Thoracic Medicine, National Heart and Lung Institute at the Imperial College School of Medicine, London SW3 6LY, United Kingdom
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ABSTRACT |
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Protein kinase
(PK) C is an increasingly diverse family of enzymes that has been
implicated in a range of cellular functions within the eosinophil.
Using isoform-specific polyclonal antibodies, we have explored the
expression of PKC isoforms in circulating eosinophils. Initial studies
demonstrated the presence of the ,
I,
II, and
and the low-level expression of the
,
,
, and µ isoforms but
no detectable expression of the
,
, and
isoforms in both
normal and asthmatic subjects. There was no difference in the total
protein expression between these two groups. Subsequent studies
examined the expression and activation of PKC isoforms in circulating
eosinophils from asthmatic patients before and 24 h after a late
asthmatic response to an inhaled allergen. Cellular fractionation
showed PKC-
and PKC-
II to be mainly located in the cytosol,
whereas PKC-
I was constitutively more expressed in the membrane. No
changes in expression or subcellular localization of these isoforms
were seen after allergen challenge. In contrast, PKC-
expression was
increased after allergen challenge, and we demonstrated a significant
PKC-
translocation to the membrane, in keeping with activation of
the enzyme. Our results suggest that 24 h after allergen exposure of
asthmatic patients, there is increased expression and activation of
eosinophil PKC-
that correlates with late asthmatic responses
recorded between 4 and 10 h postallergen challenge.
asthma; priming; leukocyte
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INTRODUCTION |
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THE EOSINOPHIL IS A KEY EFFECTOR CELL in the pathogenesis of allergic disease (13). Eosinophils contribute to tissue injury and inflammation after the generation of a number of toxic products in response to proinflammatory mediators. These include basic granule proteins such as eosinophil cationic protein and major basic protein and reactive oxygen species such as superoxide. Sputum and blood eosinophilia are recognized features of asthma, and eosinophilic proteins have been detected within the sputum and bronchoalveolar lavage (BAL) fluid of asthmatic patients (1, 5, 9). Furthermore, induction of an inflammatory response in the airway by allergen challenge is known to cause eosinophilia as well as the activation of both blood and BAL fluid eosinophils (16, 32). However, despite the strong association between eosinophil activation and airway pathology, little is known about the signal transduction mechanisms that link extracellular proinflammatory signals with cell activation.
Protein phosphorylation catalyzed by kinases is thought to play a
central role in the mechanisms of signal transduction.
Posttranslational modification of proteins by phosphorylation can
activate or inhibit enzyme pathways and, in turn, modify cell function.
Protein kinase (PK) C is an increasingly diverse family of enzymes that
are believed to be important to signal transduction in multiple cell
systems (24, 25) including eosinophils. Studies using the phorbol ester
phorbol 12-myristate 13-acetate, an activator of PKC, suggests a role
for this enzyme in the mechanism of eosinophil cell adhesion (8),
degranulation (17), and NADPH oxidase activation (33). To date, eleven
isoforms of PKC (,
I,
II,
,
,
,
,
,
, µ,
and
) have been identified. On the basis of molecular structure and
biochemical properties, the PKC family can been divided into three
groups. The conventional PKCs (cPKCs;
,
I,
II, and
isoforms) are Ca2+ and
phospholipid dependent. The novel PKCs (nPKCs;
,
,
, and
isoforms) lack the Ca2+-binding
region and are therefore Ca2+
independent, whereas the third group, atypical PKCs (aPKCs;
, µ,
and
isoforms) lack both the
Ca2+- and diacylglycerol- or
phorbol ester-binding sites (24, 25). At present, the biological
significance of this heterogeneity as well as the function of the
individual isoenzymes is largely unknown.
Using Western blot analysis, Bates et al. (2) have previously
demonstrated the presence of the isoform of PKC within human blood
and BAL fluid eosinophils. This study also found that the
Ca2+- and phospholipid-dependent
PKC activity (i.e., cPKCs) was increased in low-density eosinophils.
Because a decrease in the sedimentation density has been observed to
correlate with increases in a number of functional activities, it was
suggested that members of the PKC family may be important in the
mechanism of eosinophil activation (2). To extend this earlier
study, we used Western blot analysis to identify the
expression and subcellular distribution (cytosolic and membrane
fractions) of eleven PKC isoforms (
,
I,
II,
,
,
,
, µ,
,
, and
) in eosinophils. Furthermore, we have examined a possible role for PKCs during in vivo eosinophil activation by investigating the effect of allergen challenge on PKC expression and
activation in the circulating eosinophils of asthmatic patients who
demonstrated a late asthmatic response (LAR). Evans et al. (12) have previously demonstrated that the LAR correlates
with the activation of circulating eosinophils obtained 24 h after allergen challenge, resulting in the priming of NADPH oxidase activation and increased survival.
