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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous investigations have suggested that protein kinase C (PKC) may regulate guinea pig eosinophil responses through a suppressive "negative feedback" mechanism. Using the selective PKC inhibitors bisindolylmaleimide I (Bis I, GF 109203X) and calphostin C, we examined the role of PKC in platelet-activating
factor (PAF)-induced respiratory burst and generation of arachidonic acid metabolites in human peripheral
blood eosinophils. Bis I inhibited PAF-induced generation of superoxide anion with substantially lower
potency (geometric mean IC50 = 1.41 µM, 95% CI 0.94 -2.11 µM) than it exhibited against responses to
the phorbol esters 4-
-phorbol 12-myristate 13-acetate (PMA; IC50 = 0.25 µM, 0.09-0.72 µM; P < 0.01)
and 4--phorbol 12,13-dibutyrate (IC50 = 0.48 µM, 0.20-1.14 µM; P < 0.05). The production of thromboxane (measured as TxB2) induced by 1 µM PAF was increased significantly by Bis I at concentrations of 1 µM (162 ± 7.5% of control PAF response; P < 0.01) and 10 µM (194 ± 17%; P < 0.001); TxB2 release induced by PMA was unaffected by concentrations of Bis I up to 1 µM and inhibited by 10 µM Bis I
(48 ± 11%; P < 0.05). Bis I (1 µM) significantly increased both thromboxane and leukotriene C4 (LTC4)
production induced by 2 µM (P < 0.01 and P < 0.05, respectively) or 20 µM PAF (both P < 0.001). The
actions of Bis I on PAF-stimulated thromboxane and leukotriene production were mimicked by a second
PKC inhibitor, calphostin C, whereas the non-PKC-inhibitory analog, bisindolylmaleimide V, caused no
enhancement of TxB2 or LTC4 production. The increase in intracellular free calcium induced by 1 µM
PAF was heightened and prolonged in cells pre-treated with 1 µM Bis I or 1 µM calphostin C (peak increase, P < 0.05 for both drugs; level 60 s after addition of PAF, P < 0.001 and P < 0.05 for Bis I and
calphostin C, respectively; time to return to 50% of peak, P < 0.05 for Bis I). We conclude that PKC inhibition causes augmentation of thromboxane and LTC4 production in PAF-stimulated human eosinophils
despite suppressing respiratory burst activity, indicating that different signaling pathways predominate in
these two responses and that PKC mediates a suppression of an early stage in an alternative pathway of activation.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Eosinophils are the distinctive infiltrating cells in human airways in asthma. Eosinophil-derived basic proteins, lipid mediators, reactive oxygen species, and cytokines have been postulated as important factors in allergic and asthmatic reactions (1). The products of platelet-activating factor (PAF)-stimulated eosinophils have been demonstrated to induce contraction of airway smooth muscle and to damage respiratory epithelium in vitro (2, 3).
The signal transduction pathways through which eosinophil responses to inflammatory mediators are effected are incompletely understood, although the participation of phospholipase C, cytosolic free calcium ions, and protein kinase C (PKC) in the activation of guinea pig eosinophil respiratory burst by leukotriene B4 has been demonstrated (4). The role of PKC in guinea pig eosinophil activation has not been elucidated fully. Activation of PKC by phorbol esters decreases subsequent responses to PAF (5), suggesting a possible negative feedback action of PKC in regulating eosinophil activation. To date, no similar studies in human eosinophils have been published.
