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(Chest. 2004;125:617-625.)
© 2004 American College of Chest Physicians

Increased Surfactant Protein-A Levels in Patients With Newly Diagnosed Idiopathic Pulmonary Fibrosis*

David S. Phelps, PhD; Todd M. Umstead, BS; Mayra Mejia, MD; Guillermo Carrillo, MD; Annie Pardo, PhD and Moises Selman, MD, FCCP

* From the Department of Pediatrics (Dr. Phelps and Mr. Umstead), Penn State College of Medicine, Hershey, PA; National Institute of Respiratory Diseases (Drs. Mejia, Carrillo, and Selman), Mexico City, Mexico; and Faculty of Sciences (Dr. Pardo), National Autonomous University of Mexico, Mexico City, Mexico.

Correspondence to: David S. Phelps, PhD, Department of Pediatrics, Penn State College of Medicine, PO Box 850, Hershey, PA 17033; e-mail: dsp4{at}psu.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: To measure surfactant protein-A (SP-A) in the BAL of patients with idiopathic pulmonary fibrosis (IPF).

Design: We examined SP-A in BAL and lung tissue of patients with IPF who met the stricter recommended criteria for IPF at the time of diagnosis and prior to the beginning of treatment.

Patients: Twenty-six patients with IPF confirmed at biopsy and 22 patients with hypersensitivity pneumonitis (HP) were compared with 9 normal volunteers.

Interventions: All patients were subjected to pulmonary function testing, BAL, and lung biopsy prior to the beginning of treatment.

Measurements and results: We measured SP-A in BAL fluids and performed SP-A immunohistochemistry on lung specimens. Lung tissues of patients with IPF showed extensive type II cell hyperplasia, usually containing greatly increased levels of immunoreactive SP-A. By enzyme-linked immunosorbent assay, we found a twofold increase over normal values in BAL SP-A without changes in total phospholipids. These data were in agreement with semiquantitative assessments of SP-A by protein immunoblotting and by Western blotting of sodium dodecyl sulfate gels. Patients with HP exhibited a threefold increase of BAL SP-A.

Conclusions: The reasons for the difference between our results and previously published reports describing decreased SP-A levels in IPF is not clear. It may relate to the stricter criteria for diagnosis, the absence of treatment prior to BAL, differences in the patient population, or to other methodologic differences.

Key Words: hypersensitivity pneumonitis • idiopathic pulmonary fibrosis • interstitial lung disease • surfactant • surfactant protein-A


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by fibroblast proliferation, remodeling, and excessive extracellular matrix (ECM) deposition. IPF is usually fatal, and treatments are ineffective. An international consensus statement1 better defined IPF and provided physicians with guidelines for its diagnosis and management. Currently diagnosis of IPF is restricted to patients who meet several clinical, functional, and high-resolution CT criteria, and with biopsy specimens showing usual interstitial pneumonia (UIP). Previously, IPF included other idiopathic interstitial pneumonias such as nonspecific interstitial pneumonia, desquamative interstitial pneumonia, or bronchiolitis obliterans organizing pneumonia. This heterogeneity resulted in confusion about IPF pathogenesis and natural history, and reports of occasional patients responding well to usually ineffective treatments. IPF was thought to be an inflammatory condition, and anti-inflammatory agents, although often unsuccessful, have been widely used.

Surfactant protein-A (SP-A) and surfactant phospholipids are involved in the regulation of many lung host-defense functions,2 3 4 including defense against various pathogens and regulation of inflammatory processes. Studies5 6 implicate these surfactant components in the regulation of matrix metalloproteinases and ECM components and potentially in the repair and remodeling of damaged tissue. These different functions are essential for the maintenance of healthy lungs, but their abnormal regulation may adversely affect structure and function. Increased breakdown or synthesis of ECM components and excessive or insufficient inflammatory responses may occur in conditions such as ARDS, emphysema, or IPF.

