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American Journal of Respiratory and Critical Care Medicine Vol 165. pp. 1634-1639, (2002)
© 2002 American Thoracic Society

Original Article

Role of Inducible Nitric Oxide Synthase in Pulmonary Microvascular Protein Leak in Murine Sepsis

Le Feng Wang, Milan Patel, Habib Moshref Razavi, Sean Weicker, Mariamma G. Joseph, David G. McCormack and Sanjay Mehta

Departments of Medicine, Pharmacology/Toxicology, and Pathology, Division of Respirology, Lawson Health Research Institute, London Health Sciences Center, University of Western Ontario, London, Ontario, Canada

Correspondence and requests for reprints should be addressed to Dr. Sanjay Mehta, Division of Respirology, London Health Sciences Center-Victoria South Street Campus, 375 South Street, London, ON, N6A 4G5 Canada. E-mail: sanjay.mehta{at}lhsc.on.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of nitric oxide (NO) from calcium-independent NO synthase (iNOS) on microvascular protein leak in acute lung injury (ALI) are uncertain, possibly because of disparate effects of iNOS-derived NO from different cells. We assessed the contribution of iNOS from inflammatory versus parenchymal cells to pulmonary protein leak in murine cecal ligation and perforation-induced ALI. We studied iNOS+/+, iNOS-/-, and two reciprocally bone marrow-transplanted iNOS chimeric mice groups: + to - (iNOS+/+ donor bone marrow-transplanted into iNOS-/- recipient mice) and - to +. Sepsis-induced ALI was characterized by pulmonary leukocyte infiltration, increased pulmonary iNOS activity, and increased pulmonary microvascular protein leak, as assessed by Evans blue (EB) dye. Despite equal neutrophil infiltration, sepsis-induced EB-protein leak was eliminated in iNOS-/- mice and in - to + iNOS chimeras (parenchymal cell-localized iNOS) but was preserved in + to - chimeric mice (inflammatory cell-localized iNOS). EB-protein leak was also prevented by pretreatment with allopurinol and superoxide dismutase. Microvascular protein leak in sepsis-induced ALI is uniquely dependent on iNOS in inflammatory cells with no obvious contribution of iNOS in pulmonary parenchymal cells. Pulmonary protein leak is also dependent on superoxide, suggesting an effect of peroxynitrite rather than NO itself.

Key Words: nitric oxide • acute lung injury • pulmonary circulation • pulmonary edema • peroxynitrite


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sepsis-induced acute lung injury (ALI) remains an important clinical problem with significant morbidity, mortality, and use of healthcare resources (1–3). One of the defining pathophysiologic features of ALI is the leak of protein-rich fluid across the pulmonary microvasculature (1, 4–6). Pulmonary inflammation in ALI is the result of a complex interaction of a network of locally and systemically produced inflammatory mediators, such as nitric oxide (NO).

NO is produced by a family of isoenzymes, the NO synthases (NOS), which are classified as calcium-dependent (cNOS) isoforms that are generally constitutively expressed or calcium-independent (iNOS) isoforms that are usually cytokine inducible (7, 8). cNOS-derived NO is thought primarily to mediate homeostatic effects, such as modulation of pulmonary vascular tone and neurotransmission, whereas iNOS-derived NO participates in host defense but may contribute to tissue injury and inflammation (9–11). iNOS can be expressed in many different cell types, including both stromal or parenchymal cells (e.g., epithelial and endothelial) and inflammatory cells (e.g., neutrophils and macrophages) (12–15).

