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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1206-1218
Expression of Protein Kinase C Isozymes in Human Basophils:
Regulation by Physiological and Nonphysiological Stimuli
By
Katsushi Miura and
Donald W. MacGlashan Jr
From the Johns Hopkins Asthma and Allergy Center, Baltimore, MD.
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ABSTRACT |
The expression of protein kinase C (PKC) isozymes in human basophils
and the regulation of PKC isozymes during basophil activation by
phorbol 12-myristate 13-acetate (PMA) ± ionomycin,
f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human
basophils (> 98% purity), PKC I, II,  , and  were
expressed, PKC was difficult to detect, and PKC and  were
undetectable. In unstimulated basophils, PKCI and II were found
primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCI and II were translocated to the membrane
fraction (85% ± 4% and 83% ± 6%, respectively). In
resting cells, 48% ± 3% and 61% ± 10% of PKC and
, respectively, existed in the membrane fraction. Within 1 minute of
stimulation with PMA, 90% ± 6% of PKC was found in the membrane
fraction, however, no translocation of PKC was apparent. Stimulation
with FMLP caused modest translocation ( 20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of
measurement. However, concentrations of PMA and ionomycin that alone
caused no PKC translocation and little histamine release, together
caused significant histamine release but no apparent PKC translocation.
Studies with bis-indolylmaleimide analogs showed inhibition of
PMA-induced, but not anti-IgE-induced, histamine release. These
pharmacological studies suggest that PKC does not play a
prodegranulatory role in human basophil IgE-mediated secretion.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HUMAN BASOPHILS, AS WELL as human mast
cells, express high-affinity IgE receptors (Fc RI) on their cell
surface and release a variety of mediators1-3 in response
to aggregation of these receptors. Basophils may play an important role
in the allergic inflammatory reaction that follows the initial exposure
to antigen because it has been shown that these cells infiltrate and
secrete their mediators at specific reaction sites in the skin, nose, and lung.4-6 As a participant in these reactions, an
understanding of the mechanisms underlying their activation may be
useful in future therapies. Although many details of FcRI-mediated
signal transduction have become elucidated for mast cell lines such as the rat basophilic leukemia cell (eg, the RBL-2H3), much less is known
about their human counterparts.
In many cell types, protein kinase C (PKC) activation plays a central
role in the early stages of signal transduction. It is often found that
the coapplication of phorbol esters, which are thought to directly
activate PKC enzymes with significant specificity,7 and
calcium ionophores leads to functional behaviors that reasonably mimic
physiological stimulation. A similar result is found for human mast
cells and basophils but with the interesting caveat that phorbol
12-myristate 13-acetate (PMA) alone can induce marked histamine release
in human basophils.8 This does not occur in human lung or
skin mast cells.9 There are unusual characteristics to this
release.8,10 For example, only histamine release results
from PMA stimulation, no LTC4 or IL-4 secretion occurs.11,12 Furthermore, the kinetics of release are very slow compared with other natural secretagogues. PMA-induced release occurs in the absence of extracellular calcium and under conditions where intracellular Ca++ is heavily buffered by an
intracellular chelator like
1,2-bis(o-Aminophenoxy)ethane-N,N,N ,N-tetraacetic acid
(BAPTA; unpublished results). These
observations suggested that the signaling pathway that induces
degranulation in human basophils might be regulated by PKC in a
different way from that in other cells such as human mast cells and
RBL-2H3 cells. However, such results also suggested that histamine
release could result from the activation of PKC(s) alone. The amount of
histamine release associated with activation of basophils through IgE
receptor aggregation, among different preparations of basophils, was
found to be correlated to an increase in membrane-bound PKC-like
activity.13 These results also suggested that PKC
activation may have a role in IgE-mediated histamine release in human
basophils.
PKC may also have a role in downregulating cellular responses, and
studies in RBL cells support this possibility.14-16 In
human basophils, the process of desensitization has been hypothesized to result from the activity of a PKC and recent studies of IL-4 secretion from human basophils also suggest a downregulatory role for
PKC. Activation of PKC(s) with PMA is known to inhibit the cytosolic-free Ca++ ([Ca++] i)
elevation that follows receptor-mediated stimulation. Multiple roles
for PKC are thought to result, in part, from the activity of different
isoforms of PKC and studies in RBL cells support this
perspective.15-17 At present, 12 different PKC isozymes are known.18-22 They are divided into three major groups: the
calcium-dependent or conventional PKC (, I, II, and
), the calcium-independent or novel PKC (, , ,  , and
µ), and the atypical PKC ( and  ).18-22 Marked
differences in tissue distribution exist among the PKC isozymes. PKC
, I, II, , , and have a wide distribution, whereas PKC , , and are restricted to one or a few
tissues.18 Such differences in distribution suggest a
divergence in function between isozymes.23 A role for some
of the PKC isozymes in antigen-induced secretion in RBL-2H3 cells has
been demonstrated.15-17 It was suggested that
antigen-induced secretion was mediated primarily by PKC and and
feedback-inhibition of phospholipase C was mediated primarily by PKC
and .
