Interstitial Fibroblasts - JASN

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Although an excessive amount of fibroblasts is present in renal fibrosis. (2), little is known about the regulation of fibroblast numbers in the kidney. (3). Changes.
Cytokines

and

Fas

Interstitial

Fibroblasts ALBERTO RAIMUNDO

Regulate

ORTIZ,* CORINA GARCIA DEL de NefrologIa,

de Medicina,

Renal

Abstract.

ber of matrix. limit

flbroblasts Apoptotic cell

the

in the

regulation

observed

that

Granada,

by an increased

num-

an

of

insufficient

rate

of

accumulation. However, of renal fibroblast survival.

interaction of

of cytokines

apoptosis

of

and

renal

death

Fas

the

to

and

sensitivity

Fas

apoptosis

renal

fibroblasts

and

express

cell-surface

strate

a

Fas

role

for

Fas

ligand

ligand. Tumor necrosis factor a (TNFa) and agonistic anti-Fas antibodies induce apoptosis of renal fibroblasts in a time- and

cytokines

cooperate

to

dose-dependent renal fibroblasts.

manner. Serum contains Both serum deprivation

blasts

In chronic

diseases,

tion

renal

and

fibrosis

function amount

(reviewed of fibroblasts

known (3).

about

in cell

the processes mediate

ance

that

cell

ptosis

tend

of the

result

Both

most

from

the decrease

leading

an

those

(2-4).

Cell

mechanisms

and death of cell

(5-7).

in fibrobbast

number (7).

during In this

may

in

monocytes

blasts

that

may

regulate

help

death

researchers

and

the survival

understand

the

wound

progression of renal fibrosis and may design of new therapeutic strategies. Apoptosis

program death

involves

(8-b program.

I). Survival Certain

the

activation

factors cytokines

provide

of renal

prevent may

an

is followed

fibro-

persistence

and

the basis

for the

type.

and

death

the activation

of the

the

Received July 9. 1996. Accepted May 30. 1997. Correspondence to Dr. Alberto Ortiz, Laboratorio de NefrobogIa. Jim#{233}nez DIaz, Avda Reyes Cat#{243}licos2. 28040 Madrid. Spain. 1046-6673/08() 12- 1845$03.0O/0 Journal of the American Society of Nephrology Copyright 0 1997 by the American Society of Nephrology

death

pro-

when

( 1 9,20).

does

kill

of

lation

of

apoptosis

of

interstitial renal

of

not

if

death,

cells

of

(16,17).

( 1 8) and

may

subpopulafactors

fibroblasts

cultured

only

is preserved. in apoptosis

lymphocyte

protective renal

disease

lymphocytes

endothelial

of

macrophages

survival,

intracellular

susceptibility

the

by apoptosis

blood

or proliferation

levels The

by

(9,12,13),

in chronic

promotes

renal

include

ligand

of cell death does not result

peripheral

not

These

Fas

mediated apoptosis is poorly understood. plored the role of cytokines and the Fas

are to

high

receptor-

We have now exreceptor in the regu-

murine

renal

interstitial

fibroblasts.

Cells, TFB Eric

and

Methods

Cytokines, murine been

cells

have

blast

specific

(FCS), tomycin,

deprived

The antibodies

and renal

G. Neilson

(GIBCO, Fundaci#{243}n

TNFa

human

fibro-

for

mononuclear

activation

other

apoptosis.

target

mainly

the

Receptor

in activation

tions

a

intracellular

trigger

secreted

compose

of

and

in renal

receptors.

fibrosis

cell

regulation

Fas

1997)

and

accompanies

Materials of

are

that

engagement

result

relative failure in the clearance of renal fibroblasts may result in fibroblast accumulation during renal fibrosis. Knowledge of the factors

kidney,

specific

(TNFa)

mRNA

not demon-

fibroblast

1845-1854,

which

every

Fas

cell

regard,

a

in Fas Murine

ligand

that

therapeutic

8:

of

factor

in the

apoclear-

Apoptotic

activation

ftis

pathway

the integrity of the effector program Thus activation of lethal receptors

trans-

by

Nephrol

T lymphocytes

(14,15).

that

fibroblasts

and

infiltration

between

Soc

increase

autocrine

renal

a new

necrosis

which,

regulate

represent

through

tumor

excessive (2), little is

and

into

gram

the

fac-

deprivation

could

suggest

death-signaling

(J Am

both

in

data

of flis

growth

deprivation.

the authors

of the Fas

could

fibrosis.

of renal

proliferation cells

of injury

Modulation

in the kidney

number

conditions

to resolution

Fas

inflamma-

imbalances

cell

important

the

prognosis

numbers

fibroblast

pathophysiologicab

mediates

the

I ). Although in renal fibrosis

to increase

and

survival factors for and TNFa increase

of fibroblast

number

clearance.

