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The Journal of Immunology, 2002, 169: 1474-1481.
Copyright © 2002 by The American Association of Immunologists

Apical, But Not Basolateral, Endotoxin Preincubation Protects Alveolar Epithelial Cells Against Hydrogen Peroxide-Induced Loss of Barrier Function: The Role of Nitric Oxide Synthesis1

Frank Rose*, Bernd Guthmann*, Tobias Tenenbaum*, Ludger Fink*, Ardeschir Ghofrani*, Norbert Weissmann*, Peter König{dagger}, Leander Ermert*, Gabriele Dahlem*, Joerg Haenze*, Wolfgang Kummer{dagger}, Werner Seeger* and Friedrich Grimminger2,*

Departments of * Internal Medicine and {dagger} Anatomy, Justus-Liebig University, Giessen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The influence of LPS preincubation on hydrogen peroxide (H2O2)-induced loss of epithelial barrier function was investigated in rat alveolar epithelial type II cells (ATII). Both apical and basolateral H2O2 administration caused a manyfold increase in transepithelial [3H]mannitol passage. Apical but not basolateral preincubation of ATII with LPS did not influence control barrier properties but fully abrogated the H2O2-induced leakage response. The effect of apical LPS was CD14 dependent and was accompanied by a strong up-regulation of NO synthase II mRNA and protein and NO release. Inhibition of NO by NG-monomethyl-L-arginine suppressed the LPS effect, whereas it was reproduced by exogenous application of gaseous NO or NO donor agents. Manipulation of the glutathione homeostasis (buthionine-(S,R)-sulfoximine) and the cGMP pathway (1H-(1,2,4)oxadiazolo[4,3-{alpha}]quinoxaline-1-one; zaprinast) did not interfere with the protective effect of LPS. Superoxide (O2) generation by ATII cells was reduced by exogenous NO and LPS preincubation. O2 scavenging with exogenous superoxide dismutase, the intracellular superoxide dismutase analog Mn(III)tetrakis(4-benzoic acid) porphyrin, and the superoxide scavenger nitroblue tetrazolium and, in particular, hydroxyl radical scavenging with hydroxyl radical scavenger 1,3-dimethyl-thiourea inhibited the H2O2-induced epithelial leakage response. In conclusion, apical but not basolateral LPS preincubation of ATII cells provides strong protection against H2O2-induced transepithelial leakage, attributable to an up-regulation of epithelial NO synthesis. It is suggested that the LPS-induced NO formation is effective via interaction with reactive oxygen species, including superoxide and hydroxyl radicals. The polarized epithelial response to LPS may be part of the lung innate immune system, activated by inhaled endotoxin or under conditions of pneumonia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of polymorphonuclear leukocytes has long been implicated in the development of acute respiratory distress syndrome (ARDS),3 characterized by gas exchange abnormalities and pulmonary edema formation due to increased lung endothelial and epithelial permeability (1, 2, 3, 4). Hydrogen peroxide (H2O2), a reactive oxygen species, is an established polymorphonuclear leukocyte-derived mediator of epithelial injury. In alveolar epithelial cells, H2O2 effects are manyfold and include an initial depletion of intracellular ATP, a sustained inhibition of alveolar epithelial type II cell (ATII) surfactant synthesis, loss of epithelial barrier properties, DNA damage, and cell lysis at high dosage (5, 6, 7, 8, 9).

Sepsis is the leading cause of mortality in critically ill patients (10) and represents an important underlying event in the pathogenesis of ARDS. In animal models and under cell culture conditions, various clinical features of ARDS may be reproduced by endotoxin (LPS) released from cell walls of Gram-negative bacteria (1, 2, 3). The microcirculatory disturbances with subsequent cellular injury and loss of organ function induced by LPS in intact animals have been attributed to the activation of inflammatory cells and strong inflammatory mediator generation. ATII exposed to LPS demonstrated a marked depression of surfactant synthesis and ion transport as well as strong up-regulation of adhesion molecules, cytokines such as TNF-{alpha}, and NO synthesis via NO synthase II (NOSII) (11, 12, 13, 14).

