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Departments of Immunology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator synthesized by a range of cell types, including eosinophils 11, 12 . PAF binds to a G protein-coupled seven-transmembrane receptor 13 , and exerts various biological activities, such as platelet activation, airway constriction, development of bronchial hyperresponsiveness, and induction of microvascular leakage and edema (reviewed in Refs. 14 and 15). Because of these potent actions, PAF may play a role in the pathophysiology of bronchial asthma (reviewed in 15 . Moreover, in eosinophils, PAF induces chemotaxis 16 , degranulation 17 , and generation of superoxide anion 8, 18 and arachidonic acid metabolites 19 . Altogether, these observations as well as the eosinophil’s capacity to produce PAF 3, 12 suggest that PAF may act as an autocrine mediator promoting eosinophil functions.
In this study, we investigated the roles of endogenous PAF on eosinophil effector functions. Our findings suggest PAF is an essential autocrine mediator in superoxide production, degranulation, and eicosanoid formation. Furthermore, endogenous PAF may amplify the activation signals provoked by various receptors, such as cytokine receptors and Fc receptors, culminating in the full-blown functions of eosinophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAF and CV6209 were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA), dissolved in absolute ethanol at 40 and 10 mM, respectively, and stored at -20°C. 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho-(N,N,N-trimethyl)-hexanolamine (hexanolamine PAF) and PMA were purchased from Calbiochem (La Jolla, CA), dissolved in water and DMSO at 20 mM and 5 mg/ml, respectively, and stored at -20°C. Pertussis toxin (PTX) from Calbiochem was dissolved in water at 100 µg/ml and stored at 4°C. Y-24180 (israpafant) was a gift from Yoshitomi Pharmaceutical Industries (Fukuoka, Japan); it was dissolved in DMSO at 20 mM and stored at 20°C. Mepacrine was purchased from Sigma (St. Louis, MO). Human IL-5, a gift from Schering-Plough Research Institute (Kenilworth, NJ), was diluted in PBS containing 0.1% BSA at 100 µg/ml, and stored at -70°C. All agonists and antagonists were diluted in reaction medium immediately before the experiments. Neither ethanol nor DMSO altered eosinophil functions at the solvent concentrations used in this study (<0.05%). Purified human serum IgG was purchased from Organon Teknika-Cappel (Malvern, PA) and stored at 1 mg/ml in PBS at 4°C. For eosinophil activation, IgG was immobilized onto the wells of tissue culture plates, as described below. The other reagents, including catalase, superoxide dismutase, human serum albumin (HSA), taurine, L-serine, glutathione, and cytochrome c, were purchased from Sigma.
Eosinophil isolation
Eosinophil isolation was performed by a magnetic cell separation system (MACS; Becton Dickinson, San Jose, CA), as described previously, with minor modifications 20 . Briefly, venous blood anticoagulated with 50 U/ml heparin was obtained from normal volunteers and diluted with PBS at a 1:1 ratio. Diluted blood was overlaid on isotonic Percoll solution (density, 1.085 g/ml; Sigma) and centrifuged at 1000 x g for 30 min at 4°C. The supernatant and mononuclear cells at the interface were carefully removed, and erythrocytes in sediment were lysed by two cycles of hypotonic water lysis. Isolated granulocytes were washed twice with PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) with 1% defined calf serum (HyClone Laboratories, Logan, UT). An approximately equal volume of anti-CD16-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) was added to the cell pellet. After 60 min of incubation on ice, cells were loaded onto the separation column positioned in the strong magnetic field of the MACS. Cells were eluted with PIPES buffer containing defined calf serum. The purity of eosinophils counted by Randolph’s stain was regularly greater than 98%. Purified eosinophils were washed and suspended in reaction medium, then used immediately.
