Osteopontin inhibits nitric oxide production and cytotoxicity by ...

Osteopontin inhibits nitric oxide production by activated RAW264 7 macrophages

and cytotoxicity

.

Ellen

E. Rollo,

Departments Jersey

Abstract:

ofBiological

Osteopontin

macrophage

(OPN),

and

thesis. A human bacteriophage encoded protein

OPN T7-based purified coli carrying

inhibited

NO

11AW264.7 cells plus interferon-’y.

with

abundance shape

an

of of

the

a

and

tPj.coiogy

secreted

nitric

acidic

oxide

(NO)

syn-

by

the the of

cells

Rutgers

Words:

macrophage-like

resistance to Rickettsia pears to map closely

to the

some

5, consistent

of macrophages chemotactic

OPN-dependent

decrease

NO

synthase

in

mRNA.

curve,

with

the The

a maximal

macrophage-mediated

60: 397-404;

with

1996.

cytokines

.

inflammatory

response

mice

tumor

cells

the course and is a polyclonal immunoglobulin B cells this

The protein a thrombin

contains cleavage

several site

binding sequence Gly-Arg-Gly-Asp-Ser facilitates cell attachment and signaling grin. OPN is present in human plasma the

nM

primary

(J. Harris,

personal

function

of OPN

phosphoglycoprotein fluids [see refs.

and

correlation

that

ETA1/OPN

of injection smooth muscle

macrophages

[12],

metastatic

been

OPN

[9]. OPN is also cells [10]. The in MRLIMpJexpress abnorcorrelates with

the

contribute

malignant

to

phenotype

cells secrete at compared with ref.

between

of tumor

also

13 for review]

production cells

least their

[14,

and

of OPN 15].

a

and

Consistent

is the finding that the concentration of patients with disseminated carci-

is considerably in facilitating

[see

noted

capacity

individuals

may

it

transformed protein when

counterparts has

may

mechanism [5, 8]. Subresults in the infiltration

elevated

[16].

Strong

when evidence

development

compared for

with

a direct

of a malignancy

norrole

of

comes

conserved moand the integrin

(GRGDS), which via the av3 inteat about 30 ng/mL,

communication). is not

mal

1-4

site

murine autoimmune disorder. OPN also appears to augment

nomas Osteopontin (OPN) is a ubiquitous found in a variety of tissues and body

thought

severity of the disease [1 1]. Because OPN B cell activator, capable of stimulating (Ig)M and IgG production by mixtures of

with this observation of OPN in the plasma

INTRODUCTION

locus

defense of ETA1

to the vascular

of cancer cells. Many 10-fold more of this

destruction.

Ric

the

a rapid response administration for

infection, and it apon mouse chromo-

tsutsugamushi

subset of proliferating T cells (CD4, CD8) lpr inbred mice with autoimmune disease mally high levels of OPN in a manner that

the

or -1

New

the OPN cDNA has been cloned as gene-i (Etal) on the basis of its activation of T cells [5]. Moreover, alleles of Etal is associated with

macrophages toward NOcells, an action that was synthase inhibitor, NG Inhibition of NO production

inducible

Biol.

for reviews]. tifs, u 1uding

Piscataway,

In this latter context, early T cell activation rapid induction after expression of certain

nontransformed Key

University,

natural killer (NK) cells express OPN in response to activation by various cytokines or inflammatory mediators, suggesting that it may also be involved in host defense reactions [1, 2, 5-7].

constitute cutaneous

dose-response

from

J. Leukoc.

Toxicology,

with lipopolysaccharide also inhibited the cytolytic

effect over a narrow range of OPN concentrations, suggested a complex interaction of OPN with cell surface receptors. Our data support the hypothesis that tumor-cell-derived OPN functions to protect the tumor

T. Denhardt and

cDNA was cloned into vector, pET8C, and from an induced culture the plasmid. Recombinant

production

stimulated OPN

activity of the activated sensitive P815 mastocytoma blocked by the NO monomethyl-L-arginine. correlated

Sciences

and David

found in many tissues and body in increased amounts in response and after malignant transformawe examined the action of OPN on

cytotoxicity

Escherichia

L. Laskin,t

08855

phosphoglycoprotem fluids, is produced to certain infections lion. In this study

OPN

Debra

known,

Although considerable

data suggest that it plays a role in various mineralization processes, both normal and pathological. Fibroblasts, epithelial cells, osteoclasts, macrophages, T lymphocytes, and

