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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. S9-S16, (2002)
© 2002 American Thoracic Society

Article

Reactive Nitrogen Species and Cell Signaling

Implications for Death or Survival of Lung Epithelium

Yvonne M. W. Janssen-Heininger, Rebecca L. Persinger, Solange H. Korn, Cristen Pantano, Brian McElhinney, Niki L. Reynaert, Ramon C. J. Langen, Karina Ckless, Punya Shrivastava and Matthew E. Poynter

University of Vermont College of Medicine, Burlington, Vermont

Correspondence and requests for reprints should be addressed to Yvonne M. W. Janssen-Heininger, Department of Pathology, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405. E-mail: yjanssen{at}zoo.uvm.edu

ABSTRACT

Reactive nitrogen species such as nitric oxide, peroxynitrite, and nitrogen dioxide have been implicated in the pathophysiology of inflammatory lung diseases. Yet, the molecular mechanisms and cell signaling events responsible for cellular injury remain to be elucidated. Two major signaling pathways, co-ordinately regulated and responsible for cell survival and cell death, involve nuclear factor {kappa} B and c-Jun-N-terminal kinase, respectively. A review of these pathways, their modes of action, and their importance in executing oxidative stress responses in lung epithelial cells are discussed.

Key Words: oxidants • signal transduction • lung epithelium

REACTIVE NITROGEN SPECIES AND PATHOPHYSIOLOGY OF LUNG DISEASE

Inflammatory pulmonary diseases are accompanied by the production of a myriad of oxidants generated by inflammatory and structural pulmonary cells. Furthermore, in chronic obstructive pulmonary disease (COPD), where smoking is the main factor in disease etiology, damaging oxidants are also contributed directly by cigarette smoke (1). Considerable focus has been on reactive nitrogen species (RNS), including nitric oxide (NO), peroxynitrite (ONOO-), and nitrogen dioxide (NO2) (2). Induction of inducible nitric oxide synthase (iNOS), the high output form has been observed in patients with asthma. Not surprisingly, this results in an increase in the concentration of NO in their exhaled breath. This does not necessarily indicate that RNS mediate injury, for the biologic effects of NO and its protein targets are affected by the presence of other oxidants, which can consume NO. It is well known that under conditions where the superoxide anion is generated, NO is rapidly consumed to produce the highly reactive ONOO-, which can in turn decompose to generate an oxidant capable of nitration, likely NO2. Moreover, the presence of peroxidases such as eosinophil peroxidase and myeloperoxidase, released from eosinophils and neutrophils, respectively, can consume the stable end product of NO, nitrite, and the superoxide anion dismutation product, hydrogen peroxide (H2O2) to generate NO2 (2, 3). Nitrating species evoke unique chemical reactions, such as nitration of tyrosine moieties. Tyrosine nitration has been shown to occur in patients with asthma (4) (Figure 1) and other inflammatory lung diseases, including chronic obstructive pulmonary disease (5), cystic fibrosis, acute respiratory distress syndrome, and idiopathic pulmonary fibrosis (2). Interestingly, in asthma, the extent of nitration correlates with the expression of iNOS and correlates with disease severity. Corroborating evidence for a damaging effect of nitrating species in inflammatory lung diseases stems from studies of asthmatic children in whom an association has been shown between the levels of atmospheric NO2 and the severity of respiratory symptoms (6). Moreover, the addition of ONOO- also has been shown to cause epithelial injury and airway hyper-reactivity in guinea pigs (7).



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  		Figure 1. Tyrosine nitration in lung tissue from patients who died of nonpulmonary causes (A and B) or status asthmaticus (C and D) (4). Lung sections were deparaffinized, rehydrated, and incubated with a nitrotyrosine-specific antibody (Upstate Biotechnology, Lake Placid, NY) followed by incubation with a Cy5-conjugated secondary antibody. Nuclei were counterstained with propidium iodide to visualize the lung architecture. Sections were evaluated by confocal microscopy and merged images shown: green represents nitrotyrosine immunoreactivity, red represents propidium iodide. Adapted with permission from Reference 4.

