1Department of Medicine, School of Medicine, Keio University, Tokyo, 160-8582; 2Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kyoto, 602-8566; 4Department of Molecular and Internal Medicine, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, 734-8551; 5Department of Anesthesiology, School of Medicine, Keio University, Tokyo, 160-8582, Japan; 3Departments of Pediatrics and Medicine, School of Medicine, University of Utah, Salt Lake City, Utah 84132; and 6Medicine, Primary and Specialty Medicine Service Line, Veterans Affairs/Puget Sound Medical Center, Seattle, Washington 98108; and 7Medicine and Anesthesia, Cardiovascular Research Institute, University of California at San Francisco, San Francisco, California 94143
Submitted 9 December 2002 ; accepted in final form 26 August 2003
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ABSTRACT |
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alveolar type II cell; pulmonary edema; microsampling
Some biochemical markers have recently been described that predict the development of ALI/ARDS or the clinical outcome in patients with ALI/ARDS. For example, the appearance of type III procollagen peptide in edema fluid or bronchoalveolar lavage (BAL) fluid identifies patients with fatal outcomes, perhaps because it indicates that fibrosing alveolitis has begun early in the course of ALI (7, 8). Abnormalities of surfactant-associated proteins in BAL fluid and plasma have also been associated with poor clinical outcomes (3, 12). However, very few clinical studies have measured biochemical markers in ALI to assess pulmonary epithelial damage per se. The present study focused on the appearance of KL-6 in pulmonary epithelial lining fluid (ELF) and plasma. KL-6, a pulmonary epithelial mucin with low molecular weight, is an integral membrane glycoprotein classified as cluster 9 (MUC1) (14, 28), with an extracellular domain consisting mostly of tandem repeats of 20 amino acid sequences and a cytoplasmic tail (13). KL-6 splits off at the S-S bond near the epithelial membrane surface and becomes distributed in pulmonary ELF (13). This glycoprotein is mainly expressed on alveolar type II cells in the lung (20) and is expressed more prominently on proliferating, regenerating, or injured type II cells than on normal type II cells (19, 20). The presence of KL-6 has been used to monitor severity of disease in idiopathic pulmonary fibrosis (19).
The primary objectives of this study were 1) to determine the changes in KL-6 levels in plasma and ELF in patients with ALI to study injury to the lung epithelial barrier and 2) to test the hypothesis that elevated levels of KL-6 in plasma or ELF may be a useful biochemical predictor of outcome in ALI patients.
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METHODS |
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Control data were obtained from 18 men and 3 women, 3974 yr old, who underwent bronchoscopy so that causes of hemoptysis could be identified or small, solitary, peripheral pulmonary nodules could be examined. Chest computed tomography revealed no diffuse interstitial lung abnormality, and pulmonary function tests and SaO2 were normal. In addition, five patients with cardiogenic pulmonary edema (four men and one woman, 5477 yr old) that was confirmed by pulmonary artery wedge pressure >18 mmHg or echocardiogram were included as reference patients.
Bronchoscopic microsampling (BMS) was not performed if the patient met any of the criteria of ALI severity described by Steinberg et al. (29): 1) PaO2 < 80 mmHg with FIO2 = 1.0, 2) systolic blood pressure <90 mmHg, 3) complex ventricular arrhythmias, or 4) endotracheal tube diameter <7.0 mm.
In ALI patients, BMS of pulmonary ELF was performed on days 0 (onset of ALI) and 1, unless the patient was extubated or had died. Day 0 was defined as the day when the first BMS was done within 24 h after the diagnosis of ALI. In addition, blood was sampled on days 0, 1, 3, 5, 7, and 10. ELF and plasma were not sampled in two clinically unstable ALI patients on days 0 and 1. Control patients and patients with cardiogenic pulmonary edema underwent a single blood sampling and BMS.
BMS procedure.
