The effects of nitric oxide in acute lung injury

w.elsevier.com/locate/vph

Vascular Pharmacology 43

The effects of nitric oxide in acute lung injury

Sanjay Mehta *

Centre for Critical Illness Research, Lawson Health Research Institute, Division of Respirology, London Health Sciences Center and Department of Medicine,

University of Western Ontario, London, Ontario, Canada N6A 4G5

Received 11 July 2005; accepted 3 August 2005

Abstract

Acute lung injury (ALI) is a common clinical problem associated with significant morbidity and mortality. Ongoing clinical and basic research

and a greater understanding of the pathophysiology of ALI have not been translated into new anti-inflammatory therapeutic options for patients

with ALI, or into a significant improvement in the outcome of ALI. In both animal models and humans with ALI, there is increased endogenous

production of nitric oxide (NO) due to enhanced expression and activity of inducible NO synthase (iNOS). This increased presence of iNOS and

NO in ALI contributes importantly to the pathophysiology of ALI. However, inhibition of total NO production or selective inhibition of iNOS has

not been effective in the treatment of ALI. We have recently suggested that there may be differential effects of NO derived from different cell

populations in ALI. This concept of cell-source-specific effects of NO in ALI has potential therapeutic relevance, as targeted iNOS inhibition

specifically to key individual cells may be an effective therapeutic approach in patients with ALI. In this paper, we will explore the potential role

for endogenous iNOS-derived NO in ALI. We will review the evidence for increased iNOS expression and NO production, the effects of non-

selective NOS inhibition, the effects of selective inhibition or deficiency of iNOS, and this concept of cell-source-specific effects of iNOS in both

animal models and human ALI.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Acute lung injury; Nitric oxide; Endothelial cell; Neutrophil; Macrophage; Permeability

1. Introduction

Acute lung injury (ALI) is a major clinical problem,

contributing to the mortality of up to 100,000 critically ill

patients in the United States annually. Intensive clinical and

basic science investigation has led to a greater understanding of

the pathophysiology of ALI. However, this knowledge has yet

to be translated into new anti-inflammatory therapeutic options

for patients with ALI, or into a significant improvement in the

outcome of ALI.

ALI is characterized by up-regulation of a host of

inflammatory mediators, including an increase in the expres-

sion and activity of inducible nitric oxide (NO) synthase

(iNOS), resulting in increased NO production. Although the

1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.vph.2005.08.013

Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress

syndrome; CLP, cecal ligation and perforation; cNOS, calcium-dependent NO

synthase; EC, endothelial cell; iNOS, calcium-independent NO synthase; LPS,

lipopolysaccharide; MPO, myeloperoxidase; 3-NT, 3-nitrotyrosine; NO, nitric

oxide; NOS, NO synthase; SP-A, surfactant association protein-A.

* Tel.: +1 519 667 6723; fax: +1 519 667 6552.

E-mail address: [email protected].

increased presence of iNOS and NO in ALI are thought to

contribute importantly to the pathophysiology of ALI, this

remains controversial. Indeed, non-selective inhibition of total

NO production or selective inhibition of iNOS has not been

consistently effective in the treatment of ALI. We have recently

proposed the concept of cell-source-specific effects of NO in

ALI. This idea has potential therapeutic relevance: rather than

inhibition of NO production in all cells, targeted iNOS

inhibition specifically to key individual cells may be a more

effective therapeutic approach in patients with ALI.

In this paper, we will explore the potential role for

endogenous iNOS-derived NO in ALI. We will review the

evidence for increased iNOS expression and NO production, the

effects of non-selective NOS inhibition, the effects of selective

inhibition or deficiency of iNOS, and the concept of cell-source-

specific effects of iNOS in both animal models and human ALI.

2. Acute lung injury

ALI is an important clinical problem which is characterized

by high-protein pulmonary edema and severe hypoxemia and

(2005) 390 – 403

ww

S. Mehta / Vascular Pharmacology 43 (2005) 390–403 391

which is associated with significant morbidity and mortality.

Indeed, ALI and its most severe form, the acute respiratory

distress syndrome (ARDS) have case-fatality rates of approx-

imately 40%, contributing to the mortality of up to 100,000

critically ill patients in the United States annually (Ware and

Matthay, 2000; Rubenfeld, 2003). Despite great advances in

our understanding of the inflammatory basis of the pathophys-

iology of ALI, this inflammation is not addressed by current

ALI therapy (Abraham, 1999; Glauser, 2000; Wheeler and

Bernard, 1999). Treatment of patients with ALI remains limited

to supportive care, e.g. supplemental oxygen and mechanical

ventilation, and thus, mortality has not improved significantly.

Many clinical disorders can result in ALI, either through a

direct pulmonary insult, e.g. gastric acid aspiration, or an

indirect pulmonary insult, e.g. sepsis. The most common cause

of ALI remains systemic sepsis, which occurs in 1% of

hospitalized patients (Bone et al., 1992; Angus et al., 2001).

Regardless of the initiating cause, the pathophysiology of ALI

is characterized by activation of many cells, including

endothelial cells, neutrophils, and macrophages, and increased

synthesis and release of many soluble mediators, such as TNFa

and NO (Bone et al., 1992; Bone, 1994; Neumann et al., 1999).

Neutrophils are central to the microvascular and tissue injury of

ALI (Heflin and Brigham, 1981; Tate and Repine, 1983;

Worthen et al., 1987; Downey et al., 1995; Wagner and Roth,

1999; Doerschuk, 2001). Pulmonary neutrophil infiltration is

the result of a cascade of steps including reduced neutrophil

deformability, pulmonary vascular sequestration (McCormack

et al., 2000), neutrophil–EC adhesion, and trans-EC migration

(Wagner and Roth, 1999; Doerschuk, 2001). Neutrophil

infiltration is facilitated by endothelial cell (EC) barrier

dysfunction (Furie et al., 1987; Garcia et al., 1998; Dull and

Garcia, 2002).

A key step in pulmonary neutrophil infiltration is neutro-

phil–EC adhesion, mediated through the specific interaction of

surface adhesion molecules on each cell, including neutrophil

h2-integrins (e.g. CD11b/CD18) and EC adhesions (e.g.

ICAM-1) (Hynes, 1992; Adams and Shaw, 1994; Hynes,

1992). Following neutrophil–EC adhesion, neutrophils subse-

quently migrate through inter-EC gaps in response to chemo-

taxins (e.g. IL-8 in humans, MIP-2 in mice) (Del Maschio et

al., 1996; Burns et al., 1997; Garcia et al., 1998; Haelens et al.,

1996), putatively through the homotypic interaction of

PECAM-1 on the surface of both neutrophils and EC (Muller

et al., 1993; Schenkel et al., 2004). Neutrophils may contribute

to EC barrier dysfunction and ALI through several mechan-

isms. Neutrophils release a variety of pro-inflammatory factors

(e.g. oxidants, proteolytic enzymes) that may directly injure EC

and contribute to pulmonary oxidant stress (Burg and Pillinger,

2001; Babior et al., 2002; Dull and Garcia, 2002; Allport et al.,

2002). Alternatively, neutrophil–EC adhesion may activate

intracellular signaling cascades in the EC which can result in

barrier dysfunction (Del Maschio et al., 1996; Huang et al.,

1993; Garcia et al., 1998).

The end-result of this complex, integrated inflammatory

response involving cells and soluble mediators is injury and

dysfunction of the alveolar capillary–epithelial permeability

barrier. In direct pulmonary insults, the alveolar epithelial cell

is the principal target, exhibiting features of injury and

apoptosis. In contrast, in sepsis-induced ALI, the pulmonary

microvascular EC is one of the key targets of the systemic

inflammatory response (Curzen et al., 1994; Reinhart et al.,

2002). The importance of EC injury in sepsis has recently been

highlighted, as markers of EC injury correlate with survival in

human sepsis and ALI (Ware et al., 2001).

In summary, injury and death of pulmonary microvascular

EC and alveolar epithelial cells leads to barrier dysfunction and

the leak of plasma into the pulmonary interstitium and alveolar

spaces, resulting in high-protein pulmonary edema, as well as

neutrophil infiltration into the lungs (Wang et al., 2002; Razavi

et al., 2002; Granger and Kubes, 1994; Garcia et al., 1998;

Wagner and Roth, 1999). We will consider the effects of NO on

each of the key pathophysiologic features of ALI, including EC

and alveolar epithelial cell injury and death, pulmonary oxidant

stress, pulmonary protein leak, and pulmonary neutrophil

infiltration.

3. Endogenous NO synthesis in ALI

NO in mammalian cells is produced by a family of NO

synthases (NOS). NOS isoenzymes are classified as low-

output, calcium-dependent (cNOS) or high-output, calcium-

independent, cytokine-inducible NOS (iNOS) isoforms. In the

lung, cNOS is constitutively expressed in EC (ecNOS) and in

neurones (nNOS). cNOS is important in pulmonary homeo-

stasis, including mediating direct and neurogenic pulmonary

vasodilatation, bronchodilation, and immune modulation (Scott

and McCormack, 1999a; Mehta and Drazen, 2000; Scott et al.,

1996). However, pulmonary cNOS has a lesser role in sepsis

and ALI, in which we and others have shown cNOS is down-

regulated (Razavi et al., 2002; Scott et al., 2002; Ermert et al.,

2002).

In contrast, iNOS expression is induced in the majority of

mammalian cell types upon exposure to inflammatory stimuli,

including cytokines, bacteria, and bacterial products (e.g.

lipopolysaccharide [LPS]) (Hibbs et al., 1988; Lamas et al.,

1991; Nakayama et al., 1992; Nathan, 1992; Kolls et al., 1994;

Robbins et al., 1994; Ermert et al., 2002). It should be noted

that there is evidence for constitutive expression of iNOS in

some cells, such as bronchial epithelial cells in humans (Guo et

al., 1995). With regard to ALI, all of the key cellular

participants (e.g. neutrophils, macrophages, EC, epithelial

cells) can express iNOS under inflammatory conditions

(Robbins et al., 1993; Nathan and Hibbs, 1991). Neutrophils

and macrophages express high levels of iNOS and are

important sources of iNOS-derived NO. Moreover, NO

mediates many of the actions of neutrophils and macrophages,

e.g. microbial and tumor cell killing, cytotoxicity (Hibbs et al.,

1988; McCall et al., 1989; Munoz-Fernandez et al., 1992;

Amin et al., 1995; Bratt and Gyllenhammar, 1995; MacMick-

ing et al., 1995; Evans et al., 1996; Tsukahara et al., 1998;

Boyle et al., 2000). Of relevance to ALI in humans, enhanced

iNOS expression and/or activity have been reported in

stimulated human neutrophils and macrophages (Munoz-

S. Mehta / Vascular Pharmacology 43 (2005) 390–403392

Fernandez et al., 1992; Amin et al., 1995; Bratt and

Gyllenhammar, 1995; Evans et al., 1996; Nicholson et al.,

1996; Wheeler et al., 1997; Tsukahara et al., 1998; Kobayashi

et al., 1998; Annane et al., 2000; Boyle et al., 2002; Kooguchi

et al., 2002).

3.1. Evidence for increased NO synthesis in ALI

3.1.1. Animal studies

The intense inflammatory response that characterizes ALI is

associated with increased expression and activity of inducible

nitric oxide (NO) synthase (iNOS), leading to increased NO

production (Table 1) (Mehta et al., 1999; Webert et al., 2000;

Weicker et al., 2001; Razavi et al., 2002; Wang et al., 2002;

Hickey et al., 2002; Koizumi et al., 2004). Increased NO

production in ALI and sepsis has been most commonly

assessed indirectly by measuring the levels of the oxidative

metabolites of NO (nitrites, nitrates) in plasma and bronch-

oalveolar lavage fluid. For example, induction of sepsis in rats

and mice by cecal ligation and perforation (CLP) is associated

with increased plasma nitrites/nitrates (Pheng et al., 1995; Scott

et al., 2002; Wang et al., 2002). Increased BAL nitrites/nitrates

have also been reported in various animal models of ALI, such

as intraperitoneal or intratracheal LPS injection (Pheng et al.,

1995; Li et al., 1998).