 
Despite the aforementioned study in patients with asthma, tyrosine nitration and induction of iNOS are not necessarily linked as was recently demonstrated in iNOS knockout mice. These mice showed similar nitration as did wild-type mice in a model of ovalbumin-induced allergic airway inflammation, even though the NO levels present in bronchoalveolar lavage were significantly lower (8). Importantly, however, ovalbumin-induced airway inflammation was significantly attenuated in iNOS knockout mice, suggesting an important pro-inflammatory role for iNOS (9) independent of tyrosine nitration (8). A dissociation between NOS induction and tyrosine nitration was also apparent in New Zealand White mice that lack eosinophil peroxidase. In these mice, the inflammatory responses to ovalbumin, and the NO levels in bronchoalveolar lavage were similar compared with those in two other strains, but tyrosine nitration was markedly diminished (8). Although it thus appears that tyrosine nitration and iNOS induction are effectively uncoupled in models of allergic airway disease in mice, other NOS isoforms, in particular neuronal NOS, which is believed to play a role in asthma (10), remain to be evaluated for tyrosine nitration. Whether nitrating species contribute to pathophysiology of inflammatory lung diseases or merely reflect an epiphenomenon (2) remains to be determined because many critical targets and signaling pathways that mediate their outcomes remain to be identified.

The oxidant responsible for tyrosine nitration in lung tissue likely is NO2 or a molecule with similar reactivities. Because tyrosine nitration is found in lungs from patients with asthma (Figure 1) and other inflammatory lung diseases, understanding the mechanism by which NO2 evokes cell damage may ultimately provide a better understanding of how nitrating species contribute to the pathophysiology of asthma. In animal studies, diverse outcomes of NO2 inhalation that depend on the doses and duration of exposure, the species under investigation, pre-existing disease, nutritional status, and genetic susceptibility have been reported (11). Inhaled NO2 is absorbed all along the respiratory tract, and depending on the concentration and dose, injury can occur in the trachea, bronchi, bronchioles (the main site of deposition), and alveolar ducts (12). Concentrations ranging from 50 to 150 ppm (94–282 mg/m3) can cause death in animals, due to extensive pulmonary injury, edema, hemorrhage, and pleural effusion (11). Short-term exposure to NO2 results in a biphasic response with an initial injury phase followed by repair accompanied by increases in DNA and protein synthesis (13). Exposure to 10 ppm NO2 for greater than 24 hours causes damage to cilia and hypertrophy of the bronchiolar epithelium (14). In addition, hyperplasia of alveolar Type II epithelial cells has also been reported after inhalation of 15 or 20 ppm of NO2 (15). Other reported changes include emphysematous and fibrotic changes within the lung (11, 16). An understanding of how this model nitrating species causes injury of pulmonary epithelial cells is important because damage of the epithelium appears to be a critical initiation step in the exacerbation of pre-existing lung disease, increased susceptibility to infection, and pulmonary remodeling and fibrosis.

SIGNALING EVENTS REGULATING CELL DEATH IN RESPONSE TO RNS

The chemical reactivity of NO2 is explained by its free radical nature. Consequently, lipid peroxidation, sulfhydryl oxidation, and nitration reactions all have the potential to contribute to cellular injury. Although inhalation studies addressing the pulmonary effects of NO2 have been performed both in humans and laboratory animals, studies of gas phase exposures of pulmonary cells to NO2 are scant. This largely is the result of the high chemical reactivity of NO2, which makes these experiments particularly challenging. Our laboratory has examined the patterns of epithelial cell death in response to exposure to pure NO2 to elucidate the potential mechanisms by which NO2 causes epithelial cell injury. These studies pointed out the unique effects of nitrating species (NO2, ONOO-, or the ONOO- generator, 3-morpholinosydnonimine) in comparison with H2O2 or NO. Whereas H2O2 or NO have the potential to kill lines of rat or mouse alveolar Type II epithelial cells, independently of their growth status, NO2 or ONOO- selectively kill log phase cells. Confluent cells are strikingly resistant to cell death induced by NO2 or ONOO- (Figure 2) (17). Similarly, in experiments modeling wound healing, death is limited to cells present in the leading edge of the wound, whereas cells in the confluent area appear resistant to cell death (17). The use of 3-morpholinosydnonimine in the presence of superoxide dismutase that generates NO and H2O2, instead of ONOO- was also associated with generalized cell death (18) that was not restricted to the leading edge of the wound (17), again illustrating that H2O2 or NO kill lung epithelial cells independent of their growth status. It can be argued that the active DNA synthesis of dividing cells may enhance their susceptibility to DNA damage, making them exquisitely sensitive to killing by NO2. Alternatively, cell migration and altered contact with the extracellular matrix may also contribute to enhanced oxidant sensitivity of log phase lung epithelial cells.



