Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: Analysis with indo-1

Vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 29, 1986

Pages 500-506

Martin K. Bijsterbosch, National

Institute

Received April

Kevin P. Rigley and Gerry G.B.

for Medical Resarch, Mill

Hill,

Klaus

London I%77lAA, U.K.

1, 1986

SUMMARY:The new Ca2+-probe indo-l has a high fluorescence intensity, which allows low intracellular dye loadings. Stimulation of indo-l-loaded muse B cells with anti-Ig antibodies provoked. rapid rise of free cytoplasmic Ca2t from 100 r@l to >l p, followad by a decline to a plateau at 300-400 no. The initial rapid rise was not detected in guin2-loaded cells, presumably due to the Ca2+-buffering effects of the dye. The sustained Ca2+ increase was due to influx, whereas the initial rise was caused by release from intracellular stores. The magnitudes of Ca2+ release and inositol trisphosphate release were closely correlated. Concanavalin A does not provoke inositol trisphosphate release in mouse B cells. It did not induce a rapid initial Ca2+ rise in it&-l-loaded B cells either, but only a sustained increase to 200-300 nM. Ca2+ influx induced by both anti-Ig and concanavalin A were not Finally, 0 1986 Academic Press, Inc. affected by membrane depolarization

A wide variety

of ligands induce in their

cytoplasmic free Ca2+ ([Ca2’li). of phosphatidylinositol trisphosphate

This is generally

bisphosphate

(PIP*)

target

cells

a rapid rise

associated with

to diacylglycerol

in

degradation and inositol

(IP3),

and recent evidence indicates that IP3 causes (part of) 2t stores [ll . the increase in [Ca2+] i by releasing Ca frcm intracellular The most popular current

method for measuring [Ca2+li is t0 monitor the

fluorescence

of intracellularly

limitations,

the most serious being a low fluorescence

cell

autofluorescence,

it

is therefore

amounts of the dye into cells, Recently,

trapped guin2 [21. !&in2 has, however, scane

Ca2+ indicators

intensity.

To overcane

neccessary to load relatively

which significantly

buffers

with high fluorescence

ICa2+li transients.

intensities

developed, which can be used at much lower concentrations

large

[3].

have been We have now

ABBFUWIATIONSUSED; Anti-Ig, anti-imunoglobulin antibodies; [Ca2+1ir free cytoplasmic Ca concentration; Con A, concanavalm A; IP3, inOSitO1 tr isphosphate; PIP2, phosphatidyl inositol bisphosphate . 0006-291X/86 Copyright All rights

$1.50 0 1986 by Academic Press, Inc. of reproduction in any form reserved.

500

Vol.

137,

No.

BIOCHEMICAL

1, 1986

used one of them,

indo-1,

to study

with anti-ixununcglobulin

antibodies

Ig stimulates

B cells

antigen

quiescent

receptors.

in these cells

It

Klaus,

the ]Ca2+]i

BIOPHYSICAL

(anti-Ig) to enter

induces rapid

with

submitted]. increase

minimal

or concanavalin cell

cycle

studies,

PIP2

their

and increases but only

provokes

intracellular

and

suggested

due to influx.

demonstrated

[Ca2+Ji

Bijsterbosch

[M.K.

induced by both agents was largely

which can be readily

A (Con A). Anti-

using quin2,

IP3-mediated

incubated

by cross-linking

activation,

breakdown of

a rapid,

COMMUNICATIONS

B lymphocytes

breakdown op PIP2,

These earlier

show that guin2 does not detect Ca2+ induced by anti-Ig,

RESEARCH

[Ca2+] i in murine

(41. Con A also causes B cell

Ca2+ mobilization, G.G.B.

AND

that

We now

release

of

with indo-1.

