ATP Release From Activated Neutrophils Occurs via
Connexin 43 and Modulates Adenosine-Dependent
Endothelial Cell Function
Holger K. Eltzschig, Tobias Eckle, Alice Mager, Natalie Küper, Christian Karcher,
Thomas Weissmüller, Kerstin Boengler, Rainer Schulz, Simon C. Robson, Sean P. Colgan
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Abstract—Extracellular ATP liberated during hypoxia and inflammation can either signal directly on purinergic receptors
or can activate adenosine receptors following phosphohydrolysis to adenosine. Given the association of polymorphonuclear leukocytes (PMNs) with adenine-nucleotide/nucleoside signaling in the inflammatory milieu, we
hypothesized that PMNs are a source of extracellular ATP. Initial studies using high-performance liquid chromatography and luminometric ATP detection assays revealed that PMNs release ATP through activation-dependent pathways.
In vitro models of endothelial barrier function and neutrophil/endothelial adhesion indicated that PMN-derived ATP
signals through endothelial adenosine receptors, thereby promoting endothelial barrier function and attenuating
PMN/endothelial adhesion. Metabolism of extracellular ATP to adenosine required PMNs, and studies addressing these
metabolic steps revealed that PMN express surface ecto-apyrase (CD39). In fact, studies with PMNs derived from
cd39⫺/⫺ mice showed significantly increased levels of extracellular ATP and lack of ATP dissipation from their
supernatants. After excluding lytic ATP release, we used pharmacological strategies to reveal a potential mechanism
involved in PMN-dependent ATP release (eg, verapamil, dipyridamole, brefeldin A, 18-␣-glycyrrhetinic acid,
connexin-mimetic peptides). These studies showed that PMN ATP release occurs through connexin 43 (Cx43)
hemichannels in a protein/phosphatase-A– dependent manner. Findings in human PMNs were confirmed in PMNs
derived from induced Cx43⫺/⫺ mice, whereby activated PMNs release less than 15% of ATP relative to littermate
controls, whereas Cx43 heterozygote PMNs were intermediate in their capacity for ATP release (P⬍0.01). Taken
together, our results identify a previously unappreciated role for Cx43 in activated PMN ATP release, therein
contributing to the innate metabolic control of the inflammatory milieu. (Circ Res. 2006;99:1100-1108.)
Key Words: nucleotide 䡲 nucleoside 䡲 adenosine 䡲 endothelia 䡲 inflammation 䡲 ATP 䡲 connexin
䡲 inflammation 䡲 hypoxia
P
ast studies have revealed a central role of extracellular
nucleotide phosphohydrolysis and nucleoside signaling
in innate immune responses during conditions of limited
oxygen availability (hypoxia) or during acute inflammation.
For example, metabolic enzymes and vascular nucleotide
levels are consistently increased during hypoxia.1,2 The contribution of individual nucleotides (ATP, ADP, AMP) to
these innate responses remain unclear. Polymorphonuclear
granulocytes (PMNs) function as a first line of cellular
response during acute inflammatory episodes.3 Previous reports have suggested that PMNs may release ATP during
conditions of inflammation or hypoxia.1 Such extracellular
ATP can either signal directly to vascular ATP receptors4 or
may function as a metabolite following conversion via
ecto-apyrase (CD39, conversion of ATP to AMP) and ecto5⬘-nucleotidase (CD73, conversion of AMP to adenosine).
In the present study, we aimed to identify molecular
mechanisms involved in ATP release from activated PMNs
and detail consecutive metabolic and signaling pathways to
modulate endothelial cell function. For this purpose, we used
pharmacological and genetic approaches to inhibit ATP
release from human or rodent PMNs.
Materials and Methods
Isolation of Human PMNs
After approval by the Institutional Review Board and obtaining
written informed consent from each individual, PMNs were freshly
isolated as described previously.5,6
Original received July 8, 2006; resubmission received August 28, 2006; accepted October 3, 2006.
From the Department of Anesthesiology and Intensive Care Medicine (H.K.E., T.E., A.M., N.K., C.K., T.W.), Tübingen University Hospital, Germany;
Institut für Pathophysiologie (K.B., R.S.), Zentrum für Innere Medizin, Universitätsklinikum Essen, Germany; Transplantation Center (S.C.R.),
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School; and Center for Experimental Therapeutics and Reperfusion
Injury (S.P.C.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Holger K. Eltzschig, MD, PhD, Department of Anesthesiology and Intensive Care Medicine, Tübingen University Hospital,
Waldhörnle Str. 22, D-72072 Tübingen, Germany. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000250174.31269.70
1100
Eltzschig et al
Cx43-Dependent Neutrophil ATP Release
1101
Preparation of Activated PMN Supernatants and
Measurement of ATP or Myeloperoxidase Content
Activated PMN supernatants were prepared and ATP content measured as described previously.1
Cell Viability Assay
To evaluate lytic cell death of PMNs, lactate dehydrogenase (LDH)
activity was measured in the supernatant (Roche Diagnostics).
PMN Granule Isolation
The granule fraction from PMNs was purified from resting neutrophils as previously described.7
Immunoblotting Experiments
PMNs were freshly isolated from human donors and lysed and
blotted as described previously.1
Measurement of CD39 and CD73 Activity
on PMNs
Surface enzyme activity was measured as described previously.1
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Flow Cytometric Analysis of PMN Surface
Expression of CD39 and CD73
Surface expression of CD73 and CD39 on PMNs were measured as
described previously.8
Macromolecule Paracellular Permeability Assay
Endothelial permeability of cultured endothelia was measured as
described previously.1
PMN Adhesion Assay
PMN adhesion was measured as described previously.5
Isolation and Activation of Murine PMNs
In subsets of experiments, PMNs were isolated from previously
described mice with targeted gene deletion of cd399 or induced
ablation of connexin 43 (Cx43).10 These protocols were in accordance with NIH guidelines for the use of live animals and were
approved by the Institutional Animal Care and Use Committee at
Brigham and Women’s Hospital and of the University of Essen,
Germany.
Data Analysis
Data were compared by 2-factor ANOVA, or by Student’s t test
where appropriate. In experiments on the kinetics of ATP release, 1
representative experiment of 3 is displayed (n⫽3), with triplicate
measurement of the ATP concentration. Values are expressed as the
mean⫾SD from at least 3 separate experiments.
An expanded Materials and Methods section is available in the
online data supplement at http://circres.ahajournals.org.
Results
PMNs Release ATP on Activation
We have previously shown that PMNs have the capacity to
release adenine nucleotides in the form of ATP,1 although the
molecular details of nucleotide release from PMNs remain
largely unknown. Here, we sought to understand details of
ATP release from PMNs. Initially, we determined whether
ATP release was activation dependent. For these purposes,
we distinguished extracellular ATP levels in the presence and
absence of the potent PMN activator fMLP (N-formyl MetLeu-Phe). In fact, ATP was readily detected in supernatants
of freshly isolated PMNs (based on biophysical criteria such
as retention time, coelution with internal ATP standards, and
UV absorption spectra [data not shown]). In fact, ATP release
increased by greater than approximately 6-fold on fMLP
Figure 1. ATP release from PMNs is activation dependent. A,
Based on coelution with ATP standards (ATP Standard), the
supernatant of resting PMNs contains small amounts of ATP
(Non Activated). In other samples, freshly isolated PMNs were
activated with fMLP (100 nmol/L). B, Quantification of ATP content within the supernatant of PMNs using a standard luminometric ATP detection assay. Freshly isolated PMNs were kept
on ice in calcium free buffer (4°C, no Calcium) and the ATP content was measured in the supernatant at indicated time points
after centrifugation to discard the cellular compounds of the
supernatant. In other samples, PMNs were warmed to 37°C and
rotated end-over-end in calcium containing HBSS (Non Activated). In a third series of experiments, PMNs were warmed to
37°C in calcium-containing HBSS (Hank’s plus), activated with
100 nmol/L fMLP (⫹fMLP), and rotated end-over-end (n⫽3). C,
In additional experiments, PMNs were repeatedly stimulated
with 100 nmol/L fMLP (second, third, n⫽3).
activation (area under curve of the high-performance liquid
chromatographic [HPLC] tracing; Figure 1A). These findings
from HPLC-based detection were confirmed using a luminometric ATP detection assays. As shown in Figure 1B, ATP
release from freshly isolated PMNs was 4.2⫾1.6 nmol/107
PMNs without activation at 4°C in Ca2⫹-free Hank’s balanced
salt solution (HBSS). Higher ATP levels were observed at
37°C in Ca2⫹-containing buffer (maximal levels 13.2⫾6.3
nmol/107 PMNs; P⬍0.01 by ANOVA). With fMLP activation, extracellular ATP concentrations profoundly increased,
with a rapid ATP peak as early as 1 minute after fMLP
activation (89⫾7.7 nmol/107 PMNs; P⬍0.001 by ANOVA),
and progressively dissipated to control levels within 15
minutes. As shown in Figure 1C, repeated stimulation with
fMLP results in an attenuated ATP response, with almost no
stimulated ATP release on a third fMLP stimulation (Figure
1C). These results indicate a metabolic and activationdependent release of ATP from human PMNs.