The conventional PKC , I, II, and contain the
putative Ca2+-binding region C2 in the regulatory domain
and are Ca2+-responsive and dependent on Ca2+
for activity.18,24,25 A role for Ca2+ in the
translocation of conventional PKC induced by phorbol esters was
indicated by the study of the binding of the kinase to plasma membranes
in a cell-free system26 and the study of PKC isozyme translocation induced by calcium ionophore or calcium ionophore plus
phorbol esters in intact cells.27 In human basophils,
phorbol esters and calcium ionophore operate synergistically such that subeffective concentrations of both stimuli induce significant histamine release.10 As a starting point for understanding
the role of PKC in the human basophil response, we have examined which enzymes are present in basophils and characterized the response of the
PKC profile to direct stimulation with phorbol esters. This report then
explores whether changes in the location of these isozymes can be
detected after receptor-mediated stimulation.
Previous studies by our group and others have noted that first
generation PKC inhibitors (eg, H7 and staurosporine) inhibit histamine
release from human basophils stimulated through the IgE receptor.
However, these first generation inhibitors are nonselective, eg,
staurosporine was also found to be a potent inhibitor of tyrosine kinases. Because IgE-mediated secretion in basophils is likely to use
tyrosine kinases early in the signal transduction cascade, as has been
found for RBL cells,28-34 these older studies become ambiguous. One group has also examined a newer PKC inhibitor, calphostin C, and found inhibition of IgE-mediated
release.35 To determine whether some PKC isozymes have
either a positive or negative role in IgE-mediated secretion, the
current studies examine a more recent generation of PKC inhibitors.
With the exception of calphostin C, these inhibitors competively bind
to the catalytic domain of PKC (presumably competing with ATP).
Calphostin C is thought to bind to the regulatory domain, possibly
interfering with activation of PKC by diacylglycerols or phorbol
esters.
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MATERIALS AND METHODS |
Reagents.
The following were purchased:
piperazine-N,N-bis-2-ethanesulfonic acid (PIPES),
bovine serum albumin (BSA), ethyleneglycoltetrcetic acid (EGTA),
ethylenediamine tetraacetic acid (EDTA),
D-glucose, human IgG, Phorbol 12-myristate 13-Acetate (PMA),
-mercaptoethanol, NP-40 (Sigma, St Louis, MO); crystallized human
serum albumin (HSA) (Miles Laboratories, Elkhart, IN); fetal calf serum
(FCS) and RPMI 1640 containing 25 mmol/L HEPES (GIBCO-BRL, Grand
Island, NY); Percoll, (Pharmacia, Piscataway, NJ); Tris
(hydroxymethyl)-aminomethane, Tween-20 (Bio-Rad, Hercules, CA);
leupeptin, dithiothreitol, phenylmethylsulfonyl fluoride (PMSF)
(Boehringer Mannheim, Indianapolis, IN); 4-Phorbol 12-Myristate
13-Acetate (4-PMA) (LC Laboratories, Woburn, MA); ionomycin
(Calbiochem, La Jolla, CA). BPO (benzylpenicilloyl)-human serum albumin
(HSA) and BPO-EACA (e-aminocaproic acid) were synthesized as previously
described 36 and the antigen gp120(HIV)-OVA (ovalbumin) was
the gift of Dr Frances Davis of Tanox, Corp (Houston, TX).
Bis-indolylmaleimide I and II, Ro 31-8820, calphostin C and Go-6976
were purchased from Calbiochem.
Antibodies.
Rabbit peptide-specific antibodies (IgG fraction) against PKC I
and and blocking peptides for these antibodies were from GIBCO-BRL.
Rabbit peptide-specific antibodies against , II, , ,
and and blocking peptides for these antibodies were obtained from
Oxford Biomedical Research, Inc (Oxford, MI). The specificity of
antibody binding to specific bands on the Western blots was determined
by either excluding the primary antibodies or blocking primary antibody
binding with the peptides provided by the manufacturer. Two commercial
anti-PKC antibodies were used. They recognize different epitopes
corresponding to residues #662-672 (Oxford) of PKC and #313-326
(GIBCO). Partial genomic analysis has indicated that I and
II isozymes are derived from a single RNA transcript by
alternative splicing and differ from each other only in a short range
of about 50 amino acid residues at the C-terminal V5
region.20 The antibodies we used recognized this region of
each isozyme. Peroxidase-labeled donkey antirabbit Ig antibody was from
Amersham Life Science (Arlington Heights, IL). BPO-specific IgE was
purified from the serum of penicillin allergic patients as previously
described36 and anti-gp120(HIV) IgE was the gift of Dr
Frances Davis of Tanox, Corp.
Buffers and media.
PIPES-albumin-glucose (PAG) buffer contained 25mmol/L PIPES, 110 mmol/L
NaCl, 5mmol/L KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG
supplemented with 1 mmol/L CaCl2 and 1 mmol/L
MgCl2. PAG-EGTA was PAG supplemented with 50 nmol/L EGTA.
PAG-EDTA was PAG supplemented with 4 mmol/L EDTA. Countercurrent
elutriation was conducted in PAG containing 0.25% BSA in place of
0.003% HSA.
