Infection, especially gram-negative bacterial sepsis, is a key etiological factor for acute respiratory distress syndrome (ARDS) and sepsis.1 Lipopolysaccharide (LPS), an important component of the outer wall of all gramnegative bacteria, is a highly proinflammatory molecule. It has been shown that Toll-like receptor-4 (TLR-4) recognizes LPS and mediates the LPS signal transduction together with CD14 and MD-2.2-4 This complex triggers downstream intracellular signaling pathways, most notably via NF-κB. The process of NF-κB activation includes the degradation of its inhibitory proteins (e.g. IκB-α) and NF-κB subunits (e.g. p65) are released and translocated into the nucleus. Activated NF-κB promotes the expression of many genes associated with proinflammatory mediators and subsequently promotes the produce and release of these cytokines.1,5,6
In the receptor complex of TLR-4-CD14-MD-2, only TLR-4 is a transmembrane receptor of LPS. So it is TLR-4 that initiates the signal transduction cascade of LPS inflammatory signal into the cell.1,2 And NF-κB is the key intracellular signaling molecule that mediates an array of cell inflammatory responses.1
The lung represents a site for the invasion of various bacteria or bacterial products. And alveolar epithelial cells (AECs) are the first cells to be challenged by pathogenic microorganisms.7 AECs are not only the target of inflammatory cells and mediators, but are active inflammatory and effector cells as well.8 These cells can be activated by LPS and secrete excessive inflammatory cytokines such as IL-8, IL-6, TNF-α, ICAM-1 and so on.8,9 AECs participate in the onset and progress of ARDS, a life threatening condition with mortality rates of about 40%.10,11
Perfluorocarbons (PFCs) are a kind of liquid breathing media that can be administered as liquid,12 vapor13 or gas14 into the lung, and represent an alternative approach to treat respiratory insufficiency. PFC is a promising method in treating ARDS. In addition to its high ability at carrying and dissolving oxygen, PFC has shown cytoprotective action and anti-inflammatory properties both in vitro and in vivo.15-18 However, the previous studies failed to address the molecular mechanisms regarding the possible effects of PFC on decreasing sustained inflammation in ARDS.
Therefore we examined in an in vitro model the cytoprotection of PFC on LPS-stimulated AECs. First, the effects of PFC on the proinflammatory proteins produced by LPS-stimulated AECs that are thought to be mediated by the NF-κB pathway, i.e. ICAM-1, TNF-α, and IL-8, were assessed. To elucidate the mechanisms of PFC activity, the initial signaling molecule TLR-4 and the key intracellular step NF-κB were studied in detail.
Perfluorooctanen [CF3(CF2)6CF3] (a kind of PFC) was purchased from Shanghai Huajieshi Medical Treatment Facility Company Ltd., China. Perfluorooctanen is a clear, colorless, and odorless liquid PFC and has a molecular weight of 438.06. At room temperature, its vapor pressure is 60.9 mmHg, surface tension 13.9 dynes/cm, boiling point 100°C-105°C, density 1.74 kg/L and viscosity 0.85 cst. LPS (Escherichia coli serotype 055:B5) was obtained from Sigma Aldrich. ELISA kit for detecting ICAM-1 was purchased from RapidBio. Lab, California, USA. Radioimmunological kits for detecting TNF-α and IL-8 were purchased from Puer Biotechnology, Inc., Beijing, China. TRIZOL reagent, First-Strand cDNA Synthesis Kit and SYBR Green qPCR SuperMix for real time PCR were purchased from Invitrogen. And goat polyclonal IgG anti-TLR-4 (L-14) sc-16240, rabbit polyclonal IgG anti-NF-κB p65 (C-20) sc-372 and mouse monoclonal anti-β-actin (AC-15) sc-69879 antibodies were all from Santa Cruz Biotechnology, Inc. Rabbit IgG anti-IκB-α (Ab-42, #21176) antibody was purchased from Signalway Antibody Co., Ltd, USA. All other standard reagents and chemicals were of analytical grade and obtained from different suppliers.
A549 (Human lung adenocarcinoma cell line) cells are a widely used tool for lung cell biological studies, providing a large database with which new findings can be compared.19 So they were used in our study. A549 cells were from ATCC (American Type Culture Collection, Manassas, VA, USA). The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2 atmosphere. A549 cells were plated at 80%-90% confluence in 25 cm2 tissue culture flasks. Before further manipulation, cells were incubated for 24 hours with DMEM not containing FBS to allow them to reach full confluence and a resting state.
A549 cells were divided into four groups. (1) Control group: cells did not receive any intervention. (2) PFC group: PFC was added to the cell culture medium to a final volume concentration (vol/vol) of 30%. After vortexing, the mixed liquor containing PFC and culture media in a tube was transferred into the culture flask containing cells. As PFC is not miscible with the medium, cells exposed to PFC were constantly shaking. (3) LPS group: cells were incubated with LPS at a final concentration of 10 μg/ml. (4) LPS + PFC group (coculture group): cells were incubated with both LPS and PFC according to the above-mentioned concentrations.