At the interface between external environments and the milieu interieur, the alveolar lining layer has developed a highly organized cellular polarity: surfactant components, as an example, are exclusively secreted to the apical side, whereas fibrinogen is mainly secreted basolaterally (15). Moreover, in response to inflammatory challenge, the alveolar epithelium orchestrates enhanced leukocyte traffic to the apical side by polarized chemokine secretion and up-regulation of adhesion molecules (16), whereas protein transport across rat alveolar epithelial cell monolayers displays asymmetry with predominance of the apical-to-basolateral flux (17), the same being true for the active transepithelial sodium transport (18, 19).

In the present study in ATII monolayers, we investigated the impact of apically vs basolaterally applied endotoxin on the H2O2-elicited epithelial leakage response. Interestingly, a protective role of LPS pretreatment was noted; however, it was restricted to the apical route of LPS administration. Up-regulation of epithelial NOSII-driven NO synthesis was identified as a predominant mechanism underlying the protective effect of endotoxin preincubation, and evidence is presented that NO exerts its function largely via interaction with oxygen radicals, with superoxide anion and hydroxyl radicals putatively appearing as downstream effectors of H2O2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Male CD18 Sprague-Dawley rats (180–200 g) were purchased from Charles River (Sulzfeld/Main, Germany). Elastase (type EC 134, sp. act. 135 U/mg protein) was obtained from Elastin Products (St. Louis, MO). DMEM was supplied by Life Technologies (Karlsruhe, Germany). Zaprinast, Zn-(II)-protoporphyrin, manganese-(III)-tetrakis(benzoic acid) porphyric chloride, desferrioaxamine, 1,3-dimethylthiourea, reduced glutathione (GSH), buthionine-(S,R)-sulfoximine (BSO), hydrogen peroxide (urea stabilized), HRP, 8-bromoadenosin (8-Br)-cGMP, LPSs from Salmonella typhimurium and IgG2b Ab were obtained from Sigma (Deisenhofen, Germany). S-Nitroso-N-acetyl-D-L-penicillamine (SNAP) and spermine NONOate were purchased from Calbiochem (La Jolla, CA). [3H]Methylcholine, 125I-labeled cGMP assay system was from Amersham (Dreieich, Germany). The Limulus assay system was observed from Hemochrom (Essen, Germany). The CD14 Ab (MY-4, monoclonal) was from Coulter (Krefeld, Germany). Tissue culture plastic was purchased from BD Biosciences (Heidelberg, Germany).

Isolation of ATII

ATII were isolated as previously described in detail (20). Briefly, inflated and perfused lungs from specific pathogen-free male CD18 Sprague Dawley rats were lavaged and filled to their total lung capacity with elastase (30 U/ml)- and trypsin (0.05 mg/ml)-containing solution. Lungs were minced and free cells were separated from lung tissue by sequential filtration through 100-µm and additional 10-µm sterile nylon mesh. "Panning" of the resultant cell suspension was performed on rat IgG-coated plates. Nonadherent ATII were harvested after 1 h and resuspended in DMEM containing 10% FCS. The yield of type II epithelial cells from each rat was in the range of 30–50 x 106. The percentage of freshly isolated type II cells was 94 ± 2% as assessed by modified Papanicolaou, tannic acid, and alkaline phosphatase staining. Contaminated cells included alveolar macrophages (<4%), and neutrophils (<2%). After culture of the cells for 3 days on transwells, the electric resistance of all ATII layers used was >2000 {Omega}/cm2. In this state the percentage of type II cells was virtually 100%, as assessed by electron microscopy in separate control experiments. Using MACS in additional experiments, highly purified type II cell cultures were manufactured and used to detect the NOSII protein in type II cells by immunofluorescence (anti-rat NOSII; Santa Cruz Biotechnology, Santa Cruz, CA) and the liberation of NO. MACS was performed with anti-rat CD45 (leukocyte common Ag) Ab (OX-1; BD PharMingen, San Diego, CA) and confirmed by immunofluorescence with anti-rat macrophage Ab (ED-1; DPC Biermann, Bad Nauheim, Germany) and anti-rat CD45 Ab. ATII viability, as assessed by 5-carboxyfluorescein diacetate loading and trypan blue exclusion, was persistently >95%. Experiments have been performed in the presence of 1% FCS.