Basophil purification.
Basophils were purified from residual cells of normal donors undergoing
leukapheresis as previously described.1 The leukocytes were
partially purified by Percoll density gradient and by counter-current elutriation. Basophils were placed into culture (RPMI 1640 with 2% FCS
and 20 µg/mL gentamycin) for 1 hour after elutriation and one
subsequent Percoll separation on a two-step gradient (1.066/1.079). Typically, the last step in the procedure was a two-step Percoll gradient (1.069/1.079) that results in purities greater than 70%. For
these studies, purities ranged from 71% to 90% (median = 80% ± 2%) and for the preliminary studies of basophil PKC profile, these
cells were used for positive selection using anti-IgE antibody. For the
remaining studies, the cells were used without positive selection. To
minimize concerns that the positive selection technique might induce
changes in the PKC status, the entire procedure was carefully performed
below 4°C and the cells were suspended in PAG-EGTA (50 µmol/L).
Proteases (calpains) that induce proteolytic degradation of PKC are
calcium dependent.37 The cells were incubated for 10 minutes with 1 µg/mL mouse antihuman IgE (TES-19) (provided by Tanox
Corp) in the presence of 4 mg/mL normal human IgG to block
FcR.38 After a subsequent 20-minute incubation with
rat-antimouse IgG2a+b paramagnetic beads (8 µL per 107
cells), the cells were passed through a MACS mini-column (MACS system,
Miltenyi Biotec Inc, Sunnyvale, CA). Flow-through cells were collected
and the eluted basophil purities were found to be between 98% to
99.4%. Alcian blue staining was used to assess basophil
purity.39 Cell viability was determined by trypan blue exclusion.
Stimulation of basophils for Western analysis of PKC.
To examine PKC isozyme translocation, the cell preparations were
suspended in PAGCM at a concentration of 1 × 107
cells/mL. Appropriate stimuli were added as described in the Results
and the cells were incubated at 37°C for the indicated times. The
incubations were stopped by a rapid centrifugation and the addition of
4 volumes of ice-cold PAG-EDTA. The cells were then lysed as described
below. To examine PKC isozyme downregulation, basophil preparations
were suspended in RPMI-1640 supplemented with 2% FCS at a
concentration of 5 × 106 cells/mL and incubated with
indicated concentrations of PMA for the indicated times. The cells were
then harvested by centrifugation and the pellet lysed.
Western blot analysis of whole cell lysates.
High-speed cell pellets (~14,000 G for 5 to 10 seconds) were
resuspended at 2 × 107 cells/mL in lysis buffer
(50mmol/L Tris-HCl, pH 7.5, 5 mmol/L EDTA, 10 mmol/L EGTA, 5 mmol/L
dithiothreitol, 1% Nonidet P-40, 1 mmol/L PMSF, 20 µg/mL leupeptin,
100 µg/mL aprotinine, 10 mmol/L benzamidine). After 20 seconds of
vortexing, the cell lysates were kept on ice for 20 minutes and
microfuged for 15 minutes at 4°C. Supernatant was collected as a
protein extract containing lysed cell components without
nuclei.40 Extracts containing equal numbers of basophils (2 × 105 cell equivalents/lane) were diluted with an
equal volume of 2x loading buffer (0.125 mol/L Tris-HCl, pH 6.8, 4%
sodium dodecyl sulfate (SDS) 0.005% bromophenol blue, 20% Glycerol)
(NOVEX, San Diego, CA) containing 0.05% -mercaptoethanol, and
separated by electrophoresis on 4% to 20% Tris glycine
density-gradient gels (NOVEX) (for experiments comparing basophils and
contaminating cells from the same preparations, the samples were loaded
in adjacent lanes). Gels were then transferred to pure nitrocellulose
membrane (Schleicher & Schuell, Keene, NH) by Trans Blot (NOVEX).
Electrophoresis and transfers were performed according to the
manufacturer's recommendations. After transfer, membranes were
immersed in Tris-buffered saline-Tween (TBST; 50 mmol/L Tris, pH 7.5, 0.15 mol/L NaCl, 0.05% Tween-20) containing 5% nonfat
dry skim milk (Carnation, Los Angeles, CA) overnight to block
nonspecific binding. Membranes were then washed three times for 5 minutes with TBST. Immunoreactive proteins were detected using the
isozyme-specific antibodies that were diluted in TBST containing 1%
skim milk as follows: 2 µg/mL for antibodies against I and isozymes; and 1/250 dilution for antibodies against , II,
, , and . After a 4-hour incubation, membranes were washed
with TBST and were incubated with peroxidase-labeled donkey antirabbit
Ig antibody for 1 hour. After five 10-minute washes, membrane-bound
antirabbit Ig antibody was visualized using enhanced chemiluminescence
(ECL) Western blotting detection reagents (Pierce, Rockford, IL), and Hyper-ECL chemiluminescence detection film (Amersham). The ECL films were converted to digital format with a URL
digital camera and the images analyzed with NIH Image (Wayne Rasband,
NIH).1 These studies were designed to obtain a
reasonable estimate of the relative amounts of the PKC isozymes, so the
Western blot technique was evaluated for linearity. Serial dilutions of both PKC standards and cell samples were examined and the relative band
intensities found to be linear if the ECL development was allowed to
proceed for restricted periods of time. These times were optimized for
each gel by exposing the films for several different times and the
exposure chosen that bracketed the range of band intensities. The
comparisons were made on the basis of equal numbers of cells. However,
the protein content of these samples was also found to be equal.
