Regulation of LPS-mediated inflammation in vivo and in vitro by the thiol antioxidant Nacystelyn

Frank Antonicelli,1 David Brown,2 Maryline Parmentier,1 Ellen M. Drost,1 Nik Hirani,1 Irfan Rahman,1 Ken Donaldson,2 and William MacNee1

1Edinburgh Lung and Environment Group Initiative/Colt Research Laboratories, Department of Medical & Radiological Sciences, University of Edinburgh Medical School; and 2School of Life Sciences, Napier University, Edinburgh EH8 9AG, United Kingdom

Submitted 15 September 2003 ; accepted in final form 2 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Increased levels of proinflammatory cytokines are present in bronchoalveolar lavage fluid in various lung diseases. Redox-sensitive transcription factors such as NF-{kappa}B regulate gene transcription for these cytokines. We therefore studied the effect of a new thiol antioxidant compound, Nacystelyn (NAL), on IL-8 regulation in a human macrophage-derived cell line (THP-1). LPS (10 µg/ml) increased IL-8 release compared with control levels. This LPS activation was inhibited by coincubation with NAL (1 and 5 mM). Pretreatment with cycloheximide or okadaic acid, protein synthesis, and serine/threonine phosphatase inhibitors, respectively, did not modify inhibition of IL-8 release caused by NAL. NF-{kappa}B and C/EBP DNA binding were increased after LPS treatment compared with control, an effect inhibited by cotreatment with NAL. Activator protein (AP)-1 DNA binding was unaffected. The enhanced neutrophil chemotaxis produced by conditioned media from LPS-treated cells was inhibited when cells were cotreated with NAL. The selectivity of NAL inhibition upon IL-8 expression was studied. LPS-treated THP-1 cells also had higher levels of TNF-{alpha}, transforming growth factor (TGF)-{beta}1 and -3, MIP-1{alpha} and -{beta}, and RANTES gene expression. However, only LPS-induced IL-8 and TGF-{beta}1 expressions were inhibited by NAL. An anti-inflammatory effect of NAL was confirmed in vivo as shown by a reduction in LPS-induced neutrophil recruitment to the lungs following instillation of NAL into the lungs. Our studies demonstrate that NAL has anti-inflammatory properties in vitro and in vivo, may therefore have a therapeutic role in lung inflammation, and has the advantage over other antioxidant agents in that it may be administrated by inhalation.

interleukin-8; lipopolysaccharide; THP-1 cells


MULTIPLE EVENTS ARE INVOLVED in the development of acute inflammation and injury in the lungs. A key component of the acute inflammatory response is the influx of neutrophils into the lungs in conditions such as acute respiratory distress syndrome (ARDS) (13, 20) or exacerbations of airway diseases such as chronic obstructive pulmonary disease (24) or cystic fibrosis (3). The neutrophil influx into the lungs is initiated by chemokines that are chemotactic for neutrophils such as IL-8 (14).

IL-8 is an important mediator of the pathogenesis of inflammatory lung diseases (8, 23). The major sources of IL-8 are alveolar macrophages and alveolar epithelial cells. This chemokine, which is a potent neutrophil chemoattractant and activator (10), is long lived and is induced by IL-1{beta} and TNF-{alpha} (31). Analysis of the 5-flanking region of IL-8 gene indicates that there are binding sites for the transcription factors NF-{kappa}B and NF-IL6 and activator protein (AP)-1 and CCAAT-enhancer binding protein (C/EBP), suggesting that they have a role in its transcriptional regulation (1, 2, 18, 19). The redox state of the cell is important to the transcriptional regulation of the genes for many cytokines (5, 27). However, it remains unclear how the redox balance of a cell modulates IL-8 expression.

Antioxidant therapy, by altering the redox balance in the lungs, may downregulate the transcriptional activation of genes such as IL-8 and hence reduce lung inflammation. Nacystelyn (NAL), a thiol antioxidant compound that is a lysinated derivative of N-acetyl cysteine (NAC), possesses potent mucolytic capacities and has been shown to inhibit reactive oxygen species (ROS) effects (7). It has the advantage of having a neutral pH compared with NAC, which is acidic, and thus can be administered to the airways without the local airway irritation that occurs with NAC (7).

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of gram-negative bacteria, is a potent initiator of inflammatory responses and serves as an indicator of bacterial infection. Thus LPS may be important in a number of lung diseases and is commonly used as a model for pulmonary inflammation. Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptors. The aim of this study was to determine whether the thiol antioxidant NAL can reduce LPS-mediated lung inflammation in a CD14-independent system and to characterize the molecular mechanism of the modulation of LPS activation of IL-8 by NAL in macrophages.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All of the biochemical reagents used in this study were purchased from Sigma (Poole, UK); cell culture medium was obtained from GIBCO-BRL (Paisley, UK).

