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1Department for Neonatology and Pediatric Intensive Care Medicine, Klinik für Kinderheilkunde, Universitätsklinikum Carl Gustav Carus, Medizinische Fakultät der Technischen Universität Dresden, Dresden; 2Clinic for Neonatology, Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, and 3Clinic for Neonatology, Charité Universitätsmedizin Berlin, Campus Mitte, Berlin, Germany
Submitted 30 March 2007 ; accepted in final form 4 March 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. The anti-inflammatory effect is not entirely understood. If anti-inflammatory properties are caused by a mechanical barrier, PFC in the alveoli should have no effect on the inflammatory response to intravenous LPS administration. To test that hypothesis, rats (n = 31) were administered LPS intravenously and were either spontaneously breathing (Spont), conventionally ventilated (CMV), or receiving partial liquid ventilation (PLV). Serum concentration of TNF- was measured. The pulmonary expressions of TNF- and TNF- receptor 1 protein and of TNF- and ICAM-1 mRNA were determined. LPS caused a significant (P < 0.001) increase in serum TNF-. Serum TNF- concentration was similar in LPS/Spont (525 ± 180 pg/ml) and LPS/CMV (504 ± 154 pg/ml) but was significantly (P < 0.001) lower in animals of the LPS/PLV group (274 ± 101 pg/ml). Immunohistochemical data on TNF- protein expression showed a LPS-induced increase in TNF- and TNF- receptor 1 expression that was diminished by partial liquid ventilation. PCR measurements revealed a lower expression of TNF- and ICAM-1 mRNA in LPS/PLV than in LPS/CMV or LPS/Spont animals. Semiquantitative histological evaluation revealed only minor alveolar inflammation with no significant differences between the groups. Low serum TNF- concentration in PFC-treated animals is most likely explained by a decreased production of TNF- in the lung.
fluorocarbons; tumor necrosis factor-; lipopolysaccharide; liquid ventilation; anti-inflammatory agents
and ICAM-1. TNF- is produced earlier than most other cytokines. Although major sources for TNF- are thought to be polymorphonuclear neutrophils and alveolar macrophages, recent data show that TNF- is also produced and secreted by alveolar type II pneumocytes (9, 16, 32).
Intratracheal as well as intravenous LPS administration cause a significant release of TNF-; however, the response remains largely compartmentalized (8). Because neither intravascular nor alveolar deposition of LPS resulted in any significant spillover of the endotoxin into the adjacent compartment, both the vascular and alveolar compartments seem to possess a significant capacity for the generation of TNF- (8).
Perfluorocarbons (PFC) are used for liquid ventilation (26, 33). Besides the effects on oxygenation, PFC also reduce the production of various inflammatory cytokines, including TNF- (1, 14, 18, 27, 29). The anti-inflammatory effect, however, is not entirely understood, and several explanations, such as PFC-mediated improvement in alveolar recruitment (4), have been discussed (13, 34). But PFC could also act as a physical barrier around the cells and prevent ligand-receptor interaction.
If these anti-inflammatory properties are caused by a barrier effect, intra-alveolar PFC will not affect the inflammatory response to intravenous LPS administration. If, however, the effect is mediated by an improved alveolar recruitment, inflammation should be suppressed only if it is associated with severe respiratory insufficiency. To test that hypothesis, a mild systemic inflammation was induced in rats by an intravenous administration of LPS that did not cause respiratory insufficiency but induced an increase of TNF- in alveolar type II pneumocytes (16). Rats were either spontaneously breathing, conventionally ventilated, or received partial liquid ventilation. After only 6 h, serum concentrations of TNF- and the anti-inflammatory cytokine IL-10 were measured. Furthermore, pulmonary expressions of TNF- and TNF- receptor 1 (TNFR1) protein as well as TNF- and ICAM-1 mRNA were determined.
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MATERIALS AND METHODS |
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In total, 36 male Wistar rats (age 2 mo) were studied. As healthy controls, five animals did not receive any treatment or injury. Thirty-one animals injured with LPS intravenously were randomized into three groups: LPS/Spont (spontaneously breathing; n = 11), LPS/CMV (conventional mechanical ventilation; n = 10), and LPS/PLV (partial liquid ventilation; n = 10).
