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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Airway Disease Section, National Heart and Lung Institute, Imperial College, London, United Kingdom (A.S.W., R.I., S.Y.L., P.N., I.M.A., K.F.C.) Signal Research Division, Celgene, San Diego, California (G.D.F., B.L.B.)
Received February 19, 2007; accepted April 24, 2007.
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Abstract |
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. This study highlights an important role for JNK in response to oxidative stress through modulation of specific inflammatory and redox mediators. Inhibition of JNK with small molecule kinase inhibitors may be a means of reducing ozone-induced inflammation and AHR.
; Vagaggini et al., 1999; Eiswerth et al., 2005). Experimental exposure to ozone induces airway hyper-responsiveness (AHR) to bronchoconstrictor agents and lung neutrophilia (Fabbri et al., 1984; Seltzer et al., 1986; Zhang et al., 1995; Cho et al., 2001; Shore et al., 2002). The mechanisms underlying ozone-induced AHR and inflammation are unclear, but the influx of neutrophils may result from ozone-induced expression of proinflammatory cytokines and chemokines, such as cytokine-induced neutrophil chemoattractant, MIP-2, TNF-, and IL-1
(Haddad et al., 1996; Johnston et al., 1999; Bhalla and Gupta, 2000; Inoue et al., 2000; Bhalla et al., 2002).
Activation of mitogen-activated protein kinases (MAPKs) may lead to a rapid amplification of the inflammatory response (Adcock et al., 2006). Once phosphorylated at dual-specific serine/theronine sites by its upstream kinase cascade, a change in conformation of the MAPK confers a >1000-fold increase in specific kinase activity (Cowan and Storey, 2003). There are three family members of the MAPK family that differ in signaling pathway, substrate specificity, and response: c-Jun-NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK (Johnson and Lapadat, 2002). JNK is activated by environmental insults, such as reactive oxygen species, inflammatory stimuli, and growth factors (Davis, 2000). The serine/threonine amino-terminal transactivation domain of JNK is a component of the transcription factor, activator protein-1 (AP-1), which can regulate the transcription of inflammatory genes such as TNF- and MMPs (Brenner et al., 1989; Angel and Karin, 1991). Many proinflammatory genes involved in chronic inflammatory disease are regulated by kinase activation, which lead to activation or coactivation of redox-sensitive transcription factors, such as AP-1 and nuclear factor-
B. Therefore, ozone acting as an oxidant can stimulate pathways leading to nuclear factor-B and AP-1 activation and the transcription of proinflammatory genes (Hisada et al., 1999; Laskin et al., 2002; Gohil et al., 2003). Using an acute model of ozone exposure in mice, we have examined the effect of inhibition of JNK on AHR, inflammatory cell recruitment and inflammatory gene expression in the lungs by using anthra[1,9-cd] pyrazol-6 (2H)-one (SP600125), an inhibitor of JNK (Bennett et al., 2001).
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Materials and Methods |
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SP600125. To investigate the role of JNK in ozone-induced effects, we studied the effect of a selective inhibitor of JNK catalytic activity. SP600125 is a reversible ATP-competitive inhibitor, exhibiting more than 20-fold selectivity for JNK-1, -2, and -3 against related protein kinases, such as ERK and p38 MAPK (Bennett et al., 2001). SP600125 was prepared as a 30 mg/kg solution in a vehicle of 10% ethanol, 15% polyethoxylated castor oil (Cremophor-El), 30% polyethylene glycol 400, and 20% propylene glycol (Sigma Chemical, Poole, Dorset, UK) in sterile saline. Administration of SP600125 at 30 mg/kg i.v. has been demonstrated to be efficacious in a mouse model of endotoxin-induced inflammation, with the inhibition of lipopolysaccharide-induced tumor necrosis factor- serum levels (Bennett et al., 2001). Therefore, mice were injected intraperitoneally with SP600125 (30 mg/kg; 0.16-ml volume). Control animals received vehicle alone, in the same volume as the active inhibitor.
