Transforming growth factor-1 stimulates degranulation ... - CiteSeerX

Transforming

growth

oxidant

factor-1

stimulates

release

by adherent

human

J. Balazovich, J. Suchard,

Rosemarie and Laurence

Kenneth Suzanne Department

Abstract:

The

of Pediatrics,

signal

University

transduction

by cytokines and growth their receptors on human neutrophils understood. When PMN in suspension of these primed

agonists they for subsequent that

ported

when

stimulated

tide

are

PMN

with

of Michigan

pathways

activated

cytokines

plated or with

that

but and

forming

onto librinogen the chemotactic

by releasing hydrogen granule component factor43i

growth

cells

including

PMN.

It has

stimulates chemotaxis oxide production by cells sized when

that UF-31 they were

mediated determined

by

would adherent

Ii

activate to

by PMN

adherent

In

‘RF-

this study, H202 and

to fibrinogen.

IXW-

and

rin

release.

lactoferrin

release

phospholipase

D pathway.

In

3H]phosphatidylcholine,

we

treatment caused Propranolol (150 phosphohydrolase, rin

release,

phatidic

that

suggesting

acid

to

PMN

labeled

observed

the

cliradylglycerol

with that

by

lyso-

1XF-31

Riot.

60:

mechanisms.

one

specific

members

generate

proteins,

This

heat,

latent

growth,

In

opposing

a reviewj,

apparent

paradoxes

TGF43i kocyte

are or released

adhesion,

infiltration,

synthesis

as

fibronectin

13i

administered systemically has the expression of endothelial

iting

and

decreasing

tion

[4].

blocks

In

vitro

PMN

[1-

ester PMN

expression

adhesion

Thus

PMN

step

these results activation of

conditions.

activation

lipase

D

772

Journal

.

peroxide

.

a common pathway and cytokine ac1996.

772-777;

phosphatidic

kocyte;

H202,

KRPG,

Krebs-Ringer

tidic

acid;

TNF-a,

acid

.

Biology

Volume

60,

leu-

regulating

for integrins other hand,

such TGF.

opposite effect, inhibcell adhesion proteins

in response

to phorbol

selectively

promote upon the

December

transforming

role

growth PMN,

hydrogen

peroxide;

KRP,

phosphate

lyso-[3HPC, tumor

or

inhibit

specific of

en-

TGF-1

in

buffer

factor-1i;

fMLP,

polymorphonuclear

Krebs-Ringer

phosphate

with

I3HIPA,

glucose;

N-

leubuffer;

phospha-

1O3HJoctadecyl-sn-glycero-3-phosphochohne;

necrosis

factor

a;

1996

Laurence

of Pediatrics, Dr.,

Received

phosp/io. 30,

Leukocyte

by

ELISA,

enzyme-linked

immunosorbent

assay.

secretion

of

in vivo

to promote

to sites of inflammaPMN chemotaxis but

Presently,

TGF431,

Abbreviations:

Center

hydrogen

2

is unclear.

Correspondence:

Words:

ref.

example,

depending

formyl-methionyl.leucyl-phenylalanine;

ment

Key

may

[see

these

activation

to fibninogen

responses

vironmental

For

recruitment promotes

TGF431

functional

cells

of receptors On the

leukocyte TGF-31

matrix

promotes

underlying

is said

the

by

reportedly

TGF-

[31.

laminin

other

activated

TGF-f3

same

known.

the

and

be

to

two

extracellular

the

and

a large

dimenized

with

active

locally

and

expressed as

and

mechanisms

not

applied and

in

the

are

can

cases,

is

related

synthesized

Active

activities

although

understood

and

some

membranes (TGF43i)

complex

treatment.

These

factor-31

complexes

differentiation,

synthesis.

PMN

altered

that

cells

1J.

of poorly

of which is

TGF43i

or enzyme

seemingly

three

TGF41

peptide

many

of structurally

is proteolytically

a 25-kDa

protein

growth

tissues. that

on

a variety

of a group

similar

by (PMN)

receptors

through

mammalian

released

are

leukocytes

Transforming

of five

for

factors

of phos-

important

Overall, for fMLP

PMN and suggest that in both chemoattractant

J. Leukoc.

conversion is an

PMN activation by 1XF-31. are similar to those reported

tivation.

detected

an increase in I3Hlphosphatidate. .tM), an inhibitor of phosphatidate blocked both H202 and lactofer-

in

adherent is involved

were

‘ontinued for at least 180 min. Adhesion of PMN paralleled H202 and lactoferEthanol (200 mM) blocked both H202 and release, suggesting the involvement of the

to them

regulates

Both

lactoferrin

bind activate

acid,

H202 and lactoferrin release from adin a concentration-dependent manner, seen in the range of O.i to 100 pg/mL.

