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),