| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
16 nM) and AA (EC50 6 µM) generated hydrogen peroxide
(H2O2) in a concentration-dependent manner and
at an equivalent maximum rate (5–6 nmol/min/106 cells).
LTB4 stimulated PLA2 over a similar
concentration range that activated the NADPH oxidase, although kinetic
studies revealed that the release of [3H]AA
(t1/2 2 s) preceded
H2O2 generation
(t1/2 > 30 s). Pretreatment of
eosinophils with pertussis toxin abolished the increase in
inositol(1,4,5)trisphosphate mass, [Ca2+]c,
[3H]AA release, and H2O2
generation evoked by LTB4. Qualitatively identical results
were obtained in eosinophils in which phospholipase C (PLC) was
desensitized by 4ß-phorbol 12,13-dibutyrate with the exception that
[3H]AA release was largely unaffected. Additional studies
performed with the protein kinase C inhibitor, Ro 31-8220, and under
conditions in which Ca2+ mobilization was abolished,
provided further evidence that LTB4 released
[3H]AA independently of signal molecules derived from the
hydrolysis of phosphatidylinositol(4,5)bisphosphate by PLC.
Pretreatment of eosinophils with the PLA2 inhibitor,
mepacrine, abolished LTB4-induced [3H]AA
release at a concentration that inhibited H2O2
by only 36%. Collectively, the results of this study indicate that
agonism of LTB4 receptors on guinea pig eosinophils
mobilizes AA by a mechanism that does not involve the activation of
PLC. In addition, although LTB4 effectively stimulated
PLA2, a central role for AA in the activation of the NADPH
oxidase was excluded. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We and others have reported previously that LTB4 promotes a robust, receptor-mediated activation of the NADPH oxidase in guinea pig eosinophils (7, 8). Biochemical and pharmacologic studies designed to elucidate the signaling pathway(s) responsible for this effect suggest that Ca2+ does not play an important role. Thus, although LTB4 evokes a rapid and transient increase in [Ca2+]c and Ins(1, 4, 5)P3 mass in guinea pig eosinophils (7, 9, 10), this is evident only at concentrations far in excess of those required to activate the NADPH oxidase (7). Moreover, pretreatment of eosinophils with EGTA or (1,2-bis(o-aminophenoxy)ethane-N, N, N', N' tetraacetic acid/acetoxy methyl ester, at concentrations that abolish Ca2+ mobilization, fail to reduce the rate or amount of H2O2 produced in response to a maximally effective concentration of LTB4 (7). Similarly, SK&F 96365 and its parent compound, SC 32849, which are putative inhibitors of receptor-operated Ca2+ entry, block LTB4-induced Ca2+ mobilization without affecting ·O2- generation (8).
In exploring alternative activators of oxidative metabolism, we initially assessed the role of PLD, PKC, and PtdIns 3-kinase, which have been implicated in oxidant production from human neutrophils in response to a variety of G protein receptor-coupled agonists including LTB4 (11, 12, 13, 14, 15, 16, 17, 18, 19). Studies with wortmannin have also suggested that PtdIns 3-kinase-dependent processes are recruited in human eosinophils for the activation of the NADPH oxidase (20). However, despite these data, PLD- and PtdIns 3-kinase-driven mechanisms do not appear to be involved in LTB4-induced H2O2 generation from guinea pig eosinophils (7), and an inhibitor of PKC, Ro 31-8220, suggests that PKC plays only a relatively minor role (7).
Collectively, these results suggest that an alternative signaling
pathway(s) controls the assembly and subsequent activation of the NADPH
oxidase in guinea pig eosinophils. One possibility is that arachidonic
acid (AA) plays a central role. Persuasive evidence for this proposal
derives from several sources including the general finding that AA and
other free fatty acids promote the formation of free radicals from
intact and electropermeabilized phagocytes, as well as in a cell-free
system (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31). The mechanism of action of AA is still unknown, but it
has been shown to exert a number of intracellular effects relevant to
the activation of the NADPH oxidase. In particular, AA interacts
synergistically with GTP
S in promoting the association of the small
GTP-binding protein, p21rac, with a membrane
fraction prepared from differentiated HL-60 cells (32).
A role for AA in the activation of the NADPH oxidase is also supported by pharmacologic experiments in which inhibitors of PLA2 have been shown to suppress ·O2- generation (33, 34, 35). In addition, there is now compelling evidence that PLA2 is regulated by distinct G proteins (36, 37, 38, 39, 40, 41, 42, 43), denoted as Ga (36), suggesting that certain receptors can couple directly to PLA2 and signal the release of AA in a manner that is not dependent upon second messenger molecules derived from the hydrolysis of PtdIns(4, 5)P2 by PLC (37, 42, 44).
In this article, we report the results of studies designed to investigate whether LTB4 receptors can couple directly to PLA2 in guinea pig eosinophils and the role of endogenous AA in LTB4-induced activation of the NADPH oxidase.