There are extensive data to support a role for NO in the regulation of microvascular protein leak in the systemic, bronchial, and pulmonary circulations (16–19). However, the effects of iNOS-derived NO on pulmonary microvascular protein leak in sepsis-induced ALI are controversial (9, 20–22). Limitations of previous studies include the use of poorly isoform-selective NOS inhibitors and the use of nonclinically relevant models of sepsis such as endotoxin infusion. Moreover, whether NO inhibits or potentiates pulmonary microvascular leak may depend, in part, on the cellular source of NO. However, there are no data on potentially disparate effects of iNOS-derived NO in ALI from different cellular sources (i.e., inflammatory versus pulmonary parenchymal cells). Recent work using the technique of reciprocal bone marrow (BM) transplantation (BMT) to generate iNOS chimeric mice has defined quantitative differences in iNOS activity and NO production from inflammatory versus parenchymal cells in endotoxin-induced ALI (23). In addition, an immunohistochemical study of inflammatory bowel disease has suggested that cell source–specific nitrosative and oxidative effects of iNOS-derived NO may also be important in humans (24).

Thus, we hypothesized that iNOS-derived NO mediates pulmonary microvascular protein leak in sepsis-induced ALI and that iNOS in inflammatory cells contributes more importantly to this effect. The first objective was to measure pulmonary microvascular protein leak in murine sepsis-induced ALI and to examine the effects of iNOS-derived NO through the use of iNOS-/- mice. The second objective was to examine the differential contribution of different cellular sources (pulmonary parenchymal versus inflammatory cells) to this iNOS-dependent effect through the use of BMT iNOS chimeric mice. The third objective was to explore the mechanism of NO's effects on pulmonary microvascular leak through the use of pharmacologic antagonists, including N-(3-[Aminomethyl]benzyl)acetamidine (1400W; iNOS-selective inhibitor), allopurinol (xanthine oxidoreductase inhibitor), and polyethylene glycol-encapsulated superoxide dismutase (PEG-SOD) (25).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cecal Ligation and Perforation Model of Sepsis
Male wild-type (iNOS+/+), iNOS-/- (8–10 weeks, 20–28 g; Charles River, St. Constant, PQ, Canada), or BMT C57Bl/6 mice were used. Protocols were approved by the institutional animal ethics committee.

Donor BM was infused into irradiated recipient mice, and the BM was allowed to reconstitute over 4–6 weeks as previously described (23). Based on the genotype of donor and recipient mice, two reciprocal iNOS chimeras were created, including - to + (iNOS-/- BM transplanted into iNOS+/+ recipient; iNOS localized to recipient parenchymal cells) and + to - (iNOS+/+ BM transplanted into iNOS-/- recipient; iNOS localized to donor inflammatory cells), as well as two control groups (+ to + and - to -).

Animals were randomized to either sepsis (cecal ligation and perforation [CLP]) or sham/anesthesia (26, 27). All mice received buprenorphine 0.1 µg/g body weight (t = 0 and 12 hours) and were volume resuscitated with 1.5 ml of potassium phosphate-buffered saline (PBS, 0.9%, pH 7.4; t = 0, 6, and 12 hours) subcutaneously.

Assessment of Pulmonary Microvascular Protein Leak via Evans Blue Dye
Pulmonary Evans blue (EB)-protein leak was assessed as previously described (28). After EB infusion, mice were sacrificed (intravenous sodium pentobarbitol, 100 mg/kg) and blood was aspirated via cardiac puncture into a heparinized syringe for isolation of plasma. The pulmonary circulation was flushed (10 ml of PBS, pH 7.4, 4°C). The lungs were excised, rinsed in PBS, blotted dry, snap frozen in liquid nitrogen, and stored at -80°C. In some animals, lungs were used for biochemical assays.

For EB-protein leak, frozen tissue was homogenized in PBS (4°C) and incubated in formamide (60°C, 16 hours). After centrifugation (7,000 x g, 25 minutes), supernatant absorbances at 620 (A620) and 740 nm (A740) were recorded. Tissue EB content (µg EB/g lung/minute) was calculated by correcting A620 for the presence of heme pigments: A620 (corrected) = A620 - (1.426 x A740 + 0.030) and comparing this value to a standard curve of EB in formamide/PBS (28).

