zymosan-.induced lysosomal enzyme secretion in ... - CiteSeerX

Protein kinase C and intracellularpH regulate

zymosan-.induced macrophages Hans

Tapper

Department

Abstract:

Binding

phage to

-glucan

tents. PKC-, to

and Roger

ofCell

and

of

zymosan

receptors

trigger

of

investigated. secretion directly naling

of many other of cytosolic response, nor

also

a

However,

poor

when

stimuli could

be

bation marked particles,

stimulus

approach.

extrusion

of

acid

soluble stimuli tory response

PKC

activity

activation

of

elevation

can We

of lysosomal

sary

signals,

effect tion

on some pathway

J.

PKC.

Leukoc.

while

Biol.

sponse gered

by an

prevailing

tein

Words: kinase

C

.

be

of secretory secretion

studies of pH.

enhanced PKC

.

brane, tions

of the

contents armament

[4],

cell

macrophages a rapid

of vacuolar-type been shown to be

lysosomal

pH

and

pH.

zymosan

Also,

receptors

[7],

evoke

in macrophages [4, preformed lysosomal

could

traversing The

modulated

secretory

basis in

for

the

regulated

vesicular

and [8].

be

at either

achieved

rapid by a

of tranplethora

constitutive

and cytosol components

could

by secre-

7]. Massive and enzyme occurs

the

proteins

by

particles,

a lysosomal

molecular

include

messenger

re-

When trigionophores

[6], an inhibitor secretion has

elevated

achieved.

and

protease inhibicells. By bulk se-

resident that allows

phosphorylation/dephosphorylation, pH

secreting

degradative demands. and H+-translocating

plasma membrane, with cytoskeletal

mem-

their interacControl by level

a via

Ca2 + dependence,

or

sensitivity.

further

by

activity

and

a modulatory

has

Abbrev

neces-

FD,

rich

C-kinase

Reprint 94,

S-221 Hans

.

lysosomal

secretion

pro-

Received

NAG,

saline;

MARCKS,

fluorescein-labeled

myristoylated

alanine-

N-acetyl--o-glucosaminidase;

PDBu,

PBS,

4-phorbol-12,13-dibutyrate;

12,13-didecanoate;

12-myristate

FZ,

dehydrogenase;

substrate;

4-phorbol

2’,7’-bis(carboxyethyl)-5(6)-carboxyfluo-

isothiocyanate-dextran;

lactate

phate-buffered

Institute,

1995.

: BC EC F,

fluorescein

LDH,

zymosan;

Biology,

in the signal transducsecretory apparatus itseLf.

lysosomaipll

iat ions

rescein;

response to on basal

pH

macrophage .

range

by, for example, uptake by other

using

and

485-494;

pH

an

products is

second

the secreWe suggest

as independent

cytosolic

component(s) or in the

zymosan cytosolic

of sub[3] are

secretory pathway and not by modulation and translation, whereby control of the

Ontario

Key

macro-

number enzymes

close

to b-glucan

regulated scription

on

a stimulus

defense,

cytosolic

tory response exocytosis of

lysosomal pH. was furthermore

homeostasis

[5] or by bafilomycin Al proton pumps, lysosomal

by

incu-

and

to extracellular by weak amines

binding

soluble

within

cretion of lysosomal -are bestowed with

secretion.

effect

secretory ( 1 ) is dependent

consider pH

58:

to act

when stimuli that that activated PKC relevance for the

Earlier

lysosomal pH (2)

Sweden

a reduction of extracellular that impair macrophage

equivalents.

and

Lund,

to secrete a large these, the hydrolytic

their action is restricted tors and clearance via

pathway

have shown a sensitivity to changes in cytosolic

a model in which the an elevation oflysosomal

likely

triggered

response

particles,

University,

to host

can be induced [1, 2]. Among

the

a synergistic

zymosan

inhibited by conditions

response diester

prolonged

both activates PKC and elevates secretory response to zymosan

shown to be pH or [Na],

Sigdiffers

secretory

was obtained pH and stimuli This is of likely to

was

considered. secretion

a prior

Lund

relevance

phages stances

Such treatment also had and uptake of zymosan was made possible by a

Furthermore,

response

zymosan

or

Pathogenesis,

Of great

conof Ca2+, signaling

lysosomal the

by

in

shown

of lysosomal agents that

zymosan

pH,

down-regulated

secretory that The

by

lysosomal

lysosomal secretion elevated lysosomal were combined.

been

secretory by phorbol

of

with phorbol diester. effects on the binding the study of which

novel

was

lysosomal activation

secretion

INTRODUCTION

macro-

lysosomal

by

Molecular

secretory systems, because Ca2+ did not trigger a large did attempts to reduce cy-

triggered

raising

previously

pH lysosomal

tosolic Ca2+ affect the to other stimuli. PKC was

to

PKC dependence to some soluble

alters intracellular to macrophage

from that an elevation secretory

particles

secretion

Also, the response

Sectionfor

the involvement processes in the

lysosomal in

Biology,

preformed

In the present study, and pH-dependent

enzyme

Sundler

Molecular

has

exocytosis

macrophage

lysosomal

PKC,

protein

kinase

phosPDD,

C; PMA,

4-phorbol

13-acetate. requests: Section 00

Roger for

Lund,

Tapper’s

for

1X8,

March

Sundler,

Department

Pathogenesis,

of Cell Lund

and

University,

Molecular P.O.

Box

Sweden. present

Hospital M5G

Molecular

address: Sick

Children,

Division 555

of

Cell

Univetsity

Biology,

Research

Avenue,

Toronto,

Canada.

2,

1995;

revised

June

12,

1995;

accepted

June

15.

1995.

