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The discovery of the diffusible mediator of endothelial-dependent vascular relaxation, nitric oxide (·NO), has revolutionized how oxidative inflammatory reactions are viewed. The
discovery of nitric oxide unified the fields of oxygen radical
and ·NO biology, because O2·
reacts extremely rapidly with
·NO to form the potent secondary oxidizing and nitrating species peroxynitrite (ONOO) (1). Thus, much of the toxicity of
O2·, ·NO, and ·OH is now often ascribed to the reactions of
ONOO. Peroxynitrite also forms a reactive adduct with adventitious CO2 (nitrosoperoxocarbonate, ONOOCO2), with
this species viewed as being responsible for most ONOO-
mediated nitration reactions (2). Consequently, 3-nitrotyrosine (NO2Tyr) formation has been used as a probe for ·NO-mediated oxidative reactions in vivo, with lung cell hypercapnia accelerating this pathway (3).
In this issue of the AJRCCM (pp. 503-510), Sittipunt and
coworkers report that the stable oxidation products of ·NO,
nitrite (NO2) and nitrate (NO3), were present in elevated
concentration in bronchoalveolar lavage (BAL) fluid of patients with acute respiratory distress syndrome (ARDS) (4).
They also observed that NO2Tyr content was increased in
both BAL proteins and cytospin preparations of BAL cells of patients with ARDS, as was BAL cell-inducible nitric oxide
synthase immunoreactivity. BAL NO2 and NO3 concentrations were increased in patients both at risk for and with
ARDS, and was greater in patients who died. From these observations, the authors concluded that "oxidant stress mediated by ·NO appears to be an important factor in the pathogenesis of lung injury in patients at risk, as well as in patients
with established ARDS." Herein we address critical issues related to the potential toxicity of ·NO and its metabolic by-products in inflammatory lung diseases.
The detection of NO2Tyr has found utility as a marker of reactive nitrogen species (RNS) activity in a wide range of inflammatory processes (5). Sittipunt and colleagues utilized an enzyme-linked immunosorbent assay (ELISA) to quantitate protein-associated NO2Tyr in BAL and found that the levels of this modified amino acid are significantly higher both in subjects at risk for ARDS and in those with established ARDS, compared with healthy control subjects. This finding is not surprising, given the previous detection of NO2Tyr in lung and plasma proteins of patients with ARDS (6, 7). What is revealing, however, is that NO2Tyr was detected in patients "at risk" for ARDS and that this could potentially be used to predict the incipience of ARDS prior to its full clinical manifestation. Thus, this is the first report demonstrating protein NO2Tyr as a prognostic indicator of disease development.
Crucial questions emerge from this study as to whether NO2Tyr is simply a biochemical dosimeter of RNS production or if this posttranslational protein modification occurs at levels that can significantly impact cell and organ function. The present results and those of most other clinical studies to date do not clarify these issues. Numerous in vitro biochemical studies have demonstrated that nitration of protein tyrosine residues, per se, can alter protein function (8). This would be expected, because incorporation of the bulky NO2 group imposes both steric and electronic perturbations in tyrosine that affect its capacity to catalytically participate in enzymatic reactions and maintain structural integrity within the protein. Although NO2Tyr is formed in diverse inflammatory diseases, only a few specific proteins have been identified as in vivo targets of this NO-dependent posttranslational modification (7- 13). Importantly, a causal relationship between tyrosine nitration and disease pathology, conceivable on the basis of in vitro studies, remains to be firmly established. The observation of nitration of specific plasma proteins in patients with ARDS, an event that occurs in concert with changes in antiprotease and clotting protein function, is the most significant advance in this direction (7). While exposure of selected proteins to RNS inevitably results in an NO2Tyr "footprint," concurrent oxidative modification of other, more susceptible protein targets (i.e., cysteine) can actually be responsible for loss of protein function (14, 15). Thus, tyrosine nitration may parallel enzyme/protein dysfunction, but may not be responsible. In summary, a causal role for tyrosine nitration in inflammatory tissue injury is not warranted unless accompanied by evidence that the specific tyrosine modified is critical for protein function and that other oxidative, nitrosative, and/or nitrative amino acid modifications are ruled out.
Using human serum albumin as an exemplary protein in BAL fluids, coupled with the data presented by Sittipunt and coworkers, it is calculated that the extent of BAL protein NO2Tyr adducts represents ~ 0.01-0.05% of total protein tyrosines, consistent with previous estimates (16). Because NO2Tyr represents a small fraction of total protein tyrosines, its contribution to impaired lung cell protein function is unlikely. However, the selectivity of protein tyrosine nitration may be of significance if NO2Tyr adducts preferentially occur at critical residues in a few highly susceptible proteins. Indeed, consensus motifs that facilitate tyrosine nitration have been suggested (17). Thus, the low quantitative yield of NO2Tyr may not exclude a potential contribution to altered protein function, because the low yields of tissue NO2Tyr formation in inflammatory events are of similar magnitude to extents of tyrosine phosphorylation that occur during cell signaling processes. Alternatively, nitration may mimic tyrosine phosphorylation or adenylation, thus constituting a regulatable mechanism of modulating protein function (18). The identification of an "NO2Tyr denitrase" activity in rat tissues (19), akin to protein phosphatases, suggests that tyrosine nitration is a reversible process, further supporting the notion of nitration as a potential signaling event. It is not known whether tyrosine nitration increases or decreases rates of protein turnover. Thus, the identification of proteins susceptible to tyrosine nitration, its impact on their function, and the fate of nitrated proteins remain meritorious challenges for future investigation.