1102
Circulation Research
November 10, 2006
Mechanism of Extracellular ATP Metabolism
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
In the course of these studies, we addressed the rapid loss of
extracellular ATP following PMN activation. In our experimental setting of 107 PMN/mL, extracellular ATP concentrations were as high as 100 nmol/L, whereas cytoplasmic
concentrations were as high as 5 mmol/L (see later), thereby
resulting in a 50 000-fold transmembrane ATP gradient,
making passive ATP reuptake highly unlikely. As a second
possibility, we considered extracellular ATP phosphohydrolysis by PMNs. A primary source of extracellular ATP
phosphohydrolysis is cell surface CD39, and, therefore, we
determined whether PMNs express surface CD39. For these
purposes, we used a nonnative exogenous substrate (ethenoATP), which could be distinguished from endogenous ATP
via HPLC analysis. To measure CD39 activity, we quantified
etheno-ATP conversion to etheno-AMP by intact PMNs
(107/mL) in the presence and absence of the CD73 inhibitor
␣-␤-methylene-ADP (APCP) (10 ␮mol/L). Previous studies
have indicated that etheno-ATP (and etheno-AMP for CD73)
can be used for measuring CD39/73 activity as they show
similar conversion rates as their native compounds.1 As
shown in Figure 2A, isolated PMNs rapidly metabolized
etheno-ATP to etheno-AMP, suggesting high levels of CD39activity. Surprisingly, etheno-AMP was stable in the supernatant independent of the presence of APCP (10 ␮mol/L),
suggesting that PMNs express little or no CD73. To confirm
this hypothesis, we measured CD73 activity on PMNs (conversion of etheno-AMP to etheno-adenosine).1 This analysis
confirmed our inhibitor experiments and revealed no detectable CD73 on intact PMNs (Figure 2B). To confirm these
results, we used fluorescence-activated cell sorting (FACS)
analysis for CD39 and CD73 on various leukocyte populations. As shown in Figure 2C, PMNs and monocytes express
high levels of CD39, whereas lymphocytes lack CD39
surface expression. By contrast, PMNs and monocytes express no detectable CD73, whereas CD73 is highly expressed
on lymphocytes. These experiments in PMNs were repeated
following fMLP stimulation and did not influence the pattern
of CD39 and CD73 expression (data not shown).
Different Kinetics of ATP Levels Within the
Supernatant of Activated PMNs Derived From
cd39ⴚ/ⴚ Mice
We next extended the above findings with human PMNs to
murine PMNs. For these purposes, we compared CD39
activity on isolated PMNs from cd39-null mice9 and
littermate controls. Whereas PMNs from littermate controls readily converted etheno-ATP to etheno-AMP, such
activity was completely absent on PMNs from cd39⫺/⫺
mice (date not shown). We then measured ATP concentration in the supernatant of activated PMNs from cd39⫺/⫺
mice and compared them to littermate controls. Because
murine PMNs express little or no surface fMLP receptors,
we used leukotriene B4 (LTB4) (100 nmol/L) for activation
of PMNs.11 As shown in Figure 2D, freshly isolated PMNs
from wild-type mice released ATP in an activationdependent manner (maximal 6.7⫾0.57-fold increase), with
similar kinetics as human PMNs (see Figure 1B). Similar
to wild-type mice, PMNs from cd39⫺/⫺ mice also released
Figure 2. Expression and function of the ecto-apyrase (CD39)
and 5⬘-nucleotidase (CD73) on the surface of PMNs. A, CD39
activity on the surface of PMNs. PMNs were freshly isolated
from the blood of human volunteers and CD39 activity was
determined by HPLC analysis of etheno-ATP (E-ATP) conversion
to etheno-AMP (black bars). These experiments were also performed in the presence of the CD73 inhibitor APCP (10 ␮mol/L,
gray bars) to prevent further metabolism of etheno-AMP
(E-AMP) to etheno-adenosine (n⫽3). Results are expressed as
percentage of E-ATP conversion to E-AMP⫾SD (*P⬍0.01 compared with 0 minutes). B, CD73 activity on the surface of PMNs.
PMNs were freshly isolated from the blood of human volunteers,
and CD73 activity was determined by HPLC analysis of E-AMP
conversion to etheno-adenosine (black bars). These experiments
were also performed in the presence of APCP (10 ␮mol/L, gray
bars, n⫽3). Results are expressed as percentage of E-AMP
conversion to etheno-adenosine⫾SD. C, Flow cytometric analysis of CD39 and CD73 on PMNs, monocytes, and lymphocytes
(bold lines, isotype control). D, ATP release of activated PMNs
from cd39⫺/⫺ mice or littermate controls (cd39⫹/⫹). PMNs were
freshly isolated by double density centrifugation following cardiac puncture from cd39⫺/⫺ mice or littermate controls (cd39⫹/⫹).
PMNs were warmed to 37°C and rotated end-over-end in
calcium-containing HBSS (non activ.) or activated with 100
nmol/L LTB4 (⫹fMLP) (n⫽6). E, Reconstitution of activated
supernatants from cd39⫺/⫺ PMNs with soluble apyrase (0.1 U
per experiment; n⫽6)
ATP in an activation-dependent manner. Moreover, the
lack of extracellular metabolism through surface CD39
resulted in accumulation of ATP (1.6⫾0.09-fold increase
in maximal ATP levels compared with PMNs from wildtype mice; P⬍0.05). Similarly, ATP levels in the supernatant of nonactivated PMNs from cd39⫺/⫺ mice were
higher and stayed close to their peak concentration
throughout the experiment compared with wild-type PMNs
Eltzschig et al
TABLE 1.
Cx43-Dependent Neutrophil ATP Release
1103
LDH Release From PMNs
1 Minute
5 Minutes
10 Minutes
15 Minutes
fMLP0
0.12⫾0.03
0.24.⫾0.05
0.44⫾0.03
0.51⫾0.04
⫹fMLP
0.13⫾0.03
0.37⫾0.08
0.45⫾0.09
0.52⫾0.07
⫺LTB4
0.23⫾0.08
0.51⫾0.07
0.72⫾0.09
0.94⫾0.04
⫹LTB4
0.18⫾0.07
47⫾0.06
64⫾0.10
83⫾0.08
⫺LTB40
0.19⫾0.04
0.50⫾0.03
0.68⫾0.10
0.87⫾0.09
⫹LTB4
0.18⫾0.03
0.5⫾0.07
0.71⫾0.05
0.90⫾0.11
⫺LTB4
0.22⫾0.06
0.49⫾0.04
0.71⫾0.12
0.91⫾0.03
⫹LTB4
0.21⫾0.09
0.47⫾0.09
0.69⫾0.08
0.88⫾0.12
⫺LTB4
0.21⫾0.07
0.44⫾0.05
0.68⫾0.10
87⫾0.07
⫹LTB4
0.23⫾0.06
0.45⫾0.030
0.71⫾0.13
0.90⫾0.10
Human PMN
Murine PMN
Wild type
cd39⫺/⫺
fl/lfl
Cx43
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Cx43⫺/⫺
Determination of LDH activity in the supernatant from PMNs at indicated
time points (expressed as percentage of maximal release after cell lysis
induced by Triton X-100 treatment).
(Figure 2D; P⬍0.05 by ANOVA). Moreover, reconstitution with 0.1 U of apyrase of supernatant from cd39⫺/⫺
PMNs resulted in restoration of ATP metabolism (Figure
2E; P⬍0.01 by ANOVA). In addition, measurement of
LDH release into the supernatant from cd39⫺/⫺ or littermate controls was not different, suggesting that there is no
difference in lytic ATP release (Table 1).
