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PJ, Van Eeden G, Ralph. P. Altrock. BW, Falkenburg .... activation with phytohemagglutinin. J Immunol. 141:3868,. 1988. 35. Metcalf. D,. Nicola. NA: Proliferative.

From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

1989 74: 2398-2404

Interleukin-1 synergizes with granulocyte-macrophage colony-stimulating factor on granulocytic colony formation by intermediate production of granulocyte colony-stimulating factor MR Schaafsma, JH Falkenburg, N Duinkerken, J Van Damme, BW Altrock, R Willemze and WE Fibbe

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From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

InterleukinFactor

I Synergizes With Granulocyte-Macrophage Colony-Stimulating on Granulocytic Colony Formation by Intermediate Production Granulocyte Colony-Stimulating Factor

M. Ronald

By

Schaafsma,

J.H.

Frederik

Falkenburg,

Roel lnterleukin-1

(Il-i

) was

found

granulocyte-macrophage CSF)

on granulocytic

marrow

cells.

cells

colony

depleted

lymphocytes.

Using

we

demonstrated

effect

of

intermediate

cells an

ll-6

did

Furthermore.

this factor to

not

by itself

I

NTERLEUKIN-1 (IL-i) regulation of hematopoiesis. phase response and mediates neutrophils,

clear

phagocytes,3

bone marrow stimulating

factor

and

the

T

was

cells.

not

a

but

by

an

residual experi-

that

observed

Il-i

-

synergy.

a direct

forms, IL-i endothelial

cells’3’4 (G-CSF),

to produce macrophage

CSF- I ), and granulocyte-macrophage whereas T lymphocytes produce

cells,8’2

CSF

to

IL-l.’5

Another nized

hematopoietic

in mice,

proliferation

activity

of IL-i

has been

consisting of a synergy with of early hematopoietic progenitor

which could not yet respond to CSF lL-l was first described in combination

alone.

CSF cells

This

with

bone marrow

cells.20’2’

GM-CSF act cytic/monocytic

synergistically colonies

It was

bone marrow

adherent

IL-i

of but

with IL-3,’7 synergistic IL-i

low

as 0.1

but

not

tion

of

IL-i

Leary

of action. displayed

human

blast

In this

does

tion

From

the

Laboratory

Hematology,

lands;

ofExperimental

University

Laboratory

Hematology.

Medical

cells,

Research,

University

Thousand

Oaks,

of Leuven,

May

Supported

in part

Radiopathology

Normal

Department The

Nether-

marrow

Rega

Institute

for

Medical

hernia

Belgium;

and

cells

Amgen,

/6, and

J. V.D. is research

/989;

accepted

by grants Radiation

July

25,

the

J.A.

from

of the Belgian

Cohen

Institute

for

National

ment

of

requests

Hematology,

to M. Ronald

Bldg

1, C 2-R.

Schaafsma, University

The Neiherland. were defrayed

charge

therefore

payment.

“advertisement” indicate

This

article

in accordance

must with

this fact.

/989 by Grune & Stratton, Inc. 0006-4971/89/7407-OO/9$3.OO/O ©

2398

18 U.S.C.

MD. Medical

Rijnsburgerweg /0, 2333 AA Leiden. The publication costs ofthis article

be section

bone

of IL- 1 was caused effect

between

evidence to synergy cells

IL-i

of

normal phagothat this between

in response

to

our results indicate that proliferation and differentiamyeloid

progenitor

cells.

human

bone

nuclei

AND

After

marrow.

by aspiration

pulposi

patients

collected

in

from

who

informed

consent,

the posterior

iliac

underwent

balanced

Hanks’

heparin

METHODS

(HBSS).

laminectomy.

salt

After

bone crest

solution

dilution

of The

containing

in

RPMI-1640

5% fetal bovine serum (FBS; Hyclone, UT), the cell suspension incubated with 125 mg sterile carbonyl iron powder (Grade SF; Aristopharm, DeIft, The Netherlands) in Erlenmeyer glass flasks was

of reprint

IL-i (CD34indirect

CD34-enriched

accessory

Furthermore, the in vitro

was obtained were

(45

Research

Fund. Address

between

with

/989.

