hypersensitivity to erythropoietin using serum free ...

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1982 59: 447-451

Erythroid progenitors in polycythemia vera: demonstration of their hypersensitivity to erythropoietin using serum free cultures N Casadevall, W Vainchenker, C Lacombe, G Vinci, J Chapman, J Breton-Gorius and B Varet

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CONCISE

REPORT

Erythroid

Progenitors

in Polycythemia

Hypersensitivity N. Casadevall,

By

In order

to demonstrate

colonies

observed

standard

(epo)

used

methyl completely

Ill

epo-independent in

colonies

were in

culture

of PV In PV,

the

after

with

first

BFU-E

plating

CFU-E

the

The

compared

and

of0.0O1-0.O1

colonies

lU/mI

of epo,

was

PV

able

to

the

bone

exhibit

epo

cannot

the

normal

controls.

itself,

observed

excluded.

were

a

degrees

Using

plasma

blasts

and in the

Since

were

able

of added

absence

culture

to differentiate

systems

the latter containing trace nature of the abnormality progenitors abnormal either sensitive

in PV population

to

Zanjani from and

epo

PV

To answer dure allowing

this

in fact sitely

Blood.

amounts of epo, exhibited by

only

dependent sensitive

Vol.

59,

amounts

the exact erythroid Indeed could

question, we have growth of erythroid results from PV

hormone.

No. 2 (February).

1982

fulfilled

ies4’4

have

all

in standard

culture

hypersensitive

abnormal

progeni-

population,

but

hypersensitivity

present

to

in the

normal

spontaneous

other

serum

AND

METHODS

that

patients

epo.

serum

colonies

by

cannot

be

factors

bone

epo

samples

the

of

PV

blood

of patients

studied. stud-

mononuclear

cells

formation

conditions either

were

group;’#{176}previous colony

standard obtained

marrow

aspirates

preservative-free

separated

using

serum-free

medium

et al.9

of

in

the

of culture.

from

the

undergoing

Ficoll

transferrin

blood

of normal

thoracic

grown

surgery.

0.8.10

low

density

and

absence

of lipoproteins,

erythroid,

restored

the

Louis,

described

M a-thioglycerol (Merck

(CalbioWest

Indeed, colonies

observed.

in

the

(granulo-

Addition This

(I

human

Darmstadr,

(LDL).

colonies.

modified

albumin

purified

l0

in

by Aye

in serum

Fed3

was

of hemopoietic

were

technique

Mo.),

of hemopoietic

or megacaryocyte) growth

bovine

lipoproteins

no growth

that

in 200

cells

methylcellulose

St. M

collected

density culture

from

deionized

(Sigma),

Ca.),

Germany),

derived

Sigma,

zg/ml)

iolla,

The

in 0.8%

‘including

were

Light

technique.’4

V fraction,

(300 La

heparin.

was directly were

(Cohn

or 20 ml of blood

sterile the

Cultures

chem

cyte,

and

erythroid

ribs

of

study

Assay

PV bone

mg/mi)

the

PV

marrow

under were

or from

as cases

of the

exhibited

of added

Normal

diagnosed

criteria

shown

these

absence

U

clinically the

Dulbecco’s

of LDL

lipoprotein

class

it is for

employed a proceprogenitors in

provide evidence able to differenin serum-containing culture are on epo and, in addition, are exqui-

to the

Our

the be

erythroid sensitive of

patients

They

Colony

or exquisitely an antierythropoietin

small

in the progeni-

Samples

serum,

to differentiate into erythroblasts. has not been confirmed, since antibodies that are both specific for erythroid culture.

serum-free conditions.8’9 that erythroid progenitors “spontaneously”

(epo).

al.7 concluded that were in fact abnormally

required

MATERIALS

volunteers

erythro-

some

remains uncertain. of erythroid progenitors

et

hormone in order This experiment difficult to obtain epo and nontoxic

tiate

include

completely epo-independant to the hormone. Using

globulin, progenitors

into

erythropoietin

typically

higher

present

and

these

of

to

curve much

erythroid

homogeneous

growth

a hypersensitivity

Six

(PV).

clot2 or methyl cellulose56 culture systems, it was shown that certain erythroid progenitors from patients with PV, in contrast to normal or secondary polycythemia, exhibited an abnormal behavior

PV

of epo that

and

cultures

in controls

N VITRO STUDIES using clonal assays for study ofthe growth oferythroid progenitors’ have clearly advanced our knowledge of the pathophysiology of vera

are

of epo

that

of epo

the

I

polycythemia

amount

in their

amount

0.01 in

dose-response

spontaneously show

small

epo

dependent

They

explain

between colonies

cultures

small

represent

different

to

while

not

the

confirm

in fact

hormone.