Protocol. We studied the effect of SP600125 or of vehicle alone in air- or ozone-exposed mice at two time points. Three hours after exposure to air or ozone, lung tissues were collected for microarray analysis (Affymetrix, Santa Clara, CA). Real-time polymerase chain reaction, Western blot analysis, and bronchoalveolar were performed. Twenty to 24 h after exposure, lung resistance (RL) was measured and bronchoalveolar was performed. Mice were exposed to ozone produced by an ozonizer (model 500 Sander Ozonizer; Erwin Sander GmbH, Uetz-Eltz, Germany), mixed with air for3hata concentration of 3 ppm within a sealed Perspex container (Lucite, Southampton, UK). Ozone concentration was continually monitored with an ozone probe (ATi Technologies, Oldham, UK). Control animals received medical air only. Mice received either SP600125 or vehicle 2 h before air or ozone exposure, 8 h after exposure, and 2 h before measurement of RL.
Measurement of Lung Resistance. Twenty to 24 h after exposure, mice were anesthetized with an intraperitoneal injection of anesthetic solution containing midazolam (Roche Products Ltd., Welwyn Garden City, UK) and Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone; Janssen Animal Health, Wantage, UK). Mice were tracheostomized and ventilated (Mini Vent type 845; rate, 250 breaths/min and tidal volume, 250 µl; Hugo Sach Electronic, Germany). Mice were monitored in a whole-body plethysmograph with a pneumotachograph connected to a transducer (EMMS, Hants, UK). Transpulmonary pressure was assessed via an esophageal catheter (EMMS). Instantaneous calculation of pulmonary resistance (RL) was obtained. Increasing concentrations of acetylcholine chloride (ACh, 4-256 mg/ml; Sigma Chemical) were administered with an ultrasonic nebulizer, and RL was recorded for a 5-min period following each concentration. After each concentration, RL was expressed as percentage change from baseline RL measured following nebulized phosphate-buffered saline (PBS) (Sigma Chemical). The concentration of acetylcholine required to increase RL by 150% from baseline was calculated (PC150).
Bronchoalveolar Lavage. We used a previously published method (Nath et al., 2005). In brief, after an overdose of anesthetic, mice were lavaged with a six 0.5-ml aliquots of PBS via the endotracheal tube and retrieved as the bronchoalveolar lavage fluid. Total cell counts and differential cell counts from Cytospin preparations (Shandon Cytospin 4; Thermo Electron Corporation, Waltham, MA) stained by May-Grünwald-Giemsa stain were determined under an optical microscope (Olympus BH2; Olympus Optical Company Ltd., Tokyo, Japan). At least 400 cells were counted per mouse and identified as macrophages, eosinophils, lymphocytes, and neutrophils according to standard morphology under 400x magnification.
DNA Microarray. Total RNA was extracted from frozen lung tissue using TRIzol reagent (Invitrogen, Paisley, UK) followed by RNeasy columns (QIAGEN, Crawley, UK) for additional purity. Quantitation and quality assessment of the RNA preparations were performed on a spectrophotometer (Nanodrop Technologies, Wilmington, DE) and bioanalyzer (Agilent Technologies, Palo Alto, CA), respectively. All samples had an Agilent RNA integrity value of 10. Biotinylated antisense-RNA was generated with the MessageAmp II Kit (Ambion, Austin, TX). In brief, first-strand cDNA was synthesized from 1 µg of total RNA with T7-oligo(dT) primers for2hat 42°C. Second-strand cDNA was synthesized at 16°C for 2 h and then purified on Ambion cDNA filter cartridges. In vitro transcription was conducted at 37°C for 14 h with T7 RNA polymerase and biotinylated CTP and UTP (Enzo, Farmingdale, NY). The biotinylated antisense-RNA was purified on filter cartridges (Ambion) and fragmented at 94°C for 30 min before use on DNA microarray. Fragmented antisense-RNA (10 µg) was hybridized to Affymetrix 430A Mouse GeneChips version 2 for 16 h at 45°C according to manufacturer's instructions (Affymetrix). Mouse GeneChips were washed on the Fluidics station and imaged on a high-resolution Scanner 3000 (Affymetrix). The above procedures were performed in duplicate for each sample. Gene expression data analysis was performed using GeneSpring software version 7.2 (Agilent Technologies). Data normalization included the following steps, as recommended by Affymetrix: i) values below 0.01 were set to 0.01; ii) each gene measurement was divided by the 50th percentile of all measurements in that sample; and (iii) each gene measurement was divided by the median of its measurements in all samples. Genes included in the analysis were scored as "present" on at least 18 of 32 chips and were above the control strength reliability index established by the GeneSpring software. -Fold change analysis was performed on average values from each pair of replicates. Clustering and generation of a condition tree were performed using the Standard Correlation parameter within GeneSpring.
