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Hypoxia modulates lipopolysaccharide induced TNF-α expression in murine macrophages
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Hypoxia modulates lipopolysaccharide induced TNF-α expression in murine macrophages FengQin Liu a,1 , Yan Liu a , Vincent C.H. Lui a , Jonathan R. Lamb b , Paul K.H. Tam a , Yan Chen a,⁎ a
Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, P.R. China b Easter Bush Veterinary Centre, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, UK
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-α) together with tissue
Received 9 October 2007
hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the
Revised version received
primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-
8 January 2008
α biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known.
Accepted 8 January 2008
Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-
Available online 16 January 2008
induced TNF-α expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38
Keywords:
MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced
p38 MAPK
endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-α.
SB203580
Furthermore, hypoxia inducible factor-1alpha (HIF-1α) was detected in vivo after LPS
Lipopolysaccharide
administration but its expression was not affected by SB203580. Our data indicate that LPS
TNF-α
induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-α
Hypoxia
secretion. Furthermore, the induction of HIF-1α in mice with endotoxemia suggested a
HIF-1α
synergistic effect on p38 mediated TNF-α expression. These findings provide new insights on
Macrophages
the pathophysiological effects of hypoxia in sepsis and septic shock. © 2008 Elsevier Inc. All rights reserved.
Introduction Tumor necrosis factor-alpha (TNF-α) is an important proinflammatory cytokine that induces anti-microbial activity in response to bacterial infection [1,2]. The large amounts of TNF-α, observed in sepsis and septic shock, cause epithelial cell apoptosis and lung injury [2–5]. TNF-α also promotes the biosynthesis of other inflammatory cytokines and chemokines which contribute to disease progression. The tissue damage that occurs
during sepsis is largely mediated by macrophages [6]. Gramnegative bacteria initiates the transduction of inflammatory signal after the binding of the bacterial membrane component lipopolysaccharide (LPS) to pattern recognition receptors, Tolllike receptor (TLR) 2 and TLR4. Then via the intracellular coreceptor MD88, signaling results in the activation of the nuclear transcription factor NF-κB, which subsequently triggers mitogen-activated protein kinases (MAPKs) cascades [7–9]. MAPKs are dual-phosphorylated at threonine and tyrosine by a conserved
⁎ Corresponding author. Department of Surgery, The University of Hong Kong, Pokfulam, L9-56, Faculty of Medicine Building, 21, Sassoon Road, Hong Kong, SAR, China. Fax: +86 852 2819 9621. E-mail address: [email protected] (Y. Chen). 1 Present address: Paediatrics Department of Provincial Hospital affiliated to Shandong University, Shandong University, Jinan, Shandong, P.R. China. 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.01.007
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protein kinase cascade and transduce signals to the nucleus [10,11]. Among these MAPK pathways, p38 MAPK (p38) is critical in the biosynthesis of LPS-induced TNF-α. Previous studies have demonstrated that p38 MAPK controls the expression of TNF-α via both transcriptional and post-transcriptional mechanisms [12–14]. Inhibition of this pathway has attracted interest as a therapeutic target in the treatment of inflammatory diseases. Pyridinylimidazole compounds, such as SB203580, have been identified as selective inhibitors of p38 MAP kinase [14]. The effects of SB203580 on p38 MAPK signaling have been extensively investigated using in vitro cell systems [12,15,16], however, its in vivo effect on systemic levels of TNF-α in animal models of septic shock are still controversial [17–20]. Septic shock, systemic inflammatory response syndrome (SIRS) and organ–system failure are clinical complications of sepsis that are not only the result of systemic infection but also the ensuing multi-organ dysfunction that may affect the cardiovascular, respiratory, renal, hepatic and hematopoietic systems [21]. Of these, the commonest organ affected is the lung and patients with sepsis frequently develop acute lung injury or its extreme manifestation, the acute respiratory distress syndrome (ARDS) [22]. Inflammation in the lung and cardiovascular system as well as disturbances in blood coagulation system all contribute to abnormal lung function. The failure of lung gas exchange and defects in oxygen delivery result in tissue and cellular hypoxia [22–24]. Although hypoxia and septic shock are related, the mechanism through which hypoxia exacerbates septic shock has not been fully investigated. In this study, the pathophysiological relationship between hypoxia and TNF-α biosynthesis has been explored using SB203580, a specific p38 MAPK inhibitor. We observed that hypoxia enhanced LPS induced p38 activity and increased TNF-α secretion. The expression of p38 MAPK was unaltered but that of p-MK2 was increased. Exposure to SB203580 failed to inhibit TNF-α production in the mouse macrophage cell line RAW264.7 under hypoxic conditions, whereas under conditions of normal oxygen tension TNF-α synthesis was completely blocked. Similarly, in an experimental mice model of sublethal dose LPS induced endotoxemia the levels of TNF-α in the serum were not suppressed by SB203580 and HIF-1α was induced. These observations are relevant to the identification of novel molecular targets and strategies for the clinical management of septic shock.
Materials and methods Reagents Lipopolysaccharide (LPS, Salmonella typhosa) was purchased from Sigma (Sigma-Aldrich Co, Saint Louis, USA). SB203580 was purchased from Tocris Bioscience (Bristol, UK) and the stock solution was dissolved in DMSO at 25 mM and stored at −20 °C. Before use in experiments the stock solution of SB203580 was diluted in culture medium or saline for in vivo injection. As a control DMSO was used at the same volume as SB203580 and in cell culture this never exceeded 0.05% (v/v), a concentration at which it has no immune modulatory effects. Actinomycin D was purchased from GIBCO (Invitrogen, New York, USA).
Cell culture The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cells were maintained at 37 °C in a 5%CO2 humidified incubator in Dulbecco's modified Eagles medium (DMEM,GIBCO) with 10% (v/v) heat inactivated fetal bovine serum (GIBCO) and penicillin (100 U/ml), and streptomycin (100 μg/ml, GIBCO). The medium was changed every 2 to 3 days. In the hypoxia experiments, RAW264.7 cells were seeded 5 × 105 cells/well and were used at 80%–90% confluence. Hypoxic conditions was maintained either in a hypoxia chamber (Billups-Rothenberg, Inc. CA, USA) with 0.5% O2 or with cobalt chloride 100 μM (CoCl2, Sigma, USA), which is widely used to mimic hypoxia both in vitro and in vivo [25,26].