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  		Figure 2. NO2, ONOO-, or the ONOO- generator, 3-morpholinosydnonimine (SIN-1) selectively kill log phase lung epithelial cells. (A) A line of rat alveolar Type II epithelial cells (21) was used and grown to log phase or confluence and exposed to 5 ppm of NO2 for 4 hours, and cell death was assessed immediately or 4, 24, 48 or 72 hours after cessation of exposure. (B) Log phase or confluent rat alveolar Type II epithelial cells cells were exposed to 1 mM ONOO- or 1 mM SIN-1 and death was assessed after 2, 4, and 24 hours. Cell death was assessed using an antibody recognizing single-stranded DNA (17), and results are expressed at the percentage of APOPTAG positive cells. Adapted with permission from Reference 17. *Significantly different from air controls (p < 0.05).

 
SIGNALING PATHWAYS IN RNS-INDUCED CELL DEATH

The main question about the mechanisms of cell death caused by nitrating species is whether they are regulated by intracellular signaling cascades as opposed to unregulated necrosis. Studies from our laboratory have demonstrated that inhibition of protein synthesis rescues NO2-induced single-stranded DNA formation (17), suggesting that death effector molecules are produced in a regulated manner. Alternatively, it can be asked why cell survival mechanisms are not activated, in response to nitrating species, allowing death effector pathways to predominate, leading to cell killing. Two major pathways known to be involved in stress-induced cell death and survival are c-Jun N-terminal kinase (JNK) and nuclear factor {kappa} B (NF-{kappa}B), respectively. Their modes of action and potential involvement in executing oxidant stress responses is discussed in further detail.

JNK

The mitogen-activated protein kinases represent a family of signaling molecules that respond to cellular and environmental stimuli. In mammalian cells three distinct family members are known: extracellular signal-related kinase, JNK, and p38 (19, 20). In general it appears that, depending on the cell type and stimulus, the extent and duration of activation of the various mitogen-activated protein kinase pathways appears to control proliferation, transformation, apoptosis, cell migration, survival, and development (19, 20). Activation of the mitogen-activated protein kinases causes changes in gene transcription that contribute to the phenotypic response. The activation of JNK has been documented to be important in stress responses including apoptosis, cell survival decisions, and cell transformation. Despite a role in stress responses, JNK also plays an integral role in physiologic processes such as embryonic morphogenesis and, in some models, mitogenic signaling (19). Oxidants, including H2O2 and ONOO- (21) and other cellular stresses such as cytokines, osmotic stress, ultraviolet and {gamma}-irradiation, and arsenite are known inducers of the JNK pathway (19). Three genes encode the various JNKs, which include JNK1, JNK2, and JNK3. Although JNK1 and JNK2 are ubiquitously expressed, JNK3 is selectively expressed in the heart, testis, and brain (19). All forms of JNK are activated by the dual phosphorylation of the Thr-Pro-Tyr motif by either MAPK kinase 7 or MAPK kinase 4 (19). On phosphorylation, JNK can bind the transcription factor c-Jun and phosphorylate serines 63 and 73 that increase its transcriptional activity (22). JNK can also bind and phosphorylate other transcription factors in the activator protein-1 family, such as ATF-2, JunB, and Jun D (19). A causal relationship between JNK activation and the induction of apoptosis has now been reported in a variety of tissues in response to multiple stresses. For instance, antisense strategies to inhibit JNK1 or JNK2 revealed that blocking JNK1 but not JNK2 prevents apoptosis after ischemia and reoxygenation of cardiac myocytes (23). Murine embryonic fibroblasts isolated from the JNK1-/- and JNK2-/--deficient embryos, were resistant to ultraviolet radiation-induced apoptosis (24). JNK also has been demonstrated to play a causal role in neuronal cell apoptosis after the withdrawal of nerve growth factor (25) or after exposure to excitotoxins (26). Furthermore, a knock-in mouse in which the endogenous c-jun gene was replaced with a mutant refractory to phosphorlyation by JNK (JunAA mice) was protected from apoptosis induced by excitotoxic stress (27). Taken together, these studies underscore the role of JNK activation in apoptosis, which under some conditions appears to require the activation of c-Jun by JNK and consequently c-Jun-dependent gene transcription.