Membrane and cytosol preparation of cells.
After stimulation, medium was removed for assay of histamine (see
below). Cells were suspended at 2 × 107 cells/mL in
ice-cold hypotonic lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 5 mmol/L
EDTA, 5 mmol/L EGTA, 5 mmol/L dithiothreitol, 1 mmol/L PMSF, 200 µg/mL leupeptin, 100 µg/mL aprotinine, 10 mmol/L benzamidine). The
cell suspension was sonicated 3 times at 5-second bursts by an
ultrasonic cell disruptor (Heat System Ultrasonics, Inc, Farmingdale,
NY). After centrifugation at 500g for 5 minutes at 4°C to
remove unlysed cells and the nuclei, the supernatant was centrifuged at
100,000g for 10 minutes at 4°C. The supernatant was
collected as the cytosol preparation (2 × 107 cell
equivalent/mL). The pellet was resuspended at 2 × 107
cell equivalent/mL of hypotonic lysis buffer containing 1% Nonidet P-40 and sonicated. The samples were subjected to Western blot analysis
as described above.
Lactate dehydrogenase (LDH) determination.
To determine the cross-contamination of membrane fractions with
residual cytosolic proteins, the two fractions were analyzed for LDH
content, which should be a cytosolic marker. LDH was measured with a
commercial kit obtained from Sigma. LDH activity in cytosol and
membrane fractions was 14.7 ± 0.5 and 0.28 ± 0.12 mU/106 basophils, respectively (n = 2), indicating that
very little cytosolic protein (1.9% ± 0.7% of total) contaminated
the membrane fraction.
Histamine release and sensitization.
For the pharmacological experiments, basophils were obtained by
venipuncture and partially enriched over a single-step Percoll gradient. The cells were challenged and supernatants were harvested for
analysis by automated fluorimetry.41 Histamine release is expressed as a ratio of sample to total histamine, obtained by lysis of
an equivalent number of cells with perchloric acid, after subtracting
spontaneous release. Purified basophils were processed the same way
when histamine release data were needed. Calphostin C requires
activation by incubation under strong fluorescent lights.42 The experiments with calphostin C were performed in a water bath that
was covered with two 40-watt fluorescent bulbs. Lighting was used
during the preincubation of the cells with the drug as well as during
the challenge phase. Preliminary experiments established that this
lighting was required for adequate potency. For the data analysis in
the pharmacological experiments, the relevant analysis within each
experiment required expressing the amount of inhibition as a fraction
of the control histamine release (cells treated with equivalent
carrier). After expressing the data this way (histamine release from
all samples calculated as a fraction of the response of untreated cells
to stimulus, expressed as a percentage), the results were averaged for
the replicate experiments.
Cytosolic calcium measurements.
Purified basophils were labeled with fura-2AM (1 mmol/L for 25 minutes
at 37°C in RPMI-1640 containing 2% FBS and 0.3 mmol/L EDTA),
washed once with PAG and kept on ice before loading into a
Dvorak-Stotler chamber for observation under the microscope. A field of
30 to 50 cells was monitored by sequential dual excitation, 352 and 380 nm, and ratios of the images were converted to calcium concentrations
according to methods and parameters we have previously published.43 Ratio images were acquired every 3 seconds
early in the reaction and every 10 seconds later in the reaction. The data were compiled for the average of the 50 to 100 cells under observation. In the plots shown, the stimulus was added at the time
point marked zero after obtaining several images before stimulation. Cell perimeters were measured for these fura-2 labeled cells using algorithms previously published.44,45
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RESULTS |
Expression of PKC isozymes in human basophils.
To clarify which PKC isozymes were present in basophils, basophils that
were very close to 100% purity were examined; these cells were
obtained by positive selection as described in Materials and Methods.
These studies included a comparative analysis with the contaminating
cells that typically contaminate enriched basophil preparations.
Starting with basophil preparations of 70% to 90% and collecting the
cells that wash through the magnetized mini-MACs column yields
contaminating cells that contain  1% basophils. The subsequently
collected basophils ranged in purity between 98% and 99.4%. The cells
were lysed at a density of 20 × 106/mL and the
equidense solutions were immunoblotted with antibodies specific for
each of the seven isozymes (Fig 1). The
basophil data shown in Fig 1 (right) were expressed as a percentage of the contaminating cell band intensities. Both basophils and
contaminating cells expressed essentially equivalent levels of PKC
I, II and . PKC was expressed at a much higher level
in basophils (452% ± 142%) than that observed in contaminating
cells. Interestingly, PKC was barely detectable in these basophil
preparations (7% ± 3%) compared with contaminating cells. Two
commercial anti-PKC antibodies yielded similar results. PKC was
undetectable in basophils but was present in contaminating cells. PKC
was undetectable in both populations of cells. The data are
normalized for equal cell numbers but an analysis of the protein
content also showed essentially equal levels of protein in the basophil
and contaminating cell lysates. To place the relative values in a more
general context, we compared the band intensities from one preparation
with serial dilutions of PKC isozyme standards. We estimate that PKC
was present at 100 ng/106 basophils and at 14 ng/106 contaminating cells, and PKC was present at 26 ng/106 contaminating cells but not detectable in the
basophil sample used for this particular quantitative comparison.