RNA extraction and real-time PCR analysis Total RNA was prepared by Trizol® (Invitrogen) according to the manufacturer's instruction. Reverse transcription (RT) was performed with 1 μg RNA using Superscript II (Invitrogen). For real-time PCR, a primer master mix was prepared using 1 μM of forward and reverse primer in 2 × SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, USA). Each reaction was performed in triplicate. Samples were amplified with a twostep reaction 95 °C for 15 s, 64 °C for 1 min for 40 cycles using ABI PRISM 7700 Sequence Detector (Applied Biosystems). Primers used for TNF-α, forward 5′-CCA CAT CTC CCT CCA GAA AA-3′; reverse 5′-AGG GTC TGG GCC ATA GAA CT-3′; HIF-1α forward, 5′-AAA CTT CTG GAT GCC GGT GGT-3′; reverse 5′-ACA TGA ATA TGG CCC GTG CAG-3′ and house keeping gene β-actin, forward 5′-GAG AGG GAA ATC GTG CGT GAC-3′ and reverse 5′-GCT CAG TAA CAG TCC GCC TA-3′. Signal of a gene was normalized with β-actin using the formula ΔCT = CT target − CT reference. The differential expression signal was calculated as ΔΔCt = ΔCt (gene of LPS treated group) − ΔCt (gene of untreated group) and expressed as relative fold of change using the formula: 2− ΔΔCT. Statistic was done according to User manual (Applied Biosystems).
Western blot analysis Cells were lysed in lysis buffer (50 mM Tris–HCl;150 mM NaCl; 1% Triton X-100, pH 8.0; 1 mg/ml each of leupeptin, pepstatin, and antipain; 1 mM PMSF; 1 mM NaF; and 1 mM Na3VO4), and protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA) as recommended by the manufacturer. 25 μg of protein was loaded from each sample in 12% polyacrylamide gels. Proteins were separated electrophoretically and transferred to PVDF membranes (Millipore, Bedford, USA) using the Bio-Rad MiniGel system with 100 volts for 1 h. on ice. For immunoblotting the membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (25 mM Tris buffer, pH 7.6 containing 137 mM NaCl) with 0.1% Tween-20 (TBS/T) for 1 h. Immunostaining was performed with polyclonal rabbit anti-mouse p38 (Cell Signalling Technology Inc. Danvers, USA), phosphor-p38 (Cell Signalling Technology Inc.), phospho-MAPKAPK2 (Upstate, Temecula, USA), actin (Bethyl Laboratories, Montgomery, USA) and HIF-1α (Santa Cruz Biotechnology Inc, Santa Cruz, USA) antibodies diluted 1:1000. After washing with TBS/T, the blots were incubated with secondary HRP conjugated antibody (Zymed
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Laboratories, San Francisco, USA) for 1 h. Blots were washed the signal determined by enhanced chemiluminescence reagents (Amersham ECL-Plus, GE Healthcare UK Ltd, Buckinghamshire,
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UK). All photographs were analyzed using Scion Image (Scion Corporation, Frederick, USA). Results were expressed as the OD ratio between target protein and actin bands.
Fig. 1 – SB203580 inhibition of TNF-α production and transcription in RAW264.7 cells. (A) TNF-α levels in the supernatant of different dosage match between SB203580 and LPS under normoxia. Cells were pretreated with 10 μM SB203580 or 1 μM SB203580 for 2 h before 0.1 ng/ml, 1 ng/ml and 10 ng/ml LPS was added. (B) TNF-α levels before and after LPS stimulation in the presence and absence of SB203580 under normoxia, CoCl2 treatment and 0.5% oxygen. RAW264.7 cells were treated with SB203580 (10 μM) 2 h before LPS (1 ng/ml) exposure under different oxygen conditions. For (A) and (B), supernatants were collected at 18 h and analyzed for TNF-α by ELISA. Values are mean ± SD from three separate experiments. (C) The effect of SB203580 on TNF-α transcription under normoxia, CoCl2 treatment and 0.5% oxygen. RAW264.7 cells were stimulated as described above in (B). Total RNA was prepared for real-time PCR at 2 h after LPS stimulation. * p = 0.02.
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Fig. 2 – Kinetics of SB203580 inhibition of TNF-α production by macrophages. Cells were pretreated with SB203580 (10 μM) and then exposed to LPS (1 ng/ml) under conditions of normoxia or treated with CoCl2 for various times. The lines are representative of cells stimulated with LPS/normoxia (●), LPS/CoCl2 mimic (■), SB203580/LPS/normoxia (▲), and SB203580/LPS/ CoCl2 treatment (♦). TNF-α levels in the supernatants was measured by ELISA. Results are means ± SD from two different experiments with triplets.
Measurement of TNF-α protein expression by ELISA
In vivo mouse model of LPS induced endotoxemia
Cell culture supernatants and serum were collected and the level of TNF-α was determined using an ELISA kit (BD Biosciences Pharmingen, San Diego, USA). Each sample was measured in triplicate.
Inbred male BALB/c male mice (10 to 11 weeks old) were used in these experiments. The mice were purchased from the Animal Resources Centre, Australia. Animals were allowed free access to food and water in a 12-hour light, 12-hour dark cycled room.
Fig. 3 – Effect of SB203580 on p38 MAPK activation. RAW264.7 cells were treated with 10 μM SB203580 for 2 h before exposure to 1 ng/ml LPS. Cells were harvested 10, 30 and 60 min after the addition of LPS. Cellular lysates were analyzed in Western blot as described in Materials and methods. The upper panel in (A) is p38 and p-p38 bands for LPS stimulation without SB203580 pretreatment and the lower panel for with SB203580 treatment. (B) and (C) are for p-MAPKAPK2 and analysis by Scion Image. These figures are representatives of three independent experiments that yielded similar results.
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Fig. 4 – Effect of SB203580 on TNF-α mRNA stability under conditions of normoxia and treatment with CoCl2. RAW264.7 cells were either treated or untreated with 10 μM SB203580 for 2 h before exposed to 1 ng/ml LPS for an additional 2 h under different oxygen conditions. Actinomycin D (2 μg/ml) was then added. Total RNA was prepared at the time shown and processed for real-time PCR described in Materials and methods. The above lines show cells stimulated with LPS/normoxia (●), LPS/CoCl2 mimic (■), SB203580/LPS/normoxia (▲), and SB203580/LPS/CoCl2 mimic (♦). The experiment was repeated three times with similar results and this is one representative.
The experimental protocol was approved by the Committee on the Use of Live Animals in Teaching and Research, University of Hong Kong. The endotoxemia was achieved by i.p. injection of LPS (0.5 mg/kg). SB203580 (15 mg/kg) was administrated in saline by i.v. injection and as controls, animals received the same volume of DMSO as present in the SB203580 preparation or just saline. Each group had three mice. The drugs were administrated 2 h before LPS injection based on results of pilot experiments and as previously reported [20,27]. Then mice were sacrificed to obtain blood and lung tissue samples at 90 min and 240 min after LPS injection.