Initially, all rats were anaesthetized with ketamine (10 mg/kg) and pentobarbital (20 mg/kg) intraperitoneally. A catheter was placed intravenously, and a glucose-electrolyte mixture (50 ml) containing midazolam (5 mg) was given at 2 ml/h. In ventilated animals (LPS/PLV and LPS/CMV), the glucose-electrolyte mixture also contained fentanyl (50 µg) and pancuronium (1 mg). Furthermore, a line was placed in the tail artery to obtain arterial blood gas measurements. A tube with a side port for PFC application was inserted via tracheostomy. Animals were placed on a pressure-controlled ventilator (BP 2001; Bear Medical Systems, Palm Springs, CA) with the following settings: positive inspiratory pressure (PIP) 10, positive end-expiratory pressure (PEEP) 3 cmH2O, FIO2 0.5, inspiratory time 0.4 s, and frequency 60/min. After each blood gas, PIP was adjusted to maintain PaCO2 within a target range of 30–50 Torr. ECG was measured continuously with a Servo SMV 178 monitor (Hellige). For constant body temperature, animals were placed in an incubator and had a rectal thermosensor.
Spontaneously breathing animals (control, LPS/Spont) were placed in an incubator with additional oxygen supply to achieve a FIO2 of 0.5. Because control animals were spontaneously moving, neither ECG, temperature, nor blood-gas monitoring was possible.
Experimental protocol. LPS-treated animals (Spont, CMV, PLV) received Escherichia coli LPS (Sigma-Aldrich, Taufkirchen, Germany) intravenously at a dosage of 1 mg/kg body wt dissolved in 5 ml sterile saline and administered over a period of 30 min. According to a pilot study, that dosage induces only mild systemic inflammation without any severe pulmonary involvement or respiratory insufficiency.
In the PLV group, PF-5080 (3M Germany, Neuss, Germany) was administered intratracheally via the side port at a rate of 30 ml/h until a liquid meniscus was visible in the tube at end expiration. Thereafter, PFC was given at 
9 ml/h to compensate for evaporative losses and to achieve a continuous PFC filling of the lung (20). To measure the tidal volume in ventilated animals, a flow sensor (CO2SMO; Novametrix) was placed between the T piece of the ventilator and the endotracheal tube before injury and every hour thereafter.
Samples (150 µl) of arterialized blood were drawn to determine blood gases (ABL 505; Radiometer) at baseline and at 1, 4, and 6 h.
Animals were killed by an overdose of potassium chloride and pentobarbital at the end of the experiment at 6 h. The peritoneal cavity was opened, and blood was drawn from the inferior vena cava. Blood samples were stored for 2 h at 4°C and were centrifuged at 2,000 g thereafter. Serum aliquots were stored at –70°C for further analysis.
Rat lungs were prepared in situ while a constant PEEP of 5 cmH2O was maintained. A sternotomy was made to expose lungs and heart. The left atrium was incised to allow sufficient drainage of the perfusate. Phosphate-buffered saline was injected into the right ventricle to flush the pulmonary circulation free of blood. Thereafter, the left lung was removed, visible airways were removed, and the lung was shock frozen in liquid nitrogen and stored at –70°C until further analysis. For perfusion fixation of the right lung, 4% paraformaldehyde was instilled via the right ventricle with a constant pressure of 20 cmH2O for 20 min. The lung was removed and submersed in fixative for 24 h (9).
Histology and immunohistochemistry. Histological preparations were performed according to our standardized protocol as previously described (24, 31). In short, lung tissues were embedded in paraffin and were transversely cut in sections of 4-µm thickness. From each animal, five sections that were an equal distance apart and thus representative for the entire lung were examined in a blinded manner by light microscopy. From each section 10 areas were chosen in a randomized manner, and alveolar expansion pattern and recruitment of inflammatory cells into the airspaces were evaluated as previously described (24) after staining with hematoxylin and eosin. Alveolar inflammation was graded semiquantitatively as absent, mild, or severe. Alveolar expansion was estimated according to a four-grade scale representing expansion of either 0–25%, 26–50%, 51–75%, or 75–100% of the total alveolar spaces.