Increased NO production in ALI can also be directly

assessed by measuring levels of NO in exhaled breath (Mehta

et al., 1999; Weicker et al., 2001; Zegdi et al., 2002). For

example, systemic LPS administration was associated with

increased exhaled NO in spontaneously breathing mice and

Table 1

Evidence of increased NO production in animal models and in humans with septic

Reference Species Model of ALI

Pheng et al., 1995 Rat i.v. LPS+FNLP

Laszlo et al., 1995 Rat i.v. LPS

Hickey et al., 1997 Mouse i.v. LPS

Kobayashi et al., 1998 Human ARDS

Li et al., 1998 Rat i.t. LPS

Mehta et al., 1999 Pig i.v. LPS

Greenberg et al., 1999 Mouse i.t. LPS

Webert et al., 2000 Rat i.t. Pseudomonas

Okamoto et al., 2000 Rat CLP

Weicker et al., 2001 Mouse i.p. LPS

Sittipunt et al., 2001 Human ARDS

Zhu et al., 2001 Human ARDS

Razavi et al., 2002 Mouse CLP

Scott et al., 2002 Rat CLP

Hickey et al., 2002 Mouse i.v. LPS

Scumpia et al., 2002 Rat i.v. LPS

Soop et al., 2003 Human i.v. LPS

Mikawa et al., 2003 Rabbit i.v. LPS

Raykova et al., 2003 Rat i.p. heat-inactivated GBS

Tsubochi et al., 2003 Rat i.t. LPS

Baron et al., 2004a Mouse Nebulized LPS

Koizumi et al., 2004 Sheep i.v. LPS

Razavi et al., 2005 Mouse CLP

NO, nitric oxide; i.p., intraperitoneal; i.v., intravenous; NOS, NO synthase; iNOS

phenylalanine; ARDS, acute respiratory distress syndrome; i.t., intratracheal; BAL

streptococci; EPR, electron paramagnetic resonance; cGMP, cyclic guanosine mono

anesthetized rats (Weicker et al., 2001; Scumpia et al., 2002).

Similarly, in ALI due to intravenous hydrochloric acid

infusion, exhaled NO was significantly increased in association

with increased pulmonary iNOS expression (Haque et al.,

2003). However, exhaled NO may not necessarily correlate

with the degree of ALI, as suggested in a rat study of ALI due

to extracorporeal circulation (Zegdi et al., 2002).

In most animal models of ALI, especially in small

mammals, this increased NO production is associated with

evidence for increased iNOS expression and/or iNOS activity

in lung tissue (Table 1) (Webert et al., 2000; Razavi et al.,

2002; Wang et al., 2002; Scott et al., 1996; Ermert et al.,

2002; Hickey et al., 2002; Baron et al., 2004a), although this

may not be the case in large animals, e.g. pigs (Mehta et al.,

1999). Importantly, increased iNOS expression and/or activ-

ity have been documented in individual cell populations

including inflammatory cells, e.g. neutrophils, macrophages,

and also pulmonary parenchymal cells, e.g. epithelial,

endothelial, in animal models of ALI (Wang et al., 2001;

Hibbs et al., 1988; Haddad et al., 1994; Liu et al., 1997;

Wheeler et al., 1997; Kobayashi et al., 1998; Ermert et al.,

2002; Baron et al., 2004a). Moreover, there is evidence for

marked cell-specific differences in the regulation of iNOS vs.

cNOS expression in response to inflammatory stimuli, such

as LPS, in different pulmonary cell types (Ermert et al.,

2002).

3.1.2. Human studies

As in animals, there is evidence for increased NO

production in septic humans, as reflected by increased plasma

acute lung injury

Evidence

Increased plasma and BAL nitrites/nitrates

Increased pulmonary NOS activity

Increased pulmonary iNOS mRNA

Increased iNOS protein in alveolar macrophages

Increased BAL nitrites/nitrates

Increased exhaled NO

Increased lung homogenate nitrites/nitrates

Increased plasma nitrites/nitrates and pulmonary iNOS activity

Increased plasma nitrites/nitrates, pulmonary iNOS protein and activity


Increased BAL nitrites/nitrates, increased iNOS in alveolar macrophages

Increased BAL nitrites/nitrates


Increased plasma nitrites/nitrates, pulmonary iNOS mRNA and activity

Increased plasma nitrites/nitrates, pulmonary iNOS mRNA and protein

Increased exhaled NO, pulmonary iNOS mRNA and protein


Increased BAL and plasma nitrites/nitrates

Increased pulmonary iNOS mRNA

Increased pulmonary NO EPR signal, cGMP content, and iNOS protein

Increased iNOS protein in pulmonary parenchymal and inflammatory cells

Increased plasma and lung lymph nitrites/nitrates


, inducible NO synthase; LPS, lipopolysaccharide; FNLP, formyl–norleucly–

, bronchoalveolar lavage; CLP, cecal ligation and perforation; GBS, Group B

phosphate.


levels of nitrites/nitrates (Ochoa et al., 1991; Evans et al.,

1993; Groeneveld et al., 1996). Moreover, intravenous

infusion of LPS in healthy volunteers was associated with

increased exhaled NO levels (Soop et al., 2003). In patients

with sepsis, plasma levels of nitrites/nitrates correlate with

severity of illness (APACHE III score) and with measures of

multiple organ dysfunction, such as the Sequential Organ

Failure Assessment (SOFA) score (Ochoa et al., 1991;

Groeneveld et al., 1996; Rixen et al., 1997; Mitaka et al.,

2003).

However, there are few reports of increased NO production

in humans specifically with ALI or ARDS. Two studies have

reported increased nitrites/nitrates in bronchoalveolar lavage

(BAL) fluid in humans either at risk for ARDS or with

confirmed ARDS (Table 1) (Sittipunt et al., 2001; Zhu et al.,

2001).

There is also a paucity of data directly confirming that the

increased NO production in humans with sepsis and ALI is

derived from enhanced iNOS expression and/or activity in

human cells or lung tissue. The increase in BAL nitrites/

nitrates reported in humans either at risk for ARDS or with

confirmed ARDS was associated with increased immunohis-

tochemical staining for iNOS in macrophages isolated from

BAL fluid (Sittipunt et al., 2001). This confirms other reports

of increased human macrophage iNOS expression in ARDS

and pulmonary tuberculosis (Kobayashi et al., 1998; Choi et

al., 2002). Moreover, increased macrophage iNOS was also

found by immunohistochemistry in skin and subcutaneous

tissue samples from humans with sepsis due to soft-tissue

infection (Annane et al., 2000). Although increased neutro-

phil iNOS expression has not been specifically assessed in

patients with ALI or ARDS, it has been recognized that

human neutrophils can increase iNOS expression under

inflammatory conditions, such as sepsis and urinary tract

infection (Wheeler et al., 1997; Amin et al., 1995; Tsukahara

et al., 1998).

Thus, there is evidence for increased pulmonary NO

production due to enhanced pulmonary iNOS expression and/

or activity in both animal models of ALI and in human patients

with ALI/ARDS.

4. Effects of NO in ALI

As reviewed above, there is much evidence that NO

production is increased in ALI. Based on this evidence,

many studies have assessed the effects of NOS inhibition

in animal models of sepsis and ALI (Table 2). The earliest

studies used l-arginine analogues, such as l-nitro-l-

arginine methyl ester (l-NAME) and l-monomethyl-l-

arginine (l-NMMA), which are non-isoform selective

NOS inhibitors and thus, inhibit both cNOS and iNOS.

Subsequently, the effects of iNOS-selective inhibitors, such

as aminoguanidine and 1400W, were assessed in ALI.

Moreover, the severity and characteristics of ALI have been

compared between wild-type and iNOS�/� mice in order to

address the effects of iNOS in ALI. More recently, the

concept of cell-source-specific effects of iNOS has been

supported by studies dissecting out the effects of iNOS

from different cells in ALI.

4.1. General effects of NO in ALI


The earliest studies pursued a role for NO in ALI through

assessment of the effects of non-selective NOS inhibitors. In

one of the first studies to suggest a role for NO in ALI, l-

NAME and l-NMMA attenuated pulmonary microvascular

protein leak in a rat model of ALI due to hindlimb ischemia–

reperfusion (Seekamp et al., 1993). Similarly, non-selective

NOS inhibitors attenuated pulmonary vascular permeability in

various models of ALI including pulmonary radiation, com-

plement deposition, or oxidant stress (Mulligan et al., 1991;

Nozaki et al., 1997; Akai et al., 1998). Interestingly, non-

selective NOS inhibition also attenuated the increase in

alveolar epithelial permeability following intratracheal LPS in

rats (Li et al., 1998).

However, the effects of NO in ALI are controversial, as non-

selective NOS inhibition worsened lung permeability and

injury in other animal models of ALI (Terada et al., 1996;

Pheng et al., 1995; Aaron et al., 1998; Becker et al., 1998; Liu

et al., 2001). Moreover, NOS inhibition may have differential

effects on the different pathophysiologic features of ALI. For

example, although NOS inhibition reduced pulmonary micro-

vascular protein leak and edema in several models of ALI,

pulmonary neutrophil infiltration was not reduced by NOS

inhibition in these models (Mulligan et al., 1991; Seekamp et

al., 1993; O’Donovan et al., 1995; Li et al., 1998). Such

conflicting data on the effects of NO in ALI suggested that

endogenous NO may either prevent or contribute to ALI,

depending on concentration of NO, local physicochemical

conditions, and perhaps the exact cellular sources of NO.

As a result, non-selective NOS inhibition has been of little

overall benefit in animal models of ALI, especially in sepsis.

Non-selective NOS inhibitors improve mean arterial pressure

in septic animals (Kilbourn and Belloni, 1990; Cobb et al.,

1992; Walker et al., 1995; Tracey et al., 1995), but total NOS

inhibition reduces tissue viability, impairs organ function, and

often increases mortality (Harbrecht et al., 1992; Cobb et al.,

1992; Minnard et al., 1994; Lopez et al., 2004). The adverse

effects of non-selective NOS inhibition therapy in sepsis and

ALI are likely due to loss of the beneficial, homeostatic

effects of cNOS-derived NO. Indeed, ecNOS overexpression

in transgenic mice was associated with less mortality and

hypotension, as well as less severe ALI, including reduced

pulmonary neutrophil infiltration, edema, and histologic

injury following intraperitoneal LPS administration (Yama-

shita et al., 2000).