is one

healing,

reference is present

Fas

of interstitial

with

of tubular epithelial to increase their number

under

death

in

degree well

the regulation

Changes

differentiation contribute

the

correlates

express

but

expression

the

serum

constitutiveby

These

fibroblasts

Faeultad

serum

prevents by

ligand,

the

by both

partially

induced

survival.

renal

and

insulin-like

induced

and

expression

and

By contrast,

fibroblast

murine

EGIDO*

Spain;

death

Fas receptor.

activation

receptor

have

to Fas-induced

and

tor- 1 decreases and

may

receptor

fibroblasts

Aut#{243}noina, Madrid,

Spain.

little is known The authors

the

Universidad

mRNA

extraceblular mechanism

Renal

SILVIA GONZALEZCUADRADO,* FRANCISCO O’VALLE,t and JESUS

de Granada,

and excessive deposition cell death is a physiological

to fibroblast regulation

studied

Universidad

and

LORZ,* MORAL,t

in Murine

Fundaci#{243}n Jirn#{233}nezDIaz,

is characterized

numbers,

contribute about the have

fibrosis

Apoptosis

Grand

fibrobbasts

(University

of Pennsylvania,

extensively

characterized, (2,21 ). They

protein

2 mM

Antibodies

interstitial

FSP-1

Island,

glutamine,

NY), 100

10%

were a generous Philadelphia.

penicillin,

fetal

and

37#{176}C in 5% CO2 (2,21). In some of serum (0% FCS) for 24 to 72 h. at

io-2

(a hamster (22).

control

IgG) hamster

clone IgG,

from These

and they express the fibrowere cultured in RPMI 1640

decomplemented

U/mI

gift PA).

studies,

of anti-murine and

calf

1(X) g/ml

FITC-rabbit

cells

serum

strepwere

Fas monocbonal anti-hamster

Journal

1846

of the American

IgG were obtained is

an agonistic Recombinant

rived

growth

(mTNFa

from Pharmingen

antibody

(22).

that

factor-BB

raised

against

peptide

were

CA).

The

Polycbonab

a peptide

at the

purchased

from

(24).

(Frankfurt,

Germany),

chem

Diego.

(San

Santa

Cruz

196 through

220

rat

1gM

CA).

Soluble

(LXR

and

Fas

(23),

binds

was

and streptavidin

Dr. Paul Fitzpatrick

ligand

from

TNFa

tion

19

control

(Santa

Cruz,

ligand

(Laufelfingen, 1gM

from

were

from

1K

Calbio-

a kind

Richmond,

DNA.

gift

from

Fas

product

in total

blot

Both

been

of

Cell

Dear/i

with

mixed

with

spun

ethylenediaminetetraacetate

cells

resuspended A,

incubated

and

present

in

0.05%

(EDTA)

in the supernatant

100

g/ml

NP-40

at 4#{176}C br

(26).

propidium

1 h. and

analyzed

Cells

iodide,

in phosphate-buffered

and were

10 j.g/ml

saline

on a flow

(PBS),

cytometer

(EPICS-

ent

cells

albumin

counted.

For the morphological assessment of apoptosis. 8-well Labtek glass slides (Nunc Inc. Napersville. anti-Fas

antibody. with

or control

10%

buffered

stained with I j.tg/ml propidium pH 7.2 for 30 mm at 37#{176}C. After

were

mounted

med

in a fluorescence

Cells

using

exposed

described

were

then

washed

washed

with

PBS,

solution

in PBS and then exam-

(26).

in 0.2%

presence

obtained

stimuli

of DNA

by lysing

were

fixed

for electron

in glutaraldehyde/para-

microscopy

1% SDS.

2(X) g/mL

was extracted,

RNAse DNA

was and (28).

then

was

as previously

K in PBS,

precipitated.

resuspended,

at 37#{176}C, and

transferred

hybridized

in genomic

pH 7.2, treated

separated

to Genescreen at low

studied

DNA

10 mM Tris, 25 mM EDTA,

proteinase

A for 30 mm

branes DNA

fragmentation in 100 mM NaC1,

cells

in PBS

sample

g/ml

ciiid Northern RNA

method

gels

containing

RNA Diego.

was

was (29).

for

18h at 37#{176}C.

with

in a 1 .5% (NEN.

stringency

with

10 .tg/mL agarose

Boston,

MA)

radiolabeled

gel. memmurine

2.3%

isolated

CA).

using

RNA

Hybridization

isolated Total

was

donated

SJL

mice,

identities.

of Fas

and

but

is a cloned

PCR murine

detects

a fas

T cells

by Northern

by Eric

their

ligand

G. Neilson

Fas

and

Ligand

using RNA

(25

the

right-angle

acid-guanidine-phenol-chboro-

formaldehyde.