cDNA Synthesis, Reverse Transcription, and Real-Time Polymerase Chain Reaction. RNA extracted from each sample in the previous step was used for the following experiment. Total RNA (5 µg per sample) from lung tissue was used to synthesize single-stranded cDNA using random hexamers and an avian myeloblastosis virus reverse transcriptase (Promega, Southampton, UK). The cDNA generated was used as a template in subsequent real-time polymerase chain reaction (PCR) analyses. Transcript levels were determined by real-time PCR (Rotor Gene 3000; Corbett Research, Sydney, NSW, Australia) using SYBR Green PCR Master Mix Reagent (QIAGEN). The murine forward and reverse primers (0.5 µM) used are specified in Table 1. Cycling conditions were as follows: step 1, 15 min at 95°C; step 2, 20 s at 94°C; step 3, 20 s at 60°C; and step 4: 20 s at 72°C, with step 2 to step 4 repeated 45 times. The standard curves used to determine relative expression for each primer were obtained by running real-time PCR for a diluted sample, for example, to 1:1, 1:10, 1:100, and 1:1000. Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase.
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Measurement of c-Jun Phosphorylation in Lung Tissue. JNK activity was assessed in the lung tissue by measuring c-jun phosphorylation, using a previously published method (Eynott et al., 2004). In brief, 30 µg of total lung protein per lane were separated through 12% denaturing-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in the following buffer (Tris 20 mM, pH 7.6, NaCl 140 mM, and 0.1% Tween 20) and then incubated overnight with affinity-purified rabbit polyclonal antibodies, antinonphosphorylated c-jun anti-phosphoserine-63 (Cell Signaling Technology, Beverly, MA) and antiphosphorylated c-jun (p-c-jun) (Cell Signaling Technology) as markers of JNK activity. Horseradish peroxidase-conjugated anti-rabbit (diluted to 1:2000 from Cell Signaling Technology) was used as a secondary antibody, and enhanced chemiluminescence (GE Healthcare, Little Chalfont, Buckinghamshire, UK) reagent was used for detection. The bands, which were visualized by autoradiography, were quantified using a densitometer with Grab-It and Gel-Works software (UVP, Cambridge, UK).
Data Analysis. Data are presented as the mean ± S.E.M. For multiple comparisons of different groups, we used the Kruskal-Wallis test for analysis of variance. We then performed the Dunn's test for comparison between two individual groups. A P value of less than 0.05 was accepted as significant.
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Results |
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Bronchoalveolar Lavage Fluid
Exposure to ozone caused a significant increase in total cells (Fig. 3A). Neutrophils were significantly increased (P < 0.01; n = 6) compared with air-exposed controls. There was no difference in the total number of cells recovered between either SP600125- or vehicle-treated mice exposed to air. SP600125 caused a significant decrease in ozone-induced neutrophil numbers compared with controls (P < 0.01, n = 6) (Fig. 3B). There were no lymphocytes or eosinophils in the bronchoalveolar lavage fluid.
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92-fold increase, almost 4-fold higher than the next most abundant gene, CXCL1 (KC), which was induced by 25-fold. Other up-regulated genes include ADAMTS (a disintegrin-like and metalloprotease with thrombospondin) (20-fold), a cartilage proteoglycan (20-fold), the related matrix metalloprotease inhibitor MMP-8 (7-fold), serine protease 22 (7-fold), and the chemokine, CCL11 (eotaxin) (9.5-fold). Multifunctional antioxidant response genes, such as metallothionein-1 (MT-1) (14-fold), metallothionein-2 (MT-2) (8-fold), and hemopexin (HPXN) (6-fold), were up-regulated after ozone exposure. More than 30 genes related to oxidative stress were induced, and notable antioxidant genes outside the top 20 include hypoxia-inducible factor (HIF1) (3-fold) and hemoxygenase-1 (2-fold). Ozone also caused the down-regulation of >500 genes that were mostly involved in cell signaling and cell cycling (Table 3). Angiopoietin was most affected by ozone, with an 11-fold decrease in expression following ozone. IL-6 signal transducer (gp130), which facilitates rapid signaling through the IL-6 receptor, was down-regulated 5-fold by ozone.