Immunohistochemical staining of HIF-1α expression in mice lung Tissues were fixed in 10% formalin at 4 °C for 24 h, dehydrated in alcohol and cleared in xylene before embedding in paraffin. Sections (5 μm) were dewaxed in xylene, hydrated and processed for the immunohistochemical staining. Antigen retrieval was performed by 10 minutes incubation at 95 °C in 10 mM citrate buffer, pH 6.0. The sections were treated with 3% hydrogen peroxide in methanol for 10 minutes to quench endogenous peroxidase activity. Then sections were incubated with rabbit anti-HIF-1α antibody in PBS + 2% BSA for 16 h at 4 °C and the DAKO EnVision™+ System with 2% 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Dako Corp, Glostrup, Denmark.) were used for revealing positive signal.
Statistical analysis Data are presented as means ± SD. Statistical analysis was performed using SPSS software (SPSS Inc. Chicago, USA). The differences were determined by one-way ANOVA followed by LSD test of all groups and p b 0.05 was considered as significant.
Results The effects of SB203580 on TNF-α production under normal and hypoxic conditions The mouse macrophage cells RAW264.7 were pretreated with 10 μM SB203580 or 1 μM SB203580 for 2 h before different dosage of LPS was added and cultured under normoxia for 18 h. As showed in Fig. 1A, the administration of 10 μM SB203580 and 1 μM SB203580 or more to 1 ng/ml and 0.1 ng/ml LPS treated macrophage cultures completely abrogated TNF-α production in supernatant (p N 0.05, n = 3). In contrast, the same concentration of SB203580 had negligible inhibition on TNF-α production induced by 10 ng/ml of LPS. Single administration of SB203580 did not affect the baseline level of TNF-α. The dosage of 10 μM SB203580 and 1 ng/ml LPS was further investigated in hypoxia, for this the cultures were maintained in conditions of normoxia and hypoxia (0.5% oxygen) or treated with CoCl2 (100 μM) to mimic hypoxia for 18 h and the supernatant was collected. Under conditions of normal oxygen tension, biosynthesis of TNF-α was markedly increased from baseline (1237.33 ± 132.61 pg/ml, n = 3) to N10 fold (18777 ± 1481.68 pg/ml, n = 3) after exposure to LPS (1 ng/ml, Fig. 1B). Hypoxia and CoCl2 treatment in the absence of LPS increased the level of TNF-α to nearly two fold (p b 0.05, n = 3) above the baseline. In contrast, the TNF-α production was only slightly increased (statistically not significant) with LPS stimulation under conditions of hypoxia (Fig. 1B). Exposure to SB203580 also reduced hypoxia induced TNF-α to baseline level. However, the inhibitory effects of SB203580 on TNF-α expression by LPS stimulation were not observed under conditions of hypoxia or treatment with CoCl2.
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The effects of SB203580 on TNF-α mRNA expression were also investigated. SB203580 treatment effectively inhibits TNF-α transcription under conditions of normal oxygen tension normoxia
(p b 0.05, n = 3). Hypoxia and CoCl2 treatment augment LPSinduced TNF-α mRNA expression (Fig. 1C) which was resistant to the inhibitory effects of SB203580.
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Kinetics of TNF-α biosynthesis under conditions of hypoxia and treatment with SB203580 RAW264.7 cells were exposed to LPS in the presence and absence of SB203580 under conditions of normoxia or hypoxia (CoCl2) and TNF-α level was measured in supernatants collected at 1, 2, 4, 6 and 8 h. Under conditions of normoxia, TNF-α was detectable at 4 h and had peaked at 6 h after LPS stimulation (Fig. 2). Hypoxia accelerated TNF-α secretion by 2 h and the peak level was observed at 4 h after LPS stimulation. But in the presence of SB203580 this effect was abrogated and the kinetics of TNF-α synthesis paralleled those observed under conditions of normoxia. As reported above, SB203580 completely blocked TNF-α expression under normoxic conditions.
The effect of hypoxia on p38 MAPK activity and the down-stream target gene MAPKAPK2 Activation of p38 MAPK is a key event in TNF-α biosynthesis. Therefore we investigated the effects of hypoxia on its activity. RAW264.7 cells were pretreated with SB203580, exposed to normoxic or hypoxic (CoCl2) conditions and harvested at 10, 30 and 60 min after LPS activation. Under conditions of normal oxygen tension, the active and phosphorylated form of p38 MAPK (p-p38) was detected 10 min after LPS stimulation peaked at 30 min and had declined by 60 min. A similar pattern of p-p38 expression was observed following CoCl2 treatment and exposure to SB203580 induced no additional changes in p-p38 expression (Fig. 3A). For the inactive form of p38 MAPK neither hypoxia nor SB203580 altered its expression level. MAPKAPK2 (MK2) is a downstream gene of p38 MAPK signaling. We observed that phosphorylated MK2 was first detectable at 10 min and reached to the maximum level at 30 min after addition of LPS and declined at 60 min in the end under normoxia condition (Fig. 3B). Hypoxia increased expression of pMK2 markedly compared to that under condition of normal oxygen tension at 30 and 60 min (upper panel in Figs. 3B and C) and the pattern of expression was only minimally altered by exposure to SB203580 (Figs. 3B and C).
TNF-α mRNA stability under conditions of normoxia and hypoxia The post-transcriptional regulation is an important step in the control of TNF-α biosynthesis. RAW 264.7 cells were exposed to SB203580 and LPS as the above. After 2 h following the addition of the latter they were incubated with actinomycin D (2 μg/ml), a transcriptional inhibitor [28,29]. Total RNA was obtained at 10, 30 and 60 min after actinomycin D treatment. From the comparison of the levels of specific transcripts for TNF-α after
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quantitative PCR, we observed that mRNA stability was enhanced under conditions of hypoxia about 20% at 30 min and 10% at 60 min compare to that in normoxia condition (Fig. 4). The effect of SB203580 was minimized at 60 min both in normoxia and hypoxia condition compare to that in normoxia mRNA level although at 30 min, less than 10% difference can be observed in these three groups.
The in vivo effects of SB203580 on the serum level of TNF-α in LPS induced endotoxemia in mice The in vivo administration of SB203580 to mice with LPS induced endotoxemia had no effect on serum level of TNF-α (Fig. 5A) compare to vehicle control (p N 0.05, n = 3). The expression of HIF1α mRNA was detected by RT-PCR and the expression level was compared by real-time PCR at 90 min after LPS injection. LPS induced the expression of HIF-1α with no significant difference in SB203580 treated mice or vehicle control (p = 0.34, Fig. 5). The expression of HIF-1α protein was detected in mouse lung both at 90 min and reduced at 240 min after LPS injection. No effect of SB203580 can be found on HIF-1α expression compared with that in vehicle control mice (Fig. 5C). The immunohistochemical staining further confirmed the observation that at 90 min after LPS treatment, the protein had been detected in lung interstitial tissue, most likely presented in mononuclear cells (Fig. 5D). However, the number of immuno-positive cells reduced at 240 min post-LPS treatment, and similarly to that in western blot, no obvious effect of SB203580 on HIF-1α expression was observed.