LPS instillation into the lungs. All animal procedures were carried out humanely under license from the UK Home Office. Male Wistar rats ~3 mo old were used in these studies. Animals were anesthetized with halothane and cannulated by a laryngoscope to expose the trachea, and 0.5 ml of each treatment contained in saline was instilled into the lungs. All animals regained consciousness within minutes of this procedure and suffered no ill effects. Treatments consisted of the following.

1) Intratracheal instillation of 1 µg of LPS. Animals were killed at 6, 16, and 24 h. Control animals received 0.5 ml of saline.

2) On the basis of the results obtained in 1) above, rats were instilled with LPS at concentrations of 10, 25, and 50 ng. Animals were killed after 6 h. The standard time point and dose of LPS used in subsequent experiments were 25 ng for 6 h (see RESULTS).

3) A further series of experiments was performed by coinstillation of 25 ng of LPS plus NAL (0.5, 2.5, and 10 mM), and the animals were killed after 6 h.

Bronchoalveolar lavage. Rats were killed with a single intraperitoneal injection of Pentobarbitone, and the abdomen was opened to expose the abdominal aorta. Blood (8 ml) was removed, placed in 15-ml centrifuge tubes, and allowed to clot, after which the tubes were centrifuged at 2,000 rpm for 5 min, and serum was removed and stored at –80°C for subsequent analysis. The lungs were removed and were serially lavaged with 4x 8-ml volumes, and the cells were pooled. Total cells were counted, and cytocentrifuge smears, prepared for differential cell counts, were stained with Diff-Quik (Raymond A. Lamb, London, UK). Three hundred cells per slide were counted, and the results were expressed as a percentage of the total number of neutrophils in the lung lavage.

Endotoxin assay. A commercially available Limulus amebocyte assay kit (Associates of Cape Cod, Liverpool, UK) was used according to the manufacturer's instructions. Standards were constructed with the standard endotoxin supplied with the kit. All subsequent calculations were based on the standard curves, and data were expressed as the number of endotoxin units per milliliter. A standard dose of 25 ng of LPS was chosen for this series of experiments based on previous results. LPS, in combination with various combinations of NAL ranging from 0.15 to 10 mM, was assayed by a standard protocol. To determine whether NAL could directly affect LPS or assay reagents, we incubated NAL at concentrations ranging from 0.0625 to 10 mM with either the assay reagent or LPS before testing the endotoxin assay.

NF-{kappa}B staining. Macrophages were isolated from bronchoalveolar lavage of untreated rats. Briefly, lungs were removed from rats killed by pentobarbital overdose. Lungs were serially lavaged with 4x 8-ml volumes of sterile saline, and the cells were pooled. Cells were counted and adjusted to 1 x 106/ml in MEM containing 0.2% BSA. Five hundred microliters of cell suspension were added to each well in a 24-well plate, each well containing a 13-mm-diameter sterile glass coverslip. Cells were cultured for 2 days before treatment. Treatments consisted of LPS at a concentration of 1 µg/ml alone or in combination with NAL at 10 mM in F-10 medium without BSA. The control consisted of F-10 medium alone. Coverslips were incubated at 37°C for 2 h before staining by the method outlined below.

Formaldehyde (Sigma), ammonium chloride, Triton (Sigma), and fish gelatin (Sigma) solutions were made up to the required concentration in calcium- and magnesium-free PBS (Life Technologies, Paisley, UK). Calcium- and magnesium-free PBS was used throughout as a washing buffer.

Coverslips were washed twice with PBS and fixed with 3% formaldehyde for 20 min at room temperature. Coverslips were then washed three times with PBS, and excess aldehyde groups were quenched with 50 mM ammonium chloride for 10 min at room temperature. Cells were permeabilized with 0.1% Triton for 4 min, washed three times with PBS and three times with 0.2% fish gelatin solution, and finally given three washes with PBS over a 5-min period.

A sheet of parafilm was stretched over a 24-well plate and used as a base for the antibody staining. Rabbit polyclonal anti-NF-{kappa}B p50 subunit (Santa Cruz) was diluted 1:200 in 0.2% fish gelatin solution, 50 µl were placed on the surface of the parafilm, and the coverslips were floated on the surface to cover the cells. Coverslips were incubated in a humidified chamber for 1 h at room temperature, after which they were washed as before, three times with PBS, 0.2% fish gelatin, and PBS over 5 min. Cells were treated with a second antibody, FITC-labeled anti-rabbit IgG (SAPU Carluke, Lanarkshire, UK) diluted 1:500 in 0.2% fish gelatin solution. Coverslips were treated with 50 µl of the secondary antibody placed on parafilm, and the cells were stained for 1 h at room temperature and washed as before. Coverslips were mounted in Citifluor mounting medium (Agar Scientific, Stansted, UK) and allowed to dry before being viewed by UV microscopy at x100 magnification. Cells were scored on the basis of the staining characteristics as either diffuse or punctuate and expressed as a percentage of the total cell number.

