From the
The rat mast cell line RBL-2H3.1 contains an 85-kDa cytosolic
phospholipase A
The 85-kDa cytosolic phospholipase A
The cDNA that encodes the
cPLA
cPLA
Mast cells contain
cPLA
In this report, studies are
presented showing that cPLA
Cells were fixed by covering the
multi-well slides in the dishes with 10 ml of freshly prepared 2%
formaldehyde (Aldrich, 25,254-9) in PBS and incubating for 20 min
at 37 °C. All subsequent steps were done at ambient temperature.
The multi-well slides were removed from the dishes and washed twice
with PBS (5 min/wash in Coplin jars), and then permeabilized for 5 min
in a Coplin jar containing freshly prepared 0.1% Triton X-100 (Sigma)
in PBS. Cells were washed five times with PBS (5 min/wash in Coplin
jars). The slides were removed from the Coplin jars and placed on a
stack of moist paper towels inside of a plastic basin fitted with an
air-tight lid. The lid was kept closed except during the brief
manipulation of the samples. The liquid between the wells on the slide
was carefully removed by wiping with a paper towel. PBS was
periodically applied to the wells with a pipette so that the wells did
not become dry. The liquid in the wells was removed with a pipette, and
then 3% BSA in PBS (10 µl) was applied to each well with a pipette.
After 15 min, R11683 antiserum (10 µl of serum diluted 1000-fold
with 3% BSA in PBS) was applied to each well. Controls were carried out
in which either the same volume of preimmune serum was used or serum
was omitted. After 2 h the cells were washed four times with PBS (5
min/wash in Coplin jars). Liquid was removed between the wells, and the
cells were treated with 3% BSA in PBS as described above.
FITC-conjugated anti-IgG F(ab`)
As an additional control, a
20-µl portion of R11683 antisera (diluted 500-fold in PBS with 3%
BSA, approximately 0.6 µg of IgG) was mixed with an equal volume of
purified cPLA
To stain nuclei, cells were processed as
described above. Just prior to mounting coverslips, the wells were
treated with 4`,6-diamidino-2-phenylindole (1 µg/ml in PBS
containing 3% BSA, Molecular Probes) for 1 min. The cells were washed
four times in PBS (5 min/wash in Coplin jars) and coverslips were
mounted as above.
Slides were immediately viewed by microscopy or
wrapped with aluminum foil and stored at 4 °C for up to 24 h prior
to microscopy. Fluorescence microscopy was carried out with a Nikon
Microphot-FXA epifluorescence microscope (300
Cells were processed as described for fluorescence microspopy
(using 2 ml of solutions per well) up to and including the washing
steps following the treatment with anti-cPLA
The cells were
then processed for electron microscopy. PBS was removed from the wells,
and the cells were covered with anti-rabbit IgG-labeled 15-nm gold
particles (Janssen Life Science Products, Olen, Belgium, diluted
10-fold in PBS). After 16 h at 4 °C, the fluid was removed, and the
cells were covered with 2% glutaraldehyde in PBS for 20 min at 20
°C. The cells were washed several times with PBS, and then
post-fixed in 1% osmium tetroxide in water (Ted Pella, Inc.),
dehydrated with ethanol, and embedded in Medcast (Ted Pella, Inc.) The
samples were cut by a diamond knife at an oblique angle of 30°.
Sections were stained in uranyl acetate and lead citrate, and then
viewed with a JEOL 100B electron microscope at 60 kV as described
previously(43) .
To visualize the Golgi apparatus, cells were grown
on multi-well slides. The medium was removed, and the cells were washed
twice with PBS. Cells were covered with PBS containing 40 µM C
Arachidonate Release-Cells were grown in 12-well
plates as described above. To each well was added 0.25 µCi of
[
Cell suspension (0.33 ml) was transferred to a 1-ml
fluorescence cuvette containing 0.66 ml of BSS, 0.1% BSA, 1 mM CaCl
In
some cases high background staining was seen on the immunoblots. This
problem was alleviated by submerging the membrane in stripping buffer
(100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH
6.7) at 50 °C for 30 min with occasional agitation. The membrane
was rinsed twice (10 min each) with TTBS on a rotary shaker at ambient
temperature. Blocking was carried out with TTBS, 5% nonfat dry milk for
1 h at ambient temperature on a rotary shaker. The membrane was treated
with primary antibody and processed for ECL detection as described
above.