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METHODS |
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Study protocol. We studied 14 steroid-naive asthmatic patients and 5 nonasthmatic control subjects. Each of the asthmatic patients underwent allergen challenge, and all showed an LAR. Levels of PKC isoform expression in eosinophils were examined in the normal subjects and asthmatic patients (n = 8) both before and after allergen challenge. In six other subjects, the subcellular distribution of PKC isoforms was determined. Eosinophils were purified from peripheral blood immediately before and 24 h after allergen challenge.
Subjects and baseline lung function.
All asthmatic patients demonstrated clinical features of asthma and had
a provocative concentration of histamine causing a 20% fall in forced
expiratory volume in 1 s (FEV1)
of <4 mg/ml. The mean baseline percent predicted FEV1 was 94.3 ± 1.4% (range
87-103%). All subjects had positive skin prick tests (>6 mm) to
at least one of a number of common aeroallergens. None of the subjects
had previously taken inhaled or oral glucocorticosteroids, and the only
medication used by any of the group was albuterol on an "as
required" basis (Table 1).
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Allergen challenge. The subjects were admitted to the Royal Brompton Hospital (London, UK) Clinical Studies Unit for a 24-h period. On arrival, the subjects underwent clinical assessment before venesection. Allergen inhalation tests were performed with a nebulizer attached to a breath-activated dosimeter (dosimeter MB3, MEFAR Electromedical, Bovezzo, Italy). The nebulizer delivered particles with an aerodynamic mass median diameter of 3.5-4 µm at an output of 9 µl/breath. The nebulizer was set to nebulize for 1 s, with a pause time of 6 s, at a pressure of 22 psi. Freeze-dried allergen extracts (Aquagen SQ, Allergologisk Laboratium, Horsholm, Denmark) were used. Known dilutions of the allergen were made to give final concentrations of 200, 1,000, 2,500, 5,000, 12,500, 25,000, and 50,000 IU/ml. The initial dose for the allergen inhalation test was 200 IU/ml, and FEV1 was measured 5 and 10 min after each allergen dose. Serially increasing doses of allergen were inhaled, and the cumulative dosage resulting in a 15% reduction within 10 min was recorded and constituted an adequate challenge. The FEV1 was recorded every 15 min for the first hour and hourly thereafter. The LAR was defined as a fall in FEV1 of 15% from the postsaline FEV1 between 4 and 10 h. Twenty-four hours after allergen challenge, the subjects were venesected for a second time.
Chemicals and materials. Rabbit
polyclonal antibodies to the PKC isoforms were obtained from Santa Cruz
Biotechnology (Autogen Bioclear UK, Wilts, UK). Recombinant PKC was
obtained from Calbiochem (Nottingham, UK), whereas donkey anti-rabbit
horseradish peroxidase-linked IgG, rainbow molecular-weight markers,
enhanced chemiluminescence (ECL) Western blotting detection agents, and
[-32P]ATP were
purchased from Amersham Life Sciences (Little Chalfont, UK).
Polyacrylamide gels (10% Mini-PROTEAN II ready gels) were purchased
from Bio-Rad Laboratories (Hemel Hempstead, UK). Percoll was supplied
by Pharmacia Biotech (St. Albans, UK), and the CD16 immunomagnetic
beads were supplied by Eurogenetics (Teddington, UK). Orthophosphoric
acid was purchased from British Drug House (Poole, UK), and
phosphocellulose was from Whatman (Maidstone, UK). Penicillin,
streptomycin, and RPMI 1640 medium were supplied by GIBCO BRL (Paisley,
UK), and interleukin (IL)-5 was supplied by R&D Systems (Abingdon, UK).
All other chemicals were obtained from Sigma (Poole, UK). Kodak
X-OMAT-S film was supplied by Kodak-Pathe.
