Expression of the macrophage specific colony-stimulating factor (CSF-1) during human monocytic differentiation

Vol. 141, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 30, 1986

Pages 924-930

E X P R E S S I O N OF THE M A C R O P H A G E S P E C I F I C C O L O N Y - S T I M U L A T I N G F A C T O R (CSF-I) DURING H U M A N M O N O C Y T I C D I F F E R E N T I A T I O N

Junko

Horiguchi,

Laboratory

M. Kim Warren*,

Peter Ralph*, and Donald

of C l i n i c a l Pharmacology, D a n a - F a r b e r C a n c e r and H a r v a r d M e d i c a l School, Boston, M A 02115 * D e p a r t m e n t of Cell Biology, Corporation, Emeryville, CA

Cetus

Kufe

Institute,

94608

Received November 4, 1986 We and others h a v e p r e v i o u s l y d e m o n s t r a t e d e x p r e s s i o n of the c-fms p r o t o - o n c o g e n e during human monocytic differentiation. The c-fms gene has since b e e n shown to e n c o d e for the m a c r o p h a g e s p e c i f i c c o l o n y s t i m u l a t i n g factor (CSF-I) receptor. The p r e s e n t results demonstrate that b o t h CSF-I and c-fms t r a n s c r i p t s are induced d u r i n g m o n o c y t i c d i f f e r e n t i a t i o n of h u m a n HL-60 leukemia cells. The results further demonstrate that normal human monocytes express CSF-I RNA and that the level of these t r a n s c r i p t s i n c r e a s e s u p o n t r e a t m e n t w i t h p h o r b o l ester. Finally, the d e t e c t i o n of CSF-I RNA in HL-60 cells and in m o n o c y t e s is associated with production of the CSF-I gene product. These findings w o u l d s u g g e s t that m o n o c y t e s are c a p a b l e of r e g u l a t i n g their own survival, growth and d i f f e r e n t i a t i o n through CSF-I production. ® 1986 Academic Press, Inc.

The m a c r o p h a g e CSF)

is

required

mononuclear precursor

for

phagocytes cells

macrophages dividing

specific

(2).

to

binding

to the CSF-I

kinase

activity

(i).

(3).

also

These

receptor,

(4).

CSF-I

CSF-I

and

thus

colonies

factor

macrophages

stimulating

proliferation

form

This

colony

containing

of

(CSF-I,

differentiation

stimulates

regulates

effects

factor

the CSF-I

Mof

hematopoietic monocytes

survival are

and

of non-

mediated

by

a 165 kd g l y c o p r o t e i n

with

tyrosine

molecules

cell

surface

bound

to

Abbreviations used are: CSF-I, macrophage specific colonystimulating factor; kd, kilodalton; kb, kilobase; TPA, 12-0tetradecanoylphorbol-13-acetate; TNF, tumor n e c r o s i s factor. 0006-291x/86 $1.50 Copyrig,ht ~5 1986 t~y Academic Press, Inc'. All rights Qf reproduction in an)' ,/orm reserved.

924

Vol. 141, No. 3, 1986

receptors role

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

internalized

of i n t e r n a l i z e d

CSF-I

and

degraded

or r e c e p t o r

in

lysosomes

in g r o w t h

(5).

control,

The

however,

is uncertain. The

CSF-I

phagocytes

and

demonstrated cells of

receptor

that

the

c-fms

gene

molecules

recent

immunologically fms gene

CSF-I,

the

have

We

proto-oncogene

(7) is

mononuclear

and

also

others

(8)

expressed

in

the

monocytic

lineage.

The

product

and

the

receptor

suggested

either

have

CSF-I

closely

shown

related

that

and

functionally

certain

insights

endogenous

present

studies

differentiation with

(1,6).

on

the

related

or

similarity

identical.

CSF-I

that

Indeed,

receptor

to the p r o d u c t

is

of the

c-

(4).