Questions remain about whether surfactant alterations cause or contribute to these disturbances or they result from them. Measurement of surfactant components, primarily SP-A and surfactant protein-D (SP-D), in BAL fluid of patients with lung disease may help resolve these questions.4 7 8 9 10 SP-A is a collagenous lectin or collectin involved in the regulation of lung host-defense function. Both SP-A and phospholipids affect the function of alveolar macrophages, other immune cells, and lung fibroblasts.5 11 Often these two surfactant components exert opposing effects on cells they contact, raising speculation that surfactant alterations may significantly affect lung homeostasis.4

SP-A measurements in IPF and hypersensitivity pneumonitis (HP) in previous studies12 13 14 have found elevated SP-A in the BAL fluid of HP, while IPF findings have varied.15 16 17 18 Apparent discrepancies in IPF studies may result from differences in diagnostic criteria for IPF, the status of disease and/or treatments, or technical issues. Some of these studies may have included patients with other idiopathic interstitial pneumonias, in addition to UIP. Importantly, in some cases BAL samples were obtained from patients who were already undergoing treatment with corticosteroids,15 16 which inhibit SP-A production in humans.19 20

To resolve these discrepancies we assessed SP-A BAL levels by several methods and performed immunohistochemistry on biopsy specimens in untreated patients with newly diagnosed IPF on their initial presentation. Diagnoses were subsequently confirmed by histopathologic examination of open-lung biopsy specimens.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
Patients were tested during their initial visit to the National Institute of Respiratory Diseases in Mexico City. The project was approved by the Ethics Committee, and informed consent was obtained from all subjects. For reference, healthy volunteers with normal chest radiography and spirometry findings were subjected to BAL. For histology, normal lung tissue was obtained from grossly normal margins of tumors or patients who died from causes unrelated to pulmonary disease.

We studied 26 patients with IPF whose diagnoses were supported by clinical, radiologic, high-resolution CT, and functional findings and corroborated by lung biopsy specimens showing features of UIP.1 21 Twenty-two patients with HP induced by avian antigens were also studied. Diagnosis of HP was obtained as described,22 including the following: (1) pigeon exposure preceding disease and serum antibodies against avian antigens; (2) dyspnea with partial improvement on avian antigen avoidance; (3) clinical, radiologic, and functional features compatible with HP; (4) BAL lymphocytes > 40%; and (5) biopsy morphology consistent with HP. No IPF or HP patients were treated with corticosteroids or other immunosuppressive drugs before BAL or biopsy.

BAL
BAL with six 50-mL aliquots of saline solution was performed. Pooled BAL was filtered through gauze to remove mucus and centrifuged (250g for 10 min) at 4°C; the supernatant frozen at - 70°C until assay. There were no significant differences in the recovered fluid volumes between patients and control subjects (Table 1 ). Cell pellets were resuspended in 1 mL of phosphate-buffered saline solution (PBS), and differential cell counts were performed. Other aliquots were fixed in 50% ethanol and 20% Carbowax (50% polyethylene glycol) [Richard-Allan Scientific; Kalamazoo, MI], and two slides per sample were stained with hematoxylin-eosin and used for differential cell counts. Total BAL protein was determined using the Micro BCA assay (Pierce; Rockford, IL) using ribonuclease A as a standard. Phospholipid content in BAL was measured using the Phospholipids B assay (Wako Chemicals; Richmond, VA) as recommended. When this assay is used to measure the phospholipid content of commercially available surfactant lipid preparations, the values obtained are identical to those provided by the manufacturer of the surfactant preparation. SP-A content of the BAL fluid was assessed by three different methods as described below.


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  		Table 1. Baseline Characteristics of the Study Populations*

 
SP-A Enzyme-Linked Immunosorbent Assay
SP-A was assayed by indirect enzyme-linked immunosorbent assay (ELISA). The preparation of the rabbit polyclonal antibody to human SP-A used in the ELISA and the method for purifying the SP-A for use as a standard protein have been described.23 The antibody recognizes SP-A with high affinity and does not appear to cross-react with SP-D or the mannose binding protein. For the ELISA, flat-bottom Immulon 2 ELISA plates (Dynex; Chantilly, VA) were used. Standard wells were coated with serial dilutions of SP-A starting at 50 ng of SP-A in 100 µL of 0.05 mol/L carbonate-bicarbonate buffer (pH 9.6). BAL samples were diluted by adding 5 µL of BAL sample to 995 µL of coating buffer and applying 100 µL of the diluted BAL to duplicate wells on the plate. After coating, the plates were washed and then blocked with 0.5% Tween-20 in PBS. IgG fraction was prepared from the polyclonal antiserum to SP-A by protein A affinity chromatotography and 100 µL of PBS, 0.05% Tween-20 containing 0.2 ng of IgG was added to each well. After a 1-h incubation at 37°C, the plate was washed and 100 µL of goat anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad; Hercules, CA; diluted 1:2500) was added to each well. After incubating for 1 h, the plate was washed again, color development performed with an O-phenylene diamine dihydrochloride substrate, and the reaction terminated by the addition of sulfuric acid. Optical density was measured immediately at 490 nm, and the SP-A concentration was determined using Sigma Plot (SPSS; Chicago, IL) by comparison of the samples with the SP-A standard curve.

