rapid communication - Lung Cellular and Molecular Physiology

Am J Physiol Lung Cell Mol Physiol 279: L994–L1002, 2000.

rapid communication Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway JOHN D. LANG, JR.,1,2 PHILLIP CHUMLEY,1,2 JASON P. EISERICH,1,2 ALVARO ESTEVEZ,1,2 THAD BAMBERG,1,2 AHMAD ADHAMI,1,2 JOHN CROW,1–3 AND BRUCE A. FREEMAN1,3,4 Departments of 1Anesthesiology, 2Biochemistry and Molecular Genetics, and 3Pharmacology and 4The Center for Free Radical Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35233-4234 Received 18 April 2000; accepted in final form 20 July 2000

Lang, John D., Jr., Phillip Chumley, Jason P. Eiserich, Alvaro Estevez, Thad Bamberg, Ahmad Adhami, John Crow, and Bruce A. Freeman. Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway. Am J Physiol Lung Cell Mol Physiol 279: L994–L1002, 2000.— Ventilator strategies allowing for increases in carbon dioxide (CO2) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O2⫺), nitric oxide (䡠NO), and their product peroxynitrite (ONOO⫺). The reaction of CO2 and ONOO⫺ can yield the nitrosoperoxocarbonate adduct ONOOCO2⫺, a more potent nitrating species than ONOO⫺. Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of 䡠NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO2 (hypercapnia) revealed a significant increase in 䡠NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies 䡠NOdependent pathways to amplify cell injury. These results affirm the underlying role of 䡠NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.

SIGNIFICANT ADVANCES in understanding the pathophysiology of acute respiratory distress syn-

drome (ARDS), mortality remains at 40–60%, a moderate change since its description over 30 years ago (18). Injury to the lung by orchestrated inflammatory responses has devastating clinical consequences, including impairment of oxygenation and ventilation, as well as perturbation of nonrespiratory function, eventually necessitating mechanical ventilatory support. Several issues regarding causation, supportive measures, and treatment of ARDS patients are still receiving special attention. First, the genesis of lung dysfunction is multifactorial but, once established, involves the complex interplay of many of the pro- and anti-inflammatory mediators present in systemic inflammatory responses (10). Second, mechanical ventilation has now been accepted as a contributor to lung injury (ventilator-induced lung injury) resulting from a myriad of interactions between the mechanical ventilator, inspired gases, inflammatory mediators, and the lung itself (9, 43, 49). Finally, although pharmacological agents for the treatment of established lung injury are still in evolution, additional supportive and “protective” clinical strategies are being explored to reduce undesirable side effects (34). One promising approach is to allow carbon dioxide (CO2) tensions to increase in concert with decreasing tidal volume and minute ventilation, a practice referred to as “permissive hypercapnia” (5, 45). This concept represents a major departure from traditional practice because for many years, mechanical ventilation for ARDS equated to escalation in tidal volume, inspiratory pressure, end-expiratory pressure, or inspiratory time. Permissive hypercapnia or variants thereof allow for decreases in overall ventilation to take place at a time when alveolar-capillary integrity is particularly vulnerable, resulting in per-

Address for reprint requests and other correspondence: J. D. Lang, Jr., Dept. of Anesthesiology, The Univ. of Alabama at Birmingham, 845M Jefferson Tower, 619 19th St., Birmingham, AL 35233-6810 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

carbon dioxide; nitration; inflammation; superoxide; free radical; peroxynitrite

DESPITE

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1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society

