in de- tectable levels of NO. Macrophages from. TGF-1' mice appeared to produce. NO in a manner ...... R. K., Fleurdelys,. B. E., Stevenson,. H. C., Miller,.
Control of nitric oxide production by endogenous TGF-1 and systemic nitric oxide in retinal pigment epithelial cells and peritoneal
macrophages
Yoram Vodovoz, Janet Sparrow Laborazory
VLcion New
Abstract: demonstrate (TGF4h) form of
Research
suppresses oxide
Ii’
and
mice were
in
expression synthase
nitric
and
Andrew
ofChemoprevention, Naiional Intiuues Irutitute, Department ofOphthalmology,
Both in vivo and that transforming
the effects on retinal
resident
using cells age-matched (TGF4Eh +1-)
J. Letterio,
of
the In
(iNOS).
and
Maryland; Hospital-Cornell
peritoneal
oxide paradoxical
B. Roberts,
tTj. Margaret Medical Center,
stimulation
macrophages
littermates
and
of NO. treatment
tectable levels mice appeared
potentially
endo(RPE) ex
produced positive for
in the
The
absence
expression IFN-y,
with
to the
of the mice from such correlation Treatment
serum which existed
of RPE
both TGF-14’ TGF-31 decreased These
and
content
vivo
NO iNOS
the cells were in TGF-1 cells
TGF-31’ both iNOS
findings
or
and
N03
obtained; or TGF-j1’
[1-3].
types
and
ity of iNOS
no
An isoform
enzyme tional
produces
expression, regulation
agents
that
three
mammalian are the or smooth
isoforms
phages
of nitric
quantities
regulated
although also exist
suppress
the
mechanisms [1-3]. Of production
allele
the
we have
of TGF-1
role
demonstrate
of NO
lipopolysaccharide
in
of
vitro,
the
(TGF-1’) level (TGF-i)
mice [12].
than
by reducing the posttranscniplevels [6-1 1]. TGF431
that
express
These
littermate controls,
mice
normal associated
on a null
express
(TGF-f31) with four-
reaction products of NO, of iNOS and systemic in TGF-1’ during the
from
a novel
role
of
Abbreviations:
NOS,
nitric
oxide
synthase;
iNOS,
inducible +1+
cells
.
interferon-y
NOS
(NOS2;
.
NOS +1-
iype it); ur-p, transmormznggrowtn Iactor-p; iir-pm , wan-type; lur-pi heterozygous for a null TGF-i allele; TGF-f3(’, homozygous for a null TGF-3i allele; PCR, polymerase chain reaction; LPS, bacterial lipopolysaccharide; RPE, retinal pigment epithelial cells; IFN-y, interfemn-y, PBS, phosphate-buffered saline;
DMEM,
immunosorbent
Reprint
requests:
Institute, address
Endocrinology, Received
Journal
Dulbecco’s
modified
Eagle’s
medium;
EUSA,
enzyme-linked
assay.
Present
epithelial
of
for posttranslathe large array
of endogenous
utilized
fold elevated serum levels of the NO2-, and NO3 [13]. Expression levels of NO2- + NO3 are highest
Cancer pigment
activlevel
transforming growth factor-3 (TGF-) most potent [4-1 1]. Treatment of macromuscle cells with TGF-1 has been re-
to examine
production,
The
at the
ported to suppress NO production directly expression of iNOS at the transcriptional, tional, translational, and posttranslational NO
synthase products in many
of NO.
primarily
.
retinal
oxide
are and
with exogenous and NO pro-
endogenous TGF-31 in coupling systemic NO production to the production of NO by macrophages, and demonstrate that endogenous and exogenous TGF-f1 can act differently to suppress NO production. J. Leukoc. Biol. 60: 261-270; 1996.
Words:
free radicals for survival
and microbial can be expressed
large
is negatively
iNOS to a higher or heterozygous
TGF-1’ inversely
macrophages
mice protein
(iNOS, cell
In order
of iNOS was resulting in de-
of N02
lethal
inducible by cytokines NOS2, NOS Type II),
(NOS),
of detect-
of NO. Macrophages from to produce NO in a manner
proportional
Key
and
M. Dyson New York,
(NO) and its derived nitrogen in that they are both vital
Nitric
inducible this study,
of exogenous and pigment epithelial
and TGF-1 immunocytochemically
able production increased by
duction.
Anita
INTRODUCTION
vitro experiments growth factor-ni
from TGF-1 null (TGF-[1”) mice or wild-type (TGF-1’) or heterozygous littermates. RPE cells from both TGF-
without
mice.
Loums Chesler,
ofHealzh, Bethesda, The New York
protein only following treatment with interferon-y (IFN-y) and bacterial lipopolysaccharide (LPS); however, RPE cells from TGF-31’ mice produced 40% more NO than cells from TGF-f31 mice. In contrast, resident peritoneal macrophages from both TGF-31’ and TGF-1 mice expressed iNOS protein
G. Gemser,
York
we examined genous TGF-1 cells
John
of
Dr.