Protection.

associate

from

lineage-specific

preservative-free

CA.

Submitted

as

unclear a direct

mono-

Leiden.

Leuven,

the

as well

colony growth depleted of mononuclear

by residual

GM-CSF. not affect

in

it was shown that IL-6, but not support of IL-3--dependent

We present and attributed

is indirect,

of committed,

Center.

of Immunobiology.

of synergy

the synergistic

MATERIALS

of

M-CSF

of highly purified cells, indicating an

in

granulocytic

produced and

proliferacells

an indirect effect due to an factor produced by accessory a lack

lymphocytes.

Il-i.

cells.

with

formation

marrow T

of IL-i

IL-I IL-6

or

we show

as

that

progenitor

in combination

on

bone

G-CSF

indicate

suggesting that the synergy production of IL-6.

report

synergistic of

From these studies it remained between IL-I and CSF was

cell colony

and

this

results

pro-

GM-CSF.

Inc.

Furthermore, synergy

GM-CSF

human

GM-CSF

of accessory

on HPC, intermediate

marrow cells, by intermediate

and

G-CSF with

GM-CSF-dependent

on colony growth human progenitor

mode IL-l,

colony

-induced

synergy

myeloid

et a122 reported

IL-3 enriched) and

synergisti-

an anti-G-CSF

concentrations

& Stratton.

or IL-3.2’ the synergy of

the

numbers

growth

colony

with Il-i

Our

mature

it act monocytic

the

at

U/mI).

stimulates

by Grune

G-CSF,

and

(10

of small

GM-CSF whether

and

detected

ng/ml

did or

that for

of G-CSF

be

11-6,

1989

cytic

showed

relatively

activity

of granule. human non-

enhances

could

cytes

on human

that

proliferation of normal

cells,#{176} and that

on the (HPC),

M-CSF,’6

demonstrated

on the in cultures

recog-

synergy

analogous activities have now been documented GM-CSF,’8 and G-CSF.’9 Recently, a similar effect between IL-I and CSF has been reported

combinations

activity

cells.

and

(GM-CSF), in response

GM-CSF

Using

nor

granulocytic

responsible

IL-I-induced

granulocyte colonyCSF (M-CSF or

W. Altrock.

experiments

antibody was

action

during mononu-

Bruce

growth.

on

Neutralization

monoclonal

0

Damme,

colony

GM-CSF

presence

stimulatory

observed induces

on CFU-GM with

duction

has several activities in the IL-l is involved in the acute the increase in circulating

fibroblasts,7

stromal

formation.

showed

neither

immature Furthermore,

including

infections.’

bone

. Neutralization

to

had

human

produced

Il-i

contribute

effect cally

marrow

Jo Van

E. Fibbe

with

of Il-i

antiserum

Duinkerken,

Willem

(GM-

bone

activity

and

factor phagocytes

progenitor

anti-ll-6

11-6

microbial

of normal

hematopoietic in response

using

induced

growth

that

on

ments

synergistically

of mononuclear

action

accessory

act

CD34/HIA-DR-enriched

direct

an

to

colony-stimulating

Nelleke

Willemze.

of

minutes; the

Depart-

mononuclear

Center,

suspension myeloid

in part by page hereby 1734

solely

to

particles

were

removed i,000

harvested serum

rosetted

with

and (FBS).

magnet The

Blood,

was

added

cells were iron-loaded

by sedimentation

through

g; 20 minutes,

20#{176}C),and

washed

times

three

T lymphocytes and sedimentation

Vol 74. No 7 (November

in

were

RPM!