As in animal models, several studies of non-selective NOS

inhibition in humans with sepsis have been reported. A

consistent observation has been an increase in mean arterial

pressure, which may be associated with a reduced requirement

for exogenous vasopressors (Nava et al., 1991; Avontuur et al.,

Table 2

Effects of NOS inhibition or deficiency in animal models of septic acute lung injury

Reference Species Model of ALI Method of NOS inhibition/

deficiency

Effects of NOS inhibition/Deficiency

Pheng et al., 1995 Mouse i.p. LPS+FNLP i.v. AG Enhanced septic increase in BAL protein blunted septic increase in

pulmonary neutrophil infiltration

Laszlo et al., 1995 Rat i.v. LPS s.c. l-NAME/l-NMMA Attenuated LPS-induced increase in lung microvascular albumin leak

Hickey et al., 1997 Mouse i.v. LPS iNOS�/� mice Increased septic pulmonary neutrophil infiltration

Li et al., 1998 Rat i.t. LPS i.p. l-NMMA Attenuated LPS-induced increase in lung epithelial permeability

Aaron et al., 1998 Rat i.v. LPS i.v. l-NAME Greater septic increase in interstitial cellular infiltrate

Kristof et al., 1998 Mouse i.v. LPS iNOS�/� mice Attenuated LPS-induced increase in lung microvascular protein leak,

lung W/D weight ratio, and lung 3-NT

Greenberg et al., 1999 Mouse i.t. LPS+Klebsiella iNOS�/� mice, i.p. l-NIL Increased LPS- and Klebsiella-induced pulmonary neutrophil

infiltration, and enhanced Klebsiella clearance

Hinder et al., 1999 Sheep i.v. LPS i.v. l-NAME Aggravated LPS-induced increase in extravascular lung water

Okamoto et al., 2000 Rat CLP s.c. ONO-1714 Attenuated septic increase in histologic peribronchiolar edema and

interstitial area, no effect on lung W/D weight ratio

Evgenov et al., 2000 Sheep i.v. LPS i.v. AG Enhanced LPS-induced increases in lung lymph flow and protein

clearance, but unchanged lung water; improved pulmonary gas

exchange

Wang et al., 2002 Mouse CLP iNOS�/� mice, i.v. 1400W Abrogated septic increase in pulmonary microvascular albumin leak;

unchanged septic increase in pulmonary neutrophil infiltration

Volman et al., 2002 Mouse i.p. LPS+zymosan iNOS�/� mice, i.p. AG No effect on LPS/zymosan-induced increases in lung weight and

histologic ALI score

Speyer et al., 2003 Mouse i.t. LPS iNOS�/� mice Enhanced LPS-induced increases in pulmonary neutrophil infiltration

and pulmonary MCP-1 production

Mikawa et al., 2003 Rabbit i.v. LPS i.v. ONO-1714 Attenuated LPS-induced increases in lung W/D weight ratio,

histologic ALI score, and BAL neutrophils and albumin

Raykova et al., 2003 Rat i.p. heat-inactivated GBS i.p. AG Attenuated septic increases in lung IL-6 and MIP-2 mRNA expression

Tsubochi et al., 2003 Rat i.t. LPS s.c. AG Reversed the LPS-induced early decrease and late increase in alveolar

fluid clearance

Razavi et al., 2004 Mouse CLP iNOS�/� mice Attenuated septic pulmonary microvascular neutrophil sequestration;

enhanced septic increase in pulmonary neutrophil infiltration

Baron et al., 2004a Mouse Nebulized LPS iNOS�/� mice Attenuated LPS-induced increases in lung tissue resistance and

elastance attenuated LPS-induced surfactant dysfunction and loss of

SP-B expression

Koizumi et al., 2004 Sheep i.v. LPS i.v. ONO-1714 Attenuated LPS-induced impaired oxygenation and increase in lung

lymph flow

Razavi et al., 2005 Mouse CLP iNOS�/� mice Eliminated septic increase in pulmonary 8-isoprostanes and 3-NT

ALI, acute lung injury; AG, aminoguanidine; l-NAME, l-nitro-arginine methyl ester; l-NMMA, l-N-monomethyl arginine; W/D, wet-to-dry weight ratio; 3-NT, 3-

nitro-tyrosine; l-NIL, l-N6-iminoethyllysine; ONO-1714, (1S, 5S, 6R, 7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4,1,0]heptane HCl; 1400W, N-(3-

(aminomethyl)benzyl) acetamidine; MCP-1, monocyte chemoattractant peptide-1; IL-6, interleukin-6; MIP-2, macrophage inflammatory protein-2; SP-B,

surfactant-association protein-B; for others, see legend in Table 1.


1998; Grover et al., 1999). However, increased mean arterial

pressure appears to be mediated by systemic vasoconstriction,

often in concert with reduced blood flow, impaired cardiac

function, and possibly increased mortality (Nava et al., 1991;

Avontuur et al., 1998; Grover et al., 1999; Watson et al., 2004).

Unfortunately, there are no data on the effects of non-selective

NOS inhibition on the overall severity or the individual

pathophysiologic features of ALI in humans. A role for NO

in human pulmonary artery EC injury has been suggested in

vitro. When these EC were co-cultured with LPS-activated

monocytes, EC injury was attenuated by addition of carboxy-

PTIO, a NO scavenger (Hidaka et al., 1997). In contrast, when

stimulated neutrophils were co-cultured with these human EC,

treatment with either carboxy-PTIO or l-NAME exacerbated

neutrophil-dependent EC injury, putatively due to loss of

ecNOS-derived NO. Thus, as in animal models of ALI, NO

may worsen or improve human pulmonary EC injury, depend-

ing on the cellular sources and amount of NO released.

4.2. Specific effects of iNOS-derived NO in ALI


As reviewed above, iNOS expression is increased in many

cells in the lung and endogenous NO production is markedly

increased in ALI. The role of iNOS in ALI has been addressed

in various animal models of ALI through study of the effects of

selective iNOS inhibition and through characterization of

differences in ALI in wild-type mice vs. iNOS�/� ‘‘knockout’’

mice. Most studies demonstrate that iNOS-derived NO is

clearly involved in the pathophysiology of ALI. However, the

precise role of iNOS in ALI remains controversial. Indeed,

some studies suggest that iNOS-derived NO may not play a

role in ALI, or may attenuate the severity of ALI (Pheng et al.,

1995; Volman et al., 2002). For example, iNOS inhibition or

deficiency had no effect on increased lung weight and

macroscopic pulmonary hemorrhage in mice treated with

intraperitoneal LPS and zymosan (Volman et al., 2002).


It is generally accepted that the weight of the published

evidence suggests that iNOS contributes significantly to the

key pathophysiologic features of ALI, such as protein-rich

pulmonary edema, oxidant stress, and surfactant dysfunction

(Mikawa et al., 1998; Scott and McCormack, 1999b; Mehta et

al., 1999; Bateman et al., 2001; Wang et al., 2002; MacMicking

et al., 1995; Groeneveld et al., 1996; Ruetten et al., 1996;

Kristof et al., 1998; Vincent et al., 2000; Fakhrzadeh et al.,

2002; Baron et al., 2004a; Razavi et al., 2005). Many studies

have reported that iNOS mediates microvascular injury and

protein-rich pulmonary edema in a variety of animal models of

ALI, such as endotoxemia, sepsis, ozone inhalation, intra-

pleural carrageenan, and combined cutaneous burn and smoke

inhalation (Table 2) (Kristof et al., 1998; Cuzzocrea et al.,

2000; Wang et al., 2002; Fakhrzadeh et al., 2002; Enkhbaatar et

al., 2003). For example, in mice undergoing CLP, sepsis-

induced pulmonary protein leak was completely iNOS-depen-

dent, as it was abolished both by selective iNOS inhibition with

1400W in wild-type (iNOS+/+) mice, and by iNOS deficiency

in iNOS�/� mice (Wang et al., 2002). Similarly, pulmonary

oxidant stress, reflected by lung 8-isoprostane levels, was

evident in septic wild-type but not iNOS�/� mice (Razavi et

al., 2005). Moreover, in a recent study of mice exposed to

nebulized LPS, iNOS�/� mice were protected from LPS-

induced pulmonary physiologic dysfunction (increased tissue

resistance and elastance), surfactant dysfunction, and reduced

SP-B mRNA and protein expression (Baron et al., 2004a).

The effects of iNOS inhibition or deficiency on pulmonary

leukocyte infiltration in ALI are unresolved. In our studies of

septic mice, although iNOS deficiency or inhibition reduced

the severity of ALI, e.g. decreased pulmonary microvascular

protein leak and oxidant stress, pulmonary leukocyte infiltra-

tion was not attenuated (Wang et al., 2002; Razavi et al., 2004,

2005). Indeed, some studies have even demonstrated increased

pulmonary leukocyte infiltration in ALI following iNOS

inhibition or in iNOS�/� mice (Hickey et al., 1997; Razavi et

al., 2004). These conflicting data may be due to the technique

for measurement of pulmonary neutrophil infiltration in ALI.

For example, a common technique is the measurement of

pulmonary tissue myeloperoxidase (MPO) activity. However,

recent evidence suggests that neutrophils release MPO into the

circulation or directly at the surface of EC (Baldus et al., 2001;

Eiserich et al., 2002). Thus, increased pulmonary MPO in ALI

may reflect, in part, adherence of circulating or locally released

MPO to pulmonary microvascular EC, and may not be due

solely to pulmonary neutrophil infiltration.

MPO in a homogenate of pulmonary tissue also ignores the

potential differential presence of neutrophils in discrete

compartments, such as the pulmonary microvasculature,

interstitial space, and bronchoalveolar airspace. Indeed, in

mice with CLP-induced sepsis, we have consistently found

similar pulmonary tissue MPO activity in wild-type and

iNOS�/� mice (Wang et al., 2002; Razavi et al., 2004,

2005). However, when we analyzed neutrophil presence in

the different compartments, there were striking differences

between septic wild-type and iNOS�/� mice (Razavi et al.,

2004). Blood neutrophil counts were similar in septic iNOS�/�

mice vs. wild-type mice. However, pulmonary intravital

videomicroscopy during mechanical ventilation demonstrated

a significant increase in pulmonary microvascular sequestered

neutrophils in septic wild-type mice, which was attenuated in

iNOS�/� mice. Moreover, the septic increase in bronchoalveo-

lar lavage neutrophil counts in wild-type mice was significantly

enhanced in iNOS�/� mice. Thus, the presence of iNOS in

septic ALI appears to increase pulmonary microvascular

sequestration and possibly EC adhesion, and appears to retard

the trans-EC migration and pulmonary tissue infiltration of

neutrophils.

A few studies have reported variable effects of NO and

NOS inhibition, depending on the time point at which ALI is

assessed. For example, selective inhibition of iNOS with

ONO-1714 improved oxygenation, lung mechanics, pulmo-

nary edema, and leukocyte sequestration when administered

prior to or within 2 h after induction of ALI by intravenous

LPS injection in mechanically ventilated rabbits, but not if

administered 3 or 4 h after LPS (Mikawa et al., 2003). In

contrast, iNOS inhibition with ONO-1714 in rats with CLP-

induced ALI improved survival and pulmonary histology,

including edema, leukocyte infiltration, and microvascular

thrombi, when administered 12 h after CLP, but not when

administered immediately or 6 h after CLP (Okamoto et al.,

2000). Moreover, a recent study reported that iNOS reduced

alveolar fluid clearance at 6 h, but increased it at 24 h after

intratracheal LPS in rats (Tsubochi et al., 2003). The

conflicting data on the effects of NO at different time points

in the natural history of ALI remain unresolved, but may also

reflect the different effects of iNOS-derived NO from discrete

cell populations.

The effect of iNOS inhibition or iNOS deficiency on

mortality in the setting of ALI is also controversial. For

example, in septic models of ALI, iNOS inhibition or

deficiency does not improve and may even increase mortality

(Laubach et al., 1995; MacMicking et al., 1995; Cobb et al.,

1999). In contrast, others have suggested improved mortality

due to iNOS inhibition in septic rats and mice (Hollenberg et

al., 2000; Okamoto et al., 2000; Baron et al., 2004b).


iNOS-dependent pathophysiologic changes in human ALI

have been difficult to demonstrate directly. Indeed, the effects

of iNOS-selective inhibitors have not been reported in

humans with ALI/ARDS. However, there are data that

suggest that human cells mediate injury through iNOS

expression and activity, similar to animal cells. For example,

as described for animal cells, human neutrophils also mediate

bacterial killing through iNOS (Malawista et al., 1992). In

addition, in human pulmonary artery EC co-cultured with

human neutrophils, neutrophil-dependent EC injury was

iNOS-dependent (Hidaka et al., 1997). In our own studies

of microvascular EC isolated directly from human lungs and

co-cultured with human neutrophils, we have found that

cytomix-stimulated trans-EC protein leak was clearly iNOS-

dependent, being eliminated by pre-treatment with either

1400W or l-NAME.


Thus, iNOS-derived NO likely contributes significantly to

the pathophysiology of human ALI, although direct evidence is

not yet available.