Abbreviations: FBS, fetal bovine serum; IFN, interferon; iNOS, inducible nitric oxide synthase; IPTG, isopropyl -D-thingalactopyranoside; LPS, lipopolysaccharide; i-NMMA, NCmonomethyl.L-arginine; OPN, osteopontin; NK, natural killer; Ig, immunoglobulin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium. Correspondence: Dr. David Denhardt, Nelson Biological Laboratories, P0 Box 1059, Rutgers University, Piscataway, NJ 08855. Present address of Ellen E. Rollo: VAMC-Northport, Building 62 (151), 79 Middleville Rd., Northport, NY 11768. Received March 14, 1996; revised June 3, 1996; accepted June 7, 1996.

Journal

of Leukocyte

Biology

Volume

60,

September

1996

397

from the observation that a reduction in OPN expression in transformed cells by means of an anti-OPN antisense RNA reduced their tumorigenicity and their ability to form cobnies in soft agar [17, 18]. In addition, antisense-mediated down-regulation of OPN expression in JB6 mouse epidermal cells inhibited TPA-induced anchorage-independent growth [i9]. The question addressed in this study concerns mechanisms by which tumor cells evade host immune defenses, in particular nitric oxide (NO). NO is a highly reactive free radical

implicated

and the produced

inhibition of cellular proliferation from L-arginine by NO synthase

in

macrophage-mediated

of which

(Type

II, or iNOS)

Western

blotting

human

OPN

and

immunoprecipitation

antibody

LF7

with

confirmed

the

the

identity

bone-derived

ofthe

Western blotting Proteins fate

were

electrophoretically

(SDS)

washed,

blocked

TrisHC1,

pH

The

transferred

polyacrylamide with 7.5,

primary

gels

0.5

antibody

3%

to

gelatin

M NaC1, was

from

sodium

nitrocellulose. and

and

0.1%

Tween

incubated

anti-2arC

(for

dodecyl 20

with

detecting

mouse

systems,

[20-24]. (NOS), one

by various

induction

of

iNOS

in

for

the

targets. metastasis

It is form

(A)

kidney

cytolytic

proximal

concomitant

cytotoxicity

the

NO-sensitive

P815

MATERIALS

kQa 97.4

action

tubule

rOPN

of

facilithe

31.0

epithelial

OPN may protect cytotoxicity by into synthesize NO [26].

of the

(B)

kQa

action of OPN on macrophages and

activated

mastocytoma

AND

or LF7

that

To test this possibility, we analyzed the NO production by activated RAW264.7 the

mM

4

cytokines

Because OPN and suppresses

cells [25], it has been proposed tumor cells from macrophage-mediated hibiting the ability of macrophages

20

antibody.

cytotoxicity

is induced

tumor and

in OPN)

at least

model

were

primary

42.7

toward formation

sul-

Membranes

662

in some

protein

as OPN.

and inflammatory mediators. This enzyme effects the high output of NO by activated macrophages that is responsible, macrophages tates tumor

anti-

induced

cells

97.4

.4 66.2

toward

rOPN

662..

cells.

31.0

METHODS

21.5

Reagents Restriction

endonucleases,

and

BamHI

erly,

MA),

linkers

other

were

Pharmacia

LKB

Mannheim (Indianapolis, (4.00 units/pt was a kind Brunswick,

NJ).

028:B12, MO). by

was The

Dr.

against

and column.

(Arlington

C-terminal

Sodium

Cloning

CA),

portion

in

E.

All

cell

and expression

(no.

generously

was

media

with

Limuiws

of 60-100

provided

was

fused

to

purified

from free

662*

raised

OPN

affinity

were

97

Louis,

antiserum

CJS1)

culture

by assay

at a sensitivity

(St.

of mouse

coli [27]

[5 Crjchromate

IL).

was

IFN-y (New Serotype

Company

Anti-2arC

kQa

Boehringer-

coli,

E.

(C)

(Bev-

WI). Murine Laboratories

from

LF7

MD).

NcoI

and

BioLabs NJ),

Chemical

antibody

Bethesda,

as determined Mesa,

(LPS)

OPN

England

(Madison, Biomedical

Sigma

synthesized

Heights,

endotoxin, Costa

bone

80-amino-acid

-galactosidase an OPN

IN), or Promega gift from Pestka from

(NIH,

enzymes,

New

(Piscataway,

Lipopolysaccharide

Fisher

the

from

Biotechnology

purchased

anti-human L.

