Abstract
The anti–granulocyte-macrophage colony–stimulating factor (GM-CSF) autoantibody is inferred to cause idiopathic pulmonary alveolar proteinosis (iPAP): the antibody neutralizes GM-CSF and thereby impairs differentiation of alveolar macrophages. Administration of GM-CSF improves respiratory function of patients with iPAP, as confirmed in this study using aerosolized GM-CSF. To elucidate its mechanism, we characterized bronchoalveolar lavage fluid and alveolar macrophages obtained from three patients with iPAP who were treated successfully with aerosolized GM-CSF. Cell number, expressions of surface mannose receptor and the transcription factor PU.1, and phagocytic ability of alveolar macrophages were all restored to control levels. With treatment, the neutralizing capacity of GM-CSF activity was reduced markedly, concomitant with the decreasing autoantibody levels. Interestingly, the amount of GM-CSF autoantibody complex also decreased. In one case in which the complex was analyzed, the majority of GM-CSF binding the complex was endogenous protein, suggesting that the complex is removed immediately from the lung after treatment. Our study shows that GM-CSF administration engenders a decrease in the neutralizing capacity against the protein in the lungs. Thereby, it facilitates restoration of the normal function of alveolar macrophages.
Pulmonary alveolar proteinosis (PAP) is an uncommon lung disease characterized by an accumulation of surfactant that fills terminal airways and alveoli, thereby impairing gas exchange and engendering respiratory insufficiency (1–3). Three clinically and etiologically distinct forms of PAP are acknowledged (congenital, secondary, and idiopathic), but more than 90% of cases are idiopathic (iPAP). In iPAP, respiratory symptoms initiate insidiously, with no precipitating event or illness. Alveolar macrophages from patients with iPAP show impaired chemotactic activity, reduced adhesion to glass, and poor phagocytosis (4). Dysfunction that impairs surfactant clearance of alveolar macrophages is considered responsible for iPAP (2–4).
A serendipitous observation first suggested that abnormalities of GM-CSF signaling may be involved pathogenically in iPAP: mice lacking the hematopoietic growth factor granulocyte-macrophage colony–stimulating factor (GM-CSF) or its receptor develop histologic changes similar to those seen in PAP (5–7). In mice, GM-CSF regulates the terminal differentiation of alveolar macrophages. It is necessary for normal catabolism of surfactant lipids and proteins (8). Genetic abnormalities of GM-CSF or its receptor were reported in a small number of patients with congenital PAP (9), but were not found in iPAP (2). Instead, all patients with iPAP evaluated so far have high titers of neutralizing anti–GM-CSF autoantibody. No more than trace amounts of the antibody were detected in patients with congenital or secondary PAP, other lung diseases, or healthy volunteers (10, 11). Taken together, loss of GM-CSF activity caused by the autoantibody cripples normal functions of alveolar macrophages, thereby reducing surfactant clearance.
Recombinant human GM-CSF is used clinically to stimulate bone marrow recovery in neutropenic patients and after bone marrow transplantation. Several investigators have administered GM-CSF subcutaneously to patients with PAP and have observed varied responses (12–15). A single case report describes a patient who was treated successfully using aerosolized GM-CSF (16). A recent study demonstrated that extrinsic GM-CSF administration restored expression of a transcriptional factor, PU.1, in alveolar macrophages, and thereby improved the maturation of alveolar macrophages in patients with PAP (17, 18). Considering the preexisting autoantibody, which binds GM-CSF with high avidity and specificity (19), it is unlikely that administered GM-CSF can directly stimulate immature alveolar macrophages by binding their GM-CSF receptors.
To investigate mechanisms of action of administered GM-CSF, we observed changes in the function of alveolar macrophages, together with changes in the neutralizing activity against GM-CSF and the amount of autoantibody in bronchoalveolar lavage fluid (BALF) of three patients treated with aerosolized GM-CSF. Results suggested that inhaled GM-CSF reduced the neutralizing capacity of BALF against GM-CSF with decreased concentration of both free autoantibody and the immune complex. Consequently, inhaled extrinsic GM-CSF might condition the alveolar microenvironment in the lung, allowing alveolar macrophages' functional recovery and clearance of proteinaceous materials. Some of the results of this study have been reported previously in the form of an abstract (20).