A preliminary account of some of these data was presented to the American Thoracic Society/American Lung Association (45).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following drugs and analytical reagents were used. 4
- and
4ß-PDBu were purchased from Scientific Marketing Associates (Barnet,
Kent, U.K.); Percoll was from Pharmacia (Uppsala, Sweden); and HBSS was
from Flow Laboratories (Rickmansworth, Hertfordshire, U.K.). Fura-2-AM
(pentapotassium salt), Ro 31-8220, and ionomycin were obtained from
Calbiochem (Nottingham, U.K.), and D-myo
Ins(1, 4, 5)P3 (hexasodium salt) was from Semat
Technical (St. Albans, Hertfordshire, U.K.).
D-[inositol,1-3H(N)]
Ins(1, 4, 5)P3 (21 Ci/mmol) and
[5,6,8,9,11,12,14,15(n)-3H]AA
(74 Ci/mmol) were from NEN/DuPont (Stevenage, Hertfordshire, U.K.).
LTB4 and ZM 230,487 were generously donated by Bayer (Stoke
Poges, Slough, U.K.) and Zenecca (Macclesfield, Cheshire, U.K.)
respectively. Flurbiprofen, pertussix toxin (PTX), unlabeled AA, and
all other drugs and reagents were purchased from Sigma Chemical (Poole,
Dorset, U.K.).
Induction, harvesting, and purification of eosinophils
Eosinophils were elicited into the peritoneum of male
Dunkin-Hartley guinea pigs (1 kg) by weekly i.p. injection of human
serum (1 ml/animal), obtained as a by-product of human granulocyte
isolations. This procedure led to the production of
eosinophil/macrophage-rich peritoneal exudates, essentially devoid of
neutrophils and platelets, within 2 to 6 wk.
Three to six days after plasma injection, guinea pigs were anesthetized with ketamine (25 mg/kg body weight) and xylazine (5 mg/kg) and the peritoneal cavity of each animal lavaged with 50 ml sterile glucose (5% w/v) injected via a 17G cannula. The lavage fluid was aspirated into conical polypropylene centrifugation tubes and centrifuged at 240 x g for 10 min at 4°C to pellet cells. These were then washed in HBSS, pooled, and finally resuspended in Percoll (1.070 g/ml)-containing buffer A (in mM: PIPES (pH 7.2), 25; NaCl, 110; KCl, 5; NaOH, 40; and glucose, 5.4) supplemented with FCS (20% v/v).
Eosinophils were separated from other cell types by centrifuging the pooled cell preparation at 1600 x g for 20 min at 18°C over discontinuous Percoll density gradients (1.080, 1.085, 1.090, and 1.100 g/ml) in buffer A according to Gartner (46). Using this procedure, eosinophils were recovered from the 1.085/1.090 g/ml and 1.090/1.100 g/ml Percoll interfaces and were >97% pure and >95% viable as assessed by trypan blue exclusion. Cells were pooled, washed twice in HBSS, and resuspended in the appropriate assay buffer (see below).
Treatment of eosinophils with PTX
Purified eosinophils were suspended at 107/ml in buffer B (in mM: HEPES, 10; NaCl, 124; KCl, 4; NaH2PO4, 0.64; K2HPO4, 0.66; NaHCO3, 5.2; CaCl2, 1.6; MgCl2, 1; and glucose, 5.6) divided equally and incubated (37°C) with gentle mixing for 120 min with PTX (500 ng/ml) or its vehicle (0.1% v/v DMSO). Eosinophils were pelleted (500 x g for 10 min) and washed three times in buffer B before use.
Desensitization of PLC with 4ß-PDBu
Purified eosinophils were suspended at 107/ml
in buffer B, divided equally, and incubated (37°C) with gentle mixing
for 5 min with 4-PDBu or 4ß-PDBu (1 to 100 nM). Eosinophils were
pelleted (500 x g for 10 min) and washed three times
in buffer B before use.
Measurement of respiratory burst
The ability of eosinophils to generate H2O2 was used as a sensitive index of respiratory burst activity and was measured as the horseradish peroxidase-catalyzed oxidation of scopoletin using a modification of that described by Root et al. (47). Measurement of H2O2 from granulocytes is an accurate surrogate marker of ·O2- generation; it is released in parallel with the respiratory burst and is not detected in cells taken from patients with X-chromosome-linked chronic granulomatous disease (47, 48).
Eosinophils (106 cells) were resuspended at 108
cells/ml in buffer C (in mM: HEPES (pH 7.4) 10; NaCl, 138; KCl, 6;
NaH2PO4, 1; NaHCO3, 5; and glucose,
5.5) made 0.1% w/v with BSA, and 10 µl were added to 990 µl of
buffer C supplemented with CaCl2 (1 mM), MgCl2
(1 mM), superoxide dismutase (30 U), horseradish peroxidase (1 U), and
scopoletin (4 µM) in polystyrene cuvettes and incubated for 5 min at
37°C. When inhibitors were used, the cell suspension was incubated
for a further 5 min before the addition of LTB4, AA, or
4ß-PDBu. Hydrogen peroxide generation was measured fluorimetrically
(
excitation = 350 nm; emission = 460 nm;
slit width = 5 nm) using a thermostatically controlled recording
spectrophotofluorimeter fitted with a Peltier stirrer. Changes in
fluorescence were monitored continuously for 5 to 20 min, and negative
first derivative plots of the reduction in fluorescence were
constructed to obtain the peak rates of scopoletin extinction. These
values were converted to rates of H2O2
generation, which preliminary experimentation identified as being the
most reproducible measure of oxidase activity, and quantified by
interpolation from a standard curve constructed to known concentrations
of H2O2.