EXPERIMENTAL Reqer~tm: Indo-l (acetoxymethyl ester and potassium salt) was from Molecular Probes, Junction City, Or, U.S.A. Quin2 acetoxymethyl ester was from Amersham International, Amersham, U.K. Quin2 (free acid) and Con A (type V) were from Sigma, St. Louis, MO, U.S.A. Molar absorption coefficients of indo-l and guin2 were taken from refs. 3 and 5. Affinity-purified F(ab')2 fragements of rabbit anti-mouse Fab antibodies, henceforth called anti-Ig, were prepared as described previously (41. All other chemicals were analytical grade. cell preparations: Splenic B cells (ca. 90% surface Ig-positive) were prepared from 3-6 months old male (CBA x C57BL/lO)Fl mice by killing T cells and removing adherent cells as described earlier [4]. Aff"u of~imsilml trispbsphate: Levels of [3H]IP3 in B cells prelabelled with [ Hlinositol were measured as described in detail earlier (41. '&in2 and indo-l were dissolved in terminatim of free ca2+ in vitro: Offers containing 120 rrM KCl, 20 nM NaCl, 1 ITM MgC12, 10 rrM MOPS, and 1 no EGIA plus 0.1-0.9 mM CaC12 (@I 7.05) to final concentrations of 0.5 and 6.5 PM, respectively. Their fluorescences were measured using a Perkin Elmer MPF-4 spectrofluorimeter in a cell kept at 37 oc. Excitation and emission wavelengths were 339 and 500 ran for guin2, and 340 and 390 mn for indo-1. The CaC12 to approx. 0.5 x@l excess (for Fmax), nM by adding EGTA and Tris to 10 and 40 m concentrations in the buffers were calculated from the Ca2+= Kd x ((F-Fmin)/(F--F) I The Kd values for guin2 and indc+l under these conditions were taken to be 115 nM and 250 nM, respectively [2,3]. Cells at 50 x 106/ml in Hanks' balanced salt Deemtion of [Ca2+li: solution, containing 0.5% gelatin and 20 rrM HEPES (pH 7.2), received the acetoxymethyl esters of guin2 or indo-l to final concentrations of 2 or 15m, respectively. Both ware added from stock solutions in DMSO (final F&SC concentration <0.25%). Af er 45 min at 37 Oc, the cells were washed twice and resuspended to 5-10 x 10 t /ml in Hanks' solution containing 0.5% bovine serum albumin and 10 RM HEPFS (pH 7.3). Cell viability was >98%. The suspensions were stored on ice until use. Under these conditions, leakage of guin2 and indo-l from the cells was (1 % per hour. Shortly before fluorescence measurements, aliquots were washed and resuspend& to 5-10 x 106/ml in Hanks' solution plus 10 nEf HEPBS (pH 7.3). After 5-10 min preincubation at 37 Oc, fluorescence was measured as described above. The suspensions were stirred 501

Vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

periodically during measurements. After each run, FmaX was obtained by lysing the cells with 0.05% Triton X-100 in the presence of 10 uM diethylenetriaminepentaacetic acid. Fmin was determined.by subsequent addition of WIA and Tris as above (for in o-l) or MnC12 to 0.2 I@! (for quin2, Fmin corrected for effect of Mg2+) .[Ca 9+I i was calculated with the formula given above. Intracellular dye loads were determined ccrnparing fluorescences of cell lysates and Known concentrations of the dyes and assuming a cellular volume of 110 um3 (61. RFSJM!S The free Ca2+ concentrations in buffers

in which free Ca2+

measured by

levels

were

Fig. 1 shows that over the biologically Ca2+ concentrations

measured

quin2 and indo-l by altering

Varied

important

by indo-l

were ccmpared

Ca2+/EGTA ratios.

range of 40-1000 nM the free

and quin2

are strongly

Levels measured with quin2 were, however, 16 + 2% (means +S.E.M., than those measured with minor inaccuracies

ind+1.

This

in the Kd values

Subsequently,

B cells

nM, respectively

were stimulated

were loaded with either

quin2-loaded transient loading

declined

cells, increase

[Ca2+li to a plateau

[Ca2+li was not

is probably

quin2 or indo-1.

were very similar:

(means+S.E.M.,

with anti-Ig,

and then rapidly

discrepancy

n=18) lower due to

of the dyes.

values measured with quin2 and indo-l 114 +3

small

correlated.

107 + 4 nM and

n=12). When cells rose within

detected.

This

(1.42 + 0.14 mM; means + S.E.M.,

2A, left

panel).

n=4),

because the dye

and thus Ca'tbuffering,

(nM)

and i.d+linvitro. The levels Fiqure 1: Pree C!a2+ levels aeasuredwithquin2 Qf free -2+ in a series of Ca2+@orA buffers Was measured with quirk2 and md+l as described in detail in the Experimental section. Each point gives free Ca2+ levels measured with both quin2 and indo-l in a particular buffer (results fran two separate experiments). 502

In

at 300-400 nM, but the rapid

is presumably

Free Ca*+: indo-l

loaded with indo-l

30 set to a maximum of >lp

of 300-400 nM (Fig.

rose to the plateau

The resting

in

Vol.

137.

No.

