0022-1554/90/$3.30
The
Journal Copyright
©
and
of Histochemistry 1990 by The
Cytochemistry
Histochemical
Vol. 38, No.
Society,
Changes
lonophore
A23187
NORIYUKI
SAHARA,
Laboratory
of Immunology,
Received
for
REUBEN
publication
July
14,
1989
and
Basophilic
of Dental in revised
Research, form
January
RBL-2H3 cells lease in vitro. dergo striking secretion. The the morphological the Fc receptor. lease histamine the Fc receptor
have been widely used to study histamine reIt was previously shown that these cells unmorphological changes after IgE-mediated present study was undertaken to examine if changes were dependent on activation of Therefore, the cells were stimulated to reby two different mechanisms: activation of by antigen and treatment with the calcium ionophore A23187. Cell surface and cytoskeletal changes were examined by fluorescence microscopy and scanning electron microscopy after either IgE- or ionophore-mediated histamine release. After exposure of the cells to either secretagogue, the cells spread over the surface of the culture dish and underwent rearrangement ofthe cytoskeleton. In addition, scanning electron microscopy revealed that deep ruffles developed
on
the
release.
mediated nounced
with
skeletal
elements
surface
The
the
of
the
surface
ionophore.
cells
changes The
undergoing
were
IgE-
not
distribution
as pro-
of the
was examined
by immunofluorescence using and antibodies against vimentin and tubuIn unstimulated cells stain was localized at the cell
Introduction cells and
Mast sitivity
basophils
reactions.
ators from inflammatory
play
Release
a crucial
role in immediate
ofhistamine,
senotonin,
tides
or cytokines, for
The widely
and
treatment
with
allergic and these mcdi-
them activation to certain pep-
ionophores
nat basophilic used
to
mediated
histamine
through ionophone
release
ionophore
leukemia
study
cell
line
release
[see Siraganian
RBL-2H3 in
vitro.
In
(19)
has
the
RBL-2H3
different mechanisms, both cross-linked will induce histamine release (20).
involves produces
activation a calcium
of the influx
Fcc receptor,
without
involvement
and
Institutes
of Health,
4,
accepted
February
periphery,
just
IgE IgE-
calcium
and
and
were
released.
aeased
of
lA23,
Bethesda,
to: Dr. Constance MD
20892.
Oliver,
NIH-NIDR,
Bldg.
the
stimulation in Lucifer
10, Rm.
plasma
membrane.
In the
stimu-
concentration
of ionophore
with
the
yellow
or antigen,
amount of histamine stimulation led to intracer Lucifer yellow,
ionophore
A23187
internalization.
showed
lonophore
no
A23187
produced changes similar but not identical to those seen in the RBL-2H3 cells afterlgE-mediated histamine release. The differences may be owing to the involvement of the Fcc receptor
in IgE-mediated
KEY
IgE; Cytoskeleton;
WORDS:
receptor.
that
IgE-mediated
Previous
if the release
or
the
The
if similar
it was
seen
on
current
with
cells
occurred
determined
unique when
i.e. that
,
ne-
calcium
iono-
exposed
to the
of interest
release
to
to IgE-mehistamine
the
cells
it was then
IgE-mediated
shown
morpholog-
was’ undertaken
were
mechanism,
have
striking
study
changes
changes,
microscopy.
RBL-2H3
produces
changes
exhibited
and cells
were
supplemented
onto
(16)
filaments;
electron
to com-
to those
induced
ionophone.
Materials
Cells
cells.
Once
also
Intermediate
release
by another
A23187.
ionophore by
studies
morphological
was induced
phone
Cytochem
Scanning
histamine
in these
determine lease
Actin;
Immunofluorescence;
the
diatd
secretion.(JHistochem
1990)
Microtubules;
were cells
Correspondence
on
inaease
or histamine
grown
subeultured
per well
13-mm
Methods in Eagle’s
minimum
with 15% fetal calfserum,
Inc; Naperville, I
the
20892.