EB-protein leak was assessed in wild-type mice at 1, 2, 4, 8, and 16 hours after CLP and at selected time points in sham, iNOS-/-, and BMT iNOS chimeric mice. EB-protein leak was also measured in septic wild-type mice following intravenous treatment at t = 0 with either (1) 5 mg/kg 1,400 W, (2) 50 mg/kg allopurinol (in 0.025 N NaOH/PBS), or (3) 2,000 U/kg PEG-SOD/PBS.

Biochemical Assays
Peritoneal inflammation after CLP was quantified by analyzing aliquots of peritoneal lavage for protein concentration, total cell count, and bacterial load. Plasma samples were refluxed in saturated vanadium chloride (1 N HCl) at 90°C, resulting in reduction of nitrites/nitrates (NOx-) to NO, subsequently detected by chemiluminescence (29).

Statistical Analysis
Results are expressed as mean ± SEM. Serial changes in EB-protein leak and plasma NOx- were compared by repeated-measures analysis of variance. Differences between groups were assessed by one-way analysis of variance. Post-hoc comparisons were performed with a Student-Newman-Keuls t test. Significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Endogenous iNOS Activity on Sepsis-induced Pulmonary EB-Protein Leak
In pilot studies, pulmonary microvascular protein leak was compared in septic and sham mice using various EB (25, 50, 75, and 100 mg/kg) doses infused at different time points before harvest (15, 30, and 60 minutes; data not shown). Sepsis-induced ALI was associated with an increase in pulmonary microvascular EB-protein leak in septic versus sham mice, with an optimal difference being found after administration of 50 mg/kg of EB 30 minutes before tissue harvest. Under these conditions, EB-protein leak was similar at t = 0, 4, and 16 hours after anesthesia in sham mice. In contrast, EB-protein leak increased within 2 hours after CLP and peaked at 4 to 8 hours before returning to basal levels by 16 hours (Figure 1A) . Thus, in subsequent experiments with iNOS-/- mice, BMT iNOS chimeric mice, and pharmacologic interventions, EB-protein leak was assessed at 4 hours after CLP.



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  		Figure 1. Time course of (A) EB dye-protein pulmonary microvascular leak and (B) plasma concentrations of NOx- following CLP in wild-type (iNOS+/+) and iNOS-/- mice (n = 5–8/group/time point). *p < 0.05 for CLP versus sham; #p < 0.05 for iNOS-/- versus wild-type iNOS+/+ group at same time point.

 
The basal rate of EB-protein leak in sham iNOS-/- mice was no different than in sham wild-type (iNOS+/+) mice. However, in contrast to wild-type mice, the sepsis-induced increase in EB-protein leak was completely abrogated in iNOS-/- mice following CLP (Figure 1A). Sepsis-induced ALI was also associated with increased endogenous NO production, as evidenced by increased plasma concentrations of NOx- (Figure 1B). As expected, plasma NOx- did not increase following CLP in iNOS-/- mice, remaining at basal concentrations.

To confirm these findings in septic iNOS-/- mice, wild-type mice were treated with 1,400 W, an iNOS-selective inhibitor. Inhibition of iNOS with 1,400 W completely prevented the sepsis-induced increase in EB-protein leak (Figure 2) . Moreover, 1,400 W also attenuated the sepsis-induced increase in plasma NOx- concentrations at 4 hours after CLP (29 ± 4 versus 61 ± 11 µM in PBS-treated septic wild-type mice, p < 0.05).



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  		Figure 2. Effect of inhibition of iNOS with 1,400 W on EB dye-protein pulmonary microvascular leak at 4 hours after sham surgery or CLP (n = 6–8 per group). *p < 0.05 for CLP versus sham; #p < 0.05 for 1,400 W versus respective PBS group.

 
Sepsis-induced ALI in wild-type mice was also characterized by increased pulmonary MPO activity, a marker of leukocyte infiltration (16 ± 5 to 39 ± 8 mU/mg protein, p < 0.05). Pulmonary MPO in sham iNOS-/- mice (12 ± 4 mU/mg protein) was similar to sham wild-type mice. Moreover, pulmonary MPO increased after CLP in iNOS-/- mice (46 ± 7 mU/mg protein, p < 0.05 versus sham) to a similar degree as in wild-type mice. Given this lack of difference in pulmonary MPO between septic wild-type and iNOS-/- mice, despite the abrogated sepsis-induced EB-protein leak in iNOS-/- mice, MPO was not assessed in the BMT chimeric mice.

