Abstract
This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)–binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS). Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression. Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied. These cells produced LBP in response to interleukin (IL)-1 β , IL-6, and tumor necrosis factor- α , a response that was strongly enhanced by dexamethasone. In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation. The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7. Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7–derived LBP. Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS. Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells. The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.
The acute phase protein lipopolysaccharide (LPS)–binding protein (LBP) plays an important role in the host defense to bacterial endotoxins (or LPS). LBP strongly enhances sensitivity of monocytes and neutrophils for LPS by facilitating binding of LPS to membrane CD14, a “receptor” for endotoxin (1, 2). Recent studies indicate a role for the Toll-like receptor (TLR)-2 and TLR-4 in this LBP/ CD14-dependent LPS signaling (3, 4). In addition, LBP transfers LPS to soluble CD14, resulting in activation of membrane CD14-negative cells such as endothelial and epithelial cells (5, 6). Further, LBP accelerates the transport of LPS into high-density lipoprotein (HDL), leading to neutralization of LPS (7). Binding of LBP to gram-negative bacteria has also been reported (8, 9), which results in phagocytosis and clearance of these microorganisms (10).
Endotoxins and gram-negative bacteria are ubiquitously present in both the outer and inner environments (11). Due to inhalation of airborne particles containing bacteria and LPS from the commensal flora in the nasopharynx and from the environment, the human airways are continuously exposed to these proinflammatory compounds. The lung has efficient defense mechanisms against these agents under normal exposure conditions. However, exposure to high levels of endotoxins, as occurs in pig farmers or grain handlers, results in an inflammatory response and is associated with the development of lung diseases (12, 13).
We hypothesize that local presence of LBP contributes to the defense against bacterial endotoxins in the lung. In line with this assumption is the observation that small amounts of LBP are present in bronchoalveolar lavage fluid (BALF) of healthy individuals. Moreover, these levels are increased in patients with acute respiratory distress syndrome (ARDS) and in BALF of asthmatics after allergen challenge (14-16). The origin of this LBP, which could be either produced locally or derived from the circulation (in which 5 to 10 μg/ml of this protein is normally present and even at increased levels during disease), still has to be elucidated. Although hepatocytes represent the major source of acute-phase proteins (17), increasing evidence indicates that respiratory epithelial cells are able to produce acute-phase proteins, as shown by the production of fibrinogen and α1-acid glycoprotein by these cells (18, 19).
In this study we evaluated the presence of LBP in human lung and, in addition, studied the ability of respiratory epithelial cells to produce LBP. The human lung epithelial cell line A549 and the murine lung type II–like epithelial cell line C10 were stimulated with cytokines, which are known inducers of acute-phase protein production, resulting in the production of LBP. Also, LBP expression in human primary type II pneumocytes was demonstrated. The characteristics of LBP derived from respiratory epithelial cells were studied. This study demonstrates for the first time that lung type II pneumocytes are able to produce the acute-phase protein LBP, which could play an important role in the local defense to bacteria and bacterial endotoxins.
Human (h) recombinant (r) tumor necrosis factor (TNF)-α was kindly provided by BASF/Knoll Ag (Ludwigshafen, Germany); hr interleukin (IL)-6 by Prof. W. Sebald (Physiologisch-Chemisches Institut der Universität, Würzburg, Germany); hrIL-1β by Dr. S. Gillis (Immunex, Seattle, WA); and murine TNF-α by Genentech (San Francisco, CA). hrLBP was obtained from Chinese hamster ovary (CHO) cells transfected with human LBP complementary DNA (cDNA) (CHO-LBP cells) (indicated later). Dexamethasone was a gift from Merck Sharp & Dohme, Haarlem, The Netherlands. LPS (Escherichia coli, serotype 055:B5), DNAse I, polymyxin B, Percoll, and trypsin were purchased from Sigma (St. Louis, MO). Biotin labeling of LPS was performed using biotin-LC-hydrazide, according to the instructions of the manufacturer (Pierce, Rockford, IL). RPMI-1640, CMRL-1066, CHO-S-SFM II low-protein serum-free (SF) medium, Waymouth's 752/1 medium, and fetal calf serum (FCS) were obtained from GIBCO BRL (Paisley, UK). Bovine calf serum (BCS) was purchased from HyClone (Logan, UT) and was heated for 30 min at 56°C before storage at 4°C.