All patients with lung edema were sedated and preoxygenated (FIO2 = 1.0). A flexible bronchoscope (BF-6C240; Olympus, Tokyo, Japan) was inserted into the lung through intratracheal tube for examination of the airway, and excess sputum was suctioned if present. Another identical bronchoscope was then inserted, and its tip was advanced into a segmental bronchus of the right middle lobe (S4 or S5). The BMS probe, recently developed in conjunction with Olympus (Tokyo, Japan), consists of a 1.7-mm-diameter polyethylene outer sheath and an inner fiber rod probe 1.2 mm in diameter and 30 mm in length attached to a stainless steel guide wire 100 cm in length. The BMS procedure has been described in detail previously (16). Briefly, the probe was inserted into the channel and gently advanced. While the outer sheath was held at the target in the subsegmental bronchus, the inner probe was advanced slowly into the peripheral airway until it was in contact with the mucosal surface and was maintained in that position for 57 s, thus allowing the fiber rod to absorb 20 µl of ELF. The inner probe was then withdrawn into the outer sheath, and both were removed together. The wet inner probe was cut, placed in a tube, and stored in a freezer at 80°C until analysis. The procedure was performed in triplicate from the same subsegmental bronchus. In control patients, after standard local anesthesia with lidocaine, a flexible fiber-optic bronchoscope was inserted into the right bronchus for the BMS procedure.
The stored frozen probes were weighed before the ELF saline suspension was prepared. We prepared the solution of diluted ELF for biochemical measurements by adding the three frozen probes, which had been inserted into the same lung subsegment, into a 15-ml polyethylene tube containing 3 ml of saline vortexed for 1 min. The solution was centrifuged for 15 min at 3,000 rpm, and the supernatant was collected. The probe was dried and weighed to calculate the ELF volume recovered from the BMS probes. The dilution factor was calculated as follows: ELF volume (ml)/[3 ml + ELF volume (ml)].
In vitro experiments confirmed that the absorption of 220 µl of human serum by the fiber rod probe allowed a >93% recovery of biochemical constituents. The recovery was 96.1% for albumin, 93.7% for lactate dehydrogenase (LDH), and 95.3% for KL-6.
Measurements of KL-6 and albumin. KL-6 measurements were performed by a sandwich enzyme-linked immunosorbent assay using anti-KL-6 mouse monoclonal antibodies as solid-phase and enzyme-labeled antibodies. These antibodies were prepared by intraperitoneal injection of anti-KL-6 mouse monoclonal antibody-producing cells. The mouse ascites fluid was purified with a protein A column (18). We prepared the antibody-producing cells by immunizing a mouse with pulmonary adenocarcinoma cells (VMRC-LCR cells) and fusing its splenocytes and myeloma NS1 cells by Kohler's and Milstein's method (17).
KL-6 measurements were performed in duplicate. For plasma samples, we carried out predilution by adding 10 µl of plasma specimen to 2 ml of a diluent (0.05 M Tris·HCl buffer, pH 7.5, containing 1% BSA and 0.1% wt/vol sodium azide). Then, 100 µl of a reaction solution (0.05 M Tris·HCl buffer, pH 7.5, containing 10% normal rabbit serum, 0.1% normal mouse serum, 10 mM EDTA, 0.15 M NaCl, and 0.1% wt/vol sodium azide) were added to each well of a 96-well plate coated with anti-KL-6 monoclonal antibodies. Twenty microliters of the sample and 20 µl of the standard antigen were then added. This preparation was incubated at 25°C for 2 h. A 0.05 M Tris·HCl buffer, pH 7.5, containing a detergent, was used to wash the inside of each well to remove unreacted substances. Next, 100 µl of an enzyme-labeled antibody solution were added and allowed to react at 25°C for 1 h. After being washed, 100 µl of an enzyme-substrate solution were added and allowed to react at 2030°C for 30 min. We prepared this enzyme-substrate solution by dissolving 6 mg of 2,2'-azino-di-3-ethyl-benz-thiazoline-6-sulphonic acid in 12 ml of a citrate buffer solution (0.28 M, pH 4.2) and then adding 30 µl of 3% hydrogen peroxide (H2O2). We stopped the reaction by adding 100 µl of 0.013% sodium azide, measured the absorbance (main , 405 nm; secondary
, 492 nm), and determined the concentration of KL-6 from a standard calibration curve.
The albumin was measured with colorimetric assay kits (Beckman, Fullerton, CA). The concentrations of KL-6 and albumin in ELF obtained by the BMS probe were expressed by unit volume of ELF after correction for the dilution factor.
Lung KL-6 immunostaining. Autopsy tissues from 12 patients admitted between 1996 and 2001 to the adult medical ICU of the University of Utah Hospital or Latter-Day Saints Hospital, in Salt Lake City, were analyzed. The samples were collected within 1214 h of death by standard anatomical pathology approaches optimized to preserve tissue architecture and antigen display. The tissues were collected from six patients who died with ALI or ARDS and from six patients who died of nonpulmonary causes. Patients who died of nonpulmonary disorders served as controls. The autopsy protocol was approved by the Institutional Review Board committees at both participating hospitals. The requirement for written informed consent was waived by both committees.