Effect of Cellular Source of Endogenous iNOS Activity on Sepsis-induced Pulmonary EB-Protein Leak
To assess the effect of whole-body irradiation and the BMT protocol on pulmonary microvascular protein leak, EB-protein leak was assessed 4–6 weeks after wild-type (iNOS+/+) recipient mice had undergone BMT and received iNOS+/+ donor BM (+ to +; all cells iNOS+/+, thus genetically identical to wild-type mice). EB-protein leak was similar in sham + to + BMT mice and sham, untransplanted wild-type mice (data not shown). Similarly, basal rates of EB-protein leak were similar in all the sham (nonseptic) BMT groups, including the + to - and - to + iNOS chimeric mice. Thus, the BMT protocol itself had no effect on pulmonary EB-protein leak assessed 4–6 weeks later.

Sepsis-induced ALI in + to + BMT mice was characterized by a similar increase in EB-protein leak 4 hours after CLP as in wild-type mice (2.6 ± 0.2 versus 2.5 ± 0.3 µg EB/g lung/min, p = NS). Furthermore, as in iNOS-/- mice, the sepsis-induced increase in EB-protein leak was abolished in - to - BMT mice (data not shown). Strikingly, in the BMT iNOS chimeric mice, a sepsis-induced increase in EB-protein leak was observed in + to - chimeras (iNOS only in donor BM-derived inflammatory cells) but not in - to + chimeras (iNOS only in recipient parenchymal cells; Figure 3A) .



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  		Figure 3. Effect of reciprocal BMT to generate iNOS chimeric mice on (A) EB-protein pulmonary microvascular leak and (B) plasma concentrations of NOx- at 4 hours after CLP; the + to - notation indicates iNOS+/+ donor BM transplanted into iNOS-/- recipient mice, such that iNOS is only expressed in BM-derived inflammatory cells, not host-derived parenchymal cells (n = 6–10/group). *p < 0.05 and **p < 0.01 for CLP versus sham; #p < 0.05 and ##p < 0.01 versus + to + BM-transplanted group.

 
As in wild-type septic mice, plasma NOx- concentrations were increased in BMT + to + mice (iNOS in all cells) 4 hours after CLP (Figure 3B). However, this sepsis-induced increase in plasma NOx- was attenuated in BMT iNOS chimeric mice relative to septic wild-type mice. Whereas plasma NOx- concentrations were significantly lower in septic - to + iNOS chimeric mice (recipient parenchymal cell-localized iNOS) than septic wild-type mice, there was no sepsis-induced increase in plasma NOx- in + to - chimeras (donor BM-derived inflammatory cell-localized iNOS).

Mechanism of iNOS-derived NO Effects on Sepsis-induced Pulmonary EB-Protein Leak
The sepsis-induced increase in pulmonary EB-protein leak at 4 hours after CLP was completely prevented by previous treatment of wild-type mice with allopurinol or PEG-SOD (Figure 4A) . In contrast, the sepsis-induced increase in plasma NOx- concentrations at 4 hours after CLP was not attenuated by either of these two treatments but indeed was significantly enhanced by treatment with PEG-SOD (Figure 4B).



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  		Figure 4. Effect of iNOS deficiency (iNOS-/- mice), iNOS inhibition (1,400 W), and treatment with allopurinol (Allo) or polyethylene glycol-conjugated SOD on (A) EB-protein pulmonary microvascular leak and (B) plasma concentrations of NOx- at 4 hours after CLP (n = 6–10 per group). *p < 0.05 and **p < 0.01 versus saline-treated CLP.

