CSF-1 stimulates glucose uptake in murine bone marrow-derived macrophages

8lOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages 445-454

Vol. 138, No. 1, 1986 July 16, 1986

CSF-1

STIMULATES

John

A.

GLUCOSE

Royal

of

Melbourne

Received

Gino

Hamilton,

University

May 20,

UPTAKE IN MURINE MACROPHAGES Vairo

Melbourne,

Hospital,

and

BONE

Suzanne

R.

Department

Parkville,

MARROW-DERIVED

of

Lingelbach

Medicine,

Victoria,

3050,

Australia

1986

3H-2-deoxyglucose was used as an isotopic tracer for the measurement of glucose uptake into quiescent murine bone marrow derived macrophages. A pugified colony stimulating factor (CSF1) was shown to stimulate H-P-deoxyglucose uptake in a dosedependent manner. This stimulation was rapid, with a maximal effect seen at 20-30 minutes after growth factor addition. Both the inhibition by cytochalasin B and also the relative degree of competition by high concentrations of a series of glucose analogues suggest that the basal and CSF-1 stimulated 2deoxyglucose uptake occur via a carrier facilitated D-glucose transport system. The data indicate that a purified growth factor can increase the glucose uptake in macrophages, a finding which could be relevant to the survival and/or the proliferative response of this and other haemopoietic cell types. 0 1986 Academic Press, Inc. Colony is

stimulating

specific

for

macrophage) cell Mr

cells

lineage

specific

binding

(5).

require

a growth

proliferation

can

be

a two

Murine

prepared

as

these

adherent

Cel 1 s along

the

macrophage

and

such

as

differentiation

ABBREVIATIONS: BMM, bone specitic colony stimulating 2-DOG, 2-deoxy-D-glucose, haemopoietic cell growth

from

cells bone

L-

70,000

binding

to

marrow-derived

cells

homogenous expressing

lineage, for

mouse of

via

a relatively

CSF-1,

which

(or

glycoprotein

on the

(4).

factor

isolated

subunit

>, 95% of

factor,

a growth phagocytic

factor,

effects

r eceptors

(BMM) with

This

its

is

mononuclear

is

mediates

surface

population

the

medium,

which

macrophages

of

(CSF-1)

(1,2).

conditioned (3),

factor

their

cell

CSF-1

including

BMM,

survival,

(5).

marrow-derived macrophages; CSF-1, macrophagefactor (or macrophage growth factor); FBS, fetal bovine serum; HCGF, factor; LPS, lipopolysaccharide.

445

All

Copyright 0 1986 rights of reproducriorl

0006-291X/86 $1.50 by Academic Press, Inc. !n any ,/Lwm reserved,

Vol. 138, No. 1, 1986 The

BIOCHEMICALAND

biochemical

differentiation (6)

are

events for

interaction

associated

cells

essentially

along

CSF-1

with

BMM as

events.

Several

studies

that

enhanced

glucose

transport

the

proliferative

cell

response

references

line

survival

and

(HCGF)

(9).

action

on

whether

has

These

normal

stimulates

uptake

uptake

in

study

demonstrated

cellular

change (see,

for

which

for

depends cell

It

in

by to

the

that

same

its

2-

be

CSF-1

BMM in

factor

involve

measured

here

quiescent

to

remains

behave

its

growth

appear

of

in

precursor

as

We report

the

a haemopoietic

(10,ll).

factors.

for

have

a haemopoietic

cells

lineages the

stimuli

transport

haemopoietic

glucose

early

effects

glucose

growth

an

established

HCGF-mediated

increasing

system

mitogenic

on

(2-DOG)

other

been

studying

cells

Recently,

proliferation

deoxyglucose

and

7,8).

(FDC-P2)

is to

been

other

and

haemopoietic

a model

in

proliferation

various

We have

such

example,

with

the

unknown.

of

BIOPHYSICAL RESEARCH COMMUNICATIONS

determined

way

to

HCGF

rapidly

a dose-dependent

manner.

MATERIALS

AND METHODS

Preparation

of

BMM were CBA or

marrow-derived

obtained

from

C3H/HeJ

previously prior

bone

mice

(5,12).

are

in allowed

differentiation 12-well

homogenous

population

(5).

in

medium

develop

dishes.

The

with

prior

growth

medium

marrow

as

described

proliferation BMM in

BMM are the

use

without

Linbro

446

conditioned

(Flow

Labs,

a relatively adherent

uptake

L-cell

a

and

of

for

from

after

L-cell

"starved" to

bone

cells,

containing

>/ 95% of

generally

18 hours

precursor

by

adherent

in

Parkville)

frozen

the

BMM were

approximately reculturing

to

cells

Institute,

growth

into

Australia)

CSF-1

precursor

Briefly,

incubation

medium,

(Hall

macrophages

pure cells

growth studies conditioned

and

binding

factor

for by medium.

