Basic Science Aspects
Altered Expression OF Fas Receptor on Alveolar Macrophages and Inflammatory Effects of Soluble Fas Ligand Following Blunt Chest Trauma
Seitz, Daniel H.; Palmer, Annette; Niesler, Ulrike; Braumüller, Sonja T.; Bauknecht, Simon; Gebhard, Florian; Knöferl, Markus W.
Department of Trauma Surgery, Hand, Plastic and Reconstructive Surgery, University of Ulm, Ulm, Germany
Received 3 Dec 2010; first review completed 15 Dec 2010; accepted in final form 7 Jan 2011
Address reprint requests to Markus W. Knöferl, MD, Department of Trauma Surgery, Hand, Plastic, and Reconstructive Surgery, University of Ulm, Steinhövelstr. 9,89075 Ulm, Germany. E-mail: email@example.com.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG SPP 1151: DFG KN 475/3-2, 4-1) and State of Baden Württemberg (Juniorprofessoren-Programm) to M.W.K.
None of the authors have any financial interests or affiliations with commercial organizations whose products or services are related to the subject matter of this manuscript (no existing conflicts of interest).
Part of this study was presented at the 8th World Congress on Trauma, Shock, Inflammation and Sepsis-TSIS 2010, March 9-13, 2010, Munich, Germany.
Blunt chest trauma impairs the outcome of multiply-injured patients. Lung contusion induces inflammatory alterations and Fas-dependent apoptosis of alveolar type 2 epithelial (AT2) cells has been described. The Fas/Fas ligand (FasL) system seems to exhibit a proinflammatory potential. We aimed to elucidate the involvement of the Fas/FasL system in the inflammatory response after lung contusion. Chest trauma was induced in male rats by a pressure wave. Soluble FasL concentrations were determined in bronchoalveolar lavage fluids and alveolar macrophage (AMΦ) supernatants. Alveolar macrophages and AT2 cells were isolated to determine the surface expression (FACS) of Fas/FasL, the mRNA expression (reverse transcriptase-polymerase chain reaction) of Fas, FasL, TNF-α, IL-6, and IL-10 and to measure the release of IL-6 and IL-10 after culture with or without stimulation with FasL. After chest trauma, FasL concentration was increased in bronchoalveolar lavage fluid, and AMΦ supernatants and Fas and FasL protein were downregulated on AMΦs and unchanged on AT2 cells. The mRNA expression of Fas was increased in AMΦs and AT2 cells and that of FasL only in AMΦs isolated after lung contusion. Fas ligand stimulation further enhanced IL-6 and suppressed IL-10 release in AMΦs after trauma.
The results indicate that the Fas/FasL system is activated after chest trauma, and FasL is associated with the inflammatory response after lung contusion. The proinflammatory response of AMΦs is enhanced by FasL stimulation. Both AMΦs and AT2 cells seem to contribute to the mediator release after lung contusion. These results confirm the importance of the Fas/FasL system in the inflammatory response after chest trauma.
Blunt chest trauma is a very common injury (1). It occurs in isolation, but more frequently it is part of the injury pattern seen in combined injury (2, 3). In this regard, lung contusion plays an important role with respect to morbidity and mortality of multiply-injured patients. Mortality from polytrauma rises significantly when lung contusion is part of the injury pattern (4). Acute respiratory distress syndrome (ARDS) and respiratory failure are major causes of death in severely injured patients (1), and chest trauma is an important trigger for ARDS (5). Severe local and systemic inflammatory alterations after experimental lung contusion have been described by our laboratory (6, 7) and may contribute to the previously mentioned complications. In this context, we could show that apoptosis induction in alveolar type 2 epithelial (AT2) cells after blunt chest trauma was mainly mediated by the Fas/Fas ligand (FasL) system (8). Fas/FasL-mediated apoptosis of pulmonary epithelial cells is discussed as an important trigger for the induction of ARDS (9). In addition to the induction of apoptosis, the Fas/FasL system contributes to a septic inflammatory response. Perl et al. (10) found that the inflammatory response after acute lung injury induced by hemorrhagic shock followed by polymicrobial sepsis (cecal ligation and puncture) was markedly depressed by Fas silencing. Furthermore, stimulation of pulmonary epithelial cells with Fas-activating antibodies enhanced the release of inflammatory mediators, such as the chemoattractant IL-8 (11). The intratracheal application of Jo2 as a Fas-activating antibody induced a significant increase in neutrophil counts in the bronchoalveolar lavage of alveolar macrophage (AMΦ)-depleted mice (12). All of these observations postulate that the Fas/FasL system is involved in the pulmonary inflammatory response. Nevertheless, to the best of our knowledge, the contribution of the Fas/FasL system to pulmonary inflammation after a nonseptic insult such as blunt chest trauma remains still unclear. Because AT2 cells are located in the alveolar walls, they may be influenced by mediators present in the alveolar milieu. Therefore, this study was designed to determine if the Fas/FasL system is activated by chest trauma and if Fas ligation further enhances AMΦ or AT2 cell inflammatory response after lung contusion.