 
CLP in wild-type mice was associated with purulent peritonitis characterized by increased total peritoneal bacterial load (6.2 ± 1.4 x 105 versus less than 1 x 101, p < 0.001), protein (17.8 ± 0.9 versus 1.4 ± 0.4 mg/ml, p < 0.001) and total cell count (10.6 ± 1.7 x 106 versus 0.9 ± 0.1 x 106, p < 0.01) versus naive mice. The degree of peritoneal infection and inflammation was not significantly different in septic iNOS-/- mice (7.6 ± 1.1 x 105 bacteria, 19.3 ± 0.8 mg/ml protein, 8.4 ± 2.1 x 106 total cells) versus septic wild-type mice. Furthermore, peritonitis was not attenuated by treatment of septic wild-type mice with 1,400 W, allopurinol, or PEG-SOD (data not shown). Moreover, pretreatment with the relevant vehicles or negative control solutions (PBS, 0.025 N NaOH in PBS, or heat-inactivated PEG-SOD, respectively) had no effect on either basal leak or sepsis-induced increase in EB-protein leak at 4 hours after CLP (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sepsis-induced ALI in mice was characterized by increased endogenous NO production, pulmonary neutrophil infiltration, and increased pulmonary microvascular protein leak. The sepsis-induced increase in pulmonary microvascular protein leak was completely iNOS dependent, being abrogated in iNOS-/- animals, as well as in wild-type (iNOS+/+) mice following treatment with 1,400 W, a selective iNOS inhibitor. Furthermore, data from reciprocal BMT iNOS chimeric mice suggest that protein leak in sepsis-induced ALI was dependent on iNOS activity in donor BM-derived inflammatory cells, with no apparent contribution of iNOS-derived NO from recipient pulmonary parenchymal (stromal) cells. Sepsis-induced pulmonary microvascular protein leak was also abolished following either inhibition of xanthine oxidoreductase production of superoxide or scavenging of superoxide. The dependence of the sepsis-induced increase in pulmonary microvascular protein leak on both iNOS-derived NO and superoxide strongly suggests an effect of peroxynitrite on pulmonary microvascular endothelial cells (ECs) as the mechanism of this leak.

Pulmonary protein leak was assessed by the EB dye technique, which has been extensively used to assess microvascular protein leak in the lungs and airways in many models of injury (16, 17, 21, 28, 30–32). The EB-protein assay correlates well with assessment of leak by other methods, such as radiolabeled albumin (33, 34). In this report, pulmonary EB-protein leak was reproducibly stable over time in sham animals and was unaffected by administration of three different vehicle control solutions. In addition, the time course of the evolution of EB-protein leak after induction of sepsis by CLP was highly reproducible. Thus, based on our findings, and the published observations discussed previously here, we believe that the EB dye technique is a reliable measure of basal and sepsis-induced increases in pulmonary microvascular protein leak.

Our data suggest a central role for iNOS-derived NO in mediating pulmonary microvascular protein leak in sepsis-induced ALI. These findings are consistent with recent findings in other models of ALI (9, 10, 21, 35–37). For example, pulmonary microvascular barrier dysfunction in murine endotoxemia, characterized by increased amounts of protein and lactate dehydrogenase in lung lavage fluid, was completely inhibited in iNOS-/- mice (9). Similarly, in endotoxin-induced ALI in mice, rats, dogs, and rabbits, various iNOS inhibitors reduced histologic interstitial edema, lung lavage protein, or wet-to-dry weight ratios (10, 21, 35–37).

The reciprocal BMT chimera technique permits differentiation of the relative contribution of two populations of cells: donor BM-derived cells (e.g., leukocytes) versus recipient parenchymal or stromal cells (e.g., epithelial and endothelial) to pulmonary microvascular protein leak (23). Indeed, sepsis-induced pulmonary protein leak was completely prevented by the selective absence of iNOS in donor BM-derived inflammatory cells (i.e., - to + BMT iNOS chimeras), despite the presence of iNOS in recipient parenchymal cells. This donor BM-derived inflammatory cell population comprises both mononuclear cells (e.g., lymphocytes and macrophages) and polymorphonuclear leukocytes (e.g., neutrophils and eosinophils). The most important cellular sources of iNOS contributing to pulmonary protein leak in ALI are likely pulmonary macrophages and infiltrating neutrophils (6, 11, 13, 38).