Mechanisms of PMN ATP Release
ATP exists in the cytoplasm at millimolar concentrations12
and can be released extracellularly by several mechanisms,
including exocytosis of ATP-containing vesicles, transport
via connexin hemichannels, through nucleoside transporters, direct transport through ATP-binding cassette (ABC)
proteins or lytic cell death.12 To rule out lytic cell death as
mechanism for activation-dependent ATP release, we measured LDH activity in the supernatant from activated and
nonactivated PMNs. As shown in Table 1, no difference in
LDH release between activated and nonactivated PMNs
was found. In addition, LDH release in the supernatant has
a distinctively different kinetic (nonbiphasic), suggesting
that other mechanisms than lytic cell death are responsible
for PMN-dependent ATP release. Next, we considered
exocytosis of ATP-containing granular vesicles as a possible mechanism. To inhibit vesicular secretion, we used
the general secretion inhibitor brefeldin A (BFA).13 As
shown in Figure 3A, BFA (5 ␮g/mL) influenced neither
the kinetics nor the absolute amount of ATP liberated from
activated human PMNs. BFA significantly inhibited the
activated release of the granule-bound enzyme myeloperoxidase (MPO) (Figure 2B). Consistent with these findings, isolated granules from resting PMNs contained
greater than 95% of MPO activity (data not shown), but
nearly undetectable levels of ATP, whereas cytosolic ATP
Figure 3. ATP release from PMNs is not vesicular. A, Freshly
isolated PMNs were warmed to 37°C in calcium-containing
buffer and rotated end-over-end. The ATP content in the supernatant was measured using a standard luminometric ATP detection assay following fMLP activation (100 nmol/L) (⫹fMLP) or
without fMLP activation (Non Activated). In additional experiments, the vesicular secretion inhibitor BFA (5 ␮g/mL) was
added 1 hour before fMLP activation. B, Kinetics of MPO
release from fMLP-activated PMNs. To assess the kinetics of
vesicular release from fMLP-activated PMNs, the granular
marker MPO was measured from the supernatant. Freshly isolated PMNs were warmed to 37°C in calcium-containing buffer,
activated with fMLP, and rotated end-over-end (⫹fMLP). MPO
concentrations were measured from the supernatant at indicated time points. In control experiments, PMNs were kept on
ice in calcium free buffer (4°C, no Calcium) or assessed at 37°C
with calcium but without fMLP activation (Non Activated). C,
The ATP content of the cytosolic and the granular fraction from
freshly isolated PMNs were measured using a standard luminometric ATP detection assay.
concentrations were higher than 5 mmol/L (Figure 3C).
Taking together these studies suggest that activationdependent ATP release by neutrophils does not involve
granular exocytosis.
Role of Cx43 in ATP Release From PMNs
In view of the above results that ATP is not granule bound in
PMNs, we attempted pharmacological approaches to define
mechanisms of ATP release. Based on reports suggesting a
role of nucleoside transporter function in cellular ATP release, we examined the influence of nucleoside transport
inhibitor dipyridamole (1, 10, and 100 ␮mol/L) on PMN ATP
release.14 Dipyridamole had no effect on stimulated ATP
1104
Circulation Research
TABLE 2.
Pharmacological Examination of fMLP-Stimulated ATP Release From PMNs
1 min
Dipyridamole
Verapamil
18␣ GA
Cx40 Peptide
Cx43 Peptide
␮ mol/L
␮ mol/L
␮ mol/L
␮ mol/L
␮ mol/L
0
1
10
100
0
1
10
100
0
1*
10*
100*
0
3
30
300
0
3
30
300
92.3
90.2
91.5
86.6
89.0
91.0
87.0
93.0
93.0
56.2
32.1
16.3
93.0
91.0
90.3
95.3
93.0
62.1
23.1
15.2
(⫾SD) (⫾8.1) (⫾5.9) (⫾7.1) (⫾6.5) (⫾8.1)
5 min
November 10, 2006
28.2
24.9
27.4
23.3
26.3
(⫾8.6)
22.6
(⫾9.2) (⫾9.2) (⫾8.2) (⫾8.2) (⫾5.3) (⫾1.0) (⫾6.5) (⫾11.5) (⫾12.1)
20.6
26.9
24.6
17.2
12.1
6.12
21.6
(⫾SD) (⫾2.8) (⫾1.2) (⫾4.5) (⫾2.5) (⫾5.6) (⫾11.5) (⫾8.4) (⫾8.6) (⫾8.4) (⫾7.6) (⫾3.3) (⫾3.1) (⫾4.9)
10 min
16.7
12.0
14.7
11.7
14.3
(⫾SD) (⫾1.7) (⫾1.7) (⫾2.1) (⫾1.0) (⫾4.8)
15 min
9.4
6.3
7.9
6.7
6.8
(⫾SD) (⫾2.2) (⫾0.9) (⫾1.1) (⫾0.2) (⫾3.1)
11.6
(⫾5.8)
4.2
(⫾4.3)
10.6
14.2
12.3
6.3
4.9
4.5
14.3
(⫾9.3) (⫾6.8) (⫾6.3) (⫾6.9) (⫾3.2) (⫾2.6) (⫾6.9)
5.3
6.5
5.4
2.1
3.1
3.6
6.7
(⫾7.2) (⫾3.3) (⫾5.4) (⫾6.3) (⫾1.2) (⫾2.5) (⫾5.1)
(⫾9.7)
(⫾6.5)
(⫾8.2)
19.3
18.5
23.6
21.6
11.6
(⫾7.8)
(⫾8.5)
(⫾5.8)
13.2
12.9
(⫾6.9)
(⫾6.9)
15.2
(⫾9.4) (⫾2.1)
8.6
8.0
(⫾4.9) (⫾10.3) (⫾6.8) (⫾4.9)
14.3
(⫾11.3) (⫾6.9)
5.3
(⫾4.6)
6.3
6.0
7.3
6.7
3.8
(⫾4.2)
(⫾4.6)
(⫾4.8)
(⫾5.1)
(⫾5.9)
4.3
5.5
(⫾5.8) (⫾5.8)
3.6
3.2
(⫾3.3) (⫾2.6)
ATP content release from freshly isolated human PMNs after activation with fMLP(100 nmol/L). The ATP content in the supernatant was quantified at indicated time
points using a luminometric ATP detection assay. PMNs were preincubated over 10 min with dipyridamole (0 –100 ␮mol/L), verapamil (0 –100 ␮mol/L), 18␣ GA
(1–100 ␮mol/L), and the connexin mimetic peptide specific for Cx40 (Cx40 peptide) (SRPTEKNVFIV, 0 –300 ␮mol/L) or Cx43 (Cx43 peptide) (SRPTEKTIFII, 0 –300
␮mol/L). One representative experiment is displayed (n⫽3; ATP measurements in triplicates). *P⬍0.05 by ANOVA.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
release (Table 2). Similarly, verapamil, an inhibitor of several
ABC proteins and the multidrug-resistance gene product, had
no influence on ATP release. As shown in Table 2, no
difference in stimulated ATP release was detectable between
controls and samples treated with 1, 10, or 100 ␮mol/L
verapamil.
Based on previous reports suggesting that connexin
hemichannels may serve as ATP release channels in glial
cells15 and the observation that PMNs express surface
connexins,16 we measured ATP release of PMNs in the
presence of the nonspecific gap junction inhibitor 18-␣glycyrrhetinic acid (18␣GA). As shown in Table 2, addition of 18␣GA resulted in a concentration-dependent
inhibition of ATP release from fMLP-activated PMNs
(P⬍0.01 by ANOVA). Additional experiments with the
nonspecific gap junction inhibitor anandamide14 confirmed
the above results, revealing a 4.6⫾0.62-fold decrease in
stimulated ATP release in the presence of 100 ␮mol/L
anandamide (P⬍0.01 by ANOVA; data not shown).
We extended these findings to define specific connexin
contributions to PMN ATP release. For these purposes, we
next used connexin mimetic peptides specifically directed
against Cx40 and Cx43.16 –18 As shown in Table 2, peptides
specific for Cx40 did not significantly influence ATP
liberation from activated PMNs. By contrast, the peptides
which block Cx43 showed a concentration-dependent inhibition of ATP liberation (Table 2; P⬍0.01 by ANOVA),
with a ⬎6-fold reduction of maximal ATP release at 1
minute after fMLP stimulation. These results significantly
implicate Cx43 in activated ATP release from human
PMNs.
