School,. Piscataway,. Bristol-Myers. Squibb. Pharmaceutical. Research. Institute,. Princeton,. New. Jersey, and. Philadelphia. Biomedical. Research. Institute,.
Production of nitric oxide and peroxynitrite acute endotoxemia Theresa Steven
M. Wizemann7 Carol R. Gardner, Jeffrey D. Laskin,’ Susan Quinones,’ K. Durham,’ Nancy L. Goller,’ S. Tsuyoshi Ohnishi,S and Debra L. Laskin*
*Rutgers
University
Piscataway,
Research
Abstract:
that
and
TUniversity
Bristol-Myers
Institute,
Nitric
tor that has endotoxin-induced present studies produced in found
Squibb
King
oxide
of Medicine Pharmaceutical
Dentistry
Research
is a short-lived
implicated tissue injury we determined the lung during
cytotoxic
media-
in the pathogenesis and septic shock. In whether this mediator acute endotoxemia.
injection
of
rats
with
of the is We
bacterially
oxide
present
produced
anion studies
by
to form
IMs
and
AMs
can
peroxynitrite.
demonstrate
New
Institute,
Jersey-Robert Princeton,
that
vated following acute endotoxemia nitrogen intermediates and that ute to inflammatory responses Biol. 56: 759-768; 1994.
Taken AMs
and
react
with
su-
together, IMs
to
produce both cell types in the lung. J.
the
are
dotoxin respond contribute
[3, 4], in vivo to the
Words:
polysaccharide
alveolar GM-CSF
macrop/zages
Wood
Johnson
Jersey,
and
Medical
School,
Philadelphia
Biomedical
.
interstitial
macrophages
suggesting that pulmonary macrophages to this bacterially derived toxin and may pathogenesis of tissue damage. Ou,r labora-
tony has been interested in analyzing the functional activity of lung macnophages in normal and pathophysiological states. These cells consist of two major subpopulations, alveolan macnophages (AMs), located primarily in the alveolar spaces, and interstitial macrophages (IMs), which reside within
acti-
[5,
6]. was of IMs
In previous associated recovered
studies we found with a twofold infrom the lung with
negligible effects on AMs [7]. In addition, IMs from endotoxemic rats exhibited morphologic and functional characteristics of activation including increased release of reactive oxygen intermediates and enhanced chemotactic and phagocytic activity [7]. The present studies were designed to determine whether these cells are also activated to produce nitric oxide. This reactive nitrogen intermediate is thought to play an important role in the inflammatory and vascular responses that are characteristic of endotoxemia and septic shock [8, 9]. As AMs and IMs are known to produce nitric oxide in response to inflammatory mediators released during endotoxemia [10-13], these cells may participate in nitric oxide-mediated hypotension and/or cytotoxicity.
MATERIALS
AND
METHODS
Animals Female,
specific
( 175-225
g)
NY).
pathogen-free
were
Animals
obtained were
Sprague-Dawley from
housed
Taconic
in
rats
(Germantown,
microisolator
cages
received sterile food and pynogen-free water Acute endotoxemia was induced by intravenous rats with 5 mg/kg Escherichia coli LPS (serotype Sigma Chemical Co., St. Louis, MO).
and
ad libitum. injection of 0128:B12,
reactive contribLeukoc. Abbreviations:
Key
New
the lung parenchyma that acute endotoxemia crease in the number
the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-’y and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric
of
of Prussia
been
intravenous
and
derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-’y (IFN-7) and LPS measured by nitrite accumulation in
peroxide
in the lung during
.