The objective of this study was to investigate the role of
PKC in the mediation or modulation of human eosinophil
responses to PAF. We used the selective inhibitor, bisindolylmaleimide I (Bis I, also known as GF 109203X or Gö
6850) (6, 7), to demonstrate the involvement of PKC in
eosinophil functions. Superoxide anion (O2
.) generation
was measured as a response for which phorbol esters are
potent stimuli, indicating a significant participation of PKC in mediating its activation. In addition, generation of
cyclooxygenase and 5-lipoxygenase metabolites of arachidonic acid were selected as responses for which phorbol
esters are weak stimuli (8). Finally, the PAF-induced increase in intracellular free Ca2+ concentration ([Ca2+]i)
was assessed as an indicator of early events in PAF signal transduction. Our data demonstrate that Bis I inhibits
PAF-induced O2. generation with significantly lower potency than it exhibits against phorbol ester-stimulated respiratory burst, whereas both Bis I and a second PKC inhibitor, calphostin C, enhance significantly the PAF-induced
production of thromboxane and sulfidopeptide leukotrienes in human eosinophils. Bis I and calphostin C cause an increase in PAF-induced Ca2+ mobilization, suggesting that
activation of PKC by PAF leads to an autoinhibition of
PAF responses through suppression of an early event in
signal transduction.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Eosinophil Isolation
Granulocytes were isolated from human peripheral blood
as described by Böyum (9). Blood was drawn by antecubital venepuncture from donors without asthma (20 male,
9 female) into 1/7 vol of citrate-acid-phosphate anticoagulant and mixed with 1/5 vol of dextran (6% [wt/vol] solution in normal saline). Samples were left to stand for 45 min at room temperature to allow sedimentation of erythrocytes. The leukocyte-rich supernatant was decanted and layered onto 10-ml cushions of Ficoll-Hypaque (1.077 g
ml1) in 50-ml conical polypropylene tubes and centrifuged at 400 × g for 40 min at 17-20°C. The granulocyte
pellet was washed twice in phosphate-buffered saline containing 5 mM EDTA, 0.1% NaN3, and 1% (wt/vol) bovine
serum albumin (PBS-BSA, pH 7.2) and resuspended at a
density of 109 cells ml1.
Eosinophils were purified from the granulocyte fraction
essentially as described by Hansel and coworkers (10).
The granulocyte suspension was mixed at a ratio of 3:2
with CD16 MACS microbeads and incubated at 4°C, with
intermittent mixing, for 30 min, after which the volume
was made up to 1 ml with PBS-BSA. The cell-bead mixture was loaded onto a steel fiber matrix column, prewashed with PBS-BSA, that was placed in a strong magnetic field. The column was washed at a low flow rate with
10 column volumes of cold PBS-BSA to elute CD16 cells
(predominantly eosinophils). The CD16 preparation was
washed twice with Hanks' balanced salt solution (HBSS)
and residual erythrocytes were removed by hypotonic lysis with ice-cold deionized water followed by restoration of
osmolarity with 10× HBSS. Total cell counts were made
from Turk-stained preparations in a hemocytometer. Differential leukocyte counts were made using Wright-stained
cytocentrifuge preparations. Cell preparations contained
96.1 ± 0.27% eosinophils (mean ± SEM, n = 73, preparations from 29 donors; contaminants mainly lymphocytes).
Eosinophil Function Assays
All experiments were performed in Hepes buffer consisting of: N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (Hepes), 20 mM; NaCl, 132 mM; KCl, 6 mM; KH2PO4, 1.2 mM; Mg2SO4, 1.0 mM; CaCl2, 1.0 mM; D-glucose, 5.5 mM; BSA, 0.25% (wt/vol); pH 7.4. Cells were washed and resuspended in fresh Hepes buffer 10-20 min before experimentation.
Respiratory burst measurement.
Superoxide anion generation was measured as the superoxide dismutase (SOD)-
inhibited reduction of ferricytochrome c. Eosinophils (2 × 105) were incubated in 225 µl of Hepes buffer containing
100 µM cytochrome c, with or without drugs at the indicated concentrations, at 37°C for 10 min. PAF, 4--phorbol
12-myristate 13-acetate (PMA), 4--phorbol 12,13-dibutyrate
(PDBu), or Hepes buffer was added to give a final volume
of 250 µl and the reaction mixtures were incubated for a
further 15 min at 37°C. Cells were precipitated by centrifugation (12,000 × g for 2 min) and the extinction of 200-µl
portions of the supernatants was measured at 550 nm in a
96-well microplate reader. Cytochrome c reduction was
calculated from the increase in extinction relative to a control sample to which SOD (30 U ml1) was added immediately before the stimulus. Results are expressed as
nanomoles of cytochrome c reduced per 106 cells in 15 min, based on a molar extinction coefficient for ferrocytochrome c of 21.1 × 103 M1 cm1.
Thromboxane release measurement.
