Cytokines and Fas regulate apoptosis in murine renal interstitial fibroblasts.

Abstract

Renal fibrosis is characterized by an increased number of fibroblasts and excessive deposition of extracellular matrix. Apoptotic cell death is a physiological mechanism to limit cell numbers, and an insufficient rate of death may contribute to fibroblast accumulation. However, little is known about the regulation of renal fibroblast survival. The authors have studied the interaction of cytokines and the Fas receptor in the regulation of apoptosis of renal fibroblasts and have observed that murine renal fibroblasts express Fas and the Fas ligand. Tumor necrosis factor alpha (TNFalpha) and agonistic anti-Fas antibodies induce apoptosis of renal fibroblasts in a time- and dose-dependent manner. Serum contains survival factors for renal fibroblasts. Both serum deprivation and TNFalpha increase the sensitivity to Fas-induced death and the expression of fas mRNA and Fas receptor. By contrast, insulin-like growth factor-1 decreases apoptosis induced by both serum deprivation and Fas activation and partially prevents the increase in Fas receptor expression induced by serum deprivation. Murine renal fibroblasts express constitutively both fas ligand mRNA and cell-surface Fas ligand, but the authors could not demonstrate a role for Fas ligand in the autocrine regulation of fibroblast survival. These data suggest that Fas and other cytokines cooperate to regulate renal fibroblast apoptosis. Modulation of the Fas death-signaling pathway in renal fibroblasts could represent a new therapeutic target for renal fibrosis.