Journal

of

Leukocyte

Biology

Volume

58,

October

1995

485

Many signaling events are action with zymosan particles. changes

in cytosolic

Ca2+

MATERIALS

triggered by phagocyte interRegional and generalized concentration

are

triggered

surface. This by a second

necessitates messenger,

that lysosogenerated by

receptor ligation. We have reported tex beads do not induce a lysosomal

elsewhere secretory

and also, since gered lysosomal

inhibit zymosan-trigdetail characterized

macrophage ligand, this

soluble glucan could secretion, in some

b-glucan receptor

receptor. was shown

and Binding

the

of the

receptor

and

inhibition

response to zymosan were more efficient of larger sizes, indicating that clustering receptors at the cell surface occurs. b-Glucan demonstrated [12], as well

a

12,13-didecanoate,

lasin

B. nigericin, Tritiated

and

from

culture

were

purchased

1.8

diutn)

distinct

Ch-medium

some lead

of which to divergent

isoenzymes signal effects

are

of which

are

Ca2+

dependent

and

pathways different

may PKC

not. Signal transduction responses by usage of the

[15].

Involvement

of

protein

kinase

transduction pathway can be demonstrated of down-regulation of the kinase by incubation

phorbol diesters, kinase activators that are eliminated. Such down-regulation is thought translocation to cell membrane, activation, and of PKC [16]. PKC isoenzymes exhibit extreme with with

respect phorbol

press

several

toward proteins

to down-regulation diesters. Mouse isoforms

and

down-regulation are phosphorylated

in

by

a

the with

of these

been shown [17]. Cytoskeletal as a result of activation of PKC

thioglycollate-elicited phages exhibit less zation and organelle also

been

membrane macrophages

macrophages [19]. Resident striking changes in cytoskeletal distribution, however. Phorbol

shown

flow through [20]. The

to increase the role

macroorganidiesters

macropinocytosis

endocytic of protein

and

compartment kinase C in

in the

lysosomal secretory response triggered in the macrophage by zymosan or soluble stimuli has been characterized in the present study. Because activation of protein kinase C could have multiple effects receptor expression proach was devised ing from

on cell-particle interactions (e.g., on and phagocytic capacity), a novel apin order to differentiate particle bind-

uptake.

Journal

was

Letikocyte

Biology

Volume

58,

October

1995

from

NEN

were

from

zymosan

particles

OR.

materials

All

Laboratories.

of pH

to the

mM;

mM;

127

mM; mM;

glucose,

NaC1

by pH

in

HEPES,

ionic

composition mM;

was

of

mM;

and

mM;

pH

the

mM

adjusted

at 37#{176}Cand

fluorescence

that

to

18.5

was

pressure

by comparing with

identical by

of HEPES.

Na/HCO3-mediumn

mM.

at 37#{176}C.

chloride

partial

mM;

10

was

choline

CO2

(K-me5.4

HEPES,

performed

omission the

5.4 10 mM.

NaCl,

(NaJHCO3-medium)

and

mM; con-

KCI,

mM;

5.6

value

varying

electrode

acid)

mM;

1.2

glucose,

solution

by

5.6

NaH2PO4,

mM;

con0.8

MgSO4,

(Ch-medium)

1.2

intracellular mM;

mM;

10 mM.

KH2PO4,

indicated

mM

5.4

bicarbonate

replacement

value

(Na-medium)

KC1,

HEPES,

nominal

1.8

for

108.5

(free

bicarbonate

1.2

127

CaC12,

and

nominal

ratio

obtained

of

in medium

of bicarbonate.

Cell culture and stimulation Harvest

of resident

mice

(ALAB,

described

dishes

cultures

herent

cells.

Culture

applied

30

(PMA,

added

phorbol

lasin

B),

0.5%

(dimethyl

Zymosan washes,

dispersed

Freshly

prepared

199

h. Serum-free

PDBu,

water,

and particles

were

by

vortexing,

and

particle

Efficient

ethanol)

removal

in

never

to in

were

and

shaking

three a

always

that verified

cytochaexceeding

(water).

counted

washes

sulfoxide and

a volume

were

always

in dimethyl

subjected

particles was

10% conditions

ionomycin,

or 5%

suspensions

consecutive

containing

media

A23187,

or

performed,

cover-

of nonad-

experiments.

latex

by three

dishes.

Medium

sulfoxide

were

with

by removal

14-22

NMRI previously

on 35-mm-diameter

for

experimental

([3H]PDBu),

and

removed

to the

to the

didecanoate,

ethanol

experiments

in

as

supplemented

macrophages

serum

prior

mm

were

for

outbred was

cells

experiments

was bovine

female

Copenhagen)

of peritoneal

pH

enriched

fetal

from

Bommice,

plating

(for

were

heat-inactivated Stimuli

macrophages

or

At 2 h after

culture

glasses),

were

peritoneal

Stockholm

[5].

tissue

consecutive

B#{252}rker chamber. used.

not

cell

When

chase

associated

of the

tissue

by phase-contrast

were culture micros-

copy.

Enzyme

assays

Measurements

of N-acetyl$-u-glucosarninidase

drogenase NAG

(LDH)

was

in

0.25

crease

were

determined

to 1 .8 ml of 0.25

emission

of

KC1,

indicated

devoid

was

Eugene,

Flow

with

1.8

mM;

verified

Sigma.

Warringion,

ionomycin

unlabeled

Probes,

on presumed

except

BCECF

from

Polysciences, and

and

5.6

CaCl2,

based

contained

to the

and phorbol diesters promote cellular spreading, redistribute cytoskeletal structures, and can have profound effects on the morphology of the lysosomal compartment in

486

mM;

0.8

cytocha-

purchased

([3H]PDBu)

#{149}

Bicarbonate-containing

not rapidly to occur by degradation differences

sensitivity

glucose, chloride,

Adjustment

by prolonged treatment peritoneal macrophages ex-

a differential

has

C

with KH2PO4,

choline

NaHCO3

from

of

403-phor-

(PDBu),

were

A23187

from

mM;

solution

MgSO4,

[ 18]

have

mM;

0.8

phospholipase activity, diacylglycerol is generated and acts as an activator of the serine/threonine kinase, protein kinase C. Protein kinase C exist in at least nine

were

Molecular

solution 127

Na-free

MgSO4,

.tm)

average

media

Natbased

tamed

zymosan

fluorescein-conjugated were

An

(PMA),

12,13-dibutyrate

and

lonophore

together)

NaCI,

(FD;

2’,7’-Bis(carboxyethyl)-5(6)-carboxyfluorescein

A solution

some

(4.0

UK.