Superoxide anion production
Eosinophil superoxide production was induced by various stimuli in polystyrene 96-well flat-bottom tissue culture plates (Corning CoStar, Cambridge, MA) and measured by reduction of cytochrome c, as previously described 21, 22 . To immobilize human IgG onto the wells of tissue culture plates, 50 µl of human IgG diluted in PBS at 50 µg/ml was added to the wells and incubated overnight at 4°C. The solution was aspirated, and the wells were blocked with 50 µl of 2.5% HSA in PBS for 2 h at 37°C. After incubation, wells were washed twice with 0.9% NaCl and used immediately for the experiments. The tissue culture wells used for eosinophil stimulation with soluble agonists were blocked with HSA without IgG coating. Freshly isolated eosinophils were washed, and resuspended in HBSS with 10 mM HEPES and 200 µM cytochrome c at 5 x 105 cells/ml. Fifty microliters of serial dilutions of inhibitors were added to the wells. One hundred microliters of cell suspension were dispensed onto the wells, and the reactions were initiated by adding 50 µl of soluble stimuli, including IL-5, PMA, and PAF at 25 ng/ml, 1 ng/ml, and 0.3 µM final concentrations, respectively. Wells coated with immobilized Ig received 50 µl of medium alone. In some experiments, cells were pretreated with an optimal concentration of PTX (100 ng/ml) in 15-ml conical tubes for 2 h at 37°C before addition to the wells. Immediately after addition of stimuli, the reaction wells were measured for absorbance at 550 nm in a microplate autoreader (Thermomax, Molecular Devices, Menlo Park, CA), followed by repeated readings. Between absorbance measurements, the plate was incubated at 37°C. Superoxide anion generation was calculated with an extinction coefficient of 21.1 x 103 cm-1M-1 for reduced cytochrome c at 550 nm, and was expressed as nanomoles of superoxide production/105 cells.
PAF production
Generation of PAF by eosinophils was performed in 96-well flat-bottom tissue culture plates, as described previously by van der Bruggen 23 , with minor modifications. All incubations for PAF production were performed in enriched HBSS (HBSS supplemented with 1 mM CaCl2, 5 mM glucose, and 0.5% (w/v) HSA and containing superoxide radical scavengers (2.5 mM taurine, 5000 U/ml catalase, and 380 U/ml superoxide dismutase)) to prevent lipid degradation. Freshly isolated eosinophils were suspended in enriched HBSS at 2 x 106 cells/ml, and 100-µl aliquots were added to the wells of tissue culture plates prepared as described above. Cells were stimulated with 100 µl of IL-5, PMA, or medium alone. After incubation for 15 or 45 min at 37°C, the plates were briefly centrifuged at 400 x g. Supernatants were collected and stored under nitrogen at -70°C. The concentration of PAF in the samples was determined by a commercial RIA kit (DuPont NEN, Boston, MA), following manufacturer’s protocol. The data were expressed as pg of PAF produced/106 cells. The detection limit of the assay was 100 pg PAF produced/106 cells.
Eosinophil degranulation
Eosinophil degranulation was performed in 96-well flat-bottom tissue culture plates, as described previously 21, 22 . Eosinophils were washed and suspended in RPMI 1640 medium (Celox, Hopkins, MN), supplemented with 10 mM HEPES and 0.02% HSA, at 5 x 105 cells/ml. Serial dilutions of CV6209 (50 µl) were added to the wells, followed by 100 µl of eosinophil suspension. Cells were stimulated with 50 µl of soluble agonists, and incubated for 180 min at 37°C and 5% CO2. Eosinophils incubated in the wells coated with IgG received 50 µl of medium alone. After incubation, supernatants were collected and stored at -20°C until they were assayed for eosinophil-derived neurotoxin (EDN). To quantitate eosinophil degranulation, the concentration of EDN in the sample supernatants was measured by RIA. The RIA is a double-Ab competition assay, in which radioiodinated EDN, rabbit anti-EDN, and burro anti-rabbit IgG are used, as reported elsewhere 5 . Total cellular EDN contents were measured simultaneously using supernatants from cells lysed with 0.5% Nonidet P-40 detergent. All assays were conducted in duplicate.