PAF-stimulated release of thromboxane was found to be maximal at 5 min,
as described for guinea pig eosinophils (5). Eosinophils (5 × 105) were incubated in 100 µl of Hepes buffer, in the
presence or absence of drugs at the indicated concentrations, for 10 min at 37°C. PAF, PMA, or Hepes buffer was
added to give a final volume of 125 µl and the mixtures
were incubated for a further 5 min at 37°C. Reactions were
stopped by the addition of 125 µl of ice-cold Hepes buffer
containing 12 µM flurbiprofen (an irreversible inhibitor of
cyclooxygenase) and tubes were placed on ice. Cells were
precipitated by centrifugation (12,000 × g for 2 min) and
200-µl portions of supernatants were decanted and stored
at 
20°C until required for assay.
since basal leukotriene production was
close to the limit of detectionno 5-lipoxygenase inhibitor solution was added. Tubes were placed on ice for the
shortest time possible and reactions were stopped by rapid
centrifugation (12,000 × g for 2 min). Supernatants were decanted and stored at 20°C until required for assay.
Sulfidopeptide leukotrienes (LTC4/D4/E4) were assayed
in supernatants by competitive EIA. Because the cells were
almost exclusively eosinophils, all leukotriene measured
was assumed to be LTC4 (11). Data are expressed as femtomoles of LTC4 released per 106 cells in 5 min.
Intracellular calcium measurement.
Eosinophils were
loaded with the fluorescent Ca2+ indicator, Fura-2, as described by Raible and coworkers (12) or sham-loaded with
dimethyl sulfoxide (DMSO) under identical conditions.
Cells (2 × 106) were incubated in 2 ml of Hepes buffer, in
the absence or presence of 1 µM Bis I or calphostin C, for
10 min at 37°C in the thermostatted chamber of a spectrofluorophotometer (model LS50; Perkin-Elmer GmbH,
Überlingen, Germany). Measurement of [Ca2+]i was begun midway through this incubation period. PAF (1 µM) was added and [Ca2+]i monitoring was continued for a
further 10 min at 37°C. The [Ca2+]i measurement was
performed using the "fast ratio" technique: Ca2+-bound
Fura-2 fluorescence (
ex = 340 nm, em = 505 nm, slit
widths = 10 nm) was measured at 100-ms intervals and
ratioed with isobestic fluorescence values (ex = 360 nm,
em = 505 nm, slit widths = 10 nm) interpolated from
measurements made at the beginning and end of the monitoring period. Fmax was measured after lysis of the cells
with 50 µM digitonin; Fmin was measured after the chelation of free Ca2+ with 10 mM EDTA and 20 mM Tris base.
Eosinophil autofluorescence was measured using cells that
had been sham-loaded with DMSO (the solvent for Fura-2
acetoxymethyl ester). [Ca2+]i was calculated using the
Intracellular Biochemistry application (Perkin-Elmer).
Statistical Analysis
Unless otherwise stated, data are given as arithmetic mean ± SEM or geometric mean with 95% confidence interval (CI) from six experiments for each group.
All statistical analyses were performed using InStat® (GraphPadTM Software, San Diego, CA). Groups were compared by repeated-measures analysis of variance (ANOVA). Comparisons between untreated (control) cells and cells pretreated with various concentrations of drugs were performed using Dunnett's test for multiple comparisons. Other pairwise comparisons between groups were performed using the Newman-Keuls test for multiple comparisons. When data from different sets of experiments were compared, unpaired Student t tests were performed. A probability < 0.05 was defined as significant.
Materials
Bisindolylmaleimide I (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide), bisindolylmaleimide V [2,3-bis(1H-indol-3-yl)-N-methylmaleimide, Bis
V], calphostin C (UCN-1028c, from Cladosporium cladosporioides), and PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) were supplied by Calbiochem-Novabiochem GmbH (Bad Soden [Taunus], Germany). Dextran
(mean MW 110,000, from Leuconostoc ssp.) was purchased from Fluka Biochemika (Neu-Ulm, Germany).
Colloidal anti-Fc
RIII (CD16)-coated superparamagnetic
microbeads and magnetic separation columns (MACS system) were obtained from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). HBSS was purchased from
GIBCO-BRL (Eggenstein, Germany). TxB2 EIA kits were
obtained from Neogen Corporation (Lexington, KY).