Immunoblot analysis of total protein (soluble +
membrane-bound) extracted from RBL-2H3.1 cells clearly reveals the
presence of cPLA
RBL-2H3.1 cells when activated
release a number of hyrolytic enzymes including
Despite
these concerns, subcellular fraction studies with RBL-2H3.1 cells were
carried out. Cells bathed in RBL-BSS containing 1 mM CaCl
Images obtained by conventional fluorescence microscopy
are shown in Fig. 4, and those obtained by confocal fluorescence
microscopy are shown in Fig. 5. Visualization of microtubules
with anti-
The following controls were carried out. Cells were treated with
secondary antibody (FITC-conjugated, goat anti-rabbit IgG
F(ab`)
Translocation of cPLA
The results of this study provide strong evidence for the
translocation of cPLA
Pulse-chase experiments have shown that radiolabeled arachidonate
when added to the mouse fibrosarcoma cell line
(HSDM
Further work will be needed to determine if the selective
binding of cPLA
We are grateful to C. C. Leslie for helpful
discussions and to R. L. Wright and P. M. Brunner for help with
microscopy.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(cPLA
) that is very likely
involved in liberating arachidonate from membrane phospholipid for the
synthesis of eicosanoids following stimulation with either calcium
ionophore or IgE/antigen. In this study, the intracellular location of
cPLA
was determined using immunofluorescence microscopy and
immuno-gold electron microscopy. In nonstimulated cells, cPLA
is distributed throughout the cytosol and is excluded from the
nucleoplasm. Following cell activation with calcium ionophore, most of
the cPLA
translocates to the nuclear envelope, and the
enzyme remains there during the entire period that ionophore is
present. With IgE/antigen stimulation for 5 min, approximately
20-30% of the cPLA
translocates to the nuclear
envelope, and after 30 min of stimulation, most of the enzyme returns
to the cytosol. Measurement of intracellular calcium using the dye
Fura-2/AM shows that the level of calcium rises immediately after
antigen is added, remains high for about 30 s, and then declines back
to resting levels. Activation with calcium ionophore produces a 10-fold
larger release of arachidonate than does stimulation with IgE/antigen.
Thus, the results suggest that the extent of membrane binding of
cPLA
correlates with the release of arachidonate and that
the site of arachidonate liberation is the nuclear envelope where many
of the enzymes that oxygenate this fatty acid are located.
(cPLA
)
(
)has been identified
and purified from a number of mammalian cells including rodent
macrophage cell lines(1, 2, 3) ,
platelets(4, 5, 6, 7) , human monocytic
cell lines(8, 9, 10, 11) , and rat
kidney(12) . In contrast to the well-characterized 14-kDa
secreted PLAs
that require millimolar amounts of calcium as
a catalytic cofactor(13) , cPLA
is activated by
submicromolar amounts of Ca
. Furthermore,
Ca
is required for the binding of cPLA
to
cell membranes(9, 11, 14, 15, 16, 17) and to
synthetic phospholipid vesicles(14, 18, 19) ,
but does not serve as a catalytic
cofactor(14, 18, 20) . cPLA
is found
in the soluble fraction of cellular homogenates when the Ca
concentration is low (typically <0.1 µM), and it
is found in the particulate fraction in the presence of submicromolar
to micromolar amounts of Ca
. The enzyme in cells is
also activated in part by
phosphorylation(21, 22, 23, 24, 25, 26, 27) ,
although the molecular basis of this phenomenom is not known since the
phosphorylated enzyme shows a very modest increase in catalytic
turnover when examined in vitro.
from human U937 cells shows no homologous regions to
14-kDa secreted phospholipases A
(14, 28). The amino acid
sequence reveals a stretch of 45 residues in the amino-terminal region
that shows homology to Ca
-dependent forms of protein
kinase C and other Ca
-dependent membrane binding
proteins. A 140-amino acid fragment of cPLA
that contains
the amino terminus was shown to bind to membranes in the presence of
submicromolar amounts of Ca
(14, 29) .
preferentially hydrolyzes phospholipids with an sn-2 arachidonyl chain, whereas 14-kDa phospholipases A
display essentially no acyl-chain
specificity(30, 31, 32, 33) . All of
these results suggest the cPLA
plays a role in
signal-mediated release of arachidonic acid from membrane phospholipids
for the genesis of eicosanoids, and thus cPLA
may be a good
target for antiinflammatory therapeutics.
(34) and two additional phospholipases
A
. One is a 14-kDa enzyme of the type II variety that is
secreted from activated mast cells(34, 35) , and the
other is a 30-kDa enzyme that prefers phosphatidylserine(34) .
The role of these enzymes in mast cell function is being clarified.
Inhibitors of the 14-kDa enzyme cause suppression of histamine release,
suggesting that this enzyme is a component of the mediator release
pathway(34) . Furthermore, addition of purified 14-kDa
phospholipase A
to mast cells leads to histamine
release(36) . The role of the 14-kDa enzyme in eicosanoid
generation in mast cells is controversial. One report indicates that
addition of exogeneous mast cell type II 14-kDa phospholipase A
to mast cells does not lead to PGD
production(36) , while another group provides evidence
that addition of the 14-kDa phospholipase A
from cobra
venom leads to arachidonate liberation and production of cyclooxygenase
products(35) . These differences are likely due to the different
affinities of 14-kDa phospholipases A
for mast cell
membranes. The role of mast cell cPLA
in arachidonate
liberation is not clear, but recent studies show that this enzyme
becomes phosphorylated in response to IgE
cross-linking(37, 38) . In platelets, cPLA
becomes phosphorylated in response to stimulation with
thrombin(22) , and studies with potent cPLA
inhibitors provide strong evidence for the role of this enzyme in
arachidonic acid liberation(39, 40) . Antisense RNA
technology has been used to show that cPLA
is involved in
eicosanoid generation in monocytes stimulated with bacterial
lipopolysaccharide and platelet-activating factor(41) . No role
for the phosphatidylserine-specific phospholipase A
in mast
cell function has been reported.
in rat basophilic leukemia
cells (RBL-2H3.1) is located mainly in the cytosol in unactivated
cells, and this enzyme translocates to the nuclear envelope in response
to stimulation with calcium ionophore or IgE/antigen. The results
suggests that cPLA
is involved in the release of at least
some of the arachidonic acid from membrane phospholipid of activated
mast cells.
