory
effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate
that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP
released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling
is due to its interaction with molecular species other than ATP-binding P2 receptors. The Journal of Immunology, 2000, 165:
7189 –7198.
7190
ENDOTOXIN DOES NOT INDUCE ATP RELEASE FROM MACROPHAGES
Materials and Methods
Materials
All nucleotides were purchased as either crystalline free acids or sodium
salts; stock solutions were calibrated by absorption spectroscopy at 259
nm. ATP, oATP, ␣,␤-methylene-ATP (␣,␤-meATP), apyrase grade I (for
scavenging ATP on-line) or grade III (with reduced endotoxin content, for
prolonged incubations), and firefly luciferase ATP assay mix (FL-AAM)
and ATP assay buffer were purchased from Sigma. ADP, AMP, PMSF,
leupeptin, and DTT were obtained from Roche (Indianapolis, IN). 2-Phosphoenolpyruvate (potassium salt) was purchased from Calbiochem (La
Jolla, CA). DMEM and penicillin/streptomycin were purchased from Life
Technologies (Grand Island, NY), and bovine calf serum was supplied by
HyClone (Logan, UT). HRP-coupled secondary Abs were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Pyruvate kinase (PK) and
myokinase were obtained from Sigma as ammonium sulfate suspensions
(catalogue no. P-1506 and M-3003, respectively). Endotoxin serotype
0111:B4 was purchased from List Biologicals (Campbell, CA).
Cell culture
On-line assays of extracellular ATP using firefly luciferase
On-line ATP measurements were conducted using a Turner TD-20/20 luminometer (Turner Designs, Sunnyvale, CA) with temperature control
module started at 29°C. Thermal regulation was required to prevent luciferase inactivation that occurs above 30°C.
BAC1.2F5 or RAW264.7 macrophages were studied as adherent monolayers on 35-mm dishes. For acute endotoxin treatments, adherent cells
were washed twice, and the medium was replaced with 1 ml of sterile ATP
assay buffer (130 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 5
mM NaHCO3, 1.5 mM KH2PO4, 25 mM HEPES (pH 7.5), 0.1% BSA, and
10 mM D-glucose) supplemented with 40 ␮l of concentrated soluble luciferase/luciferin and calf serum in various amounts (see figure legends).
Dishes were placed in the measurement chamber of the luminometer, and
light production was recorded at 30-s intervals using 5-s integration times.
Experiments were conducted according to the following protocol: 10-min
background recording, stimulus (or vehicle) addition with agitation, 12min observation recording, and ATP calibration (see below).
Experiments measuring extracellular ATP following chronic endotoxin
treatment were conducted as follows. Monolayers were treated with endotoxin overnight and then washed extensively. The culture medium was
replaced with phenol red-free, serum-free DMEM, and the cells were allowed to recover for various amounts of time at 37°C in a CO2 incubator.
Following recovery, 40 ␮l of concentrated luciferin/luciferase were added,
and bioluminescence was measured until the light levels reached steady
state (no longer than 10 min), after which time the measurements were
continued and averaged for at least 4 min. Calibration was performed as
described below.
Endotoxin-induced signaling is serum dependent due to a requirement
for LPS-binding protein (2). However, serum ATPases compete with luciferase for ATP and rapidly hydrolyze ATP in extracellular spaces. Therefore, some experiments were performed using low serum concentrations
(0.1%), which were sufficient to support threshold endotoxin responsiveness in these macrophages. Other experiments were conducted using 10%
serum, which supports maximally efficacious endotoxin signaling at room
temperature.
Calibration of ATP-dependent light output
To confirm that endogenously released ATP was measurable and to establish a lower limit on intracellular ATP, the pore-forming antibiotic alamethicin (28) was added to permeabilize the cells, and peak light output was
recorded. In parallel dishes, exogenous ATP was added to the medium
bathing the intact macrophages in sequential pulses, with final concentrations ranging from 10 –300 nM, thus providing a comparison of ecto-ATPase activities. Finally, apyrase was added to scavenge all extracellular
ATP, yielding a measurement of background luminescence.
Calibrations were performed in parallel using cell-free, serum-free medium containing ATP assay buffer with or without 300 ␮M ␣,␤-meATP (as
indicated) in 35-mm dishes. Sequential pulses of exogenous ATP ranging
from 10 to 300 nM were added (light production was independently ver-
Biochemical analysis of ATP, ADP, and AMP levels in
extracellular medium samples
Extracellular samples were collected by withdrawing 0.5 ml of medium
from individual culture wells, which was boiled immediately for 5 min to
inactivate soluble ATPases. After spinning to pellet debris, these extracellular medium samples were stored on ice until further use. Adherent cells
were lysed in 1 ml of 1.67 M perchloric acid/well. Following a 20-min
incubation at room temperature, the wells were scraped with a rubber policeman, then lysates were transferred to microfuge tubes, cooled on ice for
several minutes, and spun to pellet protein precipitates. Eight hundred microliters of the deproteinized samples were neutralized with 350 ␮l of 4 M
KOH and 450 ␮l of HEPES/KOH (25 mM HEPES and 15 mM KOH, pH
8), then allowed to incubate on ice for 15 min. The samples were spun
briefly to remove precipitates and then stored on ice. These cell extracts and
corresponding extracellular samples were analyzed for ATP, ADP, and AMP
contents using the rephosphorylation protocols described by Hampp (29).