Polyclonal rabbit anti-rhLBP antibody was prepared as described elsewhere (20). After protein A purification, rabbit immunoglobulin (Ig) G was biotinylated. The murine anti-rhLBP monoclonal Ab (mAb) HM14 was obtained by immunizing mice with rhLBP following classical procedures and was selected on the basis of its reactivity with both free LBP and LBP that has formed a complex with LPS. Besides specific interaction with human LBP, HM14 was also shown to react with bovine LBP.
Lung tissue was obtained from patients undergoing lobectomy or pneumectomy for lung cancer. Within minutes after resection, either a small portion (for analysis of LBP messenger RNA [mRNA] in human lung) or large portion (for isolation of type II pneumocytes) of macroscopically nontumoral tissue was cut from the surgical specimen. The study was approved by the medical ethical committee of the University Hospital of Maastricht and Leuven.
The isolation procedure of primary human type II pneumocytes was performed as described previously (21, 22). The lung tissue was first sliced (0.7-mm thickness), and aliquots of approximately 1 g of slices were washed four times at 4°C in 10 ml phosphate-buffered saline (PBS). To dissociate the cells, the washed slices were incubated four times (2 × 5 min and 2 × 20 min) at 37°C in 10 ml fresh trypsin with gentle shaking. The protease activity was stopped by the addition of 4 ml FCS and 100 μg DNAse I per gram of tissue. This primary digest was, from here on, handled under sterile conditions. It was filtered through nylon filters with meshes of 80 and 25 μm (Heleine Cavenaile PVBA/SPRL, Brussels, Belgium), and cells were resuspended in 25 ml Waymouths' medium containing 50 μg/ml DNAse, 2% FCS, 2% fungizone, and 2% penicillin-streptomycin solution, and incubated for 1 h in a bacterial Petri dish placed in a CO2 incubator (10% CO2, 37°C) to let macrophages attach. The nonadherent cells were then layered onto discontinuous Percoll layers of density 1.089 g/ml and 1.040 g/ml in 50-ml centrifuge tubes and centrifuged 20 min at 250 × g at 4°C. Finally, the creamy cell layer above the heavy gradient was collected, rinsed twice with PBS+ (PBS− with 1.9 mM CaCl2 and 1.29 mM MgSO4), and plated (300,000 cells/cm2) in six-well cell culture plates in medium consisting of RPMI 1640 supplemented with 10% BCS and antibiotics. After 2 d in culture, cells were rinsed and provided with fresh medium. At that time point confluency was > 95%, and cells were stimulated as indicated. Cultured primary type II pneumocytes were identified by staining for alkaline phosphatase activity as described elsewhere (23), which revealed > 90% purity.
The human lung epithelial cell line A549 (lung adenocarcinoma) (24) was obtained from the American Type Culture Collection (Rockville, MD; ATCC CCL-185), and the human liver epithelial cell line HuH-7 was kindly provided by Dr. K. Fearon (University of Edinburgh, Edinburgh, UK). These cells were cultured in tissue culture flasks (Costar, Cambridge, MA), in complete medium consisting of RPMI 1640 supplemented with 10% BCS and antibiotics, in a humidified CO2 incubator at 37°C. Epithelial cells were trypsinized once a week, after being > 95% confluent for the last 2 to 3 d. The nontumorigenic murine type II pneumocyte–related lung epithelial cell line C10 was kindly provided by Dr. R. J. Ruch (Medical College of Ohio, Toledo, OH). Cells were cultured in CMRL 1066 medium, supplemented with 10% FCS and antibiotics, and were passaged via trypsinization after reaching confluence. C10 cells are a clone from the NAL-1A cell line derived from normal adult mouse-lung explants, which show ultrastructural features of type II pneumocytes, including prominent surface microvilli and osmiophilic laminar bodies, and in addition stain positive with a type II pneumocyte–specific antiserum directed against a high molecular-weight lamellar body–associated protein (25, 26).
CHO-LBP cells were kindly provided by Dr. P. S. Tobias (Scripps Research Institute, La Jolla, CA), and cultured in CHO-S-SFM II medium.
Cells were plated in complete culture medium in 96-well flat-bottom tissue culture plates, in six-well plates, or in culture flasks (Costar). After 72 h, when cells had reached > 95% confluency, medium was replaced by fresh medium containing the indicated stimuli. Experiments designed to study regulation of LBP production were performed in complete medium with serum. The experiments directed at characterization of LBP produced by human epithelial cells were performed under SF conditions (RPMI 1640, containing antibiotics) to prevent the presence of bovine LBP. After stimulation for the indicated time periods at 37°C, supernatants were harvested and kept at −20°C until analysis. Viability of cells at the end of the experiments was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as described (27), revealing > 90% survival.