synthase;
lipo-
LPS,
AM,
interferon--y;
TNF-a
lipopolysaccharide;
alveolar EPR,
iNOS,
macrophages; electron
diethyldithiocarbamate;
IM,
paramagnetic N BT,
inducible
nitric
oxide
interstitial
macrophages;
IFN--y,
resonance
spectroscopy;
DETh,
nitroblue
tetrazolium;
H BSS,
Hanks’
balanced
salt solution; DNase, deoxyribonuclease; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TPA, l2-O-tetradecanoyl phorbol 13-acetate; L-NAME, M’-nitro-L-arginine methyl ester; L-NMMA, ?s-monomethyl-L-arginine; TNF-cr, tumor
INTRODUCTION Endotoxemia is associated with the release of a variety of reactive mediators and inflammatory cytokines from activated macrophages that have been implicated in tissue injury [1, 2]. The lung is highly sensitive to damage induced by en-
Journal
necrosis
factor
factor;
M-CSF,
Reprint cology,
a ;
GM-CSF,
macrophage
requests: Rutgers
Received
Debra University,
May
of Leukocyte
10,
1994;
Biology
granulocyte-macrophage colony-stimulating
Laskin, P.O.
accepted
factor;
Department Box
789,
July
Volume
colony-stimulating IL-l3,
interleukin-1j5.
of Pharmacology Piscataway,
22,
56,
NJ
and
Toxi-
08855-0789.
1994.
December
1994
759
Detection
of nitric oxide in vivo by electron
resonance
spectroscopy
Thirty minutes cutaneously mg/kg, Aldrich (40 mg FeSO4/200 cut into small
prior with
to sacrifice, animals diethyldithiocanbamate
Chemicals, Milwaukee, mg Na citrate/kg). pieces, and transferred
tubes (Wilmad Glass Electron panamagnetic were recorded using with a liquid nitrogen described [14].
paramagnetic
addition,
spin trapping were WI) Lungs into
injected (DETC,
sub400
and Fe-citrate were removed, 4-mm quartz
Measurement Cells phenol
Co., Buena, NJ) and frozen at -40#{176}C. resonance (EPR) spectra of the lung a Vanian E-109 spectrometer equipped flow system at 110 ± 2 K as previously
Frozen
sections
then wash,
rinsed and
of
lungs 0.2
were
fixed
in
ethanol-acetic
N HCI and 25 mg/ml in 4% paraformaldehyde.
with distilled prehybnidized
water, for 60
2 x NaC1-Na mm at room
acid
pnoteinase Sections citrate temperature
K at were
56,
December
for
x 105/well) were ned-free DMEM
production
inoculated supplemented
into with
96-well 10%
dishes FBS,
in 100
and northern
blot analysis
blot analysis
with a rabbit antibody against the COOH-terminal region of mouse macrophage iNOS (generously provided by Drs. Carl Nathan and Qiao-wen Xie, Cornell University, New York, NY). Antibody binding was detected using an alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G (IgG) secondary antibody and visualized with NBT and 5-bnomo-4-chloro-3-indolyl phosphate (Kirkegaard and Perry, Gaithersbung, MD).
Immunofluorescent Cells (1.5 x 105/well) chamber slides (Nunc, and then
lagenase (175 U/mI) for 60 mm had no effect on the functional activity of these cells. After purification, the AM population contained less than 5% polymorphonuclean leukocytes (PMNs) and the IM population less than 2-3% PMNs. Both macnophage populations were greater than 95% viable, as determined by trypan blue dye exclusion. In
Volume
positively
Cells (1 x 106/well), cultured overnight in 24-well dishes, were treated with IFN-y (1 U/ml) and LPS (10 ng/ml) and/or medium control. After 48 h of incubation at 37#{176}C,supernatants were removed and the cells were lysed in 100 pi of 1 mM potassium phosphate buffer, pH 6.8, containing 2 mM PMSF and 1% Nonidet P-40, v/v. Laemmli sample buffer was added to cellular extracts and samples were denatured by boiling for 5 mm. Protein concentration in macrophage extracts was determined using the method of Bradford [19] with bovine serum albumin as the standard. Cellular proteins (10 jig/well) were fractionated on 7.5% SDS polyacrylamide gels, transferred to nitrocellulose paper, and probed