Gel Electrophoresis and Western Blotting
All samples were also subject to electrophoresis on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunostained. For this method, a 20-µL aliquot of BAL fluid was mixed with 10 µL of 3 x sodium dodecyl sulfate sample buffer without 2-mercaptoethanol and 3 µL applied to 12.5 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (0.5 mm thick). Gels were poured using GelBond PAG film (Amersham Biosciences; Piscataway, NJ) as a backing. Electrophoresis was performed with a Multiphor II (Amersham Biosciences) apparatus. Samples were applied in molded silicon sample application wells placed on top of the gels, run at 20 mA for 30 min at 15°C, the molded wells removed, the buffer strip advanced, and electrophoresis continued for 50 min at 50 mA at 15°C. For Western blots, gels were removed from the apparatus, stripped from the backing, and the protein transferred to nitrocellulose using a NovaBlot (Amersham Biosciences) semi-dry transfer apparatus for 2 h at 250 mA. The nitrocellulose was blocked overnight with 1% bovine serum albumin in PBS. Following blocking, the blots were incubated in anti–SP-A IgG (0.04 ng/mL) for 2 h, washed with PBS containing 0.5% Tween-20, and then incubated in goat anti-rabbit IgG conjugated with horseradish peroxidase (1:25,000) for 1 h. After washing the blot, enhanced chemiluminescence (ECL) was performed by incubating the blot in Western Lightning Chemiluminescent Reagent (Perkin Elmer Life Sciences; Boston, MA) for 1 min. The blot was then exposed to radiographic film, the film developed, and densitometry performed on the developed film. For densitometry, we scanned individual 3 x and 6 x bands, added the values, and plotted the total for comparison of SP-A assays.

Protein Dot Blotting
Protein dot blotting was done by diluting a 5-µL aliquot of BAL in 995 µL of Tris-buffered saline solution and applying 200 µL samples to nitrocellulose under vacuum in a dot blot apparatus. We also performed a standard curve similar to the one used for ELISA by adding various amounts of purified SP-A. After washing, immunostaining and ECL were performed as described above with the Western blots. Relative amounts of SP-A were determined by densitometry of the resulting radiograph films.

Immunohistochemistry
Lung tissue samples were fixed and embedded in paraffin by routine methods. Tissue sections were deparaffinized, rehydrated, and blocked with 3% H2O2 in methanol for 30 min followed by antigen retrieval performed with citrate buffer (10 mM, pH 6.0) for 5 min in a microwave oven.22 Tissue sections were then incubated with an antibody diluent with background reducing components (Dako; Carpinteria, CA) diluted 1:100 in PBS for 45 min. Polyclonal anti–SP-A antibody diluted 1:1000 was applied and incubated overnight at 4°C. A secondary biotinylated anti-immunoglobulin antibody followed by horseradish peroxidase-conjugated streptavidin (BioGenex; San Ramon, CA) was used as recommended. 3-amino-9-ethyl-carbazole (AEC; BioGenex) in acetate buffer containing 0.05% H2O2 was used as substrate. The sections were counterstained with hematoxylin. The primary antibody was replaced by nonimmune serum or omitted completely for negative control slides.

Statistical Analysis
SP-A ELISA, lipid, and total protein data were analyzed using Sigma Plot and Sigma Stat (SPSS; Chicago, IL). In each case, data from the individual disease groups were compared to the control group by t test. Nearly all of these data sets passed normality tests permitting the parametric analysis. In one case where the normality test failed, groups were compared with the Mann-Whitney rank-sum test. For the correlations, the Pearson product moment correlation test was used to calculate R values.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Baseline Characteristics of IPF and HP Patients
Demographic data, pulmonary function test results, and BAL differential cell counts are summarized in Table 1 . IPF and HP patients exhibited clinical radiologic and functional evidence of interstitial lung disease, with variable degrees of dyspnea, decreased lung capacities, and hypoxemia at rest that worsened during exercise. In general, the patients with IPF were older, more likely to be male, and more likely to have been cigarette smokers than the patients with HP. There are no indications in the published literature that age or gender affect surfactant levels in adults. In the HP group, differential cell counts in BAL fluid were characterized by a marked lymphocytosis, usually > 40%, while in IPF most BAL inflammatory cells were macrophages.