Full text

Page 1

Cytokines
Interstitial
and
Fibroblasts
FasRegulateApoptosisinMurineRenal
ALBERTO
RAIMUNDO
?
ORTIZ,*
GARCIA
deNefrologIa,
CORINA
DEL
Fundaci#{243}n Jirn#{233}nezDIaz,
LORZ,*
MORAL,t
SILVIA
FRANCISCO
GONZALEZ?CUADRADO,*
O’VALLE,t
UniversidadAut#{243}noina, Madrid,
andJESUS
Spain;
EGIDO*
and?Faeultad
deMedicina,Universidadde Granada,Granada,Spain.
Abstract.
ber
matrix.
Renalfibrosisis characterized
excessive
death
by anincreased
of
mechanism
num-
offlbroblasts
Apoptotic
and
cell
depositionextraceblular
is a physiologicalto
limit
contribute
about
have
inthe
cellnumbers,
to fibroblast
regulation
the
regulation
andan insufficientrateof
little
death may
accumulation.
renal
However,
survival.
and
renal
is known
authors
receptor
and
the of fibroblast
of cytokines
of
The
Fasstudied interaction
of
the
apoptosisfibroblastshave
observed
ligand.
antibodies
dose-dependent
renal
thatmurine
necrosis
renalfibroblasts
a (TNFa)
of renal
Serum
serum
express
and
fibroblasts
Fas andthe
anti-Fas
Fas
Tumor factoragonistic
induceapoptosis
manner.
Both
in a time-
factors
TNFa
and
forcontainssurvival
andfibroblasts.deprivationincrease
the
mRNA
tor-
and
sensitivityto Fas-induced
receptor.
apoptosis
and
death
contrast,
andthe expressionof flis
fac-andFasBy
induced
partially
insulin-like
both
growth
deprivation 1 decreases
Fas
byserum
theactivationpreventsincrease inFas
receptorexpressioninducedbyserumdeprivation. Murine
renal
and
fibroblasts
cell-surface
express
Fas
constitutiveby
but
both
ftis
could
ligandmRNA
demon- ligand, theauthorsnot
strate
fibroblast
a rolefor Fasligandinthe
suggest
autocrineregulation
Fas
of
survival. These data thatandother
cytokines
Modulation
blasts
cooperate
of the
toregulate renalfibroblast
pathway
apoptosis.
Fasdeath-signaling
a new
in renal
target
fibro-
renalcouldrepresenttherapeuticfor
fibrosis.(J Am SocNephrol8:1845-1854,1997)
In
tion
function
amount
known
chronic
and
renaldiseases,
correlates
the degree
well
ofinterstitial
the
Although
renal
numbers
inflamma-
of
excessive
(2),
in the
fibrosis
(reviewed
offibroblasts
about
with
I ).
in
prognosisrenal
inreference
present
an
is fibrosis little
kidney
is
the regulationof fibroblast
(3).
the
mediate
differentiation
contribute
Changes
processes
in
that
clearance.
of
to increase
cellnumberresult from
cell
imbalances
number
proliferation
into
between
those
and
tendto increase
Both
epithelial
their
andthat
cell fibroblasttrans-
maytubularcellsfibroblasts
number
(2-4).Celldeathby apo-
ptosis
ance
death
healing,
relative
in fibroblast
thefactors
blasts
progression
design
isoneof the mostimportantmechanismsof cell clear-
under
mediates
pathophysiologicab
the
leading
failure in the
accumulation
that
may help
of renal
ofnew therapeutic
conditions
in fibrobbast
of
of
during
deathand
understand
fibrosisand
strategies.
(5-7).
number
(7).
fibroblasts
fibrosis.
survival
the
provide
Apoptotic
during
this
cell
decrease
resolution
clearance
wound
to injury
renal
renal
the
In regard,
may
a
result
Knowledge
of renal
persistence
the basis
of
regulate
researchers
fibro-
and
the mayfor
Apoptosis
program
death
involves
I).
the activation
factors
cytokines
ofan intracellular
the activation
trigger
death
of the
pro-
(8-b Survival
Certain
prevent
may program. thedeath
Received
Correspondence
Jim#{233}nez DIaz,
July 9. 1996.
to Dr.
Avda
Accepted
Alberto
Reyes
May30.
Laboratorio
1997.
Ortiz,de NefrobogIa.
Spain.
Fundaci#{243}n
Cat#{243}licos 2. 28040Madrid.
1046-6673/08()
Journal
Copyright
12- 1845$03.0O/0
Americanof the
0
Society of Nephrology
1997by theAmerican Societyof Nephrology
gram
tumor
through
necrosis
activation
factor
ofspecific
(TNFa)
receptors.
and
These include
aFasligand (9,12,13),
which,
and
inthekidney, are secreted
compose
mainly
the
bythemacrophages
interstitialT lymphocytesthatmononuclear
infiltration
(14,15).
the
Thus
which accompanies
activation
effector
of lethal
fibrosis
is followed
program
receptors
in chronic
by
of cell
does not
renaldisease
onlyReceptorapoptosis
death
result
if
integrity
activation
of the is preserved.
in apoptosis
in everycell type.TNFapromotessurvival,not death,of
monocytes
Fas
andhuman
does
peripheral
not
blood lymphocytes
cells
(16,17).
and engagementkillendothelial( 18)may
resultinactivationorproliferation oflymphocyte subpopula-
tions
( 19,20).
mediated
plored
whenlevels of intracellularprotective factorsare high
The
apoptosis
therole
susceptibility of renal
understood.
andthe
fibroblasts to receptor-
nowispoorlyWehaveex-
ofcytokinesFasreceptorin the regu-
lation
fibroblasts.
ofapoptosis of culturedmurinerenal interstitial
Materials
Cells,
TFB
Eric
andMethods
and
interstitial
Cytokines,
murine
G. Neilson
Antibodies
renal
(University
extensively
protein
fibrobbasts were
Philadelphia.
andthey
were cultured
decomplemented
penicillin,
(2,21). In
24to 72
cloneof anti-murine
IgG, and
a generous
giftfrom
of Pennsylvania,
characterized,
(2,21
PA). These
cells
blast
(GIBCO,
(FCS),
have
specific
beenexpressthe fibro-
FSP-1
). They
10%
U/mI
in RPMI
fetal
1(X) ?g/ml
studies,
1640
serum
strep-
were
Grand Island,NY), calf
2 mMglutamine,
37#{176}C in
serum
(a hamster
(22).
100
CO2
and
tomycin,
deprived
The
antibodies
at
of
5% some
h.
cells
(0%FCS)
IgG)
hamster
for
io-2 Fasmonocbonal
anti-hamstercontrolFITC-rabbit