See the online supplement for further details on the methods.
The institutional review board approved this study. It was conducted after obtaining written, informed consent from each participant between December 2000 and October 2002. We treated a series of three individuals with iPAP (one man and two women; age range, 51–57 years) with aerosolized GM-CSF (Table 1)
Case 1 | Case 2 | Case 3† | |
|---|---|---|---|
| Age and sex | 51-yr female | 56-yr male | 57-yr female |
| Smoking | None | Ex-smoker | Smoker‡ |
| Diagnostic procedure | BALF, TBLB, Ab | BALF, TBLB, Ab | BALF, TBLB, Ab |
| Prior treatment | Left lung lavage | Oxygen treatment immunosuppressants* | Oxygen treatment |
| A-aDO2 decrease after GM-CSF inhalation (torr) | 17 | 20 | 27 |
Three 50-ml aliquots of normal saline were instilled and suctioned sequentially from the right middle lobe under bronchoscopy and processed immediately. Cells were stained by modified Giemsa; 400 nucleated cells were counted differentially in cytocentrifuge preparations. Then 200 alveolar macrophages were measured for lengthwise diameter and classified into the following two morphologic groups based on Iyonaga and colleagues (22): (1) nonfoamy, monocyte-like cells and (2) foamy cells.
BALF was incubated in a plastic culture dish at 37°C for 1 hour. After removing nonadherent cells by gentle washing, adherent cells (alveolar macrophages) were fixed and processed for Epon-embedded sections to be observed with a transmission electron microscope.
Alveolar macrophages, isolated as previously described, were suspended in Roswell Park Memorial Institute (RPMI)/10% fetal calf serum and plated in a four-well chamber slide (LabTek Chamber; Nunc, Rosklide, Denmark). After placing at 37°C for 2 hours, cells were incubated with 0.5% phycoerythrin (PE)-labeled latex beads (Sigma-Aldrich Corp., St. Louis, MO) for 30 minutes and fixed in 4% paraformaldehyde at 4°C for 15 minutes. Cells were then stained with a 1:3,000 dilution of Syber green (Dojindo Laboratories, Kumamoto, Japan). The alveolar macrophages that had phagocytosed beads were counted using a confocal laser microscope.
Alveolar macrophages were fixed with 4% paraformaldehyde and stained with antimannose receptor antibody (Beckman Coulter, Inc., Fullerton, CA) and horseradish peroxidase-labeled antimouse IgG antibody (Nichirei Corp., Tokyo, Japan) to examine expression of the mannose binding protein, a maturation marker for macrophages. We examined PU.1 expression by double immunostaining using a rabbit polyclonal anti-PU.1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which was detected using an alkaline phosphatase-labeled antirabbit IgG (Promega Corp., Madison, WI) and a mouse anti-CD68 monoclonal antibody labeled with horseradish peroxidase. Two pathologists independently determined quantification of a ratio of macrophages expressing PU.1. They counted the number of cells stained through a binary decision. Mean values are presented (Figure 6C).
To observe localization of PU.1, alveolar macrophages were immunostained with a rabbit anti-PU.1 polyclonal antibody and PE-labeled antirabbit polyclonal antibody (DakoCytomation, Glostrup, Denmark), counterstained with 1:3,000 dilution of Syber green; they were then examined using confocal laser microscopy.
Autoantibody concentrations in BALF or in serum were measured using purified autoantibody as a standard (15, 23).
The GM-CSF bioactivity was quantified using TF-1, a GM-CSF–dependent cell line, as described elsewhere (19).
Protein samples obtained from BALF of patients with iPAP and normal control subjects using protein-A sepharose were subjected to ELISA and Western blotting to detect GM-CSF, as described previously (19).
Statistical analyses were performed using StatView version 4 software (SAS Institute, Inc., Cary, NC), using the Mann-Whitney's U test or Kruskal-Wallis rank sum procedures for nonparametric data. Correlation of variables was assessed using the Spearman rank correlation coefficient. We considered p < 0.05 to be significant.