H202

agonists and

growth

polymorphonuclear

proteins.

we lac-

Arbor

and

including

precursor

stimulated herent PMN with effects 60 min ant and spreading

Cytokines

in

that

Ann

INTRODUCTION

functionally

PMN to release H202 fibrinogen, a response

132 integrm receptors. whether ‘JXF-31 stimulated

release

toferrin

reported

Hinkovska-Galcheva,

of Medicine,

(H202) Trans..

but not exocytosis or superin suspension. We hypothe-

and

neutrophils

Vania

(fMLP)

by many

is released

been

are reand pep-

peroxide lactoferrin.

(1F431)

1i

are

rather others

N-formyl-methionyl-leucyl-phenylalanine

they respond and the specific

School

factors binding to (PMN) are poorly encounter many

not activated, activation. We

are

Fernandez, A. Boxer

degranulation

1996.

Room April

Section L-2110, 2,

1996;

A. Boxer,

University

of Michigan,

of Hematology/Oncology, Ann revised

Arbor,

MI

August

1500

DepartB.

Medical

48109-0238. 29,

1996;

accepted

August

Human

peripheral

blood

fense lenges

against bacterial primarily by

such

as

superoxide

and

ent

to plastic,

strates

and

It

is

For

PMN

versus as

the

that

lag period

suspended

PMN

agonists

that

suspension

is

monophasic

the

second

corresponding

nived

to

from

the

TGF-Ii

of

promotes

PMN

and

lactofernin

also

when

release

were

we

able

and

to activate

oxidants

if the

to release

[9, i6J. investigated

stimulate

lactoferrin

and

were

have

TGF-1-dependent to involve phosphatidate

duced

by the

activation

In

a

to extracelof

by

PMN

activation and

(3

nm

activated PMN release of H2O2 manner. FurPMN pro-

manufacturer acrylamide choline Amersham.

and

human

found

platelets.

to be

gel electrophoresis.

factor. used

assessed

was

by sodium

dodecyl

by the

sutfate-poly-

l-O-(3Hjoctadecyl-sn.glycero-3-phospho-

(lyso-platelet-activating Reagents

> 95%

Its purity

for

lipid

161

Ci/mmol)

analysis

were

was

obtained

from

by

myeloperoxiin a final

were

warmed

to 37#{176}C.Plates to

The and

by

H2O2

an

emission

for

substituted

remove

cells

in relative was

measured wavelength

spectrometer.

specific

of scopoletin

con-

indicated

reduction

fluorescence the

con-

peroxidase,

centrifuged

nm

glucose

for

generating

Omis-

decrease

in fluores-

H2O2.

Control

agonists

and

samples inhibitors.

a standard

curve

using

of H2O2.

evaluated

for

the

been

samples

diluted

before

optical

densities

also

calculated

from to

the

the

examined

for release

developed

elsewhere

within

for

Lactoferrin

ELISA

addition

fell

were

reported

was their

(ELISA5)

granules.

sensitive

have

in

release

of specific

highly

of which

assays

H2O2

a component using

Adhesion

by (8J.

Birgens

The

lacto-

a standard

assay

linear

in

curve.

order

portion

to

of the

enstan-

assay

Adhesion

assays

were

Measurement plated onto The

conducted

as

previously

of phosphatidate fibrinogen

measurements

of phosphatidic

161.

described

PMN

factor,

lyso-[3HPC)

trifuged

at 300

at

x

1

KRPG.

PMN

mM

acid

10

Veldoven

described

in PMN

were

out

carried

were

wells

and

response.

(18,

p1

Balazovich

the

KRPG,

with

200

mM

at 37#{176}Cbefore during

the

cells

were

stimuli

M

for

For

to

this

i08

were

medium

were

et al.