An aqueous solution containing phosphoenolpyruvate (PEP) (8.3 mM
PEP and 50 mM MgSO4) in HEPES/KOH was used as the basic rephosphorylation buffer (PEP mix). PK (⬃0.75 U/ml final) and myokinase
(⬃0.6 U/ml final) were obtained by spinning 10 –20 ␮l of 3.2 M ammonium sulfate suspensions in a microfuge, carefully removing the supernatants, then resuspending the pellets in an equal volume of HEPES/KOH.
ADP assay mix consisted of 0.5 ml of PEP mix and 7.5 ␮l of PK. AMP
assay mix consisted of 0.5 ml of PEP mix, 7.5 ␮l of PK, 8 ␮l of myokinase,
and 4 ␮l of 100 ␮M ATP. Twenty-five microliters of PEP mix, ADP assay
mix, or AMP assay mix was added to 150-␮l samples in luminometer tubes
for analysis. Extracellular samples were used undiluted, while lysate samples were first diluted with HEPES/KOH to bring the ATP levels into the
linear range of the luciferase assay. AMP rephosphorylation samples were
incubated for at least 1 h, while ADP rephosphorylation required only 15
min at room temperature. The ATP content of the samples was determined
immediately. For all experiments, blanks were determined by processing
cell-free buffers identically to samples.
The total ATP content of the samples was determined using a luciferasebased bioluminescent assay. FL-AAM (Sigma) was diluted by 25-fold
in firefly luciferase ATP assay buffer. Twenty-five microliters of diluted
FL-AAM was mixed with samples already in luminometer tubes, and luminescence was recorded. Internal controls were established by adding
samples of known ATP concentration.
For each nucleotide (ATP, ADP, and AMP), a concentration series of
standard rephosphorylations was established by processing samples containing known amounts of exogenously added nucleotides. Sample recoveries were judged relative to the corresponding standard curve for each
nucleotide.
Analysis of extracellular nitrite accumulation as an index of NO
release
Medium samples (100 ␮l) were mixed with 100 ␮l of Griess reagent
(Molecular Probes, Eugene, OR) in a 96-well plate, and absorbance at 548
nm was recorded. Samples were prepared in one of two ways. For some
experiments, cells were seeded in 35-mm dishes, grown overnight, then
stimulated for 12–16 h by adding reagents directly to the dishes. After
stimulation, the cells were washed, and the medium was replaced with
phenol red-free, serum-free DMEM, then the cells were placed back into an
incubator. At designated time points, 100-␮l samples were collected and
analyzed for nitrite as described above. Alternatively, macrophages were
seeded into 96-well plates and grown overnight before adding endotoxin
and/or other stimuli (medium on all cells was replaced with 100 ␮l of fresh
medium containing the appropriate reagents). At the end of the stimulation
period, Griess reagent was added directly to the wells, and the absorbance
was recorded. In practice, the presence of phenol red in culture medium did
not interfere significantly with nitrite determination (not shown).
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BAC1.2F5 macrophages were passaged by gentle scraping/resuspension
and cultured as previously described (27). RAW 264.7 mouse macrophages
were cultured and passaged in an identical fashion using DMEM, 10% calf
serum, and 1% penicillin/streptomycin. Macrophages were routinely
seeded for experiments at 106/ml (2 ml/35-mm dish, 1 ml/well in 12-well
plates, or 100 ␮l/well in 96-well plates) and allowed to grow overnight
before experimentation.
ified to be linearly related to ATP concentration from 300 pM to 1 ␮M).
The peak light output at each ATP concentration was adjusted by subtracting the reading immediately previous to the ATP addition. A calibration
curve was then constructed that related adjusted light output to ATP concentration. This curve assigned the average value of background luminescence (after apyrase addition) to 0 nM ATP. The slope and intercept of the
best-fit straight line through the points were used to convert on-line light
intensities to ATP concentrations. An estimate of the magnitude of systematic errors introduced by the linear regression was extracted. For all
reported averages, this estimate was compared with the SD of the mean,
and the larger of the two was reported as the experimental uncertainty.
The Journal of Immunology
7191
ERK phosphorylation assays
Acute activation of endotoxin signaling cascades was assessed by measuring the induced phosphorylation of ERK family mitogen-activated protein
kinases (3). BAC1.2F5 macrophages were prepared as described for ATP
assays, and the medium was replaced with ATP assay buffer with or without calf serum in various concentrations. Cells were stimulated with 2
␮g/ml 0111:B4 endotoxin (or 1 ␮l of water) for 12 min, after which time
the buffer was aspirated, and the cells were immediately disrupted in lysis
buffer (300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100,
0.5 mM DTT, 20 mM ␤-glycerophosphate, 0.1 mM Na3VO4, 25 mM
HEPES (pH 7.5), 2 mM PMSF, and 100 ␮g/ml leupeptin). ERK phosphorylation was determined by Western blotting using phospho-specific antipERK1/2 Abs (Santa Cruz Biotechnology E-4, sc-7383; used at 1/1000) or
rabbit anti-pan-ERK immune serum (a gift from M. J. Dunn, Medical College of Wisconsin, Milwaukee, WI) (30) and ECL reagents (Pierce,
Rockford, IL).