Two to three tissue cubes (>2 x 2 x 2 cm) were obtained from each of the three lobes of the right lung of each patient. The cubes were sliced (3 mm x 2 cm x 2 cm), and the slices were immersed in 10% buffered neutral formalin (VWR, Media, PA) overnight at 4°C. The slices were placed in 70% ethanol and processed immediately. Paraffin-embedded tissue sections (5 µm) were collected on PLUS slides (VWR), and the paraffin was removed from the sections before immunohistochemistry (15). We treated the tissue sections with methanol-H2O2 to block endogenous peroxidase activity. We inhibited nonspecific binding of the primary antibody by treating the tissue sections with blocking buffer. The primary anti-KL-6 monoclonal antibody was placed on the tissue sections (1:1,000 and 1:2,000 dilutions). After overnight incubation (4°C), the tissue sections were treated with a secondary antibody (horse anti-mouse). Antigen was detected by a standard peroxidase method (ABC Elite kit; Vector Laboratories, Burlingame, CA). Immunohistochemical staining controls included substitution of the primary antibody with an irrelevant, species-matched, immunoglobulin isotype-matched secondary antibody (anti-insulin), omission of the primary antibody (replaced with blocking buffer), and omission of the secondary antibody (replaced with blocking buffer). We counterstained the tissue sections with Gill's no. 3 hematoxylin and photographed them with a Zeiss AxioPhot microscope system equipped with a Jenoptik high-resolution color digital camera (ProgRes model 3012). Figure composition was performed with Adobe Photoshop, without alteration of image color or detail.
Colocalization of KL-6 protein with a marker of alveolar type II epithelial cells. KL-6 immunolocalization to the apical region of alveolar type II epithelial cells was performed by double immunofluorescence microscopy with both the KL-6 antibody described above and surfactant precursor protein B (SPPB) antibody (catalog no. NCL-SPPB; Novocastra Laboratories, Newcastle, UK). Double immunofluorescence was performed on tissue sections from the same blocks of lung tissue that were used for localization of KL-6 protein alone. All of the tissue sections were incubated with 1:50 dilution of SPPB overnight at 4°C, followed by incubation with 1:250 dilution of anti-mouse-biotin conjugate (catalog no. BA-2000, Vector Laboratories) at room temperature for 30 min. The sections were then incubated with 1:250 dilution of streptavidin-horseradish peroxidase conjugate (catalog no. NEL701, TSA Fluorescein kit; Perkin-Elmer, Boston, MA) at room temperature for 30 min. We performed signal amplification by incubating the tissue sections in a 1:250 dilution of tyramide FITC (TSA Fluorescein kit, Perkin-Elmer) at room temperature for 10 min. After several rinse steps, the same tissue sections were incubated with 1,000 dilution of the anti-KL-6 antibody at room temperature for 2 h, followed by incubation with anti-mouse IgG rhodamine conjugate (605140; Roche Molecular Biochemicals, Indianapolis, IN) at room temperature for 60 min (1). Immunofluorescence staining controls included substitution of each primary with normal horse serum, substitution of each primary antibody with an irrelevant, species-matched, isotype-matched antibody (anti-insulin), and substitution of the secondary antibody with phosphate-buffered saline (PBS). We used a Zeiss AxioPhot photomicroscope equipped for epifluorescence microscopy. Photographs were recorded on color slide film (Kodak ASA 400). The color slides were scanned (1,200 dpi) to prepare digital images for figure composition, which was then prepared for illustration using Adobe Photoshop, without alteration of image color or detail.