One candidate gene that regulates JNK-dependent cell death is the death-inducing Fas ligand (28). In studies using T cells, environmental stress caused increases in expression of Fas ligand, which occurred in a JNK-dependent manner (29). Furthermore, murine embryonic fibroblasts derived from c-jun-/- mice displayed impaired expression of Fas ligand (30). Activation of JNK with subsequent phosphorylation of c-Jun appears to regulate Fas ligand transcription through an activator protein-1 site in the promoter (28). These studies demonstrate a role for JNK/c-Jun in the initiation of apoptosis, which in some cases may be through the production of a death-inducing molecule, like Fas ligand. However, other studies suggest an alternative mechanism for JNK-dependent stress-induced apoptosis. JNK activation was associated with the mitochondrial release of cytochrome C (24) and the consequent induction of apoptosis in a caspase-9-dependent manner. In support of the induction of mitochondrial-dependent apoptosis triggered by JNK, caspase-9-/- embryonic stem cells (31) or caspase-3-/- murine embryonic fibroblasts (32) were found to be resistant to ultraviolet-induced apoptosis. These findings illustrate that JNK activation can cause stress-dependent apoptosis via multiple pathways that either involve new gene expression and production of death effector molecules or the induction of apoptosis after disruption of mitochondria. It remains to be determined whether nitrating species evoke cell death via these pathways. Furthermore, studies determining the importance of JNK activation and its death effectors in the pathophysiologic disturbances inflicted by nitrating species remain to be undertaken.

NF-{kappa}B

NF-{kappa}B is a pleiotropic transcription factor induced by a wide variety of stresses in mammalian cells. NF-{kappa}B transcriptionally activates genes for cytokine and chemokine production, proliferation, matrix degradation, and prevention of apoptosis (33–36). It is one of the best-studied mammalian transcription factors, and the molecular events that precede its activation are being investigated in great detail (34, 37). NF-{kappa}B activity is tightly controlled by the inhibitory protein, I{kappa}B{alpha} that is normally present in the cytosol complexed to NF-{kappa}B dimers, thereby preventing the nuclear localization of NF-{kappa}B and ensuring low basal transcriptional activity. Exposure to a number of stimuli (e.g., cytokines, lipopolysaccharides, viruses) results in the phosphorylation, ubiquitination, and degradation of I{kappa}B, liberating the NF-{kappa}B dimers (33, 34). The NF-{kappa}B complex translocates into the nucleus and transcriptionally activates target genes by binding to responsive elements in the DNA, called {kappa}B motifs. Phosphorylation of the inhibitory protein I{kappa}B occurs at specific serine residues by I{kappa}B kinases (IKK). IKK are present as a large (700–900 kD) complex composed of two catalytic subunits, IKK{alpha} and IKKß, and a regulatory subunit, IKK{gamma} (38). The IKK complex serves as a point of convergence for both positive and negative NF-{kappa}B regulators (39). Of the two catalytic subunits of the IKK complex, IKKß has the major role in responding to proinflammatory stimuli (39) and is sensitive to inactivation by aspirin, salicylate, cyclopentenone prostaglandins, and the thiol-reactive metal arsenite (40, 41). Upstream kinases including mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1, NF-{kappa}B-inducing kinase, and protein kinase B (Akt), activate the IKK complex through mechanisms not fully understood but result in the phosphorylation of two serine residues in the activation loop of IKK{alpha} (serines 176/180) and IKKß (serines 177/181) (38, 39). Because the IKK complex is an important mediator in the positive and negative regulation of NF-{kappa}B, it requires stringent regulation achieved through rapid activation and inactivation.

The activation of NF-{kappa}B represents an essential strategy that allows cells to survive diverse stresses (36, 42, 43). For example transfected cells with a mutant I{kappa}B construct (dominant negative I{kappa}B) to prevent activation of NF-{kappa}B demonstrated enhanced cell killing by cytokines, ionizing radiation, or cancer therapeutics, illustrating that induction of NF-{kappa}B is critical to survival (43). A number of NF-{kappa}B regulated genes with an antiapoptotic function have been identified and include cellular inhibitor of apoptosis protein-1, cellular inhibitor of apoptosis protein-2, X chromosome-linked inhibitor of apoptosis (44), the adaptor proteins, tumor necrosis factor (TNF) receptor-associated factor 1 and TNF receptor-associated factor 2 (45), the caspase-8 antagonist, c-FLIP (46, 47), and others.