Macrophages were pretreated with oxidized ATP (500 ␮M) by adding
the reagent directly to culture wells and incubating for 2 h, followed by
washout. The inhibitory effects of oATP on endotoxin-induced ERK phosphorylation were observable for at least 6 h after its removal.
Results
For the studies described in this report, we used the adherent
BAC1.2F5 murine macrophage cell line. These cells correspond to
mature, naive macrophages, are dependent for growth upon the
presence of CSF-1 in the medium (31), and express not only the
P2X7 receptor, but also P2Y family nucleotide receptors (27, 32).
As illustrated in Fig. 1, these cells exhibit standard macrophage
responses to endotoxin, both acutely (ERK phosphorylation) and
following prolonged exposure (NO production). Activation of the
c-Jun N-terminal kinase cascade and production of IL-1␤ in response to endotoxin have also been observed in this particular
macrophage line (33) (R. D. Beigi, S. B. Kertesy, and G. R.
Dubyak, unpublished observations). Most experiments and results
described below were replicated using RAW 264.7 macrophages,
another murine cell line widely used for studies of endotoxin signaling (6, 7, 17–19, 34).
Endotoxin-induced ERK phosphorylation and NO production
were attenuated in BAC1.2F5 macrophages pretreated with oATP
(Fig. 1, A and B). Because oATP is known to inhibit activation of
the P2X7 nucleotide receptor, similar results in other macrophage
types have led to the suggestion that autocrine signaling through
P2 receptors accompanies and potentiates endotoxin signaling in
macrophages (5–9). If so, then endotoxin activation should elicit
ATP release from the cells.
Effect of acute endotoxin activation on extracellular nucleotide
concentrations
To test whether endotoxin activation causes the release of ATP
from macrophages, cells were plated onto 35-mm dishes, supplemented with concentrated luciferin/luciferase and 0.1% calf serum,
then placed into the detection chamber of a luminometer that facilitated on-line detection of extracellular ATP. As illustrated in
Fig. 2, ATP released from the cells during the mixing of luciferin/
luciferase was degraded by cellular and ecto-ATPases. The cells
established a low (0.3 ⫾ 0.2 nM; n ⫽ 3), but significant, concentration of ATP in the extracellular bathing medium. Some ATP
was released during the addition of 2 ␮g/ml purified endotoxin
(Fig. 2B). However, this was also observed during the addition of
1 ␮l of vehicle (sterile water) to control cells and was judged to be
due to mixing (Fig. 2C). Moreover, extracellular ATP concentrations did not differ significantly (0.3 ⫾ 0.2 nM; n ⫽ 3) between
endotoxin-treated vs water-treated control cells (Fig. 2, B and C).
In contrast, permeabilization of the cells with alamethicin resulted
in the rapid release of endogenous ATP, producing peak concentrations in the 1– 4 ␮M range (Fig. 2A). Finally, scavenging of all
FIGURE 1. Indices of BAC1.2F5 macrophage activation by endotoxin.
A, NO production by adherent macrophages in 35-mm dishes stimulated
for 16 h with 2 ␮g/ml endotoxin in the presence or the absence of 500 ␮M
oATP. Triplicate samples were withdrawn from each dish for analysis;
uncertainties are SDs of the averaged measurements. B, Acute stimulation
of adherent macrophages prepared for on-line ATP assays in 35-mm
dishes. Cells were stimulated with vehicle (1 ␮l of water) or 2 ␮g/ml
endotoxin for 12 min at room temperature, then lysed and prepared for
Western blotting. Where indicated, cells were pretreated with 500 ␮M
oATP for 2 h before washing and medium replacement. Equivalent loading
was confirmed in a parallel blot using anti-pan-ERK Abs. C, Serum dependence of LPS signaling. Adherent cells in a 12-well plate were prepared
with ATP assay buffer with or without various concentrations of calf serum
(percentage, v/v, indicated). Cells were stimulated with 2 ␮g/ml endotoxin
(as indicated) for 12 min at room temperature, then lysed and prepared for
Western blotting (with phospho-specific anti-ERK1/2 Abs).
extracellular ATP with apyrase reduced luminescence to background levels.
To maintain full luciferase activity, it was necessary to perform
the on-line ATP assays at temperatures ⬍30°C. However, endotoxin signaling is maximally effective at 37°C. To confirm endotoxin responsiveness of the macrophages under reduced temperature conditions, parallel cultures containing varying concentrations
of serum were analyzed for phosphorylation of ERK kinases induced by acute endotoxin treatment. Modest activation was observed at room temperature using 0.1% calf serum and was significantly increased in the presence of higher serum concentrations
(Fig. 1C). To examine extracellular ATP under the conditions most
favorable for endotoxin signaling, on-line ATP assays were repeated in the presence of 10% serum.