Discussion TNF-α is a key pro-inflammatory cytokine, which if secreted during hypoxia, a condition of low tissue oxygen supply, often results in disease pathology and worsens the clinical outcome. This is well illustrated in sepsis where high levels of TNF-α under conditions of hypoxia cause lung damage which progresses towards irreversible acute respiratory distress syndrome (ARDS) and high mortality [4,5,22]. However, the effect of hypoxia on the biosynthesis of TNF-α has not been fully investigated. Here we report that increased TNF-α production under hypoxia is mediated through enhanced expression of p38 MAPK and HIF-1α. HIF-1α predominantly contributed to the up-regulation of TNF-α, especially in vivo and is independent of p38 MAPK signaling. These results imply that reducing p38 activity together with improving oxygen supply may have beneficial effects in the management of sepsis. SB203580 is a specific p38 MAPK inhibitor [30,31] that has been widely used to investigate p38 activity in the regulation of cytokine production [12,15,16,32]. SB203580 binds in the ATP
Fig. 5 – Serum TNF-α levels and lung expression of HIF-1α in an in vivo mouse model of LPS induced endotoxemia. Balb/c mice were injected SB203580 i.v. and LPS i.p. as described in Materials and methods. Blood and lung samples were collected 90 min and 240 min after LPS injection. n = 3. (A) Effect of SB203580 on serum TNF-α levels. (B) HIF-1α gene expression in lungs at 90 min after LPS injection. Vehicle control mice and SB203580 injected mice all expressed HIF-1α. Real-time PCR comparison of HIF-1α expression between vehicle control mice and SB203580 pretreated mice. (C) HIF-1α protein expression in mouse lung at 90 min and 240 min after LPS injection. The top of two bands illustrated showed HIF-1α protein; the bottom was a nonspecific band of slightly smaller size. * a nonspecific protein detected as a loading control. (D) Immunohistochemical staining of HIF-1α in mouse lung. Present is normal control, LPS treatment at 90 min and 240 min and the effect of SB203580.
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pocket of both the inactive and active forms of p38 MAPK to prevent the phosphorylation and function of the down-stream substrate MK2 [15,16,33,34]. We tried different dosage between SB203580 and LPS in vitro under normoxia and then focused on the optimized dosage. We observed that SB203580 can inhibit LPS induced TNF-α biosynthesis in macrophages RAW264.7. Similar phenomenon was also observed in the experiments performed in mouse peritoneal macrophage (data not shown) and theses results were similar to the previous observation [35]. But the inhibition effect was failed to do so under hypoxic conditions. The mechanisms underlying this effect may involve altered p38 activity or the expression of other factors that regulate TNF-α expression independent of p38. Interactions between hypoxia and p38 MAPK activity have been investigated in different cell types. For cardiac myocytes it was reported that hypoxia up-regulated the expression of p65pak [36] and in Kupffer cells modulates Src family kinases [37]. Furthermore, through its effects on NF-κB, hypoxia can increase p38 MAPK activity [38,39]. However, we failed to detect any effects of hypoxia on inactive or active p38 in macrophages, which suggests for this cell type that there is no intermediate pathway controlling p38 activity. As the result of up-regulating p-MK2, hypoxia can directly promote p-p38 phosphorylation activity possibly by modifying the binding of p-p38 with MK2 or altering conditions to facilitate phosphorylation. We also noted that inhibition of p38 MAPK was not dose-dependent under normoxic conditions, which suggests a minimum level of p-p38 is required to initiate the phosphorylation cascade in signal transduction. p38 MAPK regulates both the transcriptional and posttranscriptional levels of TNF-α via MK2 [33,34] which we demonstrated was up-regulated in macrophages under hypoxic conditions. Analysis of the kinetics revealed that the increased expression of pMK2 occurred between 30 and 60 min and the enhanced levels were resistant to the effects of SB203580. TNF-α mRNA expression levels and stability paralleled changes in MK2 expression under both normoxic and hypoxic conditions. The increase in MK2 induced by hypoxia was consistent with that SB203580 can reduce more than 50% p-MK2 from time point 30 min to 60 min under normoxia condition and no such phenomenon was observed under hypoxia condition (Figs. 3B and C). Treatment with SB203580 delayed the peak time of TNF-α biosynthesis during hypoxia but failed to restore to the base level as during normoxia conditions. These results suggest that SB203580 can modulate TNF-α expression but were unable to totally suppress p-p38 activity under hypoxic conditions. These findings indicate that hypoxia was dominant over p38 MAPK in the regulation of TNF-α biosynthesis by macrophages. During sepsis, failure in lung gas exchange and circulatory disturbances caused tissue hypoxia. Micro-environmental conditions found in areas of inflammation are characterized by low levels of O2 [40]. This put forward the question whether LPS induced endotoximia as well as sepsis has co-existed hypoxia in vivo. Thus we extended our analysis of the regulation of TNF-α to a mouse model of endotoxemia induced with LPS. Sepsis animal model has the features of pulmonary hypertension, decreased lung mechanics and arterial hypoxemia and thereby parallels human disease. The PaO2 significantly decreased 1 h after completion of endotoxin infusion and remained below
thereafter in the model [41]. Hypoxia inducible factor-1 (HIF-1) is a principal mediator of homeostasis in cells and tissues exposed to hypoxia. The HIF-1 is a hetero-dimer that consists of two helix–loop–helix proteins, HIF-1α and HIF-1β HIF-1α can generally only be detected under low oxygen concentrations [42,43]. Recent studies reported that nonhypoxic stimuli, including LPS can also induce HIF-1 complex [44,45] via a mechanism distinct from hypoxic induction [44]. The study by Peyssonnaux et al. using mice with macrophage targeted deletion of HIF-1α demonstrated that TNF-α was greatly reduced during septic shock confirming the importance of HIF-1α in TNF-α expression [45]. They proposed that the regulation was mediated through the direct binding of HIF-1α to hypoxic response elements in the TNF-α regulatory region. Here we report that HIF-1α was detected within 90 min of the induction of endotoxemia and treatment with SB203580 had no effect on its expression. Similar results were obtained in septic shock mice model induced by higher dosage of LPS (Data not shown). Taken together, our finding suggests that HIF-1α possibly functions independently of p38 MAPK signaling in the regulation of TNF-α production in vivo. Further investigation on the effect of SB203580 in HIF-1α knockout animal will help to clarify the synergistic effects of these two pathways. In summary, our study provides evidence that hypoxia can increase p38 MAPK activity, through the modulation of MK2 to increase TNF-α expression both at the transcriptional and post-transcriptional levels. In a mouse model of LPS induced endotoxemia it appears that the activation of p38 MAPK signaling and the induction of hypoxia occur. These outcomes may partly explain the varied results of p38 inhibitors in animal models of systemic sepsis. Our results suggest that hyoxia and TNF-α biosynthesis should both be considered in the development of valid therapeutic strategies for managing septic shock.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Hypoxia modulates lipopolysaccharide induced TNF-α expression in murine macrophages FengQin Liu a,1 , Yan Liu a , Vincent C.H. Lui a , Jonathan R. Lamb b , Paul K.H. Tam a , Yan Chen a,⁎ a
Division of Paediatric Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, P.R. China b Easter Bush Veterinary Centre, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, UK
ARTICLE INFORMATION
ABS T R AC T
Article Chronology:
The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-α) together with tissue
Received 9 October 2007
hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the
Revised version received
primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-
8 January 2008
α biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known.
