Abstract
Pulmonary alveolar proteinosis (PAP) is a disease characterized by the filling of alveolar spaces with periodic acid-Schiff-positive proteinaceous material and by the hypertrophy of type II pneumocytes in the alveolar interstitium. To examine if KL-6, a mucinlike glycoprotein, is useful for the diagnosis of PAP and the estimation of its activity, serum KL-6 levels in patients with PAP were measured by an enzyme-linked immunosorbent assay and compared with those of patients with other lung diseases. Furthermore, to estimate the origin of KL-6 in some patients, measurements of KL-6 levels in bronchoalveolar lavage (BAL) fluid and immunohistochemical staining of the lung tissues with a monoclonal antibody to KL-6 antigen were performed. Serum KL-6 levels in patients with PAP were extremely high and were significantly higher than those in patients with interstitial lung diseases in which elevation of serum KL-6 has been recognized. BAL-fluid KL-6 levels in patients with PAP were higher than serum levels. Both serum and BAL-fluid KL-6 levels in patients with PAP correlated well with the disease activity. Immunohistochemically, positive staining was observed in proliferating type II pneumocytes. These results suggest the usefulness of KL-6 measurement in the diagnosis and estimation of disease activity of PAP.
Pulmonary alveolar proteinosis (PAP) is a disease of unknown etiology. As for the histologic appearance of lungs with PAP, the alveolar spaces and some of the respiratory bronchioles are filled with periodic acid-Schiff-positive proteinaceous material. The alveolar septa are usually normal except for type II pneumocyte proliferation. Lymphocytes and macrophages are sometimes seen in the interstitium (1, 2). The pathogenesis of PAP has been reported to involve overproduction of surfactantlike materials by type II alveolar pneumocytes in the alveolar lumen and/or defective clearance of these materials by type II alveolar pneumocytes and macrophages (3-6). The diagnosis of PAP was established by the combination of clinical characteristics, but histologic confirmation by transbronchial or open lung biopsy is usually required. Bronchoalveolar lavage (BAL), which is also an invasive procedure, may be diagnostic in certain instances.
Recently, serum and BAL-fluid KL-6 levels have been proved to be useful for the diagnosis and estimation of the activity of diffuse interstitial lung diseases (7, 8). In addition, the origin of KL-6 in idiopathic pulmonary fibrosis (IPF) was suggested to be regenerating type II pneumocytes. Because hyperplasia of type II pneumocytes is one of the major histopathologic findings in PAP, we hypothesized that KL-6 levels may be elevated in patients with PAP. To examine this hypothesis, we measured serum KL-6 levels in patients with PAP and compared them with those in patients with other diffuse interstitial lung diseases and with those in normal healthy control subjects. Furthermore, to estimate the origin of KL-6 in PAP in some patients, measurement of KL-6 levels in BAL-fluid and immunohistochemical staining of the lung tissues with a monoclonal antibody to KL-6 antigen were also performed.
In this study, we demonstrated that serum and BAL-fluid KL-6 levels were extremely high in patients with PAP and correlated well with clinical activity.
Sera of four patients (three males and one female with a mean age of 53.3 ± 1.5 [SE] yr) who had fulfilled the diagnostic criteria of PAP were examined (9). In all the patients, the diagnosis of PAP was confirmed by phospholipid analysis of BAL fluid and histopathologic findings of lung tissues obtained by transbronchial lung biopsy (TBLB). They had no environmental history indicating secondary alveolar proteinosis (10). None of them were receiving any treatment, including corticosteroids, at the time of diagnosis. All the patients had hypoxemia (PaO2 less than 70 mm Hg) in their arterial blood gas analyses, and their mean PaO2 was 63.0 ± 4.0 (SE) mm Hg (range, 59.0 to 67.0 mm Hg) while breathing room air. BAL-fluid samples were available from two patients with PAP. Lung tissues obtained by TBLB from two patients with PAP were available for immunohistochemical examinations. In one patient who was treated with partial lung lavage by using flexible fiberoptic bronchoscopy three times once a week and showed marked improvement, serial analyses of serum and BAL-fluid KL-6 were performed.