Basal extracellular ATP concentrations (0.1 ⫾ 0.1 nM; n ⫽ 3)
were significantly reduced in the presence of increased serum
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Endotoxin signaling in BAC1.2F5 macrophages
7192
ENDOTOXIN DOES NOT INDUCE ATP RELEASE FROM MACROPHAGES
FIGURE 2. On-line measurements of extracellular ATP during acute endotoxin stimulation of BAC1.2F5 macrophages. Observation of adherent
macrophages bathed in ATP assay buffer and 0.1% calf serum. A, Time course of extracellular ATP-dependent light production during acute activation with
2 ␮g/ml endotoxin, cellular permeabilization with 20 ␮g/ml alamethicin, and ATP scavenging with 20 U/ml apyrase. B, The same data as in A, using an
expanded vertical scale. C, Identical treatment of macrophages, using 1 ␮l of sterile water as a vehicle stimulus. Similar results were obtained using
RAW264.7 macrophages.
Released ATP is subject to hydrolysis not only by soluble nucleotidases in serum, but also by cell-associated ecto-ATPases (35).
Therefore, it was conceivable that released ATP might have escaped detection by being rapidly scavenged during its release. To
test this possibility, we repeated the ATP release assays in the
FIGURE 3. On-line measurements of extracellular ATP using higher serum concentrations. A, Time course of extracellular ATP-dependent light
production during acute activation of BAC1.2F5 macrophages with 2 ␮g/ml endotoxin in the presence of 10% calf serum. B, The same data as in A, using
an expanded vertical scale. C, Identical treatment of macrophages, using 1 ␮l of sterile water as a vehicle stimulus. D, Inhibition of macrophage
ecto-ATPases with 300 ␮M ␣,␤-meATP (added at the beginning of the measurement period, as indicated by ␣␤). E and F, Experiments corresponding to
B and C, but in the presence of 300 ␮M ␣,␤-meATP.
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(Fig. 3, A–C; compare with Fig. 2). This was a reflection of the
substantial ATPase activity present in serum preparations. However, despite the fact that these conditions were more favorable for
endotoxin signaling (Fig. 1C), no significant release of ATP was
observed during acute endotoxin stimulation (Fig. 3, A–C).
The Journal of Immunology
7193
Table I. Extracellular ATP, ADP, and AMP concentrations following acute activation of macrophages at 37°Ca
BAC1.2F5
␣,␤-meATP:
ATP
ADP
AMP
RAW264.7
⫺C
⫺ endo
⫹C
⫹ endo
⫺C
⫺ endo
⫹C
⫹ endo
bkgd
35.7 ⫾ 10.8
96.7 ⫾ 15.0
bkgd
10.2 ⫾ 7.7*
21.4 ⫾ 19.0*
10.1 ⫾ 1.7
27.0 ⫾ 4.4
87.7 ⫾ 6.8
6.2 ⫾ 9.3
9.9 ⫾ 14.4
46.8 ⫾ 14.3*
2.2 ⫾ 1.0
22.6 ⫾ 4.1
42.0 ⫾ 21.0
bkgd
8.9 ⫾ 3.7*
22.3 ⫾ 9.3
8.6 ⫾ 2.4
26.0 ⫾ 4.9
33.0 ⫾ 30.4
6.4 ⫾ 1.3
19.7 ⫾ 5.3
16.0 ⫾ 7.6
a
Numerical values are concentrations in nanomolar; uncertainties are SDs for triplicate samples from a representative experiment. bkgd, Concentrations below background;
PEP mix contained on the order of 5 nM ATP. All cells were bathed in ATP assay buffer ⫹ 10% calf serum at 37°C. C, Unstimulated macrophages; endo, macrophages stimulated
with 2 ␮g/ml endotoxin for 12 min. Values for endotoxin-treated macrophages indicated with an asterisk are significantly less than the corresponding values for unstimulated
macrophages (two-sample t test at the 0.025 significance level). Columns indicated with “⫹” correspond to cells incubated in the presence of 300 ␮M ␣,␤-meATP (to inhibit
ecto-ATPases), which was added 10 min before endotoxin.
FIGURE 4. On-line measurements of extracellular ATP following prolonged endotoxin activation of macrophages. BAC1.2F5 macrophages
were pretreated with or without 10 ␮g/ml endotoxin for 16 h. A, Averages
of on-line steady-state extracellular ATP concentrations in bulk medium
bathing macrophages at various times after washing; media were replaced
with 1.3 ml of phenol red-free, serum-free DMEM, and cells were returned
to a CO2 incubator for the times indicated before assaying nitrite and extracellular ATP. At assay time, three 100-␮l samples were withdrawn for
nitrite analysis, and the remaining medium was supplemented with concentrated luciferin/luciferase. Uncertainties are SDs of steady-state averages. B, Nitrite content of triplicate samples drawn from the media of the
cells described in A. Uncertainties are SDs of the averaged triplicates.
the cells with endotoxin (average, 7.6 ⫾ 0.7 nM; n ⫽ 3; Fig. 3, E
and F).