salt solution (HBSS). For IM isolation, after lung tissue was cut into 500-jim slices using a tissue chopper (Bninkman Instruments, Westbury, NY). Tissue slices were disaggregated through a 280-jim metal sieve in 50 ml of HBSS containing 0.005% deoxynibonuclease (DNase) and then filtered through 30-sm nyIon mesh. This was followed by digestion in HBSS containing 60 U/ml collagenase type D, 0.01% DNase, and 10% fetal bovine serum (FBS) in a shaking 37#{176}Cwater bath for 45 mm, and then for 60 mm in HBSS containing 175 U/mI collagenase. The suspension was then filtered sequentially through 60- and 15-sm mesh. After washing four times (300& 4#{176}C,10 mm) with HBSS and 2% FBS, the cells were resuspended in DMEM containing 10% FBS, 100 ig/ml penicillin, and 100 U/mI streptomycin and incubated for 30 mm at 37#{176}Cin 100-mm tissue culture dishes (15-45 x 106 cells/dish). Nonadherent cells were removed from the dishes by gentle washing with warm medium. Adherent IMs were harvested by vigorous pipetting with cold HBSS and washed twice (300& 4#{176}C,10 mm). Pretreatment of AMs with col-
Biology
(2
Western
the
of Leukocyte
stained
a 1.1% agarose gel containing 2.2 M formaldehyde, blot transferred, and hybridized with [32P]dCTP-labeled munine macrophage iNOS cDNA or with a probe to /3-actin. cDNA probes were synthesized by random hexamer priming using a kit from Pharmacia (Piscataway, NJ). The hybridized filters were washed three times at 65#{176}Cin 2 x SSC, 0.1% sodium dodecyl sulfate (SDS) for 15 mm. Autoradiography was performed by exposure to Kodak X-OMAT film at - 70#{176}Cin the presence of intensifying screens.
AMs and IMs were isolated sequentially from perfused rat lung as previously described [10, 16]. AMs were obtained by lavage of the lung 12-14 times with 7-8 ml of warm Hanks
Journal
IMs,
Cells (2 x 106/well) were cultured in six-well dishes for 5 h with medium control or IFN-y (1 U/ml) and LPS (10 ng/ml). Total cellular RNA was then extracted by the guanidinium thiocyanate-phenol-chloroform method [18] using TRI reagent (Molecular Research Center, Cincinnati, OH). Aliquots containing equal amounts of RNA were electrophoresed in
in
Cell isolation
760
not
of nitric oxide
RNA isolation
(SSC)
50% Denhardt’s solution containing 0.5 mg/ml salmon sperm DNA, 0.25 mg/ml yeast tRNA, and 50% deionized formamide. Sections were then hybridized at 37#{176}Cin prehybridization solution containing dextran sulfate with and without 5 ng of digoxigenin-labeled munine macnophage inducible nitric oxide synthase (iNOS) eDNA [15] (Dr. James M. Cunningham, Harvard Medical School, Boston, MA), which was prepared using a Genius I DNA labeling and detection kit (Boehninger Mannheim, Indianapolis, IN). After 18 h, sections were washed in decreasing concentrations of SSC followed by 0.1 M Tnis-HC1, 0.15 M NaCl, pH 7.5. Immunoreactive sites were blocked with 10% normal sheep serum. Detection of the digoxigenin-labeled probe was accomplished using anti-digoxigenin-alkaline phosphatase and visualized using nitroblue tetrazolium (NBT).
balanced lavage, Mcllwain
but
tg/ml penicillin, and 100 U/ml streptomycin. This culture medium contains 84 mg/L L-anginine. After incubation overnight at 37#{176}C,the supennatants were removed and the cells nefed with various stimuli. Nitric oxide was quantified by nitrite accumulation in the culture medium using a procedune based on the Greiss reaction with sodium nitrite as the standard [17].