LTC4 production
Eosinophil production of LTC4 was performed in 96-well tissue culture plates, as described previously, with minor modifications 24 . Eosinophils were washed with HBSS supplemented with 10 mM HEPES, 20 mM L-serine, and 5 mM glutathione, and resuspended in the same medium at 1 x 106 cells/ml. Serial dilutions of CV6209 or medium (50 µl) were added to the wells of the tissue culture plate, followed by 100 µl of cell suspension. Cells were stimulated by adding 50 µl of soluble agonists. Eosinophils incubated in the wells coated with IgG received 50 µl of medium alone. After incubation for 1 h at 37°C and 5% CO2, the supernatants from each well were collected and frozen at -70°C or assayed immediately. Concentrations of LTC4 in the sample supernatants were measured by ELISA using an LTC4 kit (Cayman Chemical, Ann Arbor, MI), following the procedure recommended by the manufacturer. The sensitivity of the assay was 7.8 pg/ml. All experiments were conducted in duplicate.
Lipid body formation
Formation of lipid bodies within the activated eosinophils was examined as described previously 25 , with minor modifications. Briefly, wells of a Lab-Tek 16-well chamber slide (Nunc, Naperville, IL) were coated with or without IgG and blocked with 2.5% HSA, as described above. Eosinophils were washed in HBSS with 10 mM HEPES, and resuspended in the same medium at 1 x 106 cells/ml. Serial dilutions of CV6209 or medium (50 µl) were added to the wells, followed by 100 µl of cell suspension. Cells were stimulated by adding 50 µl of soluble agonists. Eosinophils incubated in the wells coated with IgG received 50 µl of medium alone. After incubation for 60 min at 37°C and 5% CO2, the supernatants were removed by aspiration. Eosinophils were fixed in 3.7% formaldehyde in Ca2+/Mg2+-free HBSS, pH 7.4, and rinsed in 0.1 M cacodylate buffer, pH 7.4. Cells were stained in 1.5% OsO4 (30 min), rinsed in water, immersed in 1.0% thiocarbohydrazide (5 min), rinsed in 0.1 M cacodylate buffer, restained in 1.5% OsO4 (3 min), rinsed in water, and dried. Subsequently, chamber walls were removed and the slide was coverslipped. The numbers of lipid bodies were counted with phase-contrast microscope by consecutively scanning 50 eosinophils with x630 magnification.
Statistical analysis
Data are presented as means ± SEM from the numbers of experiments indicated. Statistical significance of the differences between various treatment groups (i.e., with or without inhibitor) was assessed using the Mann-Whitney U test or paired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, eosinophil effector functions, such as superoxide
production and degranulation, were shown to be induced by a wide range
of stimuli, such as cytokines (e.g., IL-5), lipid mediators (e.g.,
PAF), and immobilized Ig (e.g., IgG) 8, 9, 10, 21 . Consistent with these
previous findings, Fig. 1
shows that PAF,
human IgG immobilized onto tissue culture plates and IL-5, as well as a
positive control, PMA, induced eosinophil superoxide production in a
time-dependent manner. As reviewed elsewhere, PAF is known to
mediate its biological effects through activation of a G
protein-coupled seven-transmembrane receptor 13 . In addition, PTX
catalyzes the ADP ribosylation of certain G protein 
-subunits, and
treatment of intact cells with PTX results in uncoupling of
PTX-sensitive G proteins from cell surface receptors 26 . As shown in
Fig. 1A, when cells were pretreated with PTX, eosinophil
superoxide production stimulated with PAF was abolished, suggesting
that eosinophils’ PAF receptor is coupled to PTX-sensitive G
protein(s). Surprisingly, as shown in Fig. 1, B and
C, superoxide production induced by IgG immobilized onto
tissue culture plates or soluble IL-5 was markedly inhibited by PTX
pretreatment. In contrast, superoxide production induced by PMA (Fig. 2D) was not affected by
pretreatment of cells with PTX. The receptors for IgG and IL-5 belong
to the families of Fc
R (reviewed in 27 and hemopoietin
receptor (reviewed in 28 , respectively; there is no evidence for
coupling of these receptors to PTX-sensitive G proteins. Furthermore,
they are structurally distinct from typical G protein-coupled
seven-transmembrane receptors. Therefore, in eosinophils, the
superoxide production stimulated by IgG or IL-5 is most likely provoked
by generation of intermediate metabolite(s) active on G proteins,
rather than as a direct consequence of FcR or IL-5R
perturbation.