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Effect of SP600125 on Ozone-Modulated Genes. SP600125 altered the gene expression of 29 genes that were induced by ozone, with eight undergoing further induction and 21 reduced by SP600125 (n = 4 per group) (Table 4). Three of these genes (IL-6, MT-1, and HPXN) were members of the top-20 ozone-induced genes. SP600125 caused a 1.8- and 1.6-fold further increase in ozone-induced expression of IL-6 and CCL2 (monocyte chemoattractant protein-1), respectively, compared with vehicle-treated mice. CCL2 was less than 2-fold up-regulated in ozone-exposed mice compared with air-exposed controls. The antioxidant genes MT-1 and HPXN were reduced 1.6- and 1.7-fold respectively. Mitogen-activated 3 kinase 6 was reduced 1.6-fold by SP600125.
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Table 5 shows the effect of SP600125 on genes that were decreased by ozone. SP600125 increased seven and further decreased eight of these genes. The majority of genes affected were involved in cell signaling and transcription. For example, oxysterol-binding protein-like 9 (OSBPL9), which is involved in lipid transport and oxidation, was reduced by ozone and further reduced by 4.8-fold after SP600125. Other ozone-decreased genes that were further reduced by SP600125 include RIKEN cDNA 1190005F20 gene, a methyltransferase (3-fold), 5830411Rik, a polymerase and splicing factor (2.1-fold), and SFRS12, which is involved in mRNA processing (2.1-fold). In contrast, SP600125 increased the expression of a small number of genes that were initially decreased after ozone. These including HIF1 and 2HLA-B-associated transcript, a euchromatic histone lysine N-methyltransferase, were increased a further 2.5-fold by SP600125 compared with vehicle dosing.
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Real-Time PCR
We confirmed that ozone increased the expression of IL-6, CCL2, CXCL1, and TNF- compared with air exposure. SP600125 decreased the expression of TNF-. The expression of ozone-induced CCL2 and IL-6 was further increased after SP600125 treatment. Ozone-induced expression of CXCL1 was not affected by SP600125, in accordance with the gene array data (Fig. 4). In each case, the -fold change was within a similar range observed in the gene arrays.
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Discussion |
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SP600125, an anthrapyrazolone, is a reversible ATP-competitive inhibitor that interacts at the ATP-binding site of JNK. SP600125 exhibits more than 20-fold selectivity for JNK-1, -2, and -3 compared with related MAPK family members, such as ERK and p38 kinase (Bennett et al., 2001). In a mouse model of endotoxin-induced inflammation, SP600125 at 30 mg/kg significantly inhibited serum levels of TNF- (Bennett et al., 2001); therefore, in this study, we used this dose. Using a similar dosing regime, SP600125 in an adjuvant arthritis model in the rat has been shown to inhibit joint inflammation by attenuating metalloproteinase expression and joint destruction with suppression of JNK activation in the synovium (Bennett et al., 2001). In a previous study, we also showed that this dose of SP600125 also inhibited allergen-induced increase in AHR (Nath et al., 2005). In the current study, phosphorylated-c-Jun expression was increased by ozone, and this was suppressed by SP600125. We have not addressed the issue of selectivity of SP600125 in this study, an issue that was raised by a study of SP600125 on enzymatic actions of many related kinases (Bain et al., 2003). We showed in the rat that the 30 mg/kg dose achieved lung concentrations of SP600125 of the order of 2 µM, which is within the range where this compound is active as an inhibitor of c-jun N-terminal kinase activity and is also reasonably selective in its kinase activity (Eynott et al., 2004). Our gene array data also indicated that mitogen-activated 3 kinase 6, which activates specifically JNK but not ERK or p38 kinase pathways, was inhibited by SP600125, further limiting the effect of this compound selectively on the JNK pathway (Wang et al., 1998). It would be of interest to determine which airway cells show evidence of activation of JNK.