Experimental

14].

isoforms,

beads

Boehringer-Mannheim.

CaCl2,

By

4-phorbol

Products,

(Universit#{228}t

isothiocyanate/mol

13-acetate

4-phorbol-12.13-dibutyrate

Research

for cell

K. Altendorf

of fluorescein

12-myristate

monensin,

latex

Prof.

isothiocyanate-dextran

9 mmol

4J3-phorbol

bol

tamed

and [13,

42,000;

residue),

by

fluorescein

weight

glucose

An

provided

4-MethyluInbe1liferyl-N-acetyl--D-glu-

NADH,

molecular

(used

with gluof glucan receptors

kindly

Germany).

(BCECF)

se-

on human monocytes [11] as on murine macrophages

A1 was

cosaminide,

UK.

lysosomal

cans

Bafiloinycin Osnabruck,

Polystyrene

also present of ligand to

cretory

have been neutrophils

that laresponse

By use of a fluorescent to be trypsin sensitive,

Ca2+/Mg2+ independent, recirculating, in an intracellular, mobilizable pooi. b-glucan

[7]

METHODS

Materials

[9],

and protein kinase C and tyrosine kinases are activated [ 10]. An increase in lysosomal pH was shown in the present study to be caused by binding of zymosan particles to the macrophage cell mal pH is regulated

AND

M

in

sodium

by adding mM

at 341

as 200

described j.tl of either

4-methylumbelliferyl

citrate

fluorescence set

(NAG)

performed

and

buffer

with

time,

447

nm,

and

lactate

dehy-

previously

L51.

Briefly,

medium

or cell

lysate

N-acetyl$-o-glucosaminide

(final

with

pH

4.8)

and

wavelengths

respectively.

Unless

measuring

for

excitation

otherwise

the

in-

and stated,

release

of

scribed,

LDH

was

indicating

negligible

under

preserved

all

cellular

experimental

integrity

conditions

de-

[21].

This

fluorescence

ated

and

of the

Measurement Binding

was

constant

of [3H]PDBu

carried

amount

each

well.

rapidly

in

the

end

three

times

harvested

in

were

taken

with

cold

binding

of unlabelled

was

PDBu

to be of PDBu

the known

specific

activity

or 1 fmol/.tg

cell

ing

[3H]PDBu

sites

for

protein.

Measurement Loading

with

bration

x

and

mm

16

and nm,

covergiass as

with

background.

coverglass

not

with

lysosornal

was

FD-containing ratios

situ

calibration

were

mounted

a special

similar

netic

stirring 1 mI/mm. 4,

and

media

the

and

emission

FD

was

of FD

from

can

1)0th

be used

ccli

surface-associ-

to calculate

the

fraction

as

F1

=

[F7.2

at which

intensity

that

of this

approach

8000C)

medium

in

with cells

30#{176} angle continuously

to the

perfused

together

are

by

light

in

beam with

at

a flow

pH

from

in latex

to the

the

in B no

the

fluorescence particles

ha’e

intensity

Figure

7.2.

the

in fluorescence

validity

blockade

in fluorescence

reduction

this

The

after

where

pH. p11 as

a fluorescence

at pII 5,

increase

being

versus

phagosotnal

at

of

was

seen

pH

6.4

on was

extracellular.

shown

same

independent

of the

known

of Ca2

ability

of zymosan

particles

to gener-

ate a phosphoinositide response in macrophages [23], we wondered whether a rise in cytosolic [Ca2] might play a role in inducing lysosomal secretion. As shown in Figure

of in

the

in

magrate

are

compares

secretion

la, NAG sensitive treatment to further

coverglasses

equipped

of lysosomal

with

obtained the

cuvette

the

obtained or

NAG In view

to

ratio

were

relative

fluorimeter

and of a

zytnosan

experiments,

for

RESULTS

subtracted

values

be (00-

7.21

‘iE(..

to determine

is demonstrate(l and

can

the

at 497/456

identically

ratio

used

similarly

particles

7.2)

curve

be

zymosan

all

which

fluorescence

in

mm

(model

and

.

fluorescein-labeled

with

the

7) 72.,,

then

of nigerkin

factor

for

Particles):

by cytochalasin

consistent

(corrected

a calibration

can

7.2i

a correction

pH

HI/FIIC.

FEC

with

T.2)]IFn,gnt,.

to genetate

_

actual

FI.(:.

7.2)

/mnl for

30

of fluorescence

untreated

presented

=

factor

addition

cali-

used

surface-associated

comparison

pH

adherent

fluorescence

treated

recordings

traces

set

be

by cell

correction

of bind-

for

measured

with

During

were

also

phagosornal

tributed

By

pmol/well

Cells

mg

the

otherwise

in

Representative

and

experimental

preincubated

nigericin.

in a standard

0.5

experiment,

cells

at an approximately

about

from

and

[5].

of

Calibration

to those

with

holder

Figure

In

can

.

sin

to determine

to control 0.1

in a SLM

by comparison

K-medium.

particles,

performed

with

cells.

achieved

the

calculated

for measurement

incubation

was

data

used

excess

saturation

described

2 ml

loaded

FD-loaded

pH

used,

with

to each

coverglass

and

The

phagocytosis

for excitation

cells

derived and

internalized

[1’Iniget

radioactivity.

to approximately

a further

were

This

were

Aliquots

binding was

incubated

by

Prior

total

cells

conditions were

wavelengths

(PBS) and

to

washed

pH

37#{176}Cwith

respectively.

added

of a 106-fold

of the

as previously

Experiments

that

2 h. A

achieved.

followed

at

[22]

conditions

experimental

h,

spectrofluorimeter 518

not

pH were

medium.

cuvette

the

were

X-100.

presence

10%

Under

was

wells

Triton

to control

amounted

covergiasses

approximately FD-free

than

of lysosomal FD

12

content

in the

and

was

of lysosomnal

to 12

of protein

bound

iCi) saline

0.2%

less

amount

the

containing

determined

The

period,

phosphate-buffered

1 ml of PBS

at 4#{176}C for

3 pmol/0.05

incubation

for determination

Nonspecific

saline

(approx

of the

particles

was

particles

binding

phosphate-buffered

of [3H]PDBu

At

cells

cells.

out

intensity

internalized

secretion in response to zymosan particles was not to a reduction of the external [Ca2+] per se or to with Ca2+ ionophore in Ca2+depleted medium reduce internal [Ca2]. Thus, it is (1) likely that

binding

of zymosan

particles

to a secretion-triggering

experi-

ment.