Measurement of Ins(1, 4, 5)P3 mass
Eosinophils were suspended at 230 x
106/ml in buffer B and stored on ice until required.
Assays, performed in duplicate, were conducted at 37°C in a total
volume of 300 µl and were initiated by the addition of 30 µl
(7 x 106 cells) of eosinophil suspension to 240 µl
of prewarmed buffer B. The cell suspensions were incubated for 5 min,
after which LTB4 (30 µl) was added. Reactions were
terminated by the addition of 300 µl TCA (1 M), and
Ins(1, 4, 5)P3 mass was subsequently extracted
(49) and measured using a competitive protein-binding assay (50). The
detection limit and IC50 of this assay are 0.4 and 1
pmol Ins(1, 4, 5)P3, respectively.
In previous studies (7, 51), we established that exposure of guinea pig eosinophils to LTB4 (1 µM) produces a transient increase in Ins(1, 4, 5)P3 mass and in the [Ca2+]c; for both indices of activation, peak responses occur 5 s after the stimulus and then decay rapidly to basal or near basal levels (7, 51). Thus, in the experiments performed herein, measurements were made at the 5-s time point.
Measurement of [Ca2+]c
Eosinophils (107/ml) were suspended at 37°C
in buffer C supplemented with 0.1% BSA (w/v) and incubated for 30 min
with fura-2-AM (1 µM). The cells were washed three times in buffer B,
and the ability of LTB4 to elevate
[Ca2+]c was determined using well-established
spectrofluorimetric methods (excitation = 340/380 nm;
emission = 510 nm; slit width = 4 nm).
Measurement of [3H]AA release
Agonist-induced release of [3H]AA from eosinophils was performed using a modification of the method detailed in Cockcroft and Stutchfield (44). Eosinophils (107/ml) were prelabeled with [3H]AA (1 µCi/ml) for 120 min in buffer C, washed three times in the same buffer, and resuspended at a concentration of 3 x 107/ml. Aliquots (100 µl) of eosinophils were transferred to Eppendorf tubes containing 80 µl buffer C (with or without Ca2+/Mg2+ and inhibitors) and incubated for 5 min at 37°C before the addition of stimulus. Samples were incubated for a further 10 min at 37°C and the reaction subsequently terminated by the addition of 500 µl of ice-cold NaCl (0.9% w/v). Eosinophils were sedimented by centrifugation (12,000 x g for 5 min) and an aliquot of the supernatant counted in 2 ml ACS II (Amersham). In the text, the term [3H]AA refers to all tritiated species released from eosinophils.
Initial experiments revealed that the uptake of [3H]AA by guinea pig eosinophils occurred in a time-dependent manner (t1/2 = 11.5 ± 2.6 min, n = 3), reached equilibrium at approximately 15 min, and remained constant for 120 min. In a study by Debbaghi and colleagues (52), analysis of radiolabeled species released from agonist-stimulated eosinophils revealed that they consisted almost entirely of free fatty acids, indicating that this method represents a satisfactory index of AA release and that it is formed following the activation of PLA2 rather than DAG lipase (44, 52).
Statistical analysis
Data in the text, tables, and figure legends refer to the mean ± SEM of "n" independent determinations taken from different cell preparations. Where appropriate, Student’s t test (two-tailed) or the one-way analysis of variance/Newman-Keuls test was used to assess significance between "control" and "treatment" groups. The null hypothesis was rejected when p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LTB4 evoked a rapid, transient, and
concentration-dependent generation of H2O2 from
eosinophils with an EC50 of 16.2 ± 3.6 nM and a
maximal rate of production of 5.51 ± 0.03
nmol/min/106 cells (Fig. 1
).