the

the cell

collected,

and

mononuclear interphase plus

removed

5%

cells fetal

by being

(AET)-pretreated

of the rosetted

15),

remove and

Ficoll-Isopaque

the

then

bromide

phagocytosis

To

cells, and residual

2-aminoethylthiouronium

erythrocytes

to allow

phagocytes.

for 5 minutes.

red blood

were

g/cm3,

agitation)

mononuclear a sterile

was shaken cells,

bovine sheep

continuous

by

phagocytes,

phagocytes (I .077

marked

37#{176}C, with

iron

1989:

cells through

pp 2398-2404

From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

IL-i

SYNERGIZES

WITH

Ficoll-lsopaque. than

The

GM-CSF

nonrosetting

1% T lymphocytes using

cence goat

anti-mouse

The

anti-CD3

(Becton

Immunological

7TD1

2% mononu-

a-naphthylbutyrate

esterase

bone

cells was obtained

forming

cells using

an anti-CD34

Mountain

View, CA),

and

a fluorescence

Dickinson). and

T lymphocytes, at 4#{176}C. The

labeled

with

and

incubated

were

incubated

MoAb

Flow Laboratories,

separation

was

performed

negative

and

cence

intensity second

50%

centrifuged

FBS,

only.

The

(150

g;

40%

(or

and

stimulated

U/mL),

were

were

0.5

were

l0

of greater

than

Normal

defined

iO bone

Reagents. provided

MA).

Recombinant

Amgen

from

purification

human

crude

other

cytokine

CSF,

and

human

as low

neutralized

by

pure

magglutinin

(PHA).24

NH2-terminal

amino

IL-6

against was

The acid

purity sequence

of

to detect

in

protein

only

(Fig

with 1).

numbers of granulocytic colonies numbers of monocytic colonies were

cultures

IL-I-stimulated

and controls

This

attributed

in the

the

of

of both

as compared

be completely

(Fig simi-

(Fig 2B).

-

100

-

I.-

IL-i

0 ‘4-

0 50

-

was

contain

control as

levels

sheep

of

IL-lfl

could Standards IL-l$.

supernatants with wa(

Recombinant

of

phytoheverified human

010

0 be

(kindly

recombinant from

the

0

from

of the

activity,

stimulated

analysis.

was

endotoxins,

for Biological

cells

U/mL),

could

numbers

presence

GM-CSF

interferons,

activity raised

obtained

mononuclear

0

IL-ifl not

IL-2,

able

human

higher

in the

100 with

.05)




in the ±

was obtained

biologic

(P

numbers

C

It did

assay

The

in an assay

by Dr S. Poole,

peripheral

lysate

and

a

.c

0 0 0

U/mg)

Institute,

IL-ia,

to be free

amoebocyte

(mean

preparations

including

aggregates

(l0

previously.4

as 60 pg/mL.

Electrophoretically human

found

as measured

and Control,

lymphokine

increase

were

obtained

stimulated

in colony

whereas for

(10

determine

of recombinant and in the presence

were

GM-CSF

cultures

To

IL-i and GM-CSF, nordepleted of mononuclear

0)

I0

plated.

U/mg)

Wil-

150-

microscope.

146

Electrophoretically

tested,

It was

by a Limulus

endotoxin preparation, completely

(l0

as reported

activities

!L-6.

(Genetics

G-CSF CA).

cells

the

using

0

of

colonies

GM-CSF

(rh)

Clark

each

number

an inverted

10 CFU-GM

mononuclear human

like

100%

cells, the

±

of !L-6. tested,

factors

or monocytic

are 503

evaluated

GM-CSF.

between cells,

Significantly

colonies

200

T lymphocyte-

days,

using

U/mL

marrow

Oaks,

10

for day

100

schedule

provided

scored

by Dr S.C.

(Thousand

obtained

were

Recombinant

kindly

After

to an

lar

into the wells of microtiter

as granulocytic

in our laboratory

of GM-CSF

23) per

well.