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| Fig 1.
Expression of PKC isozymes in basophils and contaminating
cells. Basophils (purity: 98% to 99.4%) and contaminating cells (lymphocytes and monocytes) were lysed and subjected to Western blot
analysis as described in Material and Methods. (MWM) Molecular weight
marker (upper band: 97.4 kD, lower band: 68 kD). (R) Rat brain extract.
(B) Human basophils. (C) Contaminating cells (lymphocytes and
monocytes). The blots are representative of five experiments for PKC
and and, three experiments for the other isozymes. The band
intensities were quantified by digital imaging. The data are expressed
as the amount of each isozyme present in basophils expressed as a
percent of the amount in contaminating cells (right panel). The data
represent the mean ± SEM.
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Downregulation of PKC isozymes with overnight PMA treatment.
In many cell types, prolonged treatment with phorbol esters results in
depletion of cellular PKC by proteolytic cleavage that follows
activation37 and often there is a difference in the susceptibility of the various isozymes to downregulation with phorbol
esters.15,18 Purified basophils (purity = 76% ± 3%, n = 4) were cultured for 18 hours with PMA (100 ng/mL) and the cells
lysed for Western blots. In one of these experiments, 4- and 18-hour
time points were examined. PMA completely downregulated PKC I and
II by 18 hours and ~80% downregulation occurred by 4 hours (data
not shown). The downregulation of PKC and was inconsistent
after 18 hours and the changes were not statistically significant (76% ± 18% and 73% ± 14% of the 18-hour incubation without PMA
for PKC and , respectively). The cell viability after overnight
treatment with or without PMA was 92% ± 3% and 94% ± 1%,
respectively (n = 4). As expected, 4-PMA (an inactive analog) did
not induce downregulation of PKC isozymes (data not shown). Marked
downregulation of PKC I and II also occurred with 1 ng/mL (PKC
I and II were 10% and 34% of controls, respectively).
Translocation of PKC isozymes with PMA.
Human basophils (purity = 80% ± 3%, n=4) were incubated with or
without 100 ng/mL PMA for 15 minutes and the asssociation of PKC
isozymes with either membrane or cytosolic cell fractions was examined.
In unstimulated cells, PKC I and II were present in the cytosol
(95% ± 3% of total and 98% ± 1%, respectively) (Fig 2). Fifteen minutes after stimulation,
85% ± 4% of total PKC I and 82% ± 6% of total PKC were
found in the membrane fraction (P < .001 for both I and
II). It should be pointed out that the summation of membrane and
cytosolic PKC band intensities with or without PMA stimulation was not
equal. PMA treatment decreased the total (membrane + cytosol) band
intensities of PKC I and II to 64% ± 10% and 57% ± 5% of control, respectively. However, total PKC and levels were not affected by PMA (96% ± 11% and 99% ± 27% of
control, respectively). These results indicate that the loss of PKC
I and II did not result from poorer recovery because the total
band intensities of PKC and were not changed. Therefore, some
proteolytic cleavage of PKC I and II appears to occur within 15 minutes at this high concentration of PMA.
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| Fig 2.
Translocation of PKC isozymes in basophils stimulated
with PMA. Basophils (purity = 80% ± 3%, n = 4) were incubated
with or without PMA (100 ng/mL) for 15 minutes. The membrane and
cytosol fractions were subjected to Western blot analysis and the band intensities were quantified by digital imaging. The top panel shows a
representative Western blots from one experiment and the lower panel
shows the amount of each isozyme in the cytosol or membrane expressed
as a percentage of either component divided by the sum of both
components [eg, cytoscol/(cytosol + membrane)]. Data are presented
as mean ± SEM for four experiments. * P < .05, ***
P < .001 (paired t-test).
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Compared with PKC I and II, a larger fraction of PKC (48% ± 3% of total) and (61% ± 10%) was located in the
membrane fraction of unstimulated cells. Membrane-associated PKC increased significantly (P < .05) after treatment with PMA.
