Journal

Differential

of Leukocyte

Biology

Expression of M-CSF, G-CSF, by Human Monocytes

Mei-Ting

Lee,

Kenneth

Cetus Corporation,

Kaushansky,

Emeryville,

Peter

California

(M.-T.L.,

University of Washington,

and

Ralph,

P.R., M.B.L.); Seattle (K.K.)

(1990)

and GM-CSF

Martha

Division

47:275-282

B. Ladner

of Hematology,

The colony-stimulating factors (CSF) belong to a group of proteins which regulate blood cell production. Human monocytes allowed to adhere express high levels of M-CSF transcripts and secreted protein at 24 h In the presence but not in the absence of Indomethacm (Indo), an Inhibitor of prostaglandin E (PGE) production. When induced with Iipopolysaccharide (LPS), adherent monocytes express M-CSF, G-CSF, and GM-CSF transcripts and secrete these proteins and TNF. M-CSF and GM-CSF messages increase in LPS-induced monocytes by the addition of Indo, while G-CSF mRNA appears to decrease. Exogenous addition of PGE-2 to LPS-lnduced monocytes down-modulates the expression of M-CSF and GM-CSF transcripts. G-CSF message Is elevated, suggesting an alternate pathway to G-CSF regulatIon. PGE-2 inhibits the secretion of CSFs and TNF. In contrast, LPS-induced monocytes held 24 h in nonadherent culture express G- and GMCSF but not M-CSF. Monocytes that are adhered for 24 h and then treated with LPS for an additional 24 h express only M-CSF message and secrete M-CSF and TNF. PGE-2 added with LPS during the 24-48 h Induction blocks M-CSF and TNF production, but appears to enhance M-CSF message expression, In contrast to its effect on 0 h inductions. These results suggest that adherence alone Induces M-CSF gene expression, but low levels of PGE or other arachidonic acid metabolites limit this expression. Other events in 1 dcultured monocytes block the ability to induce G-CSF and GM-CSF expression with LPS, and block the suppressive effect of PGE-2 on M-CSF expression at the RNA level. Key words:

prostaglandin, indomethacin, colony-stimulating factor

Iipopolysaccharide,

INTRODUCTION The

of immune

colony-stimulating

GM-CSF,

are

factors,

produced

and

M-CSF,

secreted

into

G-CSF,

and

the circulatory

system by many endothelial cells,

cell types. T lymphocytes, monocytes, and fibroblasts have all been found

synthesize proteins

following activation stimulate immature

CSFs specifically

to proliferate rophages cytes and

and

differentiate

[18]. M-CSF enhances the

into

[3]. These progenitor

granulocytes

increases expression

to

glycocells or mac-

the survival of monoof maturation-related

antigens (LeuM5, LeuM3, 0KM 1, Fc receptors, and HLA-DR) [1]. Both M-CSF [5] and G-CSF [27] are highly lineage specific in their action and therefore only support the proliferation of colonies consisting of predominantly tively crete

either

[18,24]. colonies

as well [19].

often

tors such prostaglandin

© 1990

Conversely, that consist

as mixed

CSFs

macrophages

colonies

stimulate

as prostaglandin E2 (PGE-2),

Wiley-Liss,

Inc.

or

neutrophils,

respec-

GM-CSF maintains both disof macrophages or neutrophils consisting monocytes

of both to secrete

cell other

fects

mRNA,

function

of PGE-2

gene

[6,13].

thus an integral which down-regulates Previous studies

T cells

[7,8,2 1] and G-CSF have been detected

[3,12].

We

that

ef-

PGE-2 and

are

of a self-regulatory mechanism CSF production. have shown human monocytes to be

Recently

in monocyte

[28].

of the

shown

stem cell proliferation [14] of CSFs [15]. Prostaglandins

now

show

mRNA

[25,29],

has not mRNA induced

CSF, G-CSF, and GM-CSF differently in LPS-induced

[9]. High in normal

GM-CSF

cultures

the protein by monocytes murine system, GM-CSF peritoneal macrophages agents

studies

have

part

a source of M-CSF of GM-CSF message detected

Extensive

on hematopoiesis

inhibits CSF-induced macrophage secretion

vated

expression,

that

but

has

levels actibeen

secretion

of

been described. In the has been detected in with inflammatory the

mRNA monocytes

expressions

of M-

are each regulated in response to ad-

types fac-

E2 [16]. A bioactive lipid, is often a negative regulator

Received

March

22,

1989;

Reprint

requests:

Mei-Ting

Street,

Emeryville,

CA

accepted Lee,

94608.