LBP concentrations in culture supernatants were determined using sandwich enzyme-linked immunosorbent assay (ELISA) (20). In short, 96-well immuno maxisorp plates (Nunc, Roskilde, Denmark) were coated with polyclonal rabbit anti-rhLBP IgG. Human rLBP produced by CHO-LBP cells was used for standard titration curves. Biotin-labeled polyclonal rabbit anti-rhLBP IgG was followed by streptavidin-peroxidase conjugate (Zymed Laboratories Inc., San Francisco, CA). 3,3′,5,5′-Tetramethylbenzidine (KPL, Gaithersburg, MD) was used as substrate. The detection limit of ELISA was 200 pg/ml. This assay specifically detected human LBP and did not cross-react with bovine LBP present in BCS.
Murine LBP was detected using an ELISA as reported (28). Maxisorp plates (Nunc) were coated with rat mAb clone 43 directed to murine LBP. Recombinant murine LBP was used for standard titration curve, and murine LBP was detected using rabbit polyclonal antimurine LBP IgG, followed by goat antirabbit IgG peroxidase conjugate (Jackson, West Grove, PA). The detection limit of the ELISA was 100 pg/ml.
LBP was purified from conditioned SF culture supernatant of A549 or HuH-7 cells, or from supernatant of CHO-LBP cells, using preparative affinity chromatography. To this end, anti-hLBP mAb HM14 was coupled to a HiTrap affinity column, according to manufacturer's instructions (Pharmacia Biotechnology, Uppsala, Sweden). Conditioned SF culture supernatant was applied to the column, and the unbound proteins were washed out with PBS. Bound LBP was eluted using glycine-HCl buffer pH 2.5 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a Phast system (Pharmacia) followed by electrophoretic transfer onto an immobilon-P membrane (Millipore Corp., Bedford, MA). Antigenic bands were detected using biotin-labeled polyclonal rabbit antihuman LBP IgG, followed by peroxidase conjugated to streptavidin and the substrate 3,3′-diaminobenzidine (Serva, Heidelberg, Germany).
The interaction of human LBP with LPS was measured by ELISA. Microtiter plates (Nunc) were coated with anti-LBP mAb HM14. Incubation with LBP, either as derived from CHO-LBP cells, which was used for the standard titration curve, or as present in conditioned SF medium of human lung epithelial cells, was sequentially followed by biotinylated LPS and peroxidase conjugated to streptavidin. To test the effect of polymyxin B on interaction of LPS with LBP, LPS-biotin was preincubated for 30 min at 37°C with polymyxin B (100 μg/ml) before applying to the plate.
To analyze mRNA levels, cells were stimulated in culture flasks or in six-well plates with indicated stimuli. Human lung obtained as described earlier was snap-frozen in liquid nitrogen immediately after resection. Total RNA of cells was isolated according to the method of Chomczynski and Sacchi (29), whereas for homogenized human lung tissue the SV total RNA isolation system from Promega (Madison, WI) was used. To exclude contamination with genomic DNA, isolated RNA was treated with deoxyribonuclease (DNAse), and subsequently cDNA was obtained by reverse transcription of RNA. For standardization of the different RNA samples glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polymerase chain reaction (PCR) was used on serial dilutions of cDNA. PCR was performed in a 25-μl reaction volume containing 100 μM of each deoxynucleotide triphosphate, 200 nM primers, and 0.5 U Taq DNA polymerase (Perkin Elmer/ Cetus, Emeryville, CA). Human LBP PCR was performed during 40 cycles under the following conditions: 92°C for 30 s; 57°C for 1 min; and 72°C for 1 min. Primers for human LBP, which are intron-spanning, were designed as described elsewhere (30): sense primer 5′-AGG GCC TGA GTC TCA GCA TCT-3′ and antisense primer 5′-CAG GCT GGC CGT GTT GAA GAC-3′, yielding a PCR product of 565 base pairs (bp). Human GAPDH PCR was performed during 26 cycles under the following conditions: 92°C for 30 s; 55°C for 45 s; and 72°C for 30 s. Primers for human GAPDH were designed sense primer 5′-CGT CTT CAC CAC CAT GGA GA-3′ and antisense primer 5′-CGG CCA TCA CGC CAC AGT TT-3′. The PCR products were separated by electrophoresis in ethidium bromide–stained 1.2% agarose gel and visualized by ultraviolet illumination (Imagemasters VDS, Uppsala, Sweden). A mock PCR (containing buffer, primers, and enzyme but no cDNA) was performed to exclude contamination.
