The appearance of phospholipase activity in the human macrophage-like cell line U937 during dimethyl sulfoxide induced differentiation

Vol. 118, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS Pages 217-224

BIOCHEMICAL

January 13, 1984

THE APPEARANCE OF PHOSPHOLIPASE ACTIVITY IN THE HUMAN MACROPHAGE-LIKE CELL LINE U937 DURING DIMETHYL SULFOXIOE INDUCED DIFFERENTIATION Robin Department

Received

F. Myers

of Allergy 60 Orange

December

and Marvin

I.

and Inflammation, Street, Bloomfield,

Siegel Schering Plough NJ 07003

Co.,

1, 1983

The human histiocyte cell line, U937, with monocyte --Sunnnary: characteristics, can be induced to differentiate into macrophage-like cells when exposed to growth medium containing 1.5% DMSO. Following three days of exposure, DMSO-treated but not control U937 cells can be stimulated to release endogenous arachidonic acid from their phospholipids. Maximum release of the unsaturated fatty acid occurs with 10 uM calcium ionophore in the presence but not in the absence of exogenously added calcium ion. In addition, DMSO-treated but not control U937 ccl 1 S exhibit phospholipase activity when exposed to human IgG and then anti-human inmunoglobulin. These data suggest that with respect i nto to arachidonic acid metabolism U937 cells differentiate functional macrophage-like cells when exposed to DMSO.

A human hematopoietic patient

with

Nilsson

in 1976

suspension monocytic stepwise described

cell

generalized (1)

These (1)

and,

morphological for research

induced

to differentiate

macrophage-like lectin-stimulated 1 mM dibutyryl The abbreviations heptadecatrienoic iocasatetraenoic icosatetraenoic

cells

when

cells

induced

into

that

CAMP (2,4-61.

maintained

cells

chemotactic

peptide

to medium

a

to that

(2,31. line

can be

sensitive

conditioned

to 1.3% dimethyl In addition

undergo

similar

U937 cell

and

imnature

to differentiate,

monocytic

a

in

to represent

the

by exposure

from

by Sundstrom

maturation

normal

lymphocytes,

used acid acid; acid,

successfully appear

has indicated

was established

lymphoma

and functional

differentiating

Further

(U937)

histiocytic

and has been

culture. cells

line

sulfoxide

to chemotaxis,

by or to the

are: HHT, 12-hydroxy-5,8,10,: 5-HETE. 5-hvdroxv-6.8.11.14-LTB 5s 12R-d i’ hy‘hroxyi6,8,10,14leuk%&ieAe B4. 0006-291X/84 Copyright

217

All

rights

0 1984 of reproduction

$1.50

by Academic Press, Inc. in any fiwm reserved.

Vol. 118, No. 1, 1984

BIOCHEMICAL

abilities

to produce

superoxide

enzymes

and to perform

been

induced

line, various

Since acid

normal

when

serve

DMSO-induced This

report

antibody

these

fatty

from

their

acid

of this

model

of

of normal

human

promyelocytic

differentiation

endogenous

leukemia

release in U937 cells

The induction

following

of U937 cells

reported growth

it

was

to release

phospholipids. activity

could

as has been previously

appearance

stimulatable

the previously

(8-151,

cell

line

to a granulocyte-like the

phospholfpids

arachidonfc

agonists

phospholfpase

of differentiation

the

complex

cells

have

the U937 cell

endogenous

appropriate ability

differentiation. with

the the

describes

endogenous

parameters

metabolize

with

the expression

with

cytolysis

to be a useful

to investigate

as a marker

observed

cellular

Therefore,

appears

macrophages

the unsaturated Moreover,

lysozomal

function.

stimulated

of interest

(4,7).

and biochemical

monocyte-macrophage

to release

dependent

agents

neoplastfc,

biological

anion,

antibody

by these

although

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

cell

of calcium

in functional

in DMSO containing

and

acid

from

DMSO-induced

of such an activity increase

(16).

ionophore

of arachfdonic during

HL60 following

is consistent responses medium

of

(4).

MATERIALS AND METHODS: Cells. The U937 cells were a generous ~fromDr.KX~ore?JX.i.ie University. The U937 cell line was cultured in RPMI 1640 with 10% heat inactivated fetal bovine serum (lot 100389, Sterile Systems, Logan UT), penicillin (50 units/ml), streptomycin (50 ug/ml), 1% MEM non-essential amino-acids, 1% sodium pyruvate and 1% L-glutamine (all from Cell cultures wgre split so6that the Gibco, Grand Island, NY). cell density was maintained between 2 x 10 and 2 x 10 cells/ml. Cells were induced to differentiate by the addition of DMSO to a final concentration of 1.5% to the above culture medium. wf;h;;p@;;pase Ass_a3r!, Cell cultures were incubated overnight arac1Taonlc acid (Amersham, 55 uCf/umole) at a concentration of 2 uM (0.1 uCf/mll. Labelled cells were harvested by centrffugation and washed twice with 50 mM Trfs-HCl buffer, pH 7.4, containing 100 mM NaCl. Washed cells were resuspended in 50 mM Tris-HCl buffer, pH 7.4, containing 100 mM The cell suspensions (1 ml) NaCl, 1 mM CaCl f, and 1 mM glucose. were incubated t 37 for various times as indicated in the Reactions were terminated by the addition of 2.4 figure legends. ml chloroform:methanol (1:l v/v) and 10 ul of 10% formic acid. The samples were cooled in ice and centrifuged. The organic 218