Since

dose-response

the

B. Varet

concentration

to differentiate

do

and

spontaneous with

results

are

Cultures

in serum-free

from

conditions tors

spontaneous

epo

compared

These

sensitivity to

BFU-E

no

Free

of

patient

expected

tors

PV,

derived) same

epo

in the

of

at an

same

of Their

J. Breton-Gorius,

Numbers

serum

in the serum.

lipoproteins.

6 cases

Serum

lU/mI.

than

serum

transferrin,

while

increase

progenitors

addition

or

serum.

a tenfold

erythroid

In

conditions,

present

in which

density

used.

(CFU-E

serum-free

showed

low

was

we

Using

appeared

0.1 with

erythro-

Demonstration

J. Chapman,

they

using

hormone,

iron-saturated

and

epo

(PV)

from the

cultures.

by

colony

observed marrow

serum-free

replaced

Step

independent to

G. Vinci,

erythroid vera

sensitive

albumin,

Connaught

“spontaneous”

were

cellulose

C. Lacombe,

in polycythemia

exquisitely

a-thioglycerol

curves

whether

assays

or

was

W. Vainchenker,

in vitro

colony

poietin

after

to Erythropoietin

Vera:

From CHU

the

Department

Cochin,

Hbpital

Paris,

Henri

Mondor,

Supported

in part

Submitted

August

Address CHU

reprint

Cochin,

© /982

of and

75674

by Grune

Hematology

INSERM

Creteil, 198!;

requests Paris

A TP accepted

to N. Cedex

& Stratton,

and

INSERM

U.152,

JNSERM

U.35,

France.

by INSERM 25,

and U.91,

78 90 and

DGRST.

September

Casadevall,

Batiment

29,

/981. G. Roussy,

14, France. Inc.

0006-4971/82/5902-0036$O1.OO/O

447


CASADEVALL

448

was

isolated

lipoproteins,

gradient

free

of very

and

of

procedure.’2

this

technique

was

95%

or more

of the

LDL

preparations

in the

a-medium

These

LDL

when

the same

batch

serum

culture

either

10%

erythroid

red.

was

Canada). cultures

were

made

more

replicate

the

incubated

blood

were

water-saturated Bone

days.

Isolated

colonies

marrow clusters

under

clusters

were

when

no

picked

colonies.

Blood

BFU-E

colonies.

was

for controls

in

optimal

serum

was

III

for added,

sheep volume.

than

2 x l0

0.01

rigidly

IU/ml,

cells

were

6 or

)

3001

could

order

were

and to

removed

be stained

exclude

after

after

I 4 days

the

or

erythroid scored

and

for

Bone

marrow

of PV

Marrow bone

The number from one

examined.

All

The

epo four cases (Fig. concentration of typically observed

Marrow

When normal bone free medium, the epo that the first CFU-E

cultures, the earliest at an epo concentration

“spontaneous”

colonies. RESULTS

Normal

the

same

on the same day of serum in methyl

in the cellu-

erythroid of 0.01

Cultures marrow

were

colonies

studied in

of these colonies patient to another

and

culture

all with

was extremely (Fig. 3). In

serum-free cultures, no epo-independent erythroid colony was observed. In three cases, rare colonies were present in the absence of epo; these were picked off

rare

benzidine and

in serum-

cultures.

Vera

cases

serum. variable

8

as CFU-E

observed, with

plated

comparison,

plated absence

in standard

exhibited

at 5% CO2

atypical

In serum observed

Six

in a

microscope. colonies

lose (Fig. 2). colonies were

were

for scoring counted

accurate

was in the

Polycythemia

cells

Cultures

maintained

removed

were

lU/mI

marrow

(Falcon

cells

either in plasma clot or in methyl erythroid colonies appeared at a

bone marrow presence and

free than

Three

0.01

bone

plated. dishes

plasma

Willowsdale, than

marrow

medium, the earliest

IU/ml. Below 0.5 IU/ml, the number of colonies was higher in cultures containing serum, while at higher epo concentrations, the number of colonies was approximately the same. The size and degree of hemoglobinization of colonies were always lower in serum-

contained

was

final

greater

lower

were

erythroid

red in

cultures

that

ml

were

off, cytospanned,

May-Grunwald-Giemsa

I

Petri

dishes

of red

an inverted

addition,

that

culture

a

omitted.