All values in the figures and tables are expressed as means ± standard deviation (SD). Groups were compared using Student's t test. Results of experiments designed to study the effect of cytokines and dexamethasone on the secretion of LBP by cell lines were analyzed by three-way analysis of variance (ANOVA). Dependent variable values have been transformed to their square root to achieve a better approach to the normal distribution. Factors used are stimulus, dexamethasone, and dose, in which dose is nested within stimulus. ANOVA was performed by dummy regression analysis to facilitate testing of differences between experimental factor means. First, all interactions were introduced into the dummy regression model. When second-order interactions tested turned out to be nonsignificant, first-order interaction was tested within a simpler model. This procedure was repeated until the best-fitting reduced model was found, and P values belonging to differences between means belonging to this last model are reported. All data were analyzed by SPSS version 9.0. Differences were considered statistically significant at P < 0.05.
First we studied whether LBP could locally be detected in human lung. To this end, lung tissue obtained from patients undergoing resection due to lung cancer was analyzed for expression of LBP mRNA using reverse transcriptase (RT)–PCR. In all lung samples analyzed (n = 8) LBP mRNA was present, as is shown for two representative samples in Figure 1. The LBP PCR product was demonstrated to be 565 bp, similar to that described elsewhere (30). In addition, when a PCR reaction was performed on RNA samples without first conducting the reverse-transcription step, no signal for LBP was detected. This makes it unlikely that trace amounts of genomic DNA that might have been present in sample were the target of the amplification reaction. This result indicates the human lung as a source of the acute-phase protein LBP.
Fig. 1. (A) LBP mRNA expression by the human lung epithelial cell line A549 and by human primary type II epithelial cells. Two representative samples of normal human lung tissue are shown (lanes 1 and 2). A549 cells were incubated for 24 h without stimulus (lane 3) or with a combination of 10 ng/ml IL-1β (IL-1), 5 ng/ml IL-6 (IL-6), and 1 μM dexamethasone (dex) (lane 4). Human primary type II pneumocytes obtained from two donors (I and II) were incubated for 24 h without stimulus (lanes 5 and 7); with a combination of IL-1 and IL-6 (lane 8); or with a combination of IL-1, IL-6, and dex (lanes 6 and 9). Total RNA was isolated and analyzed for LBP and GAPDH mRNA expression using RT-PCR. Equal loading of cDNA in the RT-PCR per set of samples was confirmed by equal intensity of GAPDH mRNA bands. Stimulation of A549 cells resulted in LBP mRNA upregulation of approximately 50 times. Lane 10 shows results of mock PCR. (B) Quantitative expression of LBP mRNA by human primary type II pneumocytes (donors I and II) expressed as the ratio LBP/GAPDH mRNA. Quantitative analysis was performed by PhosphorImaging.
[More] [Minimize]Epithelial cells are responsible for the hepatic acute-phase protein production, which is induced by cytokines such as IL-6, IL-1, and TNF. Therefore, the effect of these cytokines upon LBP production by the human lung epithelial cell line A549 was investigated. We observed that these cells spontaneously produced small amounts of the acute-phase protein LBP, which was strongly enhanced in the presence of the cytokines tested (Figure 2). Of these cytokines, IL-1β was shown to be the most potent stimulus, resulting in the release of high amounts of LBP from IL-1β concentrations as low as 10 pg/ml. A549 cells also released considerable amounts of LBP in response to IL-6, from concentrations of 1 ng/ml and higher, whereas stimulation with TNF-α resulted in minute amounts of LBP. No LBP release was induced by exposure of A549 cells to bacterial endotoxin (1 to 100 μg/ml), although cells were activated, as demonstrated by IL-8 production (data not shown). Exposure of A549 cells to combinations of two cytokines resulted in an additive increase of the LBP production (data not shown).
Fig. 2. Cytokines induce LBP production by human lung epithelial cell line A549. A549 cells were stimulated for 72 h with IL-1β (A), IL-6 (B), and TNF-α (C). LBP was measured in supernatants using a specific ELISA. One representative experiment out of five is shown. Data are means ± SD of six measurements. SD bars are omitted if they fall within the symbol. *Statistically significant difference compared with LBP production by nonstimulated cells (P values < 0.05).