Determination of paracellular permeability

For measuring epithelial permeability, [3H]mannitol (1 µCi/ml) was added to the apical side during the incubation period.

mRNA extraction

Aliquots of 1 x 106 cells were transferred into 1.5-ml reaction tubes. After centrifugation at 300 x g, the supernatant was removed and the pellet was lysed in 300 µl lysis buffer of the Dynabeads mRNA direct kit (Dynal Biotech, Oslo, Norway). For each sample, 150 µg beads were applied. Isolated mRNA was finally solved in 20 µl diethyl pyrocarbonate-treated H2O.

Relative mRNA quantitation

Relative mRNA quantitation was performed by the Sequence Detection System 7700 (PE Applied Biosystems, Foster City, CA) and real-time PCR. We used comparative quantitation ({Delta}CT) normalizing target gene to an internal standard gene, as described in detail by Fink et al. (21). For internal calibration, mRNA transcribed from the gene encoding porphobilinogen deaminase (PBGD) was used. We could show that amplification efficiency of PBGD and NOSII primer/probe sets was approximately equal and amounted to 0.95 ± 0.02 (95 ± 2%).

cDNA synthesis and real-time PCR

For cDNA synthesis and real-time PCR, reagents as well as primers and probes were applied as described by Fink et al. (22). Two microliters of cDNA were applied to each sample. Primers were added to a final concentration of 300 nM each and hybridization probes were added to a final concentration of 200 nM in a volume of 50 µl. Cycling conditions were 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 61°C for 60 s.

Measurement of cGMP

TII were cultured on 35-mm dishes at a density of 3 x 106 cells per well (plating density was typically 4 x 105/cm2). Measurement of cyclic nucleotides was performed with a commercially available radioimmunoassay in the supernatant of permeated cells, subjected to 70% ethanol for 60 min.

Measurement of O2 concentration

Superoxide production in ATII was measured by lucigenin-induced chemiluminescence as recently described by Höhler et al. (23).

Measurement of H2O2

H2O2 was measured as described previously by Dekhuijzen et al. (24). Briefly, 100 µl of 420 µM 3',3,5,5'-tetramethylbenzidine (dissolved in 0.42 M citrate buffer, pH 3.8) and 10 µl of 52.5 U/ml HRP solution were added to 100 µl of the probe. The reaction was terminated after 20 min by admixture of 10 µl 18 N sulfuric acid. Solutions were measured photometrically by 450 nm.

Measurement of cellular NO release

Cells were cultured in transwell dishes in DMEM for 24 h. For measurement of cellular NO release the gaseous headspace was forwarded to a chemiluminescence NO analyzer (Sievers 280 NOA; Sievers Instruments, Boulder, CO) for quantification of its NO concentration. Due to the very low liquid-gas partition coefficient of NO, NO released into the culture medium is expected to largely escape into the gaseous headspace (25).

Measurement of lactate dehydrogenase

Lactate dehydrogenase release as an indicator of cellular damage was quantified by standard colorimetric technique. It ranged <2% of total enzyme activity in control cells as compared with the total release in response to the pore-forming agent mellitin (100 µg/ml).

Control experiments

The passage of LPS through the semipermeable culture membrane was measured by the Limulus assay system. Independent of the route of application, ~90% of the apically or basolaterally applied LPS was found to cross the transwell within 3 h.