Usually cells

whole after

cultures

of a step

of

35-mm

atmosphere

in air.

used

The

Laboratories,

ofepo

mononuclear

at 37#{176}C in plastic

was

of epo

run.

original

in

normal

epo concentration. allow a more

lower To

solution diluted

activity

to which

batch

were

up; for concentrations l0

serum

were

for concentration

by

20#{176}C.In all experiments,

-

Connaught

Cultures

of the

in LDL

same

containing cellulose,

B. All

Hank’s

biologic

of LDL.

Fed3

son, when

density g/ml;

were

to that

In cultures

3034.1,

cultures

against

their

AB

and

separated

density

1.028-1.050

preparations

equivalent

growth.

of epo

replicate

5 x

or the

(batch

Ontario,

final lost

human

of

LDL

a

of apolipoprotein

dialyzed

The

preparation

a-thioglycerol,

source

consisted

at 4#{176}C or at

of normal

by

range

equivalent

either

progenitor

preparation

In

moiety extensively

as for the

transferrin, The

stored

the

of high

proteins

density

within

preparations

serum

lipoproteins, serum

hydrated

protein

of phenol

days

or

The

to a concentration

serum. few

density

typically were

absence

low

contaminating

ET AL.

granulocytic

or macrophagic

curve was studied appeared at an while a plateau of epo.

in epo was

was

dose-response

colonies

grown in serumcurves showed appeared at an epo

were

dose-response 3): colonies 0.01 IU/ml, at 1 .5 IU/ml

Blood

BFU-E

The serum-free markedly However, conditions

concentration slightly below 0.1 IU/ml; a sharp increase in the number of colonies occurred with increasing concentrations (Fig. 1 ). A plateau was usually achieved at about 1 .5 IU of epo. In compari-

Cultures

dose-response

curves

of

blood

BFU-E

in

cultures from normal adults did not differ from those of marrow CFU-E (Fig. 4). the growth of blood BFU-E in serum-free was highly dependent on the cellular

300

C

Ct) C

LU

z

-j

0

200

0 C-) 0 0

100 LU

0.001

0.002

Epo

GOl

0.02

CONCENTRATION

0.1

0.2

0.5

( lU/mI)

2

Fig. 1. Epo dose-response curves from three normal bone marrows in serum-free cultures. CFU-E colonies were scored at day 8. Each point represents the average value from three replicate cultures seeded at 2 x iO cells/mI.


PV ERVTHROID

COLONIES

IN SERUM

FREE

CULTURES

449

for

concentration

instance,

no

BFU-E

colonies

could

be observed when 2 x I 0 cells/mI or less were plated. In PV patients (4 cases), no BFU-E growth could be observed in serum-free cultures when epo was not added.

The

epo

dose-response

curve

was

in 3

studied

cases. Figure response lished in BFU-E equivalent marrow U)

4 shows a typical example of the dosecurves of CFU-E and BFU-E to epo estabthe same patient on the same day: the first colonies appeared at an epo concentration or even lower than that required for CFU-E; some BFU-E colonies could be

0 -J 0

C.)

observed at 0.00 1 IU/ml. The various results were finally summarized 4) and show a tenfold increase in the sensitivity

8

from

LU

z

PV erythroid

progenitors subject.

a normal

I>-

to epo

(Fig. of the

as compared

to those

DISCUSSION

LU

In

this

article

we

have

shown

that

erythroid

genitors from the blood or the bone marrow with PV are unable to give rise to erythroid without

epo

in serum-free

epo dose-response erythroid progenitors Epo CONCENTRATION

(lU/mI)

dose-response curves for cultures (dotted lines) from colonies were scored at day value from three replicate

epo.

CFU-E

The first and BFU-E

concentrations

in PV controls.

and

that some PV and exquisitely

between results

0.01 0.1

confirm

300

3.

Epo

dose-response

curves

from PV bone marrows in serum-free culture. CFU-E colonies were scored at day 8. Each point represents the average value from three replicate cultures. with the exception of epo concentrations lower than 0.01 lU/mI in which case at least 6 cultures were run. The number of “spontaneous” colonies obtained under standard conditions

of

culture

for

the

same

patients (using the same batch of normal human AB serum) are plotted on the ordinate. The results are expressed per 2 x 1O cells.

o (p.

CONCENTRATN

lU/mI

lU/mi

400

Fig.

of the cultures of and from erythroid sensitive

erythroid colonies derived were observed, respectively,

between

These

colonies

Comparison

curves in serum-free from PV patients

controls clearly demonstrated progenitors were abnormally to

Fig. 2. Comparison of the epo serum-free (solid lines) and standard the same normal bone marrow. CFU-E 8. Each point represents the average cultures seeded at 2 x 1O cells/mI.

culture.

pro-

of patients

(lU/il)

and and

0.001

from at epo lU/mi

0.01

lU/ml

in

the previous

report

by


CASADEVALL

450

450

ET AL.