Yoram
Bethesda, of Dr.
Andrew
Indianapolis, March 4, 1996;
Leukocyte
Vodovotz,
Radiation
Biology
Branch,
National
MD 20892. C. Ceiser
Eli
Lilly
IN 46285. revised May 13, 1996;
Biology
Volume
60,
& Company,
accepted
August
Department
May
1996
of
16, 1996.
261
period
between
20-28
days
days of life
15
[13].
and
17
The
of life
aberrant
and
expression
appears to be part of a general autoimmune leads to wasting and death of TGF-1’ mice ofage [12, 14, 15]. We have macrophages These cells,
focused our and retinal while different
the extent activated
decline
by
Resident
that weeks
with
and can become to T cells [16,
vated
fetal
macrophages (100 U/mL
of
iNOS
in
macrophages requires only interferon-y 23-25], whereas IFN-y and lipopolysaccharide the minimum stimulus for induction of iNOS
[20,
21,
26,
duction
27].
In order in TGF-1’
of NO
penitoneal
were
penitoneal
(IFN-y)
[6, (LPS) is RPE in cells
the dysregulated we examined
macrophages and RPE cells of age, 20-28 days of age,
days
15-17
to define mice,
proresident
iNOS
ex vivo, from mice or 5 weeks of age.
mice
N02 An
maintained
in a specific
mouse the
chow.
The
following
mice, rus,
as
aliquot
of the
6, 20]
plates
pathogen-free
mice
were
pathogens:
determined
murine virus,
cytomegalovirus,
polyoma
virus,
as
endoparasites pressing
the
performed
null
by
scribed
allele
and
Treatment
not
chain
[28,
infected
the
with
any
of
tail
or
identified
allele.
from
by ex-
cases, with
Genotyping
assay
mg/kg)
diluted
and
TGF-i
mice
(Wyeth-Ayerst
in sterile
olive
oil
were
as
injected
Research,
(Sigma,
St.
read plate
presence
Naperville,
IL) [20].
Upon
treated
with
recombinant
master
stock:
1 x 106
burg,
MD),
LPS
coli
recombinant MN).
After
N02
and
262
reaching
in 8-well
4
(1
U/mL
human
TGF-i
Chamber
Journal
of
Slides
Leukocyte
phenol
Laboratories,
(5
R&D
ng/mL; conditioned
stained
for
Biology
iNOS
as described
Volume
of
from
Es-
CA)
or
60,
was
as described
were
p1
removed
Briefly,
100
of each
supernatant
Vmax
enzyme-linked
a
(Molecular
stained
for
assay
of NO2
Griess
reagent
triplicate.
in
The
immunosorbent
Devices,
(Dynatech,
for
L
Menlo
Park,
Chantilly,
VA)
CA)
or a
550
nm
at
of NaNO2.
protein
in
PBS
and raised
was
[20]. for
stained
coupled
the to
(Pierce,
Rockford, Vecstatin
the
coverslipped
in Crystal
Preparation
cells
blocked
were
a 1:4,000
dilution
[30].
polyclonal
Corp.,
of the
staining Burlin-
Chamber Foster
of
sulfo-MBS
Laboratories,
The
(Biomedia
peptide
using
remainder (Vector
instructions.
mount
rabbit
a carrier
The
kit
pars-
0.1%
EEPKATRL as
IL)
in 2%
containing
ofa
carboxy-terminal
ABC-AP
fixed
in serum
thyroglobulin
used
by immunocytochemistry
the
mm,
with
against
determined
Briefly,
20
procedure
Mice
Slides
City,
were
CA).
of serum
were
killed
by
asphyxiation
in
tubes following centrifugation
CO2,
cardiac
of clotted
of N02
Serum
NOI
and
filings
(Fluka
Chemical
pH
in
9.6.
and
puncture.
blood
and
and N03
NO3
distilled
Each
were
blood
collected
Serum
stored
was
into obtained
at -70#{176}C.
water,
serum
M
filings the
NY)
and
0.1
.tL)
was
overnight
in were
mm,
and
(w/v)
assayed
hydroxide,
ZnSO4
clarified
tL
NO3
for NO2
content
with
solution, by centrifu-
incubated
reduce
Cd
succes-
up to 200
were
to
Briefly,
washed
M ammonium
supernatants order
[31]. were
brought
of 30%
15-30
for
The
samples
described
of 10 pi
temperature
in a microcentrifuge. Cd
HCI,
(10-50
by addition
at room
as
Ronkonkoma,
0.1
sample
in serum
assayed
Corp.,
deproteinated
reactions
with
to
NO2.
the After
by the
Griess
t-test,
paired
[4, 6, 20].
Data analysis
assayed
for
1996
were
analyzed
if P