Extracellular adenine nucleotide levels in macrophage cultures
stimulated with endotoxin at 37oC
In contrast to the reduced temperature used in our on-line assays of
extracellular ATP, most studies of endotoxin signaling use macrophages incubated under standard tissue culture conditions, i.e., at
37°C in the presence of 10% serum. Under these conditions the
overall rate of extracellular ATP hydrolysis will be increased.
Therefore, endotoxin-induced activation of macrophages at 37°C
in the presence of high serum may result in accumulation of extracellular ADP and AMP (vs ATP per se) due to very rapid hydrolysis of released ATP. Alternatively, these nucleotides might be
directly released from endotoxin-stimulated cells. To determine
extracellular ATP, ADP, and AMP concentrations in medium conditioned by macrophages under standard tissue culture conditions,
samples were removed and biochemically analyzed off-line (Table
I). The ATP concentration in these medium samples was lower
than the background, consistent with the high ecto-ATPase activity
of 10% serum. In contrast, these conditioned samples contained
higher levels of ADP (35.7 ⫾ 10.8 nM) and AMP (96.7 ⫾ 15.0
nM). However, there were no significant increases in adenine nucleotide concentrations in the presence of endotoxin. Rather, extracellular ADP and AMP concentrations were reduced following
endotoxin stimulation, suggesting the activation of an extracellular
nucleotidase. Similar observations were noted for RAW264.7
macrophages (Table I). To determine whether inhibition of ectoATPases might reveal endotoxin-induced nucleotide release, similar experiments were performed in the presence of ␣,␤-meATP.
Inhibition of ecto-ATPase activity was confirmed by the increased
ATP concentrations (Table I, compare ATP values in the absence
and the presence of ␣,␤-meATP). However, no additional accumulation of extracellular ATP, ADP, or AMP was apparent following acute endotoxin activation of either BAC1.2F5 or
RAW264.7 macrophages (Table I).
Table II. Extracellular adenine nucleotide concentrations following
prolonged endotoxin activation of BAC1.2F5 macrophagesa
Conditions
ATP (nM)
ADP (nM)
AMP (nM)
Control
Endotoxin
3.1 ⫾ 2.8
4.9 ⫾ 1.9
23.3 ⫾ 4.7
25.3 ⫾ 9.0
136 ⫾ 71
206 ⫾ 145
a
BAC1.2F5 macrophages on 35-mm dishes were treated with 2 ␮g/ml endotoxin
for 15–18 h. The cell cultures were then washed, replenished with fresh phenol redfree, serum-free DMEM, and returned to the CO2 incubator. Samples were collected
after allowing cells to reestablish steady-state extracellular conditions for 4 h. All data
are the averages of at least five replicates collected during two independent experiments; uncertainties are SDs of the mean.
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presence of ␣,␤-meATP, an ATP analogue that inhibits ecto-ATPases without substantially altering luciferase activity. It should be
stressed that BAC1.2F5 macrophages do not express P2X receptor
subtypes that can be activated by ␣,␤-meATP (36). The traces in
Fig. 3D illustrate the substantial ecto-ATPase activity of adherent
BAC1.2F5 macrophages and 10% serum under these conditions
and confirm the ability of ␣,␤-meATP to repress this activity by
⬎80%. Extracellular ATP concentrations were significantly higher
(average, 8.2 ⫾ 0.9 nM; n ⫽ 3) in the presence of ␣,␤-meATP
than in its absence (0.1 ⫾ 0.1 nM), confirming that the cells release
and metabolize ATP constitutively. Nonetheless, additional extracellular ATP accumulation was not observed during treatment of
7194
ENDOTOXIN DOES NOT INDUCE ATP RELEASE FROM MACROPHAGES
Effect of prolonged endotoxin activation on extracellular
nucleotide concentrations
Detectable amounts of inducible NO synthase, cyclooxygenase-2,
various cytokines, and NO are produced by macrophages several
hours after initial endotoxin exposure. In addition, an increase in
extracellular ATP concentration has been reported following overnight endotoxin activation of mouse microglial cells (5). This suggested that an increase in ATP release may accompany prolonged
activation of macrophages by endotoxin.
To examine this possibility, adherent macrophages were prepared for on-line extracellular ATP measurements following overnight endotoxin activation. Cultures in 35-mm dishes were
washed, placed in fresh medium, and returned to the incubator,
during which time nucleotides released from the control or endotoxin-activated cells conditioned the medium. Following the initiation of these secondary incubations, paired culture dishes (activated vs control) were removed for on-line analysis. The data
presented in Fig. 4A summarize steady-state extracellular ATP
concentrations at 0, 4, and 8 h following cell washing and medium
replacement. Extracellular ATP concentrations did not differ significantly between endotoxin-activated cells and unstimulated controls at any time point. Eight hours after medium replacement, the
average steady-state extracellular ATP concentrations for unstimulated and endotoxin-activated cells were 0.3 ⫾ 0.2 and 0.4 ⫾ 0.2
nM, respectively (Fig. 4A). That steady-state extracellular ATP
immediately after washing and medium replacement (Fig. 4A, 0 h
vs later time points) was substantially higher than the subnanomolar levels observed at 4 and 8 h probably reflects regulated, me-
chanically induced nucleotide release or acute cellular damage that
occurred during the washing and medium replacement.