The 24-week course of inhaled GM-CSF therapy showed improved oxygenation of arterial blood with no side effects. All three patients showed a 10 mm Hg decrease or more in A-aDO2 after treatment (Table 1). Serum levels of surfactant protein-D, lactate dehydrogenase, and carcinoembryonic antigen were also improved (Figure 1
Figure 1. Clinical course of Case 1. Laboratory data for PaO2 and serum markers for idiopathic pulmonary alveolar proteinosis, including lactate dehydrogenase (LDH), a mucin-like glycoprotein, KL-6, surfactant protein-D (SP-D), and carcinoembryonic antigen (CEA), are presented with clinical information. GM-CSF = granulocyte-macrophage colony–stimulating factor.
[More] [Minimize]Case 1 | Case 2 | Case 3 | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Before | After | Before | After | Before | After | ||||
| Recovery, % of 150 ml | 69 | 60 | 53 | 48 | 40 | 70 | |||
| Cell counts, × 104/ml | 2.9 | 15.9 | 4.0 | 16.2 | 7.5 | 27.1 | |||
| Macrophages, % | 62 | 76 | 66 | 95 | 47 | 24 | |||
| Lymphocytes, % | 29 | 21 | 32 | 4 | 40 | 76 | |||
| Neutrophils, % | 9 | 3 | 2 | 1 | 12 | 0 | |||
| Eosinophils, % | 0 | 0 | 0 | 0 | 0 | 0 | |||
| CD4/8 ratio | 2.2 | 2.2 | 3.2 | 1.9 | 1.7 | 5.0 | |||
Figure 2. (A–C) Wright-Giemsa staining of the cells in bronchoalveolar lavage fluid before (left) and after (right) the GM-CSF treatment (×200; scale, 40 μm). Insets show higher magnifications of the cells (×400). (D) Electron micrographs of the alveolar macrophages of Case 1 before (left) and after (right) the GM-CSF treatment (×3,000).
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Figure 3. (A) Cell counts of alveolar macrophages in bronchoalveolar lavage fluide (BALF) of the patients with pulmonary alveolar proteinosis (PAP) before (black bars) and after (gray bars) the 24-week GM-CSF inhalation. (B) The ratio of foamy macrophages to the total number of macrophages in BALF of the patients with PAP before (pre) and after (post) the 24-week GM-CSF inhalation. (C) Diameters of nonfoamy macrophages in BALF of the patients with PAP before and after the 24-week GM-CSF inhalation. Central bars show median, box plots show 25th and 75th percentiles, error bars show 10th and 90th percentiles; dots show the minima and the maxima.
[More] [Minimize]We examined alveolar macrophages before and after treatment for changes in phagocytic activity and in the expression of two molecules. Phagocytic activity, as measured using the number of the cells harboring beads, was increased after the treatment (Figure 4)
Figure 4. Phagocytosis assay using latex beads. The upper and middle panels show confocal microscopy images of alveolar macrophages from the patients (upper panel: before GM-CSF treatment; middle panel: after GM-CSF treatment) incubated with phycoerythrin (PE)-labeled latex beads. The lower panel shows confocal microscopy images of alveolar macrophages of a normal control (N.C.) incubated with PE-labeled latex beads, and the ratio of macrophages containing latex beads to total macrophages before (black bars) and after (gray bars) the GM-CSF inhalation.
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Figure 5. Immunohistochemical staining of alveolar macrophages obtained from patients with PAP and normal control subjects with mannose receptor. The upper panel shows alveolar macrophages expressing mannose receptor (red) from the patients before treatment (left) and after treatment (middle), and control staining using murine IgG (right). The lower panel shows the percentage of macrophages expressing mannose receptor to total macrophages before (black bars) and after (gray bars) the GM-CSF inhalation.
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Figure 6. (A) Alveolar macrophages expressing PU.1 (dark blue) from the patients before (left) and after (right) treatment and from a normal control subject. (B) Confocal microscopy of alveolar macrophages obtained from the patients and stained with syber green (left panel: “Nuclei”) and PE-labeled anti-PU.1 antibody (middle panel: “PU.1”). Merge images are shown in the right panel. (C) The percentage ratio of macrophages that were positive for PU.1 to the total macrophages before (black bars) and after (gray bars) GM-CSF inhalation.
