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
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ABSTRACT |
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interleukin-8; lipopolysaccharide; THP-1 cells
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 and TNF-
(31). Analysis of the 5-flanking region of IL-8 gene indicates that there are binding sites for the transcription factors NF-
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.
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MATERIALS AND METHODS |
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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-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-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 [-32P]ATP end-labeled double-stranded probes based on the sequence of AP-1, NF-
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-
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-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-
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 manufacturers 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 -actin (26). The primers for IL-8 and
-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
-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
-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
-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- and -
; lymphotoxin
; IFN-
and -
; transforming growth factor (TGF)-
1, -2, and -3 (hCK-3 kit); regulated on activation, normal T cell expressed, and presumably secreted (RANTES); interferon-
-inducible protein (IP)-10; macrophage inflammatory protein (MIP)-1
and -
; monocyte chemoattractant protein (MCP)-1; I-309; and IL-8 (hCK-5 kit) and the housekeeping genes L32 and GAPDH were labeled with [
-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 Tukeys post hoc test for multigroup comparisons.
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RESULTS |
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To confirm the reduction of the inflammation we have analyzed the NF-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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DISCUSSION |
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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-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 (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 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-
occurs predominantly through the transcription factors AP-1, NF-
B, and NF-IL6 (2, 18, 19), with a possible synergetic effect between AP-1 and NF-
B or NF-
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-
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-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-
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-
, TGF-
1 and -3, MIP-1
and -
, and RANTES were also upregulated following LPS stimulation of THP-1 cells. Among these cytokines, IL-8 and TGF-
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-B activation in macrophages.
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GRANTS |
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FOOTNOTES |
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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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REFERENCES |
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