MATERIALS AND METHODS
Male CD rats (Charles River, Sulzfeld, Germany) with a body weight of 250 to 275 g were used. Each experiment consisted of at least five animals and up to 15 animals per group and time point. Animals were kept and treated as described previously (8). All methods including animal work as well as specimen collection were reviewed and approved by the institutional review board/ethics committee of the University of Ulm as well as by the Federal Animal Care and Use Committee (permit no. 780; Regierungspräsidium Tübingen, Germany)
Blunt chest trauma
Rats were anesthetized with 4% sevoflurane (Sevorane; Abbott, Wiesbaden, Germany) in oxygen under a continuous flow of 2 L/min, fixed in supine position, and chest and abdomen were shaved. To induce the blunt chest trauma, a single blast wave, released by a pressure wave generator, was centered on the thorax as described previously (6, 13-15). Sham animals underwent the same procedures, but no blast wave was applied.
Collection of blood plasma samples
At 6, 24, and 48 h after sham procedure or chest trauma, animals were killed by exsanguination in deep anesthesia. Blood samples were centrifuged; the supernatants were removed and stored at −80°C until further use.
Collection of bronchoalveolar lavage fluids and preparation of AMΦs
Alveolar macrophages were isolated as described previously (8, 16). In brief, at 4, 6, 16, 24, 48, or 72 h after chest trauma, animals were killed, the trachea was cannulated, and the lungs were flushed once with 5 mL of cold phosphate-buffered saline (PBS) (Dulbecco's PBS; Gibco BRL, Grand Island, NY) to collect bronchoalveolar lavage fluid (BAL) for mediator analysis (BAL 1) and then another eight times with 10 mL PBS to obtain AMΦs (BAL 2). Bronchoalveolar lavage fluid was centrifuged, and the supernatants of BAL 1 were stored at −80°C for enzyme-linked immunosorbent assay (ELISA). The pellets of BAL 1 and 2 were combined, resuspended in Dulbecco's modified Eagle medium (DMEM; Gibco), counted, and transferred to 24-well culture plates (BD, Franklin Lakes, NJ). After incubation (60 min, 37°C, 5% CO2), cultures were washed with DMEM to remove nonadherent cells. Alveolar macrophages designated for RNA isolation were now ready for use. For determination of supernatant mediator levels, cells were cultured for another 24 h in DMEM + 0.5% fetal calf serum (FCS; Life Technologies, Rockville, Md). Supernatants were then removed, centrifuged, and stored at −80°C until further use.
Preparation of AT2 cells
Alveolar type 2 epithelial cells were isolated as described previously (17, 18). In brief, the lungs were digested with elastase (Worthington Biochemical, Freehold, NJ) to obtain a cell suspension. The suspension was filtered successively through polyester meshes (500-, 200-, and 105-μm pore diameter; VWR International, Darmstadt, Germany) and plated on Petri dishes coated with rat-IgG (Sigma, Steinheim, Germany) to remove lung macrophages. Then AT2 cells were carefully collected by rinsing the Petri dishes three times with DMEM. After centrifugation, cells were resuspended in DMEM + 10% FCS and passed through a 100-μm cell strainer (BD Biosciences Discovery Labware, Bedford, Mass). Cells were counted, and viability was found to be higher than 95% by trypan blue exclusion.
Purification of AT2 cells
For further purification of AT2 cells, neutrophils were removed by magnetic bead selection using the EasySep PE selection kit (StemCell Technologies, Vancouver, British Columbia, Canada) according to the manufacturer's recommendations. Briefly, cells were resuspended in selection buffer (PBS w/o Mg2+/Ca2+ + 1 mM EDTA [Sigma] + 2% FCS), and unspecific bindings were blocked with rat-IgG (Caltag Laboratories, Burlingame, Calif). Cells were then incubated with a mouse antigranulocyte antibody (BD Biosciences, Heidelberg, Germany), washed, and again resuspended in selection buffer. The cell suspension was incubated with EasySep PE Selection Cocktail and afterward with EasySep magnetic nanoparticles. For the magnetic separation, the cell suspension was diluted to a total volume of 2.5 mL by adding selection buffer, put in a FACS tube (BD), and placed in the magnet for 10 min. The supernatant, containing AT2 cells, was transferred to another FACS tube and centrifuged, and the pellet resuspended in 500 μL PBS. Cell counts and viability were determined by trypan blue.