Recent studies in other injury models and in other tissues have suggested such cell source-dependent compartmentalization of the effects of iNOS-derived NO. For example, using reciprocal BMT iNOS chimeras, we previously reported that pulmonary iNOS activity in murine endotoxin-induced ALI was predominantly localized to parenchymal cells (approximately 70%) rather than inflammatory cells (23). This is consistent with our present data, whereby plasma concentrations of NOx- were significantly greater in septic - to + iNOS chimeras (recipient parenchymal cell-localized iNOS) than + to - mice. As well, we have recently found that pulmonary lipid peroxidation in sepsis-induced ALI is completely dependent on iNOS-derived NO from inflammatory cells (39). Similarly, others have reported that the presence of iNOS in inflammatory cells was essential to survival in murine toxoplasmosis, with no obvious contribution of parenchymal cell iNOS (40). Compartmentalization of iNOS expression specifically to macrophages has also been found in necrotic skin and muscle from humans with sepsis due to cellulitis (41). As well, an immunohistochemical study has suggested cell source-specific nitrosative and oxidative effects of NO in human inflammatory bowel disease (24).

We speculate that the cell source-dependent effects of iNOS-derived NO are most likely caused by differences in the local amounts of NO in the vicinity of the pulmonary microvascular EC. Thus, liberation of iNOS-derived NO by neutrophils adherent to ECs or alveolar macrophages adjacent to ECs may lead to very high local amounts, contributing more significantly to EC dysfunction. Alternatively, it has been suggested that the effects of NO depend on the production of different redox forms of NO, such as the free radical NO, nitrosonium ion (NO+), and nitroxyl anion (NO-) (42, 43). It remains unexplored whether different cells (e.g., inflammatory versus parenchymal) may preferentially liberate different redox forms of NO.

The mechanism of inflammatory cell iNOS-dependent pulmonary microvascular leak in murine sepsis-induced ALI remains uncertain. As for microvascular leak, we recently showed in this same model of ALI that pulmonary lipid peroxidation, as reflected by tissue F2-isoprostane amounts, was also completely dependent on iNOS-derived NO from inflammatory cells (39). Thus, sepsis-induced pulmonary microvascular leak may by mediated by NO-dependent oxidative injury. Many of NO's effects are thought to be mediated through its rapid, diffusion-limited reaction with O2-, generating the highly reactive oxidant, peroxynitrite (9, 11, 44–46). The abrogation of sepsis-induced leak in the present study following either inhibition/deficiency of iNOS or scavenging or inhibition of the production of O2- suggests that pulmonary microvascular protein leak in sepsis may by mediated by peroxynitrite. Our findings implicating NO and peroxynitrite in pulmonary microvascular barrier dysfunction are consistent with the results of others (9, 47). For example, pulmonary 3-nitrotyrosine immunostaining, thought to be a "footprint" of the oxidative actions of NO and related nitrogen oxides, was induced by endotoxin administration in iNOS+/+ but not in iNOS-/- mice (9). However, 3-nitrotyrosine staining is not specific for the effects of peroxynitrite, as it can also result from the action of peroxidases (e.g., myeloperoxidase) on soluble nitrite, in the presence of oxidants such as peroxychlorous acid (48, 49). The importance of NO and peroxynitrite in human ALI are becoming increasingly apparent, given the presence of nitrated proteins, such as surfactant-associated proteins, in the lungs of patients with ALI (11, 50, 51).