July Cetus

11,

1989.

Corporation,

1400

Fifty-Third

Lee et al.

276

herence, PGE-2, and day of monocyte ticular, indomethacin (Indo), an inhibitor din synthesis, enhanced GM-CSF and expression

by

human

monocytes,

culture. In parof prostaglanM-CSF mRNA

but

not

G-CSF

gene

expression.

normal

blood mononuclear cells buffy coats or heparinized

healthy

phocyte

donors

separation

Corp.,

medium

Durham,

NC).

twice with (Pharmacia,

PBS, and Uppsala,

monocytes

were

and either on a rocker 2 h and

washed

adherent

CA),

Organon

Teknika

harvested,

washed

the

to remove

cells

nonadherent

-85% after

were

cells.

pure monocytes cytocentrifuge

>95%

monocytes.

The

oth-

CA), and St. Louis, and 1% Woodland,

AB

indomethacin MO) and

serum

(Irvine

(10-6 M, supplemented

penicillin/streptomycin CA). Cells were

chamber of cells

and allowed that adhered

calculate the total Cells were seeded Where indicated, ng/ml; human

Corp.,

Emeryville,

PGE-2 (1,000

106

(l0

M;

recombinant

or culture

from

TNF

of Total

Cellular

mRNA

(100

24 h-adhered

and

ium

cultured under by centrifugation

thiocyanate

washed

to remove

solution.

nonadherent and lysed Adherent

nonadherent

gel

electrophoresis,

branes

(Zeta-probe

mond,

CA).

Specific

mRNAs

and Blotting were

blotted

then

were lysed

in

RNA was recovered chloride [2]; 15 ig of fractionated by agarose onto

Membranes, detected

and

for

citrate);

SDS,

5 X

10

mM

at 42#{176}C. All

washed

quantified The assay

assayed

[23]

bone

in 2 x

marrow

antibodies recombinant

kidney

(BHK)

for

(limit

SSC;

activity

in a

of detection

colony

by using relies on

is 3

assay

[11].

a sandwich radioimtwo noncrossreacting

raised by GM-CSF cells

immunization produced

baby

hamster

[10].

The

antibodies,

both CSF,

IgG 1 subclass, fail to cross react M-CSF, or IL-3, and 1 pg of

with each

human staph

neutralizes GM-CSF.

microtiter is blocked

by GA-

at least 5 .o.g of recombinant The first antibody is plated

plates,

and

nonspecific

by the addition

of 2%

protein albumin.

adSerial

standards,

are added to 10% final volume, using the blocking buffer as diluent. After adsorption, ‘251-labeled second antibody (lodobead method) is added, and after binding, individual wells are washed and counted. sensitivity of the assay is 0.1-0.5 ng/ml Samples were also tested in a proliferation uses a mouse cell line that proliferates M-CSF and G-CSF (personal communication,

The limit of (10-50 U/mI). assay which in response to P. Ralph,

J. Weaver, and I. Nakoinz). Sample titers were generated by using an M-CSF standard. TNF was assayed by to actinomycin-treated

RESULTS Effects of Indomethacin

conditions in guanidin-

monocytes

cells

guanidinium thiocyanate solution. by centrifugation through cesium total RNA was loaded per lane,

and

RIA

a murine was

(5’CTand

L929

cells

[4].

Northern

Blot Analysis Monocytes were harvested

2.0%

were

M-CSF

murine monoclonal with HPLC-purified

toxicity

Preparation

saline

dilutions of culture supernatants, or GM-CSF

Sigma), recombiLot A904, Cetus human

medium

GM-CSF munoassay.

111

Factors

supernatants

and

herence

cells in the culture. adherent cells/mi. incubated with LPS

U/mI;

(Standard

formamide,

for Protein

in 96-well

Chemical Co., 1% L-glutamine

SSC

hybridized overnight at 55#{176}C[17].

purified antibody or natural human

Ana,

stocks (J . R. Scientific, added to a hemocytometer

number of adherent into flasks at monocytes were

CA),

[4]),

Sigma with

Santa

to sit for 1 mm. Counts were taken to the glass surface and were used to

Sigma), M-CSF

U/mI; Cetus monocytes.