Although

The

c-fms

expressed

along

were

studies

specifically

precursors

the

differentiated

these

of

their

is

CSF-I also

induction

in

of

the

detected CSF-I

source

of

demonstrate

expression. been

are a v a i l a b l e

human CSF-I

that

HL-60

factor

leukemia

peripheral

is

compared

cells and

the

blood to

the effects

remains

induction

transcripts

in h u m a n

expression

this

regarding

that

unclear.

of

monocytic

is

associated

gene

product

monocytes. of

the

The c-fms

proto-oncogene. MATERIALS

AND M E T H O D S

CELL CULTURE H u m a n H L - 6 0 p r o m y e l o c y t i c l e u k e m i a cells (9) w e r e m a i n t a i n e d in logarithmic growth phase. Peripheral blood monocytes were p u r i f i e d by a d h e r e n c e for 1 h and r e m o v a l of the n o n a d h e r e n t cells (i0). The a d h e r e n t cell p o p u l a t i o n was c o l l e c t e d w i t h a p l a s t i c policeman, readhered overnight and t h e n washed again with 4 c h a n g e s of medium. The second a d h e r e n t cell p o p u l a t i o n c o n s i s t e d of over 98% monocytes by morphologic examination. Monocyte p u r i f i c a t i o n and c u l t u r i n g was p e r f o r m e d in RPMI 1640 m e d i u m w i t h 10% p o o l e d h u m a n AB serum (i0). C u l t u r e d cells w e r e t r e a t e d w i t h 3.3 x 10-8M 1 2 - O - t e t r a d e c a n o y l p h o r b o l - 1 3 - a c e t a t e (TPA, Sigma). DNA PROBES The 1.6 kb f r a g m e n t of a h u m a n CSF-I c D N A was p u r i f i e d from the p c C S F - 1 2 p l a s m i d (ii). The 1.0 kb PstI f r a g m e n t of the v-fms gene was p u r i f i e d from the pSM3 p l a s m i d (7). The i.i kb PstI f r a g m e n t of a h u m a n tumor n e c r o s i s factor (TNF) c D N A was p u r i f i e d from the pE4 p l a s m i d (12).

925

Vol. 141, No. 3, 1986

RNA EXTRACTION

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND HYBRIDIZATION

The procedures used hybridization and washing were CSF-I

for RNA as r e p o r t e d

extraction, transfer, p r e v i o u s l y (7).

DETERMINATIONS

T h e c o n c e n t r a t i o n of C S F - I in t i s s u e c u l t u r e s u p e r n a t a n t s d e t e r m i n e d b y t h e C S F - I s p e c i f i c r a d i o i m m u n e a s s a y (13).

was

RESULTS We

have

expression A

during

similar

cells TPA

a

(Fig.

HL-60

4.6 i).

increase

at

transcripts Maximum then

kb

for

been

at

cells.

in

same

of c - f m s

cell

line

monocytic

used

in

the

to

after

CSF-I

study

RNA

studies. the

was

not

CSF-I

cDNA

probe

detectable

after

24

of

cells

level TPA

of

CSF-I

exposure. shown

for

RNA were

present

at

declined

at

o

is

The

48

h

h

RNA

65

h

of in

hybridized

TPA

treatment

continued

comparison

and

HL-60

found

induction

24 h of

(7).

addition

the

the

c-fms

differentiation

present

intervals

However,

65 h of

transcripts

of

expression.

transcript

the

HL-60

various

CSF-I

48 h a n d

the

induction

Furthermore,

levels

these

used

has

collected

monitor

uninduced with

TPA

approach

were

to

previously

to

of c - f m s (Fig.

i).

induction

and

(Fig.

i).

In

TPA

28S

CSF-1 18S.

28siii::i~:~;i~:~i~!~!~i!~;J~i~!!~ii~S!!~i!~ii~ iiiiG!!ii!i!iii!i!iiii!iiiiii!iiiiiiiii@iiii!iiiiiiii!iii!i!i!i!!!iii c-fms

18s-

Figure i. Levels of CSF-I and c-fms RNA during induction of HL-60 monocytic differentiation. HL-60 cells were grown (9) in the presence of 3.3 x 10-SM TPA for the indicated times. Total cellular RNA was purified by the guanidine thiocyanate-cesium chloride method, analyzed by electrophoresis of 12 ug RNA through 1% agarose-formaldehyde gels followed by Northern blot transfer to nitrocellulose (7). The RNA filters were hybridized to the 32p_ labeled nick-translated CSF-I and v-fms probes. Untreated HL-60 cells were nonadherent, while >90% of the TPA-treated cells adhered to the tissue culture flask at 24 h.