U/mL).

difference

or

mL,

CD34/HLA-DR-enriched

each

20 cells,

values

presence

tested

in

U/mL

(100

the

of 0.1 and

cytokines

were

T lymphocytes,

FBS,

lL-ij3,

no stimulatory

phagocyte-

(10

2A),

of GM-CSF

replicates

cells were plated

containing

colonies,

Six

2,000

other

in a

At a dilution

in a-MEM.

determining

control,

medium.

mononuclear

marrow

G-CSF,

concentration

in

20%

(2.25%)

controls,

As a negative

plated

CFU-GM

-

x

In cultures

plates.

n

level.

bone

GM-CSF,

a plateau

plus

humidified

containing

bone and

CFU-GM IL-l and control

culture.

in a fully

solution

as positive

to the culture

containing

cells

with

included

growth added

depleted

containing

F!TC-

in RPM!

in CFU-GM

cultured

of a methylcellulose

Cultures colony

a-MEM

40%

the

20#{176}C),resuspended

plated

of 37#{176}C and 5% CO2 in a medium

a-MEM

IL-6),

between

collected

10 minutes,

a-MEM 5% FBS, and immediately CFU-GM culture. Cells were atmosphere

were

was raised

IL-6.2’

with

and

IL-I

effect marrow

of increasing concentrations CSF (0, 1, 10, 100 U/mL), IL-i

on the fluoresthe

human

neutralize

Differences

between

phagocytes

medium

with

of rhG-CSF

pairs.

synergistic

human

mal

times,

in a CD34

point

based

of

was able

and CSF.

analysis.

Synergy

an anti-

separated

antiserum

pure

cross-react

interferons,

activity

anti-IL-6

to completely not

test for matched

possible

with 5% FBS. Cell argon-ion laser beam

cut-off

IL-l,

proliferation

Amgen)

(iSA,

RESULTS

times.

two

essential

incubated

Cells

three

washed

were

able did

!L-6-dependent

It did not show cross-reactivity

of 1:200. The

of The

30

immunoglobulin washed

was chosen, sample

for

it was

the

the colony-stimulating

5% FBS,

at 4#{176}C with

FACS, cells

fraction.

antibody

MoAb in RPM!

minimal

the

fraction

a control

conjugated

times

MoAb

electrophoretically

antiserum

coxon

using half-maximal

or rhIL-3.

against

Statistical

Becton phagocytes

anti-CD34

Scotland)

positive

negative

of

FACStar,

anti-mouse

Irvine, using

the positive

IL-la,

two

Eagle’s

HLA-DR

and a CD34

positive

Dickinson)

30 minutes

in a-modified

nm.

(Becton

to phycoerythrin,

(a-MEM;

at 488

MoAb

at 4#{176}C, and

for

conjugated

resuspended

The

(FACS;

goat

for 30 minutes

rhGM-CSF

Dickinson,

with

FITC-conjugated

cells

tuned

at a dilution

rabbit

quantitated 1 U giving

anti-G-CSF

neutralize

U/ml)

of 1:300

of progenitor

was

7TD1?’

( I ,000

Becton

of mononuclear

cells were washed

(Nordic)

HLA-DR

sorter

cells, depleted

were

minutes

Next,

cell

selection

for

line

to completely

My-lO;

(HPCA,

an anti-HLA-DR

activated

Enrichment

cells.

by positive

MoAb

Bone marrow

antiserum

marrow

IL-6

cell

Aarden (Central Laboratory Transfusion Service, Amsterdam,

Blood

cells. The murine

with

CD34/HLA-DR-enriched

of Dr L.A.

gift

Red Cross

Netherlands).