(9A1740).
also correlated with the Additionally, IgE-mediated uptake of the soluble-phase
whereas
RBL-2H3
whereas
1990
Maryland
lated cells it was associated with the cell periphery and concentrated in the surface ruffles. As the stimulated cells spread, intermediate filaments and microtubules became distributed throughout the cell body, but there was no obvious association with the membrane ruffles. These morphological changes were dependent on the presence of extracellular
pane the changes
been
Bethesda, 9,
under
Cells
OLIVER’
National 1990;
(2H3)
CONSTANCE
ical changes
mcdi-
review].
cells, working and calcium the
other
these cells is the key factor in many acute reactions. Stimulation ofcells to release
atons can be achieved in a variety of ways, among of the IgE, IgG, on complement receptor, exposure (18)
hypensen-
and
Calcium
Leukemia
38:975-983,
cyto-
FI’It-phalloidin
lin.
by the
P. SIRAGANIAN,
Institute
1990
in USA.
Article
Induced
in Rat
National
975-983,
Printed
Original
Morphological
7, pp.
Inc.
48 hr before
in eight-chamber IL). For scanning
round release
glass were
coverslips. noted
essential
penicillin, use
plastic electron No between
and
plated
Lab-Tek
medium at a density
Chamber
microscopy, differences the
glass
(EMEM)
and streptomycin Slides
the cells were in either and
plastic
(1). of 2 x (Nunc, plated
morphology substrates.
975
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
976
SAHARA,
To measure well
histamine
release,
1
x
cells
1&
were
plated
per
well
in 12-
.
plates.
ing
Release.
the cells
IgE in the
twice
same
lated of
with
lease, ranging
from
sensitized
x
IgE.
culturing
sg/ml.
l0
examining
with
..%
on
re-
of DNP-HSA
to DNP-HSA
ionophore
effect
histamine
A23187
were first
(Sigma;
#{149}.‘a.
St Louis,
of ionophore
centrations
on cell
morphology
ranging
from
of ionophore
and 1.0-1
histamine
x
10
release,
ag/ml
were
0
,#{149}., .
MO), cells were rinsed twice in EMEM-BSA and then 0.5 sg/ml ionophore was added to the cells in EMEM-B5A. To determine the effect ofthe concentration
I
of
the
and
concentrations
All cells exposed
For stimulation
isg/ml
concentrations
changes
various
0.3
‘#{149}
2 hr the cells were stimu-
In experiments with
with
7
‘s
by rins-
of IgE concentration
After
on morphological
stimulated
1.0-1
with
was achieved
with various
tg/ml.
DNP44-HSA.
were
then
the effect
0.3-0.005
concentration cells
and
release
cells were cultured from
0.1 sg/ml
the
BSA
To determine
changes,
IgE ranging
DNP-HSA
histamine
in EMEM-2% medium.
morphological anti-DNP
IgE-mediated
OLIVER
V
4#{149}
.
Histamine
SIRAGANIAN,
.f
10
con-
#{149}i
H
1
..
..,
tested.
At various time intervals, the medium was collected and the histamine content determined by the automated fluorometnic technique. To assess the
a
#{149}-:------
-
I-i...
effects
ofcalcium
stimulation
out Ca salt
on histamine
the cells were
and Mg”,
solution Light
in
ton,
VT)
for
were
rinsed
meabilized
2%
20 mm
were
fixed
and
CA),
anti-mouse was
followed
time
mm
in 2%
goat
cells
by rinsing the
cells
distilled
were
hr at room stored
osmicated
for
20’C.
.
,fr’ tj
.
..
,
0’0
FIlE-
k
*
. .
,,.
.
er
and
El
goat
Grove,
incubated
for for
.,,.
treated
at 37’C
All
specimens
were
Fort Washington,
for 30 mm.