Activation-Dependent PMN
Cx43 Dephosphorylation
It is known that hexameric assemblies of connexin 43
molecules (so called connexons) form hemichannels connecting the intracellular to the extracellular space.19 The
conductance and permeability of such Cx43 hemichannels
is regulated by modification of their cytoplasm domain,
with phosphorylation of Ser368 causing a conformational
change resulting in decreased connexon permeability.20
Therefore, we examined the influence of fMLP on Cx43
Ser368 phosphorylation in intact PMNs. As shown in
Figure 4A, Cx43 is prominently phosphorylated in resting
PMNs (Figure 4A, 0 minutes). Within 1 minute following
fMLP activation, phosphorylation of Cx43 precipitously
decreases and slowly recovers over 15 minutes. These
results are consistent with fMLP-dependent dephosphorylation of Cx43 and subsequent conformational opening of
Cx43 hemichannels.
Previous reports have implicated protein phosphatase
2A in Cx43 dephosphorylation.21 Therefore, we performed
the above experiment in the presence of the protein
phosphatase inhibitor okadaic acid (OA) (100 nmol/L). As
show in Figure 4B, fMLP-induced dephosphorylation of
Cx43 was attenuated in the presence of 100 nmol/L OA.
Based on this observation, we assessed ATP release of
PMNs in the presence of OA. As shown in Figure 4C, ATP
of PMNs was decreased 4.1⫾0.3-fold in the presence of
100 nmol/L OA. Taken together, these results reveal
activation-dependent dephosphorylation of Cx43 via protein phosphatase and resultant activation of ATP release in
human PMNs.
Biologically Active Adenosine Liberated via PMN
CD39 and Endothelial CD73
Based on the above observation that PMNs express CD39
but not CD73 on their surface, and that ATP in the
presence of PMNs is rapidly hydrolyzed to AMP, we
hypothesized that an additional cell type is necessary to
contribute CD73-dependent AMP conversion and establish
an adenosine-dependent signaling pathway.5 Because of
the close spatial relationship of PMNs to the endothelium
during transendothelial migration, its pivotal role to orchestrate PMN invasion into the underlying tissues during
inflammatory hypoxia,4 and the fact that CD73 is induced
by hypoxia on the endothelial surface,1 we examined
effects of supernatants from activated PMNs on normoxic
or posthypoxic endothelial cell function as a model for
neutrophil/endothelial crosstalk. To pursue these experiments, we activated PMNs with fMLP and exposed
HMEC-1 cells to different concentrations of the superna-
Eltzschig et al
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Figure 4. Effects of phosphatase A2 inhibitor OA on Cx43 phosphorylation and ATP release during fMLP activation of PMNs. A,
PMNs were freshly isolated from human donors and activated
with fMLP and lysed into reducing running buffer at indicated
time points; proteins separated via SDS-PAGE and probed with
a phospho-specific antibody for Cx43 phosphorylated at its
serine 368. B, After isolation, PMNs were preincubated for 1
hour with OA (100 nmol/L), an inhibitor of phosphatase A2. Following fMLP activation, PMNs were lysed at indicated time
points and assessed for Cx43 phosphorylation by Western blot.
The same blots was probed for total Cx43 expression as a control for protein loading. C, To assess the influence of OA on
fMLP-induced ATP release from PMNs, freshly isolated PMNs
were preincubated with OA or with vehicle. fMLP-induced ATP
release was measured with a highly sensitive luminometric ATP
detection assay. OA treatment (100 nmol/L) was associated with
a dramatic inhibition of ATP release (P⬍0.01 by ANOVA compared with vehicle treatment; n⫽3).
tant and measured paracellular barrier function, using a
previously described in vitro model.1 Consistent with
previous findings, endothelial exposure to the supernatant
of PMNs resulted in a concentration-dependent decrease in
paracellular permeability (P⬍0.01 by ANOVA; Figure
5A).1 Such changes in paracellular permeability were
inhibited by the nonspecific adenosine receptor antagonist
8-phenyl-theophylline (10 ␮mol/L), thereby significantly
implicating adenosine in this response. The increased
barrier responses of posthypoxic endothelia are most likely
attributable to hypoxia induction of CD39, CD73, and the
adenosine A2B receptor, resulting in increased adenosine
generation and signaling in posthypoxic endothelia.1
Role of Cx43-Dependent ATP Release by PMNs in
Modulating Endothelial Cell Function
To investigate the role of Cx43-dependent ATP release, we
next generated supernatants from fMLP-activated PMNs that
Cx43-Dependent Neutrophil ATP Release
1105
were preincubated (10 minutes) and activated in the presence
of 18␣GA (100 ␮mol/L) and connexin mimetic peptides
(100 ␮mol/L). Although 18␣GA or connexin mimetic peptides alone did not result in a change of endothelial flux rates
(data not shown), the barrier protective effects of the supernatant was absent if PMNs were activated in the presence of
18␣GA (10 ␮mol/L) or the connexin mimetic peptide specific for Cx43 (100 ␮mol/L). This suggests that connexindependent ATP release is required for the observed barrier
effects of the supernatant (Figure 5B). Taken together, these
results suggest that the known barrier protective effects of
supernatants from activated PMNs require Cx43-dependent
ATP liberation from PMNs. Moreover, these experiments
also highlight the role of PMNs and endothelia as crosstalk
partners in an adenosine dependent signaling pathway, with
PMNs liberating ATP and CD39-dependent phosphohydrolysis to AMP, whereas endothelial CD73 activity results in the
generation of adenosine and activation of endothelial adenosine receptors.
As a second model of crosstalk between PMNs and
endothelia, we investigated the role of ATP release from
PMNs for neutrophil adhesion to normoxic or posthypoxic
endothelia. Consistent with previous studies, adhesion of
fMLP-activated PMNs was increased in posthypoxic endothelia and the addition of the nonspecific adenosine
receptor antagonist 8-phenyltheopylline (8-PT) (10 ␮mol/L)
further increased PMN adhesion, suggesting that such increases in PMNs to endothelia are dependent on adenosine
signaling (Figure 5C).5 As a next step, we measured PMN
adhesion to normo- or posthypoxic endothelia in the presence
of the connexin mimetic peptide specific for Cx40 (Figure
5C) and for Cx43 (Figure 5D). Similar to using different
concentrations of the peptides alone (data not shown), the
addition of the Cx40-specific peptide did not alter PMN
adhesion to a measurable degree. In contrast, inhibition of
ATP release from fMLP-activated PMNs with the Cx43
specific connexin mimetic peptide resulted in a
concentration-dependent increase in neutrophil/endothelial
adhesion.
Activated PMNs From Mice With Induced
Deletion of Cx43 Show Decreased ATP Release
As proof of principle for biologically relevant PMN Cx43
activity, we isolated PMNs from age- and sex-matched
mice with induced deletion of Cx43 (Cx43Cre-ER(T)/fl⫹4OHT, further referred to as Cx43⫺/⫺) and the corresponding
floxed control animals (Cx43fl/fl), as well as heterozygote
Cx43-null mice (Cx43⫹/⫺). As depicted from Western blot
analysis from cardiac tissue in Figure 6A and 6B, administration of tamoxifen resulted in nearly complete deletion
of Cx43 in the Cx43⫺/⫺ mice and a corresponding 50%
decrease in Cx43⫹/⫺ mice. Floxed control animals (Cx43fl/fl)
had similar cardiac Cx43 expression to that of wild-type
animals.
Consistent with our results from pharmacological inhibition of Cx43, isolated PMN ATP release on activation
was almost completely abolished in Cx43⫺/⫺ mice
(P⬍0.001 by ANOVA compared with wild-type mice and
compared with floxed controls, Figure 6C). By contrast,
1106
Circulation Research
November 10, 2006
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Figure 5. Role of PMN-dependent ATP release in
modulating endothelial cell functions. A, Supernatants from activated PMNs decrease endothelial
paracellular permeability. Confluent HMEC-1 cells
were cultured on permeable supports under normoxic (black bars) or hypoxic conditions (gray bars)
(2% oxygen, 48 hours) and exposed (apical surface
only) to cell-free supernatants from fMLP-activated
PMNs. Supernatants from activated PMNs were
added to monolayers. Supernatants, undiluted or
diluted (as indicated), decreased transendothelial
flux of 70 kDa fluorescein isothiocyanate– dextran
(*P⬍0.05 vs control [HBSS], # P⬍0.05 vs control
and normoxia; ANOVA). Data are from 6 monolayers
in each condition. Results are expressed as mean
reduction in permeability⫾SD. In additional control
experiments, the nonspecific adenosine receptor
antagonist 8-PT (10 ␮mol/L) was used. B, Inhibition
of Cx43 abolishes barrier effects of supernatants.