To confirm endotoxin activation of the cells used for these extracellular ATP measurements, parallel samples of the medium
were assayed for nitrite content indicative of ongoing NO production. Although endotoxin-treated cells failed to exhibit an elevation
in bulk extracellular ATP relative to untreated cells (Fig. 4A), they
continued to release NO, verifying their activation by endotoxin
(Fig. 4B).
Extracellular ADP and AMP concentrations in conditioned medium samples were also measured. These concentrations were similar in the medium from control macrophages and cells exposed to
prolonged endotoxin stimulation (Table II). Together, these observations (Fig. 4 and Table II) demonstrate that no significant
changes in bulk extracellular adenine nucleotide concentrations are
apparent following prolonged activation of macrophages by
endotoxin.
Ecto-ATPase activities on activated vs control BAC1.2F5
macrophages
Extracellular nucleotide concentrations reflect a balance between
the rates of their release and subsequent breakdown (26). Hydrolysis of extracellular ATP to ADP, AMP, and adenosine is accomplished by the sequential actions of ecto-enzymes that include the
CD39 family ecto-apyrases, the PC-1/autotaxin family of ecto-nucleotide pyrophosphatases, and CD73 ecto-5⬘-nucleotidase (35,
37). Given that endotoxin activation of macrophages can alter the
expression of various ATP receptors (38), it is conceivable that the
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FIGURE 5. BAC1.2F5 macrophage ecto-ATPase activity. A, ATP-dependent luminescence before and after the addition of 10 nM ATP (final concentration). All samples were examined at room temperature using 1 ml of ATP assay medium with or without 1% calf serum as indicated. Time points were
collected every 4 min, with gentle mixing (side-side tipping) of medium before each. This was repeated until luminescence returned to basal (prestimulation)
levels, after which time no mixing was performed. Indicated values are the averages (⫾SD) for three individual samples. Sample averages were significantly
different (by one-way ANOVA at 0.05 significance level) at every time point displayed. B, The data from A presented on a logarithmic axis to illustrate
differences in light intensity. C, Ecto-ATPase activities in endotoxin-stimulated vs control preparations. Endotoxin-activated cells were pretreated with or
without 2 ␮g/ml endotoxin in 35-mm dishes for 16 h before being prepared for on-line ATP assays. The medium for all recordings was serum free. At the
indicated times, exogenous pulses of ATP were added, and luminescence was measured, with mixing, for the next 12 min. D, The same data as C presented
on a logarithmic axis to illustrate the similarity in first order decay rate constants at each ATP concentration.
The Journal of Immunology
7195
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expression of macrophage ecto-nucleotidases might similarly be
modulated. Because steady-state extracellular nucleotide concentrations did not increase following prolonged endotoxin activation
(Fig. 4 and Table II), any concerted alteration of ecto-ATPase
activity would need to be matched by a similar change in nucleotide release rates.
To measure cell-associated ecto-ATPase activities, pulses of exogenous ATP were added to serum-free macrophage cultures, and
the resulting luciferase-dependent luminescence was recorded online. As indicated in Fig. 5, A and B, 10-nM pulses of exogenous
ATP were rapidly degraded by the cells (t1/2 ⫽ ⬃15 min), resulting in the re-establishment of low nanomolar concentrations within
1 h (average of the last six time points, 1.7 ⫾ 0.2 nM). In contrast,
the luciferase-containing assay medium displayed relatively little
ATPase activity unless supplemented with serum. Constitutive,
low level ATP release from the macrophages was inferred from the
finding that ATP concentrations in cell-free, serum-containing medium were significantly less than those in medium bathing cells at
all time points (Fig. 5, A and B).
Cellular ecto-ATPase activities were determined over a range of
added ATP concentrations by adding sequential ATP pulses of
increasing concentration and monitoring the resulting luminescence. At each concentration of added ATP, decays in light output
were fitted to single exponentials, which yielded numerical values
for ATP hydrolysis rates. Decay time constants did not vary with
ATP concentration and were found not to differ significantly between control (0.08 ⫾ 0.01 min⫺1) and endotoxin-activated
(0.09 ⫾ 0.01 min⫺1) cells (Fig. 5, C and D, and Table III). This
suggests that nucleotide release rates per se remained unaffected
during endotoxin activation.
Effects of oxidized ATP and apyrase on endotoxin-induced NO
production
The hypothesis that released ATP is an autocrine potentiator of
macrophage endotoxin signaling in macrophages is primarily
based on observations that oATP and other P2 receptor antagonists
can attenuate endotoxin-stimulated macrophage responses, such as
the induction of NO synthase (5–9). A corollary of this hypothesis
is that endotoxin-induced signaling should also be reduced by exogenously added ATP scavengers, such as soluble apyrase (E.C.