DNA-modifying

purchased

rOPN 42.7

on

Amersham

of detectable

amebocyte

lysate

Fig.

(ICN,

1. Synthesis

pET8C.OPN;

of human OPN cDNA

lane

lecular-mass The

900-base

into

the

from using which OPN MD.

17

Dr.

coding

region

promoter-based Studier,

Brookhaven

and

BamHI

linkers.

lacked mRNA

exon

5 encoding

provided

by

recombinant

of human

plasmid

F. W.

NcoI

The

pair

Drs.

OPN

expression

amino

used

acids

OPN-expressing

was

no. 42-55,

and

M. Young,

plasmid

was

coli

BL21(DE3)pLysS.pET8C.OPN,

which

1 mM IPTG for 3 h at 34#{176}C [28]. a large amount of OPN protein

398

Journal

to

of

Leukocyte

was

As illustrated was produced

Biology

grown in lane by the

Volume

NY),

ards;

purification

full-length

NIH,

Bethesda, into

and

the

The

E.

coli

3, 4):

induced

with

protein;

September

side: lane

3, inclusion

6,

1996

fractions cell

on

standards; of the

purified

Confirmation

OPN PAGE

lane lane

protein

was

gel (lanes

LF7 antiserum; lane protein as described

blot

lane

4, inhibition in Materials

purified

gel.

7,

4, inclusion

purified

a Coomassie lane

Western blotting and Methods.

2,

Right: column

OPN

protein.

blue-stained

Western

OPN

body

cut.

hydroxyapatite

of the on

stand-

sulfate

by inhibition standards;

of the

lane

ammonium

analyzed

1, 2) and

1, low-molecular-mass 3, Western

pellet;

identity

pro-

polyacrylamide

2, low-molecular-mass

5, 20-40%

low-molecular-mass (C)

lane

low-mo-

osteopontin

an SDS-12%

preparation

33.

blue-stained cultures: Lane 3, uninduced

5, SDS-PAGE

of recombinant

lysate;

lane

Coomassie

and induced pET8C; lane lane

Purification

body

supernatant;

(A)

pET8C.OPN;

(B)

1, induced

of OPN.

of uninduced 2, induced

4, induced

of selected

lane

SDS-10%

strain

4 of Figure l#{192}, induced culture.

60,

Analysis

fraction

gels lane

standards.

preparation lane

of the

create then

Left

variant,

expression

BL21(DE3)pLysS

(a gift

Upton, a splice

tein.

cloned

pET8C

transfected

isopmpyl--D-thiogalactopyranoside-(IVFG)-inducible

E.

was

Laboratory,

cDNA

L. Fisher

Ia cDNA vector

National The

and

SDS-12% polyacrylamide 1, uninduced pET8C;

pg/mL.

blotting the

protein of the

(lanes

purified using

OPN

anti-OPN

purified

OPN

(for

detecting

human

OPN)

BL21(DE3)pLysS.pET8C [29].

After

washing,

anti-rabbit

IgG

Hercules,

CA).

blot

with

drogen

the

(H+L)

we

erum

To

the

inhibition

preincubated

identity

of an

Western

blotting

as

excess

of

with

1 h,

centrifuged,

recognize

OPN)

Cultures an

of

coli

E.

described

antibody.

in

resuspended

the

in water,

7.0,

and

0.01-0.5

M at

sodium

-0.3

analyzed

dialyzed

blotting.

Fractions

buffer

electrophoresed bands

on

excised,

sodium

containing

acetate/0.1%

shaking.

OPN

remove

was

traces

gels

basis

antibody

of the

to be

lysate

of

the

described

body

su-

OPN

was

sue

buffer,

[32].

culture

OPN

Cytotoxicity

were

bach

pooled, the

in

OPN 10

overnight, 90%

and

LF7

of the

a!.

was

quantified Unless

supplemented

cytotoxicity

assays.

culture

TIB

resistant/NO-sensitive can

Type

medium

GIBCO, every

Preparation analysis

TIB

to

tions

of OPN

cells

were

35-mm a

Western

days

64)

line

were

supplemented MD),

when

P815 in

with

10%

(Amen-

fetal

penicillin,

humid

atmosphere

of 5%

-80%

confluent.

