By
From the * Centre de Recherche en Rhumatologie et Immunologie,
Centre de recherche du CHUL et Université Laval, Québec, Canada; and Merck Frosst Center for Therapeutic Research, Pointe-Claire, Québec, Canada
Adenosine (Ado) has been shown to suppress several functional responses of human polymorphonuclear leukocytes (PMNs). The current study investigated whether endogenous Ado regulates the biosynthesis of leukotriene (LT)B4 in ligand-stimulated PMNs. Measurements of Ado in PMN resuspended in Hanks' buffered salt solution (HBSS) or plasma showed a cell concentration- and time-dependent accumulation of the nucleoside. The removal of endogenous Ado with either Ado deaminase or the blockade of its action by the Ado A2a receptor antagonist, 8-(3-chlorostyryl) caffeine, markedly increased LTB4 biosynthesis upon ligand stimulation in HBSS. Similarly, LTB4 synthesis by ligand-stimulated PMNs in plasma (containing recombinant LTA4 hydrolase to allow the conversion of protein-bound LTA4) was strongly enhanced by addition of Ado deaminase. Addition of red blood cells to suspensions of PMNs in plasma mimicked the effect of adding Ado deaminase and LTA4 hydrolase in enhancing LTB4 biosynthesis upon ligand stimulation. This effect of red blood cells on LTB4 biosynthesis was blocked by dipyridamole, an inhibitor of Ado transport, or captopril, an inhibitor of LTA4 hydrolase. These results demonstrate that endogenous Ado efficiently downregulates ligand-stimulated LTB4 biosynthesis in PMN suspensions, pointing out a potentially important regulatory function of Ado in inflammatory exudates. These results also unveil a dual role for red blood cells in upregulating LTB4 biosynthesis, namely, the removal of endogenous Ado and the conversion of LTA4 released by activated PMNs.
Leukotriene (LT)1 B4 is a biologically active mediator of
inflammatory processes; indeed, LTB4 is a potent activator of leukocyte functions, and in particular, is a potent
chemokinetic and chemotactic agent for neutrophils,
monocytes, and macrophages. PMNs, mast cells, monocytes, macrophages, and B lymphocytes are the main cell
types possessing the 5-lipoxygenase (5-LO) and LTA4-converting enzymes and can thus directly produce LTB4 from
endogenous arachidonic acid (1, 2). In blood, PMNs have
been found to be the predominant cell producing LTB4 (3-
5). Interestingly, it was recently demonstrated that activated
PMNs release directly in the extracellular milieu a large
proportion of the LTA4 generated (6); such release of LTA4
by PMNs points to the potential importance of transcellular metabolism in the biosynthesis of the biologically active
metabolites of LTA4, LTB4, and LTC4. Thus, many cell
types present in the neutrophil environment that do not
express the 5-LO, such as endothelial cells, RBCs, T cells,
and platelets, likely constitute important factors in the dynamics of LT synthesis in vivo (3, 7).
Adenosine (Ado) is a ubiquitous autacoid with a large
spectrum of biological activities, including the modulation
of leukocyte functions. Indeed, numerous studies have reported that acting via Ado A2 receptors, Ado suppressed
PMN functions such as superoxide anion synthesis, adhesion, and phagocytosis, as well as the synthesis of inflammatory cytokines in monocytes. In addition, numerous studies
have also demonstrated that endogenous Ado, as well as exogenous Ado or Ado analogues, exert antiinflammatory
effects in vivo in animal models (for a review see reference
10). For these reasons, Ado has recently been proposed to
act as an endogenous antiinflammatory agent (10).
In agreement with this concept, we demonstrated in a
previous study that Ado and Ado analogues are very potent
inhibitors of the biosynthesis of LTB4 both in whole blood
and isolated PMNs stimulated with physiological agents
(11). Because it is well established that Ado accumulates in
leukocyte suspensions (as a consequence of the extracellular
breakdown of ATP; reference 12) where its accumulation
reaches a concentration that exerts suppressive effects on
PMN functions, we sought to determine the putative role
of endogenous Ado in regulating LTB4 biosynthesis by
ligand-activated PMNs in various environments.