Soluble FasL stimulation of AMΦs and AT2 cells in vitro
Alveolar type 2 epithelial cells were isolated from naive animals. To determine apoptosis induction by soluble FasL (sFasL), AT2 cells were plated on eight-well chamber slides (Lab-Tek II; Nalge Nunc Int, Naperville, Ill) and stimulated with 50, 250, or 500 ng/mL sFasL (Kamiya Biomedical Company, Seattle, Wash) in DMEM + 5% FCS (18 h, 37°C, 5% CO2). Afterward, cells were fixed in 4% formaldehyde (J. T. Baker, Deventer, the Netherlands), washed, and stained with annexin V for apoptosis detection (see below).
In addition, AMΦs or AT2 cells were isolated at 24 h after chest trauma or sham procedure and cultured for 24 h in DMEM + 0.5% FCS with or without 500 ng/mL sFasL (37°C, 5% CO2). Culture supernatants were then removed, centrifuged, and stored at −80°C until determination of mediator concentrations.
Measurement of BAL and supernatant cytokine concentrations
The concentrations of IL-6, IL-10, MCP-1 (BD OptEIA ELISA Set; BD Pharmingen, San Diego, Calif), and MIP-2 (CytoSet; Biosource, Camarillo, Calif) in BAL, AMΦ, and AT2 cell culture supernatants were determined by ELISA according to the manufacturer's recommendations. The concentrations of sFasL in AMΦ supernatants were determined by direct ELISA as described previously (8). The absorbance of standards and samples was measured with a microplate reader (Tecan Austria GmbH, Gröding, Austria).
Flowcytometric (FACS) analysis of Fas or FasL protein expression on AMΦs and AT2 cells
Alveolar macrophages and AT2 cells were isolated at 4 and 16 h after chest trauma or sham procedure. Cells were fixed and permeabilized using Leucoperm (AbD Serotec, Raleigh, NC). Isotype rabbit-IgG (Beckman-Coulter, Krefeld, Germany), anti-Fas (Santa Cruz Biotechnology, Santa Cruz, Calif), or anti-FasL (Santa Cruz) was added to both cell populations. Alveolar type 2 epithelial cells were additionally stained with anticytokeratin MNF Clone 116 (Dako, Carpinteria, Calif). Cell suspensions were incubated for 30 min at room temperature. Goat anti-rabbit-PE (Beckman-Coulter) was used as a detection antibody for Fas and FasL. Alveolar type 2 epithelial cells were additionally stained with goat anti-mouse SPRD (Spectral Red; Beckman-Coulter) as a secondary antibody for the MNF 116 stain. Cells were incubated for 25 min on ice and washed, and FACS analysis was performed on a BD FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software (BD Biosciences). The cells were gated according to their SSC versus FL1 characteristics (AMΦ) or their FSC versus SSC characteristics (AT2 cells), and 10,000 events were acquired.
Annexin V and TUNEL stain of AT2 cell cultures
To detect apoptosis in AT2 cell cultures, cells were fixed as described above, and annexin V or TUNEL staining was performed as described before (8). Cells were evaluated by fluorescence microscopy (Axio Imager M1; Zeiss, Oberkochen, Germany). To quantify apoptosis, the total number of cells and the number of annexin V-positive cells in three representative high-power fields were counted by a researcher blinded to the groups. The percentage of apoptotic cells was calculated.
Isolation of RNA
Total RNA from AMΦs and purified AT2 cells was isolated using the Absolutely RNA Miniprep Kit (Stratagene, Cedar Creek, Tex) following the manufacturer's instructions including the RNase-free DNase step to avoid contamination with genomic DNA. RNA samples were purified and concentrated using the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany).
Reverse transcriptase-polymerase chain reaction
The relative quantities of mRNA for each gene of interest were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Reverse transcription was carried out in a 40-μL final volume from 2 μg of total RNA using the AffinityScript QPCR cDNA Synthesis Kit (Stratagene) according to the manufacturer's instructions. For the RT reaction, the samples were incubated for 15 min at 42°C followed by 5 min at 95°C. Samples were then diluted 1:5 with RNase-free water. Quantitative real-time PCR was performed on an Mx3000P QPCR System and software (MxPro; Stratagene) in combination with Brilliant SYBR Green QPCR Master Mix (Stratagene) and specific primers (Table 1). The thermal cycling program consisted of an initial denaturation step (95°C for 10 min) followed by 40 cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 72°C. A melting curve was generated by linear heating from 55°C to 95°C and showed the specificity by a single peak at the expected temperature. A negative control with no template and a No-RT control were included for each gene in each qPCR run. β2-Microglobulin was used as housekeeping gene. This gene was chosen for normalization because it showed the lowest variance of expression between sham and trauma in the particular cell population (data not shown). Results were expressed as the fold increase of trauma over sham animals. The logarithm (log2) of the ΔCT values was used for statistics as mentioned in the following section.