washed

bovine and

and

incubated

blot

cDNAs

probe,

used

the

fragment cells (3000

HindIH of

the

was

Plus ride, was

OH)

membrane and then

probes

of mouse

nitric

oxide

included

OPN

cDNA

synthase

Kit

from

[27],

25 tCi Arlington

cDNA

and

from

for

measured

at 540

nm

flat-bottom

tis-

cytotoxicity

man

LS5000CE

and

-

with

is

occasional

the

of 10%

Triton

in

jiL

medium

measured

wells

cells

was

P815

determined

cells. was

with

1 h, added

8 h) RAW264.7 After

incu-

removed

the

use

Instruments,

calculated

using

containing

determined

for 45

iO’

.

from

of a BeckInc.,

the

following

spontaneous release)/(Maximum % cytolysis, where spontaneous

-

100%

X

target for

(Beckman was

target

labeled

ratio

and

of culture

x

(for This

for RAW264.7

counter

was

X-100

stimulated

or

concentra-

1 h in a sterile

The 2

After with

incubated

and

of3:1.

FBS.

various

medium,

medium,

ratio

release

measured

of radioactivity

and

swirling. in

Cr release

release)

5% medium

for

scintillation

(Experimental spontaneous

IFN-y

in all

flat-bottom

mastocytoma

(E:T)

51

with

of fresh

of [5tCrjchromate

containing

.

used

for 8 h at 37#{176}C.P815

radioactivity

Specific

jiL

U/mL

in fresh

released

CA).

100

DMEM

was

in 96-well

in DMEM

with

resuspended

dish

FBS

plated

of Lors-

red-free

iCi

h at 37#{176}C,50

release

from

dish

washed,

procedure

phenol

were

100

250

petri

16

well,

target

cells

by lysing

target

cells

release

was

mm.

Spontaneous

results

show

only.

re-

Maximum with

20 piL

25-30%

of

release.

analysis

stated

obtained

NcoI

the

as the

96-well

of the

cells/well

refed

to be optimal

Statistical

a -actin

(iNOS)

random primer was labeled with 10 mCi/mL; Amersham Corp.,

isolated After

bridized

blot

(SO

nitrite

otherwise,

from one

at least

of three

the three

or more

separate similar

an

average

experiments

±

SE

of the

or triplicate

values samples

experiments.

RAW264.7

of [a32P]dCTP Heights,

IL)

RESULTS

Pharmacia.

blot analysis

Cincinnati, dehyde.

fragment

Oligolabeling

Northern RNA

Northern

inducible

[31]. Each Ci/mmol,

using

for

i0’

plus

with

of the

each

total

x

resuspended

well

Unless The

was

using

were

supernatant

sodium

heat-inactivated cells

at an effector-to-target

release SO

CO2,

samples

x

incubated which was

medium

the

with

indicated,

5%

were LPS

again,

formula: lease

and

probes for Northern

Ul-

dishes (0.75 Cells, untreated

of culture

by a modification

ng/mL

then

Fullerton,

Dulbecco’s

tg/mL

of radiolabeled

50

(TNFa)[22]

grown

LKB

IFN-’y, were stimulation, and

reader

otherwise

at 6

cells

labeled

were

to each

Type

factor-a

jiL)

reaction

of the

RAW264.7

bacterial

cells

with

(American

necrosis cell

no.

37#{176}C in a

3-4

RAW264.7

tumor

mastocytoma

(DMEM)

at

line

the

Gaithersburg,

streptomycin

subcultured

and

Collection

Eagle’s (FBS,

ig/mL

71)

mouse

Culture

modified serum

cell

no.

(75

Griess

with

dishes

100

bation

monocyte/macrophage

was intensi-

an

culture FBS.

centrifuged,

microplate

[22].

without

native

antibody

tissue without

100 U/mL 1 h before

Aliquots

absorbance

(GIBCO)

cells

Collection

OPN

Band

using

of macrophage-mediated

with

1C).

mouse

the

solution

plates.

et

tissue

anti-OPN

inhibition

solution.

autoradiograms

points,

by the

EL31O

2 h at 37#{176}Cthe

to be OPN

the

The

mM

methanol

SDS

confirmed with as

West-

were

with

a

Cell culture Culture

rehybridized prehybridization

by

M sodium

(Pharmacia).

nitrite

experimentally

The

and the

hybridization

stripped

0.015

that

fresh

blots

M NaCl,

SDS

scanning

time

Measurement

using

and

for

a Bio-Tek

gradient.

