Introduction

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There is increasing evidence implicating the alveolar epithelium as a dynamic barrier that plays an important role in regulating the inflammatory and metabolic responses to oxidative stress and the accompanying inflammatory signal (1), sepsis, endotoxemia, and other critical illnesses in the lung (6). The respiratory epithelium, in particular, is a primary target of an inflammatory/infectious condition at the epithelial-blood interface and is itself capable of amplifying an inflammatory signal by recruiting inflammatory cells and by producing inflammatory mediators (2). Many of the side effects of lipopolysaccharide (LPS)-endotoxin, derived from the cell wall of gram-negative bacteria, are secondary to the overproduction of proinflammatory mediators, such as the pleiotropic cytokines, which exacerbate the pathophysiologic condition by activating and recruiting inflammatory cells. Therefore, the suppression of a proinflammatory signaland the downstream conjugated inflammatory pathwaysand augmentation of a counter-inflammatory response has been a major focus of the approach to the treatment of inflammatory diseases. For instance, glucocorticoids (10), extracellular purines (11), phosphodiesterase-selective inhibitors (12, 13), pyrimidylpiperazine (14), and adrenoreceptor agonism/ antagonism (17) have been widely used to counteract the effects of inflammatory cytokines and subsequently suppress the protracted pathophysiologic conditions in vitro and in vivo.

During the evolution of an inflammatory process, whether it is an acute or a contracted chronic signal, the respiratory epithelium responds with the production of proinflammatory cytokines, including interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-, and antiinflammatory mediators such as IL-10. Amiloride, a pyrazinoylguanidine compound initially synthesized as a potassium-sparing diuretic (18), inhibits the plasma membrane sodium and sodium/hydrogen anti-port system, the sodium/potassium-ATPase-dependent pump, sodium-coupled transport of glucose and various amino acids, and the sodium/calcium exchange system (18, 19). In addition to its wide-spectrum effect on ion transport and the biophysical properties of cell membranes and ion transport systems, amiloride has been demonstrated to inhibit protein, RNA, and DNA synthesis (20). Of note, relatively recent evidence suggests that amiloride affects immune cell function by suppressing the production of superoxide anion (O₂^·), derived from polymorphonuclear neutrophils, and chemotaxis associated with this inflammatory signal (24). Recently, it has been reported that amiloride and its analog, 5-(N,N-hexamethylene)-amiloride (HMA), suppressed LPS-primed human alveolar macrophage-derived cytokines (19) and cytokine biosynthesis in the respiratory epithelium infected with respiratory syncytial virus (27). However, the underlying molecular mechanism of action is not well understood both in the respiratory epithelium and in other tissues and defined models.

The promoters of genes encoding cytokines contain multiple cis-acting motifs, including those that bind such transcription factors as nuclear factor (NF)-B. Furthermore, the release of free NF-B upon extracellular stimulation due to inhibitory B (IB) phosphorylation and degradation leads to DNA binding to initiate transcription of related genes, including immunoreceptors, cytokines, and, interestingly, its own inhibitor, IB (28, 29). Although the transcription factor NF-B has been originally recognized in regulating gene expression in B-cell lymphocytes (30), subsequent studies demonstrated that it is one member of a ubiquitously expressed family of Rel-related transcription factors that serve as critical regulators of many genes, including those encoding proinflammatory cytokines (31). The translocation and activation of NF-B in response to various stimuli are sequentially organized at the molecular level. In its inactive state, the heterodimeric NF-B, which is mainly composed of two subunits, p50 (NF-B₁) and p65 (RelA), is present in the cytoplasm associated with its inhibitory protein, IB (29, 32, 33). Upon stimulation, such as with cytokines and LPS, IB- undergoes phosphorylation on serine/threonine residues, ubiquitination, and subsequent proteolytic degradation, thereby unmasking the nuclear localization signal on p65 and allowing nuclear translocation of the complex. This sequential propagation of signaling ultimately results in the release of NF-B subunits from IB- inhibitor, allowing translocation and promotion of gene transcription.

This study has attempted, therefore, to explore the immunoregulatory potential of amiloride, and the underlying molecular mechanism is further unraveled. We particularly show that amiloride acts as a novel dual immunomodulator in the alveolar epithelium: It blockades proinflammatory cytokine production (IL-1 and TNF-) and amplifies an antiinflammatory loop response (IL-6 and IL-10). In addition, this biphasic regulation of cytokines mediated by amiloride is IL-10 sensitive and associated with targeting the IB-/NF-B pathway, where we have shown that amiloride and HMA suppress the phosphorylation of IB-, allow its cytosolic accumulation, and subsequently intervene with the nuclear translocation of selective NF-B subunits, thereby blockading NF-B activation.

	Materials and Methods

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Chemicals and Reagents

Unless indicated otherwise, chemicals of the highest analytical grade were purchased from Sigma-Aldrich (Dorset, UK). IL-10 (human rDNA; rhIL-10) was purchased from the National Institute for Biological Standards and Control (NIBSC, Cambridge, UK). rhIL-10 was reconstituted in deionized H₂O, temporarily stored in aliquots at 20°C, and its biologic activity was authenticated as supplied by the manufacturer (1,000 ng/ml  5,000 IU/ml). Rabbit polyclonal anti-rat IL-10 neutralizing antibody (IL-10) was obtained from BioSource International (Nivelles, Belgium), purified from rabbit serum by protein A/G affinity column chromatography. The immunoglobulin solution was constituted in phosphate-buffered saline (PBS) (pH 7.3), and the endotoxin level was verified to be less than 0.01 ng/µg of protein. For the purpose of immunoneutralization of endogenously produced IL-10, IL-10 was used at a concentration of 5 µg/ml, which is required to neutralize 5 ng/ml rat IL-10, as recommended (BioSource International). All experimental procedures involving the use of live animals were approved under the Animals Legislation (Scientific Procedures) Act, 1986 (UK).