BIOCHEMICAL

1, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1200

. MO-1 0 Qulnz

. 0

aoo-

IndoChh2

Anti-lg 1

0

-p

0

2

Time (mid

Fi ure 2. Effects of arkGIg ad Car A an [Cd2+]i in B Delis loaded with @n2 B cells containing indo-l (0) or guin2 (0) received anti-Ig and Con A as indicated (final concentrations 50 and 2.5 fig/ml, respectively). Intracellular dye loadings were 1.42 + 0.14 W4 and 0.09 + 0.01 nM for guin2 [Ca2+]i Tas calculated fran the fluorescence and indo-1, respectively. signals, which were corrected for dilution due to additions made, as decribed in the Ex rimental section. [Ca2+]i in controls (medium added) did not change appreciaby.P Besults are means + S.E.M. of 4 replicates obtained from 4 separate experiments. of normI ~~3h~d~a&&Ig a~ [caz+]ihB Oeus he* prm B cells were loaded with indo- and incubated in normal medium (1.3 nM Ca2$. At the time indicated by the arrow, anti-Ig was added to 50 pg/ml. Approx. 15 set before this addition, the cells received EGPA to 1.8 W (0), or medium (y. Addition of E%TA inmediately reduced extracellular Ca2+ to <250 r&l. [Ca +I i was calculated as in Fig. 2. Results are means + S.E.M. of 3 replicates obtained fran 3 separat& experiments. ?$kidix.

these

cells

was much higher

means -+ S.E.M., very similar

n=4).

results

We next studied to the response reduction

of

Addition in cells

in

presence

of extracellular

release causes

[Ca2+] i

reSUltS

MS

release

unaffected,

that

Ca2+ from

(0.09 + 0.01 nN;

on the other dye (Fig.

in indo-l-loaded

hand, yielded

2, right

B cells

(Fig. the

but

increase

the sustained

the rapid stores. intracellular 503

panel).

and intracellular

<250 1-84 by EGIA,

Ca2+ was canpletely

intracellular of

either

indo-l

of extracellular

CaL+ to

suggest

of Ca2+ from the

loaded with

induced by anti-Ig extracellular

containing

of Con A to B cells,

the contributions

increase

These

than in cells

Cn2+

3). After

rapid

transient seen in the

abolished. transient Recent

[Ca2+li studies

stores

rise indicate

[1,7].

is

due to that

lp3

We therefore

Vol. 137, No. 1, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

r

Anti-l0 bIQlm’)

w

Time bin)

[Ca2+]i and 1% rdB b stimup"k ated with O-50 &ml anti-Ig. [Ca2+]i reached maximumvalues after approx. 30 set, and is given (0) with control levels (mediumadded: 104+5 nM) subtracted. [3H]1p3 levels (0) were determined 30 set after adding O-SO&g/ml anti-Ig to [3H]inositol-labelled B cells. Ihe results are expressedas %of the levels found with mediumalone: 0.19 20.01% of the total cellular radioactivity (108,000 dpa). All points are means2S.E.M. of 4 replicates frcm 4 separate experiments. Am [($+]i inBc&l~ inthemFi re 5. Effects of anti-1 md h and hi extracel&l~ 9 levels. Indo-l-loaded B cells in normal medim (0; 6 mMP, 142 Wl Na ) or high K+ medium(0; 7OrtM@, 78 I@!Na+) received anti-Ig or Con A as indicated (final concentrations 50 and 2.5pg/ml, respectively). [Ca2+]i was calculated as in Fig. 2. Points are meansf_ S.E.M. of 3 replicates fran 3 separate expriments.

compared the magnitude of anti-W

of the initial

with changes in IP3 levels.

are closely

extracellular

loaded

Ca2+.

increase Since

by the cell cells

concentrations in

in

with anti-Ig,

doses

Fig. 4 shows that these two responses

containing

of K+ (Fig.

had only

in

in

[Ca2+] i

many other

membrane potential

media

normal and high

Depolarization

peak induced by increasing

correlated.

The sustained

controlled

[Ca2+li

K'

normal

is apparently cell [8],

types

due to influx

we studied

of

[Ca'+]i

(6 mM) and depolarizing

influx

of

Ca2+ is in indo-l(70 rrM)

5). Besting [Ca2+li values were the same in cells medium (103 +

a slight

effect

3 124; means + on Ca2+ influx

S.E.M.,

in cells

whereas the response induced by Con A was not affected 504

n= stimulated

at all.

9).

BIOCHEMICAL

Vol. 137, No. 1, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION Cur data clearly better

tool

demonstrate

than guin2 for measuring

show that

anti-19

induces

~a~+ from intracellular prolonged

influx

indicates

that

respond quite

detection

was not seen Ca2+ alters

with

guin2

of a rapid transient

(Fig.

2). Another major

131. However, this we employ,

method requires

measuring

and calibration, from Figures

fluorescence

to be made by measuring

emission

uses

the rapid

transient

wide variety

of cell

types

(including

permeabilized

cells

provokes

the

A canparison

(done with

mainly

Ca2+ release

mobilization

of

finding fails

is that which allows Ca2+ at two wavelengths

instrumentation.