Total Protein and Lipid Content in BAL
All BAL samples were assayed for total protein content (Fig 1 ). The processed data are presented as box plots. The boxes are defined by the 25th and 75th percentiles and divided by the median values. The error bars indicate the 10th and 90th percentiles. We measured 45.0 ± 7.73 µg/mL (mean ± SEM) of protein in samples from normal volunteers (n = 9). Samples from patients with IPF (n = 26) had 120.19 ± 18.01 µg/mL of protein, and those with HP (n = 22) had 306.73 ± 39.88 µg/mL of protein. The samples from both disease groups showed a significant increase in total protein compared with those of the normal volunteers (p < 0.001). These findings are in general agreement with previously published studies.15 17 24



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  		Figure 1. Box plot of BAL total protein data. Total protein determinations on BAL fluid from normal volunteers (n = 9), IPF patients (n = 26), and HP patients (n = 22) were calculated and are summarized by this box plot. The limits of the box plot are the 25th and 75th percentiles of the data sets, and the error bars indicate the 10th and 90th percentiles. The bar within the box represents the median for the data set.

 
Using the Phospholipids B assay, we measured the phospholipid content of the BAL fluid (Fig 2 ). Normal volunteers had 11.05 ± 1.27 µg/mL of lipid. IPF patients and HP patients had 11.1 ± 0.95 µg/mL and 12.55 ± 1.33 µg/mL, respectively. Neither of these lipid values varied significantly from the control subjects. Although these determinations show no change in amounts of total phospholipids, they do not address the possibility that the relative amounts of individual phospholipids may be altered.



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  		Figure 2. Box plot of BAL phospholipid determinations. Total phospholipid content of BAL fluid was determined with the Phospholipids B assay, and the data were analyzed. The data are summarized in the box plot as for Figure 1 .

 
SP-A Determinations
SP-A ELISAs were performed on the BAL fluid of normal volunteers, patients with IPF, and patients with HP (Fig 3 ). In all cases, the recovery volumes of lavage fluid were similar (Table 1) . ELISA SP-A levels in the patients with IPF had a mean value of 7.99 ± 0.81 µg/mL vs 4.77 ± 0.73 µg/mL in the normal volunteers. SP-A levels in patients with HP had a mean of 13.11 ± 1.01 µg/mL. Statistical analysis indicates that both of these groups are significantly different from normal values (p < 0.001). Although not shown, SP-A normalized by total phospholipids (SP-A/phospholipid) was also significantly increased in both groups of patients (0.746 in patients with IPF and 1.287 in patients with HP) compared with normal volunteers (0.420).



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  		Figure 3. Box plot of BAL SP-A levels determined by ELISA. Levels of SP-A in BAL fluid were determined by ELISA and are summarized in the box plot. *Groups that are significantly different from the normal volunteer values. Specifics of the box plot are as described in Figure 1 .

 
Because of the apparent discrepancy between our SP-A ELISA data for patients with IPF and some of the previously published studies,15 16 17 18 we also measured SP-A with the two semiquantitative methods described above and subjected the radiographs resulting from ECL to densitometry. We performed nonreducing sodium dodecyl sulfate gel electrophoresis and Western blotting on all of the samples, because this technique also provided us with some information about the oligomeric structure of SP-A. The film of the Western blots shows a number of bands representing different oligomeric forms of SP-A (Fig 4 ), and the trimer (approximately 90 kd) and hexamer (approximately 180 kd) forms in particular. One gel depicts the control samples and IPF samples. The other gel shows the control and HP samples. The positions of various SP-A forms (3 x, 6 x) are indicated. There are also some light bands that represent higher order oligomers and some others that may represent proteolytic fragments of various SP-A oligomers. We know that there is no apparent cross-reactivity between the anti-SP-A antibody and SP-D or the serum proteins present in the BAL fluid under the conditions we employed (unpublished studies). The different SP-A bands (3 x and 6 x) were scanned individually and the values added together to give an estimate of SP-A levels. It is apparent even without densitometry that the lanes containing samples from patients with IPF are darker than those from the normal subjects, and those with the HP samples are darker than the IPF lanes.