4.3. Effects of cell-source-specific iNOS in ALI


Several lines of evidence have suggested the possibility of

differential effects of iNOS-derived NO from different cells in

ALI. As above, this includes the controversial effects of iNOS

inhibition or deficiency on the individual pathophysiologic

effects of ALI, as well as on mortality. Similarly, the effects of

iNOS-derived NO from different cells may explain the

conflicting data from iNOS inhibition at different time points

after the induction of ALI. Over the past few years, we and

other groups have designed and pursued studies to dissect out

the potential differential effects of iNOS in different cells in the

setting of murine septic ALI.

In order to investigate this concept of cell-source-specific

effects of iNOS-derived NO in ALI, we have generated and

used reciprocal bone marrow transplanted iNOS chimeric mice

(Wang et al., 2001, 2002; Razavi et al., 2004, 2005). Following

bone-marrow ablative irradiation in a wild-type mouse, the

bone marrow is reconstituted over 4–6 weeks following

intravenous infusion of donor bone marrow from an iNOS�/

� mouse. In this mouse, all parenchymal cells, e.g. endothelial

and epithelial, retain the iNOS+/+ genotype of the host.

However, all bone marrow derived inflammatory cells, e.g.

neutrophils and macrophages, were depleted and replaced by

donor iNOS�/� cells. Thus, this mouse is designated a � to +

iNOS chimera, and has iNOS expression restricted to host

parenchymal cells only. The reciprocal + to � iNOS chimera is

also generated by transplantation of iNOS+/+ bone marrow into

irradiated iNOS�/� mice, thus restricting iNOS expression to

bone marrow-derived inflammatory cells only. These two

reciprocal iNOS chimeric mice are genetically identical except

for the cellular population distribution of iNOS. In order to

assess the role of iNOS specifically in parenchymal vs.

inflammatory cells, we compared pulmonary iNOS expres-

sion/activity and the pathophysiologic features of sepsis-

induced ALI between these two reciprocal iNOS chimeric

mice.

Firstly, there is good evidence that marked differences exist

in the level of iNOS expression and NO production from these

two different cell populations. In intraperitoneal LPS-treated

mice, we found that pulmonary iNOS expression/activity was

predominantly localized to parenchymal cells (¨70%) rather

than inflammatory cells (Wang et al., 2001). Moreover,

systemic NO production, reflected by plasma levels of

nitrites/nitrates, was almost exclusively derived from paren-

chymal cell iNOS: LPS treatment was associated with a

marked increase in plasma nitrites/nitrates in � to + iNOS

chimeras (iNOS localized to parenchymal cells), similar to

LPS-treated wild-type mice, with only a negligible LPS-

induced increase in plasma nitrites/nitrates in + to � iNOS

chimeras (iNOS localized to inflammatory cells). Other groups

have confirmed and supported our findings (Hickey et al.,

2002; Baron et al., 2004a). Interestingly, in a more clinically

relevant disease model of sepsis, induced by CLP, pulmonary

iNOS expression/activity were similar in septic + to � and � to

+ iNOS chimeric mice, suggesting a significant level of iNOS

expression in both inflammatory and parenchymal cells (Wang

et al., 2002; Razavi et al., 2005). Of note, systemic NO

production was still largely dependent on parenchymal cell

iNOS in CLP-induced sepsis. Thus, both pulmonary paren-

chymal and inflammatory cells express iNOS in ALI. Asses-

sing ALI in the two reciprocal iNOS chimeric mice can then

differentiate the specific effects of iNOS from these distinct

cell populations.

We initially compared pulmonary microvascular protein

leak following CLP-induced sepsis in the reciprocal iNOS

chimeric mice (Wang et al., 2002). Sepsis was associated with

significant pulmonary microvascular protein leak in + to �iNOS chimeras (iNOS localized to inflammatory cells), similar

in degree to septic wild-type mice. In contrast, sepsis-induced

pulmonary microvascular protein leak was completely abro-

gated in � to + iNOS chimeras (iNOS localized to parenchy-

mal cells), similar to iNOS�/� mice. Moreover, we recently

showed that sepsis-induced pulmonary oxidant stress, as

reflected by tissue 8-isoprostane levels, was also present in +

to �, but not in � to + iNOS chimeras (Razavi et al., 2005).

Thus, iNOS-derived NO from inflammatory cells (e.g. neu-

trophils, macrophages) totally accounted for sepsis-induced

pulmonary protein leak and oxidant stress, key pathophysio-

logic features of ALI, with no obvious contribution of

parenchymal cell iNOS. Ongoing studies in our lab are

dissecting out the individual roles of macrophage and

neutrophil iNOS in murine ALI. Preliminary evidence suggests

that both macrophage and neutrophil iNOS contribute impor-

tantly to pulmonary microvascular EC injury and protein leak

(Fig. 1).

Based on the work of others using these same bone-marrow

transplanted iNOS chimeric mice, selective iNOS expression in

parenchymal cells may also contribute to some pathophysio-

logic features of ALI. In mice exposed to nebulized LPS, lung

physiologic dysfunction and reduced SP-B expression were

specifically dependent on iNOS expression in parenchymal

cells (Baron et al., 2004a).

We have also recently reported cell-source-specific effects

of iNOS on the trans-endothelial migration and pulmonary

infiltration of neutrophils (Razavi et al., 2004). The specific

presence of iNOS in inflammatory cells alone in + to � iNOS

chimeric mice was associated with sepsis-induced pulmonary

microvascular sequestration and increased BAL neutrophils

similar to levels seen in wild-type mice. In contrast, septic

pulmonary microvascular sequestration was reduced and BAL

neutrophilia increased in � to + iNOS chimeric mice, similar

to the findings in septic iNOS�/� mice. Thus, iNOS

specifically in inflammatory cells (e.g. neutrophils and

macrophages) promotes pulmonary microvascular neutrophil

sequestration, yet inhibits the trans-EC migration and

pulmonary infiltration of neutrophils in septic ALI. Despite

the significant levels of parenchymal cell iNOS, this does not

appear to modulate pulmonary neutrophil kinetics in septic

Fig. 1. Schematic representation of the cell-source specific effects of inducible nitric oxide synthase (iNOS) in septic acute lung injury. iNOS in neutrophils (PMN)

and in alveolar macrophages stimulates (+) pulmonary microvascular leak of plasma protein. PMN iNOS also stimulates PMN sequestration in the pulmonary

microvasculature, but inhibits (�) trans-endothelial PMN migration. iNOS in types I and II alveolar epithelial cells (AEC I and II) appears to mediate surfactant

dysfunction in ALI. The role of iNOS in endothelial cells (EC) in ALI is presently unknown.


mice. It is noteworthy that previous reports have suggested a

similar inhibitory effect of neutrophil iNOS on peritoneal

neutrophil migration during peritoneal sepsis (Tavares-Murta

et al., 1998; Crosara-Alberto et al., 2002; Benjamim et al.,

2002). We have tried to corroborate our in vivo findings in

murine septic ALI by co-culturing mouse lung microvascular

EC with neutrophils in vitro (Razavi et al., 2004). Indeed,

neutrophil iNOS significantly inhibited cytokine-stimulated

trans-EC neutrophil migration, whereas EC iNOS presence

had no effect. Of note, cytokine-stimulated neutrophil

migration across a matrix in the absence of EC was similar

for wild-type and iNOS�/� neutrophils. Mechanistically, this

suggests that neutrophil iNOS inhibits trans-EC neutrophil

migration through modulation of neutrophil–EC interaction,

and not through alteration of neutrophil deformability or

intrinsic migratory capacity.


It is becoming clear that the concept of cell-source-specific

effects of iNOS has relevance to human disease as well. For

example, compartmentalization of iNOS expression specifical-

ly to macrophages has been reported in necrotic skin and

muscle from humans with sepsis due to cellulitis (Annane et

al., 2000). Moreover, an immunohistochemical study has

suggested cell-source-specific nitrosative and oxidative effects

of iNOS-derived NO in human inflammatory bowel disease

(Tomobuchi et al., 2001).

It remains unknown whether this concept of cell-source-

specific effects of iNOS has clinical relevance for human

ALI/ARDS. In our ongoing studies of human lung

microvascular EC co-cultured with human neutrophils and

macrophages, we are attempting to dissect out the effects of

iNOS in neutrophils and macrophages in septic human EC

injury.

4.4. Mechanisms of NO’s effects in ALI


NO exerts its effects through many different mechanisms,

such as the activation of soluble guanylate cyclase and

generation of cGMP, nitrosation of protein thiols, and

formation of nitrosyl complexes with metal ions in the active

sites of key proteins (Ignarro, 1991; Abu-Soud and Hazen,

2000; Mohr et al., 1999). In the setting of ALI and increased

iNOS expression and enhanced NO production, NO’s effects

are largely not mediated through cGMP.

The interaction of NO with reactive oxygen species (ROS)

is a major mechanism of NO’s effects in the setting of

inflammation associated with increased production of both NO

and ROS, as in ALI. The rapid, diffusion-limited reaction of

NO with superoxide anion, generating the potent oxidants

peroxynitrite and subsequently hydroxyl radical, may be one of

the most important mechanisms by which iNOS-derived NO

exerts pro-oxidant, pathophysiologic effects in ALI (Rubbo et

al., 1994; Radi et al., 1991a,b; Beckman et al., 1990). The

nitration of tyrosine residues in specific proteins, yielding 3-

nitrotyrosine residues, is a putative marker of NO-dependent

oxidant stress and peroxynitrite action.

The enhanced presence of 3-nitrotyrosine in pulmonary

tissue and BAL fluid has been reported in several animal

models of ALI (Cuzzocrea et al., 1999; Tsuji et al., 2000;

Fakhrzadeh et al., 2002; Chen et al., 2003; Laffey et al., 2004).

Moreover, pulmonary 3-nitrotyrosine presence in ALI appears

to be iNOS-dependent (Tsuji et al., 2000; Fakhrzadeh et al.,


2002; Chen et al., 2003; Razavi et al., 2005). Similar to

pulmonary microvascular leak and oxidant stress, we recently

showed in reciprocal iNOS chimeric mice that pulmonary 3-

nitrotyrosine presence in septic ALI was specifically dependent

on the expression of iNOS in inflammatory cells, and not in

pulmonary parenchymal cells (Razavi et al., 2005). Finally,

exogenous peroxynitrite induces EC barrier dysfunction, in

association with actin and h-catenin nitration (Knepler et al.,

2001).

NO may also contribute to the pathophysiology of ALI

through modulation of the production of other inflammatory

mediators, including cytokines and chemokines (Walley et

al., 1999; Raykova et al., 2003; Speyer et al., 2003). As

above, the precise effects of NO on pulmonary cytokine

production are unclear. For example, selective iNOS

inhibition by aminoguanidine decreased pulmonary IL-6

and MIP-2 expression in rats with sepsis-induced ALI due

to Group B Streptococci (Raykova et al., 2003). In contrast,

l-NAME increased pulmonary IL-6 and TNF-a expression in

mice with ALI following intratracheal LPS (Walley et al.,

1999). Similarly, increased lung neutrophil infiltration was

associated with greater BAL levels of CC chemokines (e.g.

MCP-1 and MCP-3) in iNOS�/� vs. wild-type mice

following intratracheal LPS (Speyer et al., 2003). Moreover,

in vitro studies supported cell-source-specific effects of

iNOS in modulation of chemokine production: iNOS�/�

dermal microvascular EC and peritoneal macrophages both

demonstrated greater chemokine production following LPS/

IFN-gamma stimulation than wild-type cells (Speyer et al.,

2003).