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Then, what is the intermediate metabolite(s) that stimulates
eosinophil functions in a G protein-dependent manner? Eosinophils are
able to generate several lipid mediators, such as PAF and
LTC4, in response to physiologic and pharmacologic stimuli
3, 12 . Eosinophils also contain fourfold higher levels of ether
phospholipid, the stored precursor of PAF, than do neutrophils,
suggesting that the eosinophil is a good PAF producer 29 . As shown in
Fig. 2
, we found that PAF was detectable in eosinophil supernatants 15
min after stimulation with immobilized IgG or soluble PMA; the amounts
of released PAF increased dramatically by 45 min. Smaller but
significant amounts of PAF (530.5 ± 127.8 pg PAF/106
cells) were also detectable in supernatants from eosinophils stimulated
with soluble IL-5 for 45 min (p < 0.05
compared with no stimulus, n = 4). The kinetics and
amounts of PAF released by eosinophils incubated with IgG immobilized
onto the tissue culture plates (2908.8 ± 428.3 pg
PAF/106 cells) were comparable with those observed
previously by eosinophils incubated with IgG immobilized to Sepharose
beads 12 . Eosinophils incubated in medium alone released minimal
levels of PAF at both times. Thus, when stimulated with immobilized IgG
or soluble IL-5, eosinophils produce and release PAF in less than 45
min.
Effects of PAF antagonists on eosinophil effector functions
If PAF produced by activated eosinophils played major roles in
effector functions of eosinophils themselves, then prevention of PAF
binding to its receptor would dampen eosinophil effector functions. To
examine this hypothesis, we used a potent and highly selective
phospholipid analogue antagonist of PAF, CV6209; CV6209 is a
competitive antagonist of PAF receptor 30 . In our preliminary study,
titration of CV6209 in an eosinophil superoxide production assay using
a series of concentrations of PAF as stimuli showed that 50%
inhibiting concentrations (IC50) of CV6209 for 0.1, 0.3, 1,
and 3 µM of PAF were 0.08, 0.23, 0.50, and 0.95 µM, respectively.
As shown in Fig. 3A,
CV6209 inhibited eosinophil superoxide production stimulated with 0.3
µM exogenous PAF in a concentration-dependent manner.
Interestingly, as shown in Fig. 3, B and C,
eosinophil superoxide production induced by immobilized IgG or soluble
IL-5 was also inhibited by CV6209, while no exogenous PAF was added to
the system. In contrast, superoxide production by eosinophils
stimulated with PMA (Fig. 3D) was not inhibited by CV6209,
with the exception of a slight change in kinetics.
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. Stimulation of eosinophils with PAF,
immobilized IgG, IL-5, or PMA consistently induced substantial amounts
of superoxide production. Unstimulated eosinophils did not produce
detectable levels of superoxide (<0.04 nmol/105 cells).
When a PAF receptor antagonist, CV6209, was added to the incubation,
the eosinophil responses to immobilized IgG and IL-5 as well as those
to exogenous PAF were inhibited in a concentration-dependent manner;
IC50 values for immobilized IgG, IL-5, or PAF were 0.8,
0.3, and 0.3 µM, respectively. Notably, CV6209 at 1 µM abolished
the eosinophil response to IL-5 and significantly inhibited the
response to immobilized IgG by 62% (p < 0.01,
n = 5). In contrast, superoxide production induced by
PMA was not affected by CV6209 at concentrations up to 1 µM. CV6209
showed no effect on eosinophil superoxide production in the absence of
any stimuli (data not shown). Thus, eosinophil superoxide production
induced by immobilized IgG, soluble IL-5, and exogenous PAF is
sensitive to the PAF receptor antagonist, suggesting that endogenous
PAF is involved in eosinophil responses to immobilized IgG or soluble
IL-5.
