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-

which

in a host

receptor

cell

results and

of changes,

phosphoinositol

such

turnover,

as

in addi-

morphology.

com-

pg/mI.

primarily

are dependent lonophore

Cited

MA, MooreJP, signal

and

259:7137,

MA,

release 4.

Smith

GA, Hesketh

phosphatidylinositol

TR, MetcalfeJC:

breakdown

in 2H3

RogensJ,

in 2H3

MooreJP,

ofthe cells.

Smith

calcium

J

Biol

signal

Chem

GA,

Hesketh

259:7129,

J

Biol

MetcalfJC:

with histamine

1984

PS, Unanue ER: Ligand-induced with the detergent-insoluble B lymphocyte. J Immunol 128:1198,

5. Burn P: Phosphatidylinositol regulation ofcytoskeletal-membrane

TR,

and correlation

BraunJ, Hochman face immunoglobulin

lxix of the

The calcells.

1984

The mechanisms

The cytoskele-

(5). There is a thirtyfold lipids to the cytoskele-

lipids

The Qf action

Bansumian EL, Isersky C, Petrino MG, Siraganian RP: IgE-induced histamine release from rat basophilic leukemia cell lines: isolation of releasing and nonreleasing clones. EunJ Immunol 11:317, 1981

Chem

with

to the phosphoinositide pathwhich results in stimulation of lipids membrane

on

Literature

directly mem-

secretion

of the

an increase

of the

ionophore.

tion to calcium influx. The multifactonial response after Fcc receptor activation may explain the more pronounced effects of DNP-HSA

cium

plasma

Fcc receptor,

of

is accom-

exposure

Lucifer

or internalization.

the

the

ruffling pronounced

In contrast,

after

IgE-mediated

yates

and

are more

with

the

or cal-

morphological

to the mode

into

that

DNP-HSA

cell spreading

exerts

present

showing

identical

due

ionophore

calcium stimulation

either

not

to perform The

(3).

than

likely

directly

need

agent.

the effects

DNP-HSA

calcium

in intracellular

induce

are most The

transporting

but

observed by of calcium.

studies

cells with

However,

with

in response

endocytosis

is indepen-

influx.

of perturbation

clustering

associated with membrane in the binding oflabeled activation.

Ca

of membrane

tal response could also be related way. Activation of blood platelets, the

and

treatment

use of

of the mor-

mobilization

similar

secretagogues

to the

biochemical

RBL-2H3

membrane.

In contrast,

the crossalong with

This

Both

dependence

be owing

calcium

produces

two compounds.

A23187.

receptor (10,13).

breakdown

ences

of IgE-

ionophore

of the

after

even

ofthe

The

of a chelating

with

requires

ionophore

the plasma

leads

difference

by binding and transporting Ca little or no internalization ofplasma

is confirmed

substantial

This

of action

ofthe

concentration

lack of internalization

the ionophore

that

also

release

Stimulation changes.

may

in agreement

the

is in contrast to that ruffling in the absence

in the presence

are

cium

the two secretaof the cells sen-

DNP-HSA.

internalization

calcium

the ionophore functions into the cells (9,17), with brane.

with

5ev-

shown

topology, directly.

to its receptor is cross-linked by antigen, on the cell surface and is internalized

ofextracellular

panied

with

histamine

A23187

in the mechanism

compared

IgE bound IgE clusters

dent

stimulated

to differences

associated

(4,12,21) or to The unusual

change in surface the IgE receptor

The major morphological distinction between gogues is the presence of deep folds on the surface sitized

findings

release.

to the cytoskeleton morphology (6-8,11).

less profound interact with

of Pfeiffer

also been

present.

was

secretion,

not sufficient to prevent the only when a chelating agent,

was

stopped

was

or EGTA,

are dependent on calmedium no changes

of IgE-mediated

alone

phological changes on calcium Pfeiffer et al. (16), who reported The

ized

case

calcium-free medium changes. The ruffling

10_I

secretion extracellular

cycle and its possible interactions.

J

association cytoskeletal 1982

of sunma-

involvement Cell Biochem

in the 36:15,

1988

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

CELLS

6. Chinkers M, McKannaJA, Cohen S: Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth facton. J Cell Biol 83:260, 1979 7. ConnollyJL, Green SA, Green LA: Pit formation in surface morphology of sympathetic neurons growth factor. J Cell Biol 90:176, 1981

4,

‘.A

.

I

rapid

changes to nerve

8.

Davis BH, Walter RJ, Pearson CB, Becker EL, OliverJM: Membrane activity and topography of f-Met-LeuPhe-treated polymorphonuclear leukocytes. Am J Pathol 108:206, 1982

9.

Diamant B, Patkar SA: Stimulation and inhibition lease from isolated rat mast cells. lnt Arch Allergy Appl

10.

Furuichi K, RiveraJ, lsersky C: The fate of lgE bound leukemia cells. III. Relationship between antigen-induced and serotonin release. J Immunol 133:1513, 1984

11.

Goshima D, Masuda A, Owanibe K: Insulin-induced formation fling membranes of KB cells and its correlation with enhancement amino acid transport. J Cell Biol 98:801, 1984

12.

Howard sembly

.

f.

and

in response

of histamine reImmunol 49:183,

1975

.4

TH, Meyer WH: and locomotion

of rufof

modulation ofactin as98:1265, 1984

J Cell Biol

Anti-immunoglobulinmast cells studied Med 142:391, 1975

by

14.

Lo TN, Saul W, Beaven MA: The action of Ca2 ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. J Biol Chem 262:4141, 1987

15.

Maeyama K, Hohman RJ, Metzger H, Beaven MA: Quantitative idationships between aggregation oflgE receptors, generation of intracellular signals and histamine secretion in rat basophihic leukemia (2H3) cells. J Biol Chem 261:2583, 1986

16.

PfeifferJR, SeagraveJC, and cytoskeletal changes

lease from

,

peptide

Lawson D, Fewtrell C, Gomperts B, RaffMC: induced histamine secretion by rat peritoneal immuno-ferritin electron microscopy. J Exp

13.

-,-.

Chemotactic in neutrophils.

to rat basophilic endocytosis

rat basophihic

Davis BH, associated

Deanin with

leukemia

cells.

RW, Landy properties.

J Cell Biol

lonophore NY Acad

Membrane serotonin re-

101:2145,

1985

17.

Pfeiffer DR, Taylor ing and transport

18.

Siraganian Snyderman lates. New

19.

Siraganian RP, McGivney A, Barsumian EL, Crews FT. Hirata F, Axelrod J: Variants of the rat basophilic leukemia cell line for the study of histamine release. Fed Proc 41:30, 1982

20.

Urata C, Siraganian RP: Pharmacologic modulation of the IgE or Ca2 ionophore A23187 mediated Ca2 influx, phospholipase activation, and histamine release in rat basophilic leukemia cells. Int Arch Allergy AppI Immunol 78:92, 1985

21.

Vale RD. Shooter EM: Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin. J Cell Biol 94:710, 1982

RP: Mast cells and R, eds. Inflammation: York, Raven Press,

HA: Ann

GG, OliverJM: IgE-mediated

A23187: cation bindSci 307:402, 1978

basophils. In GallinJI, Goldstein basic principles and clinical 1988, 513

lM, corre-

c

.

iLi d_

4

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

Morphological changes induced by the calcium ionophore A23187 in rat basophilic leukemia (2H3) cells.

RBL-2H3 cells have been widely used to study histamine release in vitro. It was previously shown that these cells undergo striking morphological chang...
2MB Sizes 0 Downloads 0 Views