Eosinophil preparation. Eosinophils were separated with the method of Hansel et al. (15). Briefly, venous blood (50 ml) was collected into 10 ml of acid citrate-dextrose anticoagulant. The blood was diluted 1:1 with Hank's balanced salt solution (HBSS), layered onto 1.082 g/l of Percoll, and centrifuged at 1,300 g for 25 min at 22°C. After centrifugation, the mononuclear cell layer was discarded, and the pellet containing the granulocytes and red blood cells was washed in HBSS. Contaminating red blood cells were lysed by hypotonic lysis. The granulocyte fraction was washed, counted, and then resuspended in 250 µl of HBSS containing 2% fetal calf serum (FCS) and 5 mM EDTA (HBSS-FCS-EDTA). The eosinophils were purified from the neutrophils with immunomagnetic anti-CD16 antibody-conjugated beads (1 µl beads/5 × 105 neutrophils). After addition of the beads, the cells were incubated at 4°C for 30 min before being resuspended in 10 ml of HBSS-FCS-EDTA. The mixture was loaded onto a separation column positioned within a magnetic field and eluted with 30 ml of HBSS-FCS-EDTA. The CD16+ cells (i.e., neutrophils) were retained on the column while the eluted eosinophils were collected, washed in HBSS, counted, and then resuspended at 107 cells/ml. Eosinophil purity was >99.9% as assessed with microscopic examination with Kimura stain.
Subcellular fractionation. Cytosolic
and membrane fractions were prepared by ultracentrifugation. The
eosinophils were resuspended at 2 × 107/ml in ice-cold lysing buffer
(50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM dithiothreitol, 4 mM EGTA, 4 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 100 µM leupeptin, 10 µg/ml of soybean trypsin inhibitor, and 100 µg/ml of bacitracin) and then lysed by sonication (4 × 5 s).
Fresh rat brain and skeletal muscle tissues were chopped, frozen in
liquid N2, and then homogenized in
lysis buffer. All samples were then centrifuged at 100,000 g for 60 min. The supernatant
(cytosolic fraction) was removed, and the pellet (membrane fraction)
was resuspended in an equal volume of lysis buffer by sonication (3 × 10 s). Both fractions were boiled in Laemmli buffer (15)
to give an equivalent final concentration of
107 cells/ml and stored at
20°C for Western blotting. The preparation of the samples
was carried out at 4°C throughout.
Western blotting analysis. PKC isoforms were identified and quantified by Western blot analysis. Protein samples containing the equivalent of 2.5 × 105 cells (25 µl) were run in parallel with rat brain or skeletal muscle protein extracts or recombinant protein, which served as positive controls for immunodetection of the PKC isoforms. For each isoform, to minimize error arising from interassay reproducibility, all samples for each individual (i.e., both pre- and postallergen eosinophils) were run in parallel on the same gel. The samples were loaded onto individual lanes of a 10% acrylamide gel (Bio-Rad ready gel) and were separated by SDS-PAGE. After electrophoresis, the protein was transferred to nitrocellulose (Hybond-ECL, Amersham) for 2 h at 1,000 mA in transblotting buffer [183 mM glycine-HCl, 25 mM Tris base, and 20% (vol/vol) methanol]. To block nonspecific antibody binding, nitrocellulose was incubated for 1 h in 25 mM Tris base, 150 mM NaCl, and 0.05% Tween 20, pH 7.4 [Tris-buffered saline-Tween 20 (TBS-T)] containing 5% (wt /vol) nonfat dry milk. After this, the nitrocellulose membranes were incubated for 1 h in TBS-T containing 5% (wt /vol) nonfat dry milk, and the specific antibody against each PKC isoform (Santa Cruz Biotechnology) was used at a dilution of 1:500. The membranes were then washed with TBS-T (5 × 5 min) and incubated with a 1:7,000 dilution of horseradish peroxidase-linked anti-rabbit IgG in TBS-T-5% nonfat dry milk for 1 h at room temperature. The blots were washed in TBS-T (5 × 5 min) and developed with ECL Western blotting detection agents (Amersham) and Kodak X-OMAT-S film. All blots for each isoform were developed together to ensure identical exposure times. Quantification of the developed blots was performed with laser densitometry.
cPKC cytosolic enzyme activity. PKC
activity was estimated by measuring the phosphorylation of histone
IIIS. Assays were performed in duplicate at 30°C and initiated by
the addition of 25 µl (2.5 × 105 cells) of the cytosolic
fraction to 75 µl of a reaction cocktail containing 20 mM MOPS, 15 mM
magnesium acetate, 10 µM ATP (supplemented with ~100
counts · min1 · pmol
[
-32P]ATP
1),
2 mg/ml of BSA, and 1 mg/ml of histone IIIS in the presence of either 2 mM EGTA or 1.5 mM CaCl2, 100 µg/ml of phosphatidylserine, and 500 nM 4-phorbol 12,13-dibutyrate.