Page 2

1846
Journalof theAmericanSocietyof Nephrology
IgG were
an
(22).
rived
(mTNFa
(Mannheim,
raised
mapping
peptide
CA).
extracellular
Switzerland)
(Frankfurt,
chem
Dr.
obtainedfrom Pharmingen
that
(San
to and
Diego,
activates
factor-
andmurine
from
rabbit
to
Fasligand
CruzBiotechnology
Al I that
220 was
1gMandbiotin-anti-rat
streptavidin
Soluble Fasreceptors
(LXRBiotechnology,
CA). The io-2
theFas
1), platelet-de-
andhuman
Boehringer
anti-Fasligand
aminoacids
(23),and
clone
is
agonistic
Recombinant
growth
and
antibodybindsreceptor
insulin-like growth1 (IGF-
factor-BB
hTNFa)
Germany).
a peptide
theamino
purchased
rat1gM
peptide
(24).
Germany),
Diego.
Fitzpatrick
(PDGF-BB).
were
Polycbonab
corresponding
terminus
from
monocbonal
196 through
control
and
CA).
TNFa
purchasedMannheim
antibodies,
2 through
the
(Santa
theFas
(Laufelfingen,
1gM
from
a kind
CA)
against19
at ofcontrol
Cruz,
ligand
wereSanta
Thebinds
from
to
Alexis
rat from
Calbio-
gift
(25).
1K
phycoerythrin
(Sanwerefrom
PaulRichmond,
StudiesofCellDear/i
Cellswere
or
culturedin 24-well
flow
mM
cells
in
0.05%
at 4#{176}Cbr
Coulter,Luton,
decreasedDNA
fragmentednuclei,
For the morphological
8-wellLabtekglass
withTNFa. anti-Fas
with PBS.fixed
platesin thepresenceor absence
to the
(EDTA)
(26).
iodide,
saline
cytometer
percentage
apoptotic
of
stimuli
collected
mixed
spun
RNAse
incubated
XL
with
FCS.
with
detached
resuspended
and
For
2.2
cytometry.
ethylenediaminetetraacetate
present in the
100 ?g/ml
NP-40in
1 h. andanalyzed
United
staining(A0),
was counted.
assessment
slides(Nunc
antibody. or control
with10% buffered
cellsattachedplatewere
and
were
j.?g/ml
(PBS),
(EPICS-
of cells
cells
with
and
supernatant
propidium
Cells
10
A,phosphate-buffered
on
Kingdom).
comprising
a flow
MCL, The
with
of apoptosis.
Napersville.
IgG.
formalin,
cells
IL) and
were
washed
were grown
stimulated
then
with
in
Inc.
Theywashed
PBS,
stained
pH 7.2
were
med
Cells
formaldehyde
described
The
with
for 30 mm
mounted
in a fluorescence
exposed
I j.tg/mlpropidiumiodide,
being
100p.g/mlRNAse
PBS,
A,in PBS
at 37#{176}C. After
usinga 90%
washed
solution
(26).
were
for electron
with
in PBS
preparations
thenglycerol and exam-
microscope
stimuli
processed
to lethalfixed in glutaraldehyde/para-
microscopyandas previously
(27).
presenceof DNA fragmentation
in 100 mM NaC1,
proteinase
precipitated.
at 37#{176}C, and
transferredto Genescreen
hybridized at
was studiedin genomic
25 mM
for
with
DNA
obtained
1%
DNA
RNAse
DNA
branes
DNA
by lysing
2(X) ?g/mL
extracted,
A for
wasthen
and
(28).
cells
10 mM Tris,
pH
treated
in a 1.5%
(NEN.
with
EDTA,
at 37#{176}C.
10 ?.tg/mL
agarose
MA) mem-
murine
SDS. K in PBS,
resuspended,
separated
7.2,18h
was
30mm gel.
Boston,
radiolabeledlow stringency
RT-PCR
Total
form
gels
RNA
Diego.
and
were
ciiid Northern
RNA was
method(29).
containing
was isolated
CA). RNA
prehybridized
strippedand
Hybridization
using
RNA (25
formaldehyde.
the FASTRACK
transferred
hybridized
subsequently
isolated
Total
2.3%
using
was
and
the
gig)
acid-guanidine-phenol-chboro-
wasseparated
Insome experiments,
mRNA
to nylonmembranes
as previously
rehybridizedwith
in I % agarose
poly-A
kit (Invitrogen,
(Genescreen)
described
a GAPDH
San
(30).Blots
probe to
account
quantified
CA),
to the
RT-PCR
described
for small
using
the
loading
a
or transfervariations.Autoradiograms