Kinetically, H2O2 was detected after a lag of
approximately 5 s in eosinophils exposed to a maximally effective
concentration of LTB4 (1 µM) and increased steadily for a
minimum of 90 s; the estimated t1/2 was
30 s (Fig. 2). Under identical
experimental conditions, exogenous AA generated
H2O2 from eosinophils at a comparable rate
(6.15 ± 0.75 nmol/min/106 cells at 30 µM) to
LTB4 but, on a molar basis, was approximately 340 times
less potent (EC50 = 5.56 ± 0.91 µM; Fig. 3). Analysis of the
concentration-response curves, which described the generation of
H2O2, revealed that the mean slope for AA (Hill
coefficient (nH) = 2.57 ± 0.77) was
significantly greater than for LTB4
(nH = 1.14 ± 0.37). The ability of
LTB4 (1 µM) and AA (10 µM) to generate
H2O2 was not affected by flurbiprofen (3 µM)
or ZM 230,487 (50 µM), non-redox inhibitors of cyclooxygenase and
5'-lipoxygenase, respectively (Table I),
at concentrations previously shown to abolish agonist-induced
thromboxane and leukotriene production from a variety of guinea pig
tissues including eosinophils (53, 54).
|
|
|
|
At rest, guinea pig eosinophils spontaneously released
[3H]AA amounting to 0.8 to 1.2% of the total
radioactivity incorporated after 10 min incubation at 37°C. In
contrast, the elaboration of [3H]AA from eosinophils
exposed to LTB4 was significantly augmented under identical
experimental conditions (Fig. 2
). This effect was very rapid
(t1/2 = 2.2 ± 1.6 s at 1 µM;
Fig. 2) and concentration dependent (EC50 = 71.6 ±
15.2 nM; Fig. 1) and resulted, maximally, in a 2.7-fold increase in the
amount of [3H]AA released (2.46 ± 0.13% of the
total radioactivity incorporated). The ability of LTB4 to
mobilize [3H]AA from eosinophils was a receptor-mediated
phenomenon, as it was abolished by U-75,302 (55), a selective
LTB4 receptor antagonist that has been shown previously to
block LTB4-induced Ca2+ mobilization,
Ins(1, 4, 5)P3 accumulation, and
H2O2 generation in guinea pig eosinophils
(7).
Temporally, the release of [3H]AA from
LTB4-challenged eosinophils preceded the generation of
H2O2 (Fig. 2). However, the
concentration-response relationship for [3H]AA release
was complex when compared with the curve that described
H2O2 production (Fig. 1). Thus, at low
concentrations of LTB4 (1 and 3 nM), AA release was
detected in the absence of H2O2 generation,
whereas at higher concentrations of LTB4 (10 nM to 3 µM)
the concentration-response curve was displaced approximately fourfold
to the right of the curve that described the activation of the NADPH
oxidase. Although these results are difficult to interpret, they do not
exclude the possibility that endogenous AA per se is a direct effector
of NADPH oxidase, as reported in other leukocytes (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 33, 34, 35), and
additional experiments were performed to assess this possibility.
Effect of PTX on LTB4-induced Ins(1, 4, 5)P3 accumulation, Ca2+ mobilization, [3H]AA, release and H2O2 generation
To understand further the way in which LTB4
activates PLA2 and the role of AA in
H2O2 generation, eosinophils were pretreated
with PTX (500 ng/ml; 120 min; 37°C); ADP ribosylates PTX and thereby
inactivates the Go and Gi families of heterotrimeric GTP-binding
proteins. The indices of PLA2, PLC, and NADPH oxidase
activation were also measured. Exposure of guinea pig eosinophils to
LTB4 (1 µM) for 5 s resulted in an approximately
sixfold increase in Ins(1, 4, 5)P3 mass and
elevated the [Ca2+]c fivefold, from 82 to 411
nM; both of these responses were absent in cells pretreated with PTX
(Figs. 4A and
5A). Similarly, PTX
abolished [3H]AA release and H2O2
generation over the entire concentration range (0.1 nM to 3 µM) in
which LTB4 was active (Figs. 6A and
7A). The concentration
of PTX used and the time of preincubation did not affect cell viability
or responsiveness, as evinced by the robust generation of
H2O2 evoked by 1 µM of 4ß-PDBu (Fig. 7A).
|
|
|
The finding that PTX abolished the increase in Ins(1, 4, 5)P3 mass, [Ca2+]c, and [3H]AA prompted us to perform additional experiments in an attempt to dissociate the activation of PLA2 from the generation of signal molecules derived from the hydrolysis of PtdIns(4, 5)P2 by PLC. To this end, eosinophils were pretreated with 4ß-PDBu, which uncouples many G protein-linked receptors from PLC-ß in a variety of cell types by a PKC-dependent process (56).
Pretreatment of eosinophils with 4ß-PDBu (1–100 nM; 5 min; 37°C)
inhibited LTB4 (1 µM)-induced
Ins(1, 4, 5)P3 accumulation and Ca2+
mobilization in a concentration-dependent manner (IC50
35 and 70 nM, respectively) under conditions in which 4-PDBu (100
nM; 5 min; 37°C) was inactive (Figs. 4B and
5B). Signaling through PLC was abolished in cells
pretreated with 100 nM of 4ß-PDBu for 5 min at 37°C and these
conditions were used in all further experiments.