Determination of cell-associated and internalized zymosan particles and measurement of corrected phagosomal pH Macrophages

adherent

to coverglasses

labeled

zymosan

particles.

transfer efficient

to and shaking of the removal of particles

inspection

of the

inspection

was set

M HC1 was

ized

cuvette

and nm

recorded

particles)

from

7.2

curves

to

6.4

in part (cell

sine-treated

intensity

a 40%

in absolute

pH 7.2,

at time

sociated

particles

reduction

can

s, the

by

C 0

that

(internalof the

From from

at 497

nm.

1

Fig. NAG

of pH

from

7.2

Thus.

from

the

.

diutn,

with

with

release.

Upon sity

addition increased

of 10 p.tM nigericin further

as

internal

at time particles

700 were

Tapper

s, the also

and

fluorescence exposed

Sundler

intento pH

7.2.

Protein

true

kinase

A23187 A23187 pH EGTA.

mM

C and

pHi

Zero lower

Ca2

salt

regulate

7.20. and

Ca2 was

used

separate

mnacropliae

Ca2

with

means the

to prepare

Ca2-

a(l(litional were

Ca2

Ca2-free symbol)

incu-

concen-

Na-medium depicts the media,

by Fura-2

(a),

in Na-me-

macrophages

(open

as determined five

Zero

denotes

trace

and In

particles

(i) denotes

In (b),

secretion

calcium.

in Na-medium

mm

The

is represented of at least

.tM).

NAG cytosolic

zymosan

pH EGTA

(2.0

7.20.

of

ing/inl

mM

for 60

a hygroscopic

are representative

1.0

0.1

0.1 content

with

concentration,

as indicated, Since Ca2

mm

elevation

Ca2

1.0 .tM

containing

to

containing

of ionophore

bated

[F472 - F6 4)]/0.40

varying

of zymosan-induced

response

for 60

Na-medium

presence

surface-as-

as

independence in

was

trations

F172,

Calcium

secretion

incubation free

of K-medium.

by cell

I (mM)

lCa2’

to poly-L-ly-

perfusion

contributed

medium

calibration fluorescein-

or attached

by subsequent

fluorescence

fluorescence

independent

a reduction

1.0

extracellular

the

obtained

for

sufficient

of pH

in medium noted

wavelengths

If such

particles).

ocular

to a holder

nm,

pH.

to manipulation

of fluorescence

be calculated

6.4. pH

pH,

Such

s, with

to pH

versus

it was

washes

transfer

at 518

be in part

immersed

fluorescence 500

200

phagosomal

should

coverglasses,

causes

medium

sensitive

particles

increase

for

surface-associated

fluorescence

zymosan

6.4

After

for emission

affect

.

fluorescein-

microscopy.

experiment.

the

with

consecutive

in particle-free medium ensured not cell adherent, as judged by

recording

largely

at pH and

for

labeled to 6.4

not

incubated three

phase-contrast

and

to acidify

does

intensity

by in every

at 497/456

infused

acidification

were

incubation,

coverglass that were

coverglass

performed

in a fluorimeter excitation

After

m-atioing.

LDII their Data

experiments.

lymmosonsal

secretion

487

NAG secretion was precluded treatment with phorbol ester After ters,

incubation their ability

by long-term

of macrophage cultures to bind [3H]PDBu was

with phorbol esrapidly impaired

(Fig. 2, filled symbols). After pretreatment with PDBu for 6 h, the level of [3H]PDBu binding was reduced to approximately 20% of the initial level. Reduced binding of

[3H]PDBu

was of protein

tion

membrane

of

Fig.

2.

Time

binding

and

nM

300

PDBu

for the

zytnosan

percent

of the

climethyl

cultures

indicated

time,

secretion

in response

prior

NAG

secretion

sulfoxide-treated

and

Results

[3H]PDBu

Results

are

exposure are

binding compiled

by from

to 200

expressed

as

untreated

or

three

ter-binding down-regulation Like

separate

experiments.

binding particles

PDBu

(Fig.

is Ca2+

independent.

Also,

secreted partment.

[Ca2+] phages,

enzyme need not pass In many cell types, triggers however,

a secretory treatment

it

event, as has is (2) likely that

comparatively little specific secretion of NAG Under the present experimental conditions, A23187 and also ionomycin were found to cause release of LDH and NAG, increasing with dose of or Ca2+

concentration

of the

tosolic [Ca2] appears ing nor the modulation lysosomal

enzyme

TABLE

studied

1.

medium.

to be involved of the rapid

(Fig. ib). ionophore a parallel ionophore

higher

Induced

by Various

Stimuli NAG

Pi-etreatment

Stimuli

PDBu

Methylamine

PDBu

PMA -PDD a-PDD

Monensin Monensin

(5 jiM) (5 tM)

and

Monensin

(5 tM)

Monensin

(5 tM)

Control (5 mM)

kinase the

(see Materials of a phorbol C not

response

to zy-

after

pretreatment

with

symbols). However, depression of the lagged behind the reduction of as it decreased approximately linearly to decrease to very low levels. that the secretory response was specific isoform of protein kinase was due of some

to the disappearcomponent down-

of amines, rise

ionophores,

to substantial cells (not

or chiorpro-

didecanoate.