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, both hexanolamine PAF
and Y-24180 inhibited IL-5-induced superoxide production in a
concentration-dependent manner; 30 µM hexanolamine PAF and 10 µM
Y-24180 completely inhibited the eosinophil response to IL-5.
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, both
immobilized IgG and soluble IL-5 induced eosinophil EDN release.
CV6209, at 1 µM, significantly inhibited degranulation of eosinophils
stimulated with immobilized IgG and IL-5 by 49%
(p < 0.0001, n = 6) and 35%
(p < 0.05, n = 6),
respectively. In the absence of stimuli, CV6209 showed no effect on
spontaneous release of EDN (Fig. 6), and it did not inhibit PMA-induced
degranulation of eosinophils (data not shown). All in all, these
findings indicate that endogenous PAF is involved in effector functions
of eosinophils stimulated with immobilized IgG or soluble IL-5.
However, this role of endogenous PAF may be different among the
functions; for example, superoxide production is likely to be more
dependent on endogenous PAF than is degranulation.
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Recent studies suggest that intracellular lipid bodies are
distinct, inducible, nonnuclear sites for eicosanoid synthesis in
granulocytes 33, 34 . Lipid body formation in eosinophils is rapidly
induced by PAF stimulation, and PAF-induced lipid body production
correlates strongly with increased production of eicosanoids 33 .
Because endogenous PAF was involved in eosinophil functions stimulated
with IL-5 or immobilized IgG, we examined whether these agonists may
also stimulate lipid body formation and eicosanoid generation in
eosinophils. As shown in Fig. 7A, immobilized IgG and
soluble IL-5 increased the numbers of intracellular lipid bodies within
1 h, similarly to the effect of exogenous PAF. Two and one-half
times more lipid bodies were found in eosinophils stimulated with
immobilized IgG, IL-5, or PAF, than in unstimulated eosinophils.
Increased numbers of lipid bodies were also accompanied by increased
production of eicosanoids, as shown by LTC4 release into
the supernatants of activated eosinophils (Table I). As shown in Fig. 7, A and
B, the PAF antagonist, CV6209, blocked lipid body formation
and LTC4 production induced by exogenous PAF. Similarly,
CV6209, in concentrations as low as 0.3 µM, significantly inhibited
lipid body formation and LTC4 production by eosinophils
stimulated with IL-5 or immobilized IgG (p <
0.05, n = 3 and 5, respectively). CV6209, at 1 µM,
inhibited lipid body formation by more than 70% and LTC4
production by more than 75%; CV6209 did not affect the number of lipid
bodies in eosinophils incubated with medium alone. Thus, endogenous
PAF, similarly to exogenous PAF, plays major roles in eicosanoid
metabolism of eosinophils.