3.6.1.5). To examine this possibility, NO production was compared in endotoxin-stimulated macrophages that were treated with
either oATP or apyrase. As shown in Fig. 6, A and B, oATP coincubation significantly attenuated the accumulation of nitrite in
the medium from endotoxin-challenged BAC1.2F5 or RAW 264.7
Table III. ATPase rate constants for control vs endotoxin-treated
BAC1.2F5 macrophagesa
Control
Added ATP (nM)
10
30
100
300
Averages ⫾ SD:
Endotoxin
k
v
k
v
0.07
0.09
0.09
0.08
0.08 ⫾ 0.01
0.7
3.1
9.8
23.6
0.08
0.10
0.09
0.08
0.09 ⫾ 0.01
0.7
3.1
8.9
23.5
a
First-order rate constants (k) are expressed in units of min⫺1, and were extracted
from single exponential fits to the observed decay curves (Fig. 5C). Hydrolysis rates
(v) are expressed as pmol ATP/min/106 cells, and were calculated for each ATP
addition as k 䡠 [ATP]obs 䡠 (1 ml). ATP accumulated in the cell-free media with each
addition so that [ATP]obs was equal to the sum of the added concentrations. This was
not true for extracellular media because of ongoing hydrolysis (Fig. 5C). “Endotoxin”
cells were stimulated overnight with 2 ␮g/ml endotoxin before being washed and
prepared for online assays. Activation of the stimulated macrophages was confirmed
by measuring nitrite accumulation in the media before washing (data not shown).
FIGURE 6. Apyrase fails to attenuate endotoxin-induced nitrite production. Oxidized ATP (500 ␮M) and apyrase (10 U/ml; grade III) were added
several minutes before endotoxin. Basal readings correspond to nitrite in the
medium bathing unstimulated cells. A, The same data as in Fig. 1A, including
nitrite levels in the medium from apyrase-treated macrophages. B, RAW264.7
macrophages were initially seeded in a 96-well plate, incubated overnight, then
stimulated with the indicated reagents or left undisturbed. After 16-h incubation, nitrite was assayed by adding 100 ␮l of Griess reagent directly to the
wells. C, ATPase activity in the medium conditioned by overnight exposure of
the cells under the conditions described in A. One milliliter of the medium
from each of the preparations was collected, centrifuged briefly to remove any
nonadherent cells, then supplemented with luciferin/luciferase and assayed for
ATPase activity by adding exogenous ATP pulses as indicated.
macrophages. However, apyrase failed to mimic this inhibitory
effect of oATP (Fig. 6, A and B). Medium conditioned overnight
by endotoxin-stimulated cells in the presence of apyrase exhibited
7196
ENDOTOXIN DOES NOT INDUCE ATP RELEASE FROM MACROPHAGES
high ATPase activity, to the extent that light production elicited by
exogenous ATP addition was negligible (Fig. 6C); this verified that
apyrase activity remained high during NO production. Thus, the
efficient scavenging of extracellular ATP has no significant effect
on the endotoxin-dependent up-regulation of inducible NO synthase expression in murine macrophage cell lines.
Discussion
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
These studies indicate that release of ATP and autocrine activation
of P2 nucleotide receptors are not part of the pleiotropic response
of macrophages to direct stimulation by endotoxin. Three different
experimental approaches support this conclusion. First, extracellular nucleotide concentrations were measured using both on-line
assays (Figs. 2– 4) and standard biochemical analysis of macrophage-conditioned medium (Tables I and II). ATP, ADP, and AMP
in the extracellular medium of macrophages were not increased as
a result of endotoxin stimulation regardless of experimental protocol or the presence of ecto-ATPase inhibitors. Second, macrophage ecto-ATPase activities were measured directly and were unchanged by prolonged endotoxin activation (Fig. 5 and Table III).
Any marked reduction in ecto-ATPase activity in response to endotoxin would have been expected to increase the steady-state concentration of extracellular ATP. Third, extracellular nucleotide
concentrations were clamped at subnanomolar levels (by addition
of exogenous apyrase) during prolonged endotoxin exposure.
Strong and rapid scavenging of extracellular ATP and ADP
throughout the endotoxin stimulation period did not reduce NO
production (Fig. 6).
Our experimental methods were chosen to preclude several
sources of systematic experimental error that might otherwise produce artifactual results. All endotoxin was explicitly tested for
ATP contamination. Serotype 0111:B4 (List Biologicals) was
found to contain very little ATP and was, therefore, suitable for
these studies. Because medium agitation or removal can cause lytic
or mechanically induced nucleotide release (23–26), cells were
allowed to recondition their media for at least 45 min after all
washes. Each on-line analysis of adherent macrophages included
direct calibration with known amounts of ATP, permeabilization to
liberate total intracellular ATP, and treatment with apyrase to determine background luminescence; these steps permitted rigorous
quantitative evaluation of any changes in extracellular ATP accumulation or catabolism. Steady-state extracellular concentrations
of ATP in the bulk medium were thus shown to correspond to
⬍0.1% of the total ATP released during cellular permeabilization,
indicative of the integrity of the cells during experimental observation. Moreover, the observed release of 1– 4 ␮M ATP during
intentional permeabilization with alamethicin agrees with an estimate of the total releasable ATP within 106 macrophage cells, each
of ⬃5-␮m radius and containing 5 mM total intracellular ATP,
lysed into a 1-ml volume of extracellular medium.