Cell culture. THP-1 human monocytic cells (gift from Dr. S. Hart, University of Edinburgh, UK) were maintained in suspension in RPMI 1640 containing 10% FCS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). For these experiments, the cells were plated in six-well culture dishes at a density of 1 x 106 cells/ml. Differentiation of THP-1 monocytes into macrophages was achieved by overnight incubation with phorbol myristate acetate (PMA) at a concentration of 10 µM. Differentiated cells adhered to the flask, whereas undifferentiated monocytic cells remained in suspension and were removed by washing with PBS. Adherent macrophage-like cells were incubated in serum-free RPMI.

Cell treatments. The cells were incubated in free serum medium alone (control) or with LPS (10 µg/ml).

The effects of NAL on IL-8 release from THP-1 cells were studied at concentrations of 1 and 5 mM with or without coincubation for 4 and 24 h with LPS (10 µg/ml).

Cycloheximide (CHX, 1 µg/ml) and okadaic acid (OA, 0.1 µM) were introduced at 60 and 90 min, respectively, before the addition of LPS (10 µg/ml) and the antioxidant compound NAL.

The effect of glutathione monoethylester (GSHmee, 5 mM) and buthionine sulphoximine (BSO, 50 µM) to increase or decrease GSH levels on IL-8 release was studied after 24 h of incubation.

Cell viability remained >95% after all of the above treatments.

Preparation of nuclear extracts. After treatments with LPS and NAL for 4 and 24 h, the medium overlying the cells was harvested for the measurement of IL-8 and replaced with ice-cold PBS. THP-1 cells were harvested by scraping, followed by centrifugation at 400 g. Nuclear extracts were prepared by the method of Staal et al. (29).

EMSA. Binding reactions were carried out in a volume of 20 µl of binding buffer (Promega) containing 7 µg of nuclear protein and 0.25 mg/ml poly(dI-dC)·poly(dI-dC). In the binding reaction, nuclear extracts were incubated for 20 min at room temperature with [{gamma}-32P]ATP end-labeled double-stranded probes based on the sequence of AP-1, NF-{kappa}B, and C/EBP sites defined in the 5'-untranslated region of the IL-8 gene with T4 polynucleotide kinase. For each of the probes, the sequence was as follows: AP-1 (–172,–155), 5'-GTGATGACTCAGGTTTGC-3', NF-{kappa}B (–118,–107), 5'-CGTGGAATTTTCCTCTGAC-3', C/EBP (–128,–116), 5'-ATCAGTTGCAAATCGT-3'.

The specificity of DNA binding was assessed by competition using a commercially available DNA-binding fragment containing the consensus sites for AP-1 (CGC TTG ATG AGT CAG CCG GAA, Promega), NF-{kappa}B (AGT TGA GGG GAC TTT CCC AGG C, Promega), and C/EBP (CTA GGG CTT GCG CAA TCT ATA TTC G, Geneka Biotechnology). The consensus binding sequences are underlined, and the unmatching bases are in bold letters. For the competition assays, the nuclear extracts were first incubated with the corresponding consensus unlabeled probe (1.5 pmol) for 15 min at room temperature before the addition of labeled probe. The samples were then loaded and electrophoresed through 6% (AP-1 and NF-{kappa}B) and 8% (C/EBP) polyacrylamide gels at a constant voltage of 180 V. The gels were dried, and autoradiography was performed. Binding activity of the shifted bands was quantified by scanning densitometry (STORM, Pharmacia) of the shifted bands.

Isolation of RNA and reverse transcription. RNA was isolated from THP-1 cells with TRIzol reagent (Life Technologies) from untreated cells and cells treated with 5 mM NAL and/or LPS (10 µg/ml) for 4 and 24 h. Total RNA was reverse transcribed according to the manufacturer’s instructions (Life Technologies, catalog no. 8025SA). The resultant cDNA was stored at –20°C until required.