Results are presented as mean ± SEM. A one-way ANOVA followed by the Student-Newman-Keuls test as a post hoc test for multiple comparisons was performed to determine significant differences between experimental means. Whenever more than one variable was tested, a two-way ANOVA was performed before the Student-Newman-Keuls test as mentioned above. P ≤ 0.05 was considered statistically significant.
FasL concentrations in BAL and AMΦ supernatants
Concentrations of sFasL were significantly increased in BAL collected at 6, 24, and 48 h after blunt chest trauma, compared with corresponding shams (Fig. 1A). Furthermore, FasL levels in BAL collected at 24 or 48 h after the insult were significantly higher than those in BAL sampled at 6 h. Alveolar macrophages isolated at 6 h after chest trauma released significantly higher levels of FasL than AMΦs from corresponding sham animals. This effect was lost at 24 and 48 h after the insult (Fig. 1B).
Apoptosis induction in AT2 cells by sFasL
Addition of sFasL (50, 250, 500 ng/mL) to AT2 cell cultures increased AT2 cell apoptosis in a dose-dependent manner when compared with unstimulated cells, as shown by annexin V stain (Fig. 1C). TUNEL stain revealed a tendency to increased counts of apoptotic AT2 cells after addition of sFasL (Fig. 1D).
Counts of isolated AMΦs and AT2 cells
At 24, 48, and 72 h after chest trauma, significantly more AMΦs were isolated than at the same time points in sham (Fig. 2A). At 4 h after chest trauma, the counts of isolated AT2 cells were significantly higher than in sham (Fig. 2B). At the later time points, there was no further difference between trauma and sham AT2 cell counts.
Mediator release of AMΦs or AT2 cells isolated after sham procedure or blunt chest trauma, cultured with or without stimulation with sFasL
Alveolar macrophages showed a significantly increased release of IL-6, IL-10, and MCP-1 at 24 h after blunt chest trauma, when compared with corresponding sham (Fig. 3, A-C). Additional FasL stimulation (500 ng/mL) of AMΦs isolated after blunt chest trauma induced a further increase in the release of IL-6 compared with AMΦs cultured without stimulation (Fig. 3A). In supernatants of AMΦs isolated after chest trauma and stimulated with sFasL IL-10, release was significantly lower than in unstimulated AMΦs from traumatized animals (Fig. 3B). Alveolar type 2 epithelial cells isolated at 24 h after blunt chest trauma released significantly more IL-6, IL-10, and MCP-1 than the corresponding sham; additional stimulation with sFasL did not attenuate or further enhance the release of these mediators (Fig. 4, A-C).
Expression of Fas, FasL, IL-6, IL-10, and TNF-α mRNA in AMΦs and AT2 cells
The mRNA expression of Fas in AMΦs isolated at 4 h after trauma was significantly downregulated when compared with sham or the 16-h time point (Fig. 5A). At 24 h after chest trauma, Fas mRNA expression in AMΦs was upregulated when compared with corresponding sham or the 48-h time point (Fig. 5A). Expression of FasL mRNA was markedly higher in AMΦs isolated at 4 h after chest trauma than in AMΦs from sham animals (Fig. 5B). In AT2 cells, Fas mRNA expression was significantly increased at 4 h after lung contusion, when compared with AT2 cells isolated at 4 h after sham procedure and compared with those isolated at 16 h after chest trauma (Fig. 5C). Alveolar type 2 epithelial cells isolated at 24 h after blunt chest trauma showed a significantly lower mRNA expression of FasL than AT2 cells from sham animals or AT2 cells isolated at 4, 16, or 48 h after chest trauma (Fig. 5D). Regarding the cytokines, blunt chest trauma induced an increased expression of TNF-α, IL-6, and IL-10 mRNA in AMΦs (Fig. 6, A-C). The peak of cytokine mRNA expression was at the 48-h time point. In AT2 cells, mRNA expression of TNF-α, IL-6, or IL-10 was detectable but did not change after blunt chest trauma (Table 2).
Flow cytometric analysis of AMΦs and AT2 cells for expression of Fas and FasL
Flow cytometric analysis revealed a significant decrease in Fas-positive (Fig. 7A) and FasL-positive (Fig. 7B) AMΦ cell counts at 4 or 16 h after lung contusion when compared with sham. The percentage of Fas-positive (Fig. 7C) or FasL-positive (Fig. 7D) AT2 cells did not change after blunt chest trauma. Nevertheless, in all the studied groups, the fraction of Fas-positive AT2 cells among the total of AT2 cells analyzed was about 50%.
The present study was designed to determine the contribution of the Fas/ FasL system to the inflammatory response after blunt chest trauma. Our results indicate that the Fas/FasL system not only induces apoptosis in alveolar epithelial cells but also represents an important factor in regulating the inflammatory response after lung contusion.