Primary Cell Cultures of Alveolar Epithelia

Fetal alveolar type II (fATII) epithelial cells were isolated from the lungs of fetuses of pregnant Sprague-Dawley rats, essentially as reported elsewhere (2, 3). Briefly, fetal rats were removed by caesarian section at Day 19 of gestation (term = 22 d). The lungs were excised, teased free from heart and upper airway tissue, and finely minced and then washed free of erythrocytes using sterile, chilled Mg²⁺- and Ca²⁺-free Hanks' balanced salt solution (HBSS; 0.5 ml/fetus). The cleaned lung tissue was resuspended in 1 ml/fetus HBSS containing trypsin (0.1 mg/ml), collagenase (0.06 mg/ml), and DNAase I (0.012% wt/vol) and was agitated at 37°C for 20 min. The solution was then centrifuged at 100 × g for 2 min to remove undispersed tissue. The supernatant was decanted into a sterile tube, and an equal volume of Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal calf serum (FCS) was added to the supernatant. After passing the supernatant through a 120-µm pore sterile mesh, the filtrate was centrifuged at 420 × g for 5 min, the pellet was resuspended in 20 ml DMEM/FCS, and the cells were placed into a T-150 culture flask for 1 h at 37°C to enable fibroblasts and non-epithelial cells to adhere. Unattached cells were washed three times by centrifugation at 420 × g for 5 min each and then seeded onto 24-mm diameter Transwell-clear permeable supports (Costar, Hereford, UK; 0.4 µm pore size) at a density of 5 × 10⁶ cells per filter and were allowed to adhere overnight at 152 mm Hg ( 21% O₂/5% CO₂) at 37°C. DMEM/ FCS was exchanged for 4 ml of pre-equilibrated serum-free PC-1 media (Biowhittaker, Walkersville, MD) 24 h later, and cells were maintained as such until the experiment. The adenylate energy charge, an index of cell viability and competence, remained  0.7, and transepithelial monolayer resistance was monitored constantly at 250-350 cm² or more (2, 3).

Assessment of Pro- and Antiinflammatory Cytokines by Enzyme-Linked Immunosorbent Assay

The extracellularly released cytokines were measured by a two-site, solid-phase, developed sandwich enzyme-linked immunosorbent assay (ELISA) (4, 5). Immunoaffinity-purified polyclonal rabbit anti-rat IL-1, IL-6, IL-10, and TNF- (2 µg/ml) primary antibodies were used to coat high-binding microtiter plates (MaxiSorp; Nunc, Hereford, UK). Recombinant rat and biotinylated immunoaffinity-purified sheep anti-rat cytokine (R&D Systems Europe Ltd., Oxon, UK) were used as standard and recognition antibodies, respectively. The color was developed incorporating streptavidin-poly-horseradish peroxidase coupled reaction with the chromagen 3,3',5,5'-tetramethyl-benzidine dihydrochloride, and the optical density (OD) was measured at 450 nm. Inter- and intra-assay coefficients of variations were < 10%, and the minimum detectable sensitivity for each cytokine was  2 pg/ml. Results interpolated from the linear regression of the standard curve are expressed as pg/ml (4, 5).

Drug Pretreatments, Western Analysis, and Electrophoretic Mobility Shift Assay

Initially, the optimum time for drug preincubation before exposure to LPS was screened as follows: Amiloride and HMA were administered 4, 2, and 1 h before LPS stimulation, concurrent with LPS, or 1, 2, 4, and 8 h after LPS exposure. The most effective time was found between 1 and 2 h; therefore, 1.5 h was subsequently chosen in further experiments. For the dose-response curves, cells were pretreated with amiloride (0-2.5 × 10⁴ M) or HMA (0-2.5 × 10⁴ M) for 1.5 h. Filters were washed twice with pre-equilibrated PC-1 medium before exposure for a further 24 h to LPS (1 µg/ml) from Escherichia coli, serotype 026:B6. Following treatments, subcellular extracts (cytosolic and nuclear) were prepared essentially as previously described. Briefly, filters were washed twice in 5 ml ice-cold, pre-equilibrated PBS (pH 7.2-7.4), and cells (1-2 × 10⁷) were collected and centrifuged at 420 × g for 5 min at 4°C. Nuclei were released by resuspending the pellet in 250 µl buffer A containing (in mM): 10 Tris-HCl (pH 7.8), 10 KCl, 2.5 NaH₂PO₄, 1.5 MgCl₂, 1 Na₃VO₄, 0.5 dithiothreitol, 0.4 4-2-aminoethyl-benzene sulfonyl fluoride-HCl, and 2 µg/ml each of leupeptin, pepstatin A, and aprotinin. The suspension was left in ice for 10 min followed by a 45-s homogenization at a moderate speed. Nuclei were collected by centrifuging the slurry at 4,500 × g for 5 min at 4°C and resuspending in 100 µl buffer B (buffer A adjusted to [in mM]: 20 Tris-HCl [pH 7.8], 420 KCl, 20% [vol/vol] glycerol). The supernatant thus formed is termed the cytosolic extract. The nuclei were then lysed at 4°C for 30 min with gentle agitation, the debris was cleared by centrifugation at 10,000 × g for an additional 30 min at 4°C, and the supernatant was frozen in liquid nitrogen and stored at 70°C until used. In all cases, protein contents were determined by the Bradford method using BSA as a standard.

Cytosolic and nuclear proteins (20-25 µg) were resolved over SDS-PAGE (7.5%) gels at room temperature and blotted onto nitrocellulose membrane, and nonspecific binding sites were subsequently blocked. Mouse monoclonal IgG₁ anti-IB- (H-4), IgG_2b anti-(phosphorylated) pIB- (B-9), rabbit polyclonal IgG anti-p50 (NLS), anti-p52 (K-27), anti-p65 (RelA; A), anti-p68 (RelB; C-19), and anti-p75 (c-Rel; N) (Santa Cruz Biotechnology, Inc., Wiltshire, UK) antibodies were used for primary detection. Anti-rabbit Ig-biotinylated antibody (Amersham plc, Buckinghamshire, UK) was employed for secondary detection followed by the addition of streptavidin-HRP conjugate and visualized on film by chemiluminescence. -Actin standard was used as an internal reference for semiquantitative loading in parallel lanes for each variable. Western blots were scanned by NIH MagiScanII and subsequently quantitated by UN-Scan-IT automated digitizing system (version 5.1; 32-bit, Packard BioScience Ltd., Berkshire, UK), and the ratio of the density of the band to that of -actin was subsequently performed.