Immunocytochemistry was carried out as previously described (9). In short, samples were stained with rabbit polyclonal anti-TNFR1 antibody (obtained from Santa Cruz Biotechnology; Heidelberg, Germany) or goat polyclonal anti-rat TNF- antibody (BioSource Europe, Nivelles, Belgium). As secondary antibodies for immunocytochemistry, anti-goat IgG and anti-rabbit IgG conjugated with Alexa 499 and Alexa 594 were obtained from Molecular Probes Europe (Leiden, Netherlands). As control, the sections were incubated with the Alexa-labeled secondary antibodies without prior incubation with the specific first antibody. No nonspecific binding of the second antibodies occurred (results not shown). The staining was evaluated in a similar semiquantitative approach to that described above. From each animal, five sections of different parts of the lung were evaluated. From each section, 10 randomly chosen fields were graded according to the amount of stained alveolar cells: <25%, 26–50%, 51–75%, or 76–100%.
For the final analysis and comparison of groups, the relative percentage of each grading was calculated for all animals. Data are presented in Fig. 2 as means and standard deviation of relative percentage for each grading.
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mRNA expression in rat lungs. Total RNA was extracted from lung homogenates (obtained from a median part of the frozen left lung) by using the RNeasy mini kit (Qiagen, Hilden, Germany) and was transcribed into cDNA by use of avian myeloblastosis virus reverse transcriptase (Invitrogen, Karlsruhe, Germany) and random hexamers (Promega, Mannheim, Germany) according to the manufacturers' recommendations. The cDNA was amplified by PCR in a microprocessor-driven thermal cycler (Fa. Landgraf, Hannover, Germany) as previously described (31) with the use of the following forward (F) and reverse (R) oligonucleotides (BioTeZ Berlin-Buch, Berlin, Germany): GAPDH rat (GenBank BC059110; product size 303 bp), F 5'-CAG TGC CAG CCT CTG CTC AT, R 5'-ATA CTC AGC ACC AGC ATC AT; TNF- rat (GenBank NM_012675; product size 359 bp), F 5'-GGG GCC ACC ACG CTC TTC TGT, R 5'-GCA AAT CGG CTG ACG GTG TGG; and ICAM-1 rat (GenBank D00913; product size 199 bp), F 5'-CCT CTT GCG AAG ACG AGA AC, R 5'-ACT CGC TCT GGG AAC GAA TA. Products were stained with ethidium bromide and were electrophoresed through a 1.5% agarose gel. After the PCR products were transferred to nylon membranes (Amersham, Braunschweig, Germany) by capillary blotting using 20x SSC as blotting buffer, the membranes were fixed by UV light and the incorporated digoxigenin-UTP was visualized by staining with anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche Diagnostics, Mannheim, Germany). Luminescence of the substrate (Lumigen PPD) was documented by short exposure to X-ray film (Kodak, Stuttgart, Germany). Densitometric calculations of digital film images were performed with the analysis program Scion Image, version Beta 4.0.2 (Scion, Frederick, MD).
Cytokine TNF- and IL-10 enzyme-linked immunosorbent assay.
Cytokine release into the serum was assayed by means of commercially available rat-specific ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The samples were measured in duplicate by using an automated microplate reader.
Statistical analysis. Data are shown as means and standard deviations of single values for each group. For the analysis of changes between two groups over time, the general linear model for repeated measures was used. Otherwise, differences between independent groups were analyzed by the nonparametric Mann-Whitney U-test. A P value <0.05 was considered significant. All tests were carried out with the SPSS for Windows statistical package (SPSS, Chicago, IL). Significant differences from baseline values for each group were marked with a #. Significant differences between groups at a given time point are marked with an *.
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RESULTS |
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and IL-10.
Intravenous LPS application caused significantly (P < 0.001) higher serum TNF- values at 6 h compared with control (Fig. 1). Serum TNF- concentration was similar in LPS/Spont (525 ± 180 pg/ml) and LPS/CMV (504 ± 154 pg/ml) but was significantly (P < 0.001) lower in animals of the LPS/PLV group (274 ± 101 pg/ml). In contrast, LPS application had no statistically significant effect on serum IL-10 levels (Fig. 1).
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and IL-10.