926

Vol. 141, No. 3, 1 986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

69

LU i

CSF-1

c-fins

28STNF 18S-

Figure 2. Levels of CSF-I, c-fms and TNF RNA during treatment of monocytes with TPA. Peripheral blood monocytes were purified as described in Materials and Methods and treated with 3.3 x 10-8M TPA for the indicated times. The HL-60 cells were treated as described in Fig. l. Total cellular RNA (12 ug) was then hybridized to the 32p-labeled nick-translated CSF-I, v-fms and TNF probes.

contrast, (7)

transcripts

were

undetectable

the g r a n u l o c y t i c The

c-fms

monocytes

is

expression shown).

in H L - 6 0

was

human

(Fig.

after

TPA

These

findings

expression

low

but

found

]Sariban,

2).

induction

in

not

induction

monocytes.

shown)

of H L - 6 0

are

CSF-I

Maximum and in

and

c-fms

cells

along

the

monocytes ]

T. and Kufe,

927

were

were

blood

undetectable

RNA

RNA

(Fig.

was

not

by

TPA

by

6 h

achieved

declined

2).

CSF-I

(data

increased

thereafter.

down-regulation (Fig.

shown

constitutive

selected

transcripts

on CSF-I

peripheral

cellular

of

expression

to

studies

previously

in

total

A+

levels

CSF-I

these

have

level

poly

RNA

contrast

TPA-treated

E., M i t c h e l l ,

against

using

CSF-I

We

transcripts

detectable

when

further

expressed

hybridizing

Furthermore,

treatment

DMSO

(data

cells prompted

contrast,

when a

CSF-I

constitutively In

cells

However,

during

normal

(7).

in t h e s e 2).

in

both

lineage.

findings

expression that

for

The

D., u n p u b l i s h e d

of

c-fms

increase

data.

in

Vol. 141, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0

×

6

¢n

=

4i

T LL

2

o

6 12

24

48

66

Hours

Figure 3. CSF-I levels in tissue culture supernatants of TPAtreated HL-60 cells and monocytes. HL-60 cells (e) and monocytes (O) were treated with TPA as described in the legends to Figs. 1 and 2. The supernatants were assayed for CSF-I using the CSF-I specific radioimmune assay (13). The values represent the CSF-I concentration for 1 x 106 cells/ml.

CSF-I

transcripts,

however,

was s i m i l a r

of T N F R N A d u r i n g m o n o c y t i c We

have

also

in T P A - t r e a t e d undetectable

in

progressively

then

TPA

to

patterns

of

CSF-I

similar,

although

those

12

h

production and h u m a n

untreated

from

declined

behind

cells

after

supernatants

differentiation

monitored

HL-60

HL-60 of

changes

observed

in

of

the

exposure.

monocytes

at

CSF-I

In

supernatant

CSF-I

until

(Fig.

in

24 h and

3).

product

CSF-I

were

increased

contrast,

and

product

levels

and

increased

RNA

gene

CSF-I

levels

the

induction

2).

supernatants

pretreatment

expression

(Fig.

monocytes.

cell

TPA

treated

near

to the t r a n s i e n t

Thus,

level

levels

were

lagged

for CSF-I t r a n s c r i p t s . DISCUSSION

The

present

studies

but not granulocytic, with

CSF-I

finding levels

in

suggest

CSF-I

human

that

survival,

differentiation

expression.

that

demonstrate

These

is a l s o

monocytes

proliferation

Taken

are and

induction

of H L - 6 0

results

expressed

monocytes.

that

are

together,

capable

of

cells

in

at b o t h

928

is a s s o c i a t e d

concert

the these

RNA

with

and

their

through

the

protein

findings

regulating

differentiation

production.

of monocytic,

would own CSF-I

Vol. 141, No, 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Activation interleukin positive in

of

2

(IL-2)

cells

of

to

differentiation

of

both

c-fms.

of

cell

factor

these

results

also

associated

monocyte The monocytes

growth

is in

are

c-fms

gene.

subpopulations.

of

stimulating on CSF-I

both

which

inducers gamma

factor

production

gene

encodes

monocytic

express for

of both

the

CSF-I

monocytopoiesis,

production same

of

lineage.