Custom deoxy-oligonucleotide probe sequences were purchased from Sigma-Genosys (Cambridge, UK): NF-B, 5'-AGTTGAGGG GACTTTCCCAGGC-3' (binding sequence underlined). Gel-purified, double-stranded DNA was end-labeled with [³²P]-ATP (NEN Life Sciences, Cambridge, UK). Identical amounts of radioactive probe (1-2 × 10⁴ counts min¹) were added to binding reactions containing 1-5 µg fATII nuclear extracts in a final volume of 40 µl in DNA binding buffer (20 mM HEPES [pH 7.9], 1 mM MgCl₂, 4% Ficoll). Reaction mixtures were incubated for 30 min at 25°C before separating on nondenaturing 4% polyacrylamide gels at RT and subjected to electrophoresis with 1:10 5× Tris-Borate-EDTA buffer. A nonspecific competitive polydeoxyinosinic-deoxycytidylic acid (poly[dI-dC]) (Amersham plc) was added to reaction mixtures after addition of labeled probe. Gels were transferred to ion-exchange chromatography paper, vacuum dried, and then electronically visualized on an Instant phosphorimager (Packard BioScience, Ltd.). Specific quantitation of the corresponding DNA gel shift bands was performed with phosphorimaging.

Pretreatment with rhIL-10 and IL-10 and Measurement of Proinflammatory Cytokines and NF-B Activation

Epithelial cells were pretreated for 30 min with rhIL-10 (0-10 ng/ml), washed twice with sterile, pre-equilibrated PBS, and exposed to LPS (1 µg/ml) for 24 h. Cell-free supernatants were assayed for proinflammatory cytokines (IL-1 and TNF-) by ELISA. Nuclear extracts were prepared as above, and the DNA binding activity of NF-B was determined by electrophoretic mobility shift assay (EMSA). In separate experiments, epithelial cells were exposed to amiloride in the absence or presence of IL-10 (5 µg/ml) antibody, before exposure to LPS (1 µg/ml) for 24 h. Supernatants were withdrawn and analyzed for cytokines, and nuclear extracts were used to determine NF-B activation.

Statistical Analysis and Data Handling

Data are presented as the means and the error bars the SEM. Statistical analysis of the difference in mean separation was performed by one-way analysis of variance, followed by post hoc Tukey's test, and the a priori level of significance at 95% confidence level was considered at P  0.05.

	Results

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Effect of Amiloride and HMA on Proinflammatory Cytokines (IL-1 and TNF-)

Pretreatment of epithelial cells with amiloride or its analog, HMA, before exposure to LPS (1 µg/ml) reduced, in a dose-dependent manner, LPS-induced biosynthesis of IL-1, as shown in Figure 1A. The minimum effective concentration was  1 × 10⁶ M (IC₅₀ = 1.02 × 10⁴ M; Figure 1A). The analog of amiloride, HMA, similarly reduced, in a dose-dependent manner, the LPS-induced release of IL-1, with minimum effective concentration  1 × 10⁶ M (IC₅₀ = 1.03 × 10⁴ M; Figure 1B). Similarly, amiloride blockaded, in a dose-dependent manner, the LPS-induced release of TNF- at doses  1 × 10⁸ M (IC₅₀ = 7.50 × 10⁵ M; Figure 1C), and HMA reduced the LPS-dependent response at concentrations  1 × 10⁸ M (IC₅₀ = 8.92 × 10⁵ M; Figure 1D). The time-dependent kinetics of the effect of amiloride (1 × 10⁴ M) and HMA (1 × 10⁴ M) pretreatments on LPS-mediated proinflammatory cytokine biosynthesis is shown in Figure 2A (IL-1; amiloride), Figure 2B (TNF-; amiloride), Figure 2C (IL-1; HMA), and Figure 2D (TNF-; HMA). Amiloride (1 × 10⁴ M) blockaded LPS-induced release of IL-1 maximally at 1-2 h pre-treatment time point (Figure 2A; 70.38-75.87% inhibition), similar to its effect on TNF- (Figure 2B; 77.36-81.25% inhibition). HMA (1 × 10⁴ M) blockaded LPS-induced release of IL-1 maximally at 1-2 h pretreatment time point (Figure 2C; 71.37-75.25% inhibition), similar to its effect on TNF- (Figure 2D; 74.00-78.59% inhibition).

View larger version (25K): [in this window] [in a new window]   Figure 1.   The effect of amiloride and its analog, HMA, on LPS-induced proinflammatory cytokine biosynthesis. (A) Amiloride blockaded LPS-mediated induction of IL-1 production at doses  1 × 106 M, with maximum inhibition ( 93%) at 2.5 × 104 M. (B) The amiloride analog, HMA, reduced, in a dose-dependent manner, LPS-induced secretion of IL-1 at doses  1 × 106 M, with maximum inhibition ( 90%) at 2.5 × 104 M. (C) The inhibitory effect of amiloride on LPS-induced TNF- biosynthesis is dose dependent, with effective concentrations  1 × 108 M and maximum inhibition ( 95%) at 2.5 × 104 M. (D) HMA, similar to the effect of amiloride, reduced LPS-induced production of TNF- at doses  1 × 108 M, with maximum inhibition ( 93%) at 2.5 × 104 M. P < 0.05, as compared with control; *P < 0.05, **P < 0.01, and ***P < 0.001, as compared with LPS alone. n = 4, which represents the number of independent experiments performed in duplicate.

View larger version (59K): [in this window] [in a new window]   Figure 2.   The time-dependent kinetics of the effect of amiloride and its analog, HMA, on LPS-induced proinflammatory cytokine biosynthesis. Alveolar epithelial cells were treated with either amiloride (1 × 104 M) or HMA (1 × 104 M) 4, 2, and 1 h before exposure to LPS (1 µg/ml), concurrent with LPS, or 1, 2, 4, and 8 h after induction with LPS. Supernatants were subsequently harvested for determining the antigenic activity of IL-1 and TNF-. (A) The biosynthesis (percentage of LPS) of IL-1 and the effect of amiloride with maximal inhibition (70.36-76.87%) at 1-2 h pre-LPS treatment. (B) Amiloride suppressed the biosynthesis of TNF- maximally (77.36-81.25%) at 1-2 h before exposure to LPS. (C) The analog of amiloride, HMA, maximally blockaded LPS-induced accumulation of IL-1 (71.37-76.25) 1-2 h before LPS exposure. (D) HMA, similar to its effect on IL-1, reduced LPS-induced production of TNF- (74.00-78.59%) most effectively at 1- 2 h before LPS stimulation. Percentages above the darker histograms represent the maximum inhibition of cytokine biosynthesis, determined relative to LPS alone. n = 4, which represents the number of independent experiments performed in duplicate.