We have shown that there is a predominantly neutrophilic inflammation in accordance with previously reported murine and rat models of ozone exposure (Haddad et al., 1995; Zhao et al., 1998; Johnston et al., 1999; Michalec et al., 2002). Our gene array results show the up-regulated expression of inflammatory genes that are involved with inflammatory cell chemotaxis and recruitment, including CXCL1 (KC), CXCL2 (MIP-2), CCL2, eotaxin, MMP-8, ADAMTS4, and metalloproteinases MT-1 and MT-2. A similar pattern of gene expression has also been reported by other investigators in mouse and rat lung, particularly with the expression of CXCL1 and CXCL2 and MT-1 and MT-2 (Bhalla et al., 2002; Gohil et al., 2003). However, the most highly regulated gene is IL-6, confirming the observations of others (Johnston et al., 1999). IL-6 may be important for the induction of ozone-induced neutrophilia but not of airway hyper-responsiveness. It can cause the induction of neutrophilic chemokines, such as MIP-2 (Johnston et al., 2005). Also of interest is the induction of potentially inhibitory pathways by ozone in relation to neutrophilic pathways, such as tumor necrosis factor--induced protein-6 [TNFAIP6 or tumor necrosis factor-stimulated gene 6 (TSG6)] and suppressor of cytokine signaling 3 (SOCS3). TSG6 reduces IL-1-induced neutrophilia in a murine air-pouch model of acute inflammation (Wisniewski and Vilcek, 1997), whereas SOCS3 is a regulator of cytokine responses and inhibits particularly IL-6 signaling by binding to the receptor complex such that IL-6 signaling and effects are more prolonged in the absence of SOCS-3 (Croker et al., 2003).
Ozone caused a 2- to 12-fold reduction in the expression of 517 genes. Specifically related to neutrophil migration is angiopoietin-1 precursor, which reduces neutrophil migration in vivo and in vitro studies (Gamble et al., 2000; Witzenbichler et al., 2005), such that inhibition of this gene could lead to a facilitation of neutrophil chemotaxis in response to IL-6, CXCL1, and CXCL2. The IL-6 receptor -chain (IL-6 signal transducer or gp130) is inhibited 5-fold by ozone. It is activated when IL-6 binds to the IL-6 receptor, leading to the recruitment of signal transducer and activator of transcription 3 (Table 3) (Inagaki-Ohara et al., 2003).
It is perhaps surprising that SP600125 only modulated a minority of the ozone-induced genes, with a further increase of eight and reduction of 21 genes (Table 4). We confirmed the further up-regulation of IL-6 found on the gene array by reverse transcription-PCR. In relation to IL-6 effects, it is of interest that, on the genes up-regulated by ozone and that could influence IL-6 expression and activity, namely TSG6, SOCS3, angiopoietin-1, and gp130, SP600125 had no effect. We infer that IL-6 would be an unlikely candidate for the reduction in neutrophil recruitment seen with SP600125, in addition to the other neutrophilic chemokines, such as CXCL1 and CXCL2, whose expression was not inhibited by SP600125 (Table 5).
SP600125 decreased the expression of 21 genes up-regulated by ozone, including two antioxidant genes, MT-1 and HPXN. MT, which can be induced by IL-6 and TNF- (Waelput et al., 2001), can protect against oxidative damage, acting as a free radical scavenger. On the other hand, MT-1 directs chemotaxis of leukocytes and regulates leukocyte trafficking (Yin et al., 2005). Therefore, this SP600125-mediated effect may contribute to the reduction in neutrophils observed. Hemopexin, which is a plasma protein with a high affinity for haem, protects cells against oxidative stress by inducing the antioxidants hemoxygenase-1, ferritin, and MT-1 (Bennett et al., 2001). Of the 15 genes decreased by ozone, an increase in expression was most notably observed in another oxidative stress-related gene, HIF1. HIF1 binds to HIF1 to form a HIF1 heterodimer, a redox-sensitive transcription factor (Haddad and Harb, 2005), activation of which leads to the expression of genes such as hemoxygenase-1, IL-6, and KC. Hemoxygenase-1, IL-6, and KC were increased in the present study following ozone exposure.