After

Preincubation

It should

with

be

Activators

(or FDA) release (‘T of total) Pretreated

noted

of Protein

that

the

Kinase

cellular

C#{176}

Time

Mediuni

(mum)

20.5

11.0

Na

60

14.5 7.0

Na 199

60 45

7.0

199 199

45 45

PMA

(80

iiM)

41.0 33.0

and

PMA

(80

nM)

33.0

and

PMA

(80

nM)

33.0

31.5

(5 mM)

and

PMA

(80

nM)

37.0

9.0

199

80

Methylamine

(5 mM)

and

PMA

(80

nM)

40.5

5.5

199

80

PDBu

Bafilomycin

A1 (1 tM)

41.0

17.5

Na

60

‘Release

488

compiled

of pieloaded

Journal

of

from

three

overnight with either 80 nM PMA, 300 and the NAG or FD secretory response experiments,

each

representative

of am least

58,

1995

FD.

Leukocyte

Biology

Volume

October

was with and

specific NAG secreshown). The secretory

Methylamnine

are

to

secretory

PDBu

cultures were incubated (a-PDD) as indicated.

and es-

sensitive

6PDBu

‘Macrophage 12,13-didecanoate assayed. Data

of has

response to PMA and to combinations of PMA with either monensin or methylamine was highly sensitive to pretreatment with PDBu, PMA, or the but not the a form of

cy-

in neither the triggersecretion of preformed

Secretion

binding presence

down-regulated

concentrations

phorbol

of NAG

2, open

mazine could give tion from pretreated

here.

Reduction

sensitivity

C to down-regulation

The lysosomal secretory response to other stimuli also severely depressed by overnight preincubation PDBu (Table 1). The inhibition was not complete

macrocaused

In conclusion,

Differential

C or that the reduced response ance, or regulatory uncoupling, stream of protein kinase C.

through a phagosomal coman increase in cytosolic

response [26, 27]. In with Ca2+ ionophore

was

for 10 h and then continued This could indicate either dependent on one or more

if phagosome-lysosome

in macrophages is a Ca2+dependent reported for neutrophils [24, 25],

the

of [3H]PDBu,

response binding

[3H]PDBu receptor

16]. kinase

isoform(s) of protein by PDBu.

mosan secretory

fusion been

[15,

of protein

above the level of nonspecific Methods) and may indicate

with

of [3H]PDBu

to a 60-mm 7.2.

of PDBu

pretreated

to determination

pH

cells.

ester

were

isoforms

caused by an increased degradasecondary to its activation and

been reported in mouse peritoneal macrophages [17] and other cell types [28, 29]. The binding of [3H]PDBu remained approximately 20% of that observed in controls even though the time of PDBu pretreatment was extended for up to 26 h (Fig. 2, filled symbols). This is significantly

(h) by 1)horbl

Macrophage

in Na-medium,

(0)

exposure

of down-regulation

secretion.

(#{149}) or NAG

binding j.ttnl

dependence NAG

PDBu

C,

association

various

Time

probably kinase

nM

PDBu,

to various three

100 stimuli

separate

nM 4-phorboI was

assayed

experiments.

12,13-didecanoame

(-PDD),

thereafter.

cultures

Parallel

or 100 not

nM

pretreated

4a-phothol were

also

TABLE

Stiiuulus NAG secietion (c- of total) Methylamnine

(2.5

Effect

of Combining

PMA

with

Other

Stimuli#{176}

PMA (80 muM) NAG secretion (% of total)

NAG secretion (91 of motal)

4.5

30.0

40

9.5

41.0

60

9.0

41.5

60

3.5

24.7

45

Na-medium

1.5

42.0

30

K-medium

3.5

19.5

45

Combination

Time

of treatment

(miii)

Medium

mM)

10.0 Monensin

2.

Na/HCO3-medium

(1 riM)

20.5 Monensin

Na-mediutn

(5 (IM)

15.0 Nigeticin

(0.5

Na/HCO3-meditmm

.t[1)

6.0

Nigericin (5 .tM) 24.0 (5 .tM)

Nigericin

18.5 Macmo)hage treatment were

NAG

cultures were incubated in the presence of either as indicated. Data compiled from three experiments,

activity

in

similar

to that

release

of FD

the

in

cultures into in

kinase

synergistic

(not

preloaded

was down-regulated (Table 1).

Protein

after

controls

the

and

also

lysosomal with

C activation

signals

down-regulation

shown)

parallel

indicated stimulus, each representative

release

response,

the

trations response

of NAG

and pH elevation

for lysosomal

was that

compartment

the

PMA or a combination of four to six separate

of protein

kinase

whereas

are

a small

indicated stimulus. are shown.

a combination

of PMA

synergized to give Also, a combination

pH

Medium

and

6.9 and

low

time

of

concen-

a large secretory of PMA with

monensin in Na+containing media or with nigericin in K-medium, pH 7.2, was synergistic. A large synergism was also seen when a combination of PMA with nigericin was applied in Na-medium. Changes in cytosolic and lysosomal pH in response to amines and ionophores have been char-

secretion

C induced

of PMA and experinuents.

of methylamine (Table 2).

acterized Activation

K-medium,

secretory

previously

not PMA

was

[5] and

present

(not

these

were

shown).

The

similar

whether

decrease

or

in cytosolic

pH by nigericin in Na-medium implied that the increase in lysosomal pH, rather than changes in cytosolic pH, would be a critical factor for synergism to occur. However, no

a

synergism was seen when nigericin and PMA were combined in K-medium of pH 6.9 despite a large increase in lysosomal pH and only a small decrease in cytosolic pH.

b

.

/

20

Nevertheless,

/

0

.

.

.

a,

/

10

0 z

synergistic

effects

of this

[30] with

./

agent

to cause

both

and

zymosan

then

secretion,

role. The be due

activation

and a rise in lysosomal pH, this interpretation was the

PMA

on lysosomal

to be dependent on both C, while, as demonstrated

her, cytosolic pH has a modulatory zymosan as a secretagogue would

.

0

Cl,

the

shown in Table 2, are likely mal pH and protein kinase

particles

(not

lysosoear-

efficiency of to the ability

of protein

as shown below. lack of synergism

kinase

C

Consistent between

shown).