Materials
Recombinant human cPLA was
prepared from baculovirus-infected Sf9 cells as described(42) .
The polyclonal antiserum R11683 to cPLA
and preimmune serum
were obtained as generous gifts from Prof. C. C. Leslie (National
Jewish Center for Immunology and Respiratory Medicine, Denver, CO).
R11683 was obtained by injection of rabbits with full-length cPLA
that had been extracted from a gel following SDS-PAGE. Affinity
purified FITC-conjugated, goat anti-rabbit IgG F(ab`)
is
from Organon Teknika Corp. (Durham, NC). Ionomycin is from Sigma.
Anti-
-tubulin IgG and anti-BiP antisera were obtained as generous
gifts from Prof. Frank Solomon (Massachusetts Institute of Technology)
and Prof. Linda Hendershot (St. Jude's Childrens Hospital),
respectively.
Cell Culture
RBL-2H3.1 cells were obtained as a
generous gift from Professor B. A. Helm (University of Sheffield,
United Kingdom). Cells were routinely cultured at 37 °C in a
humidified atmosphere of 5% CO in dishes of minimal
essential medium (Life Technologies, Inc., 330-1650-AJ) with
heat-inactivated fecal calf serum (12% by volume), NaHCO
(29.3 ml/liter of 7.5% solution), glutamine (2 mM, added
every 2 weeks). Media also contained penicillin/streptomycin/Fungizone
(JHR Biosciences). Adherent cells were routinely dislodged by treatment
with trypsin/EDTA solution except as noted below.
Preparation of Cells for Fluorescence
Microscopy
For all studies described in this report, cells were
grown to approximately 50% confluence as described above. For
fluorescence microscopy, cells were cultured in 10-cm dishes containing
multi-well glass slides (10 wells/slide, Cell Line Associates, Inc.,
Newfield, NJ). Washing and fixation of cells in dishes were carried out
by adding the appropriate solution to the cells (10 ml/10-cm dish), and
after the desired time the solution was removed by aspiration followed
by the immediate addition of the next solution. For stimulation with
the Ca ionophore A23187 (Sigma), cells were first
washed three times with Hank's BSS with CaCl
(Life
Technologies, Inc., 14025-019). Washed cells were covered with 10
ml of Hank's BSS with CaCl
and stimulated by addition
of A21387 (final concentration 10 µM, 5 mM stock
solution in Me
SO, control cells were treated with
Me
SO only). Cells were incubated at 37 °C for 30 min
and then fixed as described below.
antiserum (10 µl of
serum diluted 300-fold with 3% BSA in PBS) was applied to each well.
After 1 h, the cells were washed four times with PBS (5 min/wash in
Coplin jars), in some cases the nuclei were stained (see below), and
coverslips were mounted over the wells using Citifluor mounting
solution (Ted Pella, Redding, CA).
(0.5 µg/µl in PBS). After 30 min at
room temperature the mixture was applied to the wells containing fixed
cells as described above.
magnification,
oil immersion). Cells were observed with either a fluorescein filter
(excitation 480 nm, barrier 535 nm) or a rhodamine filter (Nikon DM580
filter; excitation 546 nm, barrier 590 nm) depending on the dye probe
used. Confocal fluorescence microscopy was carried out with a Bio-Rad
MRC-600 confocal laser scanning microscope using the 488 line of a
krypton/argon laser (for viewing fluorescein) and a 60
objective lens.
Preparation of Cells for Electron Microscopy
For
electron microscopy, cells were grown in 12-well plastic dishes as
described above. Stimulation with A23187 was carried out as described
above. For stimulation with DNP-HSA, cells were first primed by adding
anti-DNP IgE (final concentration 0.5 µg/ml, obtained as a generous
gift from Professor B. A. Helm, or purchased from Sigma) directly to
the culture medium, and the cells were incubated at 37 °C for
12-24 h. Cells were washed three times with pre-warmed RBL-BSS (1
liter contains 8.67 g of NaCl, 0.365 g of KCl, 1.64 g of D-sorbitol, 0.6 g of KHPO
, 0.14 g of
KH
PO
, 4.83 g of HEPES, pH 7.40) containing 0.1%
BSA and 1 mM CaCl
. Cells were covered with the
same solution, and DNP-HSA was added (final concentration 100 ng/ml, 3
mg/ml stock solution, obtained as a generous gift from Professor B. A.
Helm). Cells were incubated at 37 °C for various times, and then
fixed.
antisera
(R11683 antiserum was used at a dilution of 150-fold rather than the
1000-fold dilution used for fluorescence microscopy). At this stage,
the prepared cells were covered with PBS, the dish covers were sealed
to the dishes by wrapping with Parafilm, and the dishes kept at 4
°C in an air-tight container for up to 1 week.