Both

was

with

Fractions

with

above

0.15

1.0%

plating.

various

assayed

standard

saturated,

gels,

by incubation

culture

in

autoradiographed. the

of NO production

h after at

7.0. OPN

and

together,

cells were plated into 24-well in phenol red-free DMEM

2

step

temperature

that

preliminary

incubation

generated

by

washed

except with

quantified

usually

does

phosphate

of the by

and

tL)

20%

(PAGE)

solution

band

specifically

replaced

Measurement

OPN

column

in water.

signal

above,

XL densitometer

removed

inclusion

pH

eluted

at room

a single of the

induced

(Fig.

7.4,

were

procedure

containing

7.0)

majority

from

showed

the

electrophoresis

resuspended

ability

blocked

the

blotting

pH

and

of

M sodium

(pH

OPN

precipitated

of SDS,

polyacrylamide on the

the

SDS,

1B,

SDS-polyacrylamide

and

and

mm

30

containing

were

hybridized

to

induced,

not

cut

buffer, the

preparative

minced,

but

from

0.01

gel

7,

for

as described

troScan

ability

of the

a hydroxylapatite

phosphate

by SDS-polyacrylamide

grown,

a series

sulfate

on

sodium

In

OPN

phosphate

M

[30].

against

pH

were

or treated with 100 ng/mL LPS and/or with various dilutions of OPN added

in Figure

saturated,

ammonium

chromatographed

membrane

RAW 264.7 106 cells/well)

shown

40%

the

ties

protein)

its

boiling

blots

probes

coli

K.

of

iNOS

removed

antis-

induced

(depleted

As

precipitate

20-40%

LF7

induced

were

bodies that

would

Thus

hy-

hybridization,

and

probe

in a Western

follows:

by a modification

[29].

inclusion

sulfate

pernatant.

eluted

et al.

0.02%

After

-actin citrate,

washed

human OPN made

we established

ammonium

ern

as primary

preparation

accumulate

experiments

pH

supernatant

in Sambrook

signal

the

BL21(DE3)pLysS.pET8C

body

on the plus

OPN

(containing

the

of recombinant

inclusion

not

and

used

Purification

an

lysate

(Bio-Rad,

visualized

in methanol

solution.

goat

peroxidase

was

coli

antibodies

affinity-purified

to horseradish

BL21(DE3)pLysS.pET8C.OPN for

with

E.

an

coli

anti-E.

incubated material

verify

with

neutralize

4-ch.loro-1-naphthol

performed was

was

conjugated

mg/mL

peroxide.

blot,

blot

to

Immunoreactive

0.5

pretreated

lysate

using 10% added

using and

Tri-Reagent

analyzed

electrophoresis, (DuPont-NEN, 50%

deionized

(Molecular

on

1.5%

agarose

the

RNA

gel

Boston, formamide,

was

MA). 1.0%

Research gels

Center

containing

blotted

dextran sulfate for 30 mm at 42#{176}C.The at a concentration of 5 x iO dpm/mL

formal-

Kinetics cells

of NO synthesis

by activated

RAW264.7

to a GeneScreen

Membranes SDS,

Inc.,

were 1 M sodium

prehy-

RAW264.7

chlo-

periods linear

denatured probe of hybridization

Rollo

et a!.

OPN

inhibits

cells

were

incubated

up to 72 h. NO production over a 6-8 h period, reaching

induction

of iNOS

and

with

IFN-y

was found maximum

macrophage

and/or

LPS

for

to be roughly levels after

cytotoxicity

399

To confirm

24

22

that

OPN

was

responsible

of NO synthesis, the OPN with the anti-2arC antibody nab region of recombinant

.

U)

20 U

4,

Figure 0

16

the ability OPN with

14

action

incubation

of OPN

of OPN to reduce preimmune serum

of OPN,

for the

preparation was prepared against mouse OPN [27].

although

with

the

antibody

NO production. also appeared

to a lesser

inhibition

preincubated the C-termiAs shown in abrogated Incubation to inhibit

extent.

of the

Neither

anti-

0

E

OPN serum nor preimmune NO production. Figure 5 illustrates the

12

C 4)

10

4-’

8

4.’

z

--

10

10.2

100

OPN

and

1O

102

IFN-’y.

cells.

Dose-dependent

Cells

IFN-yplus of

OPN.

action

at 5

x 105/well

100

ng/mL

After

15

on NO production

in a 96-well

LPS h,

of OPN

in the

dish

were

presence

supernatants

ofthe

were

The

at about

(pM)

by RAW

264.7

activated

with

U/mL

indicated

concentrations

collected

and

100

nitrite

with

stimulation, The total or absence reaching

content

determined.

inhibitory

6 h. An

duction 2.