of

both

by

expression of

like growth

However,

monocytes

relevance

not

CSF-I

could

signals

positive

effects

have

by m o n o c y t e s

monocytes,

similar

expression

monocytes

CSF-I

A

TPA

and

these

the is

down-

findings

to

is unclear.

studies

activation

resulting

with

human

the

The

thus

Thus,

by

both

receptor

monocytes.

that

of

IL-2

(14).

activation

increased

producing

physiologic

of

of IL-2,

associated

regulated

cells

The

populations

suggest

both

certain

of

is

c-fms

that

control

present

1 production contrast,

the

in

would

production

receptors.

cell

the

indicate

of

T

cells

findings

with

regulation

IL-2

exist

since

receptor

stimulates

Furthermore,

proliferation, and

cells

in the p r e s e n c e

HL-60

genes,

receptor, T

and

these

and

specific

appears

CSF-I

T

proliferate

expansion

situation

resting

and

may

induce

we

CSF-I

interferon,

other

have

not

the

same

Thus,

CSF-

mechanism.

expression

in

CSF-I

addressed

production

in

further

only

the

issue

monocytes.

hematopoietic

requires

In

receptor

granulocyte-macrophage

or other

in m o n o c y t e s

receptor.

CSF-I

regulate

Finally,

(GM-CSF)

CSF-I

whether

act by an a u t o c r i n e

then

of

determined

growth

The

colony factors

study.

ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by PHS G r a n t s CA34183 and CA42802 a w a r d e d by the N a t i o n a l Cancer I n s t i t u t e and by an A m e r i c a n C a n c e r Society Faculty Research Award (DK). The authors thank E. Kawasaki for p r o v i d i n g the CSF-I probe, Y. Lie for t e c h n i c a l assistance and R. Stanley for reagents used in the CSF-I r a d i o i m m u n e assay. REFERENCES I.

Stanley, E., Guilbert, L., Tushinski, (1983) J. Cell. Biochem. 21, 151-159.

929

R.,

and

Bartelmez,

S.

Vol. 141, No. 3, 1986

2. 3. 4. 5. 6. 7. 8. 9. I0. Ii.

12. 13. 14.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Stanley, E. and Guilbert, L. In Mononuclear PhagocytesFunctional Aspects, Part i, R. van Furth, ed. (The Hague; Martinus Nijhoff), pp. 417-433. Tushinski, R.J., Oliver, I.T, Guilbert, L.J., Tynan, P.W., Warner, J.R. and Stanley, E.R. (1982) Cell 28, 71-81. Sherr, C., Rettenmier, C., Sacca, R., Roussel, M., Look, A. and Stanley, E. (1985) Cell 41, 665-676. Guilbert, L.J. and Stanley, E.R. (1986) J. Biol. Chem. 261, 4024-4032. Byrne, P.V., Guilbert, L. J. and Stanley, E.R. (1981) J. Cell Biol. 91, 848-853. Sariban, E., Mitchell, T. and Kufe, D. (1985) Nature 316, 64-66. Nienhuis, A.W., Bunn, H.F., Turner, P.H., Gopal, T.V., Nash, W.G., O'Brien, S.J., and Sherr, C.J. (1985) Cell 42, 421428. Collins, S., Gallo, R. and Gallagher, R. (1977) Nature 270, 347-349. Todd, R.F. and Schlossman, S.F. (1982) Blood 59, 775-786. Kawasaki, E.S., Ladner, M.B., Wang, A.M., van Arsdell, J., Warren, K., Coyne, M.Y., Schweickart, V.L., Lee, M.T., Wilson, K.J., Boosman, A., Stanley, E.R., Ralph, P. and Mark, D.F. (1985) Science 230, 291-296. Wang, A.M., Creasey, A.A., Ladner, M.B., Lin, L.S., Strickler, J., van Arsdell, J.N., Yamamoto, R., and Mark, D.F. (1985) Science 228, 149-154. Ralph, P., Warren, M.K., Lee, M.T., Csejtey, J., Weaver, J.F., Broxmeyer, H.E., Williams, D.E., Stanley, E.R., and Kawasaki, E.S. (1986) Blood 68(3), 633-639. Gillis, S., Baker, P.E., Ruscetti, F.W. and Smith, K.A. (1978) J. Exp. Med. 148, 1093-1098.

930