Visualization of Organelles
To visualize
mitochondria, cells were grown on multi-well slides. Mitotracker
(Molecular Probes) was added to the culture medium to give a final
concentration of 25 nM. After incubation at 37 °C for
15-45 min, the cells were washed twice with PBS and then fixed as
described above. Coverslips were mounted with Citifluor as described
above, and the cells were viewed by fluorescence microscopy using the
rhodamine filter.
-DMB-ceramide (Molecular Probes) and incubated for 20
min at 37 °C. Cells were washed with PBS, and coverslips were
mounted with Citifluor as described above. Cells were viewed
immediately by fluorescence microscopy using the fluorescein filter. To
visualize tubulin, cells were prepared as described above for
cPLA
visualization except that the anti-tubulin antiserum
was used (1:250 dilution).
H]arachidonic acid (60-100 Ci/mmol, DuPont
NEN), and to some of the wells was added anti-DNP IgE (final
concentration 0.5 µg/ml). Cells were incubated for 24 h at 37
°C. Cells that were to be treated with Ca
ionophore were washed three times with prewarmed Hank's BSS
containing 0.1% BSA, and those that were to be treated with DNP-HSA
were washed with prewarmed RBL-BSS containing 0.1% BSA and 1 mM CaCl
. Cells were stimulated with A23187 or DNP-HSA as
described above. Cells were incubated at 37 °C for variable times,
and then the culture media above the cells was transferred to
microcentrifuge tubes. After centrifugation of the samples to remove
small amounts of dislodged cells (20 s at 16,000
g),
90% of the supernatants were transferred to scintillation vials. To the
wells containing the remaining adherent cells was added 1 ml of
CHCl
:MeOH (2:1 by volume). This extract was transferred to
the microcentrifuge tubes containing small amounts of pelleted cells,
and after brief mixing with a vortexer, the samples were transferred to
scintillation vials. Scintillation fluid was added to all samples, and
the samples were analyzed by scintillation counting in a Beckman LS
1801 counter. Release of radiolabeled arachidonic acid is expressed as
the percentage of the total cellular radiolabeled arachidonate released
into the culture medium for each culture well.
Ca
Increase in intracellular Ca Mobilization
was measured essentially as described(44) . Cells were
grown in a 75-cm
flask as described above, and primed with
anti-DNP IgE as described above. Nonenzymatic cell dissociation
solution (5-10 ml/flask, Sigma catalog number C-5789) was added,
and after 10-15 min the cells were dislodged by banging the plate
several times against the lab bench. Cells were collected by
centrifugation and resuspended at a density of 2
10
/ml in BSS. Fura-2/AM (Molecular Probes) was added to a
final concentration of 2 µM, and the cells were incubated
at 37 °C for 20 min with periodic swirling. Cells were washed three
times with BSS containing 0.1% BSA (Sigma A-7906) and resuspended to a
density of 1
10
/ml in BSS containing 0.1% BSA and 1
mM CaCl
. Cells were kept on ice and in the dark
for up to 2 h.
. The sample was equilibrated in a thermostated
holder to 21 °C and stirred with a small Teflon stir bar.
Fluorescence was monitored with excitation at 340 nm and emission at
490 nm. Other additives are described in the legend to Fig. 3.
Figure 3:
Ca mobilization in
RBL-2H3.1 cells following stimulation with Ca
ionophore or IgE/antigen. The fluorescence emission from
Fura-2/AM is plotted as a function of time. Bottom, cells that
were not primed with anti-DNP IgE were treated with DNP-HSA (100 ng/ml)
and then with ionomycin (7 µM, final concentration) at the second arrow and finally with a second portion of ionomycin
(14 µM total final concentration). Top, anti-DNP
IgE-primed cells were treated with DNP-HSA and then with ionomycin.
Other conditions are described under ``Experimental
Procedures.''
-Hexosaminidase Release-Essentially the procedure
of Tanaka et al.(45) was followed. Cells were grown in
12-well plates. Cells were treated with anti-DNP IgE, washed, and
stimulated with DNP-HSA as described above. After 5 or 15 min at 37
°C, aliquots of culture media (200 µl) were removed and added
to an assay mixture (900 µl) consisting of 4 mMp-nitrophenyl-2-acetamido-2-deoxy-
-D-glucopyranoside
(Sigma) in 40 mM sodium citrate, pH 4.5. The mixtures were
incubated at 37 °C for 30 min, and the reactions were stopped by
adding 1 ml of 0.2 M glycine, pH 10.5. The amount of product
was determined by measuring the absorbance at 410 nm using a
calibration curve prepared by adding known amounts of p-nitrophenol to the assay mixture.
Cell Fractionation
Cells were grown in 15-cm
culture dishes, the media was aspirated, and cells were scraped into 4
ml of RBL-BSS. Cells from three plates were pooled, and the suspension
was centrifuged at 300 g for 10 min at room
temperature in a Sorvall RC-5 centrifuge using an SS-34 rotor. The cell
pellet was resuspended in 4 ml of RBL-BSS containing 1 mM
CaCl
, and divided among 4 tubes. Five µl of a 2 mM Me
SO solution of A23187 was added to two of the tubes,
and 5 µl of Me
SO was added to the remaining tubes.