In situ hybridization (3:1), treated with 37#{176}C,and postfixed
AMs,
nonspecific estenase [16]. LPS treatment of the animals had no effect on the viability, nonspecific esterase activity, on punity of the macnophage subpopulations.
by
localization were cultured Naperville,
IFN-y (1 U/ml) on medium removed and the cells fixed permeabilization
with
overnight IL) with
in LPS
eight-well (10 ng/ml)
control. Supernatants with 1% formalin
lysolecithin
lin. Cells were then preincubated bumin in phosphate-buffered lowed by overnight incubation
1994
of iNOS
(4
l/ml)
with 1% saline (PBS) with rabbit
in
were followed 1%
forma-
bovine serum alfor 30 mm folantibody against
iNOS or control pooled normal rabbit sera. Cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. After 30 mm, the cells were washed and analyzed for fluorescence intensity on
Vectastain was utilized
a Meridian ACAS ian Instruments,
Reagents
Measurement Cells with
570 anchored Okemos, MI).
cell
analysis
system
(Mend-
of peroxynitrite
(5 x 105/well) were cultured LPS (10 mg/ml) and rat
overnight in 24-well dishes IFN-’y (1 U/ml) on medium
control. 12-O-Tetnadecanoylphonbol 13-acetate (TPA; 170 nM) was added to the wells 30 mm prior to analysis. Supernatants were then removed and the cells were washed with PBS-HEPES buffer and incubated for 5 mm at room temperature with dihydrorhodamine 123 (Molecular Probes, Eugene, OR) [20]. The cells were then washed with PBSHEPES buffer and analyzed on the Meridian ACAS 570. Peroxynitnite-mediated damage was analyzed in tissue sections using a specific antibody directed against nitrotyrosine (kindly provided by Dr. Harry Ischiropoulos, University of Pennsylvania) [21]. Rat lungs were perfused sequentially with HBSS, serum-free DMEM containing 0.5 sg/ml TPA, 0.05% NBT in DMEM and TPA, followed by HBSS. The lungs were then fixed with 10% buffered formalin instilled via the trachea and embedded in paraffin. Sections (6 tm) were preincubated with 1% bovine serum albumin in PBS for 30 mm followed by overnight incubation with primary antibody (1:2000) or control pooled normal rabbit sera. A
ABC Kit (Vector Laboratories, to visualize antibody binding.
HBSS, DMEM, DNase, Pv-nitro-L-arginine (L-NAME), and M-monomethyl-L-arginine were from Sigma Chemical Co., St. Louis, type D was purchased from Boehringer
Burlingame,
CA)
methyl ester (L-NMMA) MO. Collagenase Mannheim, Indi-
anapolis, Rancho 4
IN. FBS was obtained from Biocell Laboratories, Dominguez, CA. Rat IFN-’y (specific activity 106 U/mg) was purchased from Gibco, Grand Island, Mouse tumor necrosis factor a (TNF-a, specific activity 10 U/mg), mouse granulocyte-macrophage colony-
x
NY. 4
x
stimulating U/mg), (M-CSF,
factor human specific
(GM-CSF, macnophage activity
specific activity colony-stimulating 2 x 10 U/mg), and
intenleukin-1f3 (IL-1f3, specific from Genzyme, Cambridge, used in our studies contained prior to dilution.
1 x 106 factor mouse
activity 3.5 x 10 U/mg) were MA. All of the stock reagents less than 0.4 ng/ml endotoxin
Statistics Each experiment used one to three rats pen treatment group and was repeated two on three times. Nitric oxide data were analyzed using Student’s t-test. Results were considered statistically significant at P .05.
A
D
Fig.
1. In
after
induction
nm-alkaline
situ
hybridization of
acute
phosphatase
of iNOS endotoxemia and
visualized
mRNA were
in
the
lung
hybridized by
NBT
with staining.
during
endotoxemia.
Lung
a digoxigenin-labeled AMs
are
indicated
by
sections
obtained
eDNA
probe
arrows
and
Wizemann
for
type
Nitric
from
rats
0 h (A),
iNOS.