If important to disease pathology, elucidation of the mechanisms responsible for tyrosine nitration is critical, because
the design of interventional strategies to suppress tyrosine nitration will be intimately dependent on underlying mechanisms. While ONOO has been most commonly implicated in
tyrosine nitration (1), alternative nitration pathways are also
likely operative. Myeloperoxidase (MPO)- and eosinophil
peroxidase (EPO)-catalyzed tyrosine nitration (20, 21) is also
to be expected in lung tissue, because MPO and EPO are intimately linked with inflammatory events and display spatial
and temporal colocalization with tyrosine nitration. Interestingly, acidification of airway lining fluid promotes protonation
of NO2, conferring on lung lining fluid a chemistry that can
result in tyrosine nitration in subjects with asthma (22, 23). It remains to be demonstrated, however, whether acidification
of airspace lining fluids or cell surface and intracellular microenvironments occurs in ARDS and leads to nitrosative
chemistry similar to that observed in asthma. Thus, while a
multiplicity of distinct, yet redundant, pathways for tyrosine
nitration underscores the potential significance of this process
in inflammation, unambiguous evidence of particular or predominant mechanisms leading to tyrosine nitration remains to
be rigorously presented.
The present study stresses the fact that the increased BAL
concentration of the ·NO metabolites NO2 and NO3 parallels
the severity of disease. Both in vitro and in vivo investigations
reveal it to be unresolved as to whether alveolar NO2 and
NO3 are indicative of injurious manifestations of ·NO. In support of the contention of Sittipunt and colleagues for a detrimental role of ·NO in ARDS, acute pulmonary injury provoked by
paraquat administration to pigs is markedly reduced by ·NO
synthase inhibitors (24), inferring that ·NO amplifies the oxygen
radical-dependent lung injury long associated with paraquat.
More recently, it was observed that instillation of lipopolysaccharide (LPS) in mice triggered an increase in pulmonary inducible nitric oxide synthase (iNOS) expression, BAL lactate dehydrogenase content, and epithelial permeability, whereas these
effects were blunted in iNOS knockout mice (25).
Conversely, a number of investigations show a beneficial
effect of ·NO in acute lung injury. This is not surprising, because ·NO also mediates diverse antioxidant and antiinflammatory reactions (26, 27). In animal models, inhaled ·NO protects rat lung from hyperoxia-induced apoptosis and vascular
leakage (28). Moreover, because ·NO downregulates the expression of adhesion molecules, the extravasation and tissue
accumulation of leukocytes is frequently reduced by both endogenous ·NO biosynthesis and exogenous ·NO administration. Indeed, ventilation with ·NO often results in a significantly reduced alveolar neutrophil content (29). In patients
with ARDS, inhaled ·NO displayed favorable hemodynamic
effects, decreasing pulmonary artery pressure, and increasing
arterial oxygenation efficacy (30), suggesting the net reactions
of ·NO were protective rather than injurious in this setting.
Initial clinical investigations randomizing patients with ARDS
to inhale ·NO did not show a beneficial effect of this treatment strategy with respect to ventilator requirements and mortality (31). In aggregate, whether inflammatory production of ·NO or exogenous modulation of tissue ·NO concentrations is beneficial or deleterious in patients with ARDS remains unclear because precedents for each exist. It is possible
that iNOS-derived ·NO might even represent an antiinflammatory tissue response, in that this species also downregulates
inflammatory and oxidative reactions. Indices of net tissue
production of ·NO, such as BAL NO2 and NO3 content, are
not powerful prognostic indicators of impending or ongoing
lung injury (Figures 1 and 2 in Sittipunt and colleagues [4]). In
contrast, the authors' data related to tyrosine nitration in BAL
of patients with ARDS may be more revealing, because these adducts can also reflect accelerated ·NO production in an oxidative milieu.
In conclusion, Sittipunt and colleagues have established that NO2Tyr may provide a prognostic marker for the development of ARDS in humans. In future, it will be revealing to measure multiple ·NO-dependent modifications of biomolecules that reflect the multiplicity of oxidative inflammatory reactions occurring in ARDS (nitrosative, nitrative, and oxidative chemistry), an approach that can more comprehensively reflect the inflammatory milieu in ARDS. The utility of NO2Tyr as a biomarker in clinical samples of BAL fluids will also serve as a promising foundation for formulating strategies to pharmacologically intervene at early points in acute inflammatory diseases of the lung. The provocative observations of Sittipunt and coworkers have thus broken new ground and have also catalyzed important new questions related to both the benefits and disadvantages of therapeutic use of inhaled ·NO, as well as the biochemical mechanisms underlying ARDS.
Acknowledgments: Supported by grants from the Department of Veterans Affairs (Merit Review), the National Institute of Environmental Health Sciences (ES07498 and ES09607), and the National Heart, Lung, and Blood Institute (HL62628 and HL64855).
Supported by the National Institutes of Health (RO1-HL64937, RO1-HL 58115, and P6-HL58418; to B.A.F.), the American Heart Association (J.P.E.), and the Deutsche Herzstiftung (S.B).
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