[More] [Minimize]Glucocorticosteroids enhance the production of some acute-phase proteins, including LBP, by hepatocytes (31); therefore, we analyzed the effect of dexamethasone, a glucocorticosteroid analogue, on LBP production by A549 cells. Dexamethasone induced a significant upregulation of the LBP production by A549 cells (Table 1). In addition, this corticosteroid strongly enhanced the cytokine- induced LBP production. This effect was most pronounced for IL-6–induced LBP release, resulting in a synergistic enhancement; whereas for IL-1β and TNF-α (data not shown), an additive effect was observed (Table 1). Thus, these data indicate that corticosteroids enhance LBP release by A549 cells and confirm the acute-phase nature of LBP produced by respiratory epithelial cells.
| A549 | HuH-7 | |||||||
|---|---|---|---|---|---|---|---|---|
| Dexamethasone | Dexamethasone | |||||||
| 0 | 1 μM | 0 | 1 μM | |||||
| Stimulus | ||||||||
| None | 3 ± 1† | 44 ± 6‡ | 8 ± 2 | 9 ± 5 | ||||
| IL-1β, 0.1 ng/ml | 12 ± 3‡ | 78 ± 9 | 47 ± 11‡ | N.D.§ | ||||
| IL-1β, 1 ng/ml | 70 ± 7‖ | 186 ± 18 | 79 ± 10‖ | N.D. | ||||
| IL-1β, 10 ng/ml | 79 ± 13¶ | 206 ± 34 | 81 ± 6 | N.D. | ||||
| IL-6, 1 ng/ml | 9 ± 2‡ | 148 ± 16** | 14 ± 2‡ | 32 ± 7** | ||||
| IL-6, 10 ng/ml | 19 ± 5‖ | 215 ± 32** | 21 ± 3‖ | 75 ± 9** | ||||
| IL-6, 100 ng/ml | 27 ± 3¶ | 224 ± 44** | 29 ± 3¶ | 87 ± 24** | ||||
Induction of an acute-phase response results in upregulation of LBP mRNA in hepatocytes (32). To analyze whether LBP release by respiratory epithelial cells is preceded by increased LBP mRNA expression, LBP mRNA levels in A549 cells were analyzed using RT-PCR. As shown in Figure 1 (lane 3), unstimulated A549 cells constitutively express low levels of LBP mRNA. These levels increase strongly after stimulation of cells for 24 h with combinations of IL-6, IL-1β, and dexamethasone (Figure 1, lane 4). These data indicate that unstimulated A549 cells express LBP mRNA, which is increased after stimulation of cells. The observation that stimulation of cells results in enhanced levels of both protein and mRNA suggests that LBP is newly synthesized by A549 cells.
Because liver epithelial cells are considered to be the main producers of acute-phase proteins, the LBP production by A549 cells was compared with the LBP production by the liver epithelial cell line HuH-7. Similar to that described earlier for A549 cells, HuH-7 cells produced LBP in response to IL-1β and IL-6 (Table 1) and to a much lesser extent in response to TNF-α (data not shown). Although dexamethasone, in contrast to the results obtained with A549 cells, did not induce LBP release by HuH-7 cells, this glucocorticosteroid synergistically enhanced IL-6– induced LBP production similar to results shown for A549 cells (Table 1). Further, IL-1β and IL-6 were shown to have additive effects upon HuH-7–induced LBP production, as was also observed by A549 cells (data not shown). Thus, we demonstrated that lung epithelial cells and liver epithelial cells produce LBP in response to the same stimuli. The observation that IL-1β stimulated both cell types to produce LBP indicates that LBP is produced as a type I acute-phase protein by respiratory and liver epithelial cells.
In addition, time kinetics of LBP release by A549 and HuH-7 cells were analyzed. To this end, cells were stimulated, and after various time points culture supernatants were harvested and analyzed for the presence of LBP. The results show that A549 and HuH-7 cells released LBP with similar time kinetics (Figure 3). The cytokine-induced LBP production was evident 24 h after onset of stimulation and continued until 72 to 96 h after stimulation, which is characteristic for acute-phase protein production (17).
Fig. 3. Time kinetics of LBP production by the human lung epithelial cell line A549 and the liver epithelial cell line HuH-7. A549 and HuH-7 cells were incubated for the indicated time periods without stimulus or with IL-1β (1 ng/ml). Supernatants were collected and stored at −20°C until measurement of LBP using a specific ELISA. One representative experiment is shown. Data are means ± SD of two values. SD bars are omitted if they fall within the symbol.