60 C,) LU

Fig. 4. Comparison of the epo dose-response curves in serum-free culture of the

z 0 -J

C,)

0

uJ

C.)

z 0

-J

300

40

CFU-E

and BFU-E from normal lines) and PV (solid lines) subjects. CFU-E colonies were grown from bone marrow () BFU-E colonies were grown from peripheral blood (#{149}). Each point represents the average value from three replicate cultures. BFU-E colonies are expressed per 5 x 1 0’ cells in both the normal subject and the PV patient. Erythroid colonies are expressed per 106 cells in the PV patient. while it is expressed per 3 x 1 0 cells in the normal subject in order to respect the same scale. (dotted

0

0

LU

C.)

Q 0

LU

0

I>-

150

20

LU

La.

o005

0.001

0.01

Epo Zanjani’s

in which

group

an

0.1

CONCENTRATION

antierythropoietin

anti-

body was used to neutralize the epo present in the plasma clot culture system.7 Our data therefore confirm that the abnormality of erythroid progenitors in

PV

PV

not

only

quantitative

as

by others.’3 groups

methyl three

and

is qualitative

suggested Several areas erythroid

plasma

using

cellulose5’6

culture

in the epo

clot2’4

systems

dose-response a plateau

progenitors:

or

have

usual

described

curves typical followed by

of an

increasing number of colonies and then by a second plateau. The first plateau is considered to represent the abnormal population. However, in serum-free cultures, the first plateau is not observed and the epo

dose-response that

suggests

that

patient

cells some

curve

of normal are

with

in PV patients

subjects

erythroid

made respect

exhibiting

differs

in its sensitivity

progenitors

from

of a heterogeneous to their sensitivity degrees

varying

Adamson’s

group’5”6

who

PV patients neous”

heterozygous erythroid colonies

clone and that tion

not only

cells

of the

have

the addition of epo of normal progenitors

abnormal

clone.

shown

for G6PD, belonged

tions. The estimated amounts of epo in the added serum (s0.0003 IU/ml)’7 did not account for this observation. However, it could be explained by a nonspecific “feeder effect” of the serum. Recent data’8 suggest that in mice, one or more factors present in the serum,

and

the

explain

This

of epo

PV of

hypersensitivity

of abnordata from female

which

of

differences

serum

from

present

epo,

response

colonies

curve

a

factor

might

that

the minute

amount

if one extrapolates the erythropoietin

from dose-

culture, to the epo serum (Fig. 3), then the would be between 0. 1 and 3 IU/ml the patient studied. This might be the effect of the same putative factor. conclude from our data that abnormal in

serum-free

present

amount present according to explained by We therefore

play

could not account for numbers found in cultures to which

was added. Indeed, numbers, plotted on

concentration

may

erythroblasts.

between cultures it. Furthermore, in

and those lacking

in serum

spontaneous

serum these

observed

we have observed

in the

erythroid progenitors from PV patients are dependent of epo and exquisitely sensitive to epo. Whether this abnormality is restricted to epo or includes other differentiation

factor(s)

that the “spontato the abnormal induced differentiabut also stimulated

distinct

are

role in the differentiation of CFU-E into The effect of an equivalent human

from

in two

3

Comparison of the epo dose-response curves of normal erythroid progenitors in methyl cellulose in the absence or presence of serum showed that CFU-E required more epo to differentiate in serum-free condi-

only

one given

in the epo sensitivity is in agreement with

1 15 2

to epo.

and some being normally responsive. However, since our source of epo was not highly purified, one cannot exclude the unlikely hypothesis that the hypersensitivity in the epo dose-response curve was the consequence of an impurity present in the preparation. This heterogeneity mal PV progenitors

0.5

containing PV patients,

population to erythropoietin, of

02

(lU/mi)

remains

to be determined.

ACKNOWLEDGMENT The Muller

authors

would

for excellent

Dulac for

typing

like

to thank

technical the manuscript.

J. Bouguet,

assistance,

and

M. M.

Titeux

Segear

and and

0.

A. M.


PV ERVTHROID

COLONIES

IN SERUM

FREE

CULTURES

451

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B: Is erythropoietin

differentiation?

AS, mouse

Nature

Camiscoli in the

Med the only

62:743, factor

289: 1 84,

iF:

assay

1981

and

Use

of

the

standardiza-

1963 which

regulates

late