densitometer
densitometry
were
Molecular
expressed
of GAPDH.
Fasligand
using
Dynamics
as arbitrary
(Sunnyvale.
units
and
expression
results related
firdetection
following
was carried outaspreviously
AAT (31).theprimers: 5’ CA CACTG
TGGCTACCG, 3’ GC CCATAT CTGTCCAGT AG.The resulting
PCR
product was cloned and sequenced to confirmitsidentity.It
expandsseveralexons andthus excludesthepossibility of amplifica-
tion of genomic
The
DNA.
Fasprobe is a clonedPCRproductcorrespondingtothe
intracellular domainof murineFas (30,32).It hybridizestoanap-
proximately2-kbtranscriptin thymus andliverfrom
SJL
mice, but
notfromMRL
lpr/lpr
mice. TheFas ligandprobeis a clonedPCR
productthat comprisesbases 353through 839of thepublishedmurine
FasligandeDNA(3 1,33). This probereadilydetectsa fas
ligand
transcriptin total RNAfromstimulatedJurkatT cellsby Northern
blot
have
(34).
been
Both
sequenced
probes werekindly donated
identities.
by EricG.Neilsonand
to confirm their
Flow CytomerrvAnalysis ofFas and FasLigand
Expression
For cytofluorography, cells wereculturedin the presence of control
mediumor cytokines. Afterthe culturewaswashed with PBS, adher-
entcells were detachedwith 2.2 mMEDTA, 0.2%bovineserum
albumin(BSA)in PBS andsingle cellsuspensionswere resuspended
in 0.2% BSAIPBS.TostudyFasexpression,5
X
l0?cellsfromthe
samesample wereincubated with1 .tg/mlanti-Fas antibody or1
?g/ml controlhamsterIgG for30mmat 4#{176}C, followedby incubation
with1:100FITC-rabbit-anti-hamsterIgGfor 30mm at4#{176}C. Cells
werethenanalyzedon a cytofluorograph. Deadcells anddebriswere
excluded
right-angle
from analysisby selective
10,000
gating
events
based
were
onanterior and
each scatter.At leastcollectedfor
sample,anddataweredisplayed ona logarithmic scaleof increasing
green-fluorescenceintensity(30).Mean cellfluorescence wascalcu-
bated usingLYSISII software. Meancellfluorescenceobtained with
the
Ab.
control
Data
IgGwassubtracted fromthatobtained
over
with the anti-Fas
grownare expressedas fold-increasecontrolcells in
10% FCS
study
in 2%
RPMI.
Tointracellular
glutarabdehyde/PBS,
Fas ligandexpression,
washed
I % goat
cellswere
5
X
iO?
permeabilized
in PBS
incubated
cellswere
fixed
in PBS,
serum,
and
in 0. 1% Triton X- 100,2%
BSA,for 30 mm
with 1 ?g/mlat room temperature.Thereafter,
polyclonal anti-Fasligand antibodyin 0.1%TritonX-l00,0.2% BSA,
inPBSfor60 mm,followedbyincubationwith I : 100FITC-goat-
anti-rabbitIgGfor 60mm. Controlsamples were stained withan
anti-Fas
control
ligand
Fas
antibody
peptide
that
or with
had been
nonimmune
previously incubated
IgG.
witha
ligandrabbit Cellswere
then analyzed
study
ona cytofluorograph.
cell-surface
with1 ?g/ml
ToFas
monoclonab
FCS,
with
with
acytofluorograph.
ligand expression,
Al 1 anti-Fas
BSA,
biotin-anti-rat
5
X
iO?cells
antibody
were
incubated ligand
for
1gM
or
control
followed
4#{176}C
were
rat1gM in 5%
0.2%
1:100
1:50
in PBS30 mm
for
at 4#{176}C,
mmby
by
incubation
incubation
analyzed
30at
and
then
streptavidinphycoerythrin.
these
Cells
on For experiments,
somecellswere culturedfor3 h in thepresenceof
5 mM
release
EDTA,
ofwhichinhibits metalboproteases anddecreasesthe Fas
ligandfromthecellmembrane. thusincreasingthe availabilityof this
cytokineat thecell surface (24).
StatisticalAnalyses
Resultsareexpressedas mean
±
SEM. Significanceat the 95%
level
wasestablished usingANOVA and
I test.