The regulation of pulmonary microvascular protein leak occurs at the level of the microvascular EC. The size of pores between adjacent ECs is regulated by EC contraction, which is affected by EC activation and injury (52, 53). Thus, the NO-dependent increase in pulmonary microvascular protein leak in sepsis-induced ALI may be due to EC oxidative injury or modification of the EC contractile machinery by NO or peroxynitrite. The toxic effects of both NO and peroxynitrite are primarily mediated by inhibition of a number of mitochondrial metalloproteins essential to cellular respiration, such as cytochrome c oxidase (54, 55). However, peroxynitrite may also perturb the regulation of calcium homeostasis and EC contractile function through inactivation of Ca2+-ATPase (56, 57). NO and/or peroxynitrite may also directly and covalently modify microtubules and actin filaments, either via nitration of tyrosine residues or nitrosation of sulfhydryl-containing moieties like cysteine (58, 59). Alternatively, the degree of pulmonary edema is the net result of a complex interaction of capillary hydrostatic pressure, capillary surface area, endothelial permeability, as well as lymphatic flow and alveolar sodium and water reabsorption (19, 22, 60–62). Both NO and peroxynitrite can have effects on many of these physiologic processes, which cannot easily be assessed in the present murine model of ALI.

The observed differences in sepsis-induced pulmonary microvascular protein leak could have arisen from differences in the local and systemic inflammatory response to CLP. However, this is unlikely as the degree of peritonitis following CLP was independent of the iNOS genotype and was unaffected by any of the pharmacologic treatments. Moreover, the consistent pulmonary MPO signal in septic wild-type and iNOS-/- mice supports a similar degree of pulmonary involvement by the systemic inflammatory response induced by peritonitis. Indeed, the abrogation of leak in both iNOS-/- mice and - to + BMT iNOS chimeras, despite the equal degree of neutrophil infiltration, indicated that the simple presence of neutrophils was not sufficient to provoke microvascular capillary barrier dysfunction. The presence of functional iNOS in the inflammatory cells (e.g., neutrophils) was required to yield sepsis-induced pulmonary microvascular protein leak.

Although we have defined a role for inflammatory cell iNOS-derived NO in sepsis-induced pulmonary microvascular protein leak, we have not established which inflammatory cells contribute to this leak. There is evidence in support of a possible role for both alveolar macrophages and neutrophils in pulmonary microvascular edema (6, 11, 13, 38, 47). Further elucidation of the role of neutrophils in microvascular leak through study of neutrophil-depleted mice was not feasible because sepsis-induced ALI is a remote manifestation of the systemic inflammatory response to an initial, local inflammatory response (peritonitis) to CLP. Thus, neutrophil depletion may directly alter the degree of pulmonary microvascular protein leak, but it would be impossible to tell whether this was an indirect effect because of changes in the degree and character of the peritonitis injury following CLP. The further characterization of the individual roles of macrophages and neutrophils in sepsis-induced ALI will require in vitro and in vivo approaches, such as selective macrophage depletion. Our study is also limited by its focus on the role of iNOS in sepsis-induced pulmonary leak. It should be recognized that there is evidence in support of a role of cNOS in the modulation of basal rates of leak in the pulmonary and other vascular beds (17, 20, 22).

In summary, in a murine model of sepsis-induced ALI, pulmonary microvascular protein leak was completely dependent on iNOS activity in pulmonary inflammatory cells (e.g., neutrophils and alveolar macrophages), with no obvious contribution of iNOS in pulmonary parenchymal cells. Moreover, rather than a direct effect of iNOS-derived NO on ECs, the coincident dependence of pulmonary microvascular protein leak on superoxide suggests that peroxynitrite is the proximal mediator of the EC injury. Further work both in vivo and in vitro will dissect the respective roles of neutrophil and macrophage iNOS-derived NO in sepsis-induced ALI. This work suggests that specific cell-targeted NOS inhibition approaches may be of clinical benefit in human ALI.


   Acknowledgments
 
The authors would like to acknowledge the help and expertise of Mrs. Marta Rohan in technical procedures related to the mice.

This work was supported by the Lawson Health Research Institute, the Ontario Thoracic Society, the Canadian Lung Association, and the Canadian Institute of Health Research.

Received in original form October 3, 2001; accepted in final form March 25, 2002


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 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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