Accepted 8 January 2008
Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-
Available online 16 January 2008
induced TNF-α expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38
Keywords:
MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced
p38 MAPK
endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-α.
SB203580
Furthermore, hypoxia inducible factor-1alpha (HIF-1α) was detected in vivo after LPS
Lipopolysaccharide
administration but its expression was not affected by SB203580. Our data indicate that LPS
TNF-α
induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-α
Hypoxia
secretion. Furthermore, the induction of HIF-1α in mice with endotoxemia suggested a
HIF-1α
synergistic effect on p38 mediated TNF-α expression. These findings provide new insights on
Macrophages
the pathophysiological effects of hypoxia in sepsis and septic shock. © 2008 Elsevier Inc. All rights reserved.
Introduction Tumor necrosis factor-alpha (TNF-α) is an important proinflammatory cytokine that induces anti-microbial activity in response to bacterial infection [1,2]. The large amounts of TNF-α, observed in sepsis and septic shock, cause epithelial cell apoptosis and lung injury [2–5]. TNF-α also promotes the biosynthesis of other inflammatory cytokines and chemokines which contribute to disease progression. The tissue damage that occurs
during sepsis is largely mediated by macrophages [6]. Gramnegative bacteria initiates the transduction of inflammatory signal after the binding of the bacterial membrane component lipopolysaccharide (LPS) to pattern recognition receptors, Tolllike receptor (TLR) 2 and TLR4. Then via the intracellular coreceptor MD88, signaling results in the activation of the nuclear transcription factor NF-κB, which subsequently triggers mitogen-activated protein kinases (MAPKs) cascades [7–9]. MAPKs are dual-phosphorylated at threonine and tyrosine by a conserved
⁎ Corresponding author. Department of Surgery, The University of Hong Kong, Pokfulam, L9-56, Faculty of Medicine Building, 21, Sassoon Road, Hong Kong, SAR, China. Fax: +86 852 2819 9621. E-mail address: [email protected] (Y. Chen). 1 Present address: Paediatrics Department of Provincial Hospital affiliated to Shandong University, Shandong University, Jinan, Shandong, P.R. China. 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.01.007
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protein kinase cascade and transduce signals to the nucleus [10,11]. Among these MAPK pathways, p38 MAPK (p38) is critical in the biosynthesis of LPS-induced TNF-α. Previous studies have demonstrated that p38 MAPK controls the expression of TNF-α via both transcriptional and post-transcriptional mechanisms [12–14]. Inhibition of this pathway has attracted interest as a therapeutic target in the treatment of inflammatory diseases. Pyridinylimidazole compounds, such as SB203580, have been identified as selective inhibitors of p38 MAP kinase [14]. The effects of SB203580 on p38 MAPK signaling have been extensively investigated using in vitro cell systems [12,15,16], however, its in vivo effect on systemic levels of TNF-α in animal models of septic shock are still controversial [17–20]. Septic shock, systemic inflammatory response syndrome (SIRS) and organ–system failure are clinical complications of sepsis that are not only the result of systemic infection but also the ensuing multi-organ dysfunction that may affect the cardiovascular, respiratory, renal, hepatic and hematopoietic systems [21]. Of these, the commonest organ affected is the lung and patients with sepsis frequently develop acute lung injury or its extreme manifestation, the acute respiratory distress syndrome (ARDS) [22]. Inflammation in the lung and cardiovascular system as well as disturbances in blood coagulation system all contribute to abnormal lung function. The failure of lung gas exchange and defects in oxygen delivery result in tissue and cellular hypoxia [22–24]. Although hypoxia and septic shock are related, the mechanism through which hypoxia exacerbates septic shock has not been fully investigated. In this study, the pathophysiological relationship between hypoxia and TNF-α biosynthesis has been explored using SB203580, a specific p38 MAPK inhibitor. We observed that hypoxia enhanced LPS induced p38 activity and increased TNF-α secretion. The expression of p38 MAPK was unaltered but that of p-MK2 was increased. Exposure to SB203580 failed to inhibit TNF-α production in the mouse macrophage cell line RAW264.7 under hypoxic conditions, whereas under conditions of normal oxygen tension TNF-α synthesis was completely blocked. Similarly, in an experimental mice model of sublethal dose LPS induced endotoxemia the levels of TNF-α in the serum were not suppressed by SB203580 and HIF-1α was induced. These observations are relevant to the identification of novel molecular targets and strategies for the clinical management of septic shock.
Materials and methods Reagents Lipopolysaccharide (LPS, Salmonella typhosa) was purchased from Sigma (Sigma-Aldrich Co, Saint Louis, USA). SB203580 was purchased from Tocris Bioscience (Bristol, UK) and the stock solution was dissolved in DMSO at 25 mM and stored at −20 °C. Before use in experiments the stock solution of SB203580 was diluted in culture medium or saline for in vivo injection. As a control DMSO was used at the same volume as SB203580 and in cell culture this never exceeded 0.05% (v/v), a concentration at which it has no immune modulatory effects. Actinomycin D was purchased from GIBCO (Invitrogen, New York, USA).
Cell culture The murine macrophage cell line RAW 264.7 was obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cells were maintained at 37 °C in a 5%CO2 humidified incubator in Dulbecco's modified Eagles medium (DMEM,GIBCO) with 10% (v/v) heat inactivated fetal bovine serum (GIBCO) and penicillin (100 U/ml), and streptomycin (100 μg/ml, GIBCO). The medium was changed every 2 to 3 days. In the hypoxia experiments, RAW264.7 cells were seeded 5 × 105 cells/well and were used at 80%–90% confluence. Hypoxic conditions was maintained either in a hypoxia chamber (Billups-Rothenberg, Inc. CA, USA) with 0.5% O2 or with cobalt chloride 100 μM (CoCl2, Sigma, USA), which is widely used to mimic hypoxia both in vitro and in vivo [25,26].