Reactions were terminated after 30 min by spotting 50-µl aliquots of
the reaction mixture onto 2 × 2-cm P81 phosphocellulose paper
squares that were left for 30 s and then immersed in 150 mM
orthophosphoric acid. The paper squares were then extensively washed (4 × 5 min) with fresh orthophosphoric acid to displace any
nonspecifically bound ATP and Pi,
immersed in industrial methylated spirit (5 min) and diethyl ether (5 min), and allowed to dry. Bound radioactivity (representing phosphorylated substrate) was subsequently quantified by liquid scintillation counting in 4 ml of ACS II scintillant (Amersham International).
In vitro IL-5 incubation. Eosinophils (1 × 106 in 1 ml) were suspended in RPMI 1640 medium, 2% FCS, and 100 U of penicillin-100 µg/ml of streptomycin and cultured for 24 h in the presence of 10 pM IL-5. Cells obtained at baseline and after 24 h of culture with IL-5 were prepared for Western blot analysis as described in Western blotting analysis.
Statistics. All values for lung function are expressed as means ± SE. Analysis of the optical densities from the laser densitometry was performed with the Wilcoxon nonparametric test. A P value of <0.05 was taken to be significant.
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RESULTS |
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Response to allergen challenge. All 14 asthmatic subjects (age 19-32 yr) had a well-defined dual response to allergen challenge. The mean maximum change in FEV1 for the early response was 29 ± 2.5% (range 17-49%) and for the LAR 28 ± 2.0% (range 17-37%; Table 1). The values quoted represent changes compared with the postdiluent FEV1.
Eosinophil PKC isoforms. After
SDS-PAGE, Western blot analysis identified the expression of the ,
I,
II, and
isoforms of PKC in the circulating eosinophils of
both normal and asthmatic subjects (Fig.
1). Prolonged ECL exposure also
demonstrated the presence of the
,
,
, and µ isoforms but
not the
,
, and
isoforms. Fractions prepared from rat brain
(
,
I,
II,
,
,
,
,
, and µ), skeletal muscle
extract (
), and recombinant protein (
) were employed as
positive controls. No significant difference was observed in the total
protein expression of the
,
I,
II, and
isoforms of PKC
between normal and asthmatic subjects (data not shown).
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Effect of allergen challenge and IL-5 on PKC isoform
expression. After allergen challenge, the levels of the
,
I, and
II isoforms were unchanged compared with baseline
measurements (Fig. 2).
However, there was an increase in the level of detectable
isoform
(mean band optical density 0.20 ± 0.04 preallergen and 0.33 ± 0.04 postallergen; P = 0.02). Allergen
challenge did not induce the expression of the
,
, and
isoforms (data not shown).
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As a pilot to test for the ability of IL-5 to induce a similar change
in PKC-, we compared the level of PKC-
expression before (0 h)
and 24 h after incubation with 10 pM IL-5. The studies showed there to
be no significant change in the expression of the
isoform.
Subcellular localization of PKC isoforms and the
effect of allergen challenge. After allergen challenge,
the cytosolic and particulate distributions of the PKC isoforms in
circulating eosinophils were compared (Fig.
3). The translocation of
PKC from the cytosolic to the membrane fraction, where the enzyme
becomes activated by phospholipids, was used as a measure of PKC
activation (7, 16-18). Of the total isoform proteins detected,
PKC- and PKC-
II were predominantly located within the cytosolic
fraction of preallergen eosinophils, with no change after allergen
challenge (PKC-
: 66 ± 4% preallergen cytosol and 74 ± 6%
postallergen cytosol; PKC-
II: 71 ± 10% preallergen cytosol and
68 ± 7% postallergen cytosol). For the
I isoform, there was
constitutively more of the total protein present in the membrane at
baseline, but there was no change in the relative amounts in the
cytosolic and membrane fractions after allergen challenge (preallergen:
52 ± 10% cytosol, 48 ± 10% membrane; postallergen: 56 ± 6.0% cytosol, 44 ± 6% membrane). Preallergen, the
isoform was distributed equally between the cytosolic and membrane
fractions. However, after allergen challenge, membrane PKC-
expression was found to be significantly increased after its
redistribution from the cytosol to the membrane (preallergen: 56 ± 8% cytosol, 44 ± 8% membrane; postallergen: 39 ± 8% cytosol, 61 ± 8% membrane; P = 0.03). As
with the total cellular extracts, the sum of cytosolic and membrane
PKC-
was increased in the six patients after allergen challenge,
although this did not reach significance, whereas the expression of the
,
I, and
II isoforms was unchanged.