Similar results were obtained at basophil purities ranging from 75% to
93%, indicating that the small shift was not due to changes occurring
only in contaminating cells (a potential consideration given the
similar levels of expression for this PKC isozyme). However,
translocation of PKC was not apparent and any observed changes were
not statistically significant. 4-PMA did not cause translocation of
these four isozymes nor did it induce histamine release at 100 ng/mL
and 15 minutes of incubation (data not shown). Treatment with 100 ng/mL
PMA induced almost complete translocation of PKC within 1 minute
and translocation of PKC I and II within 5 minutes (Fig 3A). As noted previously, histamine
release after challenge with PMA is slow (requiring at least 60 minutes
to reach 60%8) and the data in Fig 3A reflects this slow
release because a 15-minute incubation with 100 ng/mL resulted in
marginal histamine release (for the four experiments shown in Fig 2,
histamine release was 12% ± 3%). The concentration dependence is
shown in Fig 3B and there were minor differences among PKC , or I
and PKC II.
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| Fig 3.
Time course of PKC isozyme translocation after
stimulation with PMA and the dose-response curve for PMA. In panel A,
basophils were incubated with PMA (100 ng/mL) for the times indicated
(n = 1) and in panel B cells were incubated with various
concentrations of PMA for 15 minutes (n = 1). The membrane and
cytosol fractions were prepared, analyzed by Western blotting, and the
bands quantified by digital imaging. The data are expressed as the
amount of membrane-associated PKC as a percentage of the summed band
intensities for membrane + cytosol. PKC I ( ), II ( ), ( ), ( ), and histamine ( ).
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Receptor-mediated PKC translocation.
Figure 4 shows a PKC translocation study of
basophils stimulated with FMLP (basophil purity = 73%). There were
apparent shifts in location for all four isozymes of PKC. PKC I
showed the greatest translocation whereas there were also smaller
shifts in the location of PKC II, and . With the exception
of PKC , the timing of the changes for each of the isozymes was
similar. Most notably, PKC I, II, and did not shift during
the first 30 seconds of the reaction, whereas PKC showed shifts
that occurred more rapidly, measurable changes occurring at the
earliest time point tested in this experiment, 30 seconds. In a second
FMLP experiment (basophil purity of 81%), no increase in
membrane-associated PKC was evident after 15 seconds although an
increase was evident by 30 seconds as noted in the first experiment. In
contrast, human basophils stimulated with an optimal concentration of
anti-IgE antibody showed no measurable shifts in any of the PKC
isozymes. Figure 5 shows the results from
the best case, basophils treated overnight with IL-3 (10 ng/mL)
followed by stimulation that led to 71% histamine release, a strong
response for an IgE-mediated process. Even under these conditions, no
changes were observed. In three experiments where cells were tested
with or without the overnight incubation with IL-3 but with lesser
secretion, no shifts in the location of the four tested PKC isozymes
were observed. The summation of membrane and cytosolic PKC band
intensities following either FMLP or anti-IgE did not vary with time,
ie, there was no apparent degradation and loss of any of the isozymes
after stimulation in this time frame (data not shown).
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| Fig 4.
PKC translocation in basophils stimulated with FMLP.
Basophils (purity: 73%) were stimulated with 1 µmol/L FMLP and after the times indicated the cells were harvested, membrane and cytosol fractions were prepared, analyzed by Western blotting, and the bands
quantified by digital imaging. The data shows one of two experiments.
The data on the lower panel is expressed as the amount of
membrane-associated PKC as a percentage of the summed band intensities
for membrane + cytosol. PKC I (), II (), (), (), and histamine ( ).
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| Fig 5.
PKC translocation in basophils stimulated with anti-IgE
antibody. Basophils (purity of 71%) were stimulated with 0.2 µg/mL of anti-IgE antibody and after the times indicated the cells were harvested, membrane and cytosol fractions were prepared, analyzed by
Western blotting and the bands quantified by digital imaging. The
results shown in the top half of the figure are from one of three
experiments, the one with the highest histamine release (71%), and the
bottom half of the figure shows the average of three experiments
(average histamine release was 34%).
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From previous studies we have noted that IgE-mediated histamine release
results from changes in several early signal transduction events that
are quite modest. It has been noted in studies of RBL cells (as well as
many other cell types) that the synergistic effect of several early
signal transduction events is likely to be responsible for observed
levels of antigen-induced secretion. The combination of PMA and a
calcium ionophore such as ionomycin is often used to mimic some of this
synergism. As noted in the one experiment shown in Fig 3B, 1 ng/ml of
PMA did not induce measurable (or marginally measurable) changes in PKC
I or II location. Figure 6 shows the
average results of two experiments in which PKC translocation and
histamine release were examined for cells stimulated with 1 ng/mL PMA,
0.1 µg/mL ionomycin or a combination of the two. PMA at 1 ng/mL and
ionomycin at 0.1 µg/mL alone caused little histamine release (2% ± 1% and 4% ± 3%, respectively), whereas the combination
caused 32% ± 10% histamine release. No translocation of any PKC
isozyme was observed under these conditions, including the combination
of both PMA and ionomycin. The summation of membrane and cytosolic PKC
band intensities did not vary with the conditions, ie, there was no
apparent degradation and loss of any of the isozymes under any of the
conditions tested for the 15-minute period examined (data not shown).
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| Fig 6.