The
dehydrated
in ethanol,
last two steps
ib
then
with
and
1’
in
At var-
fixed
were
repeated
once.
with
a
in liquid
examined
in aJEOL
were
#{149}.T#{248}1.
in PBS at 37C (Ladd) pH
7.4.
tetroxide,
The
rinsed
this,
CO2, 35CF
again
the cells were
and
coated
with
scanning
1,’
in
thiocarbohydrazide
After
point-dried
$1
‘
for 20
examined
buffer,
1% aqueous
V
.,*
adding
glutaraldehyde
osmium
-
War-
30 mm
Probes).
twice
in 2%
4-
-.
‘.
1 hr at
PA). The cells were then osmicated
critically
Samples
rinsed
in 0.1 M cacodylate
for 10 mm
V
Endocy-
and
in PBS
temperature
at 4C
. #{149}‘
PA).
(Polysciences;
ImmunoResearch).
were
!F
,
formaldehyde,
stained
in EMEM-BSA
Cells
..
-
1 hr at room
West
to tubulin and
t.
,.
The cells were further
5 mm
for 2 hr in 1% buffered
and
gold-palladium.
per-
(Chemicon;
in 2%
rinsed
at 37’C.
f-actin,
with
for
Lucifer yellow (Molecular
Microscopy.
and
(EM Sciences;
-
,i
microscope.
for 2-4
water,
at
in
of vimentin,
stained
Laboratories;
twice
for
with Flit-conjugated
IgG (Jackson
mg/mI
twice
....
.
Burling.
30 mm
to vimentin
in PBS
the cells
1
formaldehyde
rinsed,
were
tron
anti-rabbit
Electron
and fixed
and
antibody
rinsed
for
For localization
for 2 mm
methanol
were
Microphot-FX
Scanning
in 2%
stained
cells
X-100
rinsed
Industries; formaldehyde,
as above
Triton
were
4’
_,l
‘.
EDTA.
To stain
antibody
with-
balanced
10 mm
ImmunoReseanch
0.1%
intervals
cells
OR).
the
sM
Research
and
Eugene,
in Hank’s
40
in PBS, and incubated
containing
ious
in PBS,
X-100,
cells
was assessed
Nikon
Triton
before
salt solution
hematoxylin.
for
with a polyclonal
The
EMEM-BSA
with
fixed
by fixing
with
FIlE-conjugated tosis
stained
in PBS,
in absolute
room temperature PA).
(Ladd
(Jackson
by 5 mm
nington,
examination,
permeabilized
localized
permeabilized
changes,
balanced
containing
formaldehyde
a monoclonal
rinsed
IgG
Tubulin
Mg’
Probes;
with
Segundo,
morphological
in Hank’s
For routine
0.1%
(Molecular
temperature
and
and
twice
with
phalloidin cells
Ca”
Microscopy.
cells
and
twice
and then the cells were stimulated
without
PBS and fixed
release
rinsed
dcc-
Figure 1. Hematoxylin blast-like appearance
)(
1I;
stained. (a) Unstimulated. and are spindle-shaped.
ABL-2H3 cells have a fibro(b) Antigen stimulated, 30 mm. The cells have spread over the surface ofthe culture dish. (C) lonophore A23187 stimulated, 30 mm. The cells have spread, but not to the extent seen with IgEmediated secretion. Original magnification x 150. Bars = 0.1 mm.
microscope. Controls.
Controls
nation
ofcells
nation
of cells
identical
in the to the
with
treated
ionophone-mediated ionophore
for IgE-mediated
sensitized
with release,
absence untreated
histamine
IgE but
not
antigen the
without
control
of calcium. RBL-2H3
release
exposed prior
consisted In all cases,
included
to antigen, exposure of exposing the
control
exami-
and
exami-
to IgE. the
For
cells
cells
to
were
( Figures
la and
2a),
with
microvilli.
When
the
cells
IgE with
DNP-HSA,
of the culture a dramatic
cells.
came
plicated,
Results Unstimulated
calcium
RBL-2H3
cells
are
elongated
and
spindle-shaped
and
(Figures
exhibiting occurred but
lb and
in the cell surface.
ionophone
spread,
surfaces
covered
stimulated
deep when
A23187
their
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
surface
folds
histamine (Figures
changes
with
to secrete
the cells flattened
dish change
in cell shape
their were
and spread 2b).