Effect of PDBu on LTB4-induced [3H]AA release
Eosinophils in which PLC was desensitized demonstrated a
significant (3.7-fold) increase in the basal elaboration of
[3H]AA (2.9 ± 0.17% of total radiolabel
incorporated) over the amount released spontaneously (0.78 ±
0.18%; Fig. 6B). Also, the addition of
LTB4 to 4ß-PDBu-treated cells further increased the
release of [3H]AA (to 3.94 ± 0.21% of the total
incorporated radioactivity at the maximally effective concentration)
indicating that LTB4 receptors can stimulate
PLA2 by a PLC-independent mechanism (Fig. 6B). This effect was concentration dependent with an
EC50 (72.3 ± 13.2 nM) not significantly different
from that obtained in eosinophils treated with vehicle (see above; Fig. 6B). The maximum change in [3H]AA
release from "control" cells (1.6%) was routinely greater
(30%) than in eosinophils in which PLC was desensitized (1.1%).
This difference was not due to depletion of "releasable"
radiolabel, since a combination of 4ß-PDBu and ionomycin (100 nM
each) liberated 8.9 ± 1.3% (n = 3) of the
total [3H]AA incorporated into membrane lipids.
Effect of PDBu on LTB4-induced H2O2 generation
Experiments were performed in eosinophils in which PLC was
desensitized to determine the extent to which activation of NADPH
oxidase was dependent upon signaling through PLA2. As
shown in Figure 7B, LTB4 failed to augment the
rate of H2O2 production in eosinophils
pretreated with 4ß-PDBu (100 nM; 5 min; 37°C) at any concentration
examined despite the fact that these cells expressed a modest basal
rate (1.3 ± 0.32 nmol/min/106 cells) of oxidative
metabolism. This low level of H2O2 production
was presumably due to the fact that phorbol diesters evoke a robust and
prolonged activation of the NADPH oxidase in guinea pig
eosinophils (7).
Role of PKC and Ca2+ in LTB4-induced [3H]AA release
To provide further evidence that the activation of
PLA2 by LTB4 occurs independently of PLC,
the role of DAG-induced PKC activation and
Ins(1, 4, 5)P3-induced Ca2+
mobilization were examined. Pretreatment (5 min) of eosinophils with
the PKC inhibitor Ro-31,8220 (1 to 20 µM) had no significant effect
on basal or LTB4 (1 µM)-induced [3H]AA
release from guinea pig eosinophils (Fig. 8). Thus, PtdIns(4, 5)P2-derived DAG is not required for
LTB4-induced [3H]AA release.
|
and Figure 9 show the results of experiments in
which the Ca2+ sensitivity of LTB4-induced
[3H]AA release was explored. In fura-2-AM-treated
eosinophils incubated in Ca2+-free medium, EGTA (100 µM)
abolished the ability of LTB4 (0.1 nM to 1 µM) to
increase the [Ca2+]c (Fig. 9). Under these
conditions, the release of [3H]AA effected by a maximally
effective concentration of LTB4 (1 µM) was partially
suppressed, indicating the involvement of Ca2+-dependent
and -independent PLA2s (Table II). The amount of
[3H]AA released by LTB4 (1 µM) in
EGTA-treated cells, expressed as a percentage of the total radiolabel
incorporated, was 0.91%, which was almost identical to the increase
(1.1%) observed from eosinophils in which PLC had been desensitized by
4ß-PDBu (Fig. 6B).
|
|
). Thus, the Ca2+ required for
[3H]AA release are mobilized by LTB4 over a
concentration range that does not detectably activate PLC. Effect of mepacrine on LTB4-induced AA release and H2O2 generation
The role of AA in LTB4-induced NADPH oxidase was
evaluated pharmacologically by the use of mepacrine (quinacrine), an
inhibitor of PLA2. Mepacrine effectively inhibited
LTB4-induced [3H]AA release with an
IC50 of 21.2 ± 4.7 µM (Fig. 10) under conditions in which
H2O2 generation was only marginally suppressed.
Indeed, mepacrine abolished [3H]AA release at a
concentration (50 µM) that suppressed the generation of
H2O2 by only 35.8 ± 8.5% (Fig. 10).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In guinea pig eosinophils, AA effected a robust generation of
H2O2 of a magnitude equivalent to that produced
by LTB4, and over a concentration range (1–100 µM) shown
to be active in neutrophils (29). Significantly, AA-induced oxidative
metabolism was not inhibited by flurbiprofen or ZM 230,487, indicating
that neither cyclooxygenase nor 5'-lipoxygenase products were involved
in the genesis of this response. Similar results have been reported in
guinea pig macrophages (22) and human neutrophils (57), which
encourages speculation that AA per se is a direct effector of the NADPH
oxidase in phagocytes in general. A highly reproducible finding was
that the concentration-response curve that described the production of
H2O2 by AA was steep
(nH 2.6). Qualitatively identical
results have been reported for human neutrophils (27), which might
suggest positive cooperation between AA molecules in the activation of
the NADPH oxidase complex.