PKC translocation in basophils stimulated with PMA
and/or ionomycin. Basophils (purity: 82% ± 2%, n
= 2 ) were incubated with or without PMA (1 ng/mL) and with
or without ionomycin (0.1 µg/mL) for 15 minutes. Membrane and cytosol
fractions were prepared, analyzed by Western blotting, and the bands
quantified by digital imaging. Data are presented as mean ± range for
two experiments. Histamine release for these samples is also plotted.
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Effects of PKC inhibitors on secretion.
Figure 7 summarizes the data for the five
different PKC inhibitors tested. PMA induces histamine release from
human basophils in the apparent absence of any other costimulus. This
provides a useful test of the ability of these five inhibitors to
inhibit secretion presumably dependent on the activation of PKC. It can be observed in Fig 7A that all of the inhibitors inhibited PMA-induced release with an IC50 of approximately 100 nmol/L (or ~400 nmol/L for
BIS I [Bis-indolylmaleimide I]). In experiments not shown, it was
determined that a 10-minute preincubation with each of these drugs
resulted in inhibition that was the same as 40 minutes of
preincubation. Figure 7B shows that for four of the inhibitors, there
was no inhibition of ionomycin-induced histamine release. Unexpectantly, calphostin C was found to inhibit ionomycin-induced histamine release with an IC50 that was somewhat lower than its IC50
for PMA-induced release. It should be noted that calphostin C also
markedly induced histamine release in the absence of any stimulus such
that at concentrations of 1 µmol/L, spontaneous release increased to
an average of 27% ± 10%. Thus, the apparent inhibition at 1 µmol/L calphostin C was a combination of increased spontaneous
release subtracted from stimulus-induced histamine release that was
also decreased in absolute magnitude at these higher concentrations.
The concentration-dependence of this enhanced spontaneous release was
quite steep such that no enhancement was observed at 300 nmol/L
calphostin C (data not shown).
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| Fig 7.
Effects of various PKC inhibitors on stimulation of
basophil histamine release by PMA (panel A), ionomycin (panel B),
anti-IgE antibody (panel C), and FMLP (panel D). In each panel,
basophils obtained by single step Percoll (low purity) were first
incubated with the inhibitor for 10 minutes before the addition of the
stimulus. After a 45-minute challenge the supernatants were harvested
for histamine analysis. Each panel represents the average of five experiments for each stimulus, () bis-indolylmaleimide I, () bis-indolylmaleimide II, () Ro 31-8220, () Go 6976, and ( ) calphostin C. Control release averaged 58%, 73%, 38%, and 32% for
PMA (30 ng/mL), ionomycin (0.5 µg/mL), anti-IgE antibody (0.2 µg/mL) and FMLP (1 µmol/L), respectively.
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The indolylmaleimide analogs were also found to inhibit the synergy
between PMA and ionomycin. In pilot experiments, basophils were
stimulated with serial twofold dilutions of ionomycin (0.0125 µg/mL
to 0.4 µg/mL) in the presence or absence of several concentrations of
PMA (0.01 ng/mL to 10 ng/mL) to establish the concentration-dependence of the synergism. In these pilot experiments, the ionomycin
dose-response curve in the presence of 0.5 ng/mL to 1 ng/mL PMA was
midway between the curves found for no PMA and 10 ng/mL of PMA. BIS II,
at concentrations of 400 nmol/L, almost completely reversed the synergy
observed when using the combination of 0.6 ng/mL of PMA and the serial dilutions of ionomycin (reducing the response of the cells to the
levels observed with ionomycin alone) and partially reduced the synergy
between 10 ng/mL of PMA and the serial dilutions of ionomycin (data not
shown).
Figure 7C and D shows a summary of the data for the five inhibitors in
cells stimulated with either anti-IgE antibody or FMLP. The surprising
result is that the three indolylmaleimide compounds caused no
inhibition of IgE-mediated histamine release. Indeed, there was an
inconsistent but sometimes marked enhancement of histamine release with
concentrations of BIS II or Ro-31-8220 in the 0.5-10 µmol/L range.
This enhancement was not observed across an anti-IgE antibody dose
response curve, ie, using a single concentration of Ro-31-8220 (1 µmol/L) and several concentrations of anti-IgE antibody, the Ro
compound slightly inhibited supraoptimal concentrations of anti-IgE and
slightly enhanced suboptimal concentrations of anti-IgE (data not
shown). As noted in Fig 7C, there is also a suggestion that Ro-31-8220
enhanced histamine release somewhat better than noted when BIS II was
used, although the variability among donor preparations did not allow
statistical significance to be achieved. This difference between
Ro-31-8220 and BIS II persisted when cells were stimulated with
antigen. The test with antigen differed from a simple dose-response
curve. Basophils were sensitized with serial dilutions of
anti-gp120-IgE such that in the absence of any drug, histamine release
after challenge with an optimal concentration of gp120-OVA conjugate
(50 ng/mL), varied from 10% and 60% according to the concentration of
IgE used for sensitization (320 ng/mL, 800 ng/mL, 2,000 ng/mL, or 5,000 ng/mL). In cells sensitized with low concentrations of IgE, enhancement
with Ro-31-8220 was marked, 2.26- ± 0.37-fold. Although BIS II did
result in statistically significant enhancement, the effects
were modest (1.5- ± 0.15-fold at the lowest sensitization condition, P = .042;
Table 1).