The
probe
II cells
by arrowheads.
oxide
and
6 h (C),
was
peroxynitrite
detected
24
h (B),
using
in the
or
48
h (D)
anti-digoxige-
lung
761
UNTREATED
RESULTS
Inducible nitric oxide synthase expression and nitric oxide production in the lung following induction of acute endotoxemia
lung,
in
particularly
high
levels
in
the
lung
in
intenstitium,
situ We inthe
ENDOTOXEMIC
RATS
30
w 0’)
In initial studies using a specific cDNA probe and hybridization, iNOS mRNA expression was examined. found that acute endotoxemia caused a time-dependent crease in expression of mRNA for iNOS throughout
RATS
AM 20
,
10 0) 0
0
.‘
.
.
9
,
.
type
II cells, and AMs. Expression of mRNA for iNOS was maximal at 24 h and returned to control levels by 48 h (Fig. 1). No mRNA for iNOS was evident in control rat lung. EPR spin trapping studies revealed that iNOS mRNA expression following induction of acute endotoxemia with nitric oxide production in the lung, within 6 h of treatment of the rats and levels by 48 h (Fig. 2 and not shown).
was correlated which was evident returned to control This was inhibited
0
1
10
100
1000
0
lFN
Fig.
3. Dose-dependent
IFN--.
g=2.039
AMs
and
of acute
endotoxemia,
_y alone
(El)
or
or 100 ng/ml and
in
LPS
analyzed
samples
from
(70-80%) the nitric
were
nitrite of
content. or
to the
trapping
agent,
signal
was
to
In further AMs and
48
1000
the
±
SE
(0),
collected
of triplicate
experiments.
animals with L-NAME,
(Fig.
10 mg/kg of 30 mm prior that
the
EPR
2).
by isolated
pulmonary
oxide production by and endotoxemic rats. In the absence of stimulation, AMs and IMs from untreated rats were found to produce low levels of nitric oxide (Fig. 3 and Table 1). IFN-y and LPS stimulated production of nitric oxide by both cell types in a doseand time-
nitric oxide and LPS,
9y
and LPS
than IMs in response IMs released more
to the nitric
in response to low concentrations (1-10 ng/ml). Induction of acute
4 mT
this increase in responsiveness 24 h, by 48 h nitric oxide production trations
6 h after
spin
were treatment
trapping
recorded of rats
EPR studies wave power,
5 x l0
quency,
100
762
and
were
Journal
of nitric
kHz
gain,
oxide
from samples with LPS (top)
conducted
using
0.32
modulation
of Leukocyte
mT
of IFN-’y endotoxemia
was induced
Although
Fig. 2. EPR EPR spectra
combination of IFNoxide spontaneously (1 U/mi) was
or as-
sociated with an increase in spontaneous, as well as IFN-yand IFN-y plus LPS-induced nitric oxide production by AMs isolated 12 h after treatment of the rats (Fig. 4). However, these effects appeared to be transient, and by 24-48 h nitric oxide production by AMs was at control levels (Fig. 4). As observed with AMs, production of nitric oxide by IMs in response to IFN-y (1 U/ml) plus LPS (10 ng/ml) was augmented 12 h after induction of acute endotoxemia.
L-NAME
+
nitric untreated
dependent manner, reaching a maximum with 100 U/ml IFN-y and 100 ng/ml LPS and after 48 h in culture (Fig. 3 and not shown). Furthermore, the combination of IFN-y and LPS was more effective in inducing nitric oxide production by AMs and IMs than either ofthese stimuli alone (Fig. 3). Although AMs were found to produce significantly more
CONTROL
LPS
we quantified isolated from
LPS
were
average
and
of IFN-
ng/ml
supernatants
are
LPS induction
concentrations
demonstrating
oxide
by h after
(#{149}), 10
LPS
similar
of the inhibitor,
production studies IMs
Data
DETC,
or
increasing
I ng/ml
four
nitric
100
production
rats
48 h of incubation,
by pretreatment oxide synthase due
with
with
three
oxide
untreated
cultured
(#{149}).After
for
of nitric from
combination
one
Nitric oxide macrophages
LPS
isolated
10
(U/mi)
induction
IMs,
1
in
the
of lungs or LPS
a Varian
modulation,
lung
during
endotoxemia.
obtained 0 h (middle) or plus L-NAME (bottom).
spectrometer:
9.22
20
GHz
mW
animals
fre-
frequency.