Effect of Amiloride and HMA on Antiinflammatory Cytokines (IL-6 and IL-10)

Amiloride augmented LPS-induced biosynthesis of IL-6 at concentrations  1 × 10⁴ M (EC₅₀ = 5.64 × 10⁵ M; Figure 3A). The effect of HMA analog was less prominent than amiloride in augmenting LPS-induced IL-6 release (EC₅₀ = 9.00 × 10⁵ M), as shown in Figure 3B. Amiloride augmented, in a dose-dependent manner, LPS-induced biosynthesis of IL-10 effective at concentrations  1 × 10⁸ M (EC₅₀ = 2.32 × 10⁴ M; Figure 3C). Similarly, HMA upregulated LPS-induced release of IL-10 at concentrations  1 × 10⁶ M (EC₅₀ = 2.93 × 10⁴ M; Figure 3D). The time-dependent kinetics of the effect of amiloride (1 × 10⁴ M) and HMA (1 × 10⁴ M) on antiinflammatory cytokines is shown in Figure 4A (IL-6; amiloride), Figure 4B (IL-10; amiloride), Figure 4C (IL-6; HMA), and Figure 4D (IL-10; HMA), respectively. Amiloride (1 × 10⁴ M) augmented LPS-induced release of IL-6 maximally at 1-2 h pretreatment time point (Figure 4A; 32.64-38.57% induction), similar to its effect on IL-10 (Figure 4B; 36.42- 42.26% induction). HMA (1 × 10⁴ M) augmented LPS- induced release of IL-6 maximally at 1-2 h pretreatment time point (Figure 4C; 54.97-58.37% induction), similar to its effect on IL-10 (Figure 4D; 32.26-37.58% induction).

View larger version (23K): [in this window] [in a new window]   Figure 3.   The effect of amiloride and its analog, HMA, on LPS-induced antiinflammatory cytokine biosynthesis. (A) Amiloride augmented LPS-induced IL-6 production at doses  1 × 104 M, with maximum induction ( 4.2-fold) at 2.5 × 104 M. (B) The amiloride analog, HMA, induced, although less prominently than amiloride, LPS-induced secretion of IL-6 at doses  1 × 104 M, with maximum induction ( 2.5-fold) at 2.5 × 104 M. (C) The stimulatory effect of amiloride on LPS-mediated IL-10 biosynthesis is dose-dependent, with effective concentrations  1 × 108 M, with maximum induction ( 1.5-fold) at 2.5 × 104 M. (D) HMA, similar to the effect of amiloride, augmented LPS- induced production of IL-10 at doses  1 × 106 M, with maximum induction ( 1.4-fold) at 2.5 × 104 M. P < 0.05, as compared with control; *P < 0.05, **P < 0.01, and ***P < 0.001, as compared with LPS alone. n = 4, which represents the number of independent experiments performed in duplicate.

View larger version (50K): [in this window] [in a new window]   Figure 4.   The time-dependent kinetics of the effect of amiloride and its analog, HMA, on LPS-induced antiinflammatory cytokine biosynthesis. Alveolar epithelial cells were treated with either amiloride (1 × 104 M) or HMA (1 × 104 M) 4, 2, and 1 h before exposure to LPS (1 µg/ ml), concurrent with LPS, or 1, 2, 4, and 8 h after induction with LPS. Supernatants were subsequently harvested for determining the antigenic activity of IL-6 and IL-10. (A) The biosynthesis (percentage of LPS) of IL-6 and the effect of amiloride with maximal induction (32.64- 38.57%) at 1-2 h pre-LPS treatment. (B) Amiloride augmented the biosynthesis of IL-10 maximally (36.42-42.26%) at 1-2 h before exposure to LPS. (C) The analog of amiloride, HMA, maximally induced LPS-mediated accumulation of IL-6 (54.79-58.37%) 1-2 h before LPS exposure. (D) HMA, similar to its effect on IL-6, augmented LPS-induced production of IL-10 (32.26-37.58%) most effectively at 1-2 h before LPS stimulation. Percentages above the darker histograms represent the maximum induction of cytokine biosynthesis, determined relative to LPS alone. n = 4, which represents the number of independent experiments performed in duplicate.

Role of Amiloride and HMA in Regulating IB- Phosphorylation and Degradation

Cellular transduction signaling pathways mediating the immunoregulatory potential of amiloride have not been well characterized. We hypothesized that the likely inhibitory effect of amiloride in regulating the cytokine pathway involves the IB, the cytosolic inhibitors of NF-B. Subsequently, we designed a series of experiments to investigate the interfering role of amiloride and its analog in regulating IB- phosphorylation and degradation within the cytosolic compartment on stimulation with exogenous LPS. Amiloride blockaded LPS-mediated degradation of IB- in the cytoplasm, thereby allowing its accumulation, in a dose-dependent manner (Figure 5A). This effect of amiloride on IB- was associated with a dose-dependent inhibition of its phosphorylation, thus suggesting the involvement of an upstream kinase (Figure 5A). As shown in Figure 5B, HMA, in a manner similar to the effect of amiloride, blockaded LPS-induced degradation of IB-, allowing its accumulation and negatively interfering in the signal transduction pathway regulating its phosphorylation (Figure 5B). The lower blots show the expression of the housekeeping protein, -actin, as an internal reference for semiquantitative loading in parallel lanes (Figures 5A and 5B).