RNA extraction and real-time PCR analysis Total RNA was prepared by Trizol® (Invitrogen) according to the manufacturer's instruction. Reverse transcription (RT) was performed with 1 μg RNA using Superscript II (Invitrogen). For real-time PCR, a primer master mix was prepared using 1 μM of forward and reverse primer in 2 × SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, USA). Each reaction was performed in triplicate. Samples were amplified with a twostep reaction 95 °C for 15 s, 64 °C for 1 min for 40 cycles using ABI PRISM 7700 Sequence Detector (Applied Biosystems). Primers used for TNF-α, forward 5′-CCA CAT CTC CCT CCA GAA AA-3′; reverse 5′-AGG GTC TGG GCC ATA GAA CT-3′; HIF-1α forward, 5′-AAA CTT CTG GAT GCC GGT GGT-3′; reverse 5′-ACA TGA ATA TGG CCC GTG CAG-3′ and house keeping gene β-actin, forward 5′-GAG AGG GAA ATC GTG CGT GAC-3′ and reverse 5′-GCT CAG TAA CAG TCC GCC TA-3′. Signal of a gene was normalized with β-actin using the formula ΔCT = CT target − CT reference. The differential expression signal was calculated as ΔΔCt = ΔCt (gene of LPS treated group) − ΔCt (gene of untreated group) and expressed as relative fold of change using the formula: 2− ΔΔCT. Statistic was done according to User manual (Applied Biosystems).
Western blot analysis Cells were lysed in lysis buffer (50 mM Tris–HCl;150 mM NaCl; 1% Triton X-100, pH 8.0; 1 mg/ml each of leupeptin, pepstatin, and antipain; 1 mM PMSF; 1 mM NaF; and 1 mM Na3VO4), and protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA) as recommended by the manufacturer. 25 μg of protein was loaded from each sample in 12% polyacrylamide gels. Proteins were separated electrophoretically and transferred to PVDF membranes (Millipore, Bedford, USA) using the Bio-Rad MiniGel system with 100 volts for 1 h. on ice. For immunoblotting the membranes were blocked with 10% nonfat dried milk in Tris-buffered saline (25 mM Tris buffer, pH 7.6 containing 137 mM NaCl) with 0.1% Tween-20 (TBS/T) for 1 h. Immunostaining was performed with polyclonal rabbit anti-mouse p38 (Cell Signalling Technology Inc. Danvers, USA), phosphor-p38 (Cell Signalling Technology Inc.), phospho-MAPKAPK2 (Upstate, Temecula, USA), actin (Bethyl Laboratories, Montgomery, USA) and HIF-1α (Santa Cruz Biotechnology Inc, Santa Cruz, USA) antibodies diluted 1:1000. After washing with TBS/T, the blots were incubated with secondary HRP conjugated antibody (Zymed
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Laboratories, San Francisco, USA) for 1 h. Blots were washed the signal determined by enhanced chemiluminescence reagents (Amersham ECL-Plus, GE Healthcare UK Ltd, Buckinghamshire,
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UK). All photographs were analyzed using Scion Image (Scion Corporation, Frederick, USA). Results were expressed as the OD ratio between target protein and actin bands.
Fig. 1 – SB203580 inhibition of TNF-α production and transcription in RAW264.7 cells. (A) TNF-α levels in the supernatant of different dosage match between SB203580 and LPS under normoxia. Cells were pretreated with 10 μM SB203580 or 1 μM SB203580 for 2 h before 0.1 ng/ml, 1 ng/ml and 10 ng/ml LPS was added. (B) TNF-α levels before and after LPS stimulation in the presence and absence of SB203580 under normoxia, CoCl2 treatment and 0.5% oxygen. RAW264.7 cells were treated with SB203580 (10 μM) 2 h before LPS (1 ng/ml) exposure under different oxygen conditions. For (A) and (B), supernatants were collected at 18 h and analyzed for TNF-α by ELISA. Values are mean ± SD from three separate experiments. (C) The effect of SB203580 on TNF-α transcription under normoxia, CoCl2 treatment and 0.5% oxygen. RAW264.7 cells were stimulated as described above in (B). Total RNA was prepared for real-time PCR at 2 h after LPS stimulation. * p = 0.02.
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Fig. 2 – Kinetics of SB203580 inhibition of TNF-α production by macrophages. Cells were pretreated with SB203580 (10 μM) and then exposed to LPS (1 ng/ml) under conditions of normoxia or treated with CoCl2 for various times. The lines are representative of cells stimulated with LPS/normoxia (●), LPS/CoCl2 mimic (■), SB203580/LPS/normoxia (▲), and SB203580/LPS/ CoCl2 treatment (♦). TNF-α levels in the supernatants was measured by ELISA. Results are means ± SD from two different experiments with triplets.
Measurement of TNF-α protein expression by ELISA
In vivo mouse model of LPS induced endotoxemia
Cell culture supernatants and serum were collected and the level of TNF-α was determined using an ELISA kit (BD Biosciences Pharmingen, San Diego, USA). Each sample was measured in triplicate.
Inbred male BALB/c male mice (10 to 11 weeks old) were used in these experiments. The mice were purchased from the Animal Resources Centre, Australia. Animals were allowed free access to food and water in a 12-hour light, 12-hour dark cycled room.
Fig. 3 – Effect of SB203580 on p38 MAPK activation. RAW264.7 cells were treated with 10 μM SB203580 for 2 h before exposure to 1 ng/ml LPS. Cells were harvested 10, 30 and 60 min after the addition of LPS. Cellular lysates were analyzed in Western blot as described in Materials and methods. The upper panel in (A) is p38 and p-p38 bands for LPS stimulation without SB203580 pretreatment and the lower panel for with SB203580 treatment. (B) and (C) are for p-MAPKAPK2 and analysis by Scion Image. These figures are representatives of three independent experiments that yielded similar results.
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Fig. 4 – Effect of SB203580 on TNF-α mRNA stability under conditions of normoxia and treatment with CoCl2. RAW264.7 cells were either treated or untreated with 10 μM SB203580 for 2 h before exposed to 1 ng/ml LPS for an additional 2 h under different oxygen conditions. Actinomycin D (2 μg/ml) was then added. Total RNA was prepared at the time shown and processed for real-time PCR described in Materials and methods. The above lines show cells stimulated with LPS/normoxia (●), LPS/CoCl2 mimic (■), SB203580/LPS/normoxia (▲), and SB203580/LPS/CoCl2 mimic (♦). The experiment was repeated three times with similar results and this is one representative.
The experimental protocol was approved by the Committee on the Use of Live Animals in Teaching and Research, University of Hong Kong. The endotoxemia was achieved by i.p. injection of LPS (0.5 mg/kg). SB203580 (15 mg/kg) was administrated in saline by i.v. injection and as controls, animals received the same volume of DMSO as present in the SB203580 preparation or just saline. Each group had three mice. The drugs were administrated 2 h before LPS injection based on results of pilot experiments and as previously reported [20,27]. Then mice were sacrificed to obtain blood and lung tissue samples at 90 min and 240 min after LPS injection.