As in animal models of ALI, there is evidence that NO-

dependent peroxynitrite production may be the mechanism of

iNOS-dependent ALI in humans. Enhanced immunohisto-

chemical staining for 3-nitrotyrosine is found in isolated cells

and tissues from humans with ARDS (Haddad et al., 1994;

Kooy et al., 1995; Kobayashi et al., 1998; Sittipunt et al.,

2001). Increased 3-nitrotyrosine residues have also been found

in proteins isolated from BAL in humans with ALI/ARDS

(Lamb et al., 1999; Zhu et al., 2001). For example, nitration of

surfactant-associated protein (SP)-A was recently identified

(Zhu et al., 2001). Importantly, protein nitration can modify the

function of many proteins, and appears to be associated with

impairment of the immune modulatory functions of SP-A, such

as mannose binding ability and opsonization of pathogens (Zhu

et al., 1996, 1998).

There is also direct evidence for peroxynitrite production by

human neutrophils following LPS treatment (Gagnon et al.,

1998). Moreover, exogenous nitrite and peroxynitrite were

found to activate the nuclear transcription factor kB (NF-kB) in

cultured human alveolar epithelial cells, similar to the

activation seen upon treatment of these cells with BAL fluid

from ARDS patients (Nys et al., 2003). Thus, endogenous

iNOS-dependent local pulmonary production of peroxynitrite

may be responsible for alveolar epithelial and capillary

endothelial injury in ALI/ARDS.

5. Summary and future directions

Over the past few years, it has become clear that NO likely

plays a significant role in the pathophysiology of ALI in both

animals and humans. There is much evidence both in animal

models and in humans with ALI for enhanced pulmonary

expression of iNOS and increased NO production. Although

non-selective NOS inhibitors can effectively reduce NO

overproduction in animals and humans with ALI, the lack of

clinical benefit of this therapeutic approach has tempered

excitement around use of these inhibitors in human ALI/

ARDS. Similarly, complete iNOS inhibition or genetic

deficiency has not been consistently beneficial in animal

models of ALI. Rapidly evolving new ideas on the concept

of cell-source-specific effects of iNOS-derived NO in ALI may

explain in large part the lack of benefit in previous studies of

non-selective NOS inhibition or complete iNOS inhibition in

all cells.

Given the clear importance of endogenous NO in ALI, there

is an increasing focus on the measurement of increased NO

levels in human ALI using novel approaches, such as collection

and analysis of exhaled breath and exhaled breath condensate.

Such approaches are very likely to advance our understanding

of the effects of NO specifically in human ALI/ARDS.

Moreover, this increasing understanding of the role of NO in

ALI will likely also push us further towards therapeutic

manipulation of the NO system in human ALI/ARDS. Indeed,

ongoing studies will identify the specific effects of iNOS from

individual cells, e.g. neutrophils, macrophages. We suggest this

knowledge will permit targeted iNOS inhibition in these

individual cells and will be more beneficial in ALI than non-

targeted, widespread iNOS inhibition in many cells. Indeed,

this potential therapeutic approach is a reality, as novel

liposomal protein-grafting technology has already permitted

specific delivery of chemotherapeutic agents to individual

cancer cells (Hatipoglu et al., 1998; Dagar et al., 2001).

References

Aaron, S.D., Valenza, F., Volgyesi, G., Mullen, J.B., Slutsky, A.S., Stewart,

T.E., 1998. Inhibition of exhaled nitric oxide production during sepsis does

not prevent lung inflammation. Critical Care Medicine 26, 309–314.

Abraham, E., 1999. Why immunomodulatory therapies have not worked in

sepsis. Intensive Care Medicine 25, 556–566.

Abu-Soud, H.M., Hazen, S.L., 2000. Nitric oxide modulates the

catalytic activity of myeloperoxidase. Journal of Biological Chemistry

275, 5425–5430.

Adams, D.H., Shaw, S., 1994. Leucocyte–endothelial interactions and

regulation of leucocyte migration. Lancet 343, 831–836.

Akai, M., Ishizaki, T., Matsukawa, S., Shigemori, K., Miyamori, I., 1998.

Leukotoxin (9,10-epoxy-12-octadecenoate) Impairs energy and redox

state of isolated perfused rat lung. Free Radical Biology & Medicine 25,

596–604.

Allport, J.R., Lim, Y.C., Shipley, J.M., Senior, R.M., Shapiro, S.D.,

Matsuyoshi, N., Vestweber, D., Luscinskas, F.W., 2002. Neutrophils from

MMP-9- or neutrophil elastase-deficient mice show no defect in transen-

dothelial migration under flow in vitro. Journal of Leukocyte Biology 71,

821–828.

Amin, A.R., Attur, M., Vyas, P., Leszczynska-Piziak, J., Levartovsky, D.,

Rediske, J., Clancy, R.M., Vora, K.A., Abramson, S.B., 1995. Expression of


nitric oxide synthase in human peripheral blood mononuclear cells and

neutrophils. Journal of Inflammation 47, 190–205.

Angus, D.C., Linde-Zwirble, W.T., Lidicker, J., Clermont, G., Carcillo, J.,

Pinsky, M.R., 2001. Epidemiology of severe sepsis in the United States:

analysis of incidence, outcome, and associated costs of care. Critical Care

Medicine 29, 1303–1310.

Annane, D., Sanquer, S., Sebille, V., Faye, A., Djuranovic, D., Raphael, J.C.,

Gajdos, P., Bellissant, E., 2000. Compartmentalised inducible nitric-oxide

synthase activity in septic shock. Lancet 355, 1143–1148.

Avontuur, J.A., Nolthenius, R.P., Vanbodegom, J.W., Bruining, H.A., 1998.

Prolonged inhibition of nitric oxide synthesis in severe septic shock — a

clinical study. Critical Care Medicine 26, 660–667.

Babior, B.M., Lambeth, J.D., Nauseef, W., 2002. The neutrophil NADPH

oxidase. Archives of Biochemistry and Biophysics 397, 342–344.

Baldus, S., Eiserich, J.P., Mani, A., Castro, L., Figueroa, M., Chumley, P., Ma,

W., Tousson, A., White, C.R., Bullard, D.C., Brennan, M.L., Lusis, A.J.,

Moore, K.P., Freeman, B.A., 2001. Endothelial transcytosis of myeloper-

oxidase confers specificity to vascular ECM proteins as targets of tyrosine

nitration. Journal of Clinical Investigation 108, 1759–1770.

Baron, R.M., Carvajal, I.M., Fredenburgh, L.E., Liu, X., Porrata, Y., Cullivan,

M.L., Haley, K.J., Sonna, L.A., De Sanctis, G.T., Ingenito, E.P., Perrella,

M.A., 2004. Nitric oxide synthase-2 down-regulates surfactant protein-B

expression and enhances endotoxin-induced lung injury in mice. The

FASEB Journal 18, 1276–1278.

Baron, R.M., Carvajal, I.M., Liu, X., Okabe, R.O., Fredenburgh, L.E., Macias,

A.A., Chen, Y.H., Ejima, K., Layne, M.D., Perrella, M.A., 2004. Reduction

of nitric oxide synthase 2 expression by distamycin A improves survival

from endotoxemia. Journal of Immunology 173, 4147–4153.

Bateman, R.M., Jagger, J.E., Sharpe, M.D., Ellsworth, M.L., Mehta, S., Ellis,

C.G., 2001. Erythrocyte deformability is a nitric oxide-mediated factor in

decreased capillary density during sepsis. American Journal of Physiology.

Heart and Circulatory Physiology 280, H2848–H2856.

Becker, K., Savvides, S.N., Keese, M., Schirmer, R.H., Karplus, P.A., 1998.

Enzyme inactivation through sulfhydryl oxidation by physiologic NO-

carriers. Nature Structural Biology 5, 267–271.

Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A.,

1990. Apparent hydroxyl radical production by peroxynitrite: implica-

tions for endothelial injury from nitric oxide and superoxide. Proceedings

of the National Academy of Sciences of the United States of America 87,

1620–1624.

Benjamim, C.F., Silva, J.S., Fortes, Z.B., Oliveira, M.A., Ferreira, S.H., Cunha,

F.Q., 2002. Inhibition of leukocyte rolling by nitric oxide during sepsis

leads to reduced migration of active microbicidal neutrophils. Infection and

Immunity 70, 3602–3610.

Bone, R.C., 1994. Sepsis and its complications: the clinical problem. Critical

Care Medicine 22, S8–S11.

Bone, R.C., Balk, R.A., Cerra, F.B., Dellinger, R.P., Fein, A.M., Knaus,

W.A., Schein, R.M., Sibbald, W.J., 1992. Definitions for sepsis and

organ failure and guidelines for the use of innovative therapies in

sepsis. The ACCP/SCCM Consensus Conference Committee. American

College of Chest Physicians/Society of Critical Care Medicine. Chest

101, 1644–1655.

Boyle, W.A., Parvathaneni, L.S., Bourlier, V., Sauter, C., Laubach, V.E., Cobb,

J.P., 2000. iNOS gene expression modulates microvascular responsiveness

in endotoxin-challenged mice. Circulation Research 87, E18–E24.

Boyle, J.J., Weissberg, P.L., Bennett, M.R., 2002. Human macrophage-induced

vascular smooth muscle cell apoptosis requires NO enhancement of

Fas/Fas –L interactions. Arteriosclerosis, Thrombosis, and Vascular Biol-

ogy 22, 1624–1630.

Bratt, J., Gyllenhammar, H., 1995. The role of nitric oxide in lipoxin A4-

induced polymorphonuclear neutrophil-dependent cytotoxicity to human

vascular endothelium in vitro. Arthritis & Rheumatism 38, 768–776.

Burg, N.D., Pillinger, M.H., 2001. The neutrophil: function and regulation in

innate and humoral immunity. Clinical Immunology 99, 7–17.

Burns, A.R., Walker, D.C., Brown, E.S., Thurmon, L.T., Bowden, R.A., Keese,

C.R., Simon, S.I., Entman, M.L., Smith, C.W., 1997. Neutrophil transen-

dothelial migration is independent of tight junctions and occurs preferen-

tially at tricellular corners. Journal of Immunology 159, 2893–2903.

Chen, L.W., Wang, J.S., Chen, H.L., Chen, J.S., Hsu, C.M., 2003. Peroxynitrite

is an important mediator in thermal injury-induced lung damage. Critical

Care Medicine 31, 2170–2177.

Choi, H.S., Rai, P.R., Chu, H.W., Cool, C., Chan, E.D., 2002. Analysis of nitric

oxide synthase and nitrotyrosine expression in human pulmonary tubercu-

losis. American Journal of Respiratory and Critical Care Medicine 166,

178–186.

Cobb, J.P., Natanson, C., Hoffman, W.D., Lodato, R.F., Banks, S., Koev, C.A.,

Solomon, M.A., Elin, R.J., Hosseini, J.M., Danner, R.L., 1992. N omega-

amino-l-arginine, an inhibitor of nitric oxide synthase, raises vascular

resistance but increases mortality rates in awake canines challenged with

endotoxin. Journal of Experimental Medicine 176, 1175–1182.

Cobb, J.P., Hotchkiss, R.S., Swanson, P.E., Chang, K., Qiu, Y., Laubach, V.E.,

Karl, I.E., Buchman, T.G., 1999. Inducible nitric oxide synthase (iNOS)

gene deficiency increases the mortality of sepsis in mice. Surgery 126,

438–442.

Crosara-Alberto, D.P., Darini, A.L., Inoue, R.Y., Silva, J.S., Ferreira, S.H.,

Cunha, F.Q., 2002. Involvement of NO in the failure of neutrophil

migration in sepsis induced by Staphylococcus aureus. British Journal of

Pharmacology 136, 645–658.

Curzen, N.P., Griffiths, M.J., Evans, T.W., 1994. Role of endothelium in

modulating the vascular response to sepsis. Clinical Science 86, 359–374.

Cuzzocrea, S., Costantino, G., Mazzon, E., Caputi, A.P., 1999. Beneficial

effects of raxofelast (IRFI 016), a new hydrophilic vitamin E-like

antioxidant, in carrageenan-induced pleurisy. British Journal of Pharmacol-

ogy 126, 407–414.