Vol.

138,

Such

No.

1, 1988

BIOCHEMICAL

factor-deprived

GO/G1

state

cells

of

typically

the

gave

measured

by

2-OOG

cell

have cycle

nuclei

performed

KCl,

0.8mM

4 x

counting

in

Amersham,

to

be

COMMUNICATIONS

in

a quiescent

unpublished).

10

The

5 adherent

cells

protocol

per

well,

(15).

a HEPES 1.8mM

were

initiated

Australia,

cultures

were

washing

NaOH.

cells

was

was

measured

from

Pierce

Sources

(DMEM)

Chemical

the

(30-60

Ci/mmole, of

cells

method

in

0.5ml

of

1 ml

of

solubilized

Cellular

using

Illinois,

stopped

buffer

solubilized

counting.

lml

the

was

HEPES

in

Lowry

Company,

Uptake

Uptake

warm

5mM

the

protein

BCA reagent

U.S.A.

CSF-1 : Serum

was

prepared

containing

300

as

Mouse

ml

Dulbecco's

supplemented

with

fetal

L-cell

principally

alterations.

-inactivated roller

with

BMM

NaCl,

pH 7.4).

bath.

scintillation

by a modified

following in

water

and

135mM

3H-2-DOG

radioactivity by

BMM cultures

grown

HEPES,

quiescent

Incubations

4 times

D-glucose

into

2-DOG,

adding

shaking

monolayers

measured

BMM growth

20mM

by

Incorporated

of

(O.lmM

CaC12,

a 37°C

5.5mM

0.2M

buffer

uptake

1 u Ci/culture).

in

the

containing

For

shown

(3H-2-DOG)

MgC12,

measurements

the

been

RESEARCH

uptake

was

For

BIOPHYSICAL

(13,14

approximately

3H-2-deoxy-D-glucose

by

AND

bovine

described

L-cells

modified O.lg/l

minimum

(FBS)

but

essential

850

for

with

strain)

sulfate, in

medium (16)

(L-929

neomycin

serum

conditioned

were medium

10% heat

cm2

Corning

plastic

bottles. uptake

measurements

conditioned Briefly

(17). free

L-cell

absorption Ultrogel

medium

was

the

AcA-44

derived

purified

to

purification

conditioned (stage

: CSF-1

2),

medium

(stage

(stage 447

serum-free

homogeneity

stages

DEAE-sepharose

chromatgraphy

from

were: l),

as

L-cell

described

concentrated

serum

calcium

phosphate

chromatography

(Stage

4),

HPLC

size

exclusion

gel 3),

Vol. 138, No. 1, 1986

8lOCHEMlCALANDBlOPHYSlCALFiESEARCHCOMMUNlCATlONS

chromatography (Stage

6)

CSF-1

(Stage and

HPLC

reverse

rechromatography

bioactivity

semi-solid

5),

was

agar

on

measured

medium

phase

reverse

using

according

phase

C57BL

to

chromatography

ref.

HPLC

bone

(Stage

marrow

7).

cells

in

6.

REAGENTS The (Flow

following

laboratories,

Company,

D-xylose

other

precautions

St.

and

Louis,

reagents for

were

Hospital

Products)

and

lysate

tests

D-glucose

analytical

made

up

endotoxin

All

pyrogen-free

levels

practical were

routinely

Serum

taken.

water

were

(Commonwealth

6-

Australia).

contamination in

FBS

Chemical

(Ajax,

grade.

endotoxin

:

3-0-methylglucose, B (Sigma

and

of

routinely

commercially

cytochalasin

minimizing

were

limulus

obtained L-glucose,

Missouri)

Solutions

by

were

Australia),

deoxyglucose,

All

reagents

(Abbott monitored

Laboratories).

RESULTS For was

linear

BMM in for

the at

absence least

30

of

CSF-1

minutes

the

uptake

(Fig.

1).

of

2-DOG

Incubation

(0.1 of

mM) BMM

; 4,0% 7430. % e 5 2.0. iE=

1.0.

O0

Figure

I.

Glucose

uptake

in

5

untreated

IO Time

15 20 (min.)

25

30

BMM

Quiescent BMM from CBA yice were washed twice with HEPES buffer and then pulsed with H-2-DOG for varying intervals. The zero time point was determined by pretreating the cells with phloretin (0.3mM) and immediately harvesting. This background value was subtracted from all of the experimental points. Each point represents the mean of triplicate cultures (t S.E.M.). 448

Vol.