Anti–GM-CSF antibodies in patients with iPAP have a wide range of target epitopes. In addition, the crude amount of the autoantibody may not be correlated with the biological effect or a state of the disease (20). Consequently, to investigate the effect of GM-CSF inhalation on autoantibody levels in the lung, we examined BALF after treatment for the following: (1) neutralization capacity, which suppresses biological activities of GM-CSF using a GM-CSF–dependent cell line, and (2) the amount of IgG binding to GM-CSF by enzyme immunoassay (EIA) (Table 3)
Anti–GM-CSF Ab (μg/ml) | ||||
|---|---|---|---|---|
| Neutralizing Capacity | ||||
| BALF | Serum | (IC50 GM-CSF ng/BALF ml) | ||
| Case 1 | ||||
| Pre | 1.38 | 30.54 | 4.13 | |
| Post | 0.10 | 21.85 | 0.47 | |
| Case 2 | ||||
| Pre | 0.58 | 57.40 | 1.33 | |
| Post | 0.03 | 33.70 | 0.32 | |
| Case 3 | ||||
| Pre | 5.40 | NA | 10.27 | |
| Post | 0.19 | NA | 0.21 | |
The effects of inhaled exogenous GM-CSF on reduction of both the neutralizing capacity and titer of the autoantibody suggested that exogenous GM-CSF bound to the free autoantibody and thereby reduced the free autoantibody detected by both ELISA and bioassay. If that occurs, GM-CSF bound to the autoantibody in BALF may increase after treatment. To elucidate this, the concentration of GM-CSF bound or unbound to the autoantibody in BALF was measured and compared before and after treatment. Unexpectedly, concentrations of GM-CSF bound to the autoantibody were reduced consistently to a level below the range of detection after treatment (Table 4)
Before Treatment | After Treatment | |||||
|---|---|---|---|---|---|---|
| Bound | Unbound | Bound | Unbound | |||
| Case 1 | 71.3 | ND | 54.1 | ND | ||
| Case 2 | 24.0 | ND | ND | ND | ||
| Case 3 | 10.7 | ND | ND | ND | ||
Alveolar macrophages in the BALF of patients with iPAP in severe cases show defective mature alveolar macrophage functions (25, 26). Surfactant catabolism and host defense immunity regulated by transcription factor PU.1 are typical of such functions (27). Our previous studies suggested that maturation arrest of alveolar macrophages is caused by abundant autoantibody against GM-CSF in the lung (19). Because the therapeutic efficacy of extrinsic GM-CSF on iPAP has been established in clinical trials over the last decade (13–16), it is plausible to hypothesize that administered GM-CSF alters the unclear balance between GM-CSF and the autoantibody in the pulmonary microenvironment.
Several investigators have addressed the mechanism of extrinsic GM-CSF action on the pathologic status of iPAP. Seymour and colleagues (28) reported that patients with iPAP who were treated with 5 μg/kg/day of GM-CSF showed an impaired hematopoietic response to GM-CSF. Schoch and coworkers (15) demonstrated that GM-CSF treatment restored morphology and adhesive function of alveolar macrophages in patients with iPAP. The serum anti–GM-CSF titer has been reported to decrease with improvement of iPAP in patients treated with GM-CSF or plasmapheresis (24, 29). Bonfield and colleagues (18) showed that suppressed expression of PU.1 and macrophage colony–stimulating factor receptor in alveolar macrophages of patients with PAP was changed to upregulation by GM-CSF treatment in both in vitro experiments and in vivo after subcutaneous injection.
These studies demonstrated that treatment accelerated maturation of alveolar macrophages, but they did not explore alterations of the pulmonary microenvironment in which alveolar macrophages reside. Consequently, we have conducted analyses that specifically address the following two points: (1) estimation of the neutralizing capacity of the BALF against GM-CSF during the treatment and (2) determination of the GM-CSF–autoantibody immune complex. We found the following: (1) the neutralizing capacities and the levels of autoantibody against GM-CSF were decreased in BALF of patients with iPAP after aerosolized GM-CSF treatment (Table 3), (2) the amounts of GM-CSF–autoantibody immune complexes were also decreased in BALF after the treatment (Table 4), and (3) GM-CSF bound to the immune complex in BALF was not extrinsic recombinant protein but rather natural glycosylated human protein in Case 1.