Statistical analysis

For statistical comparison, one-way analysis of variance was performed. The Student Newman-Keuls test was used as a posteriori tests for linear contrasts. The significance level for the test was set at p = 0.05. A level of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apical but not basolateral LPS suppress the epithelial leakage response to H2O2

Exposure of ATII to H2O2 induced a dramatic increase in epithelial permeability, as assessed by [3H]mannitol transit (Fig. 1Go). A maximum effect was observed upon basolateral H2O2 (100 µM) application, whereas the apical route of application was less effective. Control cells did not change their epithelial permeability under influence of LPS. Apical preincubation of ATII with LPS for 12 h suppressed the toxic effect of both apically and basolaterally applied H2O2 in a dose-dependent fashion (optimum concentration, 10 ng/ml LPS) (Fig. 2Go). This effect commenced after 6 h of LPS incubation, reaching a maximum within 12 h (data not given in detail). In contrast, basal pretreatment with LPS had no effect on H2O2-induced oxidative stress over the 12-h observation period.



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  		FIGURE 1. H2O2-induced increase in epithelial permeability. ATII cultured on transwells were incubated with either apically (•) or basolaterally ({circ}) applied H2O2 (for 3 h) in various concentrations, or sham incubation was performed (control). Permeability is given as cpm [3H]mannitol transport. #, Significantly different from control. Means ± SEM of six independent experiments each are given.

 


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  		FIGURE 2. Impact of LPS pretreatment on H2O2-induced permeability. Pretreatment with different concentrations of either apically or basolaterally applied LPS or sham incubation was performed for 12 h. Next, either apical or basolateral H2O2 (100 µM) was administered and the permeability was assessed after 3 h. Data are given as cpm [3H]mannitol transport. #, Significantly different from control. Means ± SEM of six independent experiments each are given.

 
Apical but not basolateral LPS application induces NOSII mRNA expression

In accordance with the protective effect on paracellular permeability, only apical incubation of ATII with LPS increased NOSII mRNA. This response was clearly dose dependent, with a maximum at 10 ng/ml LPS (23.555 ± 9% K x NOSII copies per copy of PBGD). Quantitative analysis via real-time PCR revealed an increase in NOSII mRNA up to a 145-fold level when compared with basal LPS application (1 ng/ml) (Fig. 3Go). mRNA expression in response to apical LPS incubation was markedly reduced in the presence of CD14 Abs, indicating that the LPS-induced NOSII expression proceeds via CD14-related signaling (Fig. 3Go). Correspondingly, LPS significantly increased the liberation of NO, assessed by chemiluminescence (Table IGo), and induced the expression of NOSII protein, as detected with immunofluorescence (Fig. 4Go). In contrast, in the absence of LPS, no NO release and NOSII protein expression was noted.



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  		FIGURE 3. Impact of LPS pretreatment on NOSII mRNA formation. Pretreatment with either apically or basolaterally applied LPS (1 ng/ml) was performed for 6 h. In addition, apical stimulation with LPS was compared in the presence and absence of CD14 Abs. Experiments were controlled using isotype-matched Ab IgG2b from murine myeloma. NOSII mRNA copies are normalized to standard gene PBGD mRNA copies (k = constant). Means ± SEM of three independent experiments each are given.

 

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  		Table I. Impact of LPS pretreatment on NO release in epithelial cells and macrophages1

 


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  		FIGURE 4. Immunofluorescence staining for NOSII. A and B, ATII cultures were pretreated with LPS (10 ng/ml) for 12 h and stained with anti-rat NOSII (A, red fluorescence) and with anti-rat macrophage Ab (B, green fluorescence). C, For comparison, incubation with anti-rat NOSII Ab without LPS pretreatment was performed. D, For detection of macrophages as control, ATII were enriched with macrophages and incubated with anti-rat macrophage Ab incubation. A representative experiment of four independent experiments each is given.

 
The role of NO in H2O2-induced increase in paracellular permeability

Pretreatment with the competitive NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 1 mM) significantly reduced the protective effect of LPS preincubation, whereas both incubation of ATII in a NO atmosphere (250 ppm) or pretreatment with the NO donor agents spermine NONOate and SNAP reproduced the protective effect of LPS (Fig. 5GoA). The effect was reproducible by coincubation with both NO donors, whereas posttreatment did not ameliorate H2O2-induced hyperpermeability (data not given in detail). Interestingly, in the concentration range used, gaseous NO in the absence of H2O2 exerted some permeability-enhancing effect by itself (Fig. 5GoB).



