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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