"Biotrak" LTC4/D4/E4 EIA kits were supplied by Amersham-Buchler GmbH (Braunschweig, Germany). BSA (fraction V powder), catalase (from bovine liver), citrate-
acid-phosphate, cytochrome c (from horse heart), digitonin, EDTA (tetrasodium salt), Ficoll-Hypaque (Histopaque 1077), Fura-2 acetoxymethyl ester, Hepes, PDBu,
PMA, sodium azide (NaN3), and SOD (from bovine erythrocytes) were supplied by Sigma Chemie (Deisenhofen,
Germany). All other reagents were of analytical grade.
Bisindolylmaleimide I, Bis V, and calphostin C (10 mM
stock solutions), and PDBu, PMA, and Fura-2 acetoxymethyl ester (1 mM stock solutions), were dissolved in DMSO
and stored in aliquots at 20°C until required. Catalase
was dissolved at 4,000 U ml1 in Hepes buffer. Lyophilized
PAF was dissolved at 1 mM in deionized water 15-30 min
before use. All drugs were diluted to the desired concentration in Hepes buffer.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effects of Bisindolylmaleimide I on Eosinophil Functions
Respiratory burst. PAF induced a concentration-dependent stimulation of human eosinophil O2. generation
(Figure 1) with an EC50 of 0.39 µM (geometric mean, 95%
CI 0.26-0.61 µM). A 1-µM concentration of PAF was chosen as a submaximally effective stimulus for subsequent
experiments. For comparison, 1 fM PMA was used as a
stimulus acting through direct activation of PKC: this concentration elicited respiratory burst activity of similar
magnitude to 1 µM PAF (Figure 1). A second phorbol ester (PDBu), at an approximately equieffective concentration (0.5 nM), was also studied.
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. generation was also inhibited more potently than the response to PAF (IC50 = 0.48 µM, 95% CI 0.20-1.14 µM; P < 0.05 versus PAF).
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Effects of Bisindolylmaleimide V and Calphostin C on Eosinophil Functions
To confirm that the observed actions of Bis I were effected
through inhibition of PKC, two reference compounds were
investigated: Bis V, a structural analog of Bis I that does
not inhibit PKC; and calphostin C, a PKC inhibitor that
acts at a different site on the enzyme. Bis V had no significant effect on PAF- or PMA-induced generation of O2.
or PAF-induced eicosanoid production at concentrations
up to 1 µM. At 10 µM, Bis V did cause significant inhibition of PAF- and PMA-induced respiratory burst, but also
suppressed PAF-stimulated LTC4 production (Figure 6).
This did not reflect a reduction in cell viability, which was
unaffected by Bis V at the highest concentration (Table ).
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Calphostin C, at a concentration of 1 µM, exhibited actions similar to Bis I on PAF-induced TxB2 and LTC4 production (Figure 7).
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Influence of Catalase on the Effect of Bisindolylmaleimide I on LTC4 Production
To determine whether suppression of oxidant production
might contribute to the augmentation of eicosanoid generation by Bis I, the influence of catalase on PAF-induced
LTC4 accumulation and its sensitivity to enhancement by
Bis I were investigated. Catalase catabolizes the cell-derived
oxidant, hydrogen peroxide (H2O2), and therefore H2O2
and its derivatives can play no part in responses observed
in the presence of the enzyme. The inclusion of 400 U ml1
catalase led to a small, nonsignificant increase in PAF-stimulated LTC4 production. Under these conditions, Bis I
caused an augmentation of PAF-induced LTC4 similar in
magnitude to that observed in the absence of catalase
(Figure 8). Whereas 1 µM Bis I alone caused a mean 240%
increase in the response to 20 µM PAF, Bis I plus catalase
caused a mean 210% increase above responses observed in untreated cells and a mean 160% increase above those
in cells treated with catalase only.