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HYPERCAPNIA STIMULATES NITRIC OXIDE-DEPENDENT PATHWAYS

meability pulmonary edema. The respiratory acidosis that ensues from hypercapnia is well tolerated by most patients (20, 21). This trade-off seems intuitive in that repeated ventilatory cycles during the time of alveolar vulnerability predisposes the primary injured lung (i.e., sepsis) to a secondary injury (i.e., ventilator-induced lung injury). Recent clinical trials utilizing varying degrees of hypercapnia have resulted in improved survival in patients suffering from ARDS (2, 46). Oxidant stress or the increased rate of production of reactive species including superoxide (O2⫺), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), nitric oxide (䡠NO), and peroxynitrite (ONOO⫺) occurs in pulmonary tissues of animal models of ARDS and in ARDS patients, with these and other secondary biomolecules being proposed as contributing to the induction and maintenance of lung injury (3, 27). Markers of oxidative reactions are increased, and intrinsic host antioxidant defense mechanisms are impaired as well. In patients with ARDS, plasma ascorbate, ␣-tocopherol, ␤-carotene, and selenium were decreased along with lung glutathione (GSH) content (24, 30). Biochemical evidence of oxidant injury was also indicated by increases in lipid peroxidation by-products (35). Nitric oxide is an endogenously synthesized free radical species that contributes to both oxidative and antioxidant reactions and acts to maintain cellular homeostasis by way of multiple regulatory pathways (29). Nitric oxide is principally known as a mediator of signal transduction via the stimulation of guanylate cyclase-mediated cGMP synthesis. It is now also accepted that 䡠NO plays a pivotal and diverse role in inflammatory processes as well (36). Numerous pulmonary cell types including vascular endothelium, smooth muscle cells, macrophages, neutrophils, platelets, and type 2 alveolar epithelial cells produce 䡠NO. Nitric oxide biosynthesis is enhanced during pulmonary inflammation via inducible nitric oxide synthase (iNOS) induction (19). A reaction of particular significance during inflammatory responses occurs between 䡠NO and O2⫺, yielding the potent oxidant ONOO⫺, providing an alternative pathway for oxidative injury previously ascribed to individual reactions of 䡠NO, O2⫺, and 䡠OH (4, 31, 32, 39). ONOO⫺ oxidizes a variety of biomolecules, with its reactions profoundly influenced both kinetically and mechanistically by CO2 (13). ONOO⫺ that is produced in the presence of the almost ubiquitous biomolecule CO2 readily gives rise to the nitrosoperoxocarbonate adduct ONOOCO2⫺ (25). This adduct can undergo both heterolytic and homolytic cleavage to reactive products that possess enhanced nitrating and reduced oxidizing potential. Based on chemically oriented studies, it has been postulated that the unique reactivities of ONOOCO2⫺ will either increase or decrease the probability of ONOO⫺-mediated cell injury, but these precepts have not been tested in cell or animal models (23). Although nitration of pulmonary cell protein tyrosine residues readily occurs in respiratory distress syndromes, inferring both nitration and oxidation reactions of target molecules, nei-

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ther mechanisms nor consequences of 䡠NO-mediated lung cell protein tyrosine nitration are well defined (23, 48). This posttranslational protein modification can influence diverse aspects of cell structure and function, including cytoskeletal structure and the catalytic activity of antioxidant enzymes (14, 26, 47). We hypothesized that hypercapnia would enhance cytokine plus lipopolysaccharide (LPS)-mediated inflammatory lung cell injury by amplifying ONOO⫺mediated protein nitration reactions. To test this hypothesis, fetal rat type 2 alveolar epithelial cell monolayers were exposed to either 5 or 15% CO2 in the presence and absence of cytokines plus Escherichia coli LPS. Our results revealed that in the setting of cytokine-stimulated inflammatory responses and hypercapnia, which is currently being advocated clinically, potentially deleterious reactions that can impair cell and organ function ensue. EXPERIMENTAL METHODS

Materials. Minimum essential medium (MEM) and fetal bovine serum (FBS) were from HyClone Laboratories (Logan, UT). Hanks’ balanced salt solution (HBSS) and antibioticantimycotic solution were from GIBCO BRL (Life Technologies, Grand Island, NY). Recombinant murine interleukin (IL)-1␤, interferon (IFN)-␥, and tumor necrosis factor (TNF)-␣ were from R&D Systems (Minneapolis, MN). E. coli was from American Type Culture Collection (Manassas, VA). LPS (serotype 0111:B4) and N-monomethyl-L-arginine (LNMMA) were from Sigma (St. Louis, MO). Timed-pregnant female Sprague-Dawley rats at 16 days gestation were from Charles River Laboratories (Wilmington, MA) or Harlan Laboratories (Indianapolis, IN). Cell culture of type 2 alveolar cells. Fetal rat lung type 2 alveolar epithelial (RFLE) cells were isolated and cultured as previously described (7). Briefly, 19- or 20-day-gestation fetuses were removed aseptically; the lungs were dissected, minced, and resuspended in cold HBSS (calcium and phosphate free). The minced tissue was trypsinized for 10 min, filtered, and centrifuged. After several differential adherence steps, a 95–98% pure suspension of epithelial cells was obtained as determined by cytokeratin and vimentin staining (7). The cells were cultured in MEM plus 10% heat-inactivated FBS plus antibiotic-antimycotic solution (GIBCO BRL); then, on cell confluence (24–36 h), the monolayers were washed twice with HBSS and exposed to various experimental conditions in MEM containing 10% heat-inactivated FBS. Cell treatments. The cells were preincubated with cytokines [a combination of IFN-␥ (100 U/ml), TNF-␣ (500 U/ml), and IL-1␤ (300 pM)] and LPS. In some cases, L-NMMA (1 mM) was added, and the cells were subsequently exposed to either 5 or 15% CO2 (hypercapnia) in room air. Exposure times varied from 3 to 48 h depending on the analysis being performed. The cells were exposed in either 6- or 12-well plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ), with the plate covers loosened during the incubation. The 5% CO2 group contained 26.1 mM NaHCO3, resulting in a mean pH of 7.28 and mean partial pressure of CO2 of 41 mmHg. Cells exposed to 15% CO2 contained 59.5 mM NaHCO3, resulting in a mean pH of 7.28 and mean partial pressure of CO2 of 93.5 mmHg. NOS activity. NOS activity was determined by the conversion of [14C]arginine to [14C]citrulline (6). Cell lysates (100 ␮l) were added to an equal volume of reaction buffer contain-