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To confirm the involvement of the endogenous PAF pathway in
eosinophil activation, we examined the effects of the blockade of PAF
generation on eosinophil function. Phospholipase A2
(PLA2), the enzyme that catalyzes glycerol phospholipid to
yield lysophosphatide and arachidonic acid, is one of the key enzymes
involved in PAF generation (reviewed in 35 . Mepacrine
competitively inhibits PLA2 activity by forming a stable
complex of drug and phospholipid substrate 36 . As expected from its
drug action, 100 µM mepacrine inhibited PAF release from eosinophils
stimulated with IL-5 or immobilized IgG (Table II). As shown in Fig. 8, mepacrine also significantly inhibited
eosinophil superoxide production induced by IL-5 or immobilized IgG
(p < 0.05 and p < 0.01 at 30
and 100 µM, respectively, n = 4). In contrast,
PMA-induced superoxide production was not affected by up to 100 µM
mepacrine. Thus, PLA2 is most likely involved in PAF
generation by eosinophils stimulated with immobilized IgG or soluble
IL-5, and blockade of this enzyme inhibits superoxide production
response to these stimuli.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R and
IL-5R are not coupled to G proteins, eosinophil functional responses to
IgG or IL-5 were inhibited by PTX, suggesting that they are mediated by
intermediate metabolite(s) active on PTX-sensitive G proteins, rather
than being a direct consequence of FcR or IL-5R ligation. 2) PAF
itself was detected in the supernatants of activated eosinophils. 3)
Blockade of PAF interaction with its receptor by three PAF receptor
antagonists markedly inhibited superoxide production, degranulation,
lipid body formation, and LTC4 release by eosinophils
stimulated with IL-5 or immobilized IgG. 4) The inhibitory effects of
PAF antagonists are unlikely due to nonspecific cytotoxicity because
the IC50 and the slope of concentration-response curves of
CV6209 for IL-5 or immobilized IgG were comparable with that for
exogenous PAF (Fig. 4), and because each of three structurally distinct
PAF antagonists effectively inhibited IL-5 response (Figs. 4 and 5). In
addition, the eosinophil responses to PMA were not inhibited by these
antagonists, indicating that they do not inhibit the enzymes for
cellular functions directly. 5) Finally, inhibition of PAF generation
by a PLA2 inhibitor attenuated eosinophil functional
responses to IL-5 or IgG.
Our findings are compatible with a previous report, which demonstrated
an important role of PLA2 in eosinophil functions
stimulated by FMLP plus cytochalasin B 37 . While these
investigators did not examine how PLA2 is involved in
eosinophil functions, our study suggests that PLA2 was
involved in the previous study by inducing the production of endogenous
PAF. A number of agonists are known to induce production of PAF by
eosinophils. For example, chemotactic factors, such as FMLP and
C5a, stimulate rapid (1–3 min) release of PAF from eosinophils 3 . In
addition, IgG-coated Sepharose beads stimulate eosinophils to
produce PAF 12 . We also found that eosinophils incubated with soluble
IL-5 produced PAF (Fig. 2). Furthermore, PAF provokes various
effector functions of eosinophils, including adhesion,
degranulation, and generation of superoxide anion and arachidonic
acid metabolites 8, 17, 18, 19 . Altogether, the results suggest that
endogenous PAF may play a role in inflammatory responses of eosinophils
by acting as a central switch, which bridges activation signals from
various cell surface receptors with various effector cell functions.
The disruption of the endogenous PAF pathway, either by inhibiting
production or by preventing binding to a specific receptor, may
profoundly impact eosinophil responses to a variety of stimuli.
The remaining question is how endogenous PAF stimulates effector
functions of eosinophils. As shown in Fig. 2, we were able to detect
PAF released extracellularly in the supernatants of eosinophils.
Therefore, PAF released into the outside medium could stimulate
eosinophils in a paracrine manner. However, a caveat for this
speculation is whether the concentration of PAF in the extracellular
space is high enough to induce eosinophil effector functions. For
example, if released PAF is uniformly distributed in the incubation
medium, we estimated 5 nM PAF in the eosinophil supernatants stimulated
with immobilized IgG. This concentration may not be sufficient to fully
activate eosinophils; it usually takes 100 nM or higher concentrations
of exogenous PAF to stimulate eosinophil functions 22 . However, much
higher concentrations can be expected in the microenvironment in close
proximity to the cells. Therefore, another potential mechanism is that
PAF generated by eosinophils may remain within and activate the
cells internally, similar to intracellular second messengers. Indeed,
in eosinophils stimulated with IgG-coated Sepharose beads, the kinetic
study showed that intracellular concentrations of PAF increased quickly
after the stimulation, reached a plateau by 15 min, and stayed at the
similar levels for at least 60 min 12 . In addition, Ojima-Uchiyama et
al. reported that more intracellular PAF remains than is released
extracellularly 38 . Furthermore, the presence of intracellular PAF
receptors has been suggested in nerve cells and granulocytes 39, 40, 41 .