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Effects of Bisindolylmaleimide I and Calphostin C on Eosinophil Calcium Flux
To ascertain whether PKC inhibitors were affecting early
events in PAF signal transduction, their action on PAF-
induced Ca2+ mobilization was investigated. As shown in
Figure 9, preincubation of eosinophils with 1 µM Bis I led
to a small increase in the elevation of intracellular-free calcium induced by 1 µM PAF. This increase was statistically
significant when expressed in terms of peak 
[Ca2+]i,
[Ca2+]i 60 s after addition of PAF, or time taken for
[Ca2+]i to return to 50% of its peak value (RT50). Similar
results were obtained with calphostin C, which increased
peak [Ca2+]i from 214 ± 3.63 nM to 248 ± 12.0 nM and
"plateau" [Ca2+]i from 53.1 ± 9.20 nM to 93.3 ± 2.00 nM
above baseline (both P < 0.05, n = 3) and RT50 from 16.8 ± 2.08 s to 22.4 ± 3.78 s (NS).
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The objective of this investigation was to identify the role of PKC in mediating or modulating responses of human eosinophils to the inflammatory mediator, PAF. The selective PKC inhibitor, bisindolylmaleimide I, was used to demonstrate the involvement of the enzyme in respiratory burst, eicosanoid production, and Ca2+ mobilization responses.
Eosinophil respiratory burst was potently stimulated by
phorbol esters, implying a role for PKC in the mediation
of this cell function. The response to PMA was inhibited
concentration-dependently by Bis I in the same range
demonstrated previously to inhibit PMA-induced O2.
production in murine peritoneal neutrophils (7), collagen-induced aggregation and ATP secretion of human platelets (6), and PKC-dependent protein phosphorylations in
human platelets and Swiss 3T3 fibroblasts stimulated by
-thrombin and bombesin, respectively (6). In contrast,
substantially higher concentrations were required to inhibit the response to PAF. This finding is similar to that
reported for the actions of a related PKC inhibitor, Ro 31-8220, on respiratory burst induced by PDBu and leukotriene B4 in guinea pig peritoneal eosinophils (4). PMA exhibited extraordinarily high potency in stimulating O2.
generation in the present study and it could not be assumed with confidence that this response was mediated
exclusively by PKC. For this reason, a second phorbol ester, PDBu, was also studied. The response to PDBu was
also more sensitive than the PAF response to inhibition by
Bis I. The differential potency exhibited by the PKC inhibitor suggested that the induction of respiratory burst by
PAF is only partially mediated by PKC, so that (1) inhibition of the enzyme would only partially suppress the response to PAF and (2) PKC activation may, in addition to
mediating the respiratory burst, act to suppress an alternative pathway of NADPH:O2 oxidoreductase (NADPH oxidase, the respiratory burst enzyme complex) activation. In the latter case, the inhibition of PKC would lead both to
suppression of one pathway of oxidase activation and to
amplification of a second pathway through removal of a
PKC-mediated "brake." Thus, the inhibitory action of a
PKC inhibitor on this cell function would be minimized.
To test this hypothesis, an eosinophil function was studied that is less sensitive to stimulation by phorbol esters. PMA has been reported previously to be a very weak stimulus for thromboxane production in guinea pig eosinophils (8) and this was also the case for human eosinophils in our study. In preliminary experiments, a very small but significant stimulation of thromboxane generation was observed with 1 nM PMA, although in the experiments reported here, this response did not attain statistical significance. These findings suggest that this function has little dependence on PKC for its activation. For this reason, we studied thromboxane production as an eosinophil function in which alternative signal transduction pathways are likely to exceed PKC in importance. We postulated that activation of PKC by PAF would lead predominantly to suppression of the pathways leading to thromboxane generation and, in consequence, that inhibition of PKC by Bis I would cause an enhancement of PAF-induced TxB2 production. We found that pretreatment of eosinophils with Bis I led to a substantial increase in TxB2 production induced by PAF, whereas the small response induced by PMA was unaffected by low concentrations of Bis I and decreased in the presence of the highest concentration studied. This effect was not specific to prostaglandin H synthase (cyclooxygenase) metabolites of arachidonic acid, because the 5-lipoxygenase-dependent production of LTC4 was similarly affected.