Analysis of IL-8 mRNA by PCR. We chose oligonucleotide primers using the published sequence of the human IL-8 cDNA (21) and {beta}-actin (26). The primers for IL-8 and {beta}-actin were synthesized by MWG Biotech (Milton Keynes, UK). The sequences of the primers used for the PCR were as follows: IL-8 (sense 5'-ATTGAGAGTGGACCACACTBCBCC-3' and anti-sense 5'-CACTGATTCTTGGATAC-CACAGAG-3') and {beta}-actin (sense 5'-CCACCAACTGGGACGACATG-3' and anti-sense 5'-GTCTCAAACATGATCTGGGTCATC-3'). One microliter of the reverse-transcribed mRNA mixture was added directly to the PCR mixture and used for the PCR reactions. The IL-8 and {beta}-actin PCR were run with the same program: the PCR conditions were 94°C for 10 min and then 35 cycles at 94°C for 60 s, 60°C for 60 s, 72°C for 60 s, and a final extension at 72°C for 5 min with 1 unit of Taq DNA polymerase (Promega). The resulting PCR-amplified DNA fragments were confirmed by DNA sequencing. Bands were visualized and scanned on a white/UV transilluminator, UVP (Orme Technologies, Cambridge, UK). The relative densities of the IL-8 mRNA band C (184 bp) were expressed as a percentage of the densities of the {beta}-actin bands (121 bp).

RNase protection assay. RNase protection assays were performed with kits purchased from Pharmingen (San Diego, CA). In brief, total RNA was isolated from stimulated THP-1 cells with TRIzol (Life Technologies). Multiprobe, hCK-3, and hCK-5, which contains templates for the chemokines and cytokines including TNF-{alpha} and -{beta}; lymphotoxin{beta}; IFN-{beta} and -{gamma}; transforming growth factor (TGF)-{beta}1, -2, and -3 (hCK-3 kit); regulated on activation, normal T cell expressed, and presumably secreted (RANTES); interferon-{gamma}-inducible protein (IP)-10; macrophage inflammatory protein (MIP)-1{alpha} and -{beta}; monocyte chemoattractant protein (MCP)-1; I-309; and IL-8 (hCK-5 kit) and the housekeeping genes L32 and GAPDH were labeled with [{alpha}-32P]UTP using T7 RNA polymerase. Labeled probe (3 x 105 cpm) was hybridized to 2 µg of total RNA for 16 h at 56°C. mRNA probe hybrids were treated with RNase mixture, and phenol-chloroform was extracted. Protected hybrids were resolved on a 6% denaturing polyacrylamide sequencing gel and exposed to radiographic film overnight. Band density was assessed by scanning densitometry (STORM, Pharmacia).

Enzyme-linked immunosorbent assay for IL-8. An enzyme-linked immunosorbent assay (ELISA) was used to measure IL-8 as previously described (6). All plates were read on a microplate reader (Dynatech MR 5000) and analyzed using a computer-assisted analysis program (Assay ZAP). Typically, standard curves generated with this ELISA were linear in the 50- to 2,500-pg IL-8/ml range. Only assays having standard curves with a calculated regression line value >0.95 were used for further analysis.

Chemotaxis assay. Neutrophil chemotaxis was measured with a Neuroprobe 96-well chemotaxis chamber (Novair Filtronics) (32). Neutrophils [200 µl at 10 x 106/ml in Hanks' balanced salt solution (HBSS) with 0.3% BSA added] were placed in the upper wells of the chamber, which was separated from the lower wells by a polycarbonate filter with 3-µm-diameter pores. Supernatants from untreated or treated THP-1 cells were added to the lower wells. Each treatment was performed in triplicate for each THP-1 treatment, also in triplicate. The chamber was incubated for 37 min at 37°C, 5% CO2.

The polycarbonate filter was removed and washed with HBSS, and adherent cells were scraped from the top surface of the filter. The filter was air-dried and stained with Diff-Quik stain. The number of neutrophils that had migrated into the filter was measured with an ELISA plate reader (Dynatech M5000) at an optical density of 550 nm (32). The data are expressed as absorbance.

Statistical analysis. Data are expressed as means ± SE. Data comparison were carried out with ANOVA followed by Tukey’s post hoc test for multigroup comparisons.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effects of LPS and NAL on IL-8 release from THP-1 cells. No significant change in IL-8 release was observed after 4 h of incubation with LPS compared with untreated cells in serum-free conditions (Fig. 1A). However, after 4 h of incubation, NAL decreased IL-8 release from LPS-stimulated cells below those of untreated cells (Fig. 1A). Release of IL-8 from THP-1 cells was significantly increased by treatment with LPS at a concentration of 10 µg/ml after 24 h of incubation (40% increase compared with untreated cells) (Fig. 1B). In the same conditions, 5 mM NAL alone significantly reduced IL-8 release from THP-1 cells by 28% compared with control cells (P < 0.001, n = 3, data not shown). Coincubation with NAL abolished the LPS-induced increase in secretion (Fig. 1B).