As disease control subjects, we selected patients with idiopathic pulmonary fibrosis (IPF), interstitial pneumonia associated with collagen vascular diseases (CVD), or hypersensitivity pneumonitis (HP). The IPF group was composed of 16 patients (10 male and six female with a mean age of 61.7 ± 3.9 [SE] yr) who fulfilled the diagnostic criteria described by King (11). Seven patients were receiving oral corticosteroids (prednisolone, 5 to 20 mg/d) and the others were receiving no therapy. Their mean PaO2 values while breathing room air was 74.2 ± 3.3 mm Hg. The CVD group was composed of 11 patients (all female, with a mean age of 55.2 ± 4.3 yr), four patients with concomitant Sjögren's syndrome, three with mixed connective disease, two with rheumatoid arthritis, and two with dermatomyositis. All but four patients with Sjögren's syndrome were administrated nonsteroidal anti-inflammatory drugs. The HP group was composed of five patients with farmer's lung disease (two male and three female with a mean age of 54.4 ± 3.2 yr) who fulfilled the diagnostic criteria described by Terho (12). None of them was receiving corticosteroids at the time serum samples were taken. As normal healthy controls, we used stored sera of 237 subjects (127 male and 110 female with a mean age of 49.8 ± 0.9 yr) who had received annual health checkups, including a chest roentgenogram, and were judged to have no illnesses.
Serum and BAL-fluid KL-6 levels were measured by a sandwich-type enzyme-linked immunosorbent assay (EIA) using a KL-6 antibody kit (ED046; Eisai, Tokyo) (13, 14). Polystyrene cups coated with KL-6 antibodies were incubated with 100 μl of serum diluted 201-fold or BAL fluid diluted 402-fold by dilution solution (1% bovine serum albumin, 0.1% NaN3, and 0.05 M Tris-HCl buffer; pH, 7.5) at room temperature for 2 h. They were then washed with 0.85% NaCl and incubated at room temperature for 1 h with 100 μl of horseradish-peroxidase-conjugated KL-6 antibody diluted 1,000-fold. The cups were washed again, and 100 μl of ABTS solution (1.5 mg/ml 2,2′-azino-bis 3-ethyl-benz-thiazoline-6-sulfonic acid), 0.02% H2O2, and 0.15 M citrate-phosphate buffer (pH, 4.2) were added and incubated at room temperature for 30 min. Finally, 100 μl of 0.013 M NaN3 were added to inhibit the peroxidase reaction, and the absorbance at 405 nm was measured.
To detect the localization of KL-6, we used an immunohistochemical staining technique with the anti-KL-6 antibody (15). TBLB specimens from two patients with PAP were immediately fixed and embedded in paraffin. Tissue sections 2 μm thick were deparaffinized and dehydrated through graded alcohols and then in phosphate-buffered saline (PBS). Endogenous peroxidase was blocked by the treatment with 0.3% (vol/vol) H2O2 in PBS for 20 min at room temperature. Nonspecific binding of the antibody was blocked with normal horse serum immunoglobulin. Sections were incubated in a humid chamber overnight at 4° C with 1 μg/ml KL-6 antibody. Normal mouse serum was used as a negative control. On the following day, sections were incubated at room temperature for 30 min with affinity-purified biotin-conjugated horse antimouse IgG, then incubated for 1 h at room temperature with horseradish-peroxidase-labeled streptavidin. Antibody complexes were visualized with light microscopy after incubation with diaminobenzidine. The development reaction was stopped by immersion in water. Sections were then counterstained with hematoxylin, dehydrated, mounted in Marinol (Muto Pure Chemicals LTD, Tokyo), and examined by light microscopy.
The comparisons of serum KL-6 among groups were done by one-way analysis of variance (ANOVA). Differences with a p value of less than 0.05 were considered significant.
The serum KL-6 levels in each group are shown in Figure 1. The mean KL-6 level of serum samples from four patients with the diagnosis of PAP was 6,119.3 ± 894.5 (mean ± SE) U/ml. Those for the IPF, CVD, HP, and normal control groups were 1,203.8 ± 264.0, 1,050.5 ± 416.6, 1,305.2 ± 42.0, and 206.5 ± 6.1 U/ml, respectively. The serum KL-6 level of the PAP group was significantly higher than those of the IPF, CVD, HP, and normal control groups (p < 0.001). Serum KL-6 levels of the IPF, CVD, and HP groups were significantly higher than those of normal control group (p < 0.001).
Fig. 1. Serum KL-6 levels of PAP, other disease control subjects, and normal control subjects. Mean serum KL-6 levels ± SEM are presented. Patients with PAP showed higher serum KL-6 levels than did other disease control subjects and normal control subjects. Patients with IPF, CVD, and HP showed significantly higher serum KL-6 levels than did normal control subjects. Open circles show the patients with corticosteroids, and closed circles show the patients without corticosteroids. PAP = pulmonary alveolar proteinosis; IPF = idiopathic pulmonary fibrosis; CVD = interstitial pneumonitis in association with collagen vascular diseases; HP = hypersensitivity pneumonitis. Single asterisk indicates p < 0.001 compared with normal control subjects; double asterisks indicate p < 0.001 compared with disease control subjects (IPF, CVD, and HP).