0 6.5

7.0

7.5

0

50

pH

lNa]

100

Zymosan-induced NAG secretion exhibits dependence on extracellular pH and Na

(mM) Secretion

Fig.

3.

NAG

Dependence

secretion.

Na-medium shown.

In after

In (b),

a

exttacellular

(a),

the

15-mm

the effect

concentration Ch-medium

on

a

dependence

is shown.

The

time

ofchase

means

for 7 and

13 experiments

was (a and

NAG

secretion 250

with

The

results

pH

of

zymnosan

of Na-medium

pemiod

mm.

.tg/mnl

the extracellular

in mixtures 60

on

zymosan

is Na and (250

presented

are

and Sundler

Protein

has

previously

in lysosomal

been

pH,

shown

provided

to be induced

that

cytosolic

pH

is permissive [5]. Figure 3a shows that zymosan-induced NAG secretion exhibited a modest dependence on extracellular pH in the physiological range, with larger inhibition seen NAG tion

b), respectively.

Tapper

of NAG

by an increase

of zymosan-induced

ofvarying

preincubation

pg/ml)

Na+

with

secretion

experiments

15-mm

and of

preincubation

on NAG

by performing during

pH

kinase

only at an in response

extracellular to zymosan

of extracellular

C and

pHi

[Nat]

regulate

pH below 6.7. was also impaired (Fig.

maerophage

Secretion of by a reduc-

3b).

lysosonial

secretion

489

5.5

I

0.

E 0 (I,

5.0

0 Cl) >‘

-J

5

0

0

5

0

5

Time Fig.

4.

Characterization

of the

for 15 mm

particles

of nonadherent

(b. second

trace

from

particles

for

latex,

particles top).

200

lower

jtgjmnl

traces

incubated cells

jig/mI

B only.

The

to that was

zymosan

In (e).

cells

were

Incubation

of inacrophages pH

Figure

4a.

in

When

to the

the

exposure

exposure

caused

zymosan

particles the correlation

after

ening secretory

response,

promised

in Ch-medium

at pH

6.4

Fig. 3). particles

(Fig. The was

tor ligation FD-containing

490

Journal

4c,

two

of

Leukocyte

(top

in Na-medium,

pH

7.20)

in the

were

300

otherwise

nM

treate(l

PDBu

for (from

been

reproduced

has

particles

elevated

as

shown

in

preincubation

at

of lysosomal of 200 j.tg/ml,

required

pH that

for a maximal

NAG

or the

further

elevation

pH

even

more

surface with

Biology

after

of lysosornal

pH.

than zyless than

trace from top). Strengthin lysosomal pH and the was

upper

period

NAG secretion lysosomal pH

increase

and

chase

traces;

shown

to be com-

so in Na-medium

compare caused resulted

Fig.

58,

and

by zymosan from recep-

rather than from less acid phagosomes.

Volunme

4a

October

fusion of as the

1995

bottom

tWtnl

5

trace.

live

was was

made.

(second cells.

trace

trace

to incubation

(d).

with

from the

cells

with

prior

for 60

tg/ml

with

4-jim

incubation

top).

tm-ace).

zymosan

The cells

middle were

In

mm.

200

In (c),

In (d, top B. For

bottom

zymostmn

inctmbation

was

cytoehalasin the

h Pi0

separate

particles a 15-mm

6.40

to the

with

50 j.tg, or no addition

(stitnulation after

pH

For

0. 3. 6, or 24

in at least

jig,

(b) no addition

of 10 jig/mI top

wem-e inctmbated 100

zymosan

recorded

trace of particles

presence

as for the

fig.

l)atii(les

in Na-medium,

addition

elevation cytochalasin

200

to)) (b) was

and

top trace)

200

of nonadherent

from

trace),

mm

18 h but

0

macrophages

1 mg, with

ln the

without

with

elevated lysosomal pH stable for at least 1 h and at the cell lysosomes

7.20

similar conditions [7]. Figure 4b that increasing the time of either

4b, third of a change

the

ti-ace

pamii(les. 6.40,

a 15-ruin

which cause less [7], also affected (Fig.

Third

ph

manner,

particles

a slight

memoval

in Na-medium,

7.2, the elevation concentration

under shows

particles, particles

over.

zymosa pH

after

mm

FD-loaded

downward)

incubation

and

zymosan

concentration

Latex mosan

j.tg/mnl

In (a), trace

bilizing tosis. with

to zymosan

70

pH caused by zymosan with the NAG secretory

assayed

secretory response ( two upper traces)

200

observation

a dose-dependent

37#{176}C in Na-medium, pH was maximal at a zymosan is, similar

for

Each

started

in Ch-medium,

(15

pretreated

7.20).

An elevation of Iysosomal particle binding correlates response

lysosomal

15 mm

PDBu

was

top

In ( b, top trace),

in (b) do not cross with

Ch-tnedium nM

ph

pH

particles.

of (from

pH.

5

(mm)

by zymosan

of lysosomal

traces

for

caused

at a concentration

obtained

particles

300

in Na-medium,

7.20,

registration

two upper

in

with

pH

of lysosomal

similar

recorded

pretreated

mm

15

and

particles

were

200

been

cytochalasin jig/mI.

zymosan

with

had

ratio

in (c)

pH

registration

mm).

15

at a particle/cell

with

in lysosomal

at 37#{176}C in Na-medium,

to removal zymosan

increase

0

two

were

trace treated

(d). with

particles

(200

experiments.

of lysosomal pH was large in the presence B (Fig. 4d, top trace), a microfilanient-destaagent

that

effectively

blocks

macrophage

of

phagocy-

The elevation of lysosomal pH induced by zymosan partides was down-regulated by PDBu preincubation as shown in Figure after with

4e.