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Fig. 1. Effects of LPS and Nacystelyn (NAL) on IL-8 release from THP-1 cells. THP-1 cells were incubated in serum-free medium alone (control) or with LPS (10 mg/ml) for 4 (A) and 24 (B) h. LPS-stimulated cells were coincubated with or without NAL (1 and 5 mM). IL-8 release was measured by ELISA. Results are means ± SE of n = 5. P < 0.05, P < 0.01, P < 0.001 are denoted by 1, 2, or 3 symbols, respectively, *compared with control, #compared with LPS stimulation.

 
We used the Limulus amebocyte lysate (LAL) assay to determine whether NAL directly interacts with LPS (Table 1). There was no effect on LPS activity in the LAL assay of addition of NAL to LPS, showing that NAL did not bind LPS. Similarly, addition of NAL to the assay reagent before the addition of LPS showed no effect, indicating that NAL did not inactivate the reagent (data not shown).


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Table 1. Effect of NAL on LPS in Limulus amebocyte assay

 
Effect of CHX and OA on NAL regulation of IL-8 release from THP-1 cells. To investigate the molecular mechanisms by which NAL inhibits LPS-mediated IL-8 release, we determined whether the effect of NAL was through an intermediate event. Cells were pretreated with CHX (1 µg/ml) to prevent protein synthesis. Figure 2 shows that pretreatment for 60 min with CHX decreased LPS-mediated IL-8 release. However, when protein synthesis was inhibited with CHX, coincubation with NAL showed that this antioxidant compound was still able to decrease the release of IL-8 from THP-1 cells stimulated by LPS. We next examined whether the inhibitory effect of NAL on LPS-mediated IL-8 release involved a phosphorylation step. The data in Fig. 2 demonstrate that preincubation for 90 min with OA, a specific inhibitor of serine/threonine phosphates 1 and 2A, enhanced LPS-induced IL-8 release. However, NAL still reduced LPS-induced IL-8 release from THP-1 cells pretreated with OA.



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Fig. 2. Effects of cycloheximide (CHX) or okadaic acid (OA) pretreatments on NAL inhibition of LPS-induced IL-8 release by THP-1 cells. THP-1 cells were pretreated with CHX (1 µg/ml) for 60 min or with OA (0.1 µM) for 90 min before LPS stimulation with NAL. IL-8 release in the medium was measured by ELISA. Results are means ± SE of n = 3. P < 0.001 is denoted by 3 symbols *compared with control, #compared with LPS stimulation, +compared with LPS + CHX stimulation, °compared with LPS + OA stimulation.

 
Antioxidant effect of NAL. To support the hypothesis that NAL inhibits IL-8 release by altering the thiol redox status, we compared its effect with the GSH-lowering effect of BSO, an inhibitor of the {gamma}-glutamylcysteine synthetase (Table 2). This treatment resulted in a significant increase (160% compared with untreated cells) in the basal level of IL-8 secretion at 24 h compared with untreated cells. In contrast, enhancement of GSH concentrations by GSHmee, a cell-permeable form of glutathione, induced a decrease of IL-8 release (70% compared with untreated cells). LPS-induced IL-8 release was also decreased by a cotreatment with GSHmee, and this inhibitory effect was not abolished by pretreatment either with CHX or with OA, suggesting that NAL inhibition of IL-8 release is due to its antioxidant properties and underlining the importance of intracellular thiol status for IL-8 regulation.


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Table 2. Effect of redox status on IL-8 secretion in THP-1 cells

 
Effects of LPS and NAL on IL-8 mRNA expression and transcription factor activation. Having demonstrated that NAL inhibits LPS induction of IL-8 release, we investigated the mechanism of this effect at the mRNA level by semiquantitative PCR using {beta}-actin mRNA expression as a control. LPS induced an increase of IL-8 mRNA expression (145 and 155% compared with untreated cells at 4 and 24 h, respectively), which was abolished by NAL treatment (Fig. 3).



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Fig. 3. Effects of LPS and NAL on IL-8 mRNA expression in THP-1 cells. A: total RNA was isolated from cells incubated in free serum medium alone (C) or with LPS (10 µg/ml) coincubated with or without NAL (5 mM) for 4 and 24 h. RNA was reverse transcribed and used for PCR analysis of IL-8 and {beta}-actin mRNA. B: numeric estimates of IL-8 mRNA levels compared with the subsequent {beta}-actin bands from the same sample. The relative intensities of IL-8 and {beta}-actin bands are expressed as means ± SE percentage change from control of 3 separate experiments. P < 0.01 and P < 0.001 are denoted by 2 and 3 symbols, respectively, *compared with control, #compared with LPS stimulation.