Page 3

Fasand
Apoptosis
in RenalFibroblasts
1847
Results
Serum
Antibodies
Fibro
Because
(35),
croscopy.
tibodies
the
gated
tional
were
cells
agonistic
consistent
further
and
Genomic
DNA
cultured
Apoptosis
ized,
that
and/or
decreased
fragmentation
didnot
Serum
Deprivation,TNFa,
Apoptotic
andAgonistic Anti-Fas
induceDeathofRenalInterstitial
blasts
lossof cell
studied
deprivation,
renal
detached
occurrence
studies.Pyknotic,
present among
thathad been
anti-Fas
with
features
margination
DNA
degradation,
in10%
was
propidium
a significant
treated
DNA
due
induce
contains
contact
cell
is an early
detachment
TNFa,
fibroblast
cells(Figure
of apoptosis
condensed,
formalin-fixed,
deprivedof serum
antibodies
apoptosis.Electron
of apoptosis,
andnuclear
displayed the
whichwas
FCS RPMI
quantified
iodide-stained
percentage
withTNFa
content (A0
to apoptosis
fibroblastapoptosis.
survivalfactors
feature
phase
of apoptosis
contrast
anti-Fas
andincreased
further
and
fragmented
iodide-stained
or treated with
2B). Thismorphology
microscopy
as chromatin
fragmentation
characteristic
not present
(Figure 3).
byflowcytometry
cells.Thistechnique
offibroblasts deprived
anti-Fasantibodies
peak),consistent
(Figure4). Control
wefirst
Serum
decreased
bymi-
an-or agonistic
confluency
1).
morphological
and
propidium
number
the
ofWe investi-
func-
nuclei
by
TNFa or
is (Figure
demonstrated
condensation
(Figure
internucbeosomal
incontrol
such
2C).
cells
ofpermeabil-
revealed
of
presented
with
hamster
serum
ora
nuclear
IgG
for severalcell types.Inter-
stitiab
because
manner
Therefore,
renalfibroblasts
deprivation
5A).
westudied
alsodepend onserum
in a time-dependent
forsurvival,
serum
(Figure
induceddeath
the effectof TNFaonfibroblastdeath
inthe presenceorabsence ofserum. mTNFainducedapopto-
sis ofrenal fibroblastsinthe presence of10% FCS(at 48h:
1000
P
survival
U/mb
0.05),
mTNFa,
but
factors
12 ±
effect
48
2.2%
was
1000
apoptosis:
more
U/mL
control.
in the
mTNFa,
P
<
fibroblast
only
also
2
absence
54
± 0.3%:
<
themarked of
(ath:
±
3.6%
char-
apoptosis;
acterize
cells
55-kD
serum-free
TNF
treated
control,16±2%:
0.05).To
thereceptor
with
TNF
responsible
hTNFa.
forapoptosis.
activates
induced
werehTNFa
hTNFa
the
murine receptor(36). apo-
ptosis
ner
of renalfibroblastsin a time-
lethality
anddose-dependent
induced
man-
was (Figure5, A andB). Theby hTNFx
higher
FCS
inserum-deprived
5A).
of
apoptotic
manner
was
grown
thencharacterized
cells thaninthe presence of10%
(Figure
Activation
duced
dependent
apoptosis
cells
We
the
death
Fas receptor byagonisticantibodies in-
of renal
(Figure
marked
FCS
fibroblasts
A and
in serum-deprived
(Figure
regulation
in a time-
Again.
and dose-
5. B).Fas-induced
cellsmore
10%
than in
inRPMI
the
SA).
of Fas-induced death
by
cell
50
duced
cytokines.
types
ng/mb
IGF- 1 and
We
PDGF
have
partially
fibroblasts
are survivalfactorsfirseveral
IGF-1 (37-39).
PDGF-BB
in
observed that
100
ng/ml or
prevent
(Figure
serum-deprivation-in-
SC).IGF-deathrenalI alsopro-
tectsagainstFas-induceddeath in serum-deprivedcells (Figure
Figure
fibroblasts
tumor
individually
1. Serum
grown
necrosis
and
in 10% FCS
factora (hTNFa)
detached
cytokines regulate
RPMI
(B).
renal
renal
fibroblast
fibroblasts
media
number.
cultured
orI ?g/ml
Contrast
for 48 h under
agonistic
phasemicroscopy
serum-free
anti-Fas
revealed
conditions
that when
in the presence
displayed
compared with
10(X)
control
U/mI
and
renal
(A),
control
of
human
(C).
antibodies(D)less confluencynumerous
cells.