On-line measurements were recorded in real time over many
minutes. This was necessary because released extracellular ATP
was subject to hydrolysis by macrophage and serum ecto-ATPases
(Fig. 5). Recent studies by Lazarowski et al. (26) have demonstrated that the instantaneous extracellular nucleotide concentration reflects competing rates of constitutive release and hydrolysis
that might vary over time due to changes in the intrinsic activity of
the various ecto-nucleotidases. Our comparison of macrophage
ecto-ATPase activities revealed no significant differences between
control and endotoxin-activated cells (Fig. 5, C and D, and Table
III), suggesting that expression of these ecto-enzymes was unaltered by prolonged endotoxin stimulation (in contrast, serum ATPase activity appeared to be enhanced during endotoxin stimulation (Table I)). Because steady-state extracellular nucleotide
concentrations did not differ in the media of endotoxin-activated
macrophages (Fig. 4A), this result implied that ATP release rates
per se also remained unchanged.
The ability of macrophages to maintain extracellular ATP concentrations in the nanomolar range in the presence of potent ectoATPases is indicative of ongoing, albeit low level, ATP release
(Figs. 2–5). Accumulation of extracellular ATP under basal conditions has been reported for other cell types (22–26, 39 – 42). The
0.3 ⫾ 0.2 nM ATP we measured in the bulk, serum-free extracellular medium of BAC1.2F5 macrophage cultures (Fig. 4A) was
similar to values (0.5–10 nM) previously reported for monocytic
cells (6, 22). This value is significantly less than the concentration
required to activate even the most sensitive P2X or P2Y receptors,
which exhibit EC50 values in the 0.1–1 ␮M range (43, 44). Multiplying the ecto-ATPase decay rate constant determined in Table
III (0.08 ⫾ 0.01 min⫺1) and the observed steady-state extracellular
concentration determined in Fig. 4A (0.3 ⫾ 0.2 nM) for unstimulated cells, the basal rate of ATP hydrolysis is estimated to be
about 24 fmol/min 䡠 106 cells. ATP release from the cells at this
rate is in the range of those measured for other cells types (26) and
corresponds to ⬍0.1% of the average rate of ATP production at
steady state; this constitutive release should be easily maintained
without compromising overall intracellular energy metabolism.
Our results confirm and significantly extend the findings of Grahames et al. (22), who observed no significant difference in extracellular ATP in medium samples conditioned by control vs endotoxin-stimulated human THP-1 monocytes. In contrast, other
investigators have reported elevations in extracellular ATP during
acute addition of endotoxin to suspended, perfused RAW 264.7
murine macrophages (6) and following prolonged activation of
adherent murine microglial cells (5). These differences may be due
to differences in experimental technique.
Our observation that scavenging extracellular nucleotides with
apyrase exerted no antagonistic effect on endotoxin-induced NO
production (Fig. 6) is also consistent with the previous report that
apyrase failed to attenuate endotoxin-induced IL-1␤ release from
THP-1 human monocytes (22). These findings demonstrate that the
responses of monocyte/macrophages to endotoxin per se are independent of autocrine feedback by extracellular nucleotides. This
suggests that nucleotides used for purinergic regulation of macrophages at an inflammatory locus are released from other cells, such
as neutrophils, damaged tissues, or bacteria. Neutrophils have been
reported to release relatively high amounts of nucleotides during
activation by formyl peptides (45). A recent study also described
the release of ATP from J774 macrophage cultures infected with
Mycobacterium tuberculosis (8). Given the lack of ATP release
from macrophages in response to direct endotoxin exposure (indicated by our studies), this latter observation suggests that nucleotides might accumulate as a result of either ongoing lysis of infected macrophages (as discussed in Ref. 8) or continual release
from the bacteria per se.
Autocrine feedback by released nucleotides occurs in the vicinity of cell surfaces. Measurements of bulk extracellular nucleotide
concentrations may not accurately reflect local concentrations, because diffusion into the bulk medium (⬃1 mm thickness for 1 ml
of medium in a 35-mm dish) is slow on the length scale of millimeters and may be hindered by unstirred layer effects or membrane
invaginations. For this reason, we recently developed and described the use of a cell surface-anchored, chimeric form of luciferase (protein A-luciferase), which can provide information about
extracellular ATP concentrations at the immediate surface of activated cells (46). In preliminary experiments this reagent has been
The Journal of Immunology
Acknowledgments
We thank Atossa Alavi, Lalitha Gudipaty, Benjamin Humphreys, Faramarz
Ismail-Beigi, Sheldon Joseph, Gary Landreth, and Karen Parker for helpful
discussions and critical readings of this paper while in preparation. Sylvia
Kertesy provided excellent technical assistance.
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endotoxin signaling. Our data suggest that these will prove to be
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