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  		Figure 4. Nonreducing gel electrophoresis and immunostaining for SP-A. Aliquots of BAL were run on nonreducing sodium dodecyl sulfate gels, blotted, and immunostained with the antiserum for SP-A using ECL as a detection method. Top, A: The gel depicts normal samples and samples from the IPF patients. Bottom, B: normal and HP samples. The same normal samples are shown in both panels. Their intensity varies slightly between gels, and the values obtained in bottom, B were corrected to make the intensity of the normal subjects equivalent in both gels. Although levels vary considerably, the trend is similar to the SP-A ELISA data (Fig 3) and the SP-A protein dot blot data (Fig 5) . SP-A bands were quantified by laser densitometry of the autoradiograph, corrected as described above, and are plotted against the ELISA values in Figure 6 , bottom, B. The positions of various oligomeric forms of SP-A (3 x, 6 x) are indicated.

 
The other method we used was the dot blotting protocol. A potential advantage of this method is that it should detect all forms of SP-A without the technical issues sometimes faced with electrophoresis. As with the electrophoresis protocol, a fixed volume of BAL was applied rather than a given amount of protein. We performed a standard curve for this assay by blotting dilutions of purified SP-A and used this to obtain the values for the samples. We initially did serial dilutions of standard protein and other samples to ensure that our determinations were in the linear range of the detection curve and that the load was appropriate for the binding capacity of the filter and the amount of antibody used. A representative blot containing all of the samples analyzed by ELISA is shown (Fig 5 ). As with the electrophoretic assessment (Fig 4) , the higher levels in most of the IPF samples can be readily seen and the densitometry confirms the results of the other methods. We plotted the dot blot values against the values obtained by ELISA for all samples (Fig 6 , top, A). These two sets of data correlated well with one another as the graph shows (R = 0.866). We also plotted the values obtained by scanning SP-A bands on Western blots of gels against the values from the ELISAs (Fig 6 , bottom, B). These values also correlated very well with one another (R = 0.861).



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  		Figure 5. SP-A protein dot blots. Aliquots of BAL and purified SP-A standards (S) were diluted, blotted, and immunostained with SP-A antiserum. The staining intensity is consistent with the ELISA results (HP > IPF > normal). Densitometric values were determined for each of the dots, and approximate SP-A levels were determined by comparison with the standard values from the dots on the left. The densitometric values are used in the correlations in Figure 6 .

 


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  		Figure 6. Comparison of SP-A assays. In top, A, the densitometric values (optical density [O.D.] x area) for the protein dot blot (Fig 5) are plotted against ELISA values for the same individual (Fig 3) . In bottom, B, the densitometric values (O.D. x area) for the total of the SP-A bands on the immunostained Western blot (Fig 4) are plotted against the ELISA values from the same individual. R values are indicated for both correlations are shown. Circles indicate normal, triangles indicate IPF, and squares indicate HP.

 
SP-A Immunohistochemistry
Immunoreactive SP-A localization was evaluated in seven IPF, six HP, and three normal lungs, and representative sections are shown in Figure 7 . All IPF lungs showed extensive areas of type II epithelial cell hyperplasia and hypertrophy, and these cells were usually intensely stained with the SP-A antiserum (Fig 7 , top left, A). In areas of interstitial fibroblast proliferation and fibrosis, SP-A–immunopositive elongated alveolar epithelial cells were observed (Fig 7 , top center, B). Close to these fibroblastic foci, epithelial cell dropout was also noticed. Bronchiolar epithelial cells lining early honeycomb changes where alveoli have coalesced into irregular cystic spaces were negative for SP-A (Fig 7 , top right, C). Extensive SP-A alveolar epithelial cell staining was also noticed in lung biopsy samples from patients with HP, as exemplified in Figure 7 , bottom left, D. Samples of normal lungs exhibited thin alveolar septa and scattered type II cells that were positively stained for SP-A (Fig 7 , bottom center, E). Lung samples incubated without the primary specific antibody were negative (Fig 7 , bottom right, F).