Cuzzocrea, S., McDonald, M.C., Filipe, H.M., Costantino, G., Mazzon, E.,

Santagati, S., Caputi, A.P., Thiemermann, C., 2000. Effects of tempol, a

membrane-permeable radical scavenger, in a rodent model of carrageenan-

induced pleurisy. European Journal of Pharmacology 390, 209–222.

Dagar, S., Sekosan, M., Lee, B.S., Rubinstein, I., Onyuksel, H., 2001. VIP

receptors as molecular targets of breast cancer: implications for targeted

imaging and drug delivery. Journal of Controlled Release 74, 129–134.

Del Maschio, A., Zanetti, A., Corada, M., Rival, Y., Ruco, L., Lampugnani,

M.G., Dejana, E., 1996. Polymorphonuclear leukocyte adhesion triggers the

disorganization of endothelial cell-to-cell adherens junctions. Journal of

Cell Biology 135, 497–510.

Doerschuk, C.M., 2001. Mechanisms of leukocyte sequestration in inflamed

lungs. Microcirculation 8, 71–88.

Downey, G.P., Fialkow, L., Fukushima, T., 1995. Initial interaction of

leukocytes within the microvasculature: deformability, adhesion, and

transmigration. New Horizons 3, 219–228.

Dull, R.O., Garcia, J.G.N., 2002. Leukocyte-induced microvascular perme-

ability: how contractile tweaks lead to leaks. Circulation Research 90,

1143–1144.

Eiserich, J.P., Baldus, S., Brennan, M.L., Ma, W., Zhang, C., Tousson, A.,

Castro, L., Lusis, A.J., Nauseef, W.M., White, C.R., Freeman, B.A., 2002.

Myeloperoxidase, a leukocyte-derived vascular NO oxidase. Science 296,

2391–2394.

Enkhbaatar, P., Murakami, K., Shimoda, K., Mizutani, A., Traber, L., Phillips,

G.B., Parkinson, J.F., Cox, R., Hawkins, H., Herndon, D., Traber, D., 2003.

The inducible nitric oxide synthase inhibitor BBS-2 prevents acute lung

injury in sheep after burn and smoke inhalation injury. American Journal of

Respiratory and Critical Care Medicine 167, 1021–1026.

Ermert, M., Ruppert, C., Gunther, A., Duncker, H.R., Seeger, W., Ermert, L.,

2002. Cell-specific nitric oxide synthase-isoenzyme expression and

regulation in response to endotoxin in intact rat lungs. Laboratory

Investigation 82, 425–441.

Evans, T., Carpenter, A., Kinderman, H., Cohen, J., 1993. Evidence of

increased nitric oxide production in patients with the sepsis syndrome.

Circulatory Shock 41, 77–81.

Evans, T.J., Buttery, L.D., Carpenter, A., Springall, D.R., Polak, J.M., Cohen,

J., 1996. Cytokine-treated human neutrophils contain inducible nitric

oxide synthase that produces nitration of ingested bacteria. Proceedings of

the National Academy of Sciences of the United States of America 93,

9553–9558.

Evgenov, O.V., Hevroy, O., Bremnes, K.E., Bjertnaes, L.J., 2000. Effect

of aminoguanidine on lung fluid filtration after endotoxin in awake


sheep. American Journal of Respiratory & Critical Care Medicine 162,

465–470.

Fakhrzadeh, L., Laskin, J.D., Laskin, D.L., 2002. Deficiency in inducible nitric

oxide synthase protects mice from ozone-induced lung inflammation and

tissue injury. American Journal of Respiratory Cell and Molecular Biology

26, 413–419.

Furie, M.B., Naprstek, B.L., Silverstein, S.C., 1987. Migration of neutro-

phils across monolayers of cultured microvascular endothelial cells. An

in vitro model of leucocyte extravasation. Journal of Cell Science 88,

161–175.

Gagnon, C., Leblond, F.A., Filep, J.G., 1998. Peroxynitrite production by

human neutrophils, monocytes and lymphocytes challenged with lipopoly-

saccharide. FEBS Letters 431, 107–110.

Garcia, J.G., Verin, A.D., Herenyiova, M., English, D., 1998. Adherent

neutrophils activate endothelial myosin light chain kinase: role in

transendothelial migration. Journal of Applied Physiology 84, 1817–1821.

Glauser, M.P., 2000. Pathophysiologic basis of sepsis: considerations for future

strategies of intervention. Critical Care Medicine 28, S4–S8.

Granger, D.N., Kubes, P., 1994. The microcirculation and inflammation:

modulation of leukocyte–endothelial cell adhesion. Journal of Leukocyte

Biology 55, 662–675.

Greenberg, S.S., Jie, O.Y., Zhao, X.F., Parrish, C., Nelson, S., Giles, T.D.,

1999. Effects of ethanol on neutrophil recruitment and lung host defense in

nitric oxide synthase I and nitric oxide synthase II knockout mice.

Alcoholism: Clinical & Experimental Research 23, 1435–1445.

Groeneveld, P.H., Kwappenberg, K.M., Langermans, J.A., Nibbering, P.H.,

Curtis, L., 1996. Nitric oxide (NO) production correlates with renal

insufficiency and multiple organ dysfunction syndrome in severe sepsis.

Intensive Care Medicine 22, 1197–1202.

Grover, R., Zaccardelli, D., Colice, G., Guntupalli, K., Watson, D., Vincent,

J.L., 1999. An open-label dose escalation study of the nitric oxide synthase

inhibitor, N(G)-methyl-l-arginine hydrochloride (546C88), in patients with

septic shock. Glaxo Wellcome International Septic Shock Study Group.

Critical Care Medicine 27, 913–922.

Guo, F.H., De Raeve, H.R., Rice, T.W., Stuehr, D.J., Thunnissen, F.B.,

Erzurum, S.C., 1995. Continuous nitric oxide synthesis by inducible nitric

oxide synthase in normal human airway epithelium in vivo. Proceedings of

the National Academy of Sciences of the United States of America 92,

7809–7813.

Haddad, I.Y., Pataki, G., Hu, P., Galliani, C., Beckman, J.S., Matalon, S., 1994.

Quantitation of nitrotyrosine levels in lung sections of patients and animals

with acute lung injury. Journal of Clinical Investigation 94, 2407–2413.

Haelens, A., Wuyts, A., Proost, P., Struyf, S., Opdenakker, G., Van Damme, J.,

1996. Leukocyte migration and activation by murine chemokines. Immu-

nobiology 195, 499–521.

Haque, I.U., Huang, C.J., Scumpia, P.O., Nasiroglu, O., Skimming, J.W., 2003.

Intravascular infusion of acid promotes intrapulmonary inducible nitric

oxide synthase activity and impairs blood oxygenation in rats. Critical Care

Medicine 31, 1454–1460.

Harbrecht, B.G., Billiar, T.R., Stadler, J., Demetris, A.J., Ochoa, J.B., Curran,

R.D., Simmons, R.L., 1992. Nitric oxide synthesis serves to reduce hepatic

damage during acute murine endotoxemia. Critical Care Medicine 20,

1568–1574.

Hatipoglu, U., Gao, X., Verral, S., Sejourne, F., Pitrak, D., Alkan-Onyuksel, H.,

Rubinstein, I., 1998. Sterically stabilized phospholipids attenuate human

neutrophils chemotaxis in vitro. Life Sciences 63, 693–699.

Heflin, A.C.J., Brigham, K.L., 1981. Prevention by granulocyte depletion of

increased vascular permeability of sheep lung following endotoxemia.

Journal of Clinical Investigation 68, 1253–1260.

Hibbs, J.B.J., Taintor, R.R., Vavrin, Z., Rachlin, E.M., 1988. Nitric oxide: a

cytotoxic activated macrophage effector molecule. Biochemical and

Biophysical Research Communications 157, 87–94.

Hickey, M.J., Sharkey, K.A., Sihota, E.G., Reinhardt, P.H., MacMicking, J.D.,

Nathan, C., Kubes, P., 1997. Inducible nitric oxide synthase-deficient mice

have enhanced leukocyte–endothelium interactions in endotoxemia. The


Hickey, M.J., Sihota, E., Amrani, A., Santamaria, P., Zbytnuik, L.D., Ng, E.S.,

Ho, W., Sharkey, K.A., Kubes, P., 2002. Inducible nitric oxide synthase

(iNOS) in endotoxemia: chimeric mice reveal different cellular sources in

various tissues. The FASEB Journal 16, 1141–1143.

Hidaka, K., Mitsuyama, T., Furuno, T., Tanaka, T., Hara, N., 1997. The role

of nitric oxide in human pulmonary artery endothelial cell injury

mediated by neutrophils. International Archives of Allergy and Immunol-

ogy 114, 336–342.

Hinder, F., Stubbe, H.D., Van Aken, H., Waurick, R., Booke, M., Meyer, J.,

1999. Role of nitric oxide in sepsis-associated pulmonary edema. American

Journal of Respiratory & Critical Care Medicine 159, 252–257.

Hollenberg, S.M., Broussard, M., Osman, J., Parrillo, J.E., 2000. Increased

microvascular reactivity and improved mortality in septic mice lacking

inducible nitric oxide synthase. Circulation Research 86, 774–778.

Huang, A.J., Manning, J.E., Bandak, T.M., Ratau, M.C., Hanser, K.R.,

Silverstein, S.C., 1993. Endothelial cell cytosolic free calcium regulates

neutrophil migration across monolayers of endothelial cells. Journal of Cell

Biology 120, 1371–1380.

Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell

adhesion. Cell 69, 11–25.

Ignarro, L.J., 1991. Signal transduction mechanisms involving nitric oxide.

Biochemical Pharmacology 41, 485–490.

Kilbourn, R.G., Belloni, P., 1990. Endothelial cell production of nitrogen

oxides in response to interferon-H in combination with tumor necrosis

factor, interleukin-1, or endotoxin. Journal of the National Cancer Institute

82, 772–776.

Knepler, J.L.J., Taher, L.N., Gupta, M.P., Patterson, C., Pavalko, F., Ober,

M.D., Hart, C.M., 2001. Peroxynitrite causes endothelial cell monolayer

barrier dysfunction. American Journal of Physiology 281, C1064–C1075.

Kobayashi, A., Hashimoto, S., Kooguchi, K., Kitamura, Y., Onodera, H., Urata,

Y., Ashihara, T., 1998. Expression of inducible nitric oxide synthase and

inflammatory cytokines in alveolar macrophages of ARDS following

sepsis. Chest 113, 1632–1639.

Koizumi, T., Ogasawara, H., Yamamato, H., Tsushima, K., Ruan, Z., Jian, M.,

Fujimoto, K., Kubo, K., 2004. Effect of ONO1714, a specific inducible

nitric oxide synthase inhibitor, on lung lymph filtration and gas exchange

during endotoxemia in unanesthetized sheep. Anesthesiology 101, 59–65.

Kolls, J., Xie, J., LeBlanc, R., Malinski, T., Nelson, S., Summer, W.,

Greenberg, S.S., 1994. Rapid induction of messenger RNA for nitric oxide

synthase II in rat neutrophils in vivo by endotoxin and its suppression by

prednisolone. Proceedings of the Society for Experimental Biology and

Medicine 205, 220–229.

Kooguchi, K., Kobayashi, A., Kitamura, Y., Ueno, H., Urata, Y., Onodera, H.,

Hashimoto, S., 2002. Elevated expression of inducible nitric oxide synthase

and inflammatory cytokines in the alveolar macrophages after esophagect-

omy. Critical Care Medicine 30, 71–76.

Kooy, N.W., Royall, J.A., Ye, Y.Z., Kelly, D.R., Beckman, J.S., 1995. Evidence

for in vivo peroxynitrite production in human acute lung injury. American

Journal of Respiratory and Critical Care Medicine 151, 1250–1254.