View larger version (45K): [in this window] [in a new window]   Figure 5.   The involvement of amiloride and its analog, HMA, in the signaling pathway mediating IB- phosphorylation/degradation. (A) The effect of amiloride on IB- phosphorylation/degradation within the cytosolic compartment. The constitutive expression of IB- under nonstimulating conditions reveals the abundance of this inhibitor under the control lane. Exposure to LPS (1 µg/ml; 24 h) induced its degradation and pretreatment with amiloride (2 h) before exposure to LPS reversed the effect of LPS on IB-, thereby allowing its cytosolic accumulation in a dose-dependent manner. In addition, amiloride blockaded LPS-induced phosphorylation of IB-, suggesting the involvement of an upstream kinase. (B) The effect of HMA on IB- phosphorylation/degradation within the cytosolic compartment. In a manner similar to the effect of amiloride, HMA allowed the cytosolic accumulation of IB- by reversing the degrading effect mediated by LPS. The effect of HMA was associated with the inhibition of LPS-induced IB- phosphorylation. The housekeeping protein, -actin, was used as an internal reference for semiquantitative loading in parallel lanes. n = 3, which represents the number of experiments with independent cell cultures.

Role of Amiloride and HMA in Regulating the Nuclear Translocation of Selective NF-B Subunits

To determine whether the negative interference of amiloride and its analog in IB- signaling is associated with selective inhibition of the nuclear translocation of specific NF-B subunits, we assessed the nuclear accumulation of NF-B₁ (p50), NF-B₂ (p52), RelA (p65), RelB (p68), and c-Rel (p75) in response to LPS, amiloride, and HMA. Although LPS (1 µg/ml; 24 h) upregulated the nuclear localization of p50, p65, p68, and p75, it had no apparent effect on p52 (Figures 6A and 6B). Pretreatment with amiloride abrogated the LPS-dependent response in a dose-dependent manner, thereby reducing the accumulation of p50, p65, p68, and p75 within the nucleus (Figure 6A). Similarly, HMA blockaded the stimulatory effect of LPS and blockaded, in a dose-dependent manner, the nuclear localization of p50, p65, and p75 (Figure 6B). The effect of HMA on the nuclear translocation of p68 was dose-independent (Figure 6B). The lower blots show the expression of the housekeeping protein, -actin, as an internal reference for semiquantitative loading in parallel lanes (Figures 6A and 6B).

View larger version (57K): [in this window] [in a new window]   Figure 6.   The involvement of amiloride and its analog, HMA, in LPS-mediated nuclear translocation of selective NF-B subunits. (A) Exposure of epithelial cells to LPS (1 µg/ml; 24 h) induced the translocation of NF-B1 (p50), RelA (p65), RelB (p68), and c-Rel (p75), but not NF-B2 (p52). Pretreatment with amiloride before exposure to LPS reduced, in a dose-dependent manner, the translocation of p50, p65, p68, and p75. (B) Pretreatment with HMA abrogated, in a dose-dependent manner, LPS-mediated nuclear translocation of p50, p65, and p75, and reduced in a dose-independent manner the nuclear abundance of p68. The housekeeping protein, -actin, was used as an internal reference for semiquantitative loading in parallel lanes. n = 3, which represents the number of experiments with independent cell cultures.

Effect of Amiloride and HMA on NF-B/DNA Binding Activity

In association with the ability of amiloride and its analog to blockade LPS-mediated nuclear accumulation of selective NF-B subunits, there was dose-dependent inhibition on NF-B DNA-binding activity as determined by gel shift assays. As shown in Figure 7A, amiloride blockaded, in a dose-dependent manner, LPS-induced NF-B activation (inserted histogram shows the dose-dependent inhibition mediated by amiloride, effective at concentrations  1 × 10⁶ M). Figure 7B shows the inhibitory effect of HMA on NF-B activation (inserted histogram shows the dose-dependent inhibition mediated by amiloride, effective at concentrations  1 × 10⁶ M). We have previously shown that LPS-mediated activation of NF-B involves the p50-p65 complex (28). To re-affirm and verify the constituency and the specificity of the NF-B complex, supershift and mutation experiments were subsequently performed. As shown in Figure 7C, there was no shifted band in the control lane, which contained no nuclear extracts. Incubation of nuclear extracts with an oligonucleotide that has been mutated for 3 bp of the wild-type B moiety abrogated the binding of NF-B complex to the specific DNA sequence (LPS + M22) (see MATERIALS AND METHODS and Reference 28 for more details). The supershifted band with an anti-RelA (p65) polyclonal antibody reveals the involvement of p65 subunit in LPS-mediated activation of NF-B (Figure 7C). The addition of cold competitor (LPS + COMP) reduced, in a dose-dependent manner, the NF-B/DNA-binding activity with complete obliteration of the specific band at 200× (Figure 7C). The free probe denotes the faster migrating unbound radioisotope.

View larger version (61K): [in this window] [in a new window]   Figure 7.   The involvement of amiloride and its analog, HMA, in the signaling transduction pathway regulating LPS-mediated NF-B activation. (A) Amiloride blockaded, in a dose-dependent manner, LPS-induced activation of NF-B, effective at doses  1 × 108 M, with maximum inhibition at a dose of 2.5 × 104 M. The insert shows histogram analysis of the shifted bands as determined by phosphorimaging. (B) HMA blockaded, in a dose-dependent manner, LPS-induced activation of NF-B, effective at doses  1 × 108 M, with maximum inhibition at a dose of 2.5 × 104 M. The insert shows histogram analysis of the shifted bands as determined by phosphorimaging. (C) Supershift analysis experiment showing the composition of the NF-B/DNA complex to contain RelA (p65). The control lane contained no nuclear extracts, the mutant form of NF-B oligonucleotide abrogated the specific binding of NF-B, and addition of 25-200× abolished, in a dose-dependent manner, the shifting of the specific band. The upper open arrow indicates NF-B/DNA complex, the lower open arrow indicates the faster, free migrating radioisotope, and SS denotes the supershift band. *P < 0.05, **P < 0.01, and ***P < 0.001, as compared with LPS alone. n = 3, which represents the number of experiments with independent cell cultures.