Materials.
Ado deaminase (EC 3.5.4.4., calf intestinal type
VIII), captopril, dipyridamole, N-formyl-Met-Leu-Phe (fMLP),
and LPS (Escherichia coli 01110B4) were from Sigma Chemical Co.
(St. Louis, MO). Ado deaminase was dialyzed against NaCl 0.9%
before use. 2-p-(carboxyethyl)phenetylamino-5 Cells.
Human PMNs were isolated as previously described
(4). In brief, human venous peripheral blood was collected into
heparinized tubes. RBCs were allowed to sediment at 1 g after
mixing 4 volumes of blood and 1 volume of dextran 2% in
HBSS. Mononuclear cells were removed by centrifugation on Ficoll-Paque cushions. Contaminating RBCs in the PMN pellet
were eliminated by a 20-s hypotonic lysis in water. PMNs were
resuspended in 10 mM Hepes-buffered HBSS (pH 7.4) containing 1.6 mM CaCl2. RBCs obtained from dextran sedimentation were freed of contaminating leukocytes by repeated removal of the buffy coat after four successive centrifugations (200 g, 15 min
at 20°C) and resuspensions in four volumes of 10 mM Hepes-buffered HBSS (pH 7.4) containing 1.6 mM CaCl2. In some experiments, RBCs were treated with 1 mM captopril or its diluent
(NaCl 0.9%) for 30 min at room temperature before the fourth
washing.
5-LO Product Analysis.
Incubations were stopped by adding
cold (0°C) methanol/acetonitrile (50/50; vol/vol) containing
12.5 ng each of 19-OH prostaglandin B2 and prostaglandin B2 as
internal standards to aliquots of cell suspensions and stored at
Analysis of Ado.
PMN incubations (1 ml) were stopped by
adding 100 µl of 22% TCA. NECA was added (10 ng/sample) as
an internal standard and the denatured cell suspensions were
placed at Ado Concentrations in PMN Suspensions.
Isolated PMNs in
suspension in salt buffers have been reported to release Ado
at levels that influence their functions (12). We thus performed measurements of endogenous Ado concentrations in PMN suspensions under conditions used for the assessment of leukotriene biosynthesis. After the centrifugation
and resuspension of PMNs in fresh buffers, aliquots were
removed for up to 60 min and the concentration of Ado
was measured. PMNs resuspended in HBSS or autologous
plasma released Ado in a time- and cell concentration- dependent manner (Fig. 1). Cell-depleted plasma was found
to contain 30 ± 3 nM (mean ± SE, n = 6) of Ado. The
addition of 0.1 U Ado deaminase after the incubation of
2.0 × 107 PMN/ml for 15 min in HBSS reduced the concentration of Ado within seconds to <4 nM, and remained
below this level for up to 30 min. Stimulation of PMNs
with 0.6 µM platelet-activating factor (PAF) did not have
any effect on the levels of endogenous Ado (not shown).
LTB4 Biosynthesis in HBSS.
We first examined the effect
of endogenous Ado present in PMN suspensions in HBSS
on the synthesis of LTB4. Endogenous Ado was neutralized
using two different approaches; the addition of a selective
A2a receptor antagonist, CSC (16; Fig. 2 A), or Ado deaminase (Fig. 2 B) to the incubation media. The pretreatment of TNF-
LTB4 Biosynthesis in Plasma.
We next examined whether
endogenous Ado also suppressed LTB4 biosynthesis by
PMNs resuspended in plasma. Stimulation of TNF-
We recently reported that Ado and analogues (particularly
A2a agonists) are potent inhibitors of LTB4 biosynthesis in
whole blood, as well as in isolated PMNs and monocytes
(11). It is also recognized that Ado accumulates in PMN
suspensions (12). These observations led us to hypothesize
that endogenous Ado present in PMNs suspensions might
exert a suppressive effect on the biosynthesis of LTB4.