-30

tion

kinetics

action

h of stimulation (100 [33].

with

optimal

units/mL IFN-y and As measured either

or the final

yield

of product,

concentrations

100 ng/mL by the rate

of OPN

was

as early

evident

NOS

inhibitor

L-NMMA

Effects

of OPN

Figure

2

on by

in the preswith LPS

was

first

apparent

in NO

as about

6.5

pro-

h after

with maximum inhibition of 60-65% at 8 h. yield of NO produced by the cells in the presence of OPN appeared to be approximately the same, a plateau of about 70-80 nmol per 5 X 10” cells; of OPN the cultures cultures to reach the

took plateau.

about

of in-

LPS; data not of NO produc-

the two mediators

resulted

effect

production

reduction

OPN

together

0

(pM)

0.1

1

10

yielded the most effective induction, while LPS alone was the least effective. The addition of L-arginine to the cubtures increased NO production, whereas the addition of the pendent inhibition fect of L-NMMA

any

of NO

50%

OPN

had

a 72-h period simultaneously

approximate

however, in the presence twice as long as control

ducers shown)

alone

activated RAW264.7 cells over ence or absence of OPN added

0_

Fig.

serum

s4

OS

1*-

I-&th

in a concentration-de-

of nitrite production. The could be blocked by excess

inhibitory L-arginine.

ef-

on NO production 50

shows

that

picomolar

inhibited

NO

production

with

plus

IFN-’y

LPS

by with

studies among OPN.

is

most

The

stimulated 40

of about

these OPN

conditions. synthesized

4.

a’.

3. Northern

mRNA

levels

stimulated trations

blot as

with (pM)

Northern

LPS

autoradiogram; diographic

and

of OPN

blotting

as bottom

signals

analysis

a function IFN-’y

of the of

10

(pM)

action

OPN in the

presence

for 8 h. RNA

was

described

in

Materials

panel,

normalization

to the

3-actin

signal.

of OPN

on

concentration. then

of the extracted and of the

iNOS

and

OPN

RAW264.7 indicated and

Methods. OPN

and

were concen-

analyzed Top iNOS

by panel,

autora-

production,

data

not

OPN was responsible To establish that ity,

U

‘4. 0

0

E

we

used

These

data

for the inhibition NO was responsible

L-NMMA,

an

ability

confirmed

inhibitor

of NOS, RAW264.7

The

to synthesize suppressed

NO and to kill by L-NMMA with concentration

that

of cytotoxicity. for the cytotoxic-

of activated

production.

L-NMMA

C

shown).

to block

NO

cells

p8i5

mastocytoma a similar dependence

both

cells

was on the

that

have

[33].

4., 4.,

z

DISCUSSION Among

the

been

implicated

received

4. Effect ofanti-OPN

Fig.

production.

RAW264.7

100

ng/mL

The

bars

room

LPS labeled

(0; d

mune OPN

cells

f represent

with

that

anti-2arC

e represent,

respectively,

In preliminary the

production.

effective 0.05

P

100

U/mL

or presence

was

of NO IFN-y

preincubated

for

(c)

anti-2arC

antiserum

the

dilution

2arC

or

1 h at

preparation

was the

effect

t-test).

results IFN-y,

and

NO synthesis

negligible

(3-5%).

The

cells

The ability pronounced

LPS.

OPN itself A substantial

duction

was

observed

together;

and

in

cultures

these

correlated

P815 presence quantities

cells

OPN

with a cultured or absence of nitrite

cytotoxic. in target

by

able 30-35%.

both

cell

the

cytolytic

Inhibition LPS

this

P815

X

10

has

recently in macro-

form

macrophages

of NOS kill

some

is OPN.

and

cytotoxicity

known to be synthesized as by macrophages [i , 5, to facilitate tumor growth

The present in blocking toward

cells,

by

the

studies demonmacrophage NO

NO-sensitive

suppressing

target,

the

increase

in

100

to U)

4) U 0

0

NO pro-

E C

used

4,

action

4.’ .

z

negligible cells). As

observed with NO production (Fig. 4), preincubation of OPN with anti-OPN antiserum for 1 h at room temperature prior to addition to macrophage-P8i5 cocultures, largely reversed the inhibition of cytotoxicity. The data are presented in Figure 7. Neither affinity-purified anti-OPN antiserum nor preimmune serum alone at the concentrations used had any obvious effect on cytotoxicity (or NO