Oxidative stress has been thought to play a critical role in the activation of NF-{kappa}B based on many observations that demonstrate that cytokine-induced NF-{kappa}B activation can be prevented after treatment with antioxidants or metal chelators. The role of oxidants per se in the activation of NF-{kappa}B has been the subject of considerable debate (34, 40). For instance, it appears that the ability of H2O2 to induce NF-{kappa}B depends on the cell type being investigated and may be linked to levels of antioxidants present in those cells (40). However, even within one cell type, marked variations in the ability of H2O2 to activate NF-{kappa}B are apparent. For instance, among investigated T cell lines, Wurzburg T cells, but not Jurkat T lymphocytes, are responsive to H2O2 (40). In support of this variability, our laboratory demonstrated that in a line of rat alveolar Type II epithelial cells, H2O2 caused activation of an NF-{kappa}B-driven luciferase reporter gene (21) in contrast to mouse alveolar Type II epithelial cells (48). In studies where oxidants, including pervanadate, hypoxia/reoxygenation, and ultraviolet-C were investigated, it appeared that an alternative pathway for activation of NF-{kappa}B was used. After exposure to these stresses, NF-{kappa}B was dissociated from its inhibitor, I{kappa}B{alpha} by tyrosine phosphorylation of I{kappa}B{alpha}. This alternative pathway involved phosphorylation of tyrosine 42 instead of serines 32/36 (49) and was, in some cases, associated with degradation of I{kappa}B{alpha}, which did not appear to involve the activation of IKK (50) but required the p85 subunit of phosphatidylinositol-3-kinase (51), known to be activated by oxidants. The precise mechanism by which oxidants regulate NF-{kappa}B remains to be elucidated, but it is becoming clear from the available data that oxidant-induced NF-{kappa}B activation is highly cell type-dependent (52), pointing perhaps to a lack of a uniform mechanism of activation (40).

Because the activation of IKK is an essential prerequisite to activate NF-{kappa}B in response to most stimuli, studies in our laboratory focused on the regulation of IKK and NF-{kappa}B by the model oxidant, H2O2 in the absence or presence of the inflammatory cytokine TNF{alpha}. Although TNF induced a marked activation of IKK, H2O2 failed to induce IKK despite its ability to cause activation of JNK, another serine-directed kinase. Importantly, in cells exposed simultaneously to H2O2 and TNF, the presence of H2O2 interfered with TNF-induced IKK activation. This resulted in a lack of degradation of I{kappa}B, a decreased translocation of NF-{kappa}B to the nucleus, and a decrease in NF-{kappa}B-dependent gene transcription (Figure 3) (48). We also determined that H2O2 was capable of inducing cysteine oxidation of the IKK complex that may have contributed to the decrease of enzymatic activity (48). The decrease in TNF-induced activation of IKK in the presence of H2O2 was observed in a line of rat and mouse alveolar epithelial cells (48), illustrating that intact lung epithelium may also respond to oxidants stress with a loss of IKK and consequently NF-{kappa}B activation, which may ultimately have ramifications for cell death.



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  		Figure 3. H2O2 decreases the ability of TNF{alpha} to induce IKK activation, DNA-binding activity of NF-{kappa}B and NF-{kappa}B-dependent gene transcription. A line of mouse alveolar Type II epithelial cells was exposed simultaneously to 10 ng/ml TNF{alpha} and 200 µM H2O2 for the assessment of activation of the NF-{kappa}B pathway (48). (A) Kinase assay examining the activation of IKK. IKK{gamma} was immunoprecipitated from cells after 5 minutes of exposure to TNF or TNF in the presence of H2O2 and reacted in kinase buffer containing the substrate, GST-I{kappa}B. (B) Assessment of the binding of NF-{kappa}B to its consensus DNA sequence using the electrophoretic mobility shift assay. Cells were exposed to TNF in the presence or absence of H2O2 for 15 minutes at which time nuclear extracts were prepared. (C) Assessment of NF-{kappa}B-dependent reporter gene activity in mouse alveolar Type II epithelial cells stably expressing and NF-{kappa}B-dependent luciferase construct. Cells were exposed to TNF in the presence or absence of H2O2 for 6 hours at which time the luciferase activity was determined. Shown are values normalized to protein. Sham: sham controls that received phosphate-buffered saline instead of agents. Adapted with permission from Reference 48.

