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Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF). Biochem. BiOpILyS.
Effect of recombinant

human

macrophage-colony

factor on marrow, splenic, progenitor cells in mice Takeshi Tanabe

Yamauchi,

Central

Abstract: colony-stimulating splenic, and

Research

The

Kouji Yada,

Akio

Umemura,

The

Cross

Corporation,

Laboratory,

effects factor

Green

of human (M-CSF) progenitor

peripheral and

macrophage on marrow, cells (CFU-M,

CFU-GM, administered tg/kg).

CFU-G) recombinant Single injection

were investigated human M-CSF of 4,000 igJkg

resulted

in

in

a

decrease

progenitor cells day 2 followed the

original

type

of

the

level

on

day

4

or

progenitors

5.

of

and

In

CFU-G) returning

for

800

j.tgfkg

of M-CSF CFU-G, as

marrow

in well

CFU-G.

The

present

of mice

with

pharmacological

man

M-CSF

affect

the

indicate

to

action

of

blood

and

J.

M-CSF.

cells

lineage

not

also of coincidence between cells and increase of cells suggests that from bone marrow

reseeded

Rio!.

spleen 59:

by the

296-301;

Words:

colony-forming

unit

[16-22].

the progenitor M-CSF affects

macrophage-CSF blood cells

However,

In the present human M-CSF

little

intensive (M-CSF) been

has

is known

progenitor

cells

[17,

of purified

M-CSF

about

its

19, 21]

ef-

because

in sufficient

cells in mice. We show the number of progenitor

progenitor cells from bone marrow lowed by settlement in the spleen

cheon the reported

quan-

study we utilized purified to clarify its hematopoietic

recombieffect on

here cells

mononuclear phagocyte lineage but lineage. In addition, the present results M-CSF may stimulate the mobilization

MATERIALS

that not

human only of

also of granulocyte suggest that human of hematopoietic

to peripheral in mice.

blood

fol-

AND

METHODS

Mice C3H/HeN

7- to 8-week-old

Japan)

were

Recombinant

prolferation

male

mice

(Charles

River

Japan,

Kanagawa,

used.

CSFs bridge,

1996. Key

of human of peripheral

transplantation

after

but

to the

Leukoc.

cell

stem

of hu-

ofprogemtor

only of monocyte/macrophage granulocyte lineage. Also, the decrease of marrow progenitor splenic and peripheral progenitor the progenitor cells are released to peripheral

4- to day 3 also of

blood

of granulocytopenia

unavailability

tities. nant

and Toshizumi

Japan

on hematopoietic

of the

Hanamura,

treatments

concentrations

number

Takuji

of autologous

previously

each

started

that

573,

source

fect

mice caused a decrease in as an increase in splenic

results

Osaka

The effect proliferation

on to

increase markedly on day 2, reaching a level 15-fold higher than that of the basal value on or 4. Peripheral CFU-M, CFU-GM, and CFU-G increased on day 2. In addition, administration

Eiji Asakura,

the

marrow

contrast,

tested

hematopoietic

and for treatment motherapy [9-15].

in mice (8-4,000 of M-CSF

number

(CFU-M, CFU-GM, by a gradual increase,

splenic

and peripheral

stimulating

human

MA.

from

the

cific

activity

It was

culture assay

>99% resis.

purity

with

supplied

by

units/mg mouse

bone

content

cells

cells

pg/1O

product

purified

had

a spe-

in a colony-

[23],

and gel

units/ml

Cam-

and

determined

sulfatelyacrylamide < 10

was

final

as

marrow

Institute,

ovary

The

protein,

dodecyl

Genetics

hamster

to homogeneity.

x 10

on sodium

Endotoxin

was

in Chinese

medium of 4

formation

M-CSF produced

as shown

revealed electrophoby Limlus

INTRODUCTION Colony-stimulating teins that progenitor jects and

(CSFs)

are

a family

stimulate colony formation from cells [1-4]. When administered

or experimental granulocyte-macrophage

proliferation tissues

factors

and

animals,

of the

progenitor

peripheral

blood,

hematopoietic to human sub-

granulocyte-CSF CSF (GM-CSF) cells as well

in

Abbreviations:

of glycopro-

the

(G-CSF) stimulate blood

the

296

59,

1996

Leukocyte

Biology

Volume

February

red

CSF,

colony-stimulating

granulocyte

blood

large-cell fraction white blood cells; FCS,

Reprint

cells

cells in hematopoietic important

of

RBC,

CFU;

in the peripheral blood [5-15]. Progenitor peripheral blood that are mobilized from the tissues by G-CSF or GM-CSF are clinically