Although the EC50 value of LTB4 for the generation of [3H]AA was approximately fourfold lower than for H2O2 generation, the concentration-response relationship that described [3H]AA release spanned almost four orders of magnitude, such that at low agonist concentrations, both [3H]AA and H2O2 were released. Although the reason for the shallow [3H]AA concentration-response curves is currently unclear, this observation nevertheless prompted the question of whether a causal relationship exists between these two parameters. This was particularly relevant given that the activation of the NADPH oxidase by LTB4 was not mediated by a prostaglandin, thromboxane A2, or leukotrienes (see above). Circumstantial evidence to support this hypothesis was the critical observation that the kinetics of [3H]AA release were very rapid and preceded H2O2 generation. Another important consideration was whether the level of AA achieved in LTB4-stimulated eosinophils was sufficient to activate the NADPH oxidase. Although not formally investigated in this study, Wolf et al. (58) have reported that following stimulation of pancreatic islets with glucose, the intracellular concentration of AA, measured by mass spectrometry, reached 50 to 100 µM, which if reproduced in agonist-stimulated eosinophils, is far in excess of the concentration required to promote maximally oxidative metabolism.
Having established that the release of [3H]AA by
LTB4 had the required concentration and time dependence to
activate the NADPH oxidase, experiments were performed with PTX to
determine the extent to which signaling through PLA2 and
PLC were implicated in the release of [3H]AA and
H2O2 by LTB4. Unfortunately, this
approach was unsuccessful, since PTX abolished all effects
(H2O2 generation, [3H]AA release,
Ins(1, 4, 5)P3 accumulation, and Ca2+
mobilization) evoked by LTB4 indicating the involvement of
one or more members of the Gi or Go family of heterotrimeric
GTP-binding proteins. Although we cannot state with certainty which of
the G proteins were involved in mediating the aforementioned effects of
LTB4, recent studies have established that guinea pig
peritoneal eosinophils express a number of G proteins, including
Gi3 and Go (but not Gi1), that are PTX
substrates and are known to couple to PLC-ß in a number of cells and
tissues (59). Another possible candidate is Gi2,
although it has not been formally identified in guinea pig
eosinophils.
LTB4 promotes the hydrolysis of PtdIns(4, 5)P2 in guinea pig eosinophils with the formation of Ins(1, 4, 5)P3 and, by definition, a stoichiometrically equivalent amount of diglyceride (7). Potentially, these second messengers could act upstream of PLA2 through their ability to release Ca2+ from intracellular stores and activate PKC, thereby indirectly regulating the availability of free AA. Given that PTX abolished all effects of LTB4, additional experiments were deemed necessary to establish how LTB4 generates H2O2 and the role of PLC-derived signal molecules in [3H]AA release. To this end, two studies were performed. Initially, eosinophils were incubated with 4ß-PDBu, which is known to uncouple (desensitize) certain serpentine receptors from PLC-ß (but not PLA2) (44, 56), at a concentration that abolished LTB4-induced Ins(1, 4, 5)P3 accumulation and Ca2+ mobilization. It was reasoned that if LTB4 receptors can couple directly to PLA2, then [3H]AA release should be unaffected. Treatment of cells with 4ß-PDBu significantly enhanced the basal elaboration of [3H]AA. This was somewhat unexpected given that phorbol diesters are inactive in platelets (60), HL60 cells, and neutrophils under roughly comparable conditions (44, 61). However, a survey of the literature revealed that the ability of phorbol diesters per se to release AA varies depending on cell type (see 62 , which might be dictated by the complement and/or activation requirements (e.g., Ca2+ dependence) of PLA2 isoenzymes present in the cell of interest. Of significance to this study was the finding that LTB4 increased the release of [3H]AA from eosinophils in which PLC was desensitized above that produced by 4ß-PDBu per se, with an EC50 identical to eosinophils in which PLC was not desensitized. An interpretation of these results is that LTB4 releases [3H]AA by a PLC-independent mechanism. Unfortunately, the observation that the desensitization produced by 4ß-PDBu also was associated with [3H]AA and H2O2 release did not permit unequivocal conclusions to be drawn, and a second approach was adopted to corroborate or refute this interpretation. Logic dictates that if the formation of DAG by LTB4 is necessary for the activation of PLA2, then an inhibitor of PKC should inhibit [3H]AA release. Similarly, a requirement for Ins(1, 4, 5)P3 in this response implies that the activation of PLA2 is Ca2+ dependent and that the accumulation of Ins(1, 4, 5)P3 can account, at least in part, for Ca2+ mobilization. Experiments designed to test these assumptions failed to implicate DAG and Ins(1, 4, 5)P3 in LTB4-induced [3H]AA release. Thus, pretreatment of eosinophils with the PKC inhibitor Ro 31-8220 did not inhibit LTB4-induced [3H]AA release at concentrations (1 to 20 µM) known to inhibit PKC in leukocytes (63, 64). Since LTB4 does not activate PLD (an alternative source of diglyceride) in guinea pig eosinophils (7), these data indicate that the activation of PKC by PtdIns(4, 5)P2-derived DAG cannot be involved in the activation of PLA2. Moreover, LTB4 mobilized appreciable [3H]AA from eosinophils in which Ca2+ mobilization was abolished. Therefore, by definition, this Ca2+-independent PLA2 cannot require Ins(1, 4, 5)P3-evoked Ca2+ release for activity. The regulation of the Ca2+-dependent PLA2(s) was also dissociated from the activation of PLC as evinced by the finding that the Ca2+ required for [3H]AA release were mobilized by LTB4 over a concentration range that did not detectably increase Ins(1, 4, 5)P3.