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  		FIGURE 5. Impact of NO on H2O2-induced mannitol permeability. A, Pretreatment with various concentrations of the NO donor SNAP, spermine NONOate, and L-NMMA was performed for 3 h. Next, H2O2 was admixed basolaterally and the permeability was assessed after 3 h. For comparison, LPS pretreatment (10 ng/ml for 12 h) was performed, combined with additional L-NMMA application (1 mM for 12 h) in one set of experiments. B, In another experimental setting, ATII were incubated in the presence and absence of gaseous NO, H2O2, or both agents for 3 h. Permeability is given as cpm [3H]mannitol transport. #, Significantly different from mono-treatment with H2O2. *, Significantly different from control. Means ± SEM of four independent experiments each are given.

 
To control the NO effect in an unrelated system, increased paracellular epithelial permeability was provoked by either Escherichia coli hemolysin, a membrane-perturbating bacterial exotoxin, or the polycation protamine. In contrast to H2O2, the epithelial leakage response to these two agents was not significantly influenced by NO or the NO donors NONOate and SNAP (data not given).

How does LPS-induced NO protect against the H2O2-induced permeability increase?

Independence of cGMP. Pretreatment of ATII with both the specific phosphodiesterase (PDE) type V inhibitor zaprinast (10 µM) as well as with the guanylate cyclase inhibitor 1H-(1, 2, 4)oxadiazolo[4,3-{alpha}]quinoxaline-1-one (ODQ; 10 µM) for influencing the epithelial cGMP levels did not affect the protective effect of LPS preincubation (10 ng/ml for 12 h, Fig. 6Go). In addition, application of 8-Br-cGMP over a range from 10 µM to 10 mM did not interfere with the toxic effect of H2O2, indicating that the protective mechanism of LPS/NO was independent of the cGMP-related signal transduction pathway. In preceding pilot experiments, the significant modulation of the intracellular cGMP level in the presence of zaprinast (increase in ATII ascertained) was shown.



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  		FIGURE 6. Missing effect of cGMP manipulation on H2O2-induced permeability. Pretreatment of ATII cells was performed with either the specific PDE type V inhibitor zaprinast (10 µM for 30 min) or the guanylate cyclase inhibitor ODQ (10 µM for 30 min) previous to incubation with LPS (2 ng/ml for 12 h). In separate experiments 8-Br-cGMP (100 µM) was applied for 3 h. Next, challenge with 100 µM H2O2 (basolaterally) was performed and the permeability was assessed after 3 h. Permeability is given as cpm [3H]mannitol transport. #, Significantly different from mono H2O2 treatment. Means ± SEM of five independent experiments each are given.

 
Independence of glutathione synthase and hemoxygenase activities. Pretreatment of ATII with the glutathione synthase inhibitor BSO or the hemoxygenase inhibitor Zn-(II)-protoporphyrin did not reduce the LPS-induced protection against the H2O2-induced epithelial leakage (Fig. 7Go).



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  		FIGURE 7. Manipulation of ATII glutathione levels and hemoxygenase I metabolism impact on H2O2-induced permeability. Pretreatment of ATII with BSO (500 µM for 12 h) and Zn-(II)-protoporphyrin (100 µM for 12 h) was performed in the absence (open bars) and presence (horizontal striped bars) of LPS. Next, 100 µM H2O2 was administered and the permeability was assessed after 3 h. Permeability is given as cpm [3H]mannitol transport. Means ± SEM of four independent experiments each are indicated.

 
Putative role of the superoxide anion (O2) and the hydroxyl radical (·OH). The H2O2-induced increase in paracellular [3H]mannitol flux was significantly enhanced in the presence of optimum concentrations of the superoxide dismutase (SOD) inhibitor triethylenetetramine (TETA) (Table IIGo). In contrast, SOD, the intracellular SOD analog Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP), and the O2 scavenger nitroblue tetrazolium (NBT) all reduced the H2O2-induced leakage, thus favoring a role of O2 in this event. Most prominent suppression of the H2O2-induced mannitol flux was achieved by the hydroxyl radical scavenger 1,3-dimethyl-thiourea (DMTU) (Table IIGo).