In some

the FASTRACK transferred

separated

to nylon

mRNA

in I % agarose

experiments,

poly-A

kit (Invitrogen,

membranes

San

(Genescreen)

and prehybridized were stripped and

and hybridized as previously described (30). Blots subsequently rehybridized with a GAPDH probe to

account for small quantified using

loading or transfer variations. Autoradiograms were a Molecular Dynamics densitometer (Sunnyvale.

Fas

washed

EDTA,

cell

suspensions

for 30 mm

At

and data

were

by

selective

least

10,000

displayed

for

on

on a logarithmic

II software.

Mean

cell

fluorescence

subtracted

from

that

obtained

LYSIS

the control Ab.

IgG

Data

are

FCS

was

expressed

cell

scale

bated

using

Mean

as fold-increase

and

for

each

of increasing

fluorescence

over

were

anterior

collected

intensity

1

at 4#{176}C. Cells and debris

based

green-fluorescence

the or

by incubation

cells

were

from

antibody

30 mm

gating events

(30).

cells

anti-Fas

Dead

serum

resuspended

at 4#{176}C, followed IgG

adher-

bovine

l0

X

of control PBS,

were

5

1 .tg/ml

on a cytofluorograph.

analysis

with 0.2%

expression,

with

IgG

scatter.

was

calcu-

obtained with

control

the

with anti-Fas

cells

grown

in

RPMI.

To study intracellular Fas ligand expression, 5 X iO cells were fixed in 2% glutarabdehyde/PBS, washed in PBS, and permeabilized in 0. 1% Triton X- 100, 2% BSA, I % goat serum, in PBS for 30 mm at room

temperature.

polyclonal

Thereafter,

anti-Fas

in PBS

for

60

anti-rabbit

analyzed

60

had

with

X-l00,

with

samples been

or with

I : 100

were

BSA,

FITC-goat-

stained

previously

nonimmune

1 g/ml

0.2%

with

incubated

rabbit

IgG.

an

with

a

Cells

were

cells

were

on a cytofluorograph.

cell-surface with

rat 1gM

Fas ligand

1 g/ml in 5%

FCS,

0.2% 1:100

followed

by incubation

with

by incubation

with

were

then

analyzed

some

cells

were

which

inhibits

ligand

from

on

in PBS

biotin-anti-rat

and

membrane. surface

thus

X

iO

ligand

antibody for

30 mm

phycoerythrin. For

presence

decreases increasing

or

for 30 mm at 4#{176}C, 1gM

streptavidin

3 h in the

metalboproteases

at the cell

BSA,

1 :50

for

5

Al 1 anti-Fas

a cytofluorograph.

cultured

the cell

expression,

monoclonab

4#{176}C and

cytokine

incubated Triton

incubation

Control

that

peptide

were

in 0.1%

by

mm.

antibody

Fas ligand

cells

antibody

followed

for

ligand

control

ligand

mm,

IgG

anti-Fas

control

gig) was

mM

single

in the presence

was

FITC-rabbit-anti-hamster from

sample,

and

2.2

incubated

analyzed

incubated Total

Jurkat

with

hamster

To study

form

stimulated

the culture

To study

were

control

excluded

then

RT-PCR

to an ap-

839 of the published

cells were cultured

BSAIPBS.

same

10%

(27).

The

DNA

They

iodide, 100 p.g/ml RNAse A, in PBS being washed with PBS, preparations

microscope

and processed

IgG.

cells were grown in IL) and stimulated

formalin,

a 90% glycerol

to lethal

formaldehyde

from

probe

readily

kindly

After

(BSA)

1:100

was

fixed

liver

probe

Analysis

detached

then

PBS.

were

were

were

TNFa.

and

353 through This

to the

It hybridizes

ligand

It

of amplifica-

corresponding

(30,32).

Fas

to confirm

or cytokines.

with

with

The

from

For cytofluorography, medium

fragmented

with

RNA probes

product

Fas

in thymus

(3 1 ,33).

Cytomerrv

XL MCL, Coulter, Luton, United Kingdom). The percentage of cells with decreased DNA staining (A0), comprising apoptotic cells with nuclei,

the possibility

PCR

TCC

Expression

mM

detached

and

RNAse

2.2

excludes

mice.

sequenced

CTG

CA) (25).