Concentrations of sFasL were increased in both BAL and cell culture supernatants of unstimulated AMΦs isolated after blunt chest trauma (Fig. 1, A and B). These findings are supported by clinical observations of Matute-Bello et al. (19), who described significantly increased concentrations of sFasL in BAL fluids and LPS-stimulated BAL cell culture supernatants of patients with clinically defined ARDS. According to Matute-Bello et al. (19), increased levels of sFasL in the BAL at the onset of ARDS were associated with a poor outcome. These findings emphasize the relevance of Fas/FasL activation in lung inflammation and the impact of sFasL on the development of severe pulmonary complications, such as ARDS. Because the mentioned authors have shown that FasL has a proinflammatory potential in the context of lung injury or inflammation, this study was designed to further determine the release or expression of Fas and FasL after lung contusion and to analyze Fas/FasL effects on pulmonary immunological cells after blunt chest trauma.
TUNEL stain revealed a tendency to increased apoptosis rates (Fig. 1D). Annexin V stain, however, indicates that incubation of untreated AT2 cells with different concentrations of sFasL induced apoptosis in a dose-dependent manner (Fig. 1C). The variance between these two apoptosis assays is explained by their different kinetics and sensitivity (20, 21). This observation stands in line with findings of Hagimoto et al. (11), who found a markedly increased apoptosis rate in human bronchiolar epithelial cells after incubation with a Fas-activating antibody (CH-11) or TNF-α. According to a report by Nakamura et al. (22), sFasL itself reduced the survival of human small airway epithelial cells but not normal human bronchial epithelial cells. Small airway epithelial cells are nonciliated primary lung epithelial cells that express cytokeratin and surfactant protein A and therefore are very similar to the alveolar epithelial cells used in our experiment. Interestingly, preincubation of small airway epithelial cells with TNF-α, IL-1β, or IFN-γ further enhanced apoptosis induction, showing that proinflammatory cytokines sensitize lung epithelium to Fas-induced death (22). To confirm these findings in vivo, these authors instilled a Fas-activating antibody (Jo2) in lungs of otherwise untreated mice. After 6 h of incubation, immunohistochemistry for activated caspase 3 indicated increased apoptosis induction in epithelia covering the alveolar spaces and in intraalveolar cells (22). Because sFasL was increased in BALs after blunt chest trauma in our experiments (Fig. 1A), these findings support the hypothesis that apoptosis induction after blunt chest trauma is mediated by the Fas/FasL system (8). According to our own previous findings, the counts of isolated AMΦs were significantly higher in traumatized animals than in sham at the later time points after the insult (23). In contrast to that, AT2 cell gain was significantly higher at 4 h after chest trauma than after sham procedure, but was not changed in the later time course. Interestingly, an increased apoptosis rate of AT2 cells as mentioned in a previous publication could not be reproduced by decreased AT2 cell gain during isolation (8). A reason for this discrepancy might be that early apoptotic cells were not detected by trypan blue stain and counted as viable. The release of the proinflammatory cytokine IL-6 by AMΦs isolated after chest trauma was further enhanced by additional stimulation with FasL, and the release of anti-inflammatory IL-10 by AMΦs isolated after chest trauma was suppressed by FasL stimulation in our experiment (Fig. 3, A and B). Those results indicate that FasL stimulation is associated with the enhancement of the inflammatory response, observed after blunt chest trauma. The fact that Fas activation induces a proinflammatory cytokine response is supported by a report by Park et al. (24) on Fas stimulation in human monocytes and monocyte-derived macrophages. The authors report that TNF-α and IL-8 release in naive monocytes significantly increased after 18 h of incubation with the Fas-activating antibody CH-11 as well as with sFasL. In contrast to our experiment, Park et al. (24) used only untreated cells, whereas we compared cells collected after sham injury or chest trauma. In another experiment by Perl et al. (25), acute lung injury was induced in Fas and FasL knockout mice and wild-type mice by cecal ligation and puncture, followed by hemorrhage. In vivo experiments revealed a marked decrease in proinflammatory cytokine release, such as IL-6 in lung tissue and bronchoalveolar lavage in both knockout mice examined. In contrast to Fas knockout, FasL knockout did not attenuate chemokine concentrations (KC and MIP-2) in lung tissue isolated early after lung injury in the same experiment. Findings of Perl et al. (25) and our data on distinct cell types indicate that other mechanisms besides Fas/FasL activation are involved in chemokine release after chest trauma or lung injury. It should be noted that, with regard to the study design, our data reveal only an association between Fas/FasL activation and posttraumatic immune response after blunt chest trauma. However, based on the remarkable results of this study, further experiments should focus on therapeutic effect of the modification in Fas/FasL activation by introducing knockout animals or the auspicious method of Fas silencing (10).