[More] [Minimize]To characterize LBP produced by human lung epithelial cells, LBP was purified from the conditioned SF culture supernatant of A549 cells using an affinity column labeled with antihuman LBP mAb HM14 and subsequently analyzed by Western blotting. To this end, A549 cells were stimulated in culture flasks with the combination of IL-1β, IL-6, and dexamethasone in SF medium, resulting in production of considerable amounts of LBP (up to 300 ng/ml). As positive controls, LBP derived from CHO-LBP cells and from stimulated HuH-7 cells were purified and analyzed. As shown in Figure 4, Western blot analysis of A549-derived LBP resulted in a band of approximately 60 kD, identical to the size of LBP produced by HuH-7 and CHO-LBP cells. Hence, these results show that LBP produced by lung A549 cells has a similar molecular weight as liver-derived LBP, and further establish that A549 cells produce LBP.
Fig. 4. Western blot analysis of human LBP derived from the human lung epithelial cell line A549, human liver epithelial cell line HuH-7, and CHO-LBP cells. LBP was purified from SF culture supernatants using affinity purification followed by Western blot analysis with polyclonal rabbit anti-rhLBP IgG. Lane 1: A549 cells stimulated with IL-6 (5 ng/ml), IL-1β (10 ng/ml), and dexamethasone (1 μM); lane 2: CHO-LBP cell supernatant; lane 3: HuH-7 cells stimulated with IL-6 (5 ng/ml), IL-1β (10 ng/ml), and dexamethasone (1 μM). Molecular weight standards are indicated (lane M). One representative experiment is shown out of a series of four.
[More] [Minimize]To determine whether LBP produced by lung epithelial cells has biologic activity, its ability to interact with LPS was assessed. To this end, SF conditioned supernatant from A549 cells was added to plates coated with anti-LBP mAb HM14, followed by biotin-labeled LPS. The optical density (450 nm) obtained in this ELISA represents the LPS-binding capacity of the LBP. In parallel, absolute amounts of LBP present in supernatant were analyzed by LBP ELISA. LBP derived from CHO-LBP cells was used as a positive control. Figure 5 shows that LBP produced by stimulated A549 cells interacted with LPS. A good correlation was observed between the amounts of LBP detected in conditioned medium by LBP ELISA and the ability of conditioned medium to bind LPS. No signal was observed when SF conditioned medium of A549 cells, containing high levels of LBP, was added to noncoated wells (data not shown). In addition, the presence of polymyxin B (which interacts with LPS, resulting in reduced biologic activity) prevented the interaction between A549-derived LBP and LPS-biotin, as shown in Figure 5. These data indicate that LBP produced by respiratory epithelial cells is able to interact with LPS, and strongly suggest that A549-derived LBP has biologic activity.
Fig. 5. Interaction of LBP derived from the human lung epithelial cell line A549 with LPS. A549 cells were stimulated for 72 h with IL-6 (1 to 100 ng/ml) in the absence or presence of 1 μM dexamethasone (dex) in SF medium. Culture supernatants were assessed in parallel for the presence of LBP using LBP ELISA (ng/ml) and for the interaction with LPS-biotin (optical density 450 nm). LBP derived from transfected CHO cells (CHO-LBP) was used as a positive control. To test the effect of polymyxin B (PMB) on interaction of LPS with LBP, LPS-biotin was preincubated 30 min at 37°C with polymyxin B (100 μg/ml) before being applied to the plate. One representative experiment out of three is shown. Data are given as mean values of duplo measurements.
[More] [Minimize]The human lung epithelial cell line A549, an adenocarcinoma, has been reported to be derived from type II pneumocytes (24). To further study the role of type II epithelial cells as a source of LBP in lung, the production of LBP by C10 cells (a murine nontransformed type II alveolar epithelial cell line) was studied. Data in Table 2 show that C10 cells spontaneously produced murine LBP, which was significantly enhanced in response to IL-1β and murine TNF-α but not in response to human IL-6 (data not shown). Presence of dexamethasone also stimulated C10 cells to produce LBP which, in combination with cytokines, had an additive effect upon LBP production. Similar to that observed for A549 cells, murine LBP mRNA could be detected in nonstimulated C10 cells and levels were enhanced after stimulation of cells (data not shown).