Dick-

staining. colony

was a kind

murine

and less than

with

IL-6-’

Netherlands

(FITC)-labeled

(Nordic

Netherlands),

less

immunofluores-

isothiocyanate antisera

as determined

contained

indirect

(MoAb)

and fluorescein

Tilburg,

phagocytes

cells

with

immunoglobulin

Laboratories, clear

antibody

CA)

2399

INDUCTION

interphase

as determined

monoclonal

inson, Oxnard,

BY G-CSF

by

GM-CSF

100

(U/mi)

Fig 1 . Synergistic effect of Il-i (10 U/mI) on GM-CSFsupported CFU-GM colony formation of normal human bone marrow cells. depleted of mononuclear phagocytes and T lymphocytes. Data are a percentage (mean ± SD of 1 0 experiments) of control CFU-GM growth obtained in the presence of GM-CSF 100 U/mL.

From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

SCHAAFSMA

2400

200

ET AL

200

-

.C

0

150

150-

I-

Fig2. EffectoflL-1 (10 UI mL) on (A) GM-CSF-supported CFU-G and (B) cFu-M colony formation of normal human bone marrow cells. depleted of

>1

C

0 0 100

U

100

IL-i

0

mononuclear

0 0 50

50

0

control 0

00

1

A

GM-CSF

done,

by residual using

purpose

of highly

suspensions

depleted

bone

of accessory

additional

enriched

for

HPC.

and

T

were

further

cell suspensions

and

studies

purified

phagocyte-

marrow cells

cells,

were

For

this

depleted cells,

from

cultures

GM-CSF

tion was no longer was

present

using

cells,

(Fig

the synergy

colony

3), suggesting

that

forma-

the effect

indirect. Characterization

br.

In

order

factor

responsible

CSF,

neutralization

duction

of to

of IL-6

known

hematopoietic

centrations IL-i

progenitor on granulocytic

and

the

further for

the

synergistic

characterize synergy

experiments

is induced

the between

were

IL-i-induced and GM-

performed.

we added

antiserum

Control

Since

prohas con-

IL-6

neutralizing

to cultures

measurements

.c

fac-

IL-l

by IL-i#{176} and since

activities,

of an anti-IL-6 GM-CSF.

IL-i-induced

colony

with no

effect

colony

IL-i

in the presence

of the

antiserum

by IL- 1 was found

growth

was

Control

preparations

calculated Since fined

the

not

IL-i effect

colonies, the

by

further

GM-CSF

of

Further-

addition

of

synergistically

Results

were

for rh-IL-6

(data

proved

active

and

and

GM-CSF

we considered

that

the

contained was

G-CSF

addition MoAb,

the conas the

of neutralizthe synergy

and GM-CSF was totally abrogated (Fig 5). was not due to toxicity of the antibody since

GM-CSF-stimulated To

and

effect. Following of an anti-G-CSF

not

IL-6 at concentrano direct effect on

of IL-6

biologically

data

(Fig 4A).

Also, had

IL-6

measurements

to granulocytic

between

purified

were

could

colony verify

that

growth the

be mediated

was

not diminished.

synergy

between

by IL-i-induced

150#{149}

150

-

125#{149}

125

-

IL-i

and

production

of

0

100

100-

IL-i

>‘

C 0

Fig 3. Effect of IL-i (10 UI ml) on (A) GM-CSF-supported CFU-G colony formation of normal human bone marrow cells. depleted of mononuclear ph.gocytes and T lymphocytes and on (B) CD34/HLA-DR-onriched bone marrow cells. Data are a percentage (mean ± SD of two experiments) of control CFU-G growth obtained in the presence of GM-CSF 100 U/mI.

0

75.

U

75

-

IL-i

0

control

C 0 U

50.

50

-

25

-

control

0

25#{149}

0

00

A

1

GM-CSF

10

(U/mI)

T

of anti-IL-6

enhancement

influenced

amount of the cytokine. synergy between IL-l

factor mediating ing concentrations This

human

and

U/mL,

on the

growth

for both

shown).