TGF-31

and

onto time

course iden-

At designated

times.

x

the

modification

of the

the added

with and

neutrophil

medium to each

a Teflon

combined

of

in an (8

was

or

fibrinogen.

determination

removed pelleted

cen-

ethanol

(0.64%)

entire

were

resuspended

each

procedure,

methanol

PMN

incubated

or inhibitors.

according

PMN

as

with

and

plating

present

of ice-cold

Adherent with

with

combined

191.

labeling,

twice

treatment)

without were

extracted

Bell

250

mm

(no

in buffer eight

at 37#{176}C.After

washed

were

Control

essentially

incubated

(lyso-platelet-activating

mm

5

I#{149}

(6.

formation

cells/mL

preincubated for

propranolol

and and

30

mm,

were

propranolol or

removed

for g for 6

cells/sample)

the

cells/well)

curve.

moved

high-performance

10

PMN

warmed

NJ) PMN

inhibitors

plate,

was

abolished

by

ig/mL).

H2O2

of scopoletin

was

(50

air at 37#{176}C.At the

LS-50B

their

from

from

were

of 365

were

manner

isolated

to the

wells

accomplished

concentration

cells

was

of

and/or

to cuvettes.

oxidation

KRPG

were

details

that

the

Falcon

5 mM

x

(1

agonists

immunosorbent

determined the

in

specificity

but

release,

tical MA).

the

that

Reagents (Bedford,

as

Park,

of horseradish

in humidified

peroxidase

was

experiment.

Products

viable

described

containing

5 tg

plates

wavelength

concentrations

Ethanol

Biomedical

greater

98%

Lincoln

wells

addition

and

to the

PMN

buffer

destruction

their

in a Perkin-Elmer

the Collaborative

were

resid-

polystyrene

1-O-(3Hloctadecyl-sn-glycero-3.phosphocholine

D.

of

as previously

Dickinson,

indicated

transferred

indicating

dard

on

sedi-

lysis

than

of fibrinogen

prevent the

solution

excitation

150

from

from

of dextran

Preparations greater

assayed

(Becton

incubated

ofhorseradish

sure

in

obtained

were

due

an

was

use

flat-bottomed

scopoletin,

temperature the

M ETHODS

TGF-1,

(to

supernatant

Samples

to

plated

of adherent diradylglycerol

of phospholipase

the

ferrin

adherent ability

intervals,

and

that

exocytosis

the

time

(17J.

reported

release

PMN

sup-

to undergo

fibninogen. We demonstrated that TGF-1 adherent to fibninogen, stimulating the and lactofernin in a dose- and time-dependent thermore, appeared

[15].

tM

Before

taming

was

phosphate

24

of 1 mL.

lactoferrin

to fibrinogen,

PMN

to fibrinogen-coated

and

Samples

H2O2

by

hypotonic

f6J. and

concentrations

Enzyme-linked lactoferrin

hypothesized

receptors PMN

cells

lular matrix proteins In this study we H2O2

we

blood

in 24-well, plates

azide

to room

known

promote

[141,

others

obtained

otherwise.

exclusion.

adherent

catalase),

contained

TGF-31

to

blue

and

slowly

of 473

or induce

PMN

volume

using

de-

staining

added

Q uantitation

since

ability

adherent

.32-integrin

hypothesis,

was only

activate

they

by

burst

its

were

stated

and

described

conducted

sodium and

cence,

and

whole

as previously

in Krebs-Ringer were KRPG

sion

from

culture

fluorescence

formation

However,

through

and

D [6J.

respiratory

to fibronectin

would

mediated

TNF-a and

chemotaxis i

of our

the

suspension.

binding

the

port

in

reagents

unless

centrifugation,

saturating

suspended

1 mM

fibninogen are is biphasic,

of phospholipase

with

dase

[10-131.

release

coated

taming

of

D

diradyiglycerol

lactoferrin

other

MO)

by differential

by were

tissue

(KRPG)

For excells in

C

plated on formation

of

and

to activate PMN

integrin

that

response

phase H2O2

activation

fails

exocytosis PMN

when PMN diradylglycerol

91.

isolated

trypan

released Assays

Primaria

activation

phospholipase

hand, fMLP,

All

Louis,

assay

H2O2

factor

from

PMN by

H202

in suspension,

phospholipase

98%

[6J.