Bis I, in common with other protein kinase inhibitors such as staurosporine and H7, inhibits PKC through an interaction with the ATP-binding site of the enzyme (13). That the observed actions of the drug on eosinophil function were due to PKC inhibition was confirmed by the inability of Bis V, which does not inhibit the enzyme (14), to mimic the effects of Bis I. A high concentration of Bis V (10 µM) caused a general inhibition of eosinophil functions, which may indicate either an unidentified pharmacologic action of this compound or a nonspecific suppressive effect of the indolylmaleimide class. Neither Bis I nor Bis V exhibited any effect on cell viability, suggesting that toxicity of the compounds did not account for this general suppressive action, which remains unexplained. Further confirmation of the role of PKC was provided by the ability of calphostin C, which inhibits PKC at the regulatory (DAG/phorbol ester-binding) site (15), to mimic the action of Bis I on PAF-induced thromboxane and leukotriene production. Bis I may inhibit cyclic AMP-dependent protein kinase (PKA) at higher concentrations (IC50 against catalytic subunit = 2 µM) (6) and, because PKA inhibition enhances PAF-induced LTC4 synthesis in human eosinophils (16), this action might be suspected to contribute to the augmentation of this response by Bis I. Calphostin C, however, exhibits an IC50 > 50 µM against PKA (15) and the ability of this inhibitor, at a concentration of 1 µM, to mimic the Bis I enhancement of PAF-induced generation of TxB2 and LTC4 supports the conclusion that this action is effected through PKC inhibition.
The stage of cell activation by PAF at which PKC exerts its inhibitory action was not defined in these experiments. In guinea pig eosinophils stimulated with LTB4, a
very large increase in postpeak [Ca2+]i was observed when
cells were pretreated with 10 µM Ro 31-8220 (4). In our
study, the increase in [Ca2+]i induced by PAF was both
augmented and prolonged in human eosinophils pretreated
with 1 µM Bis I, and 1 µM calphostin C also increased peak [Ca2+]i and its level 60 s post-PAF, but these effects
were much less pronounced than that reported in LTB4-stimulated guinea pig cells. Although the ability of Bis I to
increase the PAF-induced [Ca2+]i elevation implies an action at early events in cell signaling, it is impossible to say
whether this accounts fully for the inhibitory action of the
PKC inhibitor on cell functions. The PKC isoenzyme(s) involved in PAF-induced eicosanoid generation in human
eosinophils have not been identified; a recent report has
demonstrated the presence in these cells of the conventional (cPKC) isoenzymes PKC, PKCI, and PKCII, and
the atypical (aPKC) isoenzyme PKC
(17). Of these, the
cPKCs are inhibited by such drugs as calphostin C and Bis Ithe latter with IC50 values between 16 and 20 nM (6) but the ability of these substances to inhibit aPKC isoenzymes has not been characterized (13). Moreover, the substrate whose phosphorylation by PKC accounts for the
suppression of PAF-induced eicosanoid generation remains to be identified. The PAF receptor has been suggested as a putative serine/threonine kinase substrate in
view of the presence in its cytoplasmic tail of multiple
serine and threonine residues (18). Because an early event
in PAF signal transduction, namely elevation of intracellular [Ca2+], can be shown to be enhanced by a PKC inhibitor, the PKC-mediated phosphorylation of the PAF receptor may be feasible, although a receptor-associated guanine
nucleotide-binding protein (G-protein) could also be a target. Phospholipase A2, which catalyzes the hydrolytic cleavage of arachidonic acid from membrane phospholipids, may also be a target because phosphorylation by PKC of
PLA2 has been demonstrated (19, 20). It remains unclear,
however, what role this phosphorylation may play in regulating the activity of the phospholipase (21).
Prior reports have implicated PKC in suppression of receptor-mediated cell activation. Responses to PAF in rat
Kupffer cells (22), human platelets (23) and neutrophils
(24, 25), as well as guinea pig eosinophils (5) are downregulated by treatment of the cells with the PKC activator,
PMA. Formyl peptide (fMLP)-induced arachidonic acid
mobilization in human neutrophils is enhanced in the presence of PKC inhibitors, which also increase the fMLP-stimulated generation of LTB4 and PAF while suppressing
the PMA-induced production of O2. and PAF (26). Treatment of guinea pig eosinophils with PMA leads to a downregulation of subsequent responses to PAF (5) and PMA
pretreatment also causes a staurosporine- and Bis I-inhibitable suppression of Ca2+ ionophore-stimulated LTC4 generation in an eosinophilic substrain (HL-60#7) of HL-60 promyelocytes (27). Interestingly, the effects of PKC inhibitors
vary with stimulus, response, and cell type. For example,
van der Bruggen and coworkers demonstrated that treatment of human eosinophils with various PKC inhibitors
leads to an enhancement of the oxygen consumption stimulated by opsonized zymosan while suppressing the response to PMA, but has no effect on PAF generation stimulated by the opsonized particles (28). Also, whereas LTC4 production induced by Ca2+ ionophore is suppressed by
PKC activation in HL-60#7 cells, the ionophore-stimulated production of TxB2 and PGE2 is enhanced (27).