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Cytosol enzyme activity. The activity
of the cPKC enzymes present in the cytosolic fraction was measured
before and 24 h after allergen challenge and showed that there was no
significant change (cytosolic cPKC activity: 47 ± 13 pmol · min1 · 107
cells
1 preallergen; 39 ± 8 pmol · min
1 · 107
cells
1 postallergen).
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DISCUSSION |
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This study has demonstrated the complement of PKC isoforms expressed in
human eosinophils and has shown increased expression of the aPKC-
isoform 24 h after allergen challenge and a change in the relative
distribution between the cytosolic and membranes fractions, consistent
with cellular activation. We did not undertake allergen challenge on
normal subjects nor did we examine the effects of sham challenge on
asthmatic patients. Therefore, it is not possible to conclude that our
findings are definitely attributed to the effects of allergen on the
eosinophil. Furthermore, this allergen study has not defined the
population of cells that manifest evidence of activation. The question
remains whether the population examined pre- and postallergen are the
same. Allergen challenge results in increases in airway eosinophils and
possibly recruits cells from the bone marrow. Further studies looking
at differential responses of both blood and airway eosinophils before
and at various times after allergen are required to elucidate this
issue further. Nevertheless previous studies (12, 16, 32) support the
notion that eosinophils are activated postallergen challenge,
supporting our findings from this work.
The initial aim of this study was to examine the profile of PKC isoform
expression in circulating human eosinophils. A previous report by Bates
et al. (2) demonstrated the presence of the isoform (this study did
not differentiate between the
I and
II isoforms) but not the
and
isoforms of cPKCs. Curiously, although we were also unable to
identify the presence of the
isoform, we found clear evidence for
the expression of PKC-
as well as of PKC-
I and PKC-
II. The
reason for this discrepancy is uncertain, although it could be related
to differences in antibody sensitivity. Alternatively, there may be
platelet contamination of the eosinophil preparations. Electron
micrograph images demonstrate platelets closely adherent to
granulocytes, and, therefore, expression of PKC isoforms by platelets
would influence our findings. In addition to these cPKCs, we identified
the presence of the aPKCs
, µ, and
and the nPKCs
and
.
We were unable to demonstrate expression of the other two members of
the nPKC family,
and
. Because the positive controls showed the
effectiveness of the antibodies for the nPKC isoforms, it implies that
if these isoforms are indeed expressed, it is at very low levels and
probably have no role during cellular activation or metabolism.
In subsequent studies, we examined the expression and subcellular
distribution of the ,
I,
II, and
isoforms after allergen challenge. Investigation of the effect of challenge on the
,
,
, µ,
,
, and
isoforms was precluded by the their
low-level expression. Examination of the subcellular distribution of
PKCs in allergic patients showed that the
and
II isoforms were
predominantly associated with the cytosolic fraction, whereas the
I
and
isoforms were equally distributed between the cytosolic and
membrane fractions. This distribution contrasts with that described in
another study (2) but may result from nonspecific cellular activation
during separation, such as a result of the hypotonic lysis of the red cells. Alternatively, it is possible that we are examining a preprimed population of cells because they were collected from a group of atopic
asthmatic patients. The proportion of the
I and
II isoforms associated with the membrane fractions is considerably greater than
that reported by Bates et al. (2), who demonstrated that only 7% of
activity was associated with this fraction. This discrepancy between
protein expression and enzyme activity may be related to the difficulty
in solubilizing membrane-associated PKC during the determination of activity.