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  		Table II. Manipulation of reactive oxygen species: impact on H2O2-induced permeability1

 
Interestingly, cultured epithelial cells liberated substantial O2 release, as assessed by lucigenin chemiluminescence technique. This O2 liberation was suppressed by LPS treatment of the ATII cells and in the presence of the NO donor NONOate as well as the intracellular SOD analog MnTBAP (Table IIIGo).


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  		Table III. O2 release in epithelial cells: impact of the LPS pretreatment and NO donor application1

 
Putative role of H2O2 dissociation. Testing the effect of NO on Fenton reactions, we measured H2O2 by a HRP assay in the presence and absence of spermine NONOate (10 µM) in vitro: coincubation of Fe2+ (10 µM) and H2O2 (10 µM) decreased the amount of H2O2 to a level of ~35 ± 5% within 20 min as compared with controls in the absence of Fe2+. Application of spermine NONOate did not affect the degradation of H2O2 in the presence of Fe2+, indicating that this process was not directly influenced by NO.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
When investigating the impact of endotoxin preincubation on the H2O2-elicited epithelial leakage in ATII monolayers, an impressive but polarized response was noted. Whereas basolaterally applied LPS was entirely ineffective, apical LPS pretreatment provided strong protection, and this was largely attributable to enhanced epithelial NO synthesis. A CD14-dependent marked up-regulation of NOSII was found in the ATII cells and the LPS effect was blocked by L-NMMA and was fully reproduced by exogenous administration of different NO donor agents as well as gaseous NO. Interestingly, the protective effect of NO apparently did not engage the epithelial guanylate cyclase pathway, as various approaches to manipulate the cGMP levels were ineffective. In contrast, evidence was forwarded for the hypothesis that NO exerted its function largely via interaction with oxygen radicals, with a putative role of hydroxyl radicals appearing as downstream effectors of H2O2.

During inflammatory diseases and ischemia-reperfusion, reactive O2 species such as O2 and H2O2 may be produced in large quantities, exceeding endogenous antioxidant defenses and promoting tissue injury. Previous work to define the role of NO under these circumstances has produced variable results, which is particularly true for the lung. NO-related mechanisms that may protect against inflammatory injury in this organ include vasodilation (26, 27, 28) and suppression of leukocyte adhesion to the pulmonary endothelium as well as leukocyte activation (29). In lung ischemia-reperfusion models, inhalation of NO was shown to suppress the vasoconstrictor response and pulmonary edema formation, as well as ventilation-perfusion mismatch and shunt flow (30, 31, 32, 33, 34). In contrast, the rapid reaction of NO with O2 to form peroxynitrite may cause oxidation of protein and non-protein sulfhydryl groups and may enhance lipid peroxidation, thereby promoting tissue injury (35). Concerning the currently investigated alveolar epithelial cells, NO/peroxynitrite was found to suppress the activity of sodium permeant cation and L-type calcium channels on the apical surface, as well as the basolaterally located Na ± K+ ATPase (36, 37). Moreover, high concentrations of NO deteriorate the biophysical function of the type II cell-derived pulmonary surfactant system, which again was ascribed to NO-related peroxynitrite formation (38, 39), and NO was found to decrease ATP content and surfactant synthesis in freshly isolated ATII (40, 41).

Antioxidant defense systems fall into enzymatic and nonenzymatic categories. Previous examinations in ATII cells revealed the presence of SODs, catalase, and glutathione peroxidases (8, 42). The GSH-dependent pathway is, indeed, regarded as the major detoxification mechanism in the lung, because the GSH concentration in the epithelial lining layer is ~100 times higher than that commonly found in the extracellular fluid of various tissues, and {gamma}-glutamyl transpeptidase is increased by oxidative stress in alveolar epithelial cells (42, 43, 44, 45). It is thus tempting to speculate that the protective effect of LPS on the H2O2-elicited epithelial leakage response might be caused by an up-regulation of the ATII GSH-dependent antioxidant system; however, such a suggestion was not supported by the present experimental data. Pretreatment of the ATII cells with the glutathione synthase inhibitor BSO to deplete the epithelial glutathione pool did not interfere with the protective effect of LPS preincubation on the H2O2-induced epithelial leakage response.