After immunohistochemical staining, almost no TNF- or TNFR1 expression in lungs of control animals was found. LPS injection, however, was associated with an increase in TNF- and TNFR1 expression. As Fig. 2, B and C, shows, TNF- and TNFR1 expression were most prominent in LPS/CMV and LPS/Spont animals. Treatment with partial liquid ventilation reduced the expression of both proteins in the lungs. Data on TNF- protein expression was supported at the mRNA level with a lower expression of TNF- mRNA in LPS/PLV than in LPS/CMV or LPS/Spont animals (Fig. 3A). Finally, as Fig. 3B shows, mRNA expression of ICAM-1 was increased after LPS challenge. That increase was diminished in PFC-treated animals.
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Differences between groups were more prominent when looking at alveolar expansion. As shown in Fig. 2A, alveoli of animals in the LPS/Spont group were significantly less well expanded compared with control animals. Almost all alveoli were well expanded in LPS/PLV animals, and alveolar expansion in the LPS/CMV was comparable with the control group.
Ventilatory parameters and blood gases of ventilated animals (LPS/PLV and LPS/CMV). PaO2 did not change during the experiment but was slightly (not significantly) lower in PLV- than in CMV-treated animals (Table 1). PaCO2 was (not significantly) higher in CMV animals at 2 h but decreased during the experiment to reach values similar to the PLV group (Table 1).
To keep the PaCO2 within the preset range, PIP had to be increased immediately after filling the lung with PFC in PLV animals. In contrast, in the CMV group PIP was slowly increased during the experimental course (Fig. 4A), and tidal volume was significantly higher in PLV animals when compared with CMV animals during the first hours of the experiment (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. However, the rise in serum TNF- concentration after intravenous LPS administration was significantly lower in the presence of intra-alveolar PFC. The low serum TNF- concentration is most likely explained by a decreased TNF- production in the lung, because pulmonary expression of TNF- protein and mRNA was reduced in PFC-treated animals.
Direct anti-inflammatory properties of PFC.
Besides the effects on gas exchange, PFC exhibit anti-inflammatory properties that are difficult to explain (13, 28). The present study supports previous reports that question a mechanical barrier effect as a likely explanation (28). Despite of the different localization of PFC (alveolar compartment) and LPS (vascular compartment), the LPS-induced rise in systemic TNF- was significantly lower in PFC-treated animals. Furthermore, pulmonary mRNA and protein expressions of TNF- were lower in PFC-treated animals.
Even if a small spillover of PFC cannot be entirely excluded, it is unlikely to explain the observed significant effect. In liquid-ventilated lambs, a maximum of 0.3 vol% perflubron was found in blood, and in vitro studies showed a dose-dependent PFC effect starting at a minimal concentration between 1 and 5% (7, 18, 23). Even if minor intravascular PFC amounts are unlikely to cause the significant differences in serum TNF-, it cannot entirely be ruled out, because perfluobron was capable of partitioning into cell membranes when the cells and liquid are brought into close proximity without direct contact (34). For LPS, a crossover from the alveolar into the vascular compartment due to ventilatory lung injury has been described (17). However, in the present study, the LPS effect in ventilated and spontaneously breathing animals was similar.
Liquid ventilation reduces ventilator-associated lung injury (21) and could thereby reduce pulmonary inflammation. In the present study, a model of mild systemic inflammation without significant pulmonary involvement was used. Whereas LPS administration did not cause any respiratory deterioration in spontaneously breathing animals, they showed slightly reduced alveolar expansion. Conventional mechanical ventilation did prevent that deterioration in alveolar expansion but did not reduce the inflammatory response to LPS. Therefore, prevention of ventilator-induced injury is an unlikely explanation for the PFC-mediated reduction in inflammation.
The highly lipophilic PFC were found in lipid membranes (19). The subsequent stabilization of cellular membranes could suppress inflammation (3); however, the underlying pathway was not explained. It could either disturb the receptor-mediated intracellular signal transduction or the release of cytokines. In the present study, PFC treatment reduced the pulmonary TNF- protein content but also the TNF- mRNA expression. Thus PFC seem to prevent the production rather than the secretion of TNF-. An effect on the early steps of the cellular signaling cascade is the most likely explanation (27). Data published by Fernandez et al. (7) suggest a nonspecific effect on cellular activation, because tyrosine phosphorylation was reduced in neutrophils after short perflubron incubation. We have recently reported (31) that PF-5080 prevents the production of TNF- by suppressing the activation of NF-
B in isolated alveolar type II cells.