Measurements of Ado concentrations in PMN incubation media (HBSS and plasma) clearly indicated that after 15-30
min of incubation in all experimental conditions tested,
Ado reaches concentrations (25-400 nM; Fig. 1) likely to
severely impact LTB4 synthesis, given the IC50 of 80 and 60 nM measured previously for the inhibition of LTB4 synthesis in blood (11) and HBSS (our unpublished data), respectively. Accordingly, we found that the removal of endogenous Ado using Ado deaminase or the blockade of its effect
with the A2a receptor antagonist CSC, strikingly increased
the biosynthesis of LTB4 in response to ligand stimulation. For PMNs activated in the presence of plasma, however,
the addition of Ado deaminase to the incubation media was
not efficient in increasing the biosynthesis of LTB4. On the
basis of the recent report that activated PMNs release LTA4
in the extracellular milieu (6), we postulated that LTB4
biosynthesis by PMN suspensions in plasma was inhibited
by the trapping of its precursor LTA4 by the plasma proteins, which prevented the uptake and further metabolism
of LTA4 by the PMN LTA4 hydrolase. Indeed, it has been previously shown that LTA4 binds to serum albumin,
which results in a dramatic increase of its half-life (20). Accordingly, the addition of rLTA4 hydrolase and Ado deaminase, but not of either enzyme alone, resulted in a significant increase of LTB4 biosynthesis by PMNs in plasma.
Our results therefore demonstrate that LTB4 biosynthesis
by PMNs in plasma is limited by the inhibitory constraint generated by endogenous Ado which prevents the formation of LTA4. The removal of Ado (using Ado deaminase)
is, however, insufficient to allow efficient LTB4 biosynthesis since LTA4 released by PMNs is bound to plasma proteins. The addition of rLTA4 hydrolase in PMN suspensions in plasma drastically accelerates and enhances the
generation of LTB4. The mechanism by which Ado suppresses LT synthesis in PMNs remains to be defined. We
can exclude, however, the idea that the inhibitory effect of
Ado on LT biosynthesis in PMN results from an effect of
the nucleoside on the release or uptake of LTA4. Indeed, in
the experimental system used (Fig. 2), the formation of
LTB4 (and LTB4 metabolites) depends on the reuptake of
extracellular LTA4 which is then converted by the cytosolic
LTA4 hydrolase (6). Therefore, inhibition of initial LTA4
release by the activated PMNs would be expected to facilitate LTB4 formation, whereas inhibition of extracellular
LTA4 uptake would result in the formation of the 6-trans
isomers (nonenzymic hydrolysis products) of LTB4, which
was not observed in our experiments. Another possible site
of action of Ado in the regulation of LTB4 biosynthesis
could be at the level of the conversion of LTB4 to its Another finding of the current study is the involvement
of a dual mechanism in the regulation of the biosynthesis of
LTB4 by RBCs. Indeed, RBCs have previously been
shown to enhance the biosynthesis of LTB4 through the
transcellular metabolism of LTA4 by RBC LTA4 hydrolase
(8). However, the results of our studies with PMN in
plasma suggested that the presence of RBC LTA4 hydrolase cannot fully account for the increased production of LTB4,
and that the reported capacity of RBCs to efficiently take
up Ado (18) may contribute to the ability of RBCs to enhance the synthesis of LTB4 by ligand-stimulated PMNs in
plasma. In fact, both RBC-mediated events proved to be
determinant in the stimulatory effect of RBCs since both
captopril, an inhibitor of LTA4 hydrolase, and dipyridamole, an inhibitor of adenosine transport, efficiently reversed the effect of RBCs on LTB4 synthesis, in full agreement with
the data obtained by the simultaneous addition of LTA4
hydrolase and Ado deaminase to PMNs activated in plasma.