Assessment of TNF-α, IL-8 and ICAM-1 secreted by AECs
Cell culture supernatants were collected and the concentrations of ICAM-1, TNF-α and IL-8 were analyzed after sampling at 2, 6 and 12 hours by ELISA or radioimmunological methods following the manufacturer's instructions.
Relative quantitative analyses of TLR-4 mRNA by real time polymerase chain reaction (PCR)
The cell samples were harvested at 2, 6 and 12 hours for real time PCR for the expression of TLR-4 mRNA. Total cellular RNA of each sample was extracted from about 5×106 cells using TRIZOL reagent according to the manufacturer's instructions. It was then reversely transcribed directly into cDNA with random hexamers and stored at -80°C until analysis. We conducted real time PCR with the SYBR Green qPCR SuperMix kit using forward and reverse primers specific for β-actin (NM_001101, fwr 5′-CAT GTA CGT TGC TAT CCA GGC-3′, position 466, rev 5′-CTC CTT AAT GTC ACG CAC GAT-3′, position 715, 250 bp product) and TLR-4 (HSU 88880, fwr 5′-TGG AAG TTG AAC GAA TGG AAT GTG-3′, position 1969, rev 5′-ACC AGA ACT GCT ACA ACA GAT ACT-3′, position 2116, 148 bp product). The amplification conditions were 50°C 2 minutes hold (UDG incubation), 95°C 10 minutes hold (UDG inactivation and DNA polymerase activation), 40 cycles of 95°C 15 seconds (denature), 55°C 20 seconds (anneal) and 72°C 20 seconds (extension).
After amplification, we determined the threshold cycle (Ct) to obtain expression values of 2-δδct, as previously described.20 The reaction was performed in a Roche lightCycler Sequence Detection System using software version 3.5.
Levels of TLR-4, IκB-α and NF-κB p65 proteins detected by Western blotting
The cell samples were harvested at 0.5, 2, 6 and 12 hours for detection of proteins of TLR-4, IκB-α and NF-κB p65 by Western blotting. After intervention, A549 cells of each sample were collected for protein extraction. Nuclear, cytoplasmatic and membrane proteins were extracted from cells using the Nuclear-Cytosol-Mem Extraction kit (Applygen Technologies Inc, China) following the manufacturer's instructions. The protein content of each sample was determined by Bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard (Applygen Technologies Inc., China). Protein fractions were stored at -80°C. TLR-4 protein was assayed in membrane and cytoplasmatic extracts, IκB-α protein in cytoplasmatic extracts and NF-κB p65 protein in nuclear extracts of A549 cells by Western blotting. Proteins (60 μg for TLR-4, 40 μg for IκB-α, and 20 μg for p65) from each sample were mixed with 2 × sodium dodecyl sulfate (SDS) sample buffer, heated at 95°C for 5 minutes, and separated by 10% (for TLR-4, p65) and 12.5% (for IκB-α) SDS-polyacrylamide gels. The separated proteins were blotted onto PVDF membrane. Non-specific binding sites were blocked with 10% nonfat dry milk in Tris-buffer saline (TBS)-0.05% Tween. Goat IgG anti-TLR4, rabbit IgG anti-IκB-α, rabbit IgG anti-p65 and mouse anti-β-actin antibodies were used for primary detection. Peroxidase-conjugated anti-goat IgG, anti-rabbit IgG and anti-mouse IgG antibodies were employed for secondary detection. The specific protein bands were visualized on film by enhanced chemiluminescence (ECL) (Applygen Technologies Inc., China) following the manufacturer's instructions. The bands were quantified by scanning densitometry using a GS-710 Imaging Densitometer (Bio-Rad, Hercules, CA).
Statistical analysis was performed with the SPSS statistical software package for Windows, version 11.5 (SPSS. Inc., USA). All data were processed for one-sample Kolmogorov-Smirnov test and homogeneity of variance test. Data in assay were expressed as mean ± SD. Statistical comparisons were performed by one-way analysis of variance (ANOVA). P <0.05 was considered statistically significant.
PFC reduced LPS-induced TNF-α, IL-8 and ICAM-1 secretion from AECs
From Figure 1, it can be seen that in LPS group, ICAM-1 and TNF-α were significantly increased and peaked after 2 hours before gradually declining at 6 and 12 hours; IL-8 was increased after 2 hours, which continued up to 12 hours. There was no obvious impact of PFC alone on the above mediators. In the coculture group, however, all the above mediators were significantly decreased.
Expression of TLR-4 mRNA and protein after LPS and PFC treatment
After stimulation by LPS, the expression of TLR-4 mRNA and protein was significantly increased. The former increased at 2, 6 and 12 hours, and the latter increased at 0.5, 2, 6 and 12 hours. There were no significant effects of PFC alone on the expression of TLR-4 mRNA or protein. In the coculture group, however, the mRNA and protein levels of TLR-4 were all markedly decreased (Table, Figures 2 and 3).