This The
and
were
short
over the surface
was
accompanied
by
plasma
membrane
be-
ruffles.
release lc and
small,
by cross-linking
2c).
A similar
change
was induced The
less dramatic
cells
by the flattened
than
those
IONOPHORE
Figure
A23187-INDUCED
CHANGES
IN
RBL
CELLS
2. Scanning
(a) Unstimulated. are covered
electron micrographs. The surfaces of the cells with short microvilli. (b) DNP-
HSA stimulated, 30 mm. The cells have spread and their surfaces are ruffled and phcated. (C) lonophore A23187 stimulated, mm. The cells have spread somewhat, their surface morphology is intermediate
tween
the
stimulated the ruffles DNP-HSA.
Bars
=
unstimulated
and
30 and be-
DNP-HSA-
cells. The surface is ruffled, but are not as prominent as with the Original magnification x 2800. 10 pm.
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
977
978
SAHARA,
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
SIRAGANIAN,
OLIVER
IONOPHORE
seen
A23187-INDUCED
with
ridges
IgE-mediated
were
apparent
on
CHANGES
release.
With
the
surface
cell
IN
the
RBL
ionophore,
but
the
979
CELLS
membrane
folds
were
not
as
deep. The
distribution
phalloidin,
ofpolymenized
paralleled
the
actin,
as determined
morphological
changes
w
by FIlE-
seen
in the
Co
RBL-I
2H3
cells.
tnated
In unstimulated
in
the
cell
stimulation
with
bution
ance 3d),
body
adjacent DNP-HSA
the
more
actin
started
with
could
rapid surface.
with
those
ionophone
nipheny
and
These
showed
changes
release
dition
that
(Figure
cur.
with
inhibits
3b)
at
but
time
and
Cl)
I
plasma
5
on their
stimulated
point.
By
at the
by the
4).
When
mediator
release
changes
in the
(2,10),
in cell shape
sensitized
cells
cells
had
did not ocno effect
the
cells
(Figure
actin
distribution
of antigen
5) and
At optimal concentrations, lease, the most significant amount
of antigen
were
on cell
were
directly
or ionophore
correlated
release
and
there
is no histamine
morphology
used
with
the
amount
both
out
phone, Bundles
Figure
there
the
was
less
was little
or no change
degree
varying
the
ofchange,
deeper
the
surface
cells
folds
7).
Antibodies
their cells.
cells by either in the against
distribution
and
the distribution ofmicnotubules
3. FITC-phalloidin
were
stimulated
Lucifer
the ionophore (Figure 8c).
40
45
and
the cells.
and
vimentin
morphological
The
In unstimulated
distributed
uniformly
with
DNP-HSA
either
release
with
processes
are spread
Actin
between
presumably
little
Lucifer
after
the
8b),
con-
as the
IgE
stimulation
yellow
by
was sequestered
and
used
2-S
mm of exposure
cells,
which Actin
were
directly
release
was
to be the
the membrane
dependent
it can
be determined
that
tunes
at the apical
surface,
complete
actin
is associated
cytoskeletal the
concentra-
occurred
by 30
com-
body.
cell
on the
changes
element By focusing
is concentrated
whereas
and
hista-
within
mm, the point
at
complete.
cytoskeletal changes.
Actin
other
throughout
The
also
plicated
IgE-mediated
A23187.
extend
were
becomes
with
while
on ionophore.
and
surface
ionophore
vimentin,
antigen
appears with
cell
is greater
membrane,
observed
histamine
The
it is with
in the plasma tubulin
sociated
are stimulated to secrete with either crossA23187, they undergo similar but not iden-
tubulin
most
closely
through in the
plicated
and vimentin
as-
the cells, struc-
are local-
to a band just under the plasma membrane. (b) DNP-HSA stimulated, 30 mm, no Ca. in association with the plasma membrane. (C) DNP-HSA stimulated, 5 mm. Actin is localized in the plicated structures on the apical surface ofall the cells. (d) lonophore A23187 stimulated, 5 mEn. In the cells that are ruffled and spread, actin is localized in the surface ruffles. In the remainder of the cells, actin is still localized adjacent to the plasma membrane. (a) DNP-HSA stimulated, 30 mm. By this time the cells have spread. Actin is localized in the ruffles on the apical surface and at the periphery of the plasma membrane. (f) lonophore A23187 stimulated, 30 mm.