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ing 10 mM NADPH, 0.5 M Tris, 0.4 mM FAD, 0.4 mM flavin mononucleotide, 100 mM citrulline, 4 mM tetrahydrobiopterin, 1 mM cold arginine, and 1 ␮Ci/ml of [14C]arginine at pH 7.5. After 30 min, the assays were terminated by the addition of 2 ml of 20 mM HEPES, 2 mM EDTA, and 2 mM EGTA at pH 5.5. Samples were applied to 1-ml Dowex AG50WX-8 (Tris form) columns and eluted with 2 ml of 20 mM HEPES buffer. Eluant containing [14C]citrulline was quantified by liquid scintillation counting of 100-␮l aliquots of medium from the cell incubation that were assayed for the oxidation products 䡠NO, NO2⫺, and NO3⫺. The samples were first incubated with E. coli nitrate reductase to convert NO3⫺ to NO2⫺ for subsequent analysis by the Griess reaction (37). Standard solutions of NaNO3 were prepared in medium to calculate the extent of NO3⫺ reduction to NO2⫺ and total NO2⫺ yield. Determination of 3-nitrotyrosine. Protein-bound nitrotyrosine from hydrolyzed cell lysates was analyzed by HPLC (5 ␮M Waters C18 column, 4.6 ⫻ 300 mm). Briefly, 100 ␮g of protein hydrolysate were separated at a flow rate of 0.75 ml/min with an isocratic elution profile consisting of 95% eluant A (50 mM acetic acid adjusted to pH 4.7 with a concentrated solution of semiconductor grade sodium hydroxide) plus 5% eluant B (HPLC-grade methanol) over 35 min (11). Tyrosine and 3-nitrotyrosine were identified by electrochemical detection with a 12-channel electrochemical detector (Coulachem, ESA, Chelmsford, MA). Both products were quantified by comparison with external standards. For immunocytochemical analysis, cell monolayers were fixed for 30 min at room temperature with 4% paraformaldehyde. After being washed three times with PBS, the cells were incubated in 50 ␮M lysine and 0.1% Triton X-100 in PBS (pH 7.4) for 15 min followed by blocking with 10% goat serum and PBS for 1 h. The cells were then incubated overnight with anti3-nitrotyrosine polyclonal antibodies in blocking solution (1:500), rinsed three times with PBS, and then incubated for an additional 30 min with 1:100 indocarbocyanine-linked goat anti-rabbit IgG (red; Jackson ImmunoResearch) at 25°C. After three washes with PBS, the cells were postfixed in 4% paraformaldehyde for 5 min, rinsed three times with distilled water, and finally incubated for 15 min with 1 ␮g/ml of 4⬘,6-diamidino-2-phenylindole. After three washes with distilled water, the slides were mounted in ProLong antifade (Molecular Probes, Eugene, OR). Fluorescence was visualized with an Olympus IX-70 epifluorescence microscope and an OlymPix cooled digital camera (resolution 1,328 ⫻ 1,024 pixels), and ESPRIT software was used for imaging. Cell injury assessment. Cell monolayer barrier integrity was assessed by measuring the permeability to 125 I-albumin. Transwell plates (Costar, Cambridge, MA) consist of an upper well separated from a lower well by a micropore filter (2.4-cm diameter, 4.5-cm2 surface area, 0.4-␮m pore size). The cells were plated in the upper well and grown to confluence (36–48 h) in MEM plus 10% FBS. On confluence (36– 48 h), the monolayers were washed twice with HBSS and exposed to experimental conditions in MEM plus 10% FBS. The lower well contained 2.65 ml of medium, and the upper well contained 1.5 ml of medium. Subsequently, 125I-albumin [1 ⫻ 106 counts/min (cpm)] and cytokines plus LPS with and without L-NMMA were added to the lower well. The addition of 125I-albumin (1 ⫻ 106 cpm) and cytokines plus LPS with and without L-NMMA represented time 0, and, subsequently, 10-␮l samples were obtained from the upper well at 3, 24, and 48 h. Radioactivity was assessed by gamma counting (LKB-Wallac 1275 MiniGamma). Western blot analysis for iNOS. Expression of iNOS protein was determined from the cell protein lysates after incu-