The fact that endogenous Ado exerts a negative regulation of LTB4 biosynthesis by ligand-stimulated PMNs has
led to an underestimation of the potential of this cell type
to respond to stimulation by physiological agonists such as
fMLP and PAF (24) and has also contributed to the generation of controversial data concerning the ability of PMNs
to produce LTs in response to such stimuli. Indeed, it is
likely that differences in experimental conditions used by
different investigators, such as PMN concentration and preincubation temperature and time, directly impact on
Ado concentration in the cell suspensions and therefore, on
cell responsivity to the stimuli. Moreover, while LTB4 synthesis by PMNs stimulated by soluble agonists such as
fMLP and PAF is highly sensitive to inhibition by Ado, the
synthesis of LTB4 by PMNs stimulated by the ionophore
A23187 is much less sensitive to Ado inhibition (11). It
seems important to point out that in an in vivo context, the
inhibition of PMN LTA4 biosynthesis by Ado likely has
consequences not only on LTB4 formation, but also on the
biosynthesis of cysteinyl LTs and lipoxins, since these may
be generated, at least in part, from the transcellular metabolism of PMN-derived LTA4 by endothelial cells (25) and
platelets (26).
In summary, the current study demonstrates the regulatory role of endogenous Ado on ligand-stimulated LT biosynthesis by PMNs and strongly emphasizes that an elevated level of endogenous Ado in physiological settings can
have profound consequences on the ability of PMNs to
produce LTA4, the direct precursor of the lipid mediators
LTB4, LTC4, and lipoxins, which have been shown to
modulate phagocyte functional responses and inflammatory
events. Our observations also support the recently proposed concept that Ado is a natural antiinflammatory agent
(10). Indeed, it has become increasingly apparent that the
antiinflammatory mechanism of methotrexate and sulfasalazine, two potent antiinflammatory drugs, involves an increase of Ado concentration at sites of inflammation (10, 27). Most importantly, these studies showed that leukocyte
accumulation at inflammatory sites was diminished and that
these effects of the drugs could be antagonized by Ado
deaminase or Ado receptor antagonists. In view of the ability of Ado to suppress LTB4 biosynthesis, it is tempting to
speculate that the mechanism by which these antiinflammatory agents act might include the inhibition of LTB4-dependent extravasation of leukocytes. Further studies are
needed to characterize the consequences of increasing Ado
levels on LTB4-mediated inflammatory processes. Finally,
taken together, these recent observations and the previously reported inhibitory effects of Ado on PMNs and
monocyte functions support that A2a receptor agonists or
agents that can regulate Ado biosynthesis, metabolism, or
transport may represent a novel class of potent antiinflammatory agents.
-N-ethyl-carboxamido-adenosine HCl (CGS 21680) and 8-(3-chlorostyryl)
caffeine (CSC) were from Research Biochemicals International
(Natick, MA). 5
(N-ethyl)carboxamidoadenosine (NECA) was
from ICN Biomedicals Canada Ltd. (Mississauga, Canada). rLTA4
hydrolase was obtained from Sf9 cells infected with LTA4 hydrolase using the baculovirus system (13). The 100,000 g supernatant of infected Sf9 cells was used directly as the source of LTA4 hydrolase and contained a specific activity of 60 nmol LTB4/mg of
protein (12 mg of protein/ml). The 100,000 g supernatant of the
wild-type Sf9 cells (uninfected cells) was used as control. Recombinant GM-CSF and TNF-
were provided by the Genetics Institute (Cambridge, MA) and Knoll Pharmaceuticals (Whippany,
NJ), respectively.
20°C until reverse phase HPLC (RP-HPLC) analysis. Denatured
samples were centrifuged at 2,000 g for 10 min, and the supernatants were subjected to RP-HPLC using on-line extraction procedures as previously described for PMN suspensions in HBSS
(14) or samples containing plasma (15). LTB4, 6-trans isomers of
LTB4,
oxidation metabolites of LTB4, and 5(S)-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE) were measured by photometry at 280 and 229 nm, using fixed wavelength
UV detectors. The lower limits of detection were 0.5 ng at 280 nm and 1 ng at 229 nm.
20°C for at least 30 min. The samples were then centrifuged at 2,000 g for 10 min and the supernatants were extracted
on Sep Pak Cartridges (3 cc, C-18 sorbent) as follows. The samples were loaded on the cartridges which were washed with water; Ado was then eluted with 3.5 ml of methanol/water (50:50,
containing 0.1% acetic acid). The eluates were evaporated to dryness using a Speed Vac evaporator. The residues were dissolved in
200 µl of methanol/water (25:75, containing 0.05% acetic acid).