The cytoplasmic
(a) Unstimulated.
than
folds
of either
or iono-
very
of ruffling
changes
through-
Lucifer
seen
(Figure
In contrast,
changes.
degree
iion
were
were
DNP-HSA
was endocytosed,
RBL-2H3 cells IgE orionophone
The
vimen-
with
internalized.
A23187,
marker
differences
histamine release. In unstimulated was internalized. However, when
to secrete yellow
folds.
soluble-phase
Discussion
mine
correlated with the changes in cell shape. could be seen radiating from the cell cen-
stained.
35
in a more random fashion neither tubulin nor vimen-
surface
to the functional
stimulated was being
ruffled.
on ionophone
of tubulin
tubulin
within
vimentin
In cells
DNP-HSA
distribution
to the
exposed
stimulation,
receptor
tical
those
relation were
siderable
in cell stimu-
than
were
When linked
concen-
any cells
were organized unlike actin,
IgE- and ionophore-mediated cells (Figure 8a), little tracer
where
hista-
At concentrations
filaments However,
during
ponents,
in changes
tubulin
the
reduced
Although
showed
ofRBL-2H3
resulted
to examine
always
of cieffect.
ionophone.
Stimulation (Figure
there
distribution. affected
DNP-HSA
with
was
to
was maximal histamine rechanges occurred. As the
in cell shape.
release,
ofsecretagogue
treated
tin
less change
or actin
with
where there morphological
on ionophore
mine
also
showed When
yellow
slightly
of histamine released (Figure 6). Varying the concentration then IgE (data not shown) or antigen produced the same
lated
tin
a con-
spread
membrane
IgE alone
and
on the concentration
stimulate
30
(mm)
Figure 4. ABL-2H3 cells were stimulated with either DNP-HSA(O)or ionophore A23187 (A). Al various time intervals histamine release was measured as described in Materials and Methods.
ten. Intermediate in the cytoplasm.
surface.
maximum
ofcalcium,
the
plasma
cell
time
IgE
in the absence
of the cells with
changes
tration
TIME
cells
The
25
20
15
mm
30
morphology.
dependent
10
were
mm, virtually
cells
ruffles
complete
(Figure
DNP-HSA
the
Sensitization
ofthe
ruffles
ofthe
of
3c and
stimulation At 5
this
folds were
is achieved
stimulated
folds
z
appear-
(Figures
exhibited 50%
mm
1
0 DNP-HSA AA23187
w
morphological changes were comactin was localized at the cell pe-
membrane changes
within
ionophore.
about
3f), when the secnetagogues,
in the
histamine
with only
morphological
were
seen
After distni-
actin
the initial
in the
w
concen-
the
by IgE-mediated
to IgE-DNP-HSA
was
membrane.
stimulation
concentrated
In contrast,
(Figures 3e and plete with both
after
induced
cells exposed
apical
mm
S
be seen
than
plasma
of
At
actin
or ionophore,
or ionophore
The changes
all ofthe
the
the
DNP-HSA folds.
actin
membrane.
to
3a),
Redistribution
to either
of surface
(Figure
either
changed.
exposure
cells
slightly, butactin
The cells have spread and the apical surface x 200. Bars = 0.1 mm.
is localized
is still localized
is ruffled. Actin is localized
with the ruffles and in association
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
with the plasma
membrane.
Original
magnification
980
SAHARA,
;k
r,;Pj’4
.d-
,.