As previously shown, ozone induced AHR (Fabbri et al., 1984; Zhang et al., 1995; Cho et al., 2001; Shore et al., 2002). We now show that SP600125 significantly reduced ozone-induced AHR by preventing ozone-induced reductions in PC150. However, the maximal change in lung resistance to inhaled ACh following ozone exposure was not affected by SP600125. The importance of chemokines, such as CXCL1 and CXCL2, which signal through the CXCR2 receptor (Johnston et al., 2005), of TNF- signaling through tumor necrosis factor receptor 2 and of IL-1 has been emphasized (Shore et al., 2001; Bhalla et al., 2002). Whether the same modulation of genes that are involved in oxidative stress and inflammation by a JNK inhibitor that lead to the inhibition of ozone-induced neutrophilic inflammation is also responsible for the inhibition of AHR is not clear. In previous studies, for example, in tumor necrosis factor receptor 2 gene knock-out mice, an inhibition of AHR but not of neutrophilic inflammation was observed (Shore et al., 2001).
The 3-ppm level of ozone exposure used in our study is a concentration that is higher than that found in certain polluted urban areas where maximal levels of the order of 0.4 to 0.5 ppm have been reported. However, the 2 to 3-ppm level of ozone exposure induces in rodents a neutrophilic inflammation and AHR as shown in the current study, but in humans, lower levels of exposure of 0.2 to 0.6-ppm are enough to induce similar effects in the lung (Seltzer et al., 1986; Balmes et al., 1997).
Our data indicate an important contribution of JNK in ozone-induced induced inflammatory cell recruitment, modulation of inflammatory gene expression, and AHR. The gene array data point to certain important inflammatory pathways that could be responsible for ozone-induced neutrophilia and AHR. These should be confirmed in future studies. JNK inhibition may prevent the neutrophilic inflammation and AHR in airway diseases where oxidative stress is a key component, such as in asthma and chronic obstructive pulmonary disease.
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Footnotes |
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ABBREVIATIONS: AHR, airway hyper-responsiveness; SP600125, anthra[1,9-cd] pyrazol-6 (2H)-one; BAL, bronchoalveolar lavage fluid; TNF-, tumor necrosis factor ; JNK, c-jun N-terminal kinase; IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; AP-1, activator protein-1; PCR, polymerase chain reaction; MMP, matrix metalloproteinase; ACh, acetylcholine chloride; PBS, phosphate-buffered saline; HIF, hypoxia-inducible factor; p-c-jun, antiphosphorylated c-jun; MT, metallothionein; HPXN, hemopexin; SOCS3, suppressor of cytokine signaling 3; KC, keratinocyte cytokine; MIP-2, inflammatory protein-2; TSG, tumor necrosis factor-stimulated gene.
Address correspondence to: Professor Kian Fan Chung, Airway Disease Section, National Heart & Lung Institute, Imperial College, Dovehouse St, London SW3 6LY, UK. E-mail: f.chung{at}imperial.ac.uk
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); SP600125-treated, air-exposed (
); vehicle-treated, ozone-exposed (
); and SP600125-treated, ozone-exposed mice (
). SP600125-dosing had no effect on air-exposed mice. Ozone caused an increase in lung resistance compared with air-exposed mice at every dose of ACh, whereas SP600125-administration resulted in a reduction in lung resistance after ACh. Data are expressed as means ± S.E.M., n = 8 per group. *, P < 0.05; **, P < 0.01, compared with air-exposed mice of corresponding group. #, P < 0.05; ##, P < 0.01; ###, P < 0.001, compared with vehicle-dosed, ozone-exposed mice. B, -log PC150. Animals exposed to ozone, treated with SP600125, require a significantly higher concentration of ACh to elicit a 150% increase in lung resistance from baseline than those treated with vehicle. Data are expressed as mean ± S.E.M., n = 8 per group. ***, P < 0.001, compared with air-exposed mice. ###, P < 0.001, compared with vehicle-dosed, ozone-exposed mice.