 
To assess the transcriptional regulation during IL-8 gene expression, we used three separate DNA fragments of the 5'-flanking region of IL-8 gene corresponding to the putative AP-1, NF-{kappa}B, and C/EBP sites in an EMSA. The specificity of the three sequences corresponding to AP-1, NF-{kappa}B, and C/EBP from the 5'-flanking region of the IL-8 gene was confirmed by competition experiments with an excess (250-fold) of cold consensus oligonucleotide (data not shown). LPS treatment of THP-1 cells increased NF-{kappa}B and C/EBP DNA binding to their specific sites on the IL-8 promoter (Fig. 4). However, after 24 h of incubation, AP-1 DNA binding was not affected by LPS treatment. Coincubation with NAL inhibited LPS-mediated NF-{kappa}B and C/EBP DNA binding (Fig. 4).



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Fig. 4. Transcription factor DNA binding in THP-1 cells in response to treatment with LPS and NAL. EMSAs of nuclear extracts of THP-1 cells for each specific IL-8 probe. Data are representative of 3 separate extractions. Cells were incubated in free serum medium alone (control) or with LPS (10 µg/ml) cotreated with or without NAL (5 mM) for 24 h. P < 0.01 and P < 0.001 are denoted by 2 or 3 symbols, respectively, *compared with control, #compared with LPS stimulation. AP, activator protein; C/EBP, CCAAT-enhancer binding protein.

 
Effects of NAL on neutrophil chemotactic activity of THP-1 cell-conditioned media. Human neutrophil chemotaxis was measured after incubation with the conditioned media from THP-1 cells incubated for 24 h with or without LPS. Medium from THP-1 cells stimulated by LPS enhanced chemotaxis of neutrophils, an effect that was abolished by an IL-8 capture antibody (Fig. 5) and was diminished when neutrophils were incubated with media from THP-1 cells cotreated with LPS and NAL. Media from THP-1 cells incubated in the presence of NAL alone had no significant effect on neutrophil chemotaxis (data not shown).



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Fig. 5. Functional assessment of chemokine release from LPS-treated THP-1 cells: effects of NAL. THP-1 cells were treated with LPS and NAL. Medium was harvested from THP-1 cells and assessed for chemotactic properties for neutrophils (PMN). Chemotaxis was measured as described in MATERIALS AND METHODS. P < 0.01 and P < 0.001 are denoted by 2 or 3 symbols, respectively, *compared with control, #compared with LPS stimulation.

 
Effect of NAL on LPS-induced chemokine expression in THP-1 cells. Stimulated macrophages produce both CC and CXC chemokines. To determine whether LPS differentially induces other chemokines and cytokines in THP-1 cells, we examined the induction of a panel of chemokines including IP-10, I-309, RANTES, or MCP-1 by an RNase protection assay. Total RNA was isolated from THP-1 cells stimulated with LPS (10 µg/ml) with or without antioxidant treatment. LPS enhanced a range of mRNAs for chemokine expression including TNF-{alpha}, TGF-{beta}1 and -3, MIP-1{alpha} and -{beta}, RANTES, and IL-8 (Fig. 6). However, among the cytokines upregulated by LPS, only LPS enhancement of IL-8 and TGF-{beta}1 was inhibited by NAL.



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Fig. 6. Inhibitory effects of NAL on chemokines induced following LPS stimulation. THP-1 cells were treated with LPS for 24 h alone or in presence of NAL (5 mM). Total mRNA (1 µg) were analyzed by the RNase protection assay. Arrows, protected RNA. LT, lymphotoxin; TGF, transforming growth factor; RANTES, regulated on activation, normal T cell expressed, and presumably secreted; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; IP, interferon-{gamma}-inducible protein; LTn, lymphotactin.

 
"In vivo" inhibitory effect of NAL on lung inflammation. The time and dose response of lung inflammation after intratracheal instillation of LPS is shown in Fig. 7.



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Fig. 7. Effects of LPS and NAL on PMN in bronchoalveolar lavage (BAL) from rats. Time course of PMN recruitment to BAL rats following intratracheal instillation of 1 µg of LPS. At all time points LPS induced increased influx of PMN compared with control (*P < 0.001 n = 3 rats per group). Dose response of LPS-induced influx of PMN into BAL in rats at 6 h (*P < 0.05; cf control, n = 3 rats per group). Effect of concomitant NAL exposure at different concentrations on LPS (25 ng)-induced PMN influx into BAL in rats at 6 h (*P < 0.05 cf LPS alone).