Kristof, A.S., Goldberg, P., Laubach, V., Hussain, S.N.A., 1998. Role of

inducible nitric oxide synthase in endotoxin-induced acute lung

injury. American Journal of Respiratory and Critical Care Medicine 158,

1883–1889.

Laffey, J.G., Honan, D., Hopkins, N., Hyvelin, J.M., Boylan, J.F., Mcloughlin,

P., 2004. Hypercapnic acidosis attenuates endotoxin-induced acute lung

injury. American Journal of Respiratory and Critical Care Medicine 169,

46–56.

Lamas, S., Michel, T., Brenner, B.M., Marsden, P.A., 1991. Nitric oxide

synthesis in endothelial cells — evidence for a pathway inducible by TNF-

alpha. American Journal of Physiology 261, C634–C641.

Lamb, N.J., Quinlan, G.J., Westerman, S.T., Gutteridge, J.C., Evans, T.W., 1999.

Nitration of proteins in bronchoalveolar lavage fluid from patients with acute

respiratory distress syndrome receiving inhaled nitric oxide. American


Laszlo, F., Whittle, B.J., Evans, S.M., Moncada, S., 1995. Association of

microvascular leakage with induction of nitric oxide synthase: effects of

nitric oxide synthase inhibitors in various organs. European Journal of

Pharmacology 283, 47–53.

Laubach, V.E., Shesely, E.G., Smithies, O., Sherman, P.A., 1995. Mice lacking

inducible nitric oxide synthase are not resistant to lipopolysaccharide-


induced death. Proceedings of the National Academy of Sciences of the

United States of America 92, 10688–10692.

Li, X.Y., Donaldson, K., Macnee, W., 1998. Lipopolysaccharide-induced

alveolar epithelial permeability — the role of nitric oxide. American


Liu, H.W., Anand, A., Bloch, K., Christiani, D., Kradin, R., 1997. Expression

of inducible nitric oxide synthase by macrophages in rat lung. American


Liu, P., Xu, B., Hock, C.E., 2001. Inhibition of nitric oxide synthesis by l-name

exacerbates acute lung injury induced by hepatic ischemia– reperfusion.

Shock 16, 211–217.

Lopez, A., Lorente, J.A., Steingrub, J., Bakker, J., McLuckie, A., Willatts, S.,

Brockway, M., Anzueto, A., Holzapfel, L., Breen, D., Silverman, M.S.,

Takala, J., Donaldson, J., Arneson, C., Grove, G., Grossman, S., Grover, R.,

2004. Multiple-center, randomized, placebo-controlled, double-blind study

of the nitric oxide synthase inhibitor 546C88: effect on survival in patients

with septic shock. Critical Care Medicine 32, 21–30.

MacMicking, J.D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D.S.,

Trumbauer, Stevens, K., Xie, Q.W., Sokol, K., Hutchinson, N., 1995.

Altered responses to bacterial infection and endotoxic shock in mice lacking

inducible nitric oxide synthase (published erratum appears in Cell 1995 Jun

30;81(7):following 1170). Cell 81, 641–650.

Malawista, S.E., Montgomery, R.R., van Blaricom, G., 1992. Evidence for

reactive nitrogen intermediates in killing of staphylococci by human

neutrophil cytoplasts. A new microbicidal pathway for polymorphonuclear

leukocytes. Journal of Clinical Investigation 90, 631–636.

McCall, T.B., Broughton-Smith, N.K., Palmer, R.M., Whittle, B.J., Mon-

cada, S., 1989. Synthesis of nitric oxide from l-arginine by neutrophils:

release and interaction with superoxide anions. Biochemical Journal 261,

293–298.

McCormack, D.G., Mehta, S., Tyml, K., Scott, J.A., Potter, R., Rohan, M.,

2000. Pulmonary microvascular changes during sepsis: evaluation using

intravital videomicroscopy. Microvascular Research 60, 131–140.

Mehta, S., Drazen, J.M., 2000. Bronchodilator actions of nitric oxide and

related compounds. In: Belvisi, M.G., Mitchell, J.A. (Eds.), Nitric Oxide in

Pulmonary Processes: Role in Physiology and Pathophysiology of Lung

Disease. Birkhauser Verlag AG, London, pp. 127–149.

Mehta, S., Javeshghani, D., Datta, P., Levy, R.D., Magder, S., 1999. Porcine

endotoxemic shock is associated with increased expired nitric oxide.


Mikawa, K., Nishina, K., Tamada, M., Takao, Y., Maekawa, N., Obara, H.,

1998. Aminoguanidine attenuates endotoxin-induced acute lung injury in

rabbits. Critical Care Medicine 26, 905–911.

Mikawa, K., Nishina, K., Takao, Y., Obara, H., 2003. ONO-1714, a nitric oxide

synthase inhibitor, attenuates endotoxin-induced acute lung injury in

rabbits. Anesthesia and Analgesia 97, 1751–1755.

Minnard, E.A., Shou, J., Naama, H., Cech, A., Gallagher, H., Daly, J.M., 1994.

Inhibition of nitric oxide synthesis is detrimental during endotoxemia.

Archives of Surgery 129, 142–148.

Mitaka, C., Hirata, Y., Yokoyama, K., Wakimoto, H., Hirokawa, M., Nosaka,

T., Imai, T., 2003. Relationships of circulating nitrite/nitrate levels to

severity and multiple organ dysfunction syndrome in systemic inflamma-

tory response syndrome. Shock 19, 305–309.

Mohr, S., Hallak, H., de Boitte, A., Lapetina, E.G., Brune, B., 1999.

Nitric oxide induced S-glutathionylation and inactivation of glyceralde-

hyde-3-phosphate dehydrogenase. Journal of Biological Chemistry 274,

9427–9430.

Muller, W.A., Weigl, S.A., Deng, X., Phillips, D.M., 1993. PECAM-1 is

required for transendothelial migration of leukocytes. Journal of Experi-

mental Medicine 178, 449–460.

Mulligan, M.S., Hevel, J.M., Marletta, M.A., Ward, P.A., 1991. Tissue injury

caused by deposition of immune complexes is l-arginine dependent.

Proceedings of the National Academy of Sciences of the United States of

America 88, 6338–6342.

Munoz-Fernandez, M.A., Fernandez, M.A., Fresno, M., 1992. Activation of

human macrophages for the killing of intracellular Trypanosoma cruzi by

TNF-alpha and IFN-gamma through a nitric oxide-dependent mechanism.

Immunology Letters 33, 35–40.

Naakayama, D.K., Geller, D.A., Lowenstein, C.J., Chern, H.D., Davies, P., Pitt,

B.R., Simmons, R.L., Billiar, T.R., 1992. Cytokines and lipopolysaccharide

induce nitric oxide synthase in cultured rat pulmonary artery smooth

muscle. American Journal of Respiratory Cell and Molecular Biology 7,

471–476.

Nathan, C., 1992. Nitric oxide as a secretory product of mammalian cells. The


Nathan, C.F., Hibbs Jr., J.B., 1991. Role of nitric oxide synthesis in

macrophage antimicrobial activity (Review). Current Opinion in Immunol-

ogy 3, 65–70.

Neumann, B., Zantl, N., Veihelmann, A., Emmanuilidis, K., Pfeffer, K.D.,

Heidecke, C.D., Holzmann, B., 1999. Mechanisms of acute inflammatory

lung injury induced by abdominal sepsis. International Immunology 11,

217–227.

Nava, E., Palmer, R.M., Moncada, S., 1991. Inhibition of nitric oxide synthesis

in septic shock: how much is beneficial? Lancet 338, 1555–1557.

Nicholson, S., Bonecini-Almeida, M.G., Nathan, C., Xie, Q.W., Mumford, R.,

Weidner, J.R., Calaycay, J., Geng, J., Boechat, N., 1996. Inducible nitric

oxide synthase in pulmonary alveolar macrophages from patients with

tuberculosis. Journal of Experimental Medicine 183, 2293–2302.

Nozaki, Y., Hasegawa, Y., Takeuchi, A., Fan, Z.H., Isobe, K.I., Nakashima, I.,

Shimokata, K., 1997. Nitric oxide as an inflammatory mediator of radiation

pneumonitis. American Journal of Physiology 16 (L).

Nys, M., Deby-Dupont, G., Habraken, Y., Legrand-Poels, S., Kohnen, S.,

Ledoux, D., Canivet, J.L., Damas, P., Lamy, M., 2003. Bronchoalveolar

lavage fluids of ventilated patients with acute lung injury activate NF-

kappaB in alveolar epithelial cell line: role of reactive oxygen/nitrogen

species and cytokines. Nitric Oxide: Biology and Chemistry 9, 33–43.

Ochoa, J.B., Udekwu, A.O., Billiar, T.R., Curran, R.D., Cerra, F.B., Simmons,

R.L., Peitzman, A.B., 1991. Nitrogen oxide levels in patients after trauma

and during sepsis. Annals of Surgery 214, 621–626.

O’Donovan, D.A., Kelly, C.J., Abdih, H., Bouchierhayes, D., Watson, R.W.,

Redmond, H.P., Burke, P.E., Bouchierhayes, D.A., 1995. Role of nitric

oxide in lung injury associated with experimental acute pancreatitis. British

Journal of Surgery 82, 1122–1126.

Okamoto, I., Abe, M., Shibata, K., Shimizu, N., Sakata, N., Katsuragi, T.,

Tanaka, K., 2000. Evaluating the role of inducible nitric oxide synthase

using a novel and selective inducible nitric oxide synthase inhibitor in

septic lung injury produced by cecal ligation and puncture. American


Pheng, L.H., Francoeur, C., Denis, M., 1995. The involvement of nitric oxide in

a mouse model of adult respiratory distress syndrome. Inflammation 19,

599–610.

Radi, R., Beckman, J.S., Bush, K.M., Freeman, B.A., 1991a. Peroxynitrite

oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric

oxide. Journal of Biological Chemistry 266, 4244–4250.

Radi, R., Beckman, J.S., Bush, K.M., Freeman, B.A., 1991b. Peroxynitrite-

induced membrane lipid peroxidation: the cytotoxic potential of superoxide

and nitric oxide. Archives of Biochemistry and Biophysics 288, 481–487.

Raykova, V.D., Glibetic, M., Ofenstein, J.P., Aranda, J.V., 2003. Nitric oxide-

dependent regulation of pro-inflammatory cytokines in group B strepto-

coccal inflammation of rat lung. Annals of Clinical and Laboratory Science

33, 62–67.

Razavi, H.M., Werhun, R., Scott, J.A., Weicker, S., Wang, L.F., McCormack,

D.G., Mehta, S., 2002. Effects of inhaled nitric oxide in a mouse model of

sepsis-induced acute lung injury. Critical Care Medicine 30, 868–873.

Razavi, H.M., Wang, L.F., Weicker, S., Rohan, M., Law, C., McCormack, D.G.,

Mehta, S., 2004. Pulmonary neutrophil infiltration in murine sepsis: role of

inducible nitric oxide synthase. American Journal of Respiratory and


Razavi, H.M., Wang, L.F., Weicker, S., Quinlan, G.J., Mumby, S., McCormack,

D.G., Mehta, S., 2005. Pulmonary oxidative injury in murine sepsis is due

to inflammatory cell nitric oxide. Critical Care Medicine 33, 1333–1339.

Reinhart, K., Bayer, O., Brunkhorst, F., Meisner, M., 2002. Markers of

endothelial damage in organ dysfunction and sepsis. Critical Care Medicine

30, S302–S312.

Rixen, D., Siegel, J.H., Espina, N., Bertolini, M., 1997. Plasma nitric oxide

in posttrauma critical illness: a function of ‘‘sepsis’’ and the physiologic


state severity classification quantifying the probability of death. Shock 7,

17–28.