Immunohistochemical staining of HIF-1α expression in mice lung Tissues were fixed in 10% formalin at 4 °C for 24 h, dehydrated in alcohol and cleared in xylene before embedding in paraffin. Sections (5 μm) were dewaxed in xylene, hydrated and processed for the immunohistochemical staining. Antigen retrieval was performed by 10 minutes incubation at 95 °C in 10 mM citrate buffer, pH 6.0. The sections were treated with 3% hydrogen peroxide in methanol for 10 minutes to quench endogenous peroxidase activity. Then sections were incubated with rabbit anti-HIF-1α antibody in PBS + 2% BSA for 16 h at 4 °C and the DAKO EnVision™+ System with 2% 3,3′-diaminobenzidine tetrahydrochloride (DAB) (Dako Corp, Glostrup, Denmark.) were used for revealing positive signal.
Statistical analysis Data are presented as means ± SD. Statistical analysis was performed using SPSS software (SPSS Inc. Chicago, USA). The differences were determined by one-way ANOVA followed by LSD test of all groups and p b 0.05 was considered as significant.
Results The effects of SB203580 on TNF-α production under normal and hypoxic conditions The mouse macrophage cells RAW264.7 were pretreated with 10 μM SB203580 or 1 μM SB203580 for 2 h before different dosage of LPS was added and cultured under normoxia for 18 h. As showed in Fig. 1A, the administration of 10 μM SB203580 and 1 μM SB203580 or more to 1 ng/ml and 0.1 ng/ml LPS treated macrophage cultures completely abrogated TNF-α production in supernatant (p N 0.05, n = 3). In contrast, the same concentration of SB203580 had negligible inhibition on TNF-α production induced by 10 ng/ml of LPS. Single administration of SB203580 did not affect the baseline level of TNF-α. The dosage of 10 μM SB203580 and 1 ng/ml LPS was further investigated in hypoxia, for this the cultures were maintained in conditions of normoxia and hypoxia (0.5% oxygen) or treated with CoCl2 (100 μM) to mimic hypoxia for 18 h and the supernatant was collected. Under conditions of normal oxygen tension, biosynthesis of TNF-α was markedly increased from baseline (1237.33 ± 132.61 pg/ml, n = 3) to N10 fold (18777 ± 1481.68 pg/ml, n = 3) after exposure to LPS (1 ng/ml, Fig. 1B). Hypoxia and CoCl2 treatment in the absence of LPS increased the level of TNF-α to nearly two fold (p b 0.05, n = 3) above the baseline. In contrast, the TNF-α production was only slightly increased (statistically not significant) with LPS stimulation under conditions of hypoxia (Fig. 1B). Exposure to SB203580 also reduced hypoxia induced TNF-α to baseline level. However, the inhibitory effects of SB203580 on TNF-α expression by LPS stimulation were not observed under conditions of hypoxia or treatment with CoCl2.
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The effects of SB203580 on TNF-α mRNA expression were also investigated. SB203580 treatment effectively inhibits TNF-α transcription under conditions of normal oxygen tension normoxia
(p b 0.05, n = 3). Hypoxia and CoCl2 treatment augment LPSinduced TNF-α mRNA expression (Fig. 1C) which was resistant to the inhibitory effects of SB203580.
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Kinetics of TNF-α biosynthesis under conditions of hypoxia and treatment with SB203580 RAW264.7 cells were exposed to LPS in the presence and absence of SB203580 under conditions of normoxia or hypoxia (CoCl2) and TNF-α level was measured in supernatants collected at 1, 2, 4, 6 and 8 h. Under conditions of normoxia, TNF-α was detectable at 4 h and had peaked at 6 h after LPS stimulation (Fig. 2). Hypoxia accelerated TNF-α secretion by 2 h and the peak level was observed at 4 h after LPS stimulation. But in the presence of SB203580 this effect was abrogated and the kinetics of TNF-α synthesis paralleled those observed under conditions of normoxia. As reported above, SB203580 completely blocked TNF-α expression under normoxic conditions.
The effect of hypoxia on p38 MAPK activity and the down-stream target gene MAPKAPK2 Activation of p38 MAPK is a key event in TNF-α biosynthesis. Therefore we investigated the effects of hypoxia on its activity. RAW264.7 cells were pretreated with SB203580, exposed to normoxic or hypoxic (CoCl2) conditions and harvested at 10, 30 and 60 min after LPS activation. Under conditions of normal oxygen tension, the active and phosphorylated form of p38 MAPK (p-p38) was detected 10 min after LPS stimulation peaked at 30 min and had declined by 60 min. A similar pattern of p-p38 expression was observed following CoCl2 treatment and exposure to SB203580 induced no additional changes in p-p38 expression (Fig. 3A). For the inactive form of p38 MAPK neither hypoxia nor SB203580 altered its expression level. MAPKAPK2 (MK2) is a downstream gene of p38 MAPK signaling. We observed that phosphorylated MK2 was first detectable at 10 min and reached to the maximum level at 30 min after addition of LPS and declined at 60 min in the end under normoxia condition (Fig. 3B). Hypoxia increased expression of pMK2 markedly compared to that under condition of normal oxygen tension at 30 and 60 min (upper panel in Figs. 3B and C) and the pattern of expression was only minimally altered by exposure to SB203580 (Figs. 3B and C).
TNF-α mRNA stability under conditions of normoxia and hypoxia The post-transcriptional regulation is an important step in the control of TNF-α biosynthesis. RAW 264.7 cells were exposed to SB203580 and LPS as the above. After 2 h following the addition of the latter they were incubated with actinomycin D (2 μg/ml), a transcriptional inhibitor [28,29]. Total RNA was obtained at 10, 30 and 60 min after actinomycin D treatment. From the comparison of the levels of specific transcripts for TNF-α after
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quantitative PCR, we observed that mRNA stability was enhanced under conditions of hypoxia about 20% at 30 min and 10% at 60 min compare to that in normoxia condition (Fig. 4). The effect of SB203580 was minimized at 60 min both in normoxia and hypoxia condition compare to that in normoxia mRNA level although at 30 min, less than 10% difference can be observed in these three groups.
The in vivo effects of SB203580 on the serum level of TNF-α in LPS induced endotoxemia in mice The in vivo administration of SB203580 to mice with LPS induced endotoxemia had no effect on serum level of TNF-α (Fig. 5A) compare to vehicle control (p N 0.05, n = 3). The expression of HIF1α mRNA was detected by RT-PCR and the expression level was compared by real-time PCR at 90 min after LPS injection. LPS induced the expression of HIF-1α with no significant difference in SB203580 treated mice or vehicle control (p = 0.34, Fig. 5). The expression of HIF-1α protein was detected in mouse lung both at 90 min and reduced at 240 min after LPS injection. No effect of SB203580 can be found on HIF-1α expression compared with that in vehicle control mice (Fig. 5C). The immunohistochemical staining further confirmed the observation that at 90 min after LPS treatment, the protein had been detected in lung interstitial tissue, most likely presented in mononuclear cells (Fig. 5D). However, the number of immuno-positive cells reduced at 240 min post-LPS treatment, and similarly to that in western blot, no obvious effect of SB203580 on HIF-1α expression was observed.