 
The total number of neutrophils recruited into the lungs of rats after instillation of 1 µg of LPS for 6, 16, and 24 h is increased compared with control animals instilled with saline (Fig. 7A). There was no significant difference between the 6- and 16-h time points. By 24 h, inflammation in the lung had decreased compared with 6 and 16 h. We therefore chose 6 h as the time point for all subsequent experiments. Figure 7B shows the total number of neutrophils recruited into the lungs of rats 6 h postinstillation of LPS at concentrations of 10, 25, and 50 ng per animal. There was a clear dose effect of LPS. Based on the results from the time and dose responses, all subsequent experiments were carried out with a 25-ng dose at a 6-h time point. The total number of neutrophils recruited into the lungs of rats after instillation of 25 ng of LPS in combination with NAL at concentrations of 0.5, 2.5, and 10 mM at a 6-h time point is shown in Fig. 7C. There was a clear dose effect of NAL on LPS-induced inflammation. Compared with LPS alone, 0.5 mM NAL reduced the inflammation by 20%, 2.5 mM by 39%, and 10 mM by 93%.

To confirm the reduction of the inflammation we have analyzed the NF-{kappa}B staining characteristics of rat alveolar macrophages after LPS and NAL treatments. The staining in the LPS-treated cells produced a definite "punctate" appearance in the nucleus. Controls and LPS/NAL treatments exhibited a more "diffuse" staining pattern. Percentages of cells with the different staining patterns are summarized in Table 3.


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Table 3. Percentage of "diffuse" or "punctate" staining macrophages treated with LPS and LPS/NAL

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
LPSs are considered to be important in the lung inflammation in a number of conditions, such as ARDS and cystic fibrosis (11, 30). LPS is a bacterial cell wall component that stimulates many leukocyte types. Binding of LPS to its receptor, CD14, on the cell membrane induces the release of proinflammatory cytokines such as TNF-{alpha}, IL-1{beta}, or IL-8, and ROS, which are implicated in the pathogenesis of inflammation. This oxidative stress may then potentiate IL-8 release (5, 27). IL-8 is a potent neutrophilic chemoattractant and activator (10). Migration of neutrophils from the blood circulation into the lung interstitium is a key feature in the pathogenesis of many inflammatory lung diseases. Therefore, the influx of neutrophils into the lungs and their activation by IL-8 have the potential to perpetuate inflammation by producing ROS, which are not only directly injurious but may further increase IL-8 production. In this setting, it is likely that the redox regulation of LPS-induced IL-8 expression may have a role in modifying lung inflammation.

In this study we have shown that influx of neutrophils into the lungs in response to LPS is inhibited by treatment with the antioxidant NAL. This in vivo effect, which correlated with NF-{kappa}B activation from alveolar leukocytes in response to LPS, was also inhibited by NAL. Because IL-8 is the critical cytokine responsible for neutrophil influx in the lungs in response to LPS (10), we addressed the molecular mechanisms of the modulation of LPS-induced IL-8 release by NAL in an in vitro model using THP-1 cells.

In a previous study, we have shown that human THP-1 cells differentiated to macrophages with PMA respond to a dose of LPS (10 µg/ml) by producing IL-1{beta} (25). Thus the THP-1 cell model can be considered as a representative model of activated macrophages in lung inflammation. In this study, we have investigated the influence of LPS-mediated activation of IL-8 release from THP-1 cells cultured in the absence of serum. These experimental conditions were chosen to avoid any interaction with the antioxidant molecules in serum. We found that LPS (10 µg/ml) stimulation increased IL-8 release from THP-1 cells. This confirms that LPS-induced cytokines are released from mononuclear cells even under serum-free conditions (9, 15, 17, 22).