138.

No.

1. 1986

0.4 1L

BIOCHEMICALAND

0

8lOPHYSlCALRESEARCHCOMMUNlCATlONS

10

loo [CSF-11

Figure

2.

Effect

of

both

point

2-DOG

Comparison

of

uptake

the

increase

2-DOG

uptake,

and

the

presented

the

by

Figure uptake

by

partially pulsed

was

Fig.

2 was

and

BMM

curves of

the due

the

partially the

carried

two

out

that (LPS) 3 shows

CSF-1.

3 minutes

the

contamination the (For

the

CSF-1 at

in

time

course

varying

there

was

CSF-1

of

was

The

449

active

the

strain,

uptake

was

to

experiment

BMM from

C3H/HeJ

was

not

caused

samples. stimulation

experiments used.)

intervals

preparations

L-cell

the

remaining sample

that

purified

2-DOG

CSF-1 2).

CSF-1

with

7)

(Fig.

mouse

increased

(stage

purified

CSF-1.

(fS.E.M.).

manner showed

that

to

cultures

pure

a dose-dependent

(LPS)-hyporesponsive

purified for

in

triplicate

2)

indicating

medium

suggesting

endotoxin

(stage

ability

in

lipopolysaccharide

(181,

uptake

mean of

response

activity

in

the

in

dose in

conditioned

glucose

purified

difference

that

on

represents

partially

stimulated

little

dose

(U/ml)

Quiescent BMM from C3H/HeJ mice were treated with various of partially purified (stage 2) CSF-1 (x---x) or pure 7) CSF-1 (0 -0) for 1 hour in HEPES buffer and then with 3H-2-OOG for 10 minutes (Materials and Methods). dilutions were made in HEPES buffer containing 15% FBS, to a final FBS concentration in the cultures of 1.5%.

doses (stage pulsed CSF-1 leading Each

with

CSF-1

4 ~#ooo

1,~

of

the

2-006

presented, Quiescent

after

addition

the

BMM were of

the

Vol. 138, No. 1, 1986

BIOCHEMICALAND

0

5

10

BIOPHYSICAL

15

30 Time

Figure

Kinetics

3.

of

RESEARCH

stimulation

of

COMMUNICATIONS

45

120

(min)

BMM

glucose

uptake

by

CSF-1

Quiescent BMM from CBA mice were washed twice buffer and pulsed in the same buffer for 3 minutes at varying intervals after the addition of partially CSF-1 (stage 2, 1000 U/ml). (O-O), + CSF-1; (O-B), Each point represents the mean of triplicate cultures

CSF-1

sample.

DOG uptake sharply

the

within to

which

There

the

plateau and

gradual

increase over

the

120

under

experiment

presented

which strain

yet

Difco

Labs.,

again

suggesting

samples

was

in the

2-DOG

Fig.

did

to so when

that due

to

B (1 LPS, LPS

(100

was

tested

the

increased

endotoxin

not

by

2-

at

experiments, within

resulted

a new

20 in

steady

state

enhancement

exceeded

of

3-fold.

with

a

2-

The

BMM from

the

a polypeptide block

rig/ml, itself 2-DOG

contamination. 450

to

of rising

reached

out

ug/ml),

rate

minutes,

other

maximal

carried

did

the

medium

never

3 was

Polymyxin binds

The

conditions

120

was

uptake

gEPE5 H-Z-DOG purified no CSF-1. f 5.E.M.

addition,

In

glucose

period.

in

Detroit)

not

low

basal

minute

until

cultures

the

in

factor

terminated.

treated

optimal

strain.

antibiotic this

CSF-1

in

growth

30 minutes was

incubation

DOG uptake

CBA mouse

within

the

increase

after

experiment

in

minutes

a significant

5 minutes

a plateau

time

level

was

with with

the E. (data uptake

CSF-1 coli, not in

effect Olll:B4, shown),

the

CSF-1

in

Vol.

138,

BlOCHEMlCALANDBlOPHYSlCALRESEARCH

No. 1, 1986

COMMUNICATIONS

I Figure

4.

Specificity treated

of

the

glucose

uptake

system

in

untreated

and

CSF-~

BMM

Same experiment protocol as for figure 2 except that partially purified CSF-I (stage 2, 1000 U/ml) was used and the compe ing drugs were added 15 minutes before the 10 minute pulse with 5H-2-DOG. BMM were from CBA mice. Cytochalasin B (Cyt) was at a final concentration of 50 PM while the following sugars were at 10mM: 2-DOG, D-glucose (D-Glu),L-glucose (L-Glu), 6-deoxy-Dglucose ('6-DOG), 3-D-methylglucose (3-D-MeG), and D-xylose (DEach point represents the mean of triplicate cultures XYl). (+S.E.M.)