An inhibitory

3 h (second trace PDBu was required

in lysosomal treatment

pH

(Fig.

were

4e,

lysosomal related

could

bottom

trace).

with

zymosan

incubated

presence of cytochalasin 4d, middle trace). This surface

effect

be seen

If cells

first

titosati

as

such in

the

B, lysosomal pH did increase (Fig. indicated that signaling to a rise in

pH could occur by zymosan bound at the cell and prompted us to evaluate how this and other parameters were affected by treatment with PDBu.

devised particles on

information

external

after

particles

Manipulation of zymosan particle attachment, internalization, and phagosome acidification revealed by a new method We

as early

from top), but overnight incubation for total depression of the increase

and

a method using that is simple, the

total

phagocytosed,

as

fluorescein-conjugated reproducible, and

number

of cell-associated

as

well

as

zyprovides particles,

measurement

of

C,, C

a) C

10000

=

C a) 0

0.

Cl)

a) 0 LI-

0 0

500

0

500

0

500

Time (s) 5.

Fig.

Quantitation

of cell

fluorescein-labeled trace

took

place

recordings was

zymosan in the

(a-c),

attained,

syringe.

the

fluorescence

upper

corrected

and

method,

However,

washed was

the

nm.

as

data

jig/tnt).

In (c),

the

cells

addition

K-medium,

pH

ratio

of the

representative

of four

treated lies

covergiass.

less

in the

Dependence on protein particle binding, uptake, acidification

of cell-associated Sb).

particles

with more

by

ing calculations

on traces

tides was extended as many particles

3). The

number

like

from became

15 to 45 associated

of particles

than the number of those bound cells. If the time of incubation further seen

increase (not

those

in

particles

mm,

or

5, at par-

three times cells (Table

increased

to the external was extended bound

incubation zymosan

almost with the

phagocytosed

that a number

in Figure

If an .tg/ml

less

aspect of the further, no

phagocytosed

was

Tapper

and Sundler

750

Protein

s,

were and

nM)

10

jiM

200

mm.

s. After

nigem-icin

the

incubated

was

lower

of the

In all

stable

added

traces

with

recording

for 9 h. 30

at time

particles;

preincubation of

with

the

2) and the inhibition by PDBu treatment.

ditions

by

show

three

recording microliter

the

absolute

PDBu

number

of

resulted

in

cell-associated similar a gradual

a rapid particles

to controls. increase

With in the

of these inhibition

of the rise Furthermore,

at 37#{176}C)enhanced

of the rather

subsequent

with effect

number

than

cell

zymosan of PDBu

of receptors

a more

efficient

The low phagosomal is noteworthy.

was similar to secretion (Fig.

in lysosomal pH PDBu pretreatment

the

incubation that the

changes of NAG

pH

(Fig.

association

was performed treatment was or their

binding

machinery

recorded

4e) (per-

under

for phagothese

con-

DISCUSSION Zymosan-induced conditions that ered

lysosomal would reduce

extracellular

pH,

in

secretion cytosolic

The secretory response by a reduction of extracellular be

secondary

by

Nat-dependent

to impaired

kinase

secretory

C and

pHi

cytosolic

a consequence coml)onent

response,

regulate

with

earlier

findings

to zymnosan was also itnpaired [Na]. This inhibition could

mechanisms.

acid pH could be tosolic pH of some

was inhibited by pH, such as a low-

agreement

[51.

the

shown).

(300

syringe

Cells particles

experiments.

up-regulation

increased the By perform-

presented

PDBu

3). The time dependence that shown earlier for the

properties

3 were generated. pH 7.2, with 200

with

the proportion phagocytosed extended PDBu treatment,

cytosis.

the data of Table 37#{176}C in Na-medium,

time

of particles when at 16#{176}C,indicating

kinase C of zymosan and phagosome

of cell-associated particles and concomitantly proportion of particles that were internalized.

with

cell-associated

up-regulation

formed

By comparison of Figure 5b and c, it is evident prolonged preincubation with PDBu increased the

At

pH.

incubation

proportion of particles phagocytosed resulted, in parallel with an apparent reduction of pH in the phagosomes (Table

proce-

of the experimental data, which and Methods and exemplified in B was shown to block phagocytosis

the number compare Fig.

7.2.

A short of

of phagosomal In (a),

by a microliter

to six separate

contributed are

7.2.

preincubated

with

reproducibility

every

been

of 10 jil of HCI

by phase-contrast

approach

had

estimation pH

fluorescence

coverglasses

to the for

by the

perfusion

fluorescence which

and

at 37#{176}Cin Na-medium,

by are

I)articles

15 mm

s by

confirmation

undertaken of our

500 revealed

with

of zytnosan

to 6.4

shown

contributes

dure than in the handling is described in Materials Figure 5. Cytochalasin while reducing half (Fig. 5a;

adjusted

at time The

for B (10

was pH

ease

visual

novelty

internalization

jsg/ml)

for the

The

although

microscopy

medium

exchanged

pH

particles.

(200

represent

at 497

by external the

was traces

and

of cytochalasin

of the

intensity

phagosomal handled

particles

presence

pH

medium

The

association

such

macropliage

pH The

regulation inhibition

[31,

32]

caused

by

of a dependence on cyinfluencing the magnitude of as

the

lysosonial

redistribution

secretion

of

491

TABLE

3.

Effect

of Phorbol

Ester

Pietteatinent

on the

Binding

Incubation with FZ

Pmireatmuue’mut (P[)Bu, :3oo muM)

and

Uptake

of Zymosan

Cell-associaied FZ (relative fluorescence)

Particles

and

Intracellular (% of total cell

on

Phagosome

Acidificationa

FZ associated)

Phagosomal

pH

-

15 olin.

37#{176}C

6,420

45

5.8

-

45

mm,

37#{176}C

16,481

30

5.8-

15 mm,

37#{176}C

9,228

40

5.8

3 Lu

15 mm,

37#{176}C

7,531

63

5.6

6 l

15 mm,

37#{176}C

10,123

68

5.4

12 Ii

15

37#{176}C

8,889

80

5.3

25

15

37#{176}C

9,321

83

5.2

15 mm.

16#{176}C

1,590

42

5.4

46 65

4.8 4.3

30

mm

h

-

mm, mm,

1 h 45

mimi

15 mm,

16#{176}C

2,870

5 h 45

miii

15 mm,

16#{176}C

5,093

la(rophage (-uliures 7.2. Data on intracellular Metll(KlS.