[More] [Minimize]Because serum KL-6 levels might be influenced by other factors such as hypoxemia and corticosteroid treatment, we divided the patients with IPF and CVD into two groups; those with hypoxemia (PaO2 less than 70 mm Hg while breathing room air, mean PaO2 of 64.2 ± 3.5 mm Hg) and without hypoxemia (83.3 ± 2.4 mm Hg). There was no significant difference in serum KL-6 levels between the patients with and those without hypoxemia (1,544.7 ± 367.5 and 1,102.3 ± 283.7 U/ml, respectively, p > 0.15). In addition, serum KL-6 levels of patients with PAP were significantly higher than those of patients with IPF or CVD with the same degree of hypoxemia (p < 0.0002). To examine the effect of the corticosteroid treatment, serum KL-6 levels in patients with IPF with and without the corticosteroid treatment were compared. There was no significant difference in serum KL-6 levels between patients with IPF and those without corticosteroids (1,339.0 ± 313.2 and 1,208.4 ± 345.6 U/ml, respectively, p > 0.5).
The KL-6 levels of BAL fluid of two patients with PAP were 19,404.0 and 16,152.0 U/ml, and they were higher than their serum KL-6 levels (6,016.0 and 7,018.0 U/ml, respectively). In one patient, both serum and BAL-fluid KL-6 levels were measured after treatment with repeated partial lung lavages. Both serum and BAL-fluid KL-6 levels were gradually decreased after partial lung lavages (Figure 2). These changes were associated with simultaneous improvement of pulmonary functions such as Dl CO and PaO2 and chest roentgenographic findings.
Fig. 2. Correlation of serum and BAL-fluid KL-6 levels and pulmonary functions in a patient with PAP. The patient was treated with partial lung lavage every week for 3 wk. After each lavage, serum and BAL-fluid KL-6 levels and pulmonary functions were measured. Both serum and BAL-fluid KL-6 levels were decreased with the improvement of pulmonary functions. BAL = bronchoalveolar lavage; Dl CO = diffusing capacity.
[More] [Minimize]In immunohistochemical staining of the lung tissues from two patients with PAP using the anti-KL-6 antibody, alveolar lining type II pneumocytes and proteinaceous material occupying the alveolar lumen showed positive staining (Figure 3). There seemed to be no clear staining on lymphocytes and alveolar macrophages.
Fig. 3. Immunohistochemical staining of lung biopsy specimens with anti-KL-6 monoclonal antibody. (A) Alveolar lining cells and proteinaceous materials showed positive staining. Original magnification: ×100. (B) Proliferating type II pneumocytes showed positive staining. There seemed to be no positive staining of infiltrating lymphocytes and macrophages. Original magnification: ×200.
[More] [Minimize]KL-6, a mucinlike, high-molecular-weight glycoprotein, was found to be a tumor marker, and its level is known to be elevated in patients with lung cancer, especially adenocarcinoma (14, 16). Recently, KL-6 was found to be a marker of diffuse interstitial lung diseases (7, 13).
In this study, we demonstrated that patients with PAP have extremely high levels of serum and BAL-fluid KL-6. It has been reported that serum KL-6 levels are elevated in patients with diffuse lung diseases, such as IPF, CVD, and HP (7, 13). In this study, we also demonstrated that patients with IPF, CVD, and HP had significantly higher serum KL-6 levels than did normal control subjects, which confirmed the results of previous reports. Furthermore, the serum KL-6 level in patients with PAP was significantly higher than those in patients with other diffuse interstitial lung diseases. Previous reports and our own measurements indicated that KL-6 levels in patients with IPF, CVD, and HP are usually less than 3,000 U/ml (7, 8, 13). However, all the patients with PAP in this study showed more than 3,000 U/ml. Because serum KL-6 levels might be influenced by the presence of hypoxemia in patients with PAP, we examined the effect of hypoxemia in patients with IPF and CVD. However, there was no significant difference in serum KL-6 levels between the patients with and those without hypoxemia. In addition, serum KL-6 levels in patients with PAP were significantly higher than those of patients with IPF with the same degree of hypoxemia. For these reasons, extremely high levels of serum KL-6 may indicate a diagnosis of PAP. In addition, serial measurements of serum and BAL-fluid KL-6 levels in one patient with PAP revealed that they were gradually decreased with the improvement of pulmonary functions and chest roentogenographic findings (Figure 2). This also suggests that KL-6 measurements are useful for the estimation of disease activity in PAP.
Histopathologically, the proliferation of type II pneumocytes is one of the common characteristics of IPF, CVD, and HP. This finding is also one of the pathologic characteristics of PAP (1, 2). KL-6 is known to be expressed on type II pneumocytes and respiratory bronchiolar epithelial cells in the normal lung (14). Proliferating and regenerating type II pneumocytes in pneumonitis are reported to express KL-6 antigen more strongly than normal type II pneumocytes (7, 17). In this study, we were able to measure the BAL-fluid KL-6 level and perform immunohistochemical staining of the lung tissues in two patients with PAP. BAL-fluid KL-6 levels were also elevated and were higher than serum levels. KL-6 immunoreactivities were observed in type II pneumocytes and proteinaceous materials in alveolar spaces, but not in lymphocytes and alveolar macrophages. These findings suggest that the origin of the elevated KL-6 in PAP is also proliferating type II alveolar pneumocytes.