Freshly isolated PMNs were preincubated (10 minutes) and activated in the presence of 18␣GA
(100 ␮mol/L) (SN⫹18␣GA) or connexin mimetic peptides (for connexin 43, SRPTEKTIFII [SN⫹Cx43]; for
connexin 40, SRPTEKNVFIV [SN⫹Cx40]) and tested
for endothelial barrier effects. Results are expressed
as fold change in transendothelial flux rates
(100 ␮mol/L adenosine *P⬍0.01 vs control [HBSS],
#P⬍0.05 vs control and normoxia; ANOVA) (A). C
and D, Change in PMN adhesion to endothelia with
connexin mimetic peptide for Cx40 or Cx43.
HMEC-1 cells were subjected to normoxia or hypoxia (2% O2 for 48 hours) followed by determination of
fMLP-stimulated PMN adhesion in the presence or
absence of indicated concentrations of connexin
mimetic peptides. PMN adhesion was determined
by assessment of BCECF-labeled PMNs binding to
normoxic or posthypoxic HMEC-1 cells. Results are
presented as the fold change (mean⫾SD) in BCECF
fluorescence in the presence of 1 to 1000 ␮mol/L
concentrations (C) of the connexin mimetic peptide
specific for Cx40 or Cx43 (D). In additional control
experiments, the nonspecific adenosine receptor
antagonist 8-PT (10 ␮mol/L) was used (micromolar
concentrations; *P⬍0.05 compared with normoxia,
#P⬍0.01 compared with normoxia and untreated
control).
Cx43⫹/⫺ mice had higher ATP levels than Cx43⫺/⫺ knockout mice but lower than wild-type animals or floxed
controls (P⬍0.05 compared with wild-type, floxed controls, or Cx43⫺/⫺ by ANOVA). The floxed control mice
had similar ATP levels than wild-type controls. As shown
in Figure 6D, the total amount of PMN ATP release was
closely correlated with the degree of Cx43 expression
(Figure 6B). In addition, measurement of LDH release into
the supernatant from Cx43⫺/⫺ or Cx43fl/fl-control mice was
not different, suggesting that there is no difference in lytic
ATP release (Table 1). These studies provide genetic
evidence that ATP release from activated PMNs occurs in
a Cx43-dependent fashion.
Discussion
Metabolic and transcriptional responses to inflammation
are common denominators of multiple cardiovascular and
pulmonary diseases. In particular, adaptation to “inflammatory hypoxia” has become an area of intense investigation.4 Important in this regard, a consistent finding in
hypoxic tissues is increased extracellular nucleotide lev-
els.1 Here, we identified a novel role for Cx43 in
activation-dependent ATP release from PMNs. Moreover,
ATP is rapidly metabolized to AMP through catalytic
activity involving PMN surface CD39. Confirmatory studies in inducible cx43-deficient mice revealed that Cx43
expression correlated with PMN ATP release. Taken
together, these studies demonstrate nucleotide liberation at
sites of acute inflammation by PMNs and identify Cx43dependent ATP release as a central part of an innate
inflammatory response controlling adenosine-dependent
endothelial function.
We observed a barrier-protective and antiinflammatory
role of ATP released from PMNs resulting from rapid
metabolism to adenosine. In fact, previous studies using
specific adenosine receptor antagonists (eg, MRS 1754)
have revealed a pivotal role of adenosine receptors (particularly A2A and A2B) on PMNs in modulating neutrophil/
endothelial crosstalk pathways during conditions of limited oxygen availability.5 Consistent with our findings,
previous studies indicated that Cx43 phosphorylation can
be modulated by inflammation and hypoxia, resulting in an
Eltzschig et al
Cx43-Dependent Neutrophil ATP Release
1107
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
in cardioprotection by ischemic preconditioning. In fact,
this may point to a clinical role of Cx43-dependent ATP
release in myocardial ischemia. Previous studies have
demonstrated extracellular adenosine generation by the
ecto-5⬘-nucleotidase (CD73) in cardiac ischemic preconditioning.24 However, the source from which extracellular
nucleotide precursor molecules are generated and which
cells contribute to their release remains unknown. Thus,
PMN-dependent ATP release could represent an important
substrate for nucleotidase-dependent extracellular adenosine generation during cardioprotection by ischemic preconditioning. However, pharmacological strategies to
modulate Cx43-dependent ATP release and studies using a
chimeric approach or tissue-specific gene deletion of Cx43
will have to confirm a role of Cx43-dependent ATP release
from PMNs in acute myocardial ischemia.
In summary, our results highlight for the first time a
critical role of Cx43 on the surface of PMNs in releasing
ATP from inflammatory cells during activation. Such ATP
is rapidly hydrolyzed to adenosine via close association
with CD73 expressing cell types. Thus, PMN-dependent
release of ATP may play a critical role in the metabolic
control of innate inflammatory pathways.
Acknowledgments
We acknowledge Stephanie Zug, Marion Faigle, and Edgar Hoffmann for technical assistance and Shelley K. Eltzschig for the
artwork. We acknowledge Dr R. John MacLeod for valuable advice
at the initial stages of this work.
Figure 6. ATP release from murine PMNs in genetic models of
Cx43 expression. A, Cx43 expression in genetically engineered
mice. Expression of Cx43 in induced homozygote knockout of
Cx43 (Cx43⫺/⫺), corresponding floxed control animals (Cx43fl/fl),
heterozygote Cx43 knockout mice (Cx43⫹/⫺), and wild-type controls (Cx43⫹/⫹). Shown is 1 of 4 representative Western blots
from murine cardiac tissue. B, Cx43 expression relative to
GAPDH (n⫽4 to 6 animals per genotype; * P⬍0.001 compared
with Cx43⫺/⫺, #P⬍0.01 compared with all other genotypes). C,
ATP release in neutrophils isolated from Cx43⫺/⫺, Cx43⫹/⫺,
Cx43fl/fl and wild-type mice (Cx43⫹/⫹). Whole murine blood was
obtained via cardiac puncture and PMNs were isolated via
double-density centrifugation, activated, and ATP release was
measured. C, Total amount of ATP release was calculated from
the area under the curve (n⫽4 to 6 animals per genotype;
*P⬍0.001 compared with Cx43⫺/⫺, #P⬍0.01 compared with all
other genotypes).
alteration in cellular functions. For instance, dephosphorylation of Cx43 and uncoupling of myocardial gap junctions occurs during myocardial ischemia. Under such
circumstances, Cx43 may be reversibly dephosphorylated
and rephosphorylated during hypoxia and reoxygenation
dependent on fluctuations in intracellular ATP content.22
Moreover, several studies have implicated a role for Cx43
in cardioprotection by ischemic preconditioning, insomuch
as protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous Cx43-deficient
mice.23 In view of the results from the present study
showing a critical role of Cx43 as a phosphorylationdependent ATP release channel on PMNs in modulating
endothelial adenosine responses, it is tempting to speculate
that the role of Cx43 as ATP channel may also be involved
Sources of Funding
This work was supported by Fortune grant 1416-0-0 and Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Verbundprojekt
grant 1597-0-0 from the University of Tübingen (to H.K.E.);
Deutsche Forschungsgemeinschaft (DFG) grant EL274/2-2 (to
H.K.E.); IZKF Nachwuchsgruppe of the University of Tübingen
(grant 1605-0-0 to T.E.); and NIH grants HL60569 and DK50189 (to
S.P.C.).
Disclosures
None.
References
1. Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K,
Robson SC, Colgan SP. Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of
ectonucleotidases and adenosine A2B receptors. J Exp Med. 2003;198:
783–796.
2. Thompson LF, Eltzschig HK, Ibla JC, Van De Wiele CJ, Resta R,
Morote-Garcia JC, Colgan SP. Crucial role for ecto-5⬘-nucleotidase
(CD73) in vascular leakage during hypoxia. J Exp Med. 2004;200:
1395–1405.
3. Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, Mackman
N, Haase VH, Jaenisch R, Corr M, Nizet V, Firestein GS, Gerber HP,
Ferrara N Johnson RS. HIF-1alpha is essential for myeloid cell-mediated
inflammation. Cell. 2003;112:645– 657.
4. Weissmuller T, Eltzschig HK, Colgan SP. Dynamic purine signaling and
metabolism during neutrophil-endothelial interactions. Purinergic Signal.