Biology
Volume
56,
December
IFN-y
(1-10
reduced (Figs.
3
and
U/ml)
plus
compared 4).
At
with 48
h,
however,
LPS
cells
(1
ng/ml)
was
from control these cells were
more responsive to high concentrations of IFN-y (100-1000 U/mI) plus LPS (10-100 ng/ml) than control cells. In contrast to AMs, spontaneous and IFN-y-induced nitric oxide
micro-
microwave
of
significantly
maintained for by low concen-
1994
1.
TABLE
Effects
of Various
Cytokines
Alone
or in Combination
with Nitrite
Untrea Stimulus
ted
IFN--y
on
(nmol/2
x
Nitric lO’5
Oxide
Production
by AMs
IMs’
cells)
rats
Endotox
Medium
and
emic
rats
Medium
IFN1
IFN--y
AMs None
0.3
±
0.1
IL-l3
1.6
±
0.2
1.3
±
0.3
0.2
±
0.1
±
0.9’
1.6
±
0.2k
TNF-a
0.2
±
0.1
±
0.6’
0.2
±
GM-CSF M-CSF
0.5 0.1
±
0.1
3.1
±
0.1
2.3
±
0.2’
0.4
±
0.1
0.2
None
1.5
±
0.2
2.8
lL-1fl TNF-a GM-CSF M-CSF
2.1 1.6 2.0
±
0.2
3.9
±
0.2
0.2
±
±
0.1
0.4
±
±
0.1
±
0.1
3.2
±
0.1
0.2
±
4.2
±
0.1
0.4
±
2.1
±
0.1
3.7
±
0.3
0.3
±
20.2 5.0
1.7
±
0.1
19.4
±
1.3’
0.1
4.0
±
0.6’
±
0.1
3.2
±
0.5
±
0.1
3.0
±
0.3
01d
1.2
±
02d
01d
2.7
±
Old
11
Old
3.6
± ±
01d
2.5
±
IMs
‘AMa
or
IMs,
isolated
U/ml), GM-CSF for nitrite content.
(25
from
untreated
rats
U/ml), or M-CSF (12.5 Each value is the average
or 48 h after
induction
U/mI),
or in combination
±
bSignificantly
different
from
cells
Significantly dSignificantly
different different
from from
cells cultured with untreated rats.
‘
cultured
in
alone
SE
of
two
medium
to
ofacute
four
endotoxemia, with
were
IFN--y
cultured
(I U/mI).
with
Supernatants
medium,
IL-l$3
were
(10
collected
U/mI),
01d
TNF-a
48 h later
and
0.3’
(100
analyzed
experiments.
alone.
IFN-y
alone.
production
20 ARA
by IMs
was
also
reduced
12-24
h after
induction
of acute endotoxemia (Fig. 4). We also found that nitric oxide production by AMs and IMs from both untreated and endotoxemic rats was blocked by the nitric oxide synthase inhibiton L-NMMA (Fig. 5 and not shown). These effects were dose dependent and reversed by the addition of excess
rivi
10 w
20
Co +1
30
Cl)
0
w
‘1
CTRL L-NMMA L-ARG
CI)
-H
IM
Cl)
20
L_Q)
Ln
l-0 Zx C’J 0
E C
0 0
0 0
24
TIME Fig. 4. Effects rophages.
FOLLOWING
ofacute
endotoxemia
or
isolated
AMs
IMs,
LPS
on nitric from
rats
oxide 0,
12, 24,
INJECTION production or
48
(h)
by lung h after
(P