IL-6

containing

in supernatants

(

C

antHL-6

:::

.2 0

IL-i

C,

.

antHL-6

100

50

0 0110

A

100

GM-CSF

(U/mI)

GM-CSF

(U/mI)

200

-

control

IL-i

150

a::::

IL-6

IL-i

#{149} IL-6

100 Fig 4. Effect of neutralization of the Il-iinduced 11-6 production (A). and effect of addition of exogenous natural 11-6 (30 U/mL) (B) on the synergy between IL-I (10 U/mI) and GM-CSF on CFU-G colony growth of normal human bone marrow cells. depleted of mononuclear phagocytes and T lymphocytes. Data are a percentage (mean ± SD of (Al two and [BI six experiments) of control CFU-G growth obtained in the presence of GM-CSF 100 U/mL

50

0

100

0JI,

B

I 75 .c

150

-

control

0

IL-i

a-

125 >a

C

0 0

L#{149}anti-G-CSF IL-i

100

#{149} anti-G-CSF

0

I

______ _

25

0

i

GM-CSF

10

(U/mi)

100

Fig 5. Effect of addition of a neutralizing anti-G-CSF MoAb on the synergy between IL-i (10 U/mi) and GM-CSF on CFU-G colony growth of normal human bone marrow cells. depleted of mononuclear phagocytes and T lymphocytes. Data are a percentage (mean ± SD of four experiments) of control CFU-G growth

obtained

of GM-CSF

in the

100 U/mI.

presence

From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

2402

SCHAAFSMA

Table

1 . Synergy

Between

G-C SF and

GM-CSF

on Hums n CFU-G

Colony

ET AL

Growth

GM-cSF Me6um

1 U/mL

Medium

0±1

6 ± 10

G-CSF1U/mL

2±3

i3±i9

70±23

103±10

G-CSF1OU/mL

2±3

16±26

97±31

125±33

G-CSF100U/mL

27±31

G-CSF

1,000U/mL

(mean

Data are a percentage

cells

marrow

G-CSF,

depleted

were

additional

binations

±

in

CFU-G

I

present

were

performed

G-CSF and synergizes

G-CSF

growth.

concentrations

This

of G-CSF

effect

as low

as

cell suspensions

already

140

253 owth

1 had

GM-CSF

was

illuson

exogenous

IL-6,

and

was

marrow

no effect

at still

cells,

(Table

on

2).

U/mL,

study

GM-CSF

clear

from

bone

lL-l

and

colonies

and

and

enriched for CD34/HLA-DR

HPC,

cell.

mediated

by

Only

When less

anymore.

bone

2%

results

by IL-l.

IL-6

since or

was

synergizes

with

only

of IL-3

gistic

effect

enhance

GM-CSF-stimulated

colony formation. When was neutralized by a rabbit CFU-GM

of

IL-l-induced IL-6 anti-IL-6 antiserum,

in the

colonies

presence

prothe

of IL-i

and

GM-CSF this

between

both

Synergy Colony

Between

G-CSF

Growth

and GM-CSF

on Human

CFU-G

of CD34/HLA-DR-Enriched Marrow

Bone

enhanced

either

factor

Medium

1 U/mI

10 U/mI

100 U/mI

that IL-3

0

0

72

100

0

0

70

107

0

0

109

148

murine plexity

10 U/mL

G-CSF

iOOU/mL

G-CSF

i.000

U/mL

Data are a percentage presence

of GM-CSF

70

237

298

differential

430

452

maturity

of control

CFU-G (results

colony

growth

of one experiment).

obtained

in the

IL-l and GM-CSF formation, we considIndeed, antibody

in

that

attributed

to a synergy

the

neutralno syner-

increase

in

between

In accordance

with

and

growth

to support

cells,

suggests

a

with

may

colony

direct

effect

myeloid of the

activities

by different

IL-3

synergy

recent

M-CSF

formation.

that with

both IL-3

marrow,

on proliferation

progenitor regulation

The

pres-

with

G-CSF” on blast and

and cell

Leary

These factors

of early

by Bartelmez

a direct the

bone

et

studies that act HPC.