LPS+PFC coculture inhibited IκB-α degradation and NF-κB p65 translocation
After stimulation by LPS, as one of the pivotal proteins inhibiting the translocation of NF-κB into the nucleus, IκB-α was significantly degraded at 0.5, 2, 6 and 12 hours, resulting in the release of NF-κB P65 and its translocation into the nucleus. The level of P65 protein in nucleus was significantly increased at 2 and 6 hours. The changes of the two proteins were approximately synchronized. There was no obvious effect of PFC alone on the activity of NF-κB. But in the coculture groups, the activity of NF-κB was obviously decreased, which was indicated by the decreased degradation of IκB-α and the decrease of NF-κB P65 translocation into the nucleus (Figures 4 and 5).
LPS is a highly proinflammatory molecule and can activate AECs, stimulating their production of proinflammatory mediators.8,9 There is mounting evidence that TLR-4 is the most important receptor of LPS21 and is expressed on alveolar epithelial cells.7,22-24 TLR-4 is localized in the cell membrane and cytoplasm.
The present study indicates that LPS induces a significant increase of ICAM-1, TNF-α and IL-8 release from AECs in vitro. While investigating the mechanisms of LPS-induced inflammatory responses, we found that the expression of TLR-4 mRNA and protein was significantly increased after LPS stimulation. Meanwhile, NF-κB was activated, which was indicated by the rapid degradation of IκB-α and the release of NF-κB P65 and its translocation into the nucleus. These results suggest that LPS induces inflammatory responses in AECs via activation of NF-κB via TLR-4 signaling.
PFCs are currently under investigation as pure substances used for liquid ventilation and vaporized inhalation.12 The anti-inflammatory potential and cytoprotective action of these substances has been hypothesized.25,26 However, the exact mechanism underlying the cytoprotective effect of PFC is not entirely clear.
To elucidate an assumed interaction of PFC with AECs, we examined the in vitro effects of PFC on LPS-stimulated AECs, with the pathway underlying inflammatory responses studied in detail. Our study demonstrated that PFC alone did not significantly affect the release of cytokines from AECs and had no obvious effects on the expression of TLR-4 or the activatity of NF-κB. When AECs were cultured with PFC and LPS, however, the release of ICAM-1, TNF-α and IL-8 was significantly decreased, which clearly demonstrated PFC's cytoprotective action. Furthermore, the LPS-induced up-regulation of TLR-4 expression was also significantly reduced. Meanwhile, the increased activatity of NF-κB was significantly decreased, as indicated by the marked decrease in the degradation of IκB-α and the translocation of NF-κB P65 into the nucleus. The findings observed here demonstrate that PFC is able to protect AECs from LPS-induced inflammatory injury via blocking the initiation of the LPS signaling pathway.
The protective effects of PFC observed in our study suggest that two potential mechanisms could help explain our findings. One possibility is that a dense, immiscible PFC liquid with a high spreading coefficient could form a physical protective barrier, thus shielding target cells (e.g. AECs) from various proinflammatory mediators as suggested by the cell culture experiments described here. Evidence for this barrier effect is supported by other studies.27,28
Another mechanism implicated in the biological effects of PFC is related to the fact that, PFC is able to partition into the lipid component of cellular membranes19,29 and the cytoplasm,29 where PFC can execute its non-specific cell membrane stabilization by interfering with the interaction between stimulant and receptor, the transmembrane signal transduction and the transduction of intracellular signaling pathways.29 Therefore, it can be assumed that PFC directly alters the cellular surface membrane, thereby reducing the intracellular signalling cascade and the subsequent inflammatory responses. In summary, PFC not only provides a mechanical or physical barrier, but alters the signaling pathway programs of host cells and thus influences intracellular processes. The results observed in our study can be explained as the synergistic effects of the above two mechanisms.
Summarizing the experimental findings observed in the present study, we performed a more detailed and thorough investigation into the probable signaling pathway that PFC may affect. The results provide further insight in the complex process of anti-inflammatory activity of PFC. With respect to therapeutic strategies, these direct cytoprotective properties provide further evidence for a promising and safe therapeutic approach of PFC in acute lung injuries and ARDS.
However, the present study has two limitations that require further investigation. First, we need to further study the effects of PFC on the inflammatory responses of LPS-activated AECs when PFC is added into the cell media a period of time prior to or after LPS stimulation. Second, further studies are needed to verify the demonstrated in vitro effects in vivo.
The authors thank LU Ying-lin from the Chinese Military Medical Science Institute, CAO Lu and CAI Fen from the Respiratory Laboratory of Chinese People's Liberation Army General Hospital for their technical assistance.
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Keywords:© 2011 Chinese Medical Association
perfluorocarbon; lipopolysaccharide; Toll-like receptor-4; nuclear factor-κB; alveolar epithelial cells