Alveolar type 2 epithelial cells isolated after blunt chest trauma released significantly higher levels of IL-6, IL-10, and MCP-1 than AT2 cells from sham animals (Fig. 4). These data indicate that AT2 cells are activated by blunt chest trauma and contribute to posttraumatic alterations by the release of inflammatory mediators. Stimulation with sFasL did not further increase this cytokine release. In contrast to our results, human bronchiolar epithelial cells showed a significantly increased mediator release in response to stimulation with TNF-α or the Fas-activating antibody CH-11 (11). However, Hagimoto et al. (11) used naive bronchiolar epithelial cells, whereas we studied alveolar epithelial cells from rats subjected to chest trauma or sham procedure. Furthermore, the authors determined only the concentrations of the chemoattractant IL-8. Crestani et al. (26) detected that AT2 cells isolated from untreated rats released IL-6, and additional stimulation with murine IL-1β or TNF-α further augmented the IL-6 release. We have previously shown that IL-1β is increased in bronchoalveolar lavage after blunt chest trauma (8). Therefore, it is reasonable to suggest that mediators present in the alveoli after lung contusion may activate AT2 cells to increase their mediator secretion. The present findings support the hypothesis that AT2 cells serve as an important source of inflammatory mediators in the alveolar space and are associated with the regulation of the intraalveolar immune response and recruitment of circulating monocytes (26, 27). Alveolar macrophages and AT2 cells from sham animals expressed considerable amounts of Fas and FasL mRNA (Fig. 5). In AMΦs, Fas mRNA expression was upregulated late, and FasL mRNA expression was upregulated early after trauma (Fig. 5, A and B). The early increase of FasL mRNA in AMΦs is in line with the increased release of the corresponding sFasL protein by AMΦs isolated early after chest trauma (Fig. 1B). In AT2 cells, Fas mRNA expression was upregulated early after trauma, and FasL mRNA was downregulated late after the insult (Fig. 5, C and D). Comparable results were described by Nakamura et al. (22), who showed that Fas but not FasL mRNA was expressed in normal human bronchial epithelial cells and human small airway epithelial cells. In contrast to our experiment, however, Nakamura used a slightly different cell population. Our results indicate that AMΦs and AT2 cells express the Fas receptor and FasL constitutively. Chest trauma seems to increase the susceptibility of AMΦs and AT2 cells to Fas ligation in a time-dependent manner.
IL-6 and IL-10 mRNA expression in AMΦs significantly increased after blunt chest trauma (Fig. 6). Vanderbilt et al. (28), however, found that unstimulated AMΦs did not express genes for these chemokines until they were cultured for at least 2 h, and the amount of mRNA expression depended on the time in cell culture. In our experiment, AMΦs were cultured for only 1 h until they were used for RNA isolation. As IL-6 and IL-10 mRNA expressions were barely detectable in sham animals, the activating effect of the cell culture conditions on AMΦs seems to be negligible. Thus, these data confirm our own previous findings indicating that AMΦs are activated by blunt chest trauma to release inflammatory mediators (23, 29) and to express the corresponding mRNA. Alveolar type 2 epithelial cells from sham animals showed a detectable mRNA expression of those cytokines; however, blunt chest trauma did not alter this expression (Table 2). The discrepancy between the unchanged mRNA expression for those cytokines and the increased mediator release might arise from the fact that AT2 cells were cultured for determination of mediator release, whereas the mRNA expression was determined in freshly isolated cells. Furthermore, because there is evidence in the literature that cytokines can be stored intracellularly (30, 31), another possible explanation is that preformed cytokines were present in the cells before release.
Flow cytometric analysis of Fas and FasL expression on the surface of AMΦs revealed significantly decreased numbers of Fas- and FasL-positive AMΦs at 4 and 16 h after chest trauma when compared with sham. Nevertheless, a baseline surface expression of Fas and FasL was detectable in all animals (Fig. 7). Comparable results were described by Matute-Bello et al. (32), who detected a baseline expression of Fas on the surface of AMΦs from wild-type mice. Soluble FasL is known to be released by cleavage of the membrane-bound form of FasL by a metalloproteinase (33). As we observed an increase in FasL release by cultured AMΦs isolated early after blunt chest trauma when compared with sham (Fig. 1B), our data suggest that the observed decrease in membrane-bound FasL on AMΦs may be associated with an increased release of sFasL.