Rollo

Whereas

substance

mastocytoma

NO production. and IFN-y, in the

produced

review].

secreted

production

was with

were

NO

production

by a cytokine-inducible

for

and metastasis [17, 18, 36]. strate that OPN is effective

of cytotoxicity in

X-100, nmol/1.5

and

IFN-’y

NO

and was

or IFN-y

killing

killing,

by a rat colon carcinoma line of a substance that suppressed cytokine-induced NO production by a rat brain microvessel-derived endothelial cell line [35]. We suggest

of cytotoxicity in NO produc-

and

to reduce

34

The

these responses with IFN-y than

LPS

15-20% decrease with or without of Triton (0.2-0.4

LPS

amount increase

to inhibit treated

when was

16 h later.

of either

modest fourfold

of OPN in cells was not increase

assayed

6. In the absence of LPS minimal and cytotoxicity

addition

the cultures induced a ( 15%) and an approximate tion. more

were

are shown in Figure NO production was

is regulated ref.

cell

attention.

OPN is a ubiquitous cytokine by various tumor cells as well 13]. In tumor cells, it appears

The effect of OPN on RAW264.7-mediated cytotoxicity toward P815 mastocytoma cells was next examined. Macrophages were stimulated with LPS and/or IFN-y for 8 h. Target cells were then added at an E:T ratio of 3: 1 and cytotoxicity

tumor

macrophages

tumor cell targets quite effectively by secretion of NO, other tumor cells are able to avoid or escape cytotoxicity [22-24]. The reason for this differential responsiveness is

that

Effect of OPN on macrophage-mediated cytotoxicity toward P81 5 mastocytoma

in

from

unknown but may be due to the release of inhibitors by the tumor cells. Murata et a!. recently reported the secretion

preimmune or preim-

for inhibiting

(Student’s

and

(b) of OPN.

antibodies

experiments

most

inhibition

with

(a, control) OPN

either

alone. NO

stimulated

absence

to determine on

on OPN-mediated

were

h in the

c and

and

serum

calibrated

of

for8

temperature

serum

antibodies

[see

-Q’,

released

considerable

phages q

c

mediators

et al.

OPN

0

10

20

30

40

Time Fig.

5.

with

LPS

were

activated

with

or presence were collected

of 1.0 and

inhibits

Time

course

and

IFN-’y

induction

of NO in the 100 pM the

production

presence

U/mL OPN. nitrite

of iNOS

At the content

60

70

80

cells

stimulated

(hours) by RAW264.7

or absence

IFN-yand

and

50

100

ofOPN.

RAW264.7

ng/mL

LPS,

indicated time determined.

points,

macrophage

in the

cytotoxicity

cells absence

supernatants

401

(A)

production

of NO

and cytotoxicity in RAW264.7 macrophages was in response to very low concentrations of OPN,

Inhibition

observed and ap-

peared >‘

80

ti

70 60

0

50

>% U

that

40

20

iNOS

0 18

14

U

12

‘4

I E

6

4

4..

:

0

LPS

IFN-y

--

-

rOPN

-+

-+

6. OPN-induced

absence the

++

--

100

ofO.1 P815

LPS

mastocytoma Data

and/or

presented

medium

from

the

mulation

of nitrite.

Samples

Cr release

assay.

.-+

-+

killing

synthesis.

A

NO

production

and

types. Consistent of the iNOS

NO-mediated

U/mL ratio

the

were

of3:1.

of

collected

were

or

5tCr-la-

hours

action of OPN was maximal at 6-8 to previous reports of the time course

induction

in macrophages production and by our findings

[22].

That

samples

RAW264.7 inhibition

the inhibi-

cytotoxicity were due to that antibodies to OPN

cells were was observed

seen with the presence of OPN

preof

on NO

transient. Thus, at 6-8 h, by 48 h

of nitrite in the culture supernatants were similar in and OPN-treated cultures. Nevertheless, this inhiwas sufficient to reduce cytotoxicity and NO producby

the

macrophages.