In contrast, strong evidence was provided for the suggestion that an up-regulation of epithelial NO synthesis is primarily responsible for the protective effect of LPS preincubation: 1) a time dependency of the LPS effect was noted, which is compatible with a gene regulatory event; 2) expression of NOSII protein was noted in LPS-exposed type II cells (Fig. 4Go); 3) strong up-regulation of NOSII mRNA was directly demonstrated; 4) the LPS-induced increase in the NOSII message demanded the presence of CD14, indicating specific LPS signaling events, and CD14-dependent signal transduction has previously been described to be involved in the endotoxin-induced up-regulation of NOSII in different cell types (46, 47, 48); 5) the protective effect of LPS preincubation was abrogated by the NO synthase inhibitor L-NMMA, whereas protoporphyrin, used for inhibition of CO generation by hemoxygenase, was entirely ineffective; and 6) the protective endotoxin effect was fully reproduced by the administration of two different NO donor agents as well as gaseous NO. The latter effect was particularly impressive, because at the given dosage (250 ppm NO) the gaseous NO itself exerted some epithelial leakage reaction, which was then even reduced upon coapplication with the strong leakage trigger agent H2O2.

The differential sensitivity of the alveolar epithelial cells to apical vs basolateral LPS exposure is a hitherto unreported phenomenon, with underlying reasons demanding further elucidation. A polarized distribution of CD14 and/or associated signal transduction molecules, such as Toll-like receptors, with exclusive presentation to the apical cell surface might offer a ready explanation, but currently no morphological data are available to support such view. Alternatively, a "sensing" of an apical-to-basolateral vs basolateral-to-apical LPS gradient might represent the underlying mechanisms, as some transepithelial passage of LPS within the 12-h incubation period might be expected for both routes of application. It is tempting to speculate that the alveolar epithelial responsiveness to apically offered endotoxin might be part of an innate defense system, providing protection against bacterial invasion under conditions of pneumonia.

As the most ready explanation for the antipermeability effect of NO, whether derived from endogenous or exogenous sources, its downstream efficacy via guanylate cyclase activation and cGMP increase was considered. In endothelial cell monolayers, elevated cGMP levels were, indeed, demonstrated to protect against a loss of barrier properties provoked by inflammatory agents (49, 50). However, such mode of action was largely excluded for the present NO effect on alveolar epithelial barrier properties. First, LPS preincubation as well as exogenous NO supply did not provide protection against oxidant-unrelated challenges of the alveolar barrier function, as probed with the bacterial exotoxin E. coli hemolysin (being active via induction of phosphatidylinositol response and the provocation of Ca+ ± fluxes (50, 51)) and the polycation protamine (being operative via its positive charge (52)). Second, any efforts either to increase the intracellular cGMP content in ATII cells (direct application of 8-Br-cGMP; PDE V inhibition for blockage of cGMP metabolism) or to decrease epithelial cGMP levels (guanylate cyclase inhibition by ODQ) were entirely ineffective.