FACS analysis revealed that 50% of AT2 cells expressed Fas on their surface and that the ratio did not change following blunt chest trauma. These data indicate that AT2 cells express the Fas receptor constitutively. Our results are in line with findings of Fine et al. (34), who immunohistochemically detected that Fas protein was expressed on the surface of a subpopulation of AT2 cells from untreated rats. An increased surface expression of Fas on human bronchiolar epithelial cells has been described in a report by Hagimoto et al. (11). In this study, the cells were stained for Fas expression and evaluated by FACS analysis. Interestingly, preincubation with TNF-α and IFN-γ further enhanced the expression of Fas in those cells (11). Although in our own previous experiments cytokine levels were significantly increased in bronchoalveolar lavage after blunt chest trauma (8), our results indicate that mediators detected in the alveoli of animals subjected to lung contusion do not enhance Fas receptor expression on AT2 cells. Furthermore, these findings are comparable to our previous observations that showed that the numbers of Fas-expressing AT2 cells in lung sections increased only late after lung contusion (8). However, the comparison between the studies is limited by the fact that Fas receptor expression was determined by different methods. Furthermore, AT2 cell apoptosis, which is known to occur after chest trauma (8), might explain the missing increase of Fas receptor-positive AT2 cells in the present study.
Our findings suggest that the Fas/FasL system is activated after blunt chest trauma. Alveolar macrophages appear to contribute to the increased levels of sFasL in bronchoalveolar lavage by the release of this mediator. Furthermore, AMΦs were activated by sFasL in vitro. Because this mediator is increased in bronchoalveolar lavage after lung contusion, it has the potential to enhance the posttraumatic response of AMΦs in vivo. Interestingly, we could show that AT2 cells released higher levels of IL-6, IL-10, and MCP-1 after blunt chest trauma, but in contrast to AMΦ, cultured AT2 cells did not further enhance their mediator release after stimulation with sFasL in vitro. Taken together, our results indicate that the Fas/FasL system not only induces apoptosis in alveolar epithelial cells but is also associated with the regulation of the inflammatory response after lung contusion.
1. Scarborough K, Slone DS, Uribe P, Craun M, Bar-Or R, Bar-Or D: Reduced mortality at a community hospital trauma center: the impact of changing trauma level designation from II to I. Arch Surg 143(1):22-27, 2008.
2. Shorr RM, Crittenden M, Indeck M, Hartunian SL, Rodriguez A: Blunt thoracic trauma. Analysis of 515 patients. Ann Surg 206(2):200-205, 1987.
3. Pinilla JC: Acute respiratory failure in severe blunt chest trauma. J Trauma 22(3):221-226, 1982.
4. Gaillard M, Herve C, Mandin L, Raynaud P: Mortality prognostic factors in chest injury. J Trauma 30(1):93-96, 1990.
5. Guo-shou Z, Xiang-jun B, Cheng-ye Z: Analysis of high risk factors related to acute respiratory distress syndrome following severe thoracoabdominal injuries. Chin J Traumatol 10(5):275-278, 2007.
6. Knöferl MW, Liener UC, Seitz DH, Perl M, Brückner UB, Kinzl L, Gebhard F: Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a novel mouse model of blunt chest trauma. Shock 19(6):519-525, 2003.
7. Perl M, Gebhard F, Brückner UB, Ayala A, Braumüller S, Büttner C, Kinzl L, Knöferl MW: Pulmonary contusion causes impairment of macrophage and lymphocyte immune functions and increases mortality associated with a subsequent septic challenge. Crit Care Med 33(6):1351-1358, 2005.
8. Seitz DH, Perl M, Mangold S, Neddermann A, Braumüller ST, Zhou S, Bachem MG, Huber-Lang MS, Knöferl MW: Pulmonary contusion induces alveolar type 2 epithelial cell apoptosis: role of alveolar macrophages and neutrophils. Shock 30(5):537-544, 2008.
9. Matute-Bello G, Martin TR: Science review: apoptosis in acute lung injury. Crit Care 7(5):355-358, 2003.
10. Perl M, Chung CS, Lomas-Neira J, Rachel TM, Biffl WL, Cioffi WG, Ayala A: Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis. Am J Pathol 167(6):1545-1559, 2005.
11. Hagimoto N, Kuwano K, Kawasaki M, Yoshimi M, Kaneko Y, Kunitake R, Maeyama T, Tanaka T, Hara N: Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation. Am J Respir Cell Mol Biol 21(3):436-445, 1999.
12. Bem RA, Farnand AW, Wong V, Koski A, Rosenfeld ME, van RN, Frevert CW, Martin TR, Matute-Bello G: Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice. Am J Physiol Lung Cell Mol Physiol 295(2):L314-L325, 2008.
13. Irwin RJ, Lerner MR, Bealer JF, Brackett DJ, Tuggle DW: Cardiopulmonary physiology of primary blast injury. J Trauma 43(4):650-655, 1997.
14. Jaffin JH, McKinney L, Kinney RC, Cunningham JA, Moritz DM, Kraimer JM, Graeber GM, Moe JB, Salander JM, Harmon JW: A laboratory model for studying blast overpressure injury. J Trauma 27(4):349-356, 1987.