2005;1:229 –239.
5. Eltzschig HK, Thompson LF, Karhausen J, Cotta RJ, Ibla JC, Robson SC,
Colgan SP. Endogenous adenosine produced during hypoxia attenuates
neutrophil accumulation: coordination by extracellular nucleotide metabolism. Blood. 2004 2004;104:3986 –3992.
6. Eltzschig HK, Weissmuller T, Mager A, Eckle T. Nucleotide metabolism
and cell-cell interactions. Methods Mol Biol. 2006;341:73– 87.
1108
Circulation Research
November 10, 2006
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
7. Kjeldsen L, Sengelov H, Borregaard N. Subcellular fractionation of
human neutrophils on Percoll density gradients. J Immunol Methods.
1999;232:131–143.
8. Kong T, Eltzschig HK, Karhausen J, Colgan SP, Shelley CS. Leukocyte
adhesion during hypoxia is mediated by HIF-1-dependent induction of
{beta}2 integrin gene expression. Proc Natl Acad Sci U S A. 2004;101:
10440 –10445.
9. Enjyoji K, Sevigny J, Lin Y, Frenette PS, Christie PD, Esch JS 2nd, Imai
M, Edelberg JM, Rayburn H, Lech M, Beeler DL, Csizmadia E, Wagner
DD, Robson SC, Rosenberg RD. Targeted disruption of cd39/ATP
diphosphohydrolase results in disordered hemostasis and thromboregulation. Nat Med.1999;5:1010 –1017.
10. Eckardt D, Theis M, Degen J, Ott T, van Rijen HV, Kirchhoff S, Kim JS,
de Bakker JM, Willecke K. Functional role of connexin43 gap junction
channels in adult mouse heart assessed by inducible gene deletion. J Mol
Cell Cardiol. 2004;36:101–110.
11. Haribabu B, Verghese MW, Steeber DA, Sellars DD, Bock CB, Snyderman R. Targeted disruption of the leukotriene B4 receptor in mice
reveals its role in inflammation and platelet-activating factor-induced
anaphylaxis. J Exp Med. 2000;192:433– 438.
12. Novak I. ATP as a signaling molecule: the exocrine focus. News Physiol
Sci. 2003;18:12–17.
13. Maroto R, Hamill OP. Brefeldin A block of integrin-dependent
mechanosensitive ATP release from Xenopus oocytes reveals a novel
mechanism of mechanotransduction. J Biol Chem. 2001;276:
23867–23872.
14. Wang ECY, Lee JM, Ruiz WG, Balestreire EM, von Bodungen M,
Barrick S, Cockayne DA, Birder LA, Apodaca G. ATP and purinergic
receptor-dependent membrane traffic in bladder umbrella cells. J Clin
Invest. 2005;115:2412–2422.
15. Stout CE, Costantin JL, Naus CCG, Charles AC. Intercellular calcium
signaling in astrocytes via ATP release through connexin hemichannels.
J Biol Chem. 2002;277:10482–10488.
16. Oviedo-Orta E, Evans WH. Gap junctions and connexins: potential contributors to the immunological synapse. J Leukoc Biol. 2002;72:636 – 642.
17. Leybaer L, Braet K, Vandamme W, Cabooter L, Martin PE, Evans WH.
Connexin channels, connexin mimetic peptides and ATP release. Cell
Commun Adhes. 2003;10:251–257.
18. Oviedo-Orta E, Errington RJ, Evans WH. Gap junction intercellular
communication during lymphocyte transendothelial migration. Cell Biol
Int. 2002;26:253–263.
19. Goodenough DA, Paul DL. Beyond the gap: functions of unpaired
connexon channels. Nat Rev Mol Cell Biol. 2003;4:285–294.
20. Bao X, Reuss L, Altenberg GA. Regulation of purified and reconstituted
connexin 43 hemichannels by protein kinase C-mediated phosphorylation
of serine 368. J Biol Chem. 2004;279:20058 –20066.
21. Ai X, Pogwizd SM. Connexin 43 downregulation and dephosphorylation
in nonischemic heart failure is associated with enhanced colocalized
protein phosphatase type 2A. Circ Res. 2005;96:54 – 63.
22. Turner MS, Haywood GA, Andreka P, You L, Martin PE, Evans WH, Webster
KA, Bishopric NH. Reversible connexin 43 dephosphorylation during hypoxia
and reoxygenation is linked to cellular ATP levels. Circ Res. 2004;95:726–733.
23. Schwanke U, Konietzka I, Duschin A, Li X, Schulz R, Heusch G. No
ischemic preconditioning in heterozygous connexin43-deficient mice.
Am J Physiol Heart Circ Physiol. 2002;283:H1740 –H1742.
24. Kitakaze M, Hori M, Morioka T, Minamino T, Takashima S, Sato H,
Shinozaki Y, Chujo M, Mori H, Inoue M. Infarct size-limiting effect of
ischemic preconditioning is blunted by inhibition of 5⬘-nucleotidase activity
and attenuation of adenosine release. Circulation. 1994;89:1237–1246.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
ATP Release From Activated Neutrophils Occurs via Connexin 43 and Modulates
Adenosine-Dependent Endothelial Cell Function
Holger K. Eltzschig, Tobias Eckle, Alice Mager, Natalie Küper, Christian Karcher, Thomas
Weissmüller, Kerstin Boengler, Rainer Schulz, Simon C. Robson and Sean P. Colgan
Circ Res. 2006;99:1100-1108; originally published online October 12, 2006;
doi: 10.1161/01.RES.0000250174.31269.70
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2006 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/99/10/1100
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2006/10/12/01.RES.0000250174.31269.70.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
Methods
Isolation of human PMN. After approval by the Institutional Review Board and obtaining
written informed consent from each individual, PMN were freshly isolated from whole blood
obtained by venipuncture from healthy, volunteer donors. The blood was anticoagulated with
acid citrate (10 ml monovets with Na-citrate, Sarstedt, Nümbrecht, Germany) and the
platelets, plasma, mononuclear cells and erythrocytes were removed by double-density
centrifugation using the Percoll system. In short, a double density gradient with 4 ml Percoll
density 73% at the bottom and 4 ml Percoll density 63% on top was prepared. Then, 4 ml
anticoagulated whole blood was layered carefully on top. The tubes were centrifuged at 500g
for 30 min at room temperature, no break and the band with PMN was carefully separated.
From then on, the PMN were maintained at 4°C until activation. Residual erythrocytes were
removed by lysis in cold NH4Cl buffer, followed by two careful washes in HANKs minus
(400g, 10 min, 4°C, break on). Remaining cells were greater than 99% PMN as demonstrated
by microscopic evaluation. PMN were studied within 2h following isolation. In general, this
technique yielded about 0.5 - 1 x 108 PMN from 50 ml of fresh blood.
Preparation of activated PMN supernatants and measurement of ATP or
myeloperoxidase (MPO) content. To measure nucleotide release from activated PMN,
freshly isolated human neutrophils were activated with fMLP, and samples from the
supernatant were taken at different time points after activation and analyzed by HPLC. In
short, freshly isolated human PMN (107 cells/ml) were transferred from cold (4°C) calcium
free buffer (HANKS balanced salt solution without calcium, HANKS minus) into HANKs
plus with 100 nM fMLP, and rotated end-over-end at 37o C. At 1, 5, 10 and 15 min, 200 µl
samples were transferred into an ice-cold Eppendorf cup and immediately pelleted (500g for
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
30s, 4°C). The resultant cell-free supernatants were assessed by HPLC (model 1050; HewlettPackard, Palo Alto, California, USA) with an HP 1100 diode array detector by reverse-phase
on an HPLC column (Luna 5-µm C18, 150 x 4.60 mm; Phenomenex, Torrance, California,
USA) with 100% H20 mobile phase. ATP was thus identified by its chromatographic
behaviour (retention time, UV absorption spectra, and coelution with standards). To exactly
quantify the ATP content within the supernatant, a highly sensitive luciferase based technique
was used (CHRONO-LUME reagent, Chrono-log Corp, Haverton, PA). Luciferase activity
was assessed on a luminometer (Turner Designs Inc., Sunnyvale, California, USA) and
compared with internal ATP standards. In subsets of experiments, the granular marker MPO
was assessed. In short, MPO was quantified from the cell-free supernatant after adjustment of
the pH to 4.2 with 1.0 M citrate buffer (pH 4.2) and color development was assayed at 405 nm
on a microtiter plate reader after mixing equal parts of the supernatant with a solution
containing 1 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS, Sigma-Aldrich)
and 10 mM hydrogen peroxide (H2O2) in 100 mM citrate buffer (pH 4.2). After appropriate
color development the reaction was terminated by the addition of SDS to a final concentration
of 0.5% 1, 2. In subsets of experiments, PMN were preincubated and fMLP stimulated in the
presence of brefeldine A, verapamil, dipyridamole, 18-alpha-glycyrrhetinic acid (18αGA),
and anandamide (Sigma Aldrich). In addition, the effects of connexin-mimetic peptides were
tested (for connexin 43: SRPTEKTIFII; for connexin 40: SRPTEKNVFIV, Biosource,
Solingen, Germany) 3.