| Dexamethasone | ||||
|---|---|---|---|---|
| 0 | 1 μM | |||
| Stimulus | ||||
| None | 130 ± 23† | 455 ± 45‡ | ||
| Human IL-1β | ||||
| 0.1 ng/ml | 186 ± 26‡ | 583 ± 105 | ||
| 1 ng/ml | 379 ± 58§ | 932 ± 192 | ||
| 10 ng/ml | 361 ± 34 | 821 ± 138 | ||
| Murine TNF | ||||
| 1 ng/ml | 265 ± 65‡ | 821 ± 366 | ||
| 10 ng/ml | 649 ± 124§ | 1,311 ± 142 | ||
| 100 ng/ml | 489 ± 41 | 966 ± 185 | ||
Further, LBP expression in isolated human primary type II cells was analyzed. The results of two experiments (Figure 1) show that LBP is present in these cells. Stimulation of cells with a combination of IL-1β, IL-6, and dexamethasone resulted in 1.5 to 2-fold upregulation of LBP mRNA (Figure 1B). Results of the second experiment indicate that presence of dexamethasone did not further enhance the IL-1β– and IL-6–induced upregulation of LBP mRNA. These results convincingly show that type II pneumocytes are able to produce LBP and could be a source for LBP in lung.
During breathing the human lung is regularly exposed to particulate matter containing bacteria from the environment and from the commensal flora of the upper airways. The respiratory epithelium plays an important role in the defense against invading microorganisms and their related products. These cells form a physical barrier and contribute to clearance of particles trapped in mucus via ciliary activity. Increasing evidence indicates that epithelial cells have the capacity to produce antimicrobial peptides such as lysozyme and lactoferrin (33) and also factors modulating local inflammation, such as antioxidants, antiproteases, cytokines, and chemokines (34, 35). The results of this study indicate the presence of the acute-phase protein LBP in the human lung and, in addition, proof was obtained that the respiratory epithelium is able to produce this protein involved in the defense to bacteria and bacterial endotoxins. The production of LBP by lung epithelial cells will result in enhanced local levels of LBP and potentially lead to a highly specific response at the site of exposure to bacteria. Indications that LBP can be protective during local infection were obtained by the observation that intraperitoneal injection of LBP prevented lethality due to local bacteremia (36). In addition, LBP-deficient mice showed enhanced sensitivity for intraperitoneally injected Salmonella typhimurium (37).
So far, hepatocytes are considered the main source of acute-phase proteins including LBP, resulting in systemic levels of these proteins. Previous indications for local presence of LBP in lungs were obtained. In BALF of healthy individuals LBP was present at low levels, which were enhanced during disease (14-16). In addition, LBP mRNA was detected in lungs of rats exposed to inflammatory stimuli (30, 38). In the present study we extend and confirm these previous investigations by showing LBP mRNA expression in human lung, indicating that LBP detected in BALF of healthy controls and at enhanced level during disease could locally be produced. In contrast to the previously discussed results obtained in human and rats, Martin and colleagues did not observe LBP mRNA in rabbit lung during acute-phase response (14), which could be due either to the origin of the lung or to the PCR conditions used.
Because epithelial cells are responsible for the hepatic acute-phase protein production, LBP production by lung epithelial cell lines was analyzed. The respiratory cell lines A549 and C10, both of which were demonstrated to produce LBP, have been reported to be derived from type II pneumocytes (24-26). In addition, LBP mRNA was detected in isolated human primary type II alveolar cells at increased levels after stimulation of cells, confirming that type II pneumocytes are able to produce the acute-phase protein LBP. This observation is in line with recent studies which demonstrated that stimulation of respiratory type II epithelial cells resulted in production of the acute-phase proteins fibrinogen and α1-acid glycoprotein (18, 19). However, bronchial epithelial cells were also reported to be able to produce acute-phase proteins, indicated by production of α1-proteinase inhibitor (39). Therefore, production of LBP by bronchial epithelium is currently under investigation. We speculate that Clara cells, which produce surfactant protein–like type II pneumocytes (40), could be candidate cells for production of acute-phase proteins in the bronchi.
The release of LBP by A549 and C10 cells was strongly induced by IL-1β and TNF-α, characteristic for type I acute-phase protein production (17). This indicates that respiratory epithelial cells release LBP like a type I acute-phase protein, similar to liver epithelial cells as reported in this study and by others (31, 41). Corresponding to induction of an acute-phase response in hepatocytes (32), it was shown that LBP mRNA levels in A549 cells, in C10 cells, and in primary type II cells were enhanced after stimulation. The acute-phase nature of LBP production by lung epithelial cells was confirmed by the observation that dexamethasone induced LBP release, and enhanced cytokine induced LBP release. Analysis of time kinetics revealed that LBP release was evident 24 h after stimulation and continued for 72 to 96 h, characteristic for acute-phase protein production. Further, it was shown that both the lung epithelial cells and the liver epithelial cells, similar to those described elsewhere (31), released LBP as a 60-kD protein. This suggests that lung epithelial cells process LBP as do hepatocytes, which are known to produce LBP as a 50-kD protein, which in turn is released after glycosylation as 60-kD protein (42). Together, these data indicate that LBP production by lung epithelial cells is comparable to LBP production by liver epithelial cells.