Allergen challenge had no affect on either the expression or
subcellular distribution of the cPKC isoforms ,
I, and
II. The
lack of change in cytosolic enzyme activity postallergen for cPKC
supports this finding, i.e., that the
,
I, and
II isoforms do
not translocate postallergen. However, both the expression of PKC-
and its translocation from the cytosolic to the membrane fraction was
increased after allergen challenge. Unfortunately, there is no
definitive and reliable method to directly assess enzyme activity for
PKC-
to endorse our immunoblot findings. Although membrane
translocation and subsequent binding of diacylglycerol is thought to be
important to the activation of the cPKC and nPKC isoforms (24), its
relevance for aPKCs (i.e., PKC-
) is uncertain. However, there are
data that show PKC-
stimulation by membrane-associated phosphatidylinositol 3,4,5-trisphosphate (23) and ceramide (20), suggesting that translocation may also be important to PKC-
activation. In addition, a recent study (21) has identified a family of membrane-associated receptors for activated C kinases (RACKs) that are
thought to be important to PKC function. Furthermore, studies in
neutrophils and renal mesangial cells have demonstrated PKC-
membrane translocation after stimulation with
formyl-methionyl-leucyl-phenylalanine (4) and IL-1
(30),
respectively. Given this evidence, the data from these experiments
suggest that membrane translocation of PKC-
is probably related to
enzyme activation and that this could play a role in the mechanism of
eosinophil activation after the LAR.
The actual signaling mechanism responsible for increased expression and
subsequent activation of eosinophil PKC- after the LAR as well as
its cellular function is presently unknown. Increased expression
implies RNA transcription and protein synthesis. PKC-
can be
activated in vitro by phosphatidylinositol 3,4,5-trisphosphate (23),
phosphatidic acid (19), and ceramide (20), which are released after the
activation of phosphatidylinositol 3-kinase, phospholipase D, and
sphingomyelinase, respectively. In addition, the direct association of
PKC-
with Ras has recently been demonstrated both in vitro and in
vivo (7). Once activated, PKC-
has been demonstrated to stimulate
nuclear factor-
B (6, 20) as well as RNA transport and processing via
phosphorylation of heterogenous ribonucleoprotein A1 (22) and in this
manner may influence eosinophil function by promoting the expression of
genes coding for proinflammatory proteins.
IL-5 has been implicated as the predominant eosinophil-active cytokine
during the LAR (26, 31). This cytokine has been demonstrated to
regulate the growth, accumulation, and survival as well as the
activation and priming of eosinophils (3, 10, 29). An inhibitor study
(35) has shown that IL-5-induced survival is dependent on both RNA
transcription and protein synthesis. Furthermore, IL-5 has been
demonstrated to activate a number of intracellular messenger systems
including the Janus kinase (Jak2)-signal transducer and activator of
transcription (STAT1) (28) and the Ras-Raf-1-mitogen-activated protein
kinase kinase (MEK)-mitogen-activated protein kinase (MAPK) pathways
(27, 34) as well as phosphatidylinositol 3-kinase (14). However, with
our in vitro studies with IL-5, we have been unable to demonstrate the
induction of PKC- expression over a 24-h period. This implies that
under in vitro conditions, 10 pM IL-5 was unable to replicate our in
vivo observations and that an additional unidentified factor(s) may be required.
In conclusion, we have identified the presence of three conventional
(,
I, and
II), three atypical (
, µ, and
), and two novel (
and
) isoforms of PKC in circulating human eosinophils. In contrast to the
,
I, and
II isoforms, we have shown
increased expression and activation of PKC-
in patients producing an
LAR to whole lung allergen challenge. Because, under identical
experimental conditions, eosinophils have been shown to demonstrate
priming of NADPH oxidase and increased survival, PKC-
may be
involved in mediating these responses.
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ACKNOWLEDGEMENTS |
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D. J. Evans and M. A. Lindsay contributed equally toward this work.
![]() |
FOOTNOTES |
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D. J. Evans was supported by a grant from Byk Gulden (Constance, Germany). M. A. Lindsay was supported by Wellcome Trust Grant 056814.
Address for reprint requests and other correspondence: M. A. Lindsay, Dept. of Thoracic Medicine, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, Dovehouse St., London SW3 6LY, UK (E-mail: m.lindsay{at}ic.ac.uk.
Received 14 October 1997; accepted in final form 26 March 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baigelman, W.,
S. Chodosh,
D. Pizzuto,
and
L. A. Cupples.
Sputum and blood eosinophilia during corticosteroid treatment of acute exacerbations of asthma.
Am. J. Med.
75:
929-936,
1983[Medline].
2.
Bates, M. E.,
P. J. Bates,
W. J. Calhoun,
and
W. W. Busse.
Increased protein kinase C activity in low density eosinophils.
J. Immunol.
150:
4486-4493,
1993
3.