Isolation and culture of human alveolar type II cells. Alveolar epithelial type II cells were isolated by a modification of methods previously described (4, 911). Briefly, type II cells were isolated from human lungs that were not used by the Northern California Transplant Donor Network. Recent studies indicate that these lungs are in good condition physiologically and pathologically (31). Cells were isolated after the lungs had been preserved for 48 h at 4°C. The pulmonary artery was perfused with PBS solution at 37°C, and the distal air spaces were lavaged 10 times with warmed Ca2+-, Mg2+-free PBS solution containing 0.5 mM EGTA and EDTA. Then, 12.9 U/ml elastase in Ca2+-, Mg2+-free HBSS were instilled into the distal air spaces through segmental intubation. The lungs were minced finely in the presence of fetal bovine serum (FBS) and DNase (500 µg/ml). The cell-rich fraction was filtered by sequential filtration through one-layer gauze, two-layer gauze, 150-mm, and 30-mm nylon meshes. The resultant pellet was resuspended in DMEM containing 10% FBS, and then the cell suspension was incubated in tissue culture-treated plastic petri dishes in a humidified incubator (5% CO2, 37°C) for 90 min. The cell-rich solution was layered onto a discontinuous Percoll density gradient 1.041.09 g/ml solution and centrifuged at 1,500 rpm for 20 min. The recovered upper band contains a mixture of alveolar type II cells and alveolar macrophages. The cell-rich solution containing alveolar type II pneumocytes and macrophages was centrifuged at 800 rpm for 10 min. The resultant pellet was resuspended in DMEM containing 10% FCS. We then incubated the cells containing DMEM in magnet beads coated with anti-CD14 antibodies at 4°C for 40 min during constant mixing and then passed them over a magnetic column to separate alveolar macrophages (CD14 positive). The cell viability was assessed by trypan blue exclusion. The purity of isolated human alveolar type II cells was checked by Papanicolou staining or by anti-human type II cell antibody (a gift from Dr. Leland Dobbs, University of California, San Francisco) and was >90%. Freshly isolated alveolar type II cells were resuspended in cell preservation fluid and maintained at 80°C.
KL-6 secretion from human alveolar type II cells and distal lung epithelial cells.
Human alveolar type II cells were thawed and seeded in 24-well collagen I-coated plate. After 48 h, the cells are nearly confluent. At that time, we added to each well Cytomix (Boehringer Mannheim, Indianapolis, IN) containing IFN-, IL-1
, and TNF-
, each in final concentrations of 10 or 50 ng/ml (25), and the cells were incubated for 24 h. The supernatant was then recovered to measure the concentration of KL-6. The levels of LDH in the supernatant were also measured by colorimetric assay kit (Jisseikenn, Tokyo, Japan) to assess the degree of cell injury.
Distal lung epithelial cells (Clonetics, San Diego, CA) were subcultured in small airway epithelial cell growth medium (Clonetics). They were then incubated at 37°C in a humidified, 95% air-5% CO2 atmosphere. They were split upon reaching 6070% confluence and used before the 4th passage. Cells were grown on 24-well tissue culture plates (Costar, Cambridge, MA). The supernatants were removed when the distal lung epithelial cells were nearly confluent. At that time, we added Cytomix (10 ng/ml or 50 ng/ml) in each well, and the cells were incubated for 24 h. The supernatant was then recovered to measure the concentration of KL-6.
Statistical analyses. Statistical significance was defined as P < 0.05. Differences in variables between control and ALI patients and between survivors and nonsurvivors at each time point were compared by the nonparametric Mann-Whitney U-test, since the data were not normally distributed. Analyses were performed with receiver operating characteristics (ROC) curves for individual plasma KL-6 and KL-6 in ELF in predicting the prognosis of ALI. The in vitro results of KL-6 production from pulmonary cells were examined by one-way analysis of variance with multiple comparisons and Fisher's least significant difference test.
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RESULTS |
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The average recovery of ELF was 17.3 ± 1.8 µl in ALI patients, 16.2 ± 1.4 µl in patients without lung disease, and 20.1 ± 2.8 µl in patients with cardiogenic pulmonary edema (mean ± SE, not significantly different among the groups).
KL-6 and albumin levels in ELF and plasma. Hemorrhage, pneumothorax, significant changes in SaO2, or other complications were not observed during or after BMS. The albumin concentration in ELF at ALI onset was significantly higher than that in control patients (P < 0.001; Fig. 1, top). In five reference patients with cardiogenic pulmonary edema, the median concentration of albumin in ELF was 8 mg/ml.
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KL-6 and survival in ALI. In ALI patients, the KL-6 concentration in ELF was significantly higher in nonsurvivors than in survivors on day 0 and day 1 (P < 0.002, P < 0.05, respectively; Fig. 2). Plasma KL-6 concentrations were elevated on day 0 in ALI patients who died compared with survivors (P < 0.0001, Fig. 3). Also, the plasma concentration of KL-6 was significantly higher in nonsurvivors than in survivors throughout the entire clinical course (Fig. 3).