Scientific,

5 x

human

ng/ml)

as deprepara-

Unless

were SDS

specific

washed,

with T4 mRNA was

(5’CTCAGCAGCAGTGTCTCHybridization conditions

pH 7; and 200 ig/m1 yeast RNA

Monocyte

isotonic Percoll centrifugation,

interface,

blots 0.1%

112

30%

NaPO4,

Assays

stated, cells were cultured in Alpha Minimal Medium Eagle (Irvine Scientific, Santa Ana, 5% human

(100 nant

on lym-

were

from

fraction contained by morphology

erwise Essential

puof

(LSM,

layered on 49.2% Sweden). After

harvested

were blood

centrifugation

PBMC

were

Denhardt’s,

(PBMC) venous

end-labeled M-CSF

detected with DM 85 (5’GTAAlTFGGCACGAGGTCTCCATCTGACTGTC3’), G-CSF with EK GGTAGAGGAAAAGGCCGCTATGGAGTFG3’),

all probes

cultured nonadherent in polypropylene tubes [7] or allowed to adhere on plastic at 37#{176}C for then

Percoll termined tion;

by density

probes that were kinase and [32P]ATP.

GM-CSF with EK TACTCAGGT1’CAG3’).

MATERIALS AND METHODS Monocyte Isolation and Cell Culture Peripheral rified from

oligonucleotide polynucleotide

synthetic Bio-Rad,

by hybridization

memRichwith

Regulation and Fresh Monocytes

Protein

on CSF mRNA Secretion

in

Indo is a potent inhibitor of the cyclooxygenase pathway and inhibits the endogenous production of PGE by monocytes centrifugation tants were RIAs and and bone G-CSF.

[14]. Monocytes were purified by Percoil and adherence to plastic. Culture supernaassayed for M-CSF and GM-CSF by specific for bioactivity marrow cell When

cultured

by a cell line proliferation assay colony assay sensitive to M- and in Indo

for 24 h, these

monocytes

Human

Monocyte

Production

of M-, G-, and GM-CSF

123

123

123

4.4-

4.4-

2.4-

2.4-

1.4-

1.4-

.3-

.3-

Probe:

G-CSF

M-CSF

277

a

GM-CSF

b

C

Fig. 1. Indomethacin Increases the amount of LPS-induced MCSF and GM-CSF transcripts in adherent monocytes. Total RNA (15 tg) from adherent monocytes of donor 50 was analyzed by Northern blots. Lane 1: Monocytes cultured 24 h in the pres-

ence of 10 M Indo. Lane 2: Monocytes Induced with 100 ng/mI LPS for 24 h. Lane 3: Monocytes cultured In presence of Indo and LPS for 24 h. Probe: a) M-CSF (7 d), b) G-CSF (2 d), and c) GM-CSF (7 d exposure).

contained high levels of M-CSF

TABLE 1. Cytokine Production by Adherent Cultured With LPS or Indomethacina

message

(Fig.

la).

This

high

but

no G-

expression

or GM-CSF

of the

M-CSF

gene produces a ladder of transcripts from 1 .6 to 4.4 kb [17]. Supernatants from these monocyte cultures contained M-CSF but not GM-CSF protein as measured by specific RIAs (Table ony assay detected mouse

cell

line

1). The murine predominantly

NSF-60

growth

amounts of activity. Monocytes that were induced contained high levels of G-CSF and

a moderate

though

no

amount

M-CSF

tant by RIA, were revealed

protein

1,410 by

assay

detected

colthe

similar

with LPS alone for 24 h and GM-CSF transcripts mRNA detected

(Fig. in the

U/mI of G-CSF proliferative the NFS-60 assay and 5.3

1). Al-

tures

addition moderately

CSF message probing the

increased

to LPS-induced GM-CSF

decreased slightly, Northern blot (Fig.

GM-CSF proteins (Table 1). A time early into

of Indo

were course

GM-CSF

M-iG-CSF

NFS-60

RIA

colonies

TNF

Indo

(ng/ml)

(U/mI)

(ng/ml)

(U/mi)

(U/mi)

-

-

-“

N.D.