LPS-induced IL-8 secretion from THP-1 cells was inhibited by pretreatment with CHX, a protein synthesis inhibitor, which suggests a role for de novo protein synthesis. This is in keeping with a study by Lichtman et al. (16), in which they showed that de novo protein synthesis is required for serum-independent LPS activation of Kupffer cells. In contrast, cytokine production following LPS stimulation of the serum-dependent pathway is not dependent on a new protein synthesis event (4). One possible hypothesis to explain these findings is that serum-dependent preactivation modifies the protein pattern of the cells and then leads to a transient early release of IL-8 independent of new protein synthesis upon LPS stimulation, whereas serum-independent conditions associated with IL-8 release requires a de novo protein event and thus occurs at a later time point. This dual mechanism could be linked to a concentration-dependent effect of the LPS-binding protein (9). Pretreatment with OA, an inhibitor of serine/threonine phosphatase, increased LPS-dependent IL-8 secretion from THP-1 cells, revealing that protein phosphorylation mediates LPS-induced IL-8 expression. Similarly, increases in IL-1{beta} mRNA and protein release have been reported in LPS-stimulated THP-1 cells pretreated with OA (33). This supports the fact that mechanisms other than de novo protein synthesis are required for cytokine production. In THP-1 cells, LPS also regulates IL-8 mRNA expression at the transcription level. Previous studies have shown that IL-8 promoter activation by IL-1 and TNF-{alpha} occurs predominantly through the transcription factors AP-1, NF-{kappa}B, and NF-IL6 (2, 18, 19), with a possible synergetic effect between AP-1 and NF-{kappa}B or NF-{kappa}B and NF-IL6 (2, 12). However, the effects of these transcription factors on IL-8 mRNA expression depend on the stimulus (12, 28). For instance, Lakshminarayanan et al. (12) showed that only AP-1 is involved in H2O2 stimulation of IL-8 in A549 cells. In our study, we found that LPS increases nuclear binding of the transcription factors NF-{kappa}B and NF-IL6 DNA in macrophage-derived THP-1 cells cultured in the absence of serum.

The novel thiol antioxidant compound NAL decreased LPS-mediated IL-8 release. These results suggest that an antioxidant-sensitive mechanism is involved in the control of IL-8 secretion from macrophages and may help to explain the reduction in LPS-induced neutrophil influx in vivo. Modulation of the GSH content in THP-1 cells with either a GSH inhibitor or precursor supported this hypothesis. We went on to investigate the mechanism of the protective effect of NAL. First, we performed experiments to test the hypothesis that the inhibition of IL-8-release by NAL might require an intermediate event involved in LPS stimulation of THP-1 cells. It is clear that protein synthesis is required for LPS-mediated release of IL-8. However, our results suggest that de novo protein synthesis is not required for NAL-mediated inhibition of IL-8 secretion. This is in keeping with the fact that antioxidant treatment inhibits CD14-dependent and -independent enhancement of IL-8 release. Using okadaic acid, a potent inhibitor of PP-1 and PP-2 phosphatases, we demonstrated that phosphorylation is an important element in the signal transduction leading to IL-8 release, but this event was not involved in the inhibitory effect of NAL. We also showed that NAL decreases the IL-8 mRNA levels induced by LPS concomitantly with inhibition of NF-{kappa}B and C/EBP DNA binding but not on AP-1. These data were supported by ex vivo experiments in rat alveolar macrophages. Hence, our study confirms the importance of NF-{kappa}B and C/EBP for the regulation of IL-8 gene following LPS stimulation of THP-1 cells and the notion that the activation of these transcription factors can be downregulated by NAL. Using the RNase protection assay, we showed that a range of cytokines such as TNF-{alpha}, TGF-{beta}1 and -3, MIP-1{alpha} and -{beta}, and RANTES were also upregulated following LPS stimulation of THP-1 cells. Among these cytokines, IL-8 and TGF-{beta}1 were downregulated by a cotreatment with the thiol antioxidant NAL. Thus this study showed for the first time the ability of a new soluble antioxidant compound, NAL, to inhibit IL-8 release by LPS (CD14-independent)-stimulated THP-1 cells.

In chronic or inappropriate inflammation, neutrophils are important mediators of tissue damage by producing ROS by the NADPH-oxidase enzyme system during the respiratory burst. Inflammation is a multistep system in which inflammatory cytokines released from macrophages are a key feature in the interaction between leukocytes and epithelial cells. IL-8 is an important chemoattractant, inducing neutrophil migration across the alveolar-capillary membrane during lung inflammation (10, 28). LPS induced an upregulation of IL-8 production and release from THP-1 cells, and consequently medium from LPS-treated THP-1 cells potentiated human neutrophil chemotaxis. This effect was blocked with antibody against IL-8 and was also observed in experiments in which media were co-incubated with LPS and the thiol antioxidant NAL. Thus the observed increased in neutrophil chemotaxis appears, at least in part, to be due to the production of IL-8. This in vitro study is supported by in vivo evidence that NAL decreases rat neutrophil influx into lung lavage induced by LPS.

In conclusion, the thiol antioxidant NAL inhibits LPS-induced neutrophil influx in the lungs. The mechanism of this effect may be downregulation of IL-8 expression and NF-{kappa}B activation in macrophages.


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This work was supported by SMB Pharmaceuticals (Brussels, Belgium), the EUROLUNG Project, EU. BIOMED 2 Grant BMH4-CT96-0152, and the foundation Bettencourt Schueller (Paris, France).


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. MacNee, ELEGI/Colt Research Laboratories, Univ. of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, UK (E-mail: w.macnee{at}ed.ac.uk).

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.


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