Cell Viability Assay. To evaluate lytic cell death of PMN, lactate dehydrogenase (LDH)
activity was measured in the supernatant of activated and non-activated PMN using a
quantitative biochemical assay according to the manufacturer’s instructions (RocheDiagnostics). LDH release is expressed in relation to maximal release after PMN lysis
induced by 1% Triton X-100.
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
PMN granule isolation. The granule fraction from PMN was purified from resting
neutrophils as previously described 4. Briefly, neutrophils were subjected to nitrogen
cavitation followed by centrifugation to remove nuclei and non-disrupted cells. The resulting
postnuclear supernatant was applied to the top of a discontinuous, 3-layer Percoll gradient
(1.050/1.09/1.12 g/mL) and centrifuged at 37, 000g for 30 minutes at 4°C. Gradients were
aspirated from the bottom through a peristaltic pump attached to a fraction collector set to
deliver 1 mL in each fraction. The granular fraction was pooled and Percoll was removed by
centrifugation, and the biologic material was resuspended in 1mL HANKs minus. Aliquots
were assayed for the presence of the marker protein MPO. ATP content of the granular
fraction was quantified as above and compared with the ATP content of the cytosolic fraction.
Immunoblotting experiments. PMN were freshly isolated from human donors and lysed for
10 min in lysis buffer (107PMN/500 µl; 150 mM NaCl, 25 mM Tris, pH 8.0, 5 mM EDTA,
2% Triton X-100, and 10% mammalian tissue protease inhibitor cocktail; Sigma-Aldrich),
and collected into microfuge tubes. After spinning at 14,000 g to remove cell debris, the pellet
was discarded. Proteins were solublized in reducing Laemmli sample buffer and heated to
90°C for 5 min. Samples were resolved on a 12% polyacrylamide gel and transferred to
nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in PBS
supplemented with 0.2% Tween 20 (PBS-T) and 4% BSA. The membranes were incubated in
10 µg/ml polyclonal rabbit phospho-connexin 43 (ser368) antibody (Cell Signaling
Technology, Danvers, MA, USA) for 1h at room temperature, followed by 10 min washes in
PBS-T. The membranes were incubated in 1:3,000 goat anti–rabbit IgG (ICN
Biomedicals/Cappel), and conjugated to horseradish peroxidase for 1 h at room temperature.
The wash was repeated and proteins were detected by enhanced chemiluminescence. In
subsets of experiments, PMN were activated in the presence of the protein phosphatase
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
okadaic acid (OA, Sigma). To control for protein loading, blots were stripped and reprobed
for total Cx43 (Cell Signaling Technology, Danvers, MA, USA).
Measurement of CD39 and CD73 activity on PMN. We assessed surface enzyme activity
as described previously 5, 6 by quantifying the conversion of etheno-ATP (E-ATP, Molecular
Probes Inc.) to etheno-AMP (E-AMP, Sigma Adrich; CD39 activity) or e-AMP to ethenoadenosine (CD73 activity). Briefly, HBSS with or without αβ-methylene ADP (APCP) was
added to freshly isolated PMN (107/ml). After 10 minutes, E-ATP/ E-AMP (final
concentration 1µM) was added and samples were taken at indicated time points, removed,
acidified to pH 3.5 with HCl, spun (10,000 g for 20 seconds, 4°C), and frozen (–80°C) until
analysis via HPLC. This was done with an HPLC pump P680 and a Hitachi Fluorescence
Detector L-7480 on a reverse-phase column (Grom-Sil 120-ODS-ST-5µ; 150 x 3 mm Grom)
using a mobile phase gradient from 0 to 33% acetonitrile/0.3 mM KH2PO4 (pH 5) in 10 min.
CD39/CD73 activity was expressed as percent E-ATP/E-AMP conversion in this time frame.
Flowcytometric analysis of PMN surface expression of CD39 and CD73. 100 µl of whole
blood were stained with fluorescin labelled monoclonal antibodies against CD73 (Serotec)
and PE-labelled anti CD39 (Becton Dickinson) and their IgG subclass specific isotypes
respectively according to the instructions of the manufacturer. After 10 minutes of incubation
at room temperature, erythrocytes were lysed using FACS lysing solution (Becton Dickinson).
Cells were washed in HANKS minus, fixed (CellFix, Becton Dickinson) and analysed within
less than half an hour in a Beckton Dickinson FACSort equipped with CellQuest software.
Forward and right-angle light scatter were used for gating granulocytes, monocytes and
lymphocytes. Isotypes were set within the first decade of the 4-decade log scale.
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
Endothelial Cell Culture. Human microvascular endothelial cells (HMEC-1) were a kind
gift of Francisco Candal, Centers for Disease Control, Atlanta, GA7 and were cultured as
described previously 6. For preparation of experimental HMEC-1 monolayers, confluent
endothelial cells were seeded at ~1x105 cells/ cm2 onto either permeable polycarbonate inserts
or on 12-well plates. Endothelial cell purity was assessed by phase microscopic "cobblestone"
appearance and uptake of fluorescent acetylated low-density lipoprotein.
Macromolecule paracellular permeability assay. Using a modification of methods
previously described 8, HMEC-1 were grown on polycarbonate permeable supports (0.4-µm
pore, 6.5-mm diam; Costar Corp.) and studied 7–10 d after seeding (2–5 d after confluency).
Inserts were placed in HBSS-containing wells (0.9 ml), and HBSS (alone or at indicated
concentrations of the supernatant from activated PMN) was added to inserts (100 µl). At the
start of the assay (t = 0), FITC-labeled dextran 70 kD (concentration 3.5 µM) was added to
fluid within the insert. Fluid from opposing well (reservoir) was sampled (50 µl) over 60 min
(t = 20, 40, and 60 min). Fluorescence intensity of each sample was measured (excitation, 485
nm; emission, 530 nm; Cytofluor 2300; Millipore Corp., Waters Chromatography) and FITCdextran concentrations were determined from standard curves generated by serial dilution of
FITC-dextran. Paracellular flux was calculated by linear regression of sample fluorescence. In
subsets of experiments, activation of the PMN was performed after 10 min pre-incubation and
in the presence of 18αGA (10µM), or connexin mimetic peptides (for connexin 43:
SRPTEKTIFII; for connexin 40: SRPTEKNVFIV, both 100 µM). As controls, the nonspecific adenosine receptor antagonist 8-phenyl-theophyline (8-PT; Sigma Chemical, St
Louis, MO, 10µM) and adenosine was used (Sigma Aldrich, 100 µM) 6.
PMN adhesion assay. Freshly isolated PMNs were labeled for 30 minutes at 37°C with 5 µM
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
BCECF-AM (2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein-acetoxymethyl ester; 5 µM final
concentration; Calbiochem, San Diego, CA) and washed three times in calcium free HBSS.
Labeled PMN (1 × 105/monolayer) were activated with 100 nM fMLP and added to washed
normoxic or hypoxic monolayers of confluent HMEC-1. Plates were centrifuged at 150 g for
2 min to uniformly settle PMN, and adhesion was allowed for 10 min at 37°C. Monolayers
were gently washed three times with HBSS, and fluorescence intensity (485-nm excitation,
530-nm emission) was measured on a fluorescent plate reader (Cytofluor 2300, Millipore,
Bedford, MA). Adherent cell numbers were determined from standard curves generated by
serial dilution of known PMN numbers in HBSS. All data were normalized for background
fluorescence by subtraction of fluorescence intensity of samples collected from monolayers
incubated in buffer only, without addition of PMN 2. To test the influence of connexin
mimetic peptides, fMLP activation of PMN was performed after 10 min of pre-incubation and
in the presence of indicated concentrations of connexin mimetic peptides (for connexin 43:
SRPTEKTIFII; for connexin 40: SRPTEKNVFIV, 0 - 1000 µM). As a control , the nonspecific adenosine receptor antagonists 8PT was used (both PMN and HMEC-1 monolayers
were [pre-] incubated with 10 µM 8-PT).