After 5 min at room temperature, the cells were spun down and
resuspended in 250 µl of 20 mM Tris, pH 7.4, 10 mM KCl, 2 mM MgCl
, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20
µg/ml aprotinin, and either 10 mM EGTA or 0.2 mM EGTA plus 0.3 mM CaCl
. After 20 min on ice, 3
volumes of 50 mM Tris, pH 7.4, 25 mM KCl, 5
mM MgCl
, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 20
µg/ml aprotinin, and either 10 mM EGTA or 0.2 mM EGTA and 0.3 mM CaCl
was added, and the cells
were immediately homogenized using a 2-ml glass Dounce homogenizer with
a loose fitting pestle (Kontes, B pestle). Greater than 95% of the
cells were lysed after 15-20 vigorous strokes, based on trypan
blue exclusion. The lysates were centrifuged at 1000
g for 10 min, and the pellets were resuspended in 300 µl of
buffer (low speed pellet fraction). The supernatant was centrifuged at
100,000
g for 1 h, and the membrane pellet was
resuspended in 300 µl of buffer (high speed pellet fraction). Under
microscopic examination using either trypan blue or Diff-Quik Stain
(Baxter), the low speed pellet contained crude nuclei, while the high
speed pellet and high speed supernatant (cytosolic fraction) contained
no visible nuclei. Cellular fractions were flash frozen in liquid
nitrogen and stored at -80 °C. Equal volumes of cell
fractions were thawed and applied to a 10% SDS-PAGE minigel. cPLA
was detected by immunoblot analysis as described below.
Immunoblotting
Analysis of cell extracts by
SDS-PAGE/immunoblotting was carried out essentially as
described(24) . Cells were grown in 10-cm dishes and washed
twice with PBS. Cells were scrapped into 200 µl of ice-cold lysis
buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, v/v,
1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 20 µM sodium orthovanadate, 10 mM tetradsodium pyrophosphate,
100 mM sodium fluoride, 3 µMp-nitrophenyl phosphate, 10 µg/ml aprotinin, 10
µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride,
pH 7.4; protease and phosphatase inhibitors were added fresh from
frozen stocks. Lysates were incubated on ice for 30 min and then
centrifuged (10 min at 13,000 g) for 10 min. The
protein concentration in the supernatant was estimated using a BCA
protein assay kit (Pierce), Laemmli sample buffer containing
dithiothreitol was added, and 8 µg of protein was loaded on each
lane of a 7.5% SDS-polyacrylamide gel (Laemmli, Bio-Rad
Mini-Protean-II). Prestained molecular weight markers (Bio-Rad) and
known amounts of purified cPLA
(typically 3 ng) were also
loaded in separate lanes. After electrophoresis, protein was
transferred to a nitrocellulose membrane using an electroblotter
(Bio-Rad). Following transfer, the gel was stained with Coomassie Blue
to verify the efficiency of transfer. The membrane was rinsed once for
15 min with TTBS (20 mM Tris, 137 mM NaCl, 500
µl/liter Tween 20, pH 7.6) and then incubated in TTBS containing 5%
nonfat dry milk overnight at 4 °C on a rotary shaker. After
blocking, the membrane was incubated in fresh TTBS, 5% nonfat dry milk
containing R11683 antisera (diluted 12,000-fold) for 2 h at room
temperature on a rotary shaker. The membrane was rinsed four times (15
min/rinse) with TTBS on a rotary shaker at room temperature. The
immunoblot was visualized using the ECL kit from Amersham using
horseradish peroxidase-conjugated anti-rabbit IgG diluted 5,000-fold.
The blot was exposed to Kodak X-AR x-ray film for up to 10 min.
RBL-2H3.1 Cells Contain cPLA
The
RBL-2H3.1 cells studied in this report is a subclone (46) of the
well established RBL-2H3 cell line of mast cell lineage. RBL-2H3.1
cells support high levels of mediator secretion in response to
stimulation with IgE and antigen compared to the subclone RBL-2H3.2
which supports 10-fold lower mediator release(44) . Biochemical
characterization of these subclones has shown that the low secretor
displays a reduced extent of phosphorylation of the cell surface, high
affinity IgE receptor complex (44). This low level phosphorylation
leads to subnormal production of inositol phosphate and thus a reduced
increase in intracellular Ca. For the purposes of the
present study, the high secreting variant was used in order to guard
against the possibility that signal transduction between the IgE
receptor complex and cPLA
is blocked in low secreting
variants.
as a major band of apparent molecular mass
of approximately 100-kDa (Fig. 1); the actual molecular mass of
cPLA
is nominally 87-kDa(14, 28) , and its
anomalously slow migration on SDS-PAGE gels is well
documented(2, 8, 10) . The R11863
anti-cPLA
antisera used in this analysis has relatively
high affinity for cPLA
since it can be routinely used for
immunoblotting at a dilution of 12,000-fold (Fig. 1). Even
50,000-fold dilution of this antibody gives a strong immunblot response
(not shown). The additional minor bands of higher mobility seen in the
immunoblots are also seen in highly purified cPLA
expressed
as a recombinant protein in insect cells (Fig. 1), suggesting
that they are proteolytic degradation products of cPLA
. The
presence of cPLA
in RBL-2H3 cells has also been
demonstrated by Inoue and co-workers (47) who partially purified
this enzyme from these cells.