Using an ELISA we demonstrated that LBP produced by A549 cells binds LPS, which is strongly indicative for biologic activity of this protein. The role of local LBP production in lungs is potentially important. LBP was demonstrated to enhance sensitivity of alveolar macrophages for endotoxin via a CD14-dependent pathway, resulting in production of cytokines (14), a process in which the role of TLR-2 and TLR-4 has to be elucidated. This indicates that in the presence of LBP the local defense system is activated by low concentrations of LPS or as a result of a mild bacterial load. On the basis of the present findings we speculate that cytokines produced by activated alveolar macrophages stimulate the alveolar epithelium to produce increased amounts of LBP, indicating that LBP may be an inflammatory response product in the airways. This could partly explain the increase in LBP in the lungs reported in patients with ARDS and with asthma after local allergen challenge (14-16). This de novo synthesized LBP may further potentiate defense reactions mediated by alveolar macrophages and neutrophils, including phagocytosis and subsequent clearance of bacteria (10). Alternatively, inasmuch as LBP is known to function as a lipid transport protein (43), LBP in the lung could contribute to neutralization of LPS via transfer of LPS into phospholipids, which are abundantly present in surfactant lining the alveolar space. Further studies are required to study the role of LBP produced in lungs in the defense to bacterial endotoxin, and its contribution to progression or inhibition of the lung inflammatory reaction.
LBP in the circulation is reported to be associated with apolipoprotein A1, a compound of HDL (7). So far it is not known whether LBP is secreted by lung epithelial cells alone or attached to other proteins. It is tempting to speculate that the association of LBP with surfactant proteins could affect interaction between these proteins and bacteria (40).
Several reports have indicated a clear association between bacterial endotoxins and lung disorders, thus implicating a role for locally produced LBP. Studies on occupational diseases such as grain or swine dust–induced respiratory diseases have revealed that lung disorders are clearly related to endotoxin exposure (12, 13). In addition, endotoxin is a constituent of urban air particulate matter < 10 μm in aerodynamic size, causing the particle-associated inflammatory responses, and in this way could contribute to the adverse respiratory health effects of these particles (44, 45). Alternatively, the frequent bacterial colonization in lower airways of chronic bronchitis and cystic fibrosis patients (46) suggests an impaired local defense to bacteria of these patients, which could possibly be due to disregulated LBP production. On the other hand, the observation that dexamethasone has a prominent inducing effect on LBP production by the lung epithelial cell lines could have implications for patients treated with steroids, as patients with asthma are, resulting in enhanced levels of locally produced LBP. To elucidate the role of LBP in the pathogenesis of lung disorders, studies on LBP production in human lungs during health and disease will have to be performed.
In summary, this study shows local production of the endotoxin binding protein LBP in human lung, evidenced by the presence of LBP mRNA. The type II pneumocyte-like epithelial cell lines A549 and C10 were shown to produce LBP, and expression was also detected in human primary type II pneumocytes. These data confirm the ability of respiratory epithelium to produce acute-phase proteins. The local presence of LBP may contribute significantly to host defense to bacterial endotoxins and bacteria.
The authors thank Dr. J. G. Maessen for assistance in providing lung tissue, and P. Groothuis and Dr. R. Kuijer for the primer sequence of the GAPDH PCR. This work was supported by a grant from Glaxo Wellcome, The Netherlands.
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Abbreviations: bronchoalveolar lavage fluid, BALF; bovine calf serum, BCS; complementary DNA, cDNA; Chinese hamster ovary, CHO; CHO cells transfected with human LBP cDNA, CHO-LBP; enzyme-linked immunosorbent assay, ELISA; fetal calf serum, FCS; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; human, h; immunoglobulin, Ig; interleukin, IL; LPS-binding protein, LBP; lipopolysaccharide, LPS; monoclonal antibody, mAb; messenger RNA, mRNA; phosphate-buffered saline, PBS; polymerase chain reaction, PCR; recombinant, r; reverse transcriptase, RT; standard deviation, SD; serum-free, SF; Toll-like receptor, TLR; tumor necrosis factor, TNF.