Page 4

‘?.
.?
.#{149}, V.
?‘
11
I 848Journalof theAmericanSocietyofNephrology
4,
.
.,.,t
p
?.
‘
I?)f??
?
.
,-.
-
?
#{149}‘.: ;y.
Figure
fetal
antibodies
j.?g/ml
2. Death
calf serum
of renal
(FCS)
Note
antibody
interstitial
RPM!
the pyknotic.
for
fibroblasts
stained
shrunk,
has
propidium
nucleus
chromatin
morphologicalfeatures
after
center
of apoptosis.
stimulation
of panel
margination,
(A)
1 ?g/ml
Electron
initial
and(B)
control
microscopy
nuclear
Formalin-fixed
hamster
fibroblasts
(A) or agonistic
fibroblasts
grownin 10%
were with
bright
iodide
in the
condensation.
withIgGanti-Fas
with(B). B. (C)of renaltreated1
anti-Fas24 h. Note theandfragmentation.
SD).Thecombinationof
100
U/mbhTNFa and1 p.g/ml
anti-Fasantibodyfurtherincreasedthe rateofcelldeath
(Figure SD).
Renal FibroblastsExpress theFasReceptorUnder the
Regulation
The
that
of
Cvtokines
of surface
thesensitivity
amountFasexpression
of the
is one of the
induced
factors
by Fas modulatecellto death
agonists(40). Weexplored thehypothesis thatcytokinesmod-
ify
because
thesensitivity
they
transcript
(Figure
ofrenalfibroblasts
expression
to
of
Fas-induced
Fas
fibroblasts
U/mb
apoptosis
regulatethe
detected
receptors.
Aj,.s was in renal
100
by Northern
10%blot6).Stimulationwith hTNFo?FCS,
increasedfa.s mRNAexpressionsixfoldovercontrolsgrownin
10%FCS at6h,andtenfold at 24h. Serumdeprivationin-
creasedfii.smRNA
expression
by
bevels 20-fold
Fas
at24h.
Surface of the receptor
Staining
was detected
with
in renal
fibroblasts cytofluorography.anti-Fasanti-
bodiesrevealed thatfibroblastsgrownin 10%FCSRPMI
display
IgG-stained
bow levels
cells
of
(not
surface Faswhen
Fas
compared
receptor
with control
shown).expression was
increased
hTNFa
repeated
Fas
grown
by serumdeprivation
in
deprivation
from
RPMI.
orbythe addition
7, A through
for
over
100U/mb
of 100 U/mI
C).to cells
experiments,
receptor
in 10%
grown10%FCS (Figure
of
to 3.6-fold
of
In
serum48h increased
control
hTNFa
expression
FCS
1.8-cells
in theAddition
presence ofI 0% FCSincreasedFasexpression 1.5- to 2. 1-fold
over
and
controls
100
at
hTNFa
48h.The
further
combination
increased
ofserum
expression
deprivation
U/mbFas (Figure
7B).Onehundred ng/mbIGF-1partiallyprevented therise in
Fasexpressioninducedbyserumdeprivation(Figure 7B).
RenalFibroblasts ExpressFas Ligand
ligand
renal
B).
Constitutive
RT-PCR
10% FCS
expression
Northern
(Figure
ofafas
blot
8, A and
message
fibroblasts
The
was detected
by and in cultured
message,
in
RPMIsizeof the