The reason for the reduction in the magnitude of LTB4-induced [3H]AA release in eosinophils in which PLC was desensitized was not formally addressed, but it is likely that 4ß-PDBu, by abolishing Ca2+ influx, reduced the pool of Ca2+-dependent PLA2 that LTB4 would normally mobilize. This proposal is consistent with the fact that the magnitude of LTB4-induced [3H]AA release in EGTA-treated eosinophils and in cells in which PLC was desensitized were almost identical.
Collectively, these data provide persuasive evidence that LTB4 can liberate [3H]AA in eosinophils independently of signal molecules derived from the activation of PLC. Furthermore, the demonstration that PTX abolished LTB4-induced [3H]AA release from guinea pig eosinophils, and the evidence that PLA2 is regulated by distinct G proteins in many cells (36, 37, 38, 39, 40, 41, 42, 43), suggests that LTB4 releases AA from membrane phospholipids by acting at receptors that couple directly to PLA2 via a member of the Gi or Go family of heterotrimeric GTP-binding proteins.
The role of AA in LTB4-induced activation of the NADPH
oxidase was also assessed in eosinophils in which PLC had been
desensitized. A prediction, based on previous evidence that PLC-driven
processes are not involved (7), was that the capacity of eosinophils to
produce H2O2 should be preserved. However,
contrary to expectation, LTB4 failed to generate
H2O2 in 4ß-PDBu-treated eosinophils under
conditions in which the release of [3H]AA was essentially
unchanged. Thus, despite circumstantial data to the contrary (see
Introduction), these results indicate that
LTB4 activates the NADPH oxidase by a mechanism divorced
from its ability to couple to PLA2 and generate AA. This
interpretation is corroborated, in part, by the results obtained with
the PLA2 inhibitor mepacrine, which abolished
[3H]AA release at a concentration that exerted a
relatively modest effect on H2O2 generation.
However, if it is assumed that mepacrine (50 µM) inhibits
H2O2 generation by acting solely at the level
of PLA2, then these data suggest that the liberation of AA
by LTB4 does play a relatively minor role (36%) in the
activation of the NADPH oxidase. It is striking that in other cells, in
particular in human neutrophils, mepacrine effectively suppresses the
magnitude of the respiratory burst evoked by FMLP and serum-opsonized
zymosan (33, 34) at concentrations significantly lower than those used
in this study (34). Moreover, in FMLP-stimulated human eosinophils,
mepacrine inhibited ·O2- generation in
a concentration-dependent manner (35), although in that study the
release of [3H]AA was not measured. The reason(s) for
this discrepancy is unknown, but possibilities include differences in
species or cell type (eosinophil vs neutrophil) and/or, more
fundamentally, in the signaling pathways recruited for the activation
of the NADPH between agonists (51). The possibility also remains that
eosinophils "elicited" into the peritoneal cavity might not
generate H2O2 in the same way as their
unstimulated circulating counterparts.
In conclusion, the results of this study provide persuasive evidence that agonism of LTB4 receptors on guinea pig eosinophils mobilizes AA by a PTX-sensitive mechanism that does not involve the generation of signal molecules derived from the hydrolysis of PtdIns(4, 5)P2 by PLC. Moreover, despite the ability of LTB4 to stimulate PLA2, a central role for AA in the activation of the NADPH oxidase was largely excluded using both a pharmacologic and a biochemical approach. Thus, the primary cellular signaling pathways recruited by LTB4 that control the assembly and subsequent activation of the NADPH oxidase complex in guinea pig eosinophils remain to be elucidated.
|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Mark A. Giembycz, Dept. of Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, U.K. E-mail address:
3 Abbreviations used in this paper: ·O2-, superoxide; ZM 230,487, (6-[3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxymethyl]-1-ethyl-2-quinolone); U-75,302, 6-[6-(3-hydroxy-1E, 5Z-undecadien-1-yl)-2-pyridinyl]-1,5-hexanediol; PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; Percoll, polyvinylpyrrolidine-coated silica gel; [Ca2+]c, cytosolic free calcium concentration; PLA2, phospholipase A2; PLC, phospholipase C; PLD, phospholipase D; H2O2, hydrogen peroxide; LTB4, leukotriene B4; Ins(1,4,5)P3; Ins(1,4,5)P3, inositol(1,4,5)trisphosphate; PtdIns 3-kinase, phosphatidylinositol 3-kinase; PtdIns(4, 5)P2, phosphatidylinositol(4,5)bisphosphate; AA, arachidonic acid; DAG, diacylglycerol; PTX, pertussix toxin; IC50, 50% inhibitory concentration; EC50, 50% effective concentration; nH, Hill coefficient.