Collins, P. D.,
S. Marleau,
D. A. Griffiths-Johnson,
P. J. Jose,
and
T. J. Williams.
Cooperation between interleukin-5 and the chemokine eotaxin to induce eosinophil accumulation in vivo.
J. Exp. Med.
182:
1169-1174,
1995[Abstract].
4.
Dang, P. M.-C.,
S. Rais,
J. Hakim,
and
A. Perianin.
Redistribution of protein kinase C isoforms in human neutrophils stimulated by formyl peptides and phorbol myristate acetate.
Biochem. Biophys. Res. Commun.
212:
664-672,
1995[Medline].
5.
De Monchy, J. G. R.,
H. K. Kauffman,
P. Venge,
G. H. Koeter,
H. M. Jansen,
H. J. Sluiter,
and
K. DeVries.
Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions.
Am. Rev. Respir. Dis.
131:
373-376,
1985[Medline].
6.
Diaz-Meco, M. T.,
I. Dominguez,
L. Sanz,
P. Dent,
J. Lozano,
M. M. Municio,
E. Berra,
R. T. Hay,
T. W. Sturgill,
and
J. Moscat.
PKC induces phosphorylation and inactivation of I
B-
in vitro.
EMBO J.
13:
2842-2848,
1994[Abstract].
7.
Diaz-Meco, M. T.,
J. Lozano,
M. M. Municio,
E. Berra,
S. Frutos,
L. Sanz,
and
J. Moscat.
Evidence for the in vitro and in vivo interaction of Ras with protein kinase C.
J. Biol. Chem.
269:
31706-31710,
1994
8.
Dobrina, A.,
R. Menegazzi,
T. M. Carlos,
E. Nardon,
R. Cramer,
T. Zacci,
J. M. Harlan,
and
P. Patriarca.
Mechanism of eosinophil adherence to cultured vascular endothelial cells.
J. Clin. Invest.
88:
20-26,
1991[Medline].
9.
Dor, P. J.,
S. J. Ackerman,
and
G. J. Gleich.
Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory disease.
Am. Rev. Respir. Dis.
130:
1072-1077,
1984[Medline].
10.
Drazen, J. M.,
J. P. Arm,
and
K. F. Austen.
Sorting out the cytokines of asthma.
J. Exp. Med.
183:
1-5,
1996[Medline].
11.
Epand, R. M.,
and
D. S. Lester.
The role of membrane biophysical properties in the regulation of protein kinase C activity.
Trends Pharmacol. Sci.
11:
317-320,
1990[Medline].
12.
Evans, D. J.,
M. A. Lindsay,
B. J. O'Connor,
and
P. J. Barnes.
Priming of circulating human eosinophils following late response to allergen challenge.
Eur. Respir. J.
9:
703-708,
1996
13.
Gleich, G. J.
The eosinophil and bronchial asthma: current understanding.
J. Allergy Clin. Immunol.
85:
422-436,
1990[Medline].
14.
Gold, M. R.,
V. Duronio,
S. P. Saxema,
J. W. Schrader,
and
R. Aebersold.
Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine-phosphorylated proteins.
J. Biol. Chem.
269:
5403-5412,
1994
15.
Hansel, T. T.,
J. D. Pound,
D. Pilling,
G. D. Kitas,
M. Salmon,
T. A. Gentle,
S. S. Lee,
and
R. A. Thompson.
Purification of human blood eosinophils by negative selection using immunomagnetic beads.
J. Immunol. Methods
122:
97-103,
1989[Medline].
16.
Kroegel, C.,
M. C. Liu,
W. C. Hubbard,
L. M. Lichtenstein,
and
B. S. Bochner.
Blood and bronchoalveolar eosinophils in allergic subjects after segmental antigen challenge: surface phenotype, density heterogeneity, and prostanoid production.
J. Allergy Clin. Immunol.
93:
725-734,
1994[Medline].
17.
Kroegel, C.,
T. Yukawa,
G. Dent,
K. Per Venge,
F. Chung,
and
P. J. Barnes.
Stimulation of degranulation from human eosinophils by platelet-activating factor.
J. Immunol.
142:
3518-3526,
1989
18.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
19.
Limatola, C.,
D. Schaap,
W. H. Moolenaar,
and
W. J. van Blitterrwijk.
Phosphatidic acid activation of protein kinase- overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids.
Biochem. J.
304:
1001-1008,
1994[Medline].
20.