One of the most prominent histological findings in liquid-ventilated lungs is a reduction of neutrophil sequestration (5, 22, 30). Cellular adhesion is mediated by an increased expression of ICAM-1. In vitro, PFC preexposure reduced ICAM-1 expression of activated endothelial cells (34). However, one question remained unanswered: whether preexposure to perflubron exerts its effects by modifying the initial signaling pathway through the cytokine receptors, the extent of ICAM synthesis at the translational level, and/or the trafficking of newly synthesized protein to the plasma membrane (34). TNF- is known to affect the expression of ICAM-1 in human airway epithelial cells (15). According to the presented data, the PFC-induced suppression of ICAM-1 is most likely caused by a reduced TNF- production.
Lung epithelial cells seem to be responsible for a major part of systemic TNF- production.
PFC reduce LPS-induced TNF- production in isolated leukocytes (18), mononuclear blood cells (14), and human alveolar macrophages (27) in vitro. A previous in vitro (31) and the present in vivo study show that PFC also prevent TNF- production of pneumocytes. In the present study, an intratracheal PFC administration diminished the LPS-induced rise in serum TNF- concentration by 50%. Assuming no significant spillover of either PFC or LPS into the other compartment, the lower systemic TNF- concentration is most likely due to a PFC effect on cells at the alveolar-capillary boundary (type I and II pneumocytes). That assumption is supported by the low TNF- protein and mRNA expression found in lungs of liquid-ventilated animals.
Data from the present study support the important role of alveolar pneumocytes in initiating systemic inflammation. Whereas TNF- was considered to be produced mainly by inflammatory cells, evidence is rising that other cells are a source of TNF- as well. In vitro data show that TNF- is localized in the nucleus and cytoplasm of isolated rat alveolar cells (16, 25). LPS treatment caused a rapid induction of TNF- mRNA within 1 h and a release of TNF- from alveolar epithelial cells within 4 h (16). Similar effects were found in isolated type II cells after hyperoxic exposure (9). According to the present data, alveolar pneumocytes could be the origin of up to 50% of systemic TNF-. Thus the role of the lung in systemic inflammation has to be reconsidered, because it could open new therapeutic options.
Clinical implications. Intrapulmonary application of PFC improves respiratory insufficiency significantly. Despite the less-promising results of recent clinical trials (10, 11), PFC-associated therapy still has great clinical potential (6). However, to use PFC appropriately, the molecular mechanism of activity has to be understood.
PFC-mediated suppression of inflammation would be useful to treat pulmonary and systemic inflammations. In acute respiratory distress syndrome patients, mortality is mainly caused by multiple organ failure mediated by inflammation. PFC could reduce the sequel of pulmonary damage and subsequent systemic inflammation (13). Another potential application could be the prevention of chronic lung disease of the preterm infant, because current anti-inflammatory therapies are associated with severe side effects. Data on anti-inflammatory properties of inhaled PFC (2, 12) are especially promising and open a wide field of PFC applications in nonventilated patients.
However, further studies are required to elucidate the molecular mechanism and time scale of PFC action in more detail. It is still not clear whether PFC exhibit their anti-inflammatory properties only during the early phase of inflammation. If, however, PFC cause a general reduction in cytokine production, it could interfere with the healing process if applied late in inflammation. In the present study, concentrations of the anti-inflammatory IL-10 were measured. No effects of partial liquid ventilation were found. However, IL-10 was very low in all groups. Further studies are required to investigate whether anti-inflammatory chemokines are affected by liquid ventilation.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-induced interleukin-8 release from alveolar epithelial cells in vitro. Crit Care Med 28: 1113–1118, 2000.[CrossRef][Web of Science][Medline] concentration in a rat aspiration lung injury model. Crit Care Med 28: 479–483, 2000.[CrossRef][Web of Science][Medline] on expression of ICAM-1 in human airway epithelial cells in vitro. Am J Respir Cell Mol Biol 22: 685–692, 2000. and MIP-2 via NF-B pathway in rat type II pneumocytes. Respir Res 6: 51, 2005.[CrossRef][Medline]
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