Potential Role of rhIL-10 in Regulating Proinflammatory Cytokines and NF-B Activation

As shown in Figure 8, rhIL-10 reduced, in a dose-dependent manner, LPS-induced activation of NF-B (effective at doses  0.1 ng/ml) (Figure 8A), concomitant with abrogating the LPS induction of proinflammatory cytokines IL-1 ( 0.1 ng/ml) and TNF- ( 0.1 ng/ml) along the same dose-response curve (Figure 8B).

View larger version (51K): [in this window] [in a new window]   Figure 8.   The effect of rhIL-10 on LPS-induced activation of NF-B and proinflammatory cytokine production. (A) rhIL-10 reduced, in a dose-dependent manner, LPS-induced upregulation of NF-B DNA binding activity, as shown by the representative EMSA. The upper open arrow indicates the NF-B/DNA complex, and the lower open arrow indicates the faster migrating unbound free probe. (B) rhIL-10 abrogated the stimulatory effect of LPS on proinflammatory cytokine release. P < 0.05, as compared with control; *P < 0.05, **P < 0.01, and ***P < 0.001, as compared with LPS. n = 3, which represents the number of experiments with independent cell cultures.

Effect of Anti-IL-10 Antibody on Amiloride-Dependent Inhibition of Proinflammatory Cytokines and NF-B Activation

Simultaneous co-pretreatment with anti-IL-10 antibody (IL-10) and amiloride reversed the inhibitory effect of amiloride (2.5 × 10⁴ M) on LPS-induced NF-B activation (Figure 9A) and restored proinflammatory cytokine production (Figure 9B).

View larger version (51K): [in this window] [in a new window]   Figure 9.   The effect of anti-rat IL-10 neutralizing antibody (IL-10; 5 µg/ml) on amiloride-dependent inhibition of LPS-induced activation of NF-B and proinflammatory cytokine secretion. (A) IL-10 mitigated the effect of amiloride (2.5 × 104 M) and restored the NF-B DNA binding activity, as shown by the representative EMSA. The upper open arrow indicates the NF-B/DNA complex, and the lower open arrow indicates the faster migrating unbound free probe. (B) IL-10 abrogated the inhibitory effect of amiloride (2.5 × 104 M) on LPS-induced pro-inflammatory cytokine release. P < 0.05, as compared with control; ***P < 0.001, as compared with LPS (NS = nonspecific, as compared with LPS). n = 3, which represents the number of experiments with independent cell cultures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The alveolar epithelium is recognized as a dynamic barrier that plays an important role in regulating the inflammatory and metabolic responses to oxidative stress, sepsis, endotoxemia, and other critical illnesses in the lung (4, 16, 28, 34). The respiratory epithelium is a primary target of an inflammatory/infectious condition at the epithelial- blood interface and is itself capable of amplifying an inflammatory signal by recruiting inflammatory cells and by producing inflammatory mediators. Many of the side effects of LPS are secondary to the overproduction of proinflammatory mediators. Inflammatory as well as autoimmune disease is often associated with deregulated expression and biosynthesis of inflammatory cytokines, which influence a plethora of cellular functions. Therefore, the downregulation of an inflammatory signal, and the amplification of a counter antiinflammatory signal, is a major focus of the rational approach to the treatment of inflammatory diseases, such as chronic inflammation, sepsis, and rheumatoid arthritis. For instance, a novel recent study by Haskó and colleagues (11) reported a potential role for extracellular purines, including adenosine and ATP, and inosine, a degradation product of these purines, as potent endogenous immunomodulatory molecules that inhibit inflammatory cytokine secretion and protect against endotoxin-induced shock. It has also been reported that selective inhibition of phosphodiesterases, a family of enzymes involved in the degradation of cAMP/cGMP (12, 13), steroids such as glucocorticoids [10], pyrimidylpiperazine derivatives [14-16], and ERK, and p38/RK MAPK selective inhibitors (35, 36), differentially regulate the transcription and biosynthesis of inflammatory cytokines and other mediators.

The diuretic amiloride is a prototypic inhibitor of epithelial Na⁺ channels, although amiloride and its various derivatives and analogs inhibit many Na⁺-selective transport proteins (18, 38). A pyrazinoylguanidine with a plethora of other cellular functions, amiloride has recently emerged with an immunoregulatory potential regulating immune and nonimmune cell functions (23). Furthermore, amiloride has been reported to exert antiinflammatory effects given its potential to regulate and suppress the release of inflammatory mediators, including proinflammatory cytokines (19, 27, 39). In addition, it has been shown that amiloride improves postischemic contractile dysfunction in ischemic-reperfused rat hearts (40, 41). On the immunoregulatory potential of amiloride, furthermore, a novel inhibitory effect of this compound was reported in ameliorating murine tissue swelling and inflammation in response to contact sensitizing agents and ultraviolet irradiation (42, 43). Of note, the observations reported that sodium transport and sodium-containing compounds exhibited antiinflammatory role in several models. For example, the gene encoding bumetanide-sensitive cotransporter BSC2, one of the two major isoforms of Na⁺-K⁺-Cl cotransporters, has been implicated in modulating inflammatory cytokine-dependent regulation of fluid mechanical forces in the endothelium (44). In addition, TNF- was recently reported to be implicated in regulating amiloride-sensitive sodium transport across the alveolar epithelium, indicating a possible intervention in the mechanism of alveolar edema clearance in pathology (45). Recently, it has been disclosed that excessive Na⁺ intracellular accumulation may exacerbate renal interstitial inflammation (46) and cause a dysfunctional epithelium associated with chronic inflammation (47). Furthermore, amiloride and other potential interventions were recently proposed for a novel and effective way of treating the chronic cystic fibrosis disease (48), and it was proposed that intracellular pH regulated by Na⁺ potentially contributes to the pulmonary manifestation of the aforementioned condition (49).