 
BALANCING LIFE AND DEATH THROUGH THE CONTROL OF JNK AND NF-{kappa}B

The overall balance between the activation of the JNK and NF-{kappa}B pathways appears to be critical in dictating cell survival or cell death (Figure 4) . This notion is especially well studied in the context of signaling via TNF-receptor 1 (53). As stated previously, the activation of NF-{kappa}B is required for survival in response to TNF and other agents (43). Failure to activate NF-{kappa}B in cells expressing dominant negative I{kappa}B or in cells lacking IKKß or RelA was associated with a striking elevation and prolongation of JNK activation after stimulation with TNF (44, 54). Expression of a dominant-negative version of JNK in cells that were not capable of activating NF-{kappa}B prevented TNF-induced cell death, illustrating that the enhanced JNK activation contributed to TNF-induced cell death (54). Thus, it appears that repression of JNK activation by NF-{kappa}B is critical for the antiapoptotic effect (44, 54). The NF-{kappa}B-induced antiapoptotic protein, X chromosome-linked inhibitor of apoptosis appears to play a critical role in the negative regulation of JNK activation by TNF, thereby preventing apoptosis from occurring (44). Furthermore, growth arrest and the DNA damage inducible gene (gadd), 45 ß also was found to be upregulated by NF-{kappa}B in response to TNF and to inhibit JNK-dependent apoptosis (55). Lastly, TNF receptor-associated factor 1, TNF receptor-associated factor 2, cellular inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein-2, genes induced by NF-{kappa}B, act in concert to inhibit caspase 8 activation (45) that executes TNF-receptor 1-dependent apoptosis.



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  		Figure 4. A hypothetical model by which disruption in the balance of activation of NF-{kappa}B and JNK mediates death in cells exposed to RNS. The striking activation of JNK and repression of NF-{kappa}B activation tips the balance toward cell death. The involvement of death receptors of the TNF-receptor superfamily, known to regulate the activation of JNK and NF-{kappa}B after exposure to RNS remains to be determined.

 
The effect of these antiapoptotic adapter proteins on JNK activation remains to be determined. Conversely, the apoptotic pathway itself can inhibit NF-{kappa}B after caspase-dependent cleavage of receptor interacting protein (RIP) (56), IKKß, I{kappa}B (57), and p65 (58), all preventing NF-{kappa}B activation or NF-{kappa}B-dependent gene expression (59). These combined observations illustrate that apoptosis and NF-{kappa}B activation are mutually exclusive events (53). A deviation toward activation of JNK while preventing the activation of NF-{kappa}B can then set the stage for cell death. Intriguingly, the activation of different members of the TNF-receptor superfamily, including, TNF-receptor 1 and Fas, but also other receptors such as Toll-like receptor 4 and interleukin-1 receptor can lead to the co-ordinated activation of NF-{kappa}B and JNK. As was stated before, the activation of NF-{kappa}B in this context is critical in allowing cells to survive receptor activation (47, 53). It will be of great interest to determine whether oxidants can modulate the function of death receptors and whether JNK and NF-{kappa}B activation by oxidants and ensuing life–death decisions are controlled via these death receptors.

FUTURE RESEARCH

The available data make it clear that RNS can promote cell death and interfere with survival pathways. From numerous studies examining environmental stresses, the striking activation of JNK and inhibition of NF-{kappa}B by RNS may be responsible for the induced death of lung epithelial cells (Figure 4). Although cell culture models will allow investigators to assess the contribution of these pathways in cell death and survival after exposure to RNS in vitro, their relative importance in inflammatory lung diseases remains to be identified. Although knockout mice appear attractive for elucidating the importance of these signaling pathways they are also flawed with difficulties. For instance, RelA-/- mice and mice lacking both JNK1 and 2 are embryonic lethal. In addition, other knockout mice may display altered immune responses, posing potential difficulties in studying signaling events in the context of allergic airway disease.

The link between RNS-induced signaling events and profiles of gene expression in patients with inflammatory lung diseases remains to be examined. Bronchial brushings allow investigators to selectively sample bronchial epithelium and will be instrumental to gain insights into the activation of signaling cascades and the expression of survival and death effector genes that may contribute to injury, death, or repair of the airway epithelium and, potentially, to airway remodeling.

The relative contribution of NO versus more reactive nitrating species, such as NO2 or ONOO-, in airway pathobiology remains to be elucidated, as many of the molecular targets of endogenous NO or nitrating species remain to be determined. It will be especially crucial to determine whether components of the JNK and NF-{kappa}B pathways are subject to nitration or nitrosation that would explain the importance of these signaling pathways in controlling cell death versus survival. The availability of new proteomic techniques that allow investigators to examine populations of proteins modified by tyrosine nitration (60) or S-nitrosation (61) and to determine their identity, will undoubtedly shed light on the current controversy regarding the importance of RNS in the pathophysiology of inflammatory lung diseases.

FOOTNOTES

Supported by grants NIH RO1 HL60014, PHS grant P20 RR 15557 (COBRE), and a grant from the Dutch Asthma Foundation.

Received in original form June 14, 2002; accepted in final form October 2, 2002

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