2-DOG

can

it

is

is

non-utlilizable

can

be

transported

be

by

this

a

this

molecule

is

effect

inhibited

on the

Similar

basal

inhibition

inhibitor

of

facilitated not

the

by

rather

(data

which

CSF-1

was glucose

In

Fig.

inhibit

itself

by (data

2-DOG 4,

is it

451

have

uptake

found

uptake

(Fig.

by

4).

(0.3mM),

shown).

competed

for seen

and

completely

phloretin

be

found

(20).

glucose

not

can

we

unstimulated

macrophages

(21), 2-DOG

reason,

not

Others

to

caused

this

it it

However, in

by

known

transport of

used.

poorly

induced

also

is

shown).

carrier

and

For

poorly

not

is

glucose

transport L-glucose.

up

while

material,

which often

transported

B,

cellular

analogue

because

However,

kinases.

is

BMMs

Cytochalasin its

cytoplasmic

taken

transport

(19). into

glucose

is

glucose

carrier

intracellularly, analogue

of

glucose

by

CSF-l-stimulated

that

measure

incorporation

phosphorylated

phosphorylated

a

the

glucose,

that

as

by

3-O-methyl

in

used

Also, by that

another carrier-

D-glucose both

but the

basal

Vol. 138, No. 1, 1986

and

CSF-1

BIOCHEMICALAND

induced

concentrations

3H-2-DOG of

2-DOG

O-methyl-D-glucose, shown

to

system

have

the

are

some and

2-DOG

consistent

being

incorporation and

D-glucose,

specificity

but

effective

uptake

in

with

through

basal

by

D-glucose

varying

degrees The

4).

and

high

L-glucose. have

3also

been

transport in

competing

findings

CSF-l-enhanced

carrier-facilitated

by

D-xylose

the

BMM (Fig.

the

the

to

lowered

not

and with

are

RESEARCH COMMUNICATIONS

was

6-deoxy-D-glucose

(22)

with

BIOPHYSICAL

in

Fig.

2-DOG

D-glucose

4

uptake

transport

as

system.

DISCUSSION We have basal

and

provided

stereospecific

stimulated

by

have

stimulated

to

transport

energy

of

dependent action

of

transport

that as

indicate

growth

an

particular

factor

survival

the

continual

be that

for

and,

an

BMMs in

presence all

2-DOG

cells

addition, of along

the

through

available

to

that

the

in

on

for

and

cell

on

glucose factor

proliferation

monocyte-macrophage 452

lines

is

from

the

glucose

results

progenitor

meet

HCGF-dependent

type

a growth

a

process

resulting

Our cell

cells

glucose

(9-11)

uptake.

diffusion

with

that

factor

BMMs CSF-1

made

metabolism

increase

depend

have

precursor

hemopoietic

cause

As mentioned, for

by

a normal can

is

of

attendant

energy

The

peritoneal

quiescent

uptake

shown

is

a facilitated

that

glucose

growth

measured

of

their

has

in

and and

haematopoietic

increase

for

those

more

of

that

a

manner.

alveolar

recognised

evidence

a number on

of

requirements

Recently

system

previously

increase

so that

additional

survival

well

BMM possess

a dose-dependent

with

is

growth

system

(7,8,19).

to

It

in

that

transport

described

consistent

(15,20).

suggests

systems

been

characteristics

the

CSF-1

transport

macrophages

which glucose

rapidly

monosaccharide

system

data

are

that

the

first

a purified

uptake. such

as

cells (5).

CSF-1 require

It

lineage,

could CSF-

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

Vol. 138, No. 1, 1986 1 mediates

its

Further to

survival

experiments

determine

mitogenic could

are

the

the

dependent

of

same

on

in

relevance

effect be

effect

different

to

other

of

By

BMM.

glucose

answer

otherwise

on

the

enhanced

progress or

CSF-1

for

on

this our

question to

the

factors

by

grants

from

of

Australia,

the

situation

lineages

growth

and

findings

implication

hemopoietic

specific

uptake.

which

(CSFs)

are

(6).

ACKNOWLEDGEMENTS This and

work

Medical

Council

of

was

Research Victoria,

supported Council the

Utah

Ramaciotti

Foundations

D.

and

A.

Burgess

Mrs

L.

Williamson

Metcalf

Shanks

and

and

are

the

Foundation,

the

the

Jack

Brockhoff

thanked

for

for

typing

the

the

Health

Anti-Cancer Clive

and

Foundation. CSF-1

of

National

bioassays,

Vera Drs. Ms.

manuscript.

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454