[8,

were

determined

are activators of adactivated by fMLP are

results

phosphatidylinositol-specific

only

than

of H2O2

adherent PMN. in fMLP-treated

and

(St.

Ficoll/Hypaque

erythrocytes

ual

PMN

colony-stimulating

a (TNF-a), pathways

grade. Co.

PMN

mentation,

in suspen-

fMLP

PMN

phosphatidylcholine-specific other with

with

prime

Chemical

Human

sub-

adherent

detection

activated

only

chromatography

Sigma

Cells

adherother

PMN

before

also different in suspended versus ample, diradylglycerol formation

with

and

than

liquid

cytoplas-

become

proteins,

granulocyte-macrophage

On the treated

have

differently

and tumor necrosis factor herent cells. The signaling

both

of de-

is significantly longer, and the total amounts lactoferrin released are greater in adherent

Furthermore, such

line

to host chalmetabolites

secreting

documented

quite

example,

and lactoferrin of H2O2 and

by

they

matrix

well

to agonists

sion.

and

after

extracellular

[6-9].

respond

a first

respond oxygen

that contain proteolytic enzymes. to release these compounds when

in suspension

are

provide PMN toxic

H2O2,

mic granule contents PMN can be stimulated they

PMN

infection. releasing

was well

scraper. with

activation

l0 Van

the

re-

to stop PMN meth-

773

anol

extract

from

chloroform (1:2:0.8,

v/v),

were to

corresponding

and

water vortexed,

centrifuged

a clean

water

the

and

and

centrifuged

and

washed

centrifugation,

an

the

under acyl

a stream

raphy

plates

group

in a solvent

by

treatment

with

corresponding lipid

The

areas

and

assayed

by

Statistical

dried

0 Ii

20#{176}Cto mm-

-

chromatogand

(12:2:3:

D fl9j.

with

primulin

were

identified

and

0

separated

0

of ethyl

10,

v/v).

10

1

1000

100

LOG [TGFJI,]

Unla-

from

Thin-layer areas

under

acid

scintillation

at

After

and

0

pg/mi

dioleoylphosphatidylcholine

has

of the

a UV

chro-

Fig.

2.

plate

plated were

on fibrinogen-coated added to wells (10

with

fibrinogen

lamp.

a slightly

In

faster

mo-

dioleoylphosphatidylcholine.

to phosphatidate

liquid

removed

(9:1).

of a mixture

acid:water

produced

was

to thin-layer

phase

l-O-alkyl-phosphatidic

corresponding

were

scraped

from

the

plates

counting.

Dose-response

bated

for

H2O2

release.

curve

for

stimulation

plastic cells each)

containing

varying

Data

are

samples

means

release

±

PMN

three

also

Fig.

PMN coated

43

of

withdrawn

from

SE

by

with TGF431. 24-well plate

concentrations

mm at 37#{176}C, and

120

of H2O2

and stimulated of a Primaria

and

incu-

.

assayed

experiments

for

done

in

triplicate.

analysis

Statistical

significance

was

between

means

differences are

reported

RESU

upper

final

vortexed

tube

0

phases, the

were

phase

stored

w

that

as a standard,

from

sprayed

standards

lower

were

-J

two

so

Samples

applied

-J

supernatant

To obtain

I phospholipase

type

were

system.

than

Data

were

to phosphatidate

solvent

bility

Samples

generated

cabbage

plates

1 h. Samples

the

to a clean

phosphatidate

was

and

spin.

The

of

transferred

supernatant

transferred

of the

for

5

with ratio

1 M NaCl:methanol

pentane:acetic

matography this

was

consisting

phosphatidate

first

mm.

Lipids

unlabeled

mixed

chloroform:methanol:

10:10:9.

of

of nitrogen.

system

the

10

phase

acetate:2,2,4-trimethyl

beled

above.

volume

migration.

with

described to the

g for

lower

was

supernatant

with

was

equal

the

re-extracted

added

at 2,000 with

sample

temperature

g and

from

were

of chloroform:methanol:water

imize

as

supernatant

water

ratio

down

was

centrifuged

with

chloroform

at room

at 2.000

pellet

Each

a chloroform:methanol:water

kept

10 mm

The

(1:2:0.8),

wells.

obtain

and

for

tube.

combined

to

determined

by

were

as the

mean

use

of

considered values

the

Student’s

significant

±

SE

unless

1-test;

if P