The samples were analyzed by liquid chromatography-mass spectrometry using nebulizer-assisted electrospray ionization in the
positive mode and by monitoring the transitions m/z 309 and m/z
268 (protonated parent ions) to m/z 136 (protonated adenine), corresponding to the loss of the carbohydrate moieties from
NECA and Ado. The samples (1-2 µl) were injected onto a C-18
column (Ultrasphere, 2 × 150 mm, 5-µ particles; Beckman, Fullerton, CA) and eluted at a flow rate of 200 µl/min using methanol/water (40:60, containing 0.1% acetic acid) as the mobile phase.
Ado was quantitated by extrapolating the measured Ado/NECA
ratio on a calibration curve generated from standard solutions
containing 1 ng NECA and 0-4 ng Ado in 5 µl. The limit of detection for Ado was ~50 pg injected (signal to noise ratio
5).
Fig. 1.
Time course of Ado accumulation in PMN suspensions.
Freshly isolated PMNs were resuspended in HBSS (A) or autologous
plasma (B) at concentrations of 5 × 106/ml (squares) or 20 × 106/ml (circles). At various time points, 1-ml aliquots of the cell suspensions were denatured with TCA and the Ado content was measured by liquid chromatography-mass spectrometry. Results shown are the means ± SD of
triplicate incubations from one experiment representative of three. Error
bars are not shown when smaller than symbols.
[View Larger Version of this Image (14K GIF file)]
/GM-CSF-primed neutrophils with increasing
concentrations of either CSC or Ado deaminase before
stimulation with 0.6 µM PAF resulted in a progressive enhancement of 5-LO product biosynthesis, as compared to
cells stimulated in the absence of CSC or Ado deaminase. The stimulatory effect of Ado deaminase was fully reversed
by the addition of 1 µM CGS 21680, a selective A2a receptor agonist (17). Fig. 2 C illustrates typical HPLC profiles
of the 5-LO products generated by PMNs activated with
PAF in the presence or absence of CSC or Ado deaminase.
In the absence of PAF stimulation, the biosynthesis of
LTB4 was not detected.
Fig. 2.
Effects of CSC and Ado deaminase treatment of PMN suspensions on LTB4 biosynthesis. PMN suspensions in HBSS (107/ml) were
preincubated with 700 pM GM-CSF + 1.2 nM TNF- for 30 min, and
then treated with various concentrations of CSC (A) or Ado deaminase
(B) for 5 min and stimulated with 0.6 µM PAF. After 10 min of incubation in the presence (or absence) of PAF, incubations were stopped and
5-LO products were measured by RP-HPLC. In B, CGS 21680 was
added at the concentration of 1 µM. Data are the means (± SD) of triplicate incubations from one experiment representative of three. (C) RP-HPLC profiling of 5-LO products generated by TNF-
/LPS-primed
PMN suspensions stimulated (or not, control) with PAF, with or without
earlier pretreatment with either 0.1 U deaminase Ado or 1 µM CSC.
Conditions were as indicated for A and B; 5(S)-hydroxy-6,8,11,14(E,Z,Z,Z)-
eicosatetraenoic acid (5-HETE) was not detectable in all conditions
tested. Amounts of LTB4 indicated represent the sum of 20-OH LTB4,
20-COOH LTB4, and LTB4. PGB2, prostaglandin B2; ADA, adenosine
deaminase.