Lozano, J.,
E. Berra,
M. M. Municio,
M. T. Diaz-Meco,
I. Dominguez,
L. Sanz,
and
J. Moscat.
Protein kinase C isoform is critical for
B-dependent promoter activation by sphingomyelinase.
J. Biol. Chem.
269:
19200-19202,
1994
21.
Mochly-Rosen, D.
Localization of protein kinases by anchoring proteins: a theme in signal transduction.
Science
268:
247-251,
1995[Medline].
22.
Municio, M. M.,
J. Lozano,
P. Sanchez,
J. Moscat,
and
M. T. Diaz-Meco.
Identification of heterogenous ribonucleoprotein A1 as a novel substrate for protein kinase C.
J. Biol. Chem.
270:
15884-15891,
1995
23.
Nakanishi, H.,
K. A. Brewer,
and
J. H. Exton.
Activation of the isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem.
268:
13-16,
1993
24.
Nishizuka, Y.
Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
Science
258:
607-613,
1992[Medline].
25.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J.
9:
484-496,
1995
26.
Ohnishi, T.,
H. Kita,
D. Weiler,
S. Sur,
J. B. Sedgwick,
W. J. Calhoun,
W. W. Busse,
J. S. Abrams,
and
G. J. Gleich.
IL-5 is the predominant eosinophil-active cytokine in the antigen-induced pulmonary late-phase reaction.
Am. Rev. Respir. Dis.
147:
901-907,
1993[Medline].
27.
Pazdrak, K.,
D. Schreiber,
P. Forsythe,
L. Justement,
and
R. Alam.
The intracellular signal transduction mechanism of interleukin 5 in eosinophils: the involvement of lyn tyrosine kinase and the Ras-Raf-1-MEK-microtubule-associated protein kinase pathway.
J. Exp. Med.
181:
1827-1834,
1995[Abstract].
28.
Pazdrak, K.,
S. Stafford,
and
R. Alam.
The activation of the Jak-STAT1 signaling pathway by IL-5 in eosinophils.
J. Immunol.
155:
397-402,
1995[Abstract].
29.
Rothenberg, M. E.,
J. Petersen,
R. L. Stevens,
D. S. Silberstein,
D. T. McKenzie,
K. F. Austen,
and
W. F. Owen, Jr.
IL-5-dependent conversion of normodense human eosinophils to the hypodense phenotype uses 3T3 fibroblasts for enhanced viability, accelerated hypodense, and sustained antibody-dependent cytotoxicity.
J. Immunol.
143:
2311-2316,
1989
30.
Rzymkiewicz, D. M.,
T. Tetsuka,
D. Daphna-Iken,
S. Srivastava,
and
A. R. Morrison.
Interleukin-1 activates protein kinase C
in renal mesangial cells: potential role in prostaglandin E2 up-regulation.
J. Biol. Chem.
271:
17241-17246,
1996
31.
Sedgwick, J. B.,
W. J. Calhoun,
G. L. Gleich,
H. Kita,
J. S. Abrahms,
L. B. Schwartz,
B. Volovitz,
M. Ben-Yaakov,
and
W. W. Busse.
Immediate and late airway response of allergic rhinitis patients to segmental antigen challenge. Characterization of eosinophil and mask cell mediators.
Am. Rev. Respir. Dis.
144:
1274-1281,
1991[Medline].
32.
Sedgwick, J. B.,
W. J. Calhoun,
R. F. Vrtis,
M. E. Bates,
P. K. McAllister,
and
W. W. Busse.
Comparison of airway and blood eosinophil function after allergen challenge.
J. Immunol.
149:
3710-3718,
1992
33.
Sedgwick, J. B.,
K. M. Geiger,
and
W. W. Busse.
Superoxide generation by hypodense eosinophils from hypodense eosinophils from patients with asthma.
Am. Rev. Respir. Dis.
142:
120-125,
1990[Medline].
34.
Van der Bruggen, T.,
E. Caldenhoven,
D. Kanters,
P. Coffer,
J. A. M. Raaijmakers,
J.-W. J. Lammers,
and
L. Koenderman.
Interleukin-5 signaling in human eosinophils involves JAK2 tyrosine kinase and STAT1.
Blood
85:
1442-1448,
1995
35.
Yamaguchi, Y.,
T. Suda,
S. Ohta,
K. Tominaga,
Y. Miura,
and
T. Kasahara.
Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils.
Blood
78:
2542-2547,
1991[Abstract].