In

that G-CSF, but not IL-6, GM-CSF on relatively mature

studies

exert on

activity

with

on HPC.

bone

we demonstrate

also

signifi-

in comparison

CD34/HLA-DR-enriched

of murine

study

previous

GM-CSF

effect was already observed at low (10 U/mL), which, in the absence on

in the

and

348

the

to the cells. It in the

a similar effect of IL-6 in humans. that IL-6 and probably G-CSF are

IL-i

43

whether

T lymof the

colony

synergy

In addition,

276

100 U/mL

failed

synergistically

IL-i G-CSF

CFU-G

development

HPC.

clear

is a co-stimulant

In mice, Ikebuchi et al described IL-63’ have a synergistic activity

Medium 1OU/mL

IL-6

of G-CSF

this

of

is involved GM-cSF

This

T cells, and activated for the production between colony

was tested.

alone. This of G-CSF

of GM-CSF,

the present

Cells

CSFs

cantly

colony

cells.

indicating that on CFU-GM

indicating

reports,3#{176} the combination

ence

colony and

and IL-l-induced G-CSF. Further evidence for obtained in additional studies, in which synergy

was

suggest 2.

was

IL-6

be attributed of accessory

this activity. an anti-G-CSF

found,

colonies

a122 found Table

was

granulocytic

marrow

not

that

to mediate experiments with

ization

the

it is not

synergistic activity. the synergistic effect seen on granulocytic

indicate

did

recently

ered G-CSF

and IL-6

production mononuclear

present

of

30 to i ,000

progenitor

recent studies,333 with GM-CSF

of PHA-stimulated may be responsible

Since

that

indirect factor.

of myeloid

At

from

GM-CSF-stimulated synergy between

no

reported

concentrations

number

Bone

addition

ranging

in

formation of early blast cell colonies?1 and a similar synergistic effect with GM-CSF has been reported on myeloid (CFU-GM) colony growth.3U3 However, our results CFU-GM duction

U/mL.

100

Furthermore,

has been phocytes

mononuclear were further

suggest

GM-CSF was of an intermediate

diminished.

in concentrations

presence

observed

on the

IL-6

GM-CSF

of

activity of IL-6 could or absence of small numbers

of

marrow

not

formation.

of

antibodies against the sorting, no synergistic

that we considered, endothelial cells?

phagocytes#{176} is induced

formation

these

than

These and

production

was one of the factors IL-6 by fibroblasts,28

that

± 185

302

presence

on proliferation

activation

of mononu-

by the combination effect on a committed,

monoclonal and cell

IL-i

the

depleted

1% T lymphocytes,

using antigens

between

and GM-CSF act human CFU-GM

lymphocytes.

less than

found

was

synergy

T

containing

suspensions,

effect

suspensions

progenitor

phagocytes

IL-l of

was enhanced suggesting an

GM-CSF,

lineage-specific cell

marrow

phagocytes

granulocytic

in the

finding is in contrast with IL-6 may act synergistically differences

we demonstrate that on the proliferation

synergistically

colonies

184±47

i76

±

did not influence Thus, we found

growth.

colony DISCUSSION

In this

obtained

com-

detectable

10 U/mL bone

of IL-

using

GM-CSF. As with GM-CSF

was

on CD34/HLA-DR-enriched

which

±

100

145±34

SD of three experiments) of control CFU-G colony phagocytes and T lymphocytes.

experiments

Table

colony

198

100 U/mI

10

±

of mononuclear

of recombinant

trated

45±23

± 102

161

10 U/mI. 69

et a142 suggest

synergistic

proliferation

of

activity very

with

primitive

cells. This illustrates the comof hematopoiesis, suggesting a

of various

cytokines

depending

on

level of the target cell. Thus, the indirect synergistic of IL-I on primitive and mature HPC are mediated intermediate

factors.

the

From bloodjournal.hematologylibrary.org by guest on June 9, 2013. For personal use only.

IL-i

SYNERGIZES

WITH

GM-CSF

BY G-CSF

2403

INDUCTION

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2404

SCHAAFSMA

granulocytic

supports cells

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