The underlying signaling transduction mechanism involved in mediating the immunoregulatory effect of amiloride is, however, largely unknown and has yet to be ascertained in vitro and in vivo. Therefore, we designed this investigation to unravel the molecular pathway that is likely to be a target for the antiinflammatory role that amiloride plays in the alveolar epithelium. In particular, amiloride downregulates LPS-mediated secretion of proinflammatory cytokines (IL-1 and TNF-) and upregulates LPS-mediated production of antiinflammatory cytokines (IL-6 and IL-10). Amiloride, in addition, mediated its immunoregulatory effects on pro- and antiinflammatory cytokines with similar kinetics to its analog, HMA. Of note, amiloride blockaded LPS-dependent phosphorylation/degradation of IB-, abrogated the nuclear translocation of selective NF-B subunits, and reduced the DNA-binding activity of this transcription factor. Collectively, amiloride acts as a novel dual immunoregulator by exhibiting biphasic, antagonistic effect on pro- and antiinflammatory signals, mediated through the targeting of the IB-/NF-B signaling transduction pathway.

Although the inflammatory signals mediated by LPS are well recognized in other systems and cell models, this role in the fetal alveolar epithelium is not well characterized. Administration of LPS differentially regulated NF-B nuclear subunit translocation in vitro (28). Despite the observation that LPS has no influence on the unit composition of NF-B₂ (p52), its stimulatory effect on NF-B₁ (p50), RelB (p68), c-Rel (p75), and RelA (p65), the major transactivating member of the Rel family (28, 29, 31), is prominent. Although the transcription factor NF-B was originally recognized in regulating gene expression in B-cell lymphocytes (30), subsequent studies demonstrated that it is one member of a ubiquitously expressed family of Rel-related transcription factors that serve as critical regulators of many genes, including those of inflammatory cytokines (31, 37). The translocation and activation of NF-B in response to various stimuli are sequentially organized at the molecular level. In its inactive state, the heterodimeric NF-B, which is composed mainly of two subunits, p50 (NF-B₁) and p65 (RelA), is present in the cytoplasm associated with its inhibitory protein, IB (31, 37). Upon stimulation, such as with cytokines and LPS, IB- undergoes phosphorylation on serine/threonine residues, ubiquitination, and subsequent proteolytic degradation, thereby unmasking the nuclear localization signal on p65 and allowing nuclear translocation of the complex. This sequential propagation of signaling ultimately results in the release of NF-B subunits from IB- inhibitor, allowing translocation and promotion of gene transcription, especially those encoding inflammatory cytokines. The novel interference of the inhibitor of Na⁺/H⁺ exchange, amiloride, in regulating the phosphorylation of IB-, the major cytosolic inhibitor of NF-B, suggests the involvement of an upstream kinase. In this respect, signals emanating from membrane receptors, such as those for IL-1 and TNF-, activate members of the MEKK-related family, including NF-B-inducing kinase (NIK) and MEKK₁, both of which are involved in the activation of IB kinases, IKK₁ and IKK₂, components of the IKK signalsome (33). IKK₁ and IKK₂ were identified as components of the high-molecular weight complex containing a number of proteins involved in NF-B regulation (29, 33). These kinases phosphorylate members of the IB family, including IB-, at specific serines within their amino termini, thereby leading to site-specific ubiquitination and degradation by the 26S proteasome. Therefore, the immunoregulatory potential of amiloride to downregulate the phosphorylation of IB- and its subsequent degradation strongly implicate an upstream kinase, probably the IKK complex, as a potential target for the antiinflammatory action of this novel compound. Moreover, the selective interference mediated by amiloride in regulating the activation of NF-B and the expression of its inhibitor IB- is of particular interest because it suggests that this compound's antiinflammatory role within the alveolar space resides within and/or above the upstream pathway regulating the phosphorylation of IB-, thereby regulating the downstream pathway governing NF-B translocation and activation and subsequently interfering with the regulation of cytokine signaling. However, the possibility of amiloride directly interacting with the NF-B complex, thus preventing its binding to the B DNA moiety, cannot be excluded.

The promoters of genes encoding cytokines, particularly IL-1, IL-1, IL-2, IL-3, IL-6, IL-8, IL-12, and TNF- (31), contain multiple cis-acting motifs including those that bind such transcription factors as NF-B. Furthermore, the release of free NF-B upon extracellular stimulation due to IB phosphorylation and degradation leads to DNA binding to initiate transcription of related genes, including immunoreceptors, cytokines, and, interestingly, its own inhibitor, IB (28, 29). Two unique features of the NF-B/IB complex system are deduced from its feedback regulation. The transcriptional activation of NF-B triggers the synthesis of IB, and NF-B-activated transcription is maintained by continuous degradation of IB, which is sustained by an extracellular stimulus (28, 29, 33). Thus, the expression of IB parallels both NF-B activity and the duration of the activating extracellular stimulation, suggesting that this temporal parallelism between IB accumulation/degradation and an effective external stimulation is a mechanism allowing dual, biphasic regulation of NF-B within the alveolar space. Of particular interest, unraveling the downstream cytokine pathway that is regulated by NF-B/IB remains a major target for the search for novel therapeutic agents that tend to suppress the inflammatory signal mediated but have the potential to upregulate a counterinflammatory response. One such novel approach is the discovery reported in this investigation of the potential immunoregulatory role that amiloride exhibits in suppressing an inflammatory signal, yet up-regulating an antiinflammatory loop, in an IB-/NF-B dependent mechanism.

The so-called "inflammatory" cytokines, which include IL-1 and TNF-, are involved in a plethora of cellular actions, particularly inflammatory/infectious conditions. Locally, these cytokines stimulate leukocyte proliferation, cytotoxicity, synthesis, and release of proteolytic enzymes and synthesis of prostaglandins that initiate a cascade of "secondary" cytokine transcription/biosynthesis, including the amplification of IL-1/TNF- and other inflammatory mediators. These cytokines, for instance, have the ability to raise the thermoregulatory set point systemically and, via differential influences on the expression of iron-binding proteins, mediate a redistribution of iron from extracellular to intracellular sites and stores. Moreover, inflammatory mediators orchestrate a metabolic reaction that reduces any energy consumption not directed at repelling the microbial pathogen, redirects host resources to the defense effort, and sets up a defense network that protects nonleukocyte cells from collateral damage by antimicrobial effectors. One such secondary cytokine, IL-6, is often referred to as an inflammatory cytokine because of its temporal association with the aforementioned processes. However, many actions of IL-6 (including downregulation of IL-1 and TNF- production) are counterinflammatory, thereby acting to keep potentially destructive inflammatory responses from overshooting (50). In a novel recent study reported by Ward and colleagues (51), the antiinflammatory potential exhibited by IL-6 was supported by unequivocal evidence implicating this cytokine in mechanisms mediating, for example, cytoprotection in hyperoxic acute lung injury. Further evidence supported the antiinflammatory potential exercised by IL-6 conferring a protective role in LPS-galactosamine septic shock model (52) and ameliorating acute inflammation in vivo (53). Moreover, it was observed that there was a defective inflammatory response in IL-6^/ knockout mice (54) and that IL-6 deficiency exacerbated inflammatory bone resorption and destruction (55). On the mechanism of the antiinflammatory action of IL-6, it was reported that IL-6 inhibits LPS-mediated TNF- secretion in vitro and in vivo (56), induces IL-1 receptor antagonist (IL-1ra) and soluble TNF- receptor (p55) (57), and immunoneutralization of endogenous IL-6 suppresses the induction of IL-1ra (58). We therein reported for the first time that amiloride is capable of augmenting LPS-mediated biosynthesis of IL-6 in the alveolar epithelium, possibly indicating an antiinflammatory potential, an observation consistent with the immunoregulatory capacity and the ability of amiloride to upregulate a counter antiinflammatory loop.