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|
Table 1.
Enhancement of Histamine Release by Two
Indolylmaleimides From Basophils Sensitized With Anti-gp120 IgE and
Stimulated With gp120-OVA
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In contrast, both calphostin C and Go-6976 completely inhibited
histamine release. Go-6976 inhibited IgE-mediated histamine release
with an IC50 that was threefold lower than required for inhibition of
PMA-induced release. This observation is readdressed below. All of the
five compounds inhibited FMLP-induced histamine release to varying
extents although, with the exception of calphostin C, the
concentrations required were markedly higher than required for
inhibition of PMA-induced release.
The disparity in results with the indolylmaleimide compounds versus
Go-6976 suggested a problem with the specificity of Go-6976. We
examined whether these compounds had any effect on the IgE-mediated cytosolic calcium response, [Ca++]i. Our
expectation was that there would be no inhibition if the compounds were
acting in ideal manner, inhibition of PKC only. Figure 8A shows that this held true for BIS
II, the kinetic curves for the IgE-mediated
[Ca++]i response were very similar in the
presence or absence of the compound. In a single experiment not shown,
treatment with 2 µmol/L Ro-31-8220 also had no effect on the
[Ca++]i response. On the other hand, Go-6976
completely inhibited the IgE-mediated [Ca++]i
response (Fig 8B). The ability of BIS II to reverse the PMA-induced inhibition of the [Ca++]i elevation that
follows stimulation with anti-IgE antibody was also examined. Figure 8C
shows that BIS II at 400 nmol/L partially reversed this inhibition and
1 µmol/L BIS II almost completely reversed the inhibition. For these
experiments, it was possible to measure another distinct PMA-induced
response: PMA induces morphological changes that are manifested as
flattening and spreading of the cells. Before the stimulation with
anti-IgE antibody in these experiments, cells were incubated ± BIS
II for 10 minutes followed by ± PMA for 5 minutes (see Fig 8
legend). Both preceeding and following the 5 minutes ± PMA, the
cell perimeters were measured. We have previously used this measure to
study shape change in basophils.44,45 For a 5-minute
incubation, 10 ng/mL of PMA induced modest but consistent changes in
the cell perimeters: expressed as a fraction of the starting cell
perimeter, the increase averaged 1.45 ± .02 after PMA. Buffer alone
for 5 minutes shows a slight increase (due to continued cell
attachment) of 1.21 ± .01 whereas pretreatment with BIS II led to a
change similar to buffer alone, 1.23 ± .02. For this endpoint, 400 nmol/L BIS II completely reversed the effect of PMA.
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| Fig 8.
Effect of PKC inhibitors on the
[Ca++]i response to anti-IgE antibody in
basophils. Purified basophils (88% ± 4%) were labeled with fura-2
and analyzed by digital microscopy for changes in [Ca++]i in the presence or absence of the
noted drugs. Panel A shows the results for 400 nmol/L
bis-indolylmaleimide II and panel B the results for 100 nmol/L Go 6976 where the basophils were stimulated with 0.5 µg/mL anti-IgE antibody
(n = 2 for each drug tested). Panel C shows the average results for
three experiments in which basophils were incubated with or without 400 nmol/L BIS II for 10 minutes followed by a 5-minute treatment with or
without 10 ng/mL PMA and then a stimulation with 0.5 µg/mL anti-IgE
antibody. The conditions were (1) neither BIS nor PMA before challenge
with anti-IgE antibody, (2) BIS II before incubation with anti-IgE antibody, (3) PMA before anti-IgE antibody, (4) BIS II (400 nmol/L) before PMA that preceeded anti-IgE antibody, and (5) same as 4 but with
1 µmol/L BIS II.
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DISCUSSION |
These studies first established the presence or absence of
several PKC isozymes in human basophils. PKC is expressed in many
cells,18,19 however, PKC was barely detectable in human basophils compared with contaminating cells (lymphocytes and
monocytes). Basophils expressed only 7% ± 3% of the PKC in
contaminating cells. Studies in human neutrophils and
eosinophils48-50 indicate that PKC is also absent and
the authors of these studies suggest that low levels of platelet
contamination probably contribute to faint banding sometimes observed
for this isozyme. We did not explicitly determine the amount of
platelet contamination in our preparations and although it should be
quite low given the nature of the preparative technique, there is a
possibility that the low level we observed was derived from platelets.
Devalia et al48 has suggested that the PKC isozyme is
specifically downregulated during human neutrophil terminal
differentiation. A similar process may apply to basophils and
eosinophils during their terminal differentiation.
In studies of RBL cells, PKC and PKC appear to have different
roles. Washing permeabilized RBL cells resulted in the loss of PKC
isozymes and the secretory response to antigen. A full secretory
response could be reconstituted by the subsequent addition of PKC and , which suggested that PKC and promoted
exocytosis.15 However, these same studies also suggested
that PKC and had an inhibitory effect on phospholipid
hydrolysi |
Abstract
Introduction
Abstract