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DISCUSSION |
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The BMS procedure detected significant changes in alveolar epithelial permeability. The albumin concentration in the ELF of patients with ALI was significantly higher than in controls (Fig. 1), confirming prior results using BAL in patients with ARDS (26). Although ELF sampling with the BMS probe was done in distal airways 23 mm in diameter, the presence of high KL-6 levels at that level demonstrates that the BMS procedure can identify pathophysiological changes associated with damage to the alveolar epithelial barrier.
The high-molecular-weight pulmonary epithelial mucin KL-6 is an integral membrane glycoprotein that appears to be predominantly expressed on injured and/or activated alveolar type II cells (19). Recently, we reported that the localization of KL-6 protein appeared primarily on the apical surface of alveolar type II cells in postmortem ARDS patients (1). Accordingly, in the present study, we studied the production of KL-6 with isolated human alveolar type II cells in vitro. These studies indicate that KL-6 release significantly increased by stimulation by a mixture of proinflammatory cytokines, Cytomix. In this setting, LDH levels in the supernatant were normal even after Cytomix was added, suggesting that the Cytomix had not induced cell death. We did not directly measure cell proliferation in this assay, but it is well known that it is difficult to induce proliferation of primary adult type II cells in culture. Thus it is unlikely that cell proliferation was responsible for the elevation of KL-6 in the in vitro experiments, although we cannot rule out the possibility that proliferation of some alveolar type II cells occurred in the injured lungs, which conceivably contribute to elevated KL-6 levels in clinical ALI. Therefore, these results suggest that inflammatory cytokines that accumulate in the lung during ALI might induce the production of KL-6. The in vitro study of distal human lung epithelial cells indicated that these cells are also capable of producing KL-6 when stimulated by Cytomix. However, the production of KL-6 was nearly 70 times greater in alveolar type II cells. This result suggests that the distal lung epithelial cells are not the main source of KL-6. The postmortem immunohistochemical differences support this interpretation. We found prominent expression of KL-6 in alveolar type II cells confirmed by colocalization of SP-B in the ALI lungs. Alveolar barrier integrity to albumin was disrupted at the onset of ALI. However, despite the nearly 10-fold higher concentration of KL-6 in ELF of patients with ALI than in controls, the plasma levels at the onset of ALI were similar to controls, perhaps because of the relatively large molecular size of KL-6. The molecular mass of KL-6 antigen, which includes large amounts of saccharides, is estimated to be at least 1,0002,000 kDa, whereas that of albumin is 67 kDa. This molecular size difference may explain the slower diffusion of KL-6 through the paracellular pathway of lung endothelium and alveolar epithelium compared with albumin. The extent of alveolar septal barrier injury responsible for the leakage of albumin into the alveolar lumen may not have been sufficient to cause significant alveolar KL-6 leakage into the bloodstream. In some patients, increased plasma KL-6 concentrations were present at the onset of ALI. This early increase in plasma KL-6 concentration especially in nonsurvivors suggests that the alveolar barrier might be more disrupted in those patients.
The significant increase in KL-6 concentrations in ELF and plasma at the onset of ALI in nonsurvivors further supports the hypothesis that alveolar epithelial cell injury may be a crucial determinant of prognosis of ALI and that measurements of KL-6 might be useful to predict its outcome. From the ROC curve analyses, KL-6 levels in both ELF and plasma were sensitive and specific markers of fatal outcomes. Because these cut-off values were chosen by a post hoc analysis and the number of patients was rather small, the hypothesis should be validated in a large prospective study.
In conclusion, the BMS procedure is a safe and practical method to identify pathophysiological changes in the alveolar space in patients with ALI. KL-6 concentrations in ELF of patients with ALI were significantly increased at the onset of the disorder, and KL-6 in ELF at ALI onset was significantly higher in nonsurvivors than in survivors. These observations suggest the participation of proliferating, stimulated, and/or injured pulmonary epithelial cell in the pathogenesis of ALI, which may help define the prognosis of the disorder. Furthermore, plasma KL-6 in nonsurvivors remained significantly higher than in survivors throughout the 10-day observation period. This finding suggests that both the production of pulmonary KL-6 and disruption of the alveolar barrier could be associated with a poor prognosis.
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GRANTS |
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ACKNOWLEDGMENTS |
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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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