Isolation and activation of murine PMN. Isolation and activation of murine PMN. In
subsets of experiments, PMN were isolated from previously described mice with targeted
gene deltion of cd399 or induced ablation of Cx43.10 In the later mice, the 4-OHT-inducible
Cre-recombinase is targeted to the endogenous Cx43 locus, thereby replacing one Cx43 allele
(Cx43Cre-ER(T)/+ mice). These mice were mated with Cx43fl/fl mice, in which the Cx43 coding
region is flanked by loxP sites, resulting in Cx43Cre-ER(T)/fl mice. Thus, Cx43 can be ablated by
injection of 4-OHT in all cells expressing Cx43. Successful deletion of the Cx43 at 13 days
after the first 4-OHT treatment has been demonstrated in heart, lung, brain and tail. 10In
subsets of experiments, PMN were isolated from mice with induced ablation of Cx43. For this
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
purpose, adult Cx43Cre-ER(T)/fl mice that received intraperitoneal injections of 3 mg 4hydroxytamoxifen (4-OHT) once per day on five consecutive days as previously described
were used 10. The animals were sacrificed at day 12 after the first injection. For control,
Cx43fl/fl mice were used. In other experiments, PMN isolated from heterozygote Cx43+/- 11
and cd39-/- mice were used 9. In short, age and gender matched knockout mice and littermate
controls received intraperitoneal heparin (300 i. E./kg) and pentobarbital (100 mg/kg). After
induction of anesthesia, whole blood was obtained by cardiac puncture (500 – 800 µl per
animal) and the animals were sacrificed. PMN were isolated with a double density gradient
using 4 ml Percoll 73% at the bottom and 4 ml Percoll 63% above, with the heperanized
blood of one animal carefully layered on top. The tubes were centrifuged at 500g for 30 min
at room temperature, no break and the band with PMN was carefully separated. PMN were
studied within 2h of their isolation. In general, this technique yielded about 1 - 5 x 106 PMN
per animal. Due to low expression rates of fMLP receptors on murine PMN, activation was
performed with leukotriene B4 (LTB4 100 nM, Calbiochem) 12. In short, freshly isolated PMN
were transferred from cold (4°C) calcium free buffer (HANKS minus) into HANKs plus with
100 nM LTB4 at a concentration of 106 cells/ml, and rotated end-over-end at 37o C. At 1, 5,
10 and 15 min, 200 µl samples were transferred into an ice-cold Eppendorf cup and
immediately pelleted (500g for 30s, 4°C). The resultant cell-free supernatants were assessed
for ATP content with a standard luciferase based technique (CHRONO-LUME reagent,
Chrono-log Corp, Haverton, PA) as above (n = 4-6 animals per condition). Cx43 expression
was assessed by Western blot analysis from cardiac tissue 13. In short, mouse myocardial
extracts were snap frozen, homogenized with a mortar in liquid nitrogen and transferred to 1×
Cell Lysis buffer (Cell Signaling, Beverly, MA, containing in mM: Tris pH 7.5 20, NaCl 150,
EDTA 1, EGTA 1, sodium pyrophosphate 2.5, β-glycerolphosphate 1, Na3VO4 1, Triton X100 1%, Leupeptin 1µg/ml, Complete Protease Inhibitor Cocktail 1× (Roche, Basel,
Switzerland)). Subsequently, the samples were sonicated for 20s and centrifugated at 14, 000g
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
for 10min. The supernatants were collected and the protein concentrations were determined
using Dc protein assay (Biorad, Hercules, CA). 25 µg total proteins were electrophoretically
separated on 10% SDS-PAGE and transferred to nitrocellulose membrane. Cx43 was detected
using a rabbit polyclonal antibody against rat total Cx43 (Zymed, Berlin, Germany, dilution
1:1000) and GAPDH was detected using a monoclonal antibody against rabbit GAPDH
(HyTest, Turku, Finland, dilution 1:2500) Immunoreactive signals were detected by
chemiluminescence (LumiGLO, Cell Signaling) and quantified using Scion Image software.
In subset of experiments, supernatants of PMN from cd39-/- mice were reconstituted with
0.1U of apyrase/experiment (Sigma-Adrich).These protocols were in accordance with
National Institutes of Health guidelines for use of live animals and were approved by the
Institutional Animal Care and Use Committee at Brigham and Women's Hospital and of the
University of Essen, Germany.
Data analysis. Data were compared by two-factor ANOVA, or by Student’s t test where
appropriate. In experiments on the kinetics of ATP-release, one representative experiment of
three is displayed (n=3) with triplicate measurement of the ATP-concentration. Values are
expressed as the mean ± SD from at least three separate experiments.
Online Supplement
Eltzschig et al.
Cx43-dependent neutrophil ATP release
References
1.
Parkos CA, Delp C, Arnaout MA, Madara JL. Neutrophil migration across a cultured
intestinal epithelium: Dependence on a CD11b/CD18 - mediated event and enhanced
efficiency in the physiologic direction. J Clin Invest. 1991;88:1605-1612.
2.
Eltzschig HK, Thompson LF, Karhausen J, Cotta RJ, Ibla JC, Robson SC, Colgan SP.
Endogenous adenosine produced during hypoxia attenuates neutrophil accumulation:
coordination by extracellular nucleotide metabolism. Blood. 2004;104:3986-3992.
3.
Zahler S, Hoffmann A, Gloe T, Pohl U. Gap-junctional coupling between neutrophils
and endothelial cells: a novel modulator of transendothelial migration. J Leukoc Biol.
2003;73:118-126.
4.
Kjeldsen L, Sengelov H, Borregaard N. Subcellular fractionation of human neutrophils
on Percoll density gradients. J Immunol Methods. 1999;232:131-143.
5.
Synnestvedt K, Furuta GT, Comerford KM, Louis N, Karhausen J, Eltzschig HK,
Hansen KR, Thompson LF, Colgan SP. Ecto-5'-nucleotidase (CD73) regulation by
hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia. J
Clin Invest. 2002;110:993-1002.
6.
Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K, Robson SC,
Colgan SP. Coordinated adenine nucleotide phosphohydrolysis and nucleoside
signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B
receptors. J Exp Med. 2003;198:783-796.
7.
Robinson KA, Candal FJ, Scott NA, Ades EW. Seeding of vascular grafts with an
immortalized human dermal microvascular endothelial cell line. Angiology.
1995;46:107-113.
8.
Lennon, PF, Taylor CT, Stahl GL, Colgan SP. Neutrophil-derived 5'-Adenosine
Monophosphate Promotes Endothelial Barrier Function via CD73-mediated
Conversion to Adenosine and Endothelial A2B Receptor Activation. J Exp Med.
1998;188:1433-1443.
9.
Enjyoji K, Sevigny J, Lin Y, Frenette PS, Christie PD, Esch JS 2nd, Imai M, Edelberg
JM, Rayburn H, Lech M, Beeler DL, Csizmadia E, Wagner DD, Robson SC,
Rosenberg RD. Targeted disruption of cd39/ATP diphosphohydrolase results in
disordered hemostasis and thromboregulation. Nat Med. 1999;5:1010-1017.
10.
Eckardt D, Theis M, Degen J, Ott T, van Rijen HV, Kirchhoff S, Kim JS, de Bakker
JM, Willecke K. Functional role of connexin43 gap junction channels in adult mouse
heart assessed by inducible gene deletion. J Mol Cell Cardiol. 2004;36:101-110.
11.
Reaume AG, de Sousa PA, Kulkarni S, Langille BL, Zhu D, Davies TC, Juneja SC,
Kidder GM, Rossant J. Cardiac malformation in neonatal mice lacking connexin43.
Science. 1995;267:1831-1834.
12.
Haribabu B, Verghese MW, Steeber DA, Sellars DD, Bock CB, Snyderman R.
Targeted Disruption of the Leukotriene B4 Receptor in Mice Reveals Its Role in
Inflammation and Platelet-activating Factor-induced Anaphylaxis. J Exp Med.
2000;192:433-438.
13.
Boengler K, Schulz R, Heusch G. Connexin 43 signaling and cardioprotection. Heart.
2005. doi:10.1136/hrt.2005.066878.