Received for publication September 3, 1997. Accepted for publication December 30, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
receptor-mediated superoxide generation in macrophages. J. Immunol. 138:4353.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  C. H. C. Serezani, D. M. Aronoff, S. Jancar, and M. Peters-Golden Leukotriene B4 mediates p47phox phosphorylation and membrane translocation in polyunsaturated fatty acid-stimulated neutrophils J. Leukoc. Biol., October 1, 2005; 78(4): 976 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. H. C. Serezani, D. M. Aronoff, S. Jancar, P. Mancuso, and M. Peters-Golden Leukotrienes enhance the bactericidal activity of alveolar macrophages against Klebsiella pneumoniae through the activation of NADPH oxidase Blood, August 1, 2005; 106(3): 1067 - 1075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Thomas, S. L. Kunkel, and N. W. Lukacs Regulation of Cockroach Antigen-Induced Allergic Airway Hyperreactivity by the CXCR3 Ligand CXCL9 J. Immunol., July 1, 2004; 173(1): 615 - 623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Decoursey Voltage-Gated Proton Channels and Other Proton Transfer Pathways Physiol Rev, April 1, 2003; 83(2): 475 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Antczak, P. Montuschi, S. Kharitonov, P. Gorski, and P. J. Barnes Increased Exhaled Cysteinyl-Leukotrienes and 8-Isoprostane in Aspirin-induced Asthma Am. J. Respir. Crit. Care Med., August 1, 2002; 166(3): 301 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Woo, H.-J. You, S.-H. Cho, Y.-W. Eom, J.-S. Chun, Y.-J. Yoo, and J.-H. Kim Leukotriene B4 Stimulates Rac-ERK Cascade to Generate Reactive Oxygen Species That Mediates Chemotaxis J. Biol. Chem., March 1, 2002; 277(10): 8572 - 8578. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T E DeCoursey, V V Cherny, A G DeCoursey, W Xu, and L L Thomas Interactions between NADPH oxidase-related proton and electron currents in human eosinophils J. Physiol., September 15, 2001; 535(3): 767 - 781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V V Cherny, L M Henderson, W Xu, L L Thomas, and T E DeCoursey Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid J. Physiol., September 15, 2001; 535(3): 783 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. HANAZAWA, S. A. KHARITONOV, and P. J. BARNES Increased Nitrotyrosine in Exhaled Breath Condensate of Patients with Asthma Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): 1273 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. del Pozo, F. Pirotto, B. Cárdaba, I. Cortegano, S. Gallardo, M. Rojo, I. Arrieta, E. Aceituno, P. Palomino, A. Gaya, et al. Expression on human eosinophils of CD148: a membrane tyrosine phosphatase. Implications in the effector function of eosinophils J. Leukoc. Biol., July 1, 2000; 68(1): 31 - 37. [Abstract] [Full Text]
|
|
|
|
|
|
O. T. Lynch, M. A. Giembycz, I. Daniels, P. J. Barnes, and M. A. Lindsay Pleiotropic role of lyn kinase in leukotriene B4-induced eosinophil activation Blood, June 1, 2000; 95(11): 3541 - 3547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Giembycz and M. A. Lindsay Pharmacology of the Eosinophil Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-H. Woo, Y.-W. Eom, M.-H. Yoo, H.-J. You, H. J. Han, W. K. Song, Y. J. Yoo, J.-S. Chun, and J.-H. Kim Tumor Necrosis Factor-alpha Generates Reactive Oxygen Species via a Cytosolic Phospholipase A2-linked Cascade J. Biol. Chem., October 6, 2000; 275(41): 32357 - 32362. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and on [3H]AA release (•). Highly
purified (>97%) eosinophils (106) were added to buffer B
at 37°C in polystyrene fluorimeter cuvettes supplemented with
Ca2+ (1 mM), Mg2+ (1 mM), superoxide dismutase
(30 U), horseradish peroxidase (1 U), and the fluorophore scopoletin (4
µM). Eosinophils were allowed to equilibrate (5 min) and then
challenged with LTB4. The generation of
H2O2 by the cells was measured
spectrophotofluorimetrically by monitoring the reduction in
fluorescence of scopoletin from which peak rates of
H2O2 generation were calculated. In parallel
experiments, eosinophils were labeled with [3H]AA (1
µCi/ml; 2 h), and the amount of radiolabeled species released by
LTB4 (1 µM) in 10 min was measured. Data points represent
the mean ± SEM of eight experiments performed with different cell
preparations. See Materials and Methods section for further
details.
) to release
tritium was monitored for the first 60 s of the response.
LTB4-induced H2O2 generation (
) of extracellular
Ca2+ (+100 µM EGTA) was determined as described in
Materials and Methods. Data points represent peak changes
and are the mean ± SEM of four experiments performed with
different cell preparations. Note that the maximal increase in
[Ca2+]c occurred without a detectable
increase in Ins(1,4,5)P3 mass.