In summary, we measured serum and BAL-fluid KL-6 levels in patients with PAP and found the possible usefulness of KL-6 measurements in the diagnosis and estimation of the disease activity of PAP. Because the number of patients with PAP in this study was small, further studies with large numbers of the patients are required to evaluate further roles of serum KL-6 levels in PAP.
The writers thank Dr. Nobuoki Kohno (Second Department of Medicine, Ehime University, Japan) for his kind gift of the anti-KL-6 monoclonal antibody, and Dr. Yoshio Kuroki (Department of Biochemistry, Sapporo Medical University, Japan) for his kind gift of patient serum.
| 1. | Spencer, H. 1985. Pulmonary oedema and its complications and the effects of some toxic gases and substances on the lung. In H. Spencer, editor. Pathology of the Lung. Pergamon Press, Oxford. 726–731. |
| 2. | Dail, D. H. 1987. Metabolic and other disease. In D. H. Dail and S. P. Hammar, editors. Pulmonary Pathology. Springer-Verlag, New York, 535–588. |
| 3. | King, T. E., Jr. 1993. Idiopathic pulmonary fibrosis. In M. I. Schwarz and T. E. King, Jr., editors. Interstitial Lung Disease, 2nd ed. Mosby-Year Book, St. Louis, MO. 309–366. |
| 4. | Kariman K., Kylstra J. A., Spock A.Pulmonary alveolar proteinosis: prospective clinical experience in 23 patients for 15 years. Lung1621984223231 |
| 5. | Larson R. K., Gardinier R.Pulmonary alveolar proteinosis: report of six cases, review of the literature, and formulation of a new therapy. Ann. Intern. Med.621965292312 |
| 6. | Golde D. W., Territo M., Finley T. N., Clime M. J.Defective lung macrophages in pulmonary alveolar proteinosis. Ann. Intern. Med.851976304309 |
| 7. | Kohno N., Kyoizumi S., Awaya Y., Fukuhara H., Yamakido M., Akiyama M.New serum indicator of interstitial pneumonitis activity. Chest9619896873 |
| 8. | Kohno N., Awaya Y., Oyama T., Yamakido M., Akiyama M., Inoue Y., Yokoyama A., Hamada H., Fujioka S., Hiwada K.KL-6, a mucin-like glycoprotein, in bronchoalveolar lavage fluid from patients with interstitial lung disease. Am. Rev. Respir. Dis.1481993637642 |
| 9. | Claypool W. D., Rogers R. M., Matuschak G. M.Update on the clinical diagnosis, management, and pathogenesis of pulmonary alveolar proteinosis (phospholipidosis). Chest851984550558 |
| 10. | Singh G., Katyal S. L., Bedrossian C. W. M., Rogers R. M.Pulmonary alveolar proteinosis: staining for surfactant apoprotein in alveolar proteinosis and in conditions simulating it. Chest8319838286 |
| 11. | King, T. E., Jr. 1993. Idiopathic pulmonary fibrosis. In M. I. Schwarz and T. E. King, Jr., editors. Interstitial Lung Disease, 2nd ed. Mosby-Year Book, St. Louis, MO. 367–412. |
| 12. | Terho E. O.Diagnosis criteria for farmer's lung disease. Am. J. Ind. Med.101986329 |
| 13. | Kobayashi J., Kitamura S.KL-6: a serum marker for interstitial pneumonia. Chest1081995311315 |
| 14. | Kohno N., Akiyama M., Kyoizumi S., Hakoda M., Kobuke K., Yamakido M.Detection of soluble tumor-associated antigens in sera and effusions using novel monoclonal antibodies, KL-3 and KL-6, against lung adenocarcinoma. Jpn. J. Clin. Oncol.181988203216 |
| 15. | Kyoizumi S., Akiyama M., Kouno N., Kobuke K., Hakoda M., Jones S. L., Yamakido M.Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis. Cancer Res.45198532743281 |
| 16. | Kohna N., Hamada H., Fujioka S., Hiwada Y., Yamakido M., Akiyama M.Circulating antigen KL-6 and lactate dehydrogenase for monitoring irradiated patients with lung cancer. Chest1021992117122 |
| 17. | Hamada H., Kohno N., Akiyama M., Hiwada K.Monitoring of serum KL-6 antigen in patient with radiation pneumonia. Chest1011992858860 |