Page 5

C/)
0
LL?
(flLL?
!q?z
LI?I-
U-
(I)
FCS SF
iS241024
TNF
‘U
z
-J
-J
lii
Cs)
LU
>
LU
FAS
0 1024
0
1024
SF/FAS
I
SF/TNF
AX
01024
01024
Fas andApoptosisin RenalFibroblasts I849
Fasactivationinduceapoptosisin culturedrenal fibroblasts. In
this regard,renal fibroblastsdiffer from
( I 6, 17).
othercell types.TNFa
preventscelldeathin leukocytesFasis notlethalfor
endothelialcells(1 8): however, Fas andTNFa induceapopto-
sisinrenal mesangialand tubularepithelialcells (26,30.41-
43).
Int’ivo
fibroblasts
of cytokines
ofthe
areimmersed
other
ofserum
inamicroenvironment
Wethus
cytokines
composed
influence
andfactors.
and
studied
on
the
sus-presence the
ceptibility ofrenalfibroblaststoreceptor-mediatedapoptosis.
Lethalityinduced byeither TNFa orFas inrenal fibroblasts
Figure
internucleosomal
degradation
(SF)
?g/mL
3.
Deathofrenal
DNA
interstitial
fragmentation.
in fibroblasts
1000 U/mI
anti-Fas/10%
fibroblasts is associatedwith
DNA
media
or
Internucleosomal
cultured
hTNFa/l0%
(Fas)for
waspresent
with
in serum-free
FCS
48h.
ortreated
agonistic
(TNF)1
FCS
approximately
ligand
renal
Forty-one
anti-Fas
with
ligand
were
was
(clone
higher
body-stained
The
themetalloprotease
0.2%,
2 kb,corresponded
(33).
express
of the
antibody.
rabbitIgG
antibody, after
positive. Moreover,
also detected
A 1 1) (Figure
inanti-Fas
cells
percentage of positive
withthe size
demonstrated
ligand
when
2%of cells
stained
the control
expression
anti-Fas
of positive
cellsthan
4.9±
increased after
EDTAfor3 h (19
of the murine
fas
transcriptFlowcytometrythat
8C).
with
fibroblastsintracellular
cells
By
and2.4%
competition
cell-surface
anunrelated
8D).The
ligand-stained
(7.8±
Fas(Figure
stainedpercent
ligand
were
contrast,
positive
only stained
anti-Fas
peptide,
ligand
antibody
cells
controlof cellswith
with
of Fas
ligandusing
percentage was
anti-
0.05).
with
in control
P
treatment
±2 versus
1.2
cells
versus
0.3%,
<
inhibitor
5 ±
P
<
0.05),
aspreviouslydescribed inlymphoid cells
(24).
Soluble
Induced
Fas
by
Receptors
TNFx
Do NotBlockCell Death
Renal orSerumDeprivationin
Fibroblasts
The
fibroblasts
activation
which
serum-deprived
of
100
Soluble
possibleroleof Fas
autocrine
Fasreceptor
ligand
regulation
was
Fas
cultured
serum
to be protective
expressedbycultured
death
renal
in theofcellthrough
of the
to 2 jtg/ml
addressed
receptor
in the
deprivation
in experiments
(25)was
presence
for
(at 72 h: serum-free,
in
to0.2soluble added
fibroblasts
hTNFa
was not
or absence
to U/ml
Fas
or2472 h.
found
22± 2%;serum-free and 2 j.tg/mlsolubleFas, 27±5%;TNF,
59± 7%; TNFand2 j.tg/ml solubleFas,57±10% apoptotic
cells;notstatisticallysignificant [ns]).
Discussion
In this
factors
further
elude
article,
that
observed
cytokines,
we have
apoptosis
survival
as
shown that
renal
serum contains survival
We preventin fibroblasts.
renal
1, that
have
that
such
factors
and
for fibrobbasts
promote
in-
PDGF IGF-sur-
vivalin othercell types(37-39).Onthe otherhand, TNFa and
DNACONTENT
Figure
distinct
4.
A() peak
Flow cytometry
among
analysis
fibroblasts
of DNAcontentdemonstrates
stimuli.
a
renal exposedto lethal The
DNA
propidium
(the
with
in control
significant
of serum
(mTNFct)
further
contentwasassessed byflowcytometry
The
DNA
apoptosis.
10%FCS
is present
witheither
agonistic
serum-deprived
in permeabilized,
peak
consists
are
Bycontrast.
fibroblasts
U/ml murine
antibodies
were
iodide-stainedfibroblasts.
withdecreased
undergoing
grown
A0cells
or treated
1 jtg/ml
when
subdiploid
staining)
termedA0
percentage
fragmented
ofcells
nuclei
of cells
negligibleA0 cells
(FCS).fibroblasts
amount
48 h (SF)
(TNF)
increased
ina
ofamong deprived
TNFa
(Fas)
treated
for1000
or anti-Fas
fibroblasts
and
with
mTNFa (SF/TNF)or anti-Fas antibodies(SFIFas).