15. Liener UC, Knöferl MW, Sträter J, Barth TF, Pauser EM, Nüssler AK, Kinzl L, Brückner UB, Gebhard F: Induction of apoptosis following blunt chest trauma. Shock 20(6):511-516, 2003.
16. Huber-Lang M, Younkin EM, Sarma JV, Riedemann N, McGuire SR, Lu KT, Kunkel R, Younger JG, Zetoune FS, Ward PA: Generation of C5a by phagocytic cells. Am J Pathol 161(5):1849-1859, 2002.
17. Dobbs LG: Isolation and culture of alveolar type II cells. Am J Physiol 258(4 Pt 1):L134-L147, 1990.
18. Dobbs LG, Gonzalez R, Williams MC: An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134(1):141-145, 1986.
19. Matute-Bello G, Liles WC, Steinberg KP, Kiener PA, Mongovin S, Chi EY, Jonas M, Martin TR: Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J Immunol 163(4):2217-2225, 1999.
20. Zhao J, Schmid-Kotsas A, Gross HJ, Gruenert A, Bachem MG: Sensitivity and specificity of different staining methods to monitor apoptosis induced by oxidative stress in adherent cells. Chin Med J (Engl) 116(12):1923-1929, 2003.
21. Vermes I, Haanen C, Steffens-Nakken H, Reutellingsperger C: A novel assay for apoptosis Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 184(1):39-51, 1995.
22. Nakamura M, Matute-Bello G, Liles WC, Hayashi S, Kajikawa O, Lin SM, Frevert CW, Martin TR: Differential response of human lung epithelial cells to Fas-induced apoptosis. Am J Pathol 164(6):1949-1958, 2004.
23. Seitz DH, Niesler U, Palmer A, Sulger M, Braumüller ST, Perl M, Gebhard F, Knöferl MW: Blunt chest trauma induces mediator-dependent monocyte migration to the lung. Crit Care Med 38(9):1852-1859, 2010.
24. Park DR, Thomsen AR, Frevert CW, Pham U, Skerrett SJ, Kiener PA, Liles WC: Fas (CD95) induces proinflammatory cytokine responses by human monocytes and monocyte-derived macrophages. J Immunol 170(12):6209-6216, 2003.
25. Perl M, Chung CS, Perl U, Lomas-Neira J, de PM, Cioffi WG, Ayala A: Fas-induced pulmonary apoptosis and inflammation during indirect acute lung injury. Am J Respir Crit Care Med 176(6):591-601, 2007.
26. Crestani B, Cornillet P, Dehoux M, Rolland C, Guenounou M, Aubier M: Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. Regulation by alveolar macrophage secretory products. J Clin Invest 94(2):731-740, 1994.
27. Thorley AJ, Ford PA, Giembycz MA, Goldstraw P, Young A, Tetley TD: Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages. J Immunol 178(1):463-473, 2007.
28. Vanderbilt JN, Mager EM, Allen L, Sawa T, Wiener-Kronish J, Gonzalez R, Dobbs LG: CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. Am J Respir Cell Mol Biol 29(6):661-668, 2003.
29. Liener UC, Perl M, Huber-Lang MS, Seitz DH, Brückner UB, Gebhard F, Knöferl MW: Is the function of alveolar macrophages altered following blunt chest trauma? Langenbecks Arch Surg 396(2):251-259, 2010.
30. Spencer LA, Melo RC, Perez SA, Bafford SP, Dvorak AM, Weller PF: Cytokine receptor-mediated trafficking of preformed IL-4 in eosinophils identifies an innate immune mechanism of cytokine secretion. Proc Natl Acad Sci U S A 103(9):3333-3338, 2006.
31. Tsakiri N, Kimber I, Rothwell NJ, Pinteaux E: Mechanisms of interleukin-6 synthesis and release induced by interleukin-1 and cell depolarisation in neurones. Mol Cell Neurosci 37(1):110-118, 2008.
32. Matute-Bello G, Lee JS, Liles WC, Frevert CW, Mongovin S, Wong V, Ballman K, Sutlief S, Martin TR: Fas-mediated acute lung injury requires Fas expression on nonmyeloid cells of the lung. J Immunol 175(6):4069-4075, 2005.
33. Nagata S: Fas ligand-induced apoptosis. Annu Rev Genet 33:29-55, 1999.
34. Fine A, Anderson NL, Rothstein TL, Williams MC, Gochuico BR: Fas expression in pulmonary alveolar type II cells. Am J Physiol 273(1 Pt 1):L64-L71, 1997.
Lung contusion; inflammation; Fas/Fas ligand; alveolar macrophages; alveolar type 2 epithelial cells
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