B cells),

Its

addition

The method

by cell

destruction

validity

is apparent

of various

distinguish

doses of anti-Ig

intracellular

from degradation

of [l].

and J.

1,4,5-IP3,

isomers,

Heslop)

the

Cur data therefore Ca2t by anti-Ig

of PIP2.

[Ca2+Ii

505

(Fig.

(Fig.

we found that

anti-Ig

but

capable

of

inducing

support the hypothesis is

2).

4).

earlier

mediated

This view is further

rise

to induce IP3

in

isomer

that Con A, which does not provoke substantial to induce the transient

stores

a close correlation

between IP3

M. Berridge

of exogenous IP3 to

of C?a2+ from intracellular

[Ca2+l i peak revealed

release

intracellular

generated

of indc-1

ratios

followed

that

in [Ca2+li induced by anti-Ig in of Ca2+ from intracellular stores. In a

release

of the capacity

assay does not

induces

allowed

rise

is due to release

and the initial

experiments

fluorescence,

in [Ca2+li

feature

spectrofluorimeter.

cells

Our IP3

rise

the emission

sophisticated

it&o-l-loaded

release

response

1 and 2.

We found that

[1,7].

by

to stimulation.

emission,

at one wavelength

a standard

followed

lower Ca2+-buffering

The resulting

of its

of

of the initial

synchronously

we

release

massive

IP3-mediated),

cells

the wavelength

determinations

rapid,

is a much

Using indo-1,

over guin2 is its much brighter

lowar dye loadings.

in indo-l-loaded

indo-l

cells.

Ca2+. The rapidity

The main advantage of indo-l which allows

an extremely

(which is presumably

of extracellular the cells

in living

[Ca2+li

in B cells

stores

2+ the new Ca probe

that

by 1,4,5-IP3

corroborated

IP3 release

that

by the

in B cells,

Vol. 137, No. 1, 1986

BIOCHEMICAL

There are various Some cell

AND BIOPHYSICAL

mechanisms regulating

types possess voltage-gated

the influx

Ca2+ channels.

plasma membranes leads to massive Ca2+ influx, channel blockers

like

voltage-sensitive Entry

of

mentioned

verapamil

[9].

these

channels

above. In yet other

cell

types,

by membrane depolarization,

to inhibition

by Ca2+ channel

5),

plus

mouse B cells

the lack

in human B cells,

Ca2+ channel suggest,

resting

[ll].

inhibit

by Ca2+

surface

and stimulated

[lo].

Ca2+ levels

of Ca2+ susceptible experiments

(not shown), indicate

group. The results

blockers

of their

by the blockers

The depolarization

of verapamil

belong to the latter

who found that

be inhibited

181.

that open by a

on their

nor is influx

blockers

of effect

Depolarization

which can be inhibited

of receptors

can also

are unaffected

(Fig.

of Ce2+ into cells

Others have Ca2+ channels

mechanism upon ligation

Ca2+ via

RESEARCH COMMUNICATIONS

of Clevers et al.

anti-Ig-induced

[Ca2+],

that [12],

increase I

however, tbat there may be species differences.

ACKNOWLEDGMENTS: M. K. Bijsterbosch and K. Rigley were supported by fellowships from the Wellcane Trust. We wish to thank Mary Holman for her technical assistance. REFERENCES 1. 2. 3.

Berridge, M. (1984) Biochem. J. 220, 345-360 Tsien, R.Y., Pozzan, T. and Rink, T. (1982) J. Cell Biol. 94, 325-334 Grienkiewicz, G., Poenie, M. and Tsien, R.Y. (1985) J. Biol. Chem. 260, 3440-3450 4. Bijsterbosch, M.K., Meade, C.J., mrner, G.A. and Klaus, G.G.B. (1985) Cell 41, 999-1006 5. T&en, R.Y. (1980) Biochemistry 19, 2396-24046 6. De France, A.L., Kung, J.T. and Paul, W.E. (1982) Immunol. Rev. 64, 161-182 Ranscnn, J.T., Harris, L.K. and Cambier, J.C. (1986) J. Immunol., in press 2 Towart,R. and Schramm, M. (1985) Biochem. Sot. Symp. 50, 81-95 9. Taylor, W.M., van de Pol, E., van Helden, D.F., Reinhart, P.H. and Bygrave, F.L. (1985) FEBS L&t. 183, 70-74 10. Oettgen, H.C., Terhorst, C., Cantley, L.C. and Rosoff, P.M. (1985) Cell 40, 583-590 11. Doyle, V.M. and Ruegg, U.T. (1985) Biochem. Biophys. Res. Corms. 127, 161-167 F. and Ballieux, R.E. (1985) 12. Clevers, H.C., Bloem, A.C., Gmelig-Meyling, Scan. J. Immunol. 22, 557-562

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