Whvmm incubation

reprcseniati%

of at leasi

I.

lysosomes

from

a perinuclear

of cells, as has pH [33]. Upon zymosan, interface

with

zymosan,

but

is concomitantly

ate

extracellular

deed 6.7.

greatly

up-regulated

inhibited

to all

at an also

stimuli

withstands

Centrifugal

PKC-down-regulated

cells

Furthermore, soluble stimuli

a moder-

extracellular

down-regulated indicating

on PKC activity This process was

was

still

the was

elevation similar

signal

movement

and

extension

of

the

compartment

are

mediated

by

kinesin

process indicates

appears independent that the pH-dependent

of cytosolic regulation

tribution colocalizes

would be primarily with lysosomes

on a centripetal in cultured

translocation

in-

shown

of

for

lysosomes

to a lysosomal pH was

can

act

as

a barrier

controlling

this

proteins

with

ties. Secretory network and

to

in

observed

between

stimuli

that

stimuli that activated PKC. itself was a poor secretagogue, lysosonial pH elevation. Translocation along and above,

of

microtubules intermediate

filament

a redistribution

by

this

caused confluent

492

of

Journal

Leukocyte

networks of lysosomes of cytosolic organelle

parameter.

redistribution peared more

has

involvement of in the synergy

lysosomal

been

pH

and

shown

to

occur

of microfilament [36].

As

and

late

alluded

to

endosomes

Notably,

upon

centripetal

the organelles apindicating fusion.

Volume

58.

October

1995

are

bundling,

phils has

[42] no

this

number

cross-linking,

in,

Such granule

gelsolin and more, cell-free linization

of [44]. of the

the

cytosol

actin

in

substrates cytoskeleton

secretion

lysosomotropic

mechanism agents

to a lysosomal

network signaling afthat affects

depolymerization that

have and

relevance MARCKS

reversibly

of lysosomal

proteins [43]. Furthersuggested a role for an alka-

proteins phage relocate

actin-filaand baso-

by the enhancing effect Cytochalasin B

dephosphorylated alanine-rich

[45]. Of possible lysosomal secretion,

proper-

response.

by polyphosphoinositide interaction and by [Ca2]

PKC

and

linked to the bind to stor-

of a cortical neutrophils

to membranes in their MARCKS (myristoylated

dependent

severing

of the actin-filament

other actin-binding in vitro studies

filaments

with

a role for fodrin has been secretion. A prerequisite

macrophage

Regulation

can be accomplished fecting profihin-actin

for secretion network that of actin-binding

and

disintegration for example,

on

a prerequisite

to be physically proteins can

be demonstrated secretory

effect

however.

be

Associated

[41].

on the

such

contents,

can

could

a large

vesicles appear some actin-binding

age organelle membranes. implicated in chromaffin

control

pH [33, 37] indicattransport can be regu-

by alkalinization, and elongated,

Biology

pH, a role

phorbol diester by the necessity for a

to be independent

occurs upon manipulation ing that microtubule-based lated

elevated However, implying

lysosomes and

in lysosomal and a modulatory

exocytosis

network

as

phagocytosis,

One obstacle for the compartments destined would be a subplasmalemmal actin-filament

of cytochalasins

PKC

location,

frustrated

some

of lysosomal whether PKC

for an obtained

a perinuclear

during

secre-

been implicated: a triggering increase necessary PKC-dependent later step, Further evidence signaling was

motor. Dynein cells [40] and

the

for fusion would be ment network, and

pH. secretory

this This dis-

that

for to the

macrophage and

might be mediated by dynein and for a lysosomal secretory response.

down-regulated or not, but no secretory response was triggered in the down-regulated cells. Thus, for lysosomal secretion to occur, three crucial factors for many stimuli have

of cytosolic

to

macrophages

pH and each

pH [38, 39]. of organelle

below

pH, since receptor by zymosan in

could

lysosomal

indicated, in Na-medium, described in Materials from five experiments.

pH

was necessary located distal

lysosomal uptake)

in-

of the

secretion

tested,

to an increase in (without particle

increase. by

and

only

depending secretion.

signaling ligation

caused

periphery

capacity

Lysosomal

of PKC

response

process lysosomal

pH

to the

the pH-regulatory

acidification.

Down-regulation tory

localization

been reported to occur at an acidic cytosolic neutrophil or macrophage interaction with

a respiratory burst is triggered at the cell-zymosan [34, 35]. Thus, generation of metabolic acid

creases cell

isem-t inculuated witl fluoresrein-labeled zymosan particles (FZ. 2()0 pg/mull for the time and at the temperature particles (7I ) an(l phagosomal pH aeme derived from tmaces such as those presented in Figure 5, by calculations was (1imie(l out at 16#{176}C, a 30-mimi chase at 37#{176}C was allowed prior to registration of data. Data are compiled three similar, separate experiments.

been

furthermore

can form

actin in bind

C-kinase

are the substrate)

to regulation have been

of macroshown to

compartment

upon treatment (Dr. Lee-Ann

of implicated

by a PKC-

of intact cells with H. Allen, Rockefeller

University,

New

York,

CONCLUDING

personal

12.

secretory

events

but

macrophage

and

are

properties

of the

response. signals signal

secretory

to external proposed

often

fusion,

signaling

pH

and

ionic

and/or lysosomal

subject requires

composition

of the plasma

fusion

or vesicular

lysosomal

pH-regulaand also

of the

likely candidates cytoskeletal

membrane. along

could

various

also

19.

20. 21.

22.

23.

regulate

lund sity,

is

5410),

Swedish

endocytic

the

Medical

Crafoord

path-

and

the

Research

Foundation,

Foundation, the Greta & Johan Gustaf V 80 years Foundation, gratefully

Medical

Kock the

Faculty,

Coun-

the

Rapslee.

Lund

2.

3.

4.

5.

6.

7. 8.

26.

27.

Univer-

acknowledged.

28.

29.

D.A.,

sl)eti%e.

Werb,

Z. ( 1989)

Macrophage

secretions:

a functional

Inst.

Pasteur

87,

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31.

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