bation for 12 h. The cells were lysed by sonication in 40 mM Tris 䡠 HCl (pH 7.9), 10% glycerol, 1% Igepal, 5 ␮g/ml of aprotinin, 1 ␮g/ml of pepstatin, 1 ␮M leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 4 ␮M FAD, 4 ␮M flavin mononucleotide, 4 ␮M tetrahydrobiopterin, and 3 mM dithiothreitol; heated to 100°C for 5 min; and then stored at ⫺20°C. Proteins were fractionated on 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and incubated with a 1:2,000 dilution of anti-mouse macrophage iNOS. The blots were then treated with a 1:50,000 dilution of peroxidase-linked anti-rabbit IgG and developed with a maximum sensitivity chemiluminescence substrate kit (Pierce, Rockford, IL). Apoptosis quantification. Type 2 alveolar cells were exposed for 48 h as in Cell treatments and then terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick endlabeling staining was performed with the Apoptosis Detection System (Promega, Madison, WI). Each experimental condition was replicated in four wells, and six fields per well were recorded. Fluorescence was visualized with an Olympus IX-70 epifluorescence microscope and an OlymPix cooled digital camera (resolution of 1,328 ⫻ 1,024 pixels), and ESPRIT software was used for imaging. Determination of total cell thiol content. Net cell sulfhydryl content was quantified as previously described (40). Briefly, a 0.05-ml sample was mixed with 1 ml of phosphate-buffered solution, pH 8.2, and added before transfer to a cuvette containing 0.02 ml of 5,5⬘-dithio-bis(2-nitrobenzoic acid) in a 0.01 M phosphate-buffered solution. The formation of the 5-thio-2-nitrobenzoate ion was measured at 412 nm (molar extinction coefficient ⫽ 1.36 ⫻ 104 M⫺1 䡠 cm⫺1) after a 15-min incubation at 25°C. pH assessment. With combined pH and blood gas analysis monitoring (IL 1306 Instrumentation Laboratories), NaHCO3 was added to the cell culture medium (MEM) equilibrated with either 5% CO2 (26.1 mM NaHCO3) or 15% CO2 (59.5 g/l of NaHCO3), and the pH was titrated to 7.40. At pH 7.4, the 5% CO2 condition was determined to have 35–40 mmHg and 15% CO2 corresponded to 75–90 mmHg CO2 partial pressure. Type 2 alveolar epithelial cell intracellular pH measurements were made with 2⬘,7⬘-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (Molecular Probes, Eugene, OR) 24 h after cytokines plus LPS and CO2 exposure as previously described (44). Statistical analyses. Statistical analyses were performed with ANOVA with Tukey’s post hoc analysis on Systat 7.0 software (SPSS, Chicago, IL). Results with P ⬍ 0.05 were considered significant. All values are means ⫾ SD. RESULTS

Hypercapnia enhances type 2 alveolar epithelial cell 䡠NO production. To determine whether cytokine-induced 䡠NO production was altered by hypercapnia, cell NO2⫺ and NO3⫺ production was determined. Type 2 alveolar epithelial cells were exposed to 5 or 15% CO2 in the absence and presence of cytokines plus LPS for 48 h. Cell NO2⫺ and NO3⫺ production in the absence of cytokines plus LPS was enhanced by exposure to 15% CO2 (Fig. 1). Although the increase in 5% CO2-exposed cell NO2⫺ and NO3⫺ production in the presence of cytokines plus LPS was significant (P ⬍ 0.05), the greatest extent of NO2⫺ and NO3⫺ production was induced by exposure of cytokine-activated cells to 15% CO2 (P ⬍ 0.05). In this group, L-NMMA inhibited NO2⫺ and NO3⫺ production 90% (P ⬍ 0.05).


Fig. 1. Influence of cytokines plus lipopolysaccharide (LPS) exposure and hypercapnia on cell nitric oxide production. Type 2 alveolar epithelial cells were exposed for 48 h to 5 or 15% CO2 alone (solid bars) and in the presence of cytokines plus LPS (hatched bars) and cytokines plus LPS plus N-monomethyl-L-arginine (L- NMMA; open bars). Values are means ⫾ SD. * P ⬍ 0.05. † P ⬍ 0.05. ‡ P ⬍ 0.05.

NOS activity and protein expression were enhanced by hypercapnia. The rate of cell lysate arginine-tocitrulline conversion indicated changes in net NOS activity after cell exposure to hypercapnia and cytokines plus LPS for 24 h. Cytokines plus LPS enhanced NOS activity in the presence of 5% CO2 (21.6 ⫾ 3.2 nmol 䡠 min⫺1 䡠 mg protein⫺1; P ⬍ 0.05), with an even greater increase induced in cytokines plus LPS-treated cells in the presence of 15% CO2 (40.3 ⫾ 3.5 nmol 䡠 min⫺1 䡠 mg protein⫺1; P ⬍ 0.05; Fig. 2). The NOS activity of both cytokines plus LPS-treated cell groups in 5 and 15% CO2 exposure conditions was significantly inhibited by L-NMMA (1.5 ⫾ 0.30 and 1.6 ⫾ 0.45 nmol 䡠 min⫺1 䡠 mg protein⫺1, respectively; P ⬍ 0.05). Western blot analysis confirmed that during exposure to 15% CO2 in the presence of cytokines plus LPS, the level of expression of iNOS protein was maximal. Compared with 5% CO2 with cytokines plus LPS, iNOS protein expression was increased 70% by hypercapnia

Fig. 2. Influence of cytokines plus LPS and hypercapnia on cell nitric oxide synthase (NOS) activity. Cell NOS activity was measured by L-arginine to L-citrulline assay. Type 2 alveolar epithelial cells were exposed for 24 h to 5 or 15% CO2 alone (solid bars) and in the presence of cytokines plus LPS (hatched bars) and cytokines plus LPS plus L-NMMA (open bars). Values are means ⫾ SD. * P ⬍ 0.05. † P ⬍ 0.05.