Aerosolized recombinant human GM-CSF given to cynomolgus monkeys increased the total number of BAL cells more effectively than intravenous infusion of GM-CSF (30). Aerosolized GM-CSF also improved lung histology, alveolar macrophage differentiation, and surfactant protein B (SP-B) immunostaining to normal levels in GM-CSF–deficient mice (8). These results suggest that inhalation of GM-CSF might be an effective approach to affect alveolar macrophages' proliferation and functional maturation. It is notable that Case 3 demonstrated the increase of lymphocytes in BALF after GM-CSF treatment. The increase of lymphocytes was greater than in other two cases, and it might be associated with smoking cessation (Table 1). BAL lymphocytosis was also observed in a patient with PAP throughout the treatment course of subcutaneous GM-CSF injection, despite clinical improvement (15). The pulmonary infiltrates of lymphocytes in GM-CSF–deficient mice decreased but remained under successful treatment with aerosolized GM-CSF (8). Aerosolized GM-CSF itself increased lymphocytes in BALF of healthy cynomolgus macaques (30). The mechanism of the persistent BAL lymphocytosis during PAP treatment with GM-CSF remains to be elucidated.
The lungs of patients with iPAP contain abundant anti–GM-CSF antibody, and they produce GM-CSF to the comparable extent of normal lung (19). Decreased levels of the anti–GM-CSF antibody and the immune complex in BALF of the post-treatment patients suggested that aerosolized GM-CSF might affect the regulatory mechanism of production/disposition of anti–GM-CSF antibody locally or systemically. We infer that the reduced antibody restores bioactivity of intrinsic GM-CSF, engendering an increase of alveolar macrophages. To test the assumption, we attempted to demonstrate the presence of biologically active endogenous GM-CSF in BALF using TF-1, a GM-CSF–dependent cell line. However, neither BALF from normal control subjects nor BALF from the post-treatment patients sustained cell survival; their GM-CSF activities were below the detectable range (data not shown).
It remains unclear why treatment with extrinsic GM-CSF can decrease both the amount and the neutralizing capacity of autoantibody against GM-CSF in BALF of patients with iPAP (14, 15). It is a remarkable finding that the aerosolized GM-CSF therapy decreased the titer and neutralizing capacity of the anti–GM-CSF antibody in BALF during administration of immunosuppressants in Case 2. Further study should address the following: (1) the immune complex might modify a profile of T-cell population that regulates the autoantibody production and (2) apoptosis of the B cells that produce anti–GM-CSF antibody might be triggered by the immune complex of extrinsic GM-CSF and the autoantibody through Fc receptors, such as inhibitory FcγRIIB, as in the process of negative selection of B cells (31).
The clinical implication of the present study is that quantification of anti–GM-CSF antibody in BALF is useful to predict the response to GM-CSF treatment in each patient. The neutralizing capacity of GM-CSF in BALF is correlated significantly with serum markers including carcinoembryonic antigen, KL-6, and surfactant protein-D. It is also strongly correlated with the titer of anti–GM-CSF antibody in BALF and Po2. Clinical trials of GM-CSF treatment revealed the existence of patients who showed no improvement in clinical parameters such as Po2, computed tomographic, and pulmonary function tests (13, 14). Furthermore, these clinical markers often showed delayed response to GM-CSF therapy in some cases. Techniques to evaluate the amount and the neutralizing capacity of anti–GM-CSF antibody in BALF during GM-CSF treatment would be useful tools to enable prediction of the response to GM-CSF treatment.
In conclusion, the present study demonstrated the importance of evaluating microenvironments surrounding macrophages in lungs as well as functions of alveolar macrophages in patients with iPAP. Techniques for detecting the neutralizing capacity and amount of anti–GM-CSF autoantibody in BALF could contribute to optimization of treatment for patients with iPAP.
The authors thank Dr. John F. Seymour and Dr. Bruce C. Trapnell for their critical reading of the manuscript.
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