Figure 1:
Immunoblot analysis of cPLA in RBL-2H3.1 extracts probed with the R11683 anti-cPLA
antiserum. Lanes 1-3, protein (8 µg) extracted
from RBL-2H3.1 cells. Lane 4, purified cPLA
(3 ng)
expressed as a recombinant protein in insect cells. The positions of
the molecular weight markers are indicated. Other conditions are as
described under ``Experimental
Procedures.''
Response of RBL-2H3.1 Cells to Stimulation with
Ca
Several
studies have shown that RBL-2H3 cells liberate arachidonic acid in
response to stimulation with CaIonophore or IgE/Antigen
ionophore or
IgE/antigen (for example, see Refs. 48-51). In order to
demonstrate that the subclone RBL-2H3.1 is capable of stimulus-mediated
arachidonate mobilization, these cells were labeled with
[
H]arachidonate for 24 h and unincorporated fatty
acid was removed by extensive washing with buffer containing BSA. Cells
that were stimulated with antigen (DNP-HSA) were primed overnight with
anti-DNP IgE. The results are shown in Fig. 2. Stimulation with
Ca
ionophore results in arachidonate release over 30
min approaching 30% of the total radiolabeled fatty incorporated into
the cells, whereas very little release was seen in the absence of
ionophore. Compared to ionophore, stimulation with IgE/antigen resulted
in a lower level of arachidonate release over 30 min but well above
that measured in cells that were stimulated with antigen but were not
primed with IgE. Similar trends have been reported previously for
RBL-2H3 cells(49, 52, 53, 54) .
Figure 2:
Release of radiolabeled arachidonic acid
from RBL-2H3.1 cells stimulated with Ca ionophore or
IgE/antigen. Release is expressed as the percent of total cellular
radiolabeled arachidonic acid that was released into the culture
medium. Filled triangles, with A23187; open triangle,
without A23187; filled circles, anti-DNP IgE-primed cells
stimulated with DNP-HSA; open circle, stimulated with DNP-HSA
but without IgE priming. Other conditions are described under
``Experimental Procedures.''
The
results in Fig. 3show that RBL-2H3.1 cells mobilize
Ca in response to stimulation with IgE/antigen as
reported previously(44) . The fluorescence emission of
Fura-2/AM-loaded cells was monitored in real time before and after the
addition of stimulus. Stimulation of the cells that were not primed
with IgE with the Ca
ionophore ionomycin led to an
immediate rise in intracellular Ca
, and this
increased Ca
level was persistent and was not altered
by the addition of a second portion of ionomycin (Fig. 3, bottom). A23187 was not used for these experiments because of
its high autofluorescence. In the absence of ionophore, antigen
promoted an increase in intracellular Ca
but only if
the cells were primed with IgE. In this case, the Ca
level started to rise 5-10 s after the addition of antigen,
and the increase lasted 10-15 s. Ca
remained
high for about 25 s and then started to fall over 15-20 s to
nearly the unstimulated level at which point addition of ionomycin
resulted in an immediate increase in intracellular Ca
(Fig. 3, top).
-hexosaminidase.
Release of this enzyme from RBL-2H3.1 cells was monitored with a
colorimetric substrate as described under ``Experimental
Procedures.'' Released enzyme levels from cells primed with
anti-DNP IgE and stimulated with DNP-HSA for 15 min were 5-6-fold
higher than those measured in the absence of DNP-HSA and 5-6-fold
higher than those measured in the presence of DNP-HSA but in the
absence of priming with IgE (not shown).
Cell Fractionation Studies
Locating cPLA in cells by subcellular fraction studies is likely to be
problematic because the distribution of enzyme between membranes and
cytosol depends on the concentration of Ca
in the
homogenation buffer. For example, when RAW 264.7 macrophages are
homogenized in the presence of EGTA most of the cPLA
appears in the cytosol, but when the homogenation buffer contains
>200-300 µM Ca
, 60-70% of
the enzyme is recovered in the membrane fraction(15) . Similar
results have been reported for U937
cells(8, 9, 10) , neutrophils(55) , rat
liver macrophages(16) , and platelets(56) . Furthermore,
membrane-bound enzyme can be released in variable yields by treatment
with EGTA(15, 16) . Thus, translocation studies
involving cell homogenation may be more a reflection of the conditions
of the homogenation rather than of the events that occur in an intact
and stimulated cell. In addition, since the membrane binding is
reversible, there could be scrambling of cPLA
between
different cellular membranes during the fraction procedure.
were treated either with A23187 or vehicle as
described under ``Experimental Procedures.'' In both cases,
cells were homogenized either in the absence of Ca
(10 mM EGTA) or in the presence 100 µM free
Ca
, and cytosol, nuclear pellet, and crude membrane
pellet were isolated as described under ``Experimental
Procedures.'' Surprisingly, under all conditions essentially all
of the cPLA
detected by immunoblotting was found in the
cytosolic fraction even when the cells were homogenized in the presence
of 100 µM free Ca
. This result differs
significantly from results reported with other cell types as referenced
above. Given this result and the problems inherent in using subcellular
fractionation to determine the cellular site of binding of cPLA
to membranes, attempts were made to localize cPLA
in
fixed and permeabilized cells using immunocytochemistry combined with
microscopy. Such an approach has been used to localized other
Ca
-dependent membrane binding proteins such as
protein kinase C(57, 58) .