Cells were cultured in 24-well plates in the presence or absence of stimuli or FCS. For flow cytometry. cells attached to the plate were collected

its identity.

thus

bases

eDNA

(34).

to confirm

transcript lpr/lpr

transcript have

AG. The resulting

sequenced

of murine

that comprises

ligand

TAT

is a cloned

2-kb MRL

Flow Studies

probe

AGT

and

and

domain

from

Fas

cloned

of genomic

proximately not

3’ GC CCA

was

exons

intracellular

the

CCG,

several

antibodies,

phycoerythrin receptors

product

The

biotin-anti-rat

Biotechnology,

CTA

PCR

Mannheim

to the Fas Alexis

TGG expands

2 through and

Biotechnology

Al I that

control

human

acids

ligand

receptor

1 ), platelet-de-

and

to amino

of Fas

Fas

Boehringer

anti-Fas

monocbonal

peptide

Switzerland)

murine

rabbit

terminus

the

1 (IGF-

from

corresponding

amino

rat 1gM

extracellular

and

purchased

CA). The io-2 clone

activates

factor-

(PDGF-BB).

Germany).

mapping

to and

growth

were

of Nephrology

(San Diego,

binds

insulin-like

and hTNFa)

(Mannheim,

Society

these

experiments,

of 5 mM the

at

Cells

release

EDTA, of

the availability

Fas

of this

(24).

CA), and the results to the

expression RT-PCR fir described (31).

expressed as arbitrary densitometry units related of GAPDH. Fas ligand detection was carried out as previously using the following primers: 5’ CA CA AAT CTG

Statistical Results level

was

Analyses are expressed established

as mean using

ANOVA

±

SEM. and

Significance I

test.

at the

95%

Fas and

Results

stitiab

Serum

Deprivation,

TNFa,

induce

Apoptotic

Antibodies

Fibro

Agonistic

Death

of Renal

Because

loss

we

first

croscopy. tibodies

of cell studied

Serum decreased

number

contact cell

is an early

detachment

deprivation, TNFa, renal fibroblast

of detached

cells

feature by phase

(Figure

1). We

fibroblasts

serum

also

manner (Figure 5A). Therefore, we studied

of apoptosis

in the

mi-

anti-Fas anand increased further

sis

presence

of

renal

P


0

1024

0

1024

5 ±

lymphoid

cells

and

and

SF/FAS

statistically

deprivation soluble

soluble

significant

Fas,

in to

0

24

to 72

DNA

h.

(at 72 h: serum-free,

4. Flow

cytometry

Fas,

distinct

A() peak

among

27 ±

± 5%;

10%

TNF,

DNA

apoptotic

content

propidium

[ns]).

was

(the percentage with fragmented

article,

we have

shown

that

serum

contains

survival

that prevent apoptosis in renal fibroblasts. We observed that survival factors for renal fibrobbasts cytokines, such as PDGF and IGF- 1, that promote in other

cell

types

(37-39).

On

the

other

hand,

TNFa

have insurand

in control significant of serum

(mTNFct) further mTNFa

analysis renal

ofcells nuclei

by

(TNF) increased (SF/TNF)

or treated

when

in 10% is present with

agonistic

serum-deprived

or anti-Fas

antibodies

demonstrates

to lethal

cytometry The

stimuli.

peak

termed

A0

staining) consists of cells A0 cells are negligible

FCS (FCS). By among fibroblasts

either

a The

in permeabilized,

subdiploid

with decreased DNA undergoing apoptosis.

or 1 jtg/ml

content

exposed

flow

fibroblasts.

fibroblasts grown amount of A0 cells for 48 h (SF)

of DNA

fibroblasts

assessed

iodide-stained

1024

CONTENT

Figure

57

0

1024

or absence

for

to be protective 2 j.tg/ml

SF/TNF

AX

in experiments (25) was added

in the presence

serum

2 j.tg/ml

Death in Renal

Discussion In this

1024

by cultured renal cell death through

was addressed Fas receptor

cultured or

not found

59

Cell

ligand expressed regulation of

activation of the Fas receptor which 0.2 to 2 jtg/ml soluble serum-deprived

Not Block Deprivation

fibroblasts

SF

LU

Soluble Induced

Soluble

apoptosis.

in renal

Cs)

I

100

Fas

TNF

-J -J lii

(24).

of

or

iS24

z

stained

of Fas

in

TNFa

the sus-

‘U

of positive cells was than in control anti-

described

studied on the

FCS

that 8C). with

peptide,

ligand

for 3 h (19

We thus cytokines

to receptor-mediated

either

1

anti-Fas

control

anti-Fas

1 .2 versus 4.9 cells increased

inhibitor

2%

expression

an unrelated

or

of the murine

stained the

8D). The percentage ligand-stained cells

0.05),