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  		Figure 7. Lung localization of immunoreactive SPA in IPF fibrosis and HP. Top left, A: Intense intracytoplasmic SP-A labeling is observed in hyperplastic type 2 pneumocytes in IPF lung (40 x). Top center, B: IPF lung showing several positive alveolar epithelial cells closed to areas of fibroblast proliferation and collagen deposition (arrows; 40 x). Top right, C: IPF lung exhibiting an area of honeycomb change lined by SP-A–negative bronchiolar epithelium (arrows; 40 x). Bottom left, D: HP lung exhibiting SP-A–immunostained alveolar epithelial cells (40 x). Bottom center, E: Normal lung showing discrete staining in type 2 pneumocytes (40 x). Bottom right, F: negative control omitting the primary antibody (40 x).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several years ago, an international consensus statement was formulated to more tightly define the condition of IPF.1 This effort was due in part to the observed heterogeneity in IPF patient course and response to treatment and the realization that this variability could be the result of classifying several different conditions as IPF. In conjunction with this revised definition, there has also been a reconsideration of the theories behind IPF pathogenesis.25 It had been assumed that IPF was an inflammatory disease, and standard treatment had been based on the use of potent anti-inflammatory agents, particularly corticosteroids. However, responses to this therapy have been largely ineffective. The new concept of pathogenesis focuses on the alveolar epithelial cell injury and activation preceding fibroblast migration and proliferation and the formation of the fibroblastic foci and disordered repair.

During the development of fibrosis, there is intra-alveolar migration of fibroblasts and re-epithelialization of the alveolar wall by type II cell migration and hyperplasia, which may bring fibroblasts in contact with the fluid lining the alveoli, and surfactant in particular. SP-A, a component of surfactant, is a member of the collectin family of proteins, with an amino-terminal collagenous domain and a carboxy-terminal carbohydrate recognition or lectin domain. SP-A, like SP-D, another collectin, can influence phagocytosis,3 play a modulatory role in the production of cytokines by immune cells,26 27 28 29 and influence their expression of surface molecules30 and proliferative activity.31 32 In many cases, the principal surfactant lipids modulate or inhibit the activity of SP-A.4 It is also important to note, particularly in the context of IPF, that SP-A and the surfactant lipids can also influence the function of fibroblasts.5 11 The ability of surfactant constituents to modulate fibroblast function directly, as well as their role in the regulation of a number of cytokines that may be involved in regulating fibroblasts, raises the possibility that surfactant may play a role in the pathogenesis of some lung fibrotic disorders. It has been recently found that mice overexpressing lung interleukin-13, developed intra-alveolar and interstitial fibrosis and exhibit alveolar type II cell hypertrophy with a significant increase in surfactant phospholipids as well as surfactant proteins.33

Several investigators have examined surfactant components in IPF patients and SP-A in particular; these studies have produced varying results, with some15 17 34 35 reporting decreased levels of SP-A and another18 reporting no change. Given these discrepancies and the changes in the description of IPF and its pathogenesis, we reexamined the levels of SP-A in the BAL fluid of a cohort of patients with newly diagnosed IPF who that met all of the American Thoracic Society consensus criteria for IPF and whose diagnosis was corroborated by open-lung biopsy.1 21 Although the levels of SP-A varied widely, we found that SP-A levels were increased nearly twofold in patients with IPF over normal individuals, and nearly threefold in another group of interstitial lung disease patients with HP resulting from exposure to birds. There were no changes in the surfactant lipid levels in these groups. Although a previous study17 of BAL lipid content in IPF and HP patients has described unchanged values, others15 36 have reported lipid levels below control values.

To ensure that the SP-A values we reported, which differed from earlier reports, were not simply due to a different, polyclonal antibody-based ELISA, we coupled the ELISA to several other methods to measure SP-A levels. We prepared Western blots of nonreducing gels of BAL samples. Most of the SP-A on these blots was in oligomers of predictable size, but there were some intermediate size SP-A forms that may have been generated by in vivo proteolytic activity. If proteolytic activity does occur, it is possible that SP-A ELISAs employing monoclonal antibodies15 16 17 37 38 could underestimate SP-A levels if the epitope recognized by the monoclonal antibody has been removed or destroyed by the proteolysis. The presence of multiple oligomeric forms of SP-A also raises the question of whether each of these forms is proportionately bound by antibody. The similar results obtained with the different methods employed here suggest that this is not a major issue in our study. However, the above issues may be more important considerations in assays involving monoclonal antibodies.