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Fig. 3. Influence of cytokines plus LPS and hypercapnia on expression of inducible NOS (iNOS) protein. A: Western blot. Lane 1, 5% CO2; lane 2, 15% CO2; lane 3, 5% CO2 with cytokines plus LPS; lane 4, 15% CO2 with cytokines plus LPS. B: densitometric analysis of iNOS expression from replicate cell preparations and their Western blot analyses (n ⫽ 3). Values are iNOS expression by cells exposed to cytokines plus LPS at 5% CO2 adjusted to 100% and increased means ⫾ SD of iNOS induced by hypercapnia (15% CO2) after 24 h. * P ⬍ 0.05 compared with 5% CO2 with cytokines plus LPS.

(Fig. 3). No detectable iNOS protein expression was observed during exposure of non-cytokine-activated cells to 5 and 15% CO2 alone. Evidence for formation of 䡠NO-derived nitrating species. Cell protein 3-nitrotyrosine content was significantly increased by hypercapnic exposure of cytokines plus LPS-activated cells. 3-Nitrotyrosine was quantified after 48 h both immunohistochemically and by HPLC separation of cell protein hydrolysates with a diode array detection. Hypercapnia and cytokines plus LPS-activated type 2 alveolar epithelial cells displayed the greatest 3-nitrotyrosine immunoreactivity, a response inhibitable by L-NMMA (Fig. 4). Cell immunoreactivity for 3-nitrotyrosine was not observed in 5% CO2-exposed cells, and only minimally increased 3-nitrotyrosine immunoreactivity was observed in activated cells exposed to 5% CO2. Cells exposed to 15% CO2 also displayed only minimal 3-nitrotyrosine immunoreactivity compared with the more intense 3-nitrotyrosine immunoreactivity of cells exposed to 15% CO2 and cytokines plus LPS. Further quantitative support for increased 3-nitrotyrosine formation by hypercapnia-activated type 2 alveolar epithelial cells was obtained by HPLC analysis (Table 1). 3-Nitrotyrosine was not detectable (less than the limit of sensitivity of 0.5 mol 3-nitrotyrosine/ 1,000 mol tyrosine residues) in 5% CO2, 15% CO2, or 5% CO2 and cytokines plus LPS-treated cells (Fig. 4). In contrast, 9 mol 3-nitrotyrosine/1,000 mol protein tyrosine residues was detected in cytokines plus LPSactivated cells exposed to 15% CO2. Transmonolayer albumin flux increased significantly in the presence of 15% CO2 with cytokines plus LPS. The transmonolayer flux of 125I-albumin from the basolateral to the apical side of type 2 alveolar epithelial cell monolayers was greatest in the cells exposed to 15% CO2 and cytokines plus LPS (P ⬍ 0.05) compared

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Fig. 4. Type 2 alveolar epithelial cell 3-nitrotyrosine content after exposure to cytokines plus LPS plus L-NMMA in the presence of 5 and 15% CO2. A: cells exposed to 5% CO2 alone. B: cells exposed to 5% CO2 and cytokines plus LPS. C: cells exposed to 5% CO2 and cytokines plus LPS plus L-NMMA after 48 h. D: cells exposed to 15% CO2 alone. E: cells exposed to 15% CO2 and cytokines plus LPS. F: cells exposed to 15% CO2 and cytokines plus LPS plus L-NMMA after 48 h.

with that in hypercapnia alone (Fig. 5). The addition of L-NMMA to the hypercapnia-activated cells reduced albumin flux 56% (P ⬍ 0.05) but increased the rate of albumin flux in cytokines plus LPS-activated cells exposed to 5% CO2. Hypercapnia enhances apoptosis of cytokines plus LPS-activated type 2 alveolar epithelial cells. The occurrence of apoptotic nuclei was minimal in cells exposed to 5% CO2 alone (4.4 ⫾ 0.8%), with cell cytokines plus LPS treatment with and without L-NMMA also inducing no significant difference (Fig. 6). In the presence of 15% CO2, a significant increase in apoptotic nuclei was observed in cells exposed to cytokines plus LPS (14.6 ⫾ 1.0%) compared with that in 15% CO2alone exposure conditions (1.1 ⫾ 0.3%; P ⬍ 0.05). The addition of L-NMMA decreased cytokines plus LPSactivated hypercapnic cell apoptotic nuclei by 63% (14.6 ⫾ 1 vs. 5.5 ⫾ 1.1%). Hypercapnia decreases cell thiol content. The total cell thiol (RSH) content (protein plus nonprotein thiols) was not significantly influenced by hypercapnia (104 ⫾