Thus, the NO effect was considered to proceed via some direct interference with reactive oxygen species, appearing under conditions of epithelial H2O2 challenge. From the literature, we are not aware of any evidence for a direct interaction of NO with H2O2, and the presently undertaken in vitro incubation of the NO donor agent NONOate with H2O2 further supported this view. Interestingly, H2O2 treatment of the epithelial cells elicited readily detectable superoxide generation, as measured by lucigenin chemiluminescence technique, and NO interaction with O2 is well established. Indeed, the appearance of O2 under conditions of ATII H2O2 challenge was reduced both upon ATII preincubation with LPS and in the presence of NONOate. Moreover, the H2O2-elicited leakage response was further enhanced by inhibition of epithelial SOD activity with TETA and was suppressed by exogenous supply with SOD and, in particular, the intracellular SOD analog MnTBAP. These observations support the view that O2, appearing in the sequence of events following epithelial H2O2 incubation, might contribute to the induction of the strong leakage response, and that O2 capturing by NO might interfere with this pathway. This suggestion is of interest against a controversial background, questioning whether NO-O2 interaction with the appearance of peroxynitrite might enhance or suppress inflammatory sequelae such as loss of barrier properties (53, 54, 55). However, a putative interaction of NO with O2 may not fully explain the protective effect of LPS preincubation and NO donor application in the present study, because the intracellular SOD analog MnTBAP provided the strongest scavenging of O2 but was clearly less effective than LPS and exogenous NO in protecting against H2O2-induced epithelial leakage. This may be explained by an MnTBAP-elicited increase in the generation of H2O2 and its downstream metabolites (e.g., ·OH). In line with this argumentation is the fact that the highest efficacy against H2O2-induced epithelial hyperpermeability was noted for the hydroxyl radical scavenger DMTU. It has long been known that ·OH may be generated from H2O2 in the presence of divalent redox iron, by the Fenton reaction. Hydroxyl radicals are highly reactive with a large variety of target molecules, and their appearance in the very vicinity of the epithelial tight junction structures might well cause loss of barrier properties. Assuming OH·-dependent injury, the protective role of NO might either again be attributed to its scavenging of superoxide (the latter providing Fe2+ from Fe3+ for the Fenton reaction) or to some direct interaction with ·OH, e.g., with formation of HNO2 (56, 57, 58). It is tempting to speculate that the ·OH-NO contact is part of the protective response as schematically depicted in Fig. 8Go. Clearly, direct proof of such radical-radical interaction as putative underlying mechanisms of NO-related protection of the epithelial barrier function will demand additional experimental work with exact discrimination of individual reactive oxygen species by electron spin resonance.



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  		FIGURE 8. Cartoon of the postulated signal transduction pathways investigated in this study. H2O2-induced epithelial permeability increase is antagonized by LPS via NO up-regulation, thereby protecting the epithelial monolayer. Expression of NOSII is critically dependent on the apical route of LPS application and the presence of CD14. The putative intermediate generation of the ·OH radical is indicated.

 
In conclusion, apical but not basolateral LPS preincubation of alveolar epithelial cells provided strong protection against a H2O2-elicited epithelial leakage response, and this was clearly attributable to a marked up-regulation of the epithelial NO synthesis. Radical-radical interactions are suggested as underlying mechanisms of the protective NO effect, whereas no evidence was obtained for a role of the NO-dependent cGMP formation. LPS-elicited induction of epithelial NOSII may be considered part of the lung innate immune system, activated under conditions of inhalative bacterial and endotoxin challenge as well as pneumonia.


   Acknowledgments
 
We thank R. M. Bohle and the project Z1 (SFB 547) for support as well as M. M. Stein for skillful technical assistance.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Grant 547, cardiopulmonary vascular system). Back

2 Address correspondence and reprint requests to Dr. Friedrich Grimminger, Department of Internal Medicine, Justus-Liebig University, Klinikstra{beta}e 36, D-35392 Giessen, Germany. E-mail address: Friedrich.Grimminger{at}innere.med.uni-giessen.de Back

3 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; ATII, alveolar epithelial type II cell; NO, nitric oxide; L-NMMA, NG-monomethyl-L-arginine; BSO, buthionine-(S,R)-sulfoximine; ODQ, 1H-(1,2,4)oxadiazolo[4,3-{alpha}]quinoxaline-1-one; TETA, triethylenetetramine; 8-Br, 8-bromoadenosin; MnTBAP, Mn(III)tetrakis(4-benzoic acid) porphyrin; NBT, nitroblue tetrazolium; SNAP, S-Nitroso-N-acetyl-D-L-penicillamine; DMTU, hydroxyl radical-scavenger 1,3-dimethyl-thiourea; PBGD, porphobilinogen deaminase; SOD, superoxide dismutase; NOSII, NO synthase II; PDE, phosphodiesterase; GSH, reduced glutathione. Back

Received for publication July 18, 2001. Accepted for publication May 28, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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