Robbins, R.A., Hamel, F.G., Floreani, A.A., Gossman, G.L., Nelson, K.J.,

Belenky, S., Rubinstein, I., 1993. Bovine bronchial epithelial cells

metabolize l-arginine to l-citrulline: possible role of nitric oxide synthase.

Life Sciences 52, 709–716.

Robbins, R.A., Springall, D.R., Warren, J.B., Kwon, O.J., Buttery, L.D.,

Wilson, A.J., Adcock, I.M., Riveros-Moreno, V., Moncada, S., Polak, J.,

1994. Inducible nitric oxide synthase is increased in murine lung epithelial

cells by cytokine stimulation. Biochemical and Biophysical Research

Communications 198, 835–843.

Rubbo, H., Radi, R., Trujillo, M., Telleri, R., Kalyanaraman, B., Barnes, S.,

Kirk, M., Freeman, B.A., 1994. Nitric oxide regulation of superoxide and

peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-

containing oxidized lipid derivatives. Journal of Biological Chemistry 269,

26066–26075.

Rubenfeld, G.D., 2003. Epidemiology of acute lung injury. Critical Care

Medicine 31, S276–S284.

Ruetten, H., Southan, G.J., Abate, A., Thiemermann, C., 1996. Attenuation of

endotoxin-induced multiple organ dysfunction by 1-amino-2-hydroxy-

guanidine, a potent inhibitor of inducible nitric oxide synthase. British

Journal of Pharmacology 118, 261–270.

Schenkel, A.R., Chew, T.W., Muller, W.A., 2004. Platelet endothelial cell

adhesion molecule deficiency or blockade significantly reduces leukocyte

emigration in a majority of mouse strains. Journal of Immunology 173,

6403–6408.

Scott, J.A., McCormack, D.G., 1999a. Nonadrenergic noncholinergic vasodi-

lation of guinea pig pulmonary arteries is mediated by nitric oxide.

Canadian Journal of Physiology and Pharmacology 77, 89–95.

Scott, J.A., McCormack, D.G., 1999b. Selective in vivo inhibition of inducible

nitric oxide synthase in a rat model of sepsis. Journal of Applied Physiology

86, 1739–1744.

Scott, J.A., Craig, I., McCormack, D.G., 1996. Nonadrenergic noncholinergic

relaxation of human pulmonary arteries is partially mediated by nitric

oxide. American Journal of Respiratory and Critical Care Medicine 154,

629–632.

Scott, J.A., Mehta, S., Duggan, M., Bihari, A., McCormack, D.G., 2002.

Functional inhibition of constitutive nitric oxide synthase in a rat model of

sepsis. American Journal of Respiratory and Critical Care Medicine 165,

1426–1432.

Scumpia, P.O., Sarcia, P.J., DeMarco, V.G., Stevens, B.R., Skimming, J.W.,

2002. Hypothermia attenuates iNOS, CAT-1, CAT-2, and nitric oxide

expression in lungs of endotoxemic rats. American Journal of Physiology.

Lung Cellular and Molecular Physiology 283, L1231–L1238.

Seekamp, A., Mulligan, M.S., Till, G.O., Ward, P.A., 1993. Requirements for

neutrophil products and l-arginine in ischemia– reperfusion injury. Amer-

ican Journal of Pathology 142, 1217–1226.

Sittipunt, C., Steinberg, K.P., Ruzinski, J.T., Myles, C., Zhu, S., Goodman, R.B.,

Hudson, L.D., Matalon, S., Martin, T.R., 2001. Nitric oxide and nitrotyr-

osine in the lungs of patients with acute respiratory distress syndrome.

American Journal of Respiratory and Critical Care Medicine 163, 503–510.

Soop, A., Sollevi, A., Weitzberg, E., Lundberg, J.O., Palm, J., Albert, J., 2003.

Exhaled NO and plasma cGMP increase after endotoxin infusion in healthy

volunteers. European Respiratory Journal 21, 594–599.

Speyer, C.L., Neff, T.A., Warner, R.L., Guo, R.F., Sarma, J.V., Riedemann,

N.C., Murphy, M.E., Murphy, H.S., Ward, P.A., 2003. Regulatory effects of

iNOS on acute lung inflammatory responses in mice. American Journal of

Pathology 163, 2319–2328.

Tate, R.M., Repine, J.E., 1983. Neutrophils and the adult respiratory distress

syndrome. American Review of Respiratory Disease 128, 552–559.

Tavares-Murta, B.M., Cunha, F.Q., Ferreira, S.H., 1998. The intravenous

administration of tumor necrosis factor alpha, interleukin 8 and macro-

phage-derived neutrophil chemotactic factor inhibits neutrophil migration

by stimulating nitric oxide production. British Journal of Pharmacology

124, 1369–1374.

Terada, L.S., Mahr, N.N., Jacobson, E.D., 1996. Nitric oxide decreases

lung injury after intestinal ischemia. Journal of Applied Physiology 81,

2456–2460.

Tomobuchi, M., Oshitani, N., Matsumoto, T., Kitano, A., Seki, S., Arakawa, T.,

2001. In situ generation of nitric oxide by myenteric neurons but not by

mononuclear cells of the human colon. Clinical and Experimental

Pharmacology and Physiology 28, 13–18.

Tracey, W.R., Tse, J., Carter, G., 1995. Lipopolysaccharide-induced changes in

plasma nitrite and nitrate concentrations in rats and mice: pharmacological

evaluation of nitric oxide synthase inhibitors. Journal of Pharmacology and

Experimental Therapy 272, 1011–1015.

Tsubochi, H., Suzuki, S., Kubo, H., Ueno, T., Yoshimura, T., Suzuki, T.,

Sasano, H., Kondo, T., 2003. Early changes in alveolar fluid clearance by

nitric oxide after endotoxin instillation in rats. American Journal of


Tsuji, C., Shioya, S., Hirota, Y., Fukuyama, N., Kurita, D., Tanigaki, T.,

Ohta, Y., Nakazawa, H., 2000. Increased production of nitrotyrosine in

lung tissue of rats with radiation-induced acute lung injury. American

Journal of Physiology. Lung Cellular and Molecular Physiology 278,

L719–L725.

Tsukahara, Y., Morisaki, T., Horita, Y., Torisu, M., Tanaka, M., 1998.

Expression of inducible nitric oxide synthase in circulating neutrophils of

the systemic inflammatory response syndrome and septic patients. World

Journal of Surgery 22, 771–777.

Vincent, J.L., Zhang, H., Szabo, C., Preiser, J.C., 2000. Effects of nitric oxide in

septic shock. American Journal of Respiratory and Critical Care Medicine

161, 1781–1785.

Volman, T.J., Goris, R.J., van der, J.M., van de Loo, F.A., Hendriks, T., 2002.

Organ damage in zymosan-induced multiple organ dysfunction syndrome in

mice is not mediated by inducible nitric oxide synthase. Critical Care

Medicine 30, 1553–1559.

Wagner, J.G., Roth, R.A., 1999. Neutrophil migration during endotoxemia.

Journal of Leukocyte Biology 66, 10–24.

Walker, T.A., Curtis, S.E., King-VanVlack, C.E., Chapler, C.K., Vallet, B.,

Cain, S.M., 1995. Effects of nitric oxide synthase inhibition on

regional hemodynamics and oxygen transport in endotoxic dogs. Shock

4, 415–420.

Walley, K.R., McDonald, T.E., Higashimoto, Y., Hayashi, S., 1999.

Modulation of proinflammatory cytokines by nitric oxide in murine acute

lung injury. American Journal of Respiratory and Critical Care Medicine

160, 698–704.

Wang, L.F., Mehta, S., Weicker, S., Scott, J.A., Joseph, M., Razavi, H.M.,

McCormack, D.G., 2001. Relative contribution of hemopoietic and

pulmonary parenchymal cells to lung inducible nitric oxide synthase

(iNOS) activity in murine endotoxemia. Biochemical and Biophysical

Research Communications 283, 694–699.

Wang, L.F., Patel, M., Razavi, H.M., Weicker, S., Joseph, M.G., McCormack,

D.G., Mehta, S., 2002. Role of inducible nitric oxide synthase in pulmonary

microvascular protein leak in murine sepsis. American Journal of


Ware, L.B., Matthay, M.A., 2000. The acute respiratory distress syndrome.

New England Journal of Medicine 342, 1334–1349.

Ware, L.B., Conner, E.R., Matthay, M.A., 2001. von Willebrand factor antigen

is an independent marker of poor outcome in patients with early acute lung

injury. Critical Care Medicine 29, 2325–2331.

Watson, D., Grover, R., Anzueto, A., Lorente, J., Smithies, M., Bellomo, R.,

Guntupalli, K., Grossman, S., Donaldson, J., Le Gall, J.R., 2004.

Cardiovascular effects of the nitric oxide synthase inhibitor NG-methyl-l-

arginine hydrochloride (546C88) in patients with septic shock: results of a

randomized, double-blind, placebo-controlled multicenter study (study no.

144-002). Critical Care Medicine 32, 13–20.

Webert, K.E., Vanderzwan, J., Duggan, M., Scott, J.A., Lewis, J.F.,

McCormack, D.G., Mehta, S., 2000. Effects of inhaled nitric oxide in a

rat model of Pseudomonas aeruginosa pneumonia. Critical Care Medicine

28, 2397–2405.

Weicker, S., Karachi, T.A., Scott, J.A., McCormack, D.G., Mehta, S., 2001.

Non-invasive measurement of exhaled nitric oxide in a spontaneously

breathing mouse. American Journal of Respiratory and Critical Care

Medicine 163, 1113–1116.

Wheeler, A.P., Bernard, G.R., 1999. Treating patients with severe sepsis. New

England Journal of Medicine 340, 207–214.


Wheeler, M.A., Smith, S.D., Garcia-Cardena, G., Nathan, C.F., Weiss, R.M.,

Sessa, W.C., 1997. Bacterial infection induces nitric oxide synthase in

human neutrophils. Journal of Clinical Investigation 99, 110–116.

Worthen, G.S., Haslett, C., Rees, A.J., Gumbay, R.S., Henson, J.E., Henson,

P.M., 1987. Neutrophil-mediated pulmonary vascular injury. Synergistic

effect of trace amounts of lipopolysaccharide and neutrophil stimuli on

vascular permeability and neutrophil sequestration in the lung. American

Review of Respiratory Disease 136, 19–28.

Yamashita, T., Kawashima, S., Ohashi, Y., Ozaki, M., Ueyama, T., Ishida, T.,

Inoue, N., Hirata, K., Akita, H., Yokoyama, M., 2000. Resistance to

endotoxin shock in transgenic mice overexpressing endothelial nitric oxide

synthase. Circulation 101, 931–937.

Zegdi, R., Fabre, O., Cambillau, M., Detruit, H., Fornes, P., Rajnoch, C.,

Shen, M., Herve, P., Carpentier, A., Fabiani, J.N., 2002. Exhaled nitric

oxide does not reflect the severity of acute lung injury: an experimental

study in a rat model of extracorporeal circulation. Critical Care Medicine

30, 2096–2102.

Zhu, S., Haddad, I.Y., Matalon, S., 1996. Nitration of surfactant protein A (SP-

A) tyrosine residues results in decreased mannose binding ability. Archives

of Biochemistry and Biophysics 333, 282–290.

Zhu, S., Kachel, D.L., Martin, W.J., Matalon, S., 1998. Nitrated SP-A does not

enhance adherence of Pneumocystis carinii to alveolar macrophages.

American Journal of Physiology. Lung Cellular and Molecular Physiology

19, L1031–L1039.

Zhu, S., Ware, L.B., Geiser, T., Matthay, M.A., Matalon, S., 2001. Increased

levels of nitrate and surfactant protein A nitration in the pulmonary edema

fluid of patients with acute lung injury. American Journal of Respiratory

and Critical Care Medicine 163, 166–172.

Documents

The effects of nitric oxide in acute lung injury