3 nmol RSH/mg protein in 5% CO2 and 138 ⫾ 12 nmol RSH/mg protein in 15% CO2; Fig. 7). Activation of cells by cytokines plus LPS caused a moderate increase in thiol content in cells maintained in 5% CO2 (138 ⫾ 12 nmol/mg), with exposure to 15% CO2 alone decreasing total cell thiols 34% to 91 ⫾ 5 nmol/mg protein (P ⬍ 0.05). DISCUSSION

It was observed that hypercapnia modulates cell inflammatory responses via reactions that were predominantly detrimental to cell function. Cells exposed

Table 1. Influence of cytokines plus LPS exposure and hypercapnia on alveolar epithelial cell protein tyrosine nitration Treatment

mol 3-Nitrotyrosine/1,000 mol Tyrosine

5% CO2 ⫹Cytokines and LPS 15% CO2 ⫹Cytokines and LPS

ND ND ND 9*

Cells were cultured for 24 h with 20 ␮g/ml of Escherichia coli endotoxin [lipopolysaccharide (LPS)], 1.0 ng/ml of tumor necrosis factor-␣, 10 pg/ml of interleukin-1␤, and 100 U/ml of interferon-␥ in the presence of 5 or 15% CO2 in humidified air. ND, not detected. * P ⬍ 0.05 compared with control.

Fig. 5. Influence of cytokines plus LPS and hypercapnia on cell monolayer barrier function. Type 2 alveolar epithelial cells were exposed for 48 h to 5 or 15% CO2 alone (solid bars) and in the presence of cytokines plus LPS (hatched bars) and cytokines plus LPS plus L-NMMA (open bars). Values are means ⫾ SD. * P ⬍ 0.05. † P ⬍ 0.05. ‡ P ⬍ 0.05.


Fig. 6. Influence of cytokines plus LPS and hypercapnia on apoptosis of type 2 alveolar cell exposure after 48 h of exposure. A: cells exposed to 5% CO2 alone. B: cells exposed to 5% CO2 and cytokines plus LPS. C: cells exposed to 5% CO2 and cytokines plus LPS plus L-NMMA. D: cells exposed to 15% CO2 alone. E: cells exposed to 15% CO2 and cytokines plus LPS. F: cells exposed to 15% CO2 and cytokines plus LPS plus L-NMMA. G: percent of apoptotic nuclei. Solid bars, CO2 only; hatched bars, CO2 and cytokines plus LPS; open bars, CO2 and cytokines plus LPS plus L-NMMA. * P ⬍ 0.05. † P ⬍ 0.05. ‡ P ⬍ 0.05. § P ⬍ 0.05.

to hypercapnic conditions produced more 䡠NO as inferred by increased iNOS protein expression and the extent of L-NMMA-inhibitable cell NO3⫺ and NO2⫺ production. Both HPLC and immunohistochemical analysis showed cell protein tyrosine nitration was increased by hypercapnia, inferring increased formation of the reactive nitrating intermediate ONOOCO2⫺. Apoptotic cells were also present in significantly larger quantities during hypercapnia with cytokines plus LPS. Of functional significance was the loss of epithelial barrier integrity when activated cells were exposed to a proinflammatory hypercapnic environment.

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A cultured fetal rat type 2 alveolar epithelial cell model was chosen based on its ability to reflect lung epithelial responses to inflammatory stimuli and because the lung epithelium is a key target of inflammatory injury. A combination of cytokines (TNF-␣, IL-1␤, and IFN-␥) plus LPS was administered on cell confluence to stimulate inflammatory responses, including oxidative and 䡠NO-mediated stress. Cells were exposed for up to 48 h in 5 (35–40 mmHg) or 15% (75–90 mmHg) CO2. Normally, tissues exposed to hypercapnia would become more acidic, secondary to bicarbonate dissociation and carbonic anhydrase-catalyzed increases in H⫹ concentration. Because we focused on testing whether CO2 reacted directly with 䡠NO-dependent oxidative pathways to then modify susceptible target molecules, we normalized the values for the ancillary actions of CO2 (i.e., acidosis). This involved additional buffering of the medium and documentation of similar extracellular and intracellular acid-base environments for all CO2 exposure conditions. Although there was relative ease in maintaining and monitoring comparable extracellular pH levels, the use of a fluorescent intracellular pH indicator probe was necessary to confirm that intracellular pH was consistent with the extracellular milieu. For all experimental conditions, it was confirmed that intracellular and extracellular pH values were within 0.08 pH units of each other after 24 h of exposure (5% CO2 mean intracellular pH of 7.31; 15% CO2 mean intracellular pH of 7.33). Thus the results herein were reflective of the chemical reactivity of CO2 and are not due to indirect responses to acidosis. The hypercapnia-induced increased formation of 3-nitrotyrosine was used to indicate CO2-stimulated nitration reactions of ONOO⫺ (e.g., ONOOCO2⫺). Because the activity of iNOS was increased significantly by cell activation with a combination of cytokines and LPS, especially during hypercapnic exposures (Figs. 1–3), it remains possible that increased cell 䡠NO production also contributes in part to CO2-stimulated nitration reactions.