..1
K#{149}
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
SIRAGANIAN,
OLIVER
IONOPHORE
A23187-INDUCED
CHANGES
IN
RBL
981
CELLS
to a-actinin, an actin binding protein. The difference in membrane changes seen with the RBL-2H3 cells may be a reflection of the 0 DNP-HSA
A
degree
A23187
ofstimulation
ofthe
timal
.(
may be utilized during IgE-mediated ionophore A23187 will also stimulate
U)
w
w w
0
in
conditions
2H3
as much
phosphoinositide
w
cells
(14),
are hydrolyzed
z
The
but
as 50%
less
pathway.
ofthe
than
membrane
Under
histamine release phosphoinositide
5%
of
seen
with
op-
phospholipids
membrane
(15). The turnover
phospholipids
(2).
morphological
changes
the RBL-2H3
cells
with
I-
U) I
either cium.
IgE- or ionophore-mediated Without calcium in the
were iO_s
10_6
iOs
iO_a
10_2
pg/ml Figure 6. ABL-2H3 cells were exposed DNP-HSA (0) or ionophore A23187 (A). described in Materials and Methods.
to various Histamine
observed.
In the
concentrations of either release was measured as
such
as EDTA
discrepancy
in findings
the experiments in the cell body.
et al. (16), with
This
who showed
membrane
enal agents
is consistent
an increase
ruffling
that
bind
after
in the
present
IgE-mediated
study
to a similar although though it does not
the findings
in polymenized
to cell surface
to induce binding of receptors produce changes in cell surface finding
with
actin
serotonin
receptors
have
is that
the
ionophore
with
may
be related
mediated When linked the
IgE
and
secretion
receptor
(16).
The
by phosphoinositol
The
ing
uptake
no uptake
with
The
accentuated
DNP-HSA
caused
the
could
by either
by the studies
ofthe
dye
with
ionophore
A23187.
infolding
of the
be
the
receptor
phosphoinositol
ponents increase
ton
after
result
cycle,
causes
Furthermore,
with
stimulation
1.
to
yellow
showand
membrane
2.
Beaven
3. Beaven
membrane
Figure 5. FITC-phalloidin stained. The morphological released. DNP-HSA: (a) 1.0 pg/mI; (b) 101 pg/mI; Bars = 0.1 mm.
in cytoskeletal
are bound
changes (C) 102
differof the
its effects
by binding
and
cells.
consequent
rise
The
then activates of histamine
other systems within the cell. release by HSA-DNP acti-
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However,
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Ca
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A23187.
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A23187
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The major morphological distinction between gogues is the presence of deep folds on the surface sitized
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less profound interact with
of Pfeiffer
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on the concentration of secretagogue and correlate with the amount of histamine A23187: (d) 0.5 pg/mI; (e) 0.1 pg/mI; (f) 0.01 pg/mI. Original magnification x 200.
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
Figure 7. a-c are stained for tubuhin; bundles of microtubules can be seen stimulated, 30 mm. (d) Unstimulated. (a) DNP-HSA stimulated, 30 mm; (f)
d-f are stained for vimentin. (a) Unstimulated. Tubuhin is localized throughout the cytoplasm. (b, C) in the stimulated cells, radiating from microtubule organizing centers to the cell periphery. (b) DNP-HSA stimulated, 30 mm; (C) ionophore A23187 The cells are stained throughout the cytoplasm. (e, f) In the stimulated cells, bundles of intermediate filaments can be seen. ionophore A23187 stimulated, 30 mm. Original magnification x 200. Bars = 0.1 mm.
982
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
IONOPHORE
A23187-INDUCED
CHANGES
IN
RBL
983
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Figure 8. Lucifer yellow. (a) Unstimulated cells have endocytosed little tracer. (b) DNP-HSA stimulated, 30 mm. After IgE-mediated histamine release considerable Lucifer yellow is present intracellularhy in small vesicles. (C) lonophore A23187 stimulated, 30 mm. There is no increase in the amount of Lucifer yellow
intemalized
after ionophore
0.1 mm.
Downloaded from jhc.sagepub.com at University of Bristol on March 16, 2015
administration.
Original magnification
x 200. Bars