Because intracellular PAF can be used immediately without involvement
of secretory processes or without potential exposure to extracellular
catalytic enzymes, this PAF hypothesis provides an economical cellular
strategy. Therefore, the traditional plasma membrane receptors may be
primarily responsible for mediating paracrine effects of PAF, while
intracellular receptors may mediate the autocrine effects of PAF.
Close examination of the kinetics of superoxide production provides
some insights regarding the roles of endogenous PAF in eosinophil
functions. As shown in Fig. 3B, at early time points (less
than 30 min), IgG-induced superoxide production was minimally affected
by pretreatment of cells with a PAF receptor antagonist, CV6209.
However, the inhibitory effect of CV6209 was pronounced when
eosinophils were producing superoxide at later time points (i.e., 60
min). In contrast, the effects of exogenous PAF were completely
inhibited by 1 µM CV6209 from the beginning of the cellular response
(Fig. 3A). The effect of PTX on PAF- and IgG-induced
superoxide production also showed similar findings (Fig. 1, A and B). These delayed onsets for the effects of
a PAF receptor antagonist or PTX suggest that, at early time points,
small amounts of superoxide may be generated by eosinophils through
alternative mechanisms (e.g., signals from IgG Fc receptors) without
the effects of endogenous PAF. Thus, at later time points, the
dependency on endogenous PAF may become greater when cells are
vigorously producing superoxide. Therefore, the role of endogenous PAF
may be to enhance the otherwise small responses of eosinophils
triggered by cytokines or Ig, and to expand to full bloom the effector
function of eosinophils.
Eosinophil activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of allergic diseases, such as bronchial asthma. Our study suggests that endogenous PAF plays an important role in mediator release by eosinophils. Furthermore, in addition to its autocrine effects, exogenous PAF exerts various biological activities relevant to bronchial asthma, such as airway constriction, development of bronchial hyperresponsiveness, and induction of microvascular leakage and edema (reviewed in Refs. 14 and 15). Indeed, severe asthma has been positively correlated with a deficiency of PAF acetylhydrolase in a Japanese population 42 . Despite the evidence for potential roles of PAF in bronchial asthma, previous studies with PAF receptor antagonists failed to show beneficial effects in patients with asthma 43, 44 . However, studies using newly developed and more potent PAF receptor antagonists have shown the effects of these antagonists on bronchial asthma. For example, Y-24180, which is also used in this study, inhibited allergen-induced eosinophilia and physiologic changes in guinea pig lungs 45 , and prevented bronchial hyperresponsiveness in patients with asthma 46 . Therefore, further studies are needed to elucidate the roles of PAF in bronchial asthma and other allergic diseases. Especially, as shown in this study, PAF antagonists inhibited eosinophil functions induced by a range of physiologic agonists. This wide spectrum of PAF antagonist effects may be advantageous from a therapeutic point of view because a number of eosinophil-active mediators produced by various immunologic or interstitial cells are likely to be involved in the pathophysiology of allergic diseases. The inhibition of only one mediator may be insufficient to inhibit eosinophil functions in such circumstances. Our study suggests that eosinophil activation and functions can be controlled effectively by managing the endogenous PAF pathways, and this concept may open a new avenue for the treatment of patients with allergic or eosinophilic disorders.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hirohito Kita, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail address:
3 Abbreviations used in this paper: LT, leukotriene; EDN, eosinophil-derived neurotoxin; HSA, human serum albumin; IC50, 50% inhibiting concentrations; PAF, platelet-activating factor; PLA2, phospholipase A2; PTX, pertussis toxin.