The ability of amiloride, furthermore, to augment the production of IL-10, a potent antiinflammatory cytokine, remains of particular interest. IL-10 was originally identified as a cytokine inhibitory factor, which suppresses the biosynthesis of an array of proinflammatory mediators, including IL-1, IL-4, IL-8, TNF-, TNF-, interferon-, and granulocyte/macrophage colony stimulating factor (59). The level of regulation by IL-10 on cytokines is both transcriptional and translational, raising the possibility of IL-10 acting on transcription factors involved in regulating inflammatory genes (59). It has also been shown that IL-10 selectively inhibited NF-B activation, a phenomenon well correlated with a dose-dependent inhibition of the release of proinflammatory cytokines (59). Moreover, we have previously shown that exogenous rhIL-10 suppressed NF-B activation, accompanied by a dose-dependent inhibition of inflammatory cytokine biosynthesis (16). On the mechanism of action of IL-10, it seems that it involves the blockade of a reaction required for the release of IB from the complex in intact cells (16, 60). For example, certain experiments using cell-free preparations have suggested that certain protein kinases phosphorylate IB, causing its release and allowing activation of the NF-B complex (60). Because IL-10 interfered with the activation of NF-B and subsequently suppressed the release of cytokines, it was suggested that this transcription factor is a target for the antiinflammatory action of this cytokine. Furthermore, immunoneutralization of endogenous IL-10 restored the activity of NF-B and augmented the release of inflammatory mediators (16). Although it is evident that the immunopharmacologic potential of amiloride is IL-10 sensitive and requires NF-B targeting, it is also possible that the effect of the Na⁺/H⁺ exchange inhibitor on NF-B is closely associated with IL-10. Furthermore, the possibility that amiloride-mediated inhibition of IB- phosphorylation and NF-B translocation/activation directly involves the endogenous upregulation of IL-10, and that the inhibitory effects of amiloride are secondary to, and mediated by, IL-10, cannot be excluded. This latter assumption is supported by the unequivocal evidence that immunoneutralization of the effect of IL-10 induced by amiloride restored the DNA-binding activity of NF-B and the secretion of proinflammatory cytokines. Therefore, the ability of amiloride to downregulate NF-B requires, at least in part, IL-10. In short, the involvement of an IL-10 sensitive pathway implicated in the immunoregulatory role of amiloride is supported by (1) the ability of exogenous rhIL-10 to suppress NF-B activation, concomitant with downregulating the biosynthesis of proinflammatory mediators, and (2) that immunoneutralization of endogenous IL-10 induced by amiloride restored LPS-dependent NF-B activation and the release of proinflammatory cytokines. Schematized pathways simulating the biphasic immunoregulatory potential of amiloride and the signal transduction pathways involved are given in Figure 10.

View larger version (26K): [in this window] [in a new window]   Figure 10.   Schematic flow diagram simulating the particular involvement of amiloride in the transduction pathways mediating IB-/NF-B signaling. Exposure to LPS, derived from the cell wall of gram-negative bacteria, induces a signaling cascade that ultimately converges onto an upstream kinase, NIK. NIK regulates the phosphorylation and activation of IKK, which in turn phosphorylates IB- within specific residues in the IB-/NF-B complex in the cytosol. The ability of amiloride to block the activity of NIK and other upstream kinases has yet to be ascertained; however, amiloride is capable of blockading the phosphorylation of IB-, suggesting direct interference in the immediate upstream kinase, IKK. The phosphorylation of IB- marks its dissociation from the IB-/NF-B complex in the cytoplasm, thereby signaling IB- ubiquitination and degradation by the proteasome complex. This frees NF-B subunits to translocate onto the nucleus and regulates the transcription of genes encoding cytokines. Amiloride has the potential to blockade NF-B translocation, thereby interfering with the capacity of the epithelium to regulate proinflammatory cytokines, showing a marked ability to positively regulate and amplify an antiinflammatory signal.

The present investigation has revealed a novel immunoregulatory potential of amiloride. The results are highlighted as follows: (1) Amiloride and its analog, HMA, suppressed LPS-mediated release of inflammatory cytokines (IL-1 and TNF-) with similar time kinetics; (2) this inhibitory effect was closely associated with the augmentation of an antiinflammatory loop (IL-6 and IL-10); (3) pretreatment with either amiloride or HMA suppressed the phosphorylation of IB-, the major cytosolic inhibitor of NF-B, thereby allowing its cytosolic accumulation; (4) analysis of the downstream NF-B pathway revealed the interference of amiloride in the nuclear translocation of selective NF-B subunits, an effect associated with blockading NF-B activation; (5) IL-10 blockaded LPS-induced secretion of IL-1 and TNF- and reduced NF-B activation; and (6) immunoneutralization of endogenous IL-10 reversed the inhibitory effect of amiloride on proinflammatory cytokines and restored the activity of NF-B. We conclude that amiloride acts as a novel dual immunoregulator by targeting the IB-/NF-B sensitive pathway in an IL-10 sensitive mechanism in the alveolar epithelium.