Visualization of cPLA2 in RBL-2H3.1 Cells by
Immunofluorescence Microscopy
Fixation and permeabilization
of RBL-2H3.1 cells for microscopy was carried out essentially as
described previously for the RBL cell line(59) . Fluorescent
anti--tubulin IgG was used to evaluate the integrity of the
prepared cells. Among the fixatives formaldehyde, paraformaldehyde, and
glutaraldehyde, formaldehyde was found to give the best results, and
permeabilization with Triton X-100 was superior to that obtained with
methanol.
-tubulin IgG (Fig. 4, upper left) shows
the expected pattern of strands radiating outward from the microtubule
organizing center located near the center of the cell. Fig. 4(upper right) shows the mitochondria (visualized
with the dye Mitotracker) scattered throughout the cytosol. Most of the
Golgi complex (visualized with the dye C
-DMB-ceramide)
appears close to and on one side of the nucleus (Fig. 4, middle left). The confocal image of the endoplasmic reticulum (Fig. 5, upper right) shows the expected fluorescence
emission from the nuclear envelope and regions radiating away from the
nucleus and throughout the cytosol. This organelle was visualized with
antisera to the immunoglobulin heavy chain binding protein (BiP)
previously shown to reside in the endoplasmic reticulum(60) .
Figure 4:
Conventional fluorescence microscopy of
RBL-2H3.1 cells. Top left, microtubules visualized with the
anti--tubulin IgG antibody; top right, mitochondria
visualized with Mitotracker; middle left, the Golgi complex
visualized with C
-DMB-ceramide; middle right,
cPLA
in A23187-activated cells visualized with
anti-cPLA
antiserum and FITC-conjugated, goat anti-rabbit
IgG F(ab`)
; lower left, nuclei visualized with
4`,6-diamidino-2-phenylindole.
Figure 5:
Confocal fluorescence microscopy of
RBL-2H3.1 cells. Top left, cPLA in resting cells
visualized with anti-cPLA
antiserum and FITC-conjugated,
goat anti-rabbit IgG F(ab`)
. The white bar has a
length of 20 µm; top right, the endoplasmic reticulum
visualized with anti-BiP antiserum and FITC-conjugated, goat
anti-rabbit IgG F(ab`)
; lower left, cPLA
in A23871-activated cells visualized with anti-cPLA
antiserum and FITC-conjugated, goat anti-rabbit IgG
F(ab`)
; lower right, same as lower left except a different cell was viewed (this image is zoomed a factor
of 1.5 relative to the other images).
Fig. 4(middle right) shows the image obtained with
the anti-cPLA antiserum in A23187-treated cells. It can be
seen that cPLA
tends to collect on the nuclear envelope;
the nucleus was viewed with the stain 4`,6-diamidino-2-phenylindole (Fig. 4, lower left). This cell has two nuclei, but
similar nuclear envelope staining was seen in fluorescence images of
cells with a single nucleus. The confocal images of cPLA
location are very striking. In the resting cell, cPLA
exists throughout the cytosol but excluded from the nucleoplasm (Fig. 5, upper left). Following stimulation with A23187,
cPLA
translocates to the nuclear envelope (Fig. 5, bottom left and right). The pattern of fluorescence
is significantly different than that seen with the endoplasmic
reticulum marker antibody in that most of the membrane-bound cPLA
appears on the perimeter of the nucleus rather than fanning out
into the cytosol. However, it is difficult to rule out that a sizeable
portion of cPLA
is bound to the endoplasmic reticulum.
) in the absence of anti-cPLA
antiserum;
cells were treated with preimmune serum followed by secondary antibody;
cells were treated with anti-cPLA
antiserum that had been
previously incubated with an excess of purified cPLA
, and
the cells were then treated with secondary antibody. In all cases,
conventional and confocal fluorescence images obtained with the same
film exposure as those shown in Fig. 4and Fig. 5were
featureless (not shown).
Visualization of cPLA2 in RBL-2H3.1 Cells by
Immuno-gold Electron Miscroscopy
RBL-2H3.1 cells were
treated with either calcium ionophore or IgE/antigen, fixed, and
permeabilized as described for the fluorescence microscopy studies, and
then further processed for electron microscopy using anti-rabbit
IgG-labeled gold particles. The images are shown in Fig. 6. In
the absence of stimulation, nearly all of the cPLA (>95%
of the gold particles, seen in three independent cell preparations)
appears to be in the cytosol (Fig. 6A). Stimulation with
IgE/antigen for 5 min results in translocation of some of the
cPLA
(20-30%, seen in five independent cell
preparations) to the nuclear envelope (Fig. 6B). After
prolonged treatment with IgE/antigen (30 min) most of the cPLA
(85-95%, seen in three independent cell preparations) is
found in the cytosol (not shown). Stimulation with calcium ionophore
for 5 min results in the translocation of most of the cPLA
(>80% the gold particles, seen in three independent cell
preparations) to regions on or near the nuclear envelope (Fig. 6C). Occasionally, clusters of cPLA
are seen in the images of stimulated cells for unknown reasons (Fig. 6, A and B).