It has been reported that eosinophil-derived LTC4 may be broken down by hypohalous ions produced from cell-derived hydrogen peroxide (H2O2) through the action of eosinophil peroxidase (11). Because it could be conceived that the increase in LTC4 generation observed in eosinophils treated with PKC inhibitors may simply reflect a suppression of oxidative breakdown owing to the reduced production of reactive oxygen species, we undertook experiments under conditions in which oxidants could not account for any changes in LTC4 production. L-Serine has been used as a scavenger of hypohalous ions and causes a significant increase in Ca2+ ionophore-induced LTC4 production by human eosinophils (11); in our hands, however, the amino acid caused a profound suppression of both basal and PAF-induced LTC4 generation (data not shown), the grounds for which have not been investigated. Catalase, which catalyzes the breakdown of H2O2 to oxygen and water, caused a small, nonsignificant increase in PAF-induced LTC4 production and, in the presence of catalase, Bis I continued to exhibit an augmentation of the leukotriene generation induced by PAF. These findings indicate that this action of the PKC inhibitor cannot result exclusively from the decreased oxidant accumulation, because oxidants had been eliminated by the inclusion of catalase. The magnitude of the augmentation produced by Bis I in the presence of catalase was similar to that produced in the absence of the enzyme, suggesting that the suppression of oxidant generation by Bis I contributes only slightly, if at all, to the enhancement of eicosanoid production.
Our study demonstrates for the first time that PAF- induced eicosanoid generation in human eosinophils is augmented by PKC inhibition and that PAF-induced respiratory burst is suppressed with lower potency than the response to phorbol esters by a selective PKC inhibitor, Bis I. These findings suggest that PKC mediates a "negative feedback" action on other signal transduction pathway(s) in PAF-stimulated eosinophils and that different pathways predominate in the mediation of respiratory burst and eicosanoid generation responses. The enhancement of PAF-induced [Ca2+]i mobilization by Bis I and calphostin C may indicate that PKC activation leads to suppression of an early event in cell signaling.
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Footnotes |
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Address correspondence to: Gordon Dent, Ph.D., Krankenhaus Grosshansdorf, Wöhrendamm 80, D-22927 Grosshansdorf, Germany. E-mail: Gordon_Dent{at}compuserve.com
(Received in original form October 22, 1996 and in revised form May 12, 1997).
Acknowledgments: This work was supported by Grant No. 4001-01 KE 9301 from the Federal Research and Technology Ministry (BMFT), Germany, and Grant No. HL-46368 from the Division of Lung Disease, National Heart, Lung, and Blood Institute, Bethesda, Maryland.
The authors thank Dr. R. A. Coleman (Pharmagene Laboratories Ltd., Royston, Hertfordshire, UK) for his critical reading of the manuscript.
Abbreviations
Bis I, bisindolylmaleimide I (GF 109203X);
Bis V, bisindolylmaleimide V;
LTC4, leukotriene C4;
PAF, platelet-activating factor;
PDBu, 4--phorbol 12,13-dibutyrate;
PKC, protein kinase C;
PLA2, phospholipase
A2;
PMA, 4--phorbol 12-myristate 13-acetate.
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References |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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1. Hamann, K. J. 1996. Inflammatory cells in airways. In Pulmonary and Critical Care Pharmacology and Therapeutics. A. R. Leff, editor. McGraw-Hill, New York. 355-369.
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Rabe, K. F.,
N. M. Muñoz,
A. J. Vita,
B. E. Morton,
H. Magnussen, and
A. R. Leff.
1994.
Contraction of human bronchial smooth muscle caused by activated human eosinophils.
Am. J. Physiol.
267:
L326-L334
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