Fig. 7. Influence of cytokines plus LPS and hypercapnia on total thiol concentration. Type 2 alveolar epithelial cells were exposed for 48 h to conditions of 5 and 15% CO2 alone (solid bars) and in the presence of cytokines plus LPS (hatched bars). Values are means ⫾ SD. * P ⬍ 0.05. † P ⬍ 0.05.

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The reaction between ONOO⫺ and CO2 has a facile rate constant (k ⫽ 5.8 ⫻ 104 M⫺1 䡠 s⫺1) (8) and is highly favored in biological milieus, especially during periods of metabolic stress when inflammatory and hemodynamic events impact on O2⫺ and 䡠NO production and CO2 removal. The net effect of ONOOCO2⫺ formation on cell and organ function could be either injurious or protective because ONOOCO2⫺ decays in a neutral aqueous solution more rapidly than ONOO⫺ (e.g., a half-life of 1 ␮s to 1 ms for ONOOCO2⫺ compared with a half-life for ONOO⫺ of 0.1–1.6 s depending on the molecular composition of the microenvironment). Importantly, ONOOCO2⫺ and its secondary products also manifest an altered reactivity relative to ONOO⫺, displaying an increased propensity for nitration reactions and less potential as an oxidant (25). Because of these characteristics, CO2-mediated formation of ONOOCO2⫺ might alternatively protect cells from cytotoxicity due to redirection of ONOO⫺ to less oxidizing and kinetically faster decay mechanisms, thus decreasing cell exposure time to reactive species. This is in contrast to a scenario where the influence of CO2 yields not only ONOO⫺ but also ONOOCO2⫺ that would react with a host of susceptible target molecules (membrane, cytoskeletal, nuclear, or metabolic components) and defense mechanisms (i.e., ascorbate, tocopherols, thiols) to both oxidize and extensively nitrate targets in a manner detrimental for cell viability. For example, recent data (42) convincingly supports the concept that ONOO⫺-mediated biological nitration reactions are predominantly CO2 dependent and ONOOCO2⫺ mediated. Based on thermodynamic measurements (free energy of activation ⫽ 7.3 ⫾ 5.5 kcal/mol), reaction A would be the predominant pathway of further ONOOCO2⫺ reaction, with the other decay reactions, reactions B–D, being either less thermodynamically feasible or such that the reactants possess half-lives much shorter in magnitude than ONOOCO2⫺ (38) ONOO⫺ ⫹ CO2 3 䡠NO2 ⫹ HCO3䡠

(A)

ONOO⫺ ⫹ CO2 3 NO2⫹ ⫹ 䡠HCO3⫺ ⫺

(B) ⫺

(C)

⫺ 3

(D)

ONOO ⫹ CO2 3 䡠NO ⫹ 䡠OOCOO ⫺

⫹

ONOO ⫹ CO2 3 NO ⫹ HOOCO

Another possibility is that ONOOCO2⫺ rearranges to form nitrocarbonate (O2NOCO2⫺), which could behave as the proximal nitrating and oxidizing species. If ONOOCO2⫺ decays according to reaction A, two longerlived nitrating and oxidizing species are generated: nitrogen dioxide (䡠NO2) and carbonate radical (HCO3䡠), both of which are toxic molecules as noted by a rich foundation of photochemistry- and radiation chemistry-based studies (1, 16). Both 䡠NO and 䡠NO2 yield nitrating species via multiple mechanisms, with CO2 serving as a critical catalyst for ONOO⫺-dependent nitration reactions (15, 33). HCO3䡠, a strong oxidant, is also a potent free radical-propagating species (8). Therefore, in the setting of hypercapnia, ONOOCO2⫺ is a potentially toxic intermediate, with 3-nitrotyrosine