Discussion TNF-α is a key pro-inflammatory cytokine, which if secreted during hypoxia, a condition of low tissue oxygen supply, often results in disease pathology and worsens the clinical outcome. This is well illustrated in sepsis where high levels of TNF-α under conditions of hypoxia cause lung damage which progresses towards irreversible acute respiratory distress syndrome (ARDS) and high mortality [4,5,22]. However, the effect of hypoxia on the biosynthesis of TNF-α has not been fully investigated. Here we report that increased TNF-α production under hypoxia is mediated through enhanced expression of p38 MAPK and HIF-1α. HIF-1α predominantly contributed to the up-regulation of TNF-α, especially in vivo and is independent of p38 MAPK signaling. These results imply that reducing p38 activity together with improving oxygen supply may have beneficial effects in the management of sepsis. SB203580 is a specific p38 MAPK inhibitor [30,31] that has been widely used to investigate p38 activity in the regulation of cytokine production [12,15,16,32]. SB203580 binds in the ATP
Fig. 5 – Serum TNF-α levels and lung expression of HIF-1α in an in vivo mouse model of LPS induced endotoxemia. Balb/c mice were injected SB203580 i.v. and LPS i.p. as described in Materials and methods. Blood and lung samples were collected 90 min and 240 min after LPS injection. n = 3. (A) Effect of SB203580 on serum TNF-α levels. (B) HIF-1α gene expression in lungs at 90 min after LPS injection. Vehicle control mice and SB203580 injected mice all expressed HIF-1α. Real-time PCR comparison of HIF-1α expression between vehicle control mice and SB203580 pretreated mice. (C) HIF-1α protein expression in mouse lung at 90 min and 240 min after LPS injection. The top of two bands illustrated showed HIF-1α protein; the bottom was a nonspecific band of slightly smaller size. * a nonspecific protein detected as a loading control. (D) Immunohistochemical staining of HIF-1α in mouse lung. Present is normal control, LPS treatment at 90 min and 240 min and the effect of SB203580.
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pocket of both the inactive and active forms of p38 MAPK to prevent the phosphorylation and function of the down-stream substrate MK2 [15,16,33,34]. We tried different dosage between SB203580 and LPS in vitro under normoxia and then focused on the optimized dosage. We observed that SB203580 can inhibit LPS induced TNF-α biosynthesis in macrophages RAW264.7. Similar phenomenon was also observed in the experiments performed in mouse peritoneal macrophage (data not shown) and theses results were similar to the previous observation [35]. But the inhibition effect was failed to do so under hypoxic conditions. The mechanisms underlying this effect may involve altered p38 activity or the expression of other factors that regulate TNF-α expression independent of p38. Interactions between hypoxia and p38 MAPK activity have been investigated in different cell types. For cardiac myocytes it was reported that hypoxia up-regulated the expression of p65pak [36] and in Kupffer cells modulates Src family kinases [37]. Furthermore, through its effects on NF-κB, hypoxia can increase p38 MAPK activity [38,39]. However, we failed to detect any effects of hypoxia on inactive or active p38 in macrophages, which suggests for this cell type that there is no intermediate pathway controlling p38 activity. As the result of up-regulating p-MK2, hypoxia can directly promote p-p38 phosphorylation activity possibly by modifying the binding of p-p38 with MK2 or altering conditions to facilitate phosphorylation. We also noted that inhibition of p38 MAPK was not dose-dependent under normoxic conditions, which suggests a minimum level of p-p38 is required to initiate the phosphorylation cascade in signal transduction. p38 MAPK regulates both the transcriptional and posttranscriptional levels of TNF-α via MK2 [33,34] which we demonstrated was up-regulated in macrophages under hypoxic conditions. Analysis of the kinetics revealed that the increased expression of pMK2 occurred between 30 and 60 min and the enhanced levels were resistant to the effects of SB203580. TNF-α mRNA expression levels and stability paralleled changes in MK2 expression under both normoxic and hypoxic conditions. The increase in MK2 induced by hypoxia was consistent with that SB203580 can reduce more than 50% p-MK2 from time point 30 min to 60 min under normoxia condition and no such phenomenon was observed under hypoxia condition (Figs. 3B and C). Treatment with SB203580 delayed the peak time of TNF-α biosynthesis during hypoxia but failed to restore to the base level as during normoxia conditions. These results suggest that SB203580 can modulate TNF-α expression but were unable to totally suppress p-p38 activity under hypoxic conditions. These findings indicate that hypoxia was dominant over p38 MAPK in the regulation of TNF-α biosynthesis by macrophages. During sepsis, failure in lung gas exchange and circulatory disturbances caused tissue hypoxia. Micro-environmental conditions found in areas of inflammation are characterized by low levels of O2 [40]. This put forward the question whether LPS induced endotoximia as well as sepsis has co-existed hypoxia in vivo. Thus we extended our analysis of the regulation of TNF-α to a mouse model of endotoxemia induced with LPS. Sepsis animal model has the features of pulmonary hypertension, decreased lung mechanics and arterial hypoxemia and thereby parallels human disease. The PaO2 significantly decreased 1 h after completion of endotoxin infusion and remained below
thereafter in the model [41]. Hypoxia inducible factor-1 (HIF-1) is a principal mediator of homeostasis in cells and tissues exposed to hypoxia. The HIF-1 is a hetero-dimer that consists of two helix–loop–helix proteins, HIF-1α and HIF-1β HIF-1α can generally only be detected under low oxygen concentrations [42,43]. Recent studies reported that nonhypoxic stimuli, including LPS can also induce HIF-1 complex [44,45] via a mechanism distinct from hypoxic induction [44]. The study by Peyssonnaux et al. using mice with macrophage targeted deletion of HIF-1α demonstrated that TNF-α was greatly reduced during septic shock confirming the importance of HIF-1α in TNF-α expression [45]. They proposed that the regulation was mediated through the direct binding of HIF-1α to hypoxic response elements in the TNF-α regulatory region. Here we report that HIF-1α was detected within 90 min of the induction of endotoxemia and treatment with SB203580 had no effect on its expression. Similar results were obtained in septic shock mice model induced by higher dosage of LPS (Data not shown). Taken together, our finding suggests that HIF-1α possibly functions independently of p38 MAPK signaling in the regulation of TNF-α production in vivo. Further investigation on the effect of SB203580 in HIF-1α knockout animal will help to clarify the synergistic effects of these two pathways. In summary, our study provides evidence that hypoxia can increase p38 MAPK activity, through the modulation of MK2 to increase TNF-α expression both at the transcriptional and post-transcriptional levels. In a mouse model of LPS induced endotoxemia it appears that the activation of p38 MAPK signaling and the induction of hypoxia occur. These outcomes may partly explain the varied results of p38 inhibitors in animal models of systemic sepsis. Our results suggest that hyoxia and TNF-α biosynthesis should both be considered in the development of valid therapeutic strategies for managing septic shock.
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