[View Larger Versions of these Images (13 + 27K GIF file)]
/ LPS-primed PMNs with fMLP resulted in the biosynthesis
of only minimal amounts of LTB4 (Fig. 3 A). The addition
of Ado deaminase to the similarly primed PMN suspensions before fMLP stimulation failed to further increase the
biosynthesis of LTB4. In contrast, the addition of both Ado
deaminase and rLTA4 hydrolase resulted in a marked enhancement of LTB4 biosynthesis. As observed with PMNs
treated with Ado deaminase alone, the addition of rLTA4
hydrolase alone failed to increase the synthesis of LTB4
upon fMLP stimulation. The effect of adding Ado deaminase and rLTA4 hydrolase to PMN suspensions in plasma
on LTB4 biosynthesis was mimicked by the addition of
RBCs, which are known to uptake extracellular Ado (18)
and contain LTA4 hydrolase activity (7; Fig. 3 B). That the
RBC LTA4 hydrolase was involved in the biosynthesis of
LTB4 was confirmed by a pretreatment of RBCs with captopril, an inhibitor of LTA4 hydrolase (19), which resulted
in a near complete inhibition of the effect of RBCs. Similarly, the involvement of RBCs in lowering extracellular
Ado was demonstrated by adding dipyridamole, an inhibitor of Ado transport (18), to the incubation media 15 min
before fMLP stimulation. Dipyridamole also caused a
marked inhibition of LTB4 biosynthesis; that the inhibitory
effect of dipyridamole was the consequence of an accumulation of Ado in the incubation media was assessed by the
coaddition of Ado deaminase, which completely restored
LTB4 biosynthesis to the level observed with RBCs (data
not shown). The pretreatment of RBCs with captopril and
addition of dipyridamole inhibited the biosynthesis of
LTB4 to the level observed in the absence of RBCs.
Fig. 3.
Effect of exogenous
LTA4 hydrolase, Ado deaminase,
and RBCs on the biosynthesis of
LTB4 by PMNs in plasma. (A)
PMN (5 × 106) in suspension in
autologous plasma (0.5 ml) were
treated with 1.2 nM TNF- and
1 µg/ml LPS for 30 min at
37°C. 10 µl of the preparation of
rLTA4 hydrolase (see Materials
and Methods) and/or 4 U of
Ado deaminase (or its diluent,
NaCl 0.9%) were added (per milliliter of incubation media) 1 and 5 min, respectively, before stimulation with 1 µM fMLP.
After 15 min of stimulation, the
incubations were stopped and
LTB4 production was measured
by RP-HPLC. (B) PMNs (5 × 106) in suspension in autologous
plasma (0.5 ml) were treated with 1.2 nM TNF-
and 1 µg/ml LPS
for 30 min at 37°C. RBCs (0.5 ml of packed cells) treated or not with
captopril (see Materials and Methods) were next added to PMNs in
plasma; the incubation media were further treated or not with 30 µM
dipyridamole for 25 min at 37°C, and then stimulated with 1 µM fMLP
for 15 min. LTB4 biosynthesis was measured by RP-HPLC. Results
shown are the means ± SD of triplicate incubations from one experiment
representative of three. LTA4-H, LTA4-hydrolase; DIPY, dipyridamole;
CAPT-RBC, captopril-treated red blood cells; ADA, Ado deaminase.
[View Larger Version of this Image (15K GIF file)]
oxidation products. However, as seen in Fig. 2 C, endogenous Ado suppresses the formation of both LTB4 and 20-OH LTB4, the sum of LTB4, and its metabolites being
two- to threefold greater in incubations performed in presence of Ado deaminase or CSC. Interestingly, previous studies
have shown that elevated extracellular concentrations of
Ado inhibits external Ca2+ influx in PMNs stimulated by
either PAF or fMLP (21). Since LTB4 biosynthesis is highly
dependent on ligand-stimulated Ca2+ influx (22), it is
therefore conceivable that the inhibitory effects of Ado
might be related to the modulation of arachidonate release
and/or 5-LO activation, both of which are Ca2+ dependent
(22, 23). Studies are in progress to assess this hypothesis.
Address correspondence to Dr. Pierre Borgeat, Centre de Recherche en Rhumatologie et Immunologie, 2705 Blvd. Laurier, Rm T 1-49, Sainte-Foy, Québec G1V 4G2 Canada. Phone: 418-654-2772; FAX: 418-654-2765.
Received for publication 16 June 1997 and in revised form 25 July 1997.
This work was supported by the Arthritis Society of Canada.
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