Received for publication August 14, 1998. Accepted for publication November 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor II and complement receptor type 3 during activation of platelet-activating factor release by cytokine-primed human eosinophils. J. Immunol. 153:2729.[Abstract]
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 N. Tedla, C. Bandeira-Melo, P. Tassinari, D. E. Sloane, M. Samplaski, D. Cosman, L. Borges, P. F. Weller, and J. P. Arm Activation of human eosinophils through leukocyte immunoglobulin-like receptor 7 PNAS, February 4, 2003; 100(3): 1174 - 1179. [Abstract] [Full Text] [PDF]
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P. Pacheco, F. A. Bozza, R. N. Gomes, M. Bozza, P. F. Weller, H. C. Castro-Faria-Neto, and P. T. Bozza Lipopolysaccharide-Induced Leukocyte Lipid Body Formation In Vivo: Innate Immunity Elicited Intracellular Loci Involved in Eicosanoid Metabolism J. Immunol., December 1, 2002; 169(11): 6498 - 6506. [Abstract] [Full Text] [PDF]
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M. Kato, H. Kimura, Y. Motegi, A. Tachibana, H. Minakami, A. Morikawa, and H. Kita Platelet-Activating Factor Activates Two Distinct Effector Pathways in Human Eosinophils J. Immunol., November 1, 2002; 169(9): 5252 - 5259. [Abstract] [Full Text] [PDF]
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 C. Bandeira-Melo, L. J. Woods, M. Phoofolo, and P. F. Weller Intracrine Cysteinyl Leukotriene Receptor-mediated Signaling of Eosinophil Vesicular Transport-mediated Interleukin-4 Secretion J. Exp. Med., September 16, 2002; 196(6): 841 - 850. [Abstract] [Full Text] [PDF]
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L. L. Thomas, W. Xu, and T. T. Ardon Immobilized Lactoferrin Is a Stimulus for Eosinophil Activation J. Immunol., July 15, 2002; 169(2): 993 - 999. [Abstract] [Full Text] [PDF]
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C. Bandeira-Melo, K. Sugiyama, L. J. Woods, M. Phoofolo, D. M. Center, W. W. Cruikshank, and P. F. Weller IL-16 Promotes Leukotriene C4 and IL-4 Release from Human Eosinophils via CD4- and Autocrine CCR3-Chemokine-Mediated Signaling J. Immunol., May 1, 2002; 168(9): 4756 - 4763. [Abstract] [Full Text] [PDF]
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T. Kobayashi, T. Miura, T. Haba, M. Sato, I. Serizawa, H. Nagai, and K. Ishizaka An Essential Role of Mast Cells in the Development of Airway Hyperresponsiveness in a Murine Asthma Model J. Immunol., April 1, 2000; 164(7): 3855 - 3861. [Abstract] [Full Text] [PDF]
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X. Zhu, N. M. Munoz, K. P. Kim, H. Sano, W. Cho, and A. R. Leff Cytosolic Phospholipase A2 Activation Is Essential for {beta}1 and {beta}2 Integrin-Dependent Adhesion of Human Eosinophils J. Immunol., September 15, 1999; 163(6): 3423 - 3429. [Abstract] [Full Text] [PDF]
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 C. Bandeira-Melo, M. Phoofolo, and P. F. Weller Extranuclear Lipid Bodies, Elicited by CCR3-mediated Signaling Pathways, Are the Sites of Chemokine-enhanced Leukotriene C4 Production in Eosinophils and Basophils J. Biol. Chem., June 15, 2001; 276(25): 22779 - 22787. [Abstract] [Full Text] [PDF]
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 P. J. Kingham, W. G. McLean, D. A. Sawatzky, M. T. Walsh, and R. W. Costello Adhesion-dependent interactions between eosinophils and cholinergic nerves Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1229 - L1238. [Abstract] [Full Text] [PDF]
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,
Significant differences (p < 0.05) from values
obtained in the absence of stimuli; *, significant differences
(p < 0.05) from values obtained with eosinophils
incubated without CV6209 in the same stimulus group. B,
The amounts of LTC4 released into the supernatants were
measured by ELISA. Data are normalized to the values in the absence of
CV6029 as 100% and presented as means ± SEM from five
independent experiments. * and **, Significant differences (*,
p < 0.05; **, p < 0.01)
from values obtained with eosinophils incubated without CV6209.