representing at least one possible “footprint” reaction product. The potent protection lent by L-NMMA to the loss of monolayer barrier function in 15% CO2-exposed, cytokine-activated cells affirms that hypercapnia deleteriously influences 䡠NO-mediated oxidative and nitration pathways, a pattern also observed in cells stained for the occurrence of apoptosis. This finding also lends support to the hypothesis that 䡠NO-derived species mediate, in part, cell responses to oxidant stress and hypercapnia. Multiple response variables revealed a difference in basal (nonactivated) cell responses to 5 and 15% CO2. For instance, there was an 87% increase in cell 䡠NO production as indicated by the increase in medium NO2⫺ plus NO3⫺ concentrations on type 2 cell exposure to 15% CO2. Impaired monolayer barrier function was also observed, with increased transmonolayer 125I-albumin flux occurring in the 15% (2,198 ⫾ 321 cpm) and 5% (1,455 ⫾ 98 cpm) CO2 groups. Immunofluorescent staining of 3-nitrotyrosine was apparent in the 15% CO2-alone group, whereas no staining was observed in the control 5% CO2 group. CO2 has historically been considered a relatively innocuous molecule during normocapnia, with limited data on the effects of CO2 in varying concentrations on cellular and humoral functions. When elevated to concentrations of 15% or greater, stimulation of the autonomic and central nervous systems, increased pulmonary vascular resistance, and elevated cardiac output have been recognized (50). Other regional vascular beds, including those of the renal-glomerular axis and splanchnic circulation, have been reported to have hypercapnia-induced increases in blood flow (12, 17). The predominant explanation for the physiological consequences of hypercapnia has been indirect effects resulting from the action of CO2 as a weak acid. Herein, pH was controlled for by additional buffering, with intracellular and extracellular acid-base environments documented to be similar in both normocapnic and hypercapnic conditions. Thus it is proposed that hypercapnia alone can alter normal cell signaling and that elevated CO2 levels serve as a catalyst for directing ONOO⫺mediated reaction pathways, leading to impairment of cell structure and function. Both protein and nonprotein thiols (e.g., GSH, which represents ⬎90% of nonprotein thiols in cells) are ubiquitous in vivo and serve as important intrinsic antioxidants. ONOO⫺ readily reacts with GSH, a reaction that is inhibited by its more facile reaction with CO2 (13, 33). Cytokines plus LPS exposure consistently enhanced cell thiol content, a not unexpected observation because the rate-limiting enzyme of GSH synthesis, ␥-glutamylcysteine synthetase, is induced by 䡠NO and redox-activated transcription factors (28). Net cell thiol content decreased after exposure to hypercapnia, a significant response when cytokine-stimulated cells maintained in 5% CO2 were compared with 15% CO2exposed cytokine-stimulated cells (P ⬍ 0.05). Potential explanations for these findings include 1) the possibility that in cell systems, hypercapnia may stimulate the


formation of secondary propagating radical species (e.g., HCO3⫺) that depletes thiols; 2) integrated metabolic reactions responsible for maintaining thiol reduction are impaired by hypercapnia; and 3) hypercapnia may also accelerate cellular production of partially reduced species of oxygen. This emphasizes the widespread metabolic and oxidative effects of hypercapnia and is in contrast to observations of decreased ONOO⫺-mediated oxidation of GSH occurring in the presence of increasing concentrations of CO2 (51). Recent clinical studies have confirmed the efficacy of protective ventilatory strategies in decreasing the expression of proinflammatory mediators (34) and increased survival in patients with ARDS (2, 21, 46). A consequence in several of these studies has been varying degrees of hypercapnia that occurred compared with that in patients treated with conventional strategies. Importantly, pulmonary cell iNOS protein expression and 䡠NO production will be elevated in the proinflammatory milieu of ARDS lungs (9, 19). This, combined with the clinical use of inhaled 䡠NO, hyperoxia, and the induction of hypercapnia, infers that a complex set of oxidative and free radical reactions can be occurring within lung cells. Last, low tidal volume strategies and accompanying normocapnia have been associated with improved survival in patients with ARDS (41). The consequences of the increased respiratory rates (versus increased tidal volume) required to maintain these traditional targeted levels of CO2 are not fully known, thus opening up an additional area for investigation. In summary, these data reveal the detrimental cellular effects of hypercapnia in an inflammatory environment that includes amplification of the production of 䡠NO and secondary nitrating species. These findings are of particular clinical significance and timing, showing a novel reactivity for CO2 in the context of emerging ventilator strategies that allow hypercapnia to occur in patients suffering from inflammatory-mediated lung injury. The current literature pertaining to hypercapnia in the setting of critical illness is both limited and in conflict (22). The present results thus encourage clinical studies prospectively evaluating the contribution of oxidative inflammatory reactions and aberrant lung cell 䡠NO-dependent signal transduction during the evaluation of protective ventilation modes that result in varying degrees of hypercapnia. This study was supported by a Foundation for Anesthesia and Education Research Grant (New Investigator Award); Arrow International (J. D. Lang); and National Heart, Lung, and Blood Institute Grants HL-58418, HL-58115, and HL-51245 (B. A. Freeman). REFERENCES 1. Adams GE. Free radicals in biology: the pulse radiolysis approach. In: Free Radicals in Biology, edited by Pryor WA. New York: Academic, 1977, p. 53–91. 2. Amato MBP, Barbas CSV, Medeiros DM, Magaldi RB, Schettino GP, Lorenzo-Filho G, Kairalla RA, Deheinzelin D, Munoz C, Oliveira R, Takagaki TY, and Carvalho CR. Effect of a protective ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 338: 347– 354, 1998.

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