Journal

G-CSF,

CSF;

cell;

WBC,

of white CFU-M,

tory, Osaka

573,

as

29,

Green

white

CFU; calf

requests:

The

Received tember

fetal

factor;

GM-CSF,

CFU-G,

M-CSF,

macrophage

granulocyte-macrophage

blood

blood cells; macrophage

granulocyte-macrophage

hematopoietic

as mature

CSF;

cell;

PLT,

CSF;

platelet;

WBC-LCC,

WBC-SCC, colony-forming

small-cell units;

granulocyte

CFU;

fraction CFU-GM,

CFU-S,

of

spleen

serum. Dr.

Cross

Takeshi

Yamauchi,

Corporation

2-25-1,

revised

September

Central

Research

Shodai-Ohtani,

LaborsHirakata,

Japan. July 1995.

17,

1995;

28,

1995;

accepted

Sep-

Table

ts of M-CSF

1. Effec

Treatment

progenitor

on the

Cells

on

Day

ber

Num

CFU-GM,

of Marrow

were

2.

and

added

or CFU-G cells

CFU-G

CFU-M

CFU-GM

x

103/femur

X l03/femur

Saline

5.13

±

6.86

±

1.82

1.29

±

8

5.10

± 0.90

6.86

±

0.48

1.12

± 0.13

80

3.84

±

4.11

± 0.71

0.81

± 0.12

800

3.30

± 0.09

3.98

±

0.33

±

Treatment (M-CGF,

gIkg)

0.85

0.30

0.11

Human

prepared

M-CSF

dilution

saline.

was

of

Control

intravenously

the

mice

original

were

injected

material

into

in

administered

an

0.

mice

2 ml

equal

Recombinant

bridge,

per

of hematological milliliter

(PLT),

WBC

of

total

(0.

2 ml)

of

determined

and

with

blood

blood

cells

small-cell

were

Genzyme

a Sysmex

blood

(WBC),

(Cam-

cells

large-cell

of WBC

Micro-cell-counter

(RBC),

fraction

of

(WBC-SCC)

F-800

day

before

three

mice

were

(day killed

obtained

by

a cardiac

spleens

were

excised

equal

volume

of

temperature (150

mm).

5

saline,

mixed ml

of

dosteum

were

with

McCoy’s

upper

McCoy’s

5A 5A

spleen

22.

on

in

resuspended

the

after

2 x

Splenic and

ice

for

suspensions in

reached

the

levels

counted,

and

McCoy’s

5A

numbers

seen

in control

Macrophage

assayed

Briefly,

bone

tured

in the

presence

medium

for

marrow,

humidified cells

made

splenic,

Petri

CO2

atmosphere

scored

as

granulocyte

pipetted

at

conditions

were

for the

determined blood.

of human

M-CSF,

added 1,

5.

0.0

;(

4.

(D :S

2.0

U.

0

were desired

0.0

0.3%

dishes.

agar The

and

colonies.

All

0.

5,

CFU-M,

10

to each and

inoculated

20%

cells

were

fetal

were

2.0

cul-

calf

serum

incubated

7 days.

cultures cell

Clones

were

ng of mouse

dish, 5

iO

and

cells/dish

for CFU-G

GM-CSF,

respectively.

x

number

except

CFU-GM,

or

In the

assay

were

4.0

semisolid

in 1 ml of McCoy’s

cultures

at 37#{176}Cfor

b

units

in

blood

or G-CSF

experimentally

For

6.0

C’)

colony-forming activities

or peripheral GM-CSF,

with

plastic were

peripheral

cells,

4 or

in

0

in a

performed

10

and the

cell

assays

CSF

.

the means

M-CSF

of the

(open

absolute

frotn

effects

Mice

were

symbols)

of marrow the

±SE

study

progenitors.

number

represent

G-CSF

Kinetic

marrow

of human

in units

5

4

of M-CSF

or saline

progenitors number

3 anitnals

of human

was point.

the

oti

intravenously (closed

counted

of progenitor

at each

M-CSF

injected

cells

days

per

P