BIOCHEMICAL

Vol. 118, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

layer was withdrawn and evaporated to dryness under a steady stream of N The dry extract was then redissolved in a small volume of c P‘1oroform:methanol (1:2 v/v) and spotted on silica thin layer plates (sil 625, without gypsum, Brinkmann). Chromatograms were developed with an ascending solvent system composed of the upper layer of ethylacetate:iso-octane:glacial acetic acid:water (90:50:20:100 v/v/v/v~. Labelled products were located by autoradiography and the appropriate regions of the chromatograms were scraped and counted in a liquid scintillation counter. Products were identified by co-chromatography with authentic standards. All assays were in duplicate and have been repeated at least twice. Values are plus or minus 10% of the mean. Since negligible metabolism of released arachidonic acid occurred, phospholipase activity was determined by measuring the free arachidonic acid produced upon stimulation. RESULTS: --

U937 cells

to divide

after

Figure

control

approximately

every

rate

divide

by the

three

cultures

is

not

As illustrated

second

day of exposure

as measured

significantly

in

double

DMSO-treated

Viability,

however,

days.

1.5% DMSO cease

hours,

by the

third.

containing

of U937 cells

seventeen

of division

exclusion,.

in medium

approximately

1, while

their

grown

in density

cells

slow and cease

by trypan

affected

to

blue

(data

not

shown). This

halt

in cell

increased

ability

acid

endogenous

from

while

both

arachidonic

acid

U937 cells

do not

presence

medium

release

ionophore

activity

activity

corresponds

DMSO-treated

ionophore

from DMSO treated

but

dependent.

As shown

A23187

maximal

with

21.

in Figure

fatty

three

cells

acid

control when exposed

grown

in the

days express

The induction

this

of this

of growth

2,

incorporate

phospholipids,

However,

at least

(Fig.

arachidonic

readily

unsaturated

to the cecession

cultures

The calcium

their

A23187.

(Fig.

by an

to release

cells

into

the

reflected

As illustrated

and DMSO-treated from

is

cells

phospholipids.

of 1.5% DMSO for

phospholipase

and division

of DMSO-treated

control

to the calcium

growth

enzymatic

observed

in

1). stimulated

not control in Figure

arachidonic

release U937 cells

3, DMSO-treated acid 219

release

of arachidonic

acid

is concentration cells occurring

respond at a

to

Vol. 118, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

---

of U937 cells in DMSO containing medium. U937 --Fig. 1. Growth cells were grown in suspension culture in the absence (o) or presence (01 of 1.5% DMSO as described in Materials and Methods. The number of cells in the culture was rneaYlZE!Xly. Asdetermined by trypan blue exclusion, viability was greater than 95% in control and DMSO containing cultures throughout the times shown. Fig. 2. Appearance of nhospholipase activity in DMSO treated IlW7Fells. U937 cells were grown as described in Fig. 1 in the absence (01 or presence (0) of 1.5% DMSO. At the times indicated, cells were assayed for 2 minutes for the oresence of phospholipase activity as described in Materials and Methods I'n thel$resence of 10 uM A2 IncorporXXniXf----ClClarachidonic acid 3&!z'cellular lipids was greater than 80% in control and 60% in DMSO-treated cells14fter overnight labelling. The amount flf esterified5[1C$rachidonic acid in the assays wgs 8.0 x 10 50 1.7 x610 dpm/lO cells for control and 1.1 x 10 to 2.5 x 10 dBm/lO cells for DMSO-treated cells. Assays contained 4 - 10 x 10 cells. Results are reported as the per cent of esterified arachidonic acid released.

concentration

of

cells

do

not

their

endogenous

ionoohore

quantity times

release

phospholipids

induced

Maximal

of unesterified (Fig.

4).

of

approximately

amounts

10

uM.

Control

of arachidonic

at concentrations

acid

from

of the calcium

as 20 uM.

ionophore

process.

ionophore significant

as high

Calcium rapid

the

This

release

release

occurs

arachfdonic decline

is

of arachidonic in two minutes acid

apparently 220

declining

acid with

is

a the

at longer

due to the re-esteri-

BIOCHEMICAL

Vol. 118, No. 1, 1984

Calcium ionophore concentration dependence of acid release Control (a) and 4 day DMSO-treated 10) assayed ior their ability to release endogenous wer [l-e4 C7arachidonic acid in the presence of 1 mM CaC12 concentrations of the calcium ionophore A23187, as in Fig. 2.

%&donic U937 cells esterified and various described Fig. 4. treated absence described

fication

Time course of arachidonic acid release. U937 cells for 4 days with DMSO were assayed in the presence 10) (e) of 10 uM A23197 in the presence of 1 mM C&l2 as in Fig. 2.

of the unsaturated

metabolism LTB4 is

observed

A23187

41.

