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Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
(1, 2, 3, 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-1ß, IL-6, platelet-activating 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 endotoxin-dependent responses.
This hypothesis is based on observations that treatment of macrophages
with ATP receptor antagonists, including periodate-oxidized ATP (oATP)
and pyridoxal-phosphate-6-azophenyl-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-1ß, NO, arachidonic
acid, and cyclooxygenase-dependent products of arachidonic acid
metabolism (5, 6, 7, 8, 9, 10, 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, 13, 14). Additionally, a subset of macrophage
responses to endotoxin is modulated by costimulation with exogenous
nucleotides (5, 15, 16, 17, 18, 19, 20, 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, 24, 25, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All nucleotides were purchased as either crystalline free acids
or sodium salts; stock solutions were calibrated by absorption
spectroscopy at 259 nm. ATP, oATP, ,ß-methylene-ATP
(,ß-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
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 µl/well in 96-well plates) and allowed to grow overnight before experimentation.
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 µl 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, 12-min 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 µl 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 µM
,ß-meATP (as indicated) in 35-mm dishes. Sequential pulses of
exogenous ATP ranging from 10 to 300 nM were added (light production
was independently verified to be linearly related to ATP concentration
from 300 pM to 1 µM). 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.
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 µl of 4 M KOH and 450 µl 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 µl
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 µl of PK. AMP assay mix consisted of 0.5 ml of PEP mix, 7.5 µl
of PK, 8 µl of myokinase, and 4 µl of 100 µM ATP. Twenty-five
microliters of PEP mix, ADP assay mix, or AMP assay mix was added to
150-µl 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 luciferase-based 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 µl) were mixed with 100 µl 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-µl 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 µl 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).
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 µg/ml 0111:B4 endotoxin (or 1 µl 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 ß-glycerophosphate, 0.1 mM Na3VO4, 25 mM HEPES (pH 7.5), 2 mM PMSF, and 100 µg/ml leupeptin). ERK phosphorylation was determined by Western blotting using phospho-specific anti-pERK1/2 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 µM) 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1ß 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, 18, 19, 34).
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, 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, 6, 7, 8, 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 µg/ml purified endotoxin (Fig. 2B). However, this was
also observed during the addition of 1 µl 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 µM range (Fig. 2A). Finally, scavenging of all extracellular ATP with
apyrase reduced luminescence to background levels.
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C). 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 (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).
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,ß-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 ,ß-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 ,ß-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
,ß-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 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 ecto-ATPases might reveal endotoxin-induced nucleotide
release, similar experiments were performed in the presence of
,ß-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 ,ß-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).
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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, mechanically induced nucleotide
release or acute cellular damage that occurred during the washing and
medium replacement.
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A),
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.
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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
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 on-line. 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).
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, C and
D, and Table III). This
suggests that nucleotide release rates per se remained unaffected
during endotoxin activation.
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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, 6, 7, 8, 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
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 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) 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, 24, 25, 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 µM
ATP during intentional permeabilization with alamethicin agrees with an
estimate of the total releasable ATP within 106
macrophage cells, each of 5-µm 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 ecto-ATPases 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, 23, 24, 25, 26, 39, 40, 41, 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 µM 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 · 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-1ß 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 tethered to BAC1.2F5
cells and used to monitor cell surface-localized extracellular ATP. No
significant differences were observed between the extracellular ATP
concentrations (35 ± 16 vs 28 ± 18 nM; n =
2) measured with luciferase-coated macrophages incubated in the absence
or the presence, respectively, of exogenously added endotoxin. However,
our current methods for production of recombinant protein A-luciferase
use bacterial expression, and residual endotoxin contamination of the
purified protein A-luciferase limits our ability to use this reagent
without preactivation of endotoxin signaling during the cell-tethering
step. Nonetheless, we find no experimental basis to support the
hypothesis of a surface-localized increase in extracellular ATP during
acute activation by endotoxin.
The P2X7 receptor is an ATP-gated ion channel expressed on monocytic cells in levels that are regulated by pro- and anti-inflammatory stimuli (12, 13, 38, 43, 47). It is also the only P2 receptor subtype known to be strongly antagonized by oATP (11). Because oATP treatment attenuates multiple functions of monocytes and macrophages, autocrine activation of the P2X7 nucleotide receptor has been implicated in the fusion of monocytes into multinucleate giant cells (48) and in various sequellae of endotoxin activation, such as the release of arachidonic acid, NO, and IL-1ß (5, 6, 7, 9, 20). However, oATP was originally developed as an affinity label for ATP-binding enzymes. When used in vitro, oATP can modify a variety of other proteins, including phosphoglycerate kinase, Na+,K+-ATPase, histone kinase, mitochondrial ATPase, and ecto-ATPases (11, 49, 50, 51, 52). Thus, effects hitherto ascribed to P2X7 receptor antagonism may be due to inhibition of other ATP-dependent enzymes or signaling proteins. The finding by Sikora et al. (8) that oATP pretreatment repressed endotoxin-induced NO production in macrophages derived from mice bearing a targeted deletion of the P2X7 gene supports this interpretation. Intracellular accumulation of oATP and de facto modification of intracellular proteins may occur via pinocytosis of extracellular fluid by macrophages. This would be facilitated by the 2- to 3-h preincubation with oATP that is required to observe inhibition of macrophage responses to endotoxin. Additional studies are required to determine the molecular targets of oATP that result in repression of endotoxin signaling. Our data suggest that these will prove to be species other than ATP-binding purinergic receptors.
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Acknowledgments |
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Footnotes |
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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.
3 Abbreviations used in this paper: ERK, extracellular regulated kinase; oATP, periodate-oxidized ATP; ,ß-meATP, ,ß-methylene-ATP; FL-AAM, firefly luciferase ATP assay mix; PEP, phosphoenolpyruvate; PK, pyruvate kinase.
Received for publication August 15, 2000. Accepted for publication September 13, 2000.
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