The three methods we used for SP-A measurement were in general agreement and seem to be borne out by the immunohistochemical data showing strong labeling of SP-A in the alveolar epithelium. The high levels of SP-A in these cells are consistent with the increases we report in BAL fluid and also suggest the possibility that there could be either secretion or leakage of SP-A into the interstitium. Findings by several groups37 38 that SP-A can be found at low levels in serum and that these levels increase in various lung diseases may be a consequence of this possibility and an additional mechanism by which fibroblast function could be modulated. Moreover, it has been shown38 that serum SP-A as well SP-D is significantly elevated in patients with IPF and that the levels of these proteins are highly predictive of survival. However, it is not clear how these proteins enter the blood and whether they have any function there. It should also be noted that SP-A levels in the lung are increased fairly rapidly after the administration of bleomycin in a rodent model of bleomycin-induced pulmonary fibrosis.39

The basis for the different SP-A results among studies is not clear. It may relate to the stricter definition of IPF. At least one of the earlier studies17 also included patients with a pathologic picture of desquamative interstitial pneumonitis. In another case,16 some of the patients were already undergoing treatment with corticosteroids at the time of sample procurement. Corticosteroids have been shown to decrease human SP-A production19 20 and may have influenced the BAL levels of SP-A. The possibility that the SP-A ELISA influenced the findings should also be considered because the studies15 16 17 reporting decreased SP-A levels used the same SP-A monoclonal antibody-based ELISA. The likelihood that these different assays may be responsible for the reported differences is increased by the fact that SP-A levels in patients with HP have consistently been reported to be elevated12 13 14 and were significantly higher than control levels in our study as well, but were reported to be decreased in one of the studies17 that also found lower SP-A levels in IPF. Polyclonal antibody-based ELISAs were used in both our investigation and in the study18 reporting no significant change in SP-A. It should be noted that in the latter study, there was a trend toward increased SP-A levels in IPF, although statistical significance was not achieved. As mentioned earlier, the greater number of epitopes recognized by the polyclonal antibodies may have allowed the recognition of forms of SP-A in the patients with IPF that were slightly altered in their chemical or structural properties or by proteolysis, although we have no data to confirm this possibility. It is also possible that other study design or technical issues played a role in the differences, such as the BAL itself, the methods used to perform the BAL and prepare it for analysis (ie, buffer, centrifugal force), and the nature of the control group, as may be the case with a recent study where BAL was performed with 10-mL aliquots (vs 50 to 100 mL in most studies) and the control group to which the patients with IPF were compared were patients with cough and hemoptysis.35

The significance of the altered SP-A levels we report is not clearly understood at this time, nor is it clear whether these increases are a cause or a consequence of this disease. SP-A is altered in a variety of conditions. However, in each of these conditions there is a unique environment consisting of variable populations of cells, a complex mixture of mediators, changing surfactant lipid levels, and many other variables. As a result, it is very possible that increased SP-A could have a very different impact under different circumstances (ie, IPF vs HP). One could speculate that the target cell for SP-A in IPF might be the fibroblast, whereas in HP it may the lymphocyte. If this were indeed the case, very different effects would be seen. The significance of the elevated SP-A in IPF is not yet clear, but given the variety of roles that SP-A and surfactant lipids play in the regulation of immune cells and their ability to affect fibroblast function, the possibility should be considered that they could play a role in the pathogenesis of IPF and their roles need to be explored more thoroughly.


   Footnotes
 
Abbreviations: ECL = enhanced chemiluminescence; ECM = extracellular matrix; ELISA = enzyme-linked immunosorbent assay; HP = hypersensitivity pneumonitis; IPF = idiopathic pulmonary fibrosis; PBS = phosphate-buffered saline solution; SP-A = surfactant protein-A; SP-D = surfactant protein-D; UIP = usual interstitial pneumonia

This study was supported in part by National Institutes of Health HL-54683, Programa Universitario de Investigacion en Salud, and National Autonomous University of Mexico.

Received for publication January 13, 2003. Accepted for publication July 1, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. . American Thoracic Society (2000) Idiopathic pulmonary fibrosis: diagnosis and treatment; international consensus statement. Am J Respir Crit Care Med 161,646-664[Free Full Text]
  2. Wright, JR Immunomodulatory functions of surfactant. Physiol Rev 1997;77,931-962[Abstract/Free Full Text]
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T. Betsuyaku, Y. Kuroki, K. Nagai, Y. Nasuhara, and M. Nishimura
Effects of ageing and smoking on SP-A and SP-D levels in bronchoalveolar lavage fluid
Eur. Respir. J., December 1, 2004; 24(6): 964 - 970.
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