(Fig.

Ionophore

induced

upon exogenous

ion concentration by

acid

with

are detected

A23187

free

is

HHT, 5-HETE

in

In the absence

of free

(Fig.

increased,

occurs.

is 51.

is

release

calcium

ion

concentrations

complexes

are

natural

of

dependent calcium

in

observed

in the

However,

as the

significant

Optimal

of

absence

the

acid

activity

phospholipase of arachidonic between

0.5

5).

Antigen-antibody function

of DMSO-treated

but

consistent

those

with

(12,13,15,171. not

stimulators

As a measure

control

U937 cells

of normal

macrophaqes,

221

or

quantities

of arachidonic

of 10 uM A23187

or

negligible

Insignificant

no phospholipase

calcium

since

to prostaqlandins,

acid

ion.

or presence

stimulation

acid

shown).

release

absence

2.0 mM (Fig.

not

calcium

assay medium,

occurs

acid

(data

arachidonic

macrophage

fatty

of arachidonic

unesterified

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

of the

to express cells

were

ability

activities tested

and

Vol. 118, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-v Y ‘-PI i / O-o-@-*-e 0.5

1.0

I.5

2.0

[Co Cl21 , mM

%iinA assayed

for

their

Dependence of arachidonic acid release on exogenous ion. U937 cells treated for 4 davs with DMSO were as described in Fig. 2 in the presence (0) or absence for 2 minutes at various concentrations of

response

illustrated

to IgG-anti-IgG

in Figure

complex

formation.

6, OMSO treated

cells,

to human IgG and then

anti-human

release

from

phospholipids

their

endogenous

phospholipase complex

is

IgG alone cells

their

(data

not

DISCUSSIO& subject

activatable acid

necessary

from

In addition, arachidonic pathways

for

not

grown

endogenous

release

arachidonic

acid

(Fig.

when

burst

6).

exposed

of

IgG-anti-IgG

of this

in DMSO - do not lipids

first

of the

the expression

As

when exposed

in a rapid

The formation

does not cause

- those

from

the

activity.

IgG,

activity,

since

In addition, release to

(01

control acid

arachidonic IgG

and

anti-IgG

shown).

Arachidonic

acid

metabolism

of extensive

research

phospholipase,

which

membrane

releases

phospholipids,

macrophages acid

recently

via

in macrophages (8-15,

17).

to metabolize

the cycle-oxygenase

(8-10,12,15,18-20). 222

An

the unsaturated

has been described

are known

has been

(13,

unesterified

and lipoxygenase

fatty 18-201.

BIOCHEMICAL

Vol. 118, No. 1, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IqG-anti-IqG stimulated arachidonic acid release a&t-treated U937 cells were preincubated for one houi a:"!;' with 1 mg/ml human IqG (Sigma) in RPM1 1640 and then washed and resuspended in 50 rr+l Tris-HCl buffer, pH 7.4, containing 100 mM NaCl, 1 mM CaCl and 1 mM glucose. The preincubated cells were assayed in the b resence (01 or absence (01 of 1 mq/ml anti-human IqG (Sigma) for the times indicated as described in Materials ------and Methods. -The present acid

metabolic

been

induced

study

describes

activity,

the appearance

phospholipase,

to differentiate

in U937 cells

by exposure

of a phospholipase

activity

cells

prevent

of endogenous

arachidonic

acid

functional

cycle-oxygenase dependent

ability

release

of normal

and peptide

growth

receptors

of U937 cells this

arachidonic

acid

metabolizing

previously

reported

that

be metabolized

challenge

of these

is

these

products.

have been

with

metabolic

zymosan, 223

(12,13,201, specific

is

in these

by

DMSO or dibutyryl induction

cells.

added

by control opsonized

only

surface

shown to be induced

of the

exogenously

mediated

of arachidonic

medium,

report

to prostaglandins cells

and

While

enzyme while

esterified

macrophages

the first

U937

or lipoxygenase

in conditioned

CAMP (2,4-6,211,

could

in undifferentiated

metabolism

cells.

have

sulfoxide.

to immunoglobulin

and subsequent

in the DMSO cultured

antigens

upon

to respond

a characteristic

present

the

the

by the

acid,

via

activities

For example, signals

metabolism

which

to dimethyl

The absence would

of an arachidonic

It

of an has been

arachidonic

acid

U937 cells, zymosan,

phorbol

Vol. 118, No. 1, 1984 myristate A23I87

BIOCHEMICAL

acetate, failed

acid cells

findings.

(221.

and its

The fact

can respond

line

may serve

arachidonic

PGE2 synthesis The lack

induction that

to activators

and IgG-anti-IgG

complex as a useful

acid

IgG or calcium ionophore

aggregated

to stimulate

arachidonic control

heat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

metabolism

from

endogenous

of phospholipase

activity

by DMSO (Fig.

DMSO-treated

but

2) explains

not control

such as the calcium formation model

suggests for

in

ionophore that

studying

in human macrophage

these

U937 cells A23I87

the U937 cell

the role

of

function.

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224