Figure 6:
Electron microscopy images of RBL-2H3.1
cells. A, unstimulated cells; B, cells stimulated for
5 min with IgE/antigen; C, cells stimulated for 5 min with
calcium ionophore. Fifteen-nm immuno-gold particles appear as black
dots (circled). Particles clearly attached to the nuclear
envelope are marked with an arrow, those that form small
aggregates are marked with an arrowhead.
to membranes was not seen
in RBL-2H3.1 cell homogenates prepared in the presence or absence of
calcium. This is in contrast to other reports with
macrophages(15, 16) , brain(17) ,
neutrophils(61) , and mouse mammary gland-derived cells (62) showing that cPLA
is found mainly in the
particulate fraction when the cells are homogenized in the presence of
micromolar amounts of calcium. The reason for this difference is not
known. Although it may be possible to identify homogenation conditions
that render cPLA
membrane bound, the physiological
relevance of such studies would be highly questionable. Thus,
microscopic studies using immunolocalization of cPLA
was
warranted.
from the cytosol to the nuclear
envelope in calcium ionophore- or IgE/antigen-stimulated cells of mast
cell lineage (RBL-2H3.1). This is supported by both fluorescence and
electron microscopies of fixed and permeabilized cells. In a recent
study using immunoblot analysis, cPLA
has been
preferentially detected in nuclei isolated from cavitated rat
peritoneal macrophages stimulated with calcium ionophore(63) .
It is interesting to note that treatment of RBL-2H3.1 cells with
calcium ionophore produced a
10-fold larger release of arachidonate
than did stimulation with IgE/antigen (Fig. 2), and that
immuno-gold electron microscopy reproducibly showed a larger fraction
of cPLA
bound to membranes in calcium ionophore- versus IgE/antigen-stimulated cells. This may be due, at least in part,
to the fact that following IgE/antigen treatment, the rise in
intracellular calcium is transient (Fig. 3); after an initial
rise, intracellular calcium returns to resting levels in about
1-2 min. In contrast, with ionophore treatment, the calcium level
remains elevated. Indeed after a 30-min treatment with IgE/antigen the
amount of membrane-bound cPLA
, as seen by electron
microscopy, is reduced to 10-20% of the total immunologically
detected enzyme (not shown). Furthermore, as seen by fluorescence
microscopy, cPLA
remains on the nuclear envelope even after
30 min of stimulation with calcium ionophore (not shown). It is also
possible that the phosphorylation state of cPLA
is
different in IgE/antigen- versus calcium ionophore-stimulated
cells and that this results in a change in the fraction of
membrane-bound enzyme. The addition of okadaic acid or phorbol
myristate, which are known to enhance the release of arachidonate in
mouse peritoneal macrophages probably by increasing the level of
cPLA
phosphorylation(24) , resulted in no change or
only a 1.7-fold increase, respectively, in released arachidonic
following a 5-min stimulation with IgE/antigen (data not shown).
Further work is needed to fully understand all of the elements that
control the translocation of cPLA
to the nuclear membrane.
C
) most rapidly is found in nuclear
membranes and that this pool is selectively hydrolyzed in response to
bradykinin-induced production of prostaglandin
E
(64) . Although this work is interesting in light
of the present studies, the enzyme responsible for arachidonate
liberation in HSDM
C
cells has not been
identified. It is interesting to note that some of the enzymes that
oxygenate liberated arachidonate are located, at least in part, in
nuclear membranes. The enzyme cyclooxygenase has been detected in
nuclear and endoplasmic reticulum
membranes(65, 66, 67) . The enzyme
5-lipoxygenase in unactivated RBL cells is located in the
nucleus(68) . In a variety of stimulated cells the enzyme is
found in the nuclear
membrane(63, 66, 69, 70) . The protein
5-lipoxygenase activating protein, which is required for the function
of 5-lipoxygenase in cells, is also located in the nuclear
membrane(69) . The enzymes 12- and 15-lipoxygenase are found in
nuclear membranes(66) , endoplasmic reticulum(66) , and
bound to chromatin(66, 71) . If the eicosanoids are
biosynthesized in the nuclear membrane and endoplasmic reticulum, as
appears to be the case, these compounds can very likely spontaneously
exchange between intracellular membranes as well as be released into
the extracellular fluid. This stems from their finite solubility in
both membranes and aqueous phases; the same is true of arachidonic
acid.
to the nuclear envelope is the result of an
endogenous protein receptor or of other factors such as the specific
phospholipid composition of this membrane. Although the binding of
cPLA
to phosphatidylcholine vesicles is enhanced by the
presence of negatively charged lipids(18, 32) , the
fraction of phospholipids that are acidic in the nuclear membrane is
not significantly different that in microsomes or plasma membrane (see,
for example, Refs. 72-74). The possibility of an asymmetric
distribution of acidic phospholipids between inner and outer leaflets
of the nuclear membrane should be considered, but there is little, if
any, data to support such a proposal.
,
85-kDa cytosolic phospholipase A
found in RBL-2H3.1 and
other mammalian cell types; BSA, bovine serum albumin; BSS, balanced
salt solution; DNP-HSA, dinitrophenol conjugated to human serum
albumin; PBS, phosphate-buffered saline (pH 7.2); PAGE, polyacrylamide
gel electrophoresis; FITC, fluorescein isothiocyanate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.