These

data

suggest

that

a

assayed

and

was

our

observation

that

there

is a 10-fold

dif-

are of

for the

accu-

those

taken

after

Of note

and

(B) Samples

immediately

in a 50%

h, of

damage in areas of inflammation. OPN synthesized by tumor cells may function as a defense against macrophagemediated destruction [26].

later

in Materials

experiments.

in (A)

presence with

Sixteen

triplicate

separate

or treated

in the

cultured

as described

mean

three

NO production

untreated

IFN-y were

ef-

with an inhibition gene is the finding

produce OPN. OPN produced by macrophages and fibroblasts may function as an autoregulatory molecule limiting cytotoxic mediator release and thus reducing tissue

and

cells,

OPN,

at an E:T

experiments

for the

++

determined

are from

++

cell

100

human was

of results

-

of tumor

cells,

cytotoxicity

representative culture

ng/mL

++

--

(A) RAW264.7

pM recombinant

percentage

Methods.

inhibition

RAW2#{212}4.7 cells.

for 8 h with

mRNA

transient increase in the local concentration of cytokines like OPN may be important in the regulation of macrophage activity. Appropriately stimulated fibroblasts and macrophages as well as tumor cells have the capacity to

2

beled

gene

tion

by stimulated

block

tory effects of NO OPN is supported

levels control bition

0

Fig.

may

production whereas

10

‘5

of iNOS

abrogated its action. The partial inhibition immune serum (Fig. 4) may result from OPN in normal serum. Kinetic studies revealed that the effects

16 Cl)

2

OPN

that the inhibitory which corresponds

*

10

-ii

IFN-y-induced

to inhibition

fects in a variety of cell by OPN of transcription

30

(B)

to be due

and

similar reduction in iNOS mRNA synthesis by OPN has been described in kidney proximal tubule epithelial cells, where it was shown that the inhibition was at least in part at the level of transcription [25, 40]. These results suggest

‘4

4.,

of LPS

4.. U ‘4

0

iNOS mRNA diators. This

abundance suggests

targets

macrophage-mediated

ing (TGF-)

from the

synthesis has

NO synthesis However, the

of

also

induced that OPN NO.

been

by the inflammatory may protect certain destruction

Transforming

reported

and cytotoxicity in mechanism underlying

0

4.. 5% U

by inhibitgrowth

to inhibit

4.,

metumor

the

factor-3

induction

macrophages the actions

of

[37, 38]. of TGF-

OPN

and OPN are distinct; TGFacts by up-regulating arginase activity, depleting the cells of the substrate for the iNOS enzyme [37, 38]. Taurine chloramine has also been reported to abrogate the induction of iNOS mRNA in RAW 264.7 cells by LPS and IFN-?, possibly by interfering with the synthesis of a protein scription of the iNOS gene

402

Journal

of

Leukocyte

required

for

the

increased

tran-

Preimmune Fig.

7.

anti-OPN

Volume

60,

September

1996

Reconstitution antibodies

P

0.05

-

+

-

+

-

-

-

+

+

-

-

-

-

-

-

+

+

of cytotoxicity before

carried out as described OPN was preincubated antiserum.

[39].

Biology

Anti-OPN

addition

by to cultures.

preincubation Cytotoxicity

in Materials and Methods for 1 h with either preimmune (Student’s

1-test).

except serum

+

of OPN

with

assays

were

that 0.1 pM or anti-OPN

ference in the concentration of OPN required to inhibit NO production and cytotoxicity in RAW264.7 cells. Thus, whereas cytotoxicity was blocked by 0.1 pM OPN, i pM was required to maximally inhibit production of NO by RAW264.7

cells.

cytotoxicity P815 cells.

assays In this

The

reduced

requirement

may be due regard, Feng

for

to production et a!. [36]

OPN

in

of OPN by have reported

that OPN expression by tumor cells is inversely correlated with resistance to macrophage-mediated killing and tumor cell survival. These data, together with the present findings, provide evidence to support the hypothesis that OPN can function to protect struction by macrophages. tects

tumor

stimulating ing focal [Lopez

cell

targets

cells from nonspecific process by which OPN

is unknown.

the phosphorylation adhesion kinase and and

Denhardt,

that OPN may ing to inhibition inhibit

tumor The

unpublished

NO-induced

OPN

Further

studies

depro-

is capable

results].

apoptosis the

P815

cells

[41].

with

necessary

some

aspect

to explore

of this these

research

Institutes Cancer

was

and

Oxide

Production

by Recombinant

tial fulfillment gers University,

11.

regulated

supported the the

by grants Nicholas Bureau

of

the

C. Palczuk Biological

S. Rittling

and

This report E. E. Rolbo and Human

H.

Passmore

is taken entitled

for a critique from

the

Inhibition

of

Macrophage-Mediated

and

Osteopontin

of the requirements New Brunswick,

for

presented a Ph.D.

19.

20.

21.

Nitric 23.

in parfrom

24.

Rut-

NJ. 25.

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