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Original Article

GM-CSF Autoantibodies and Neutrophil Dysfunction in Pulmonary Alveolar Proteinosis

List of authors.
  • Kanji Uchida, M.D., Ph.D.,
  • David C. Beck, M.D., Ph.D.,
  • Takashi Yamamoto, M.D., Ph.D.,
  • Pierre-Yves Berclaz, M.D., Ph.D.,
  • Shuichi Abe, M.D., Ph.D.,
  • Margaret K. Staudt, M.S.,
  • Brenna C. Carey, Ph.D.,
  • Marie-Dominique Filippi, Ph.D.,
  • Susan E. Wert, Ph.D.,
  • Lee A. Denson, M.D.,
  • Jonathan T. Puchalski, M.D.,
  • Diane M. Hauck, B.A., M.T.,
  • and Bruce C. Trapnell, M.D.

Abstract

Background

Increased mortality from infection in patients with pulmonary alveolar proteinosis occurs in association with high levels of autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF). We tested the hypothesis that neutrophil functions are impaired in patients with pulmonary alveolar proteinosis and that GM-CSF autoantibodies cause the dysfunction.

Methods

We studied 12 subjects with pulmonary alveolar proteinosis, 61 healthy control subjects, and 12 control subjects with either cystic fibrosis or end-stage liver disease. We also studied GM-CSF−/− mice and wild-type mice. We evaluated basal neutrophil functions, neutrophil functions after priming by GM-CSF to augment antimicrobial functions, and the effects of highly purified GM-CSF autoantibodies on neutrophil functions in vitro and in vivo.

Results

Neutrophils from subjects with pulmonary alveolar proteinosis had normal ultrastructure and differentiation markers but impaired basal functions and antimicrobial functions after GM-CSF priming. GM-CSF−/− mice also had reduced basal neutrophil functions, but functions after GM-CSF priming were unimpaired. The neutrophil dysfunction characteristic of pulmonary alveolar proteinosis was reproduced in a dose-dependent fashion in blood specimens from healthy control subjects after incubation with affinity-purified GM-CSF autoantibodies isolated from patients with pulmonary alveolar proteinosis. The injection of mouse GM-CSF antibodies into wild-type mice also caused neutrophil dysfunction.

Conclusions

The antimicrobial functions of neutrophils are impaired in patients with pulmonary alveolar proteinosis, owing to the presence of GM-CSF autoantibodies. The effects of these autoantibodies show that GM-CSF is an essential regulator of neutrophil functions.

Introduction

Pulmonary alveolar proteinosis1 is a rare disorder in which surfactant accumulates within pulmonary alveoli, causing respiratory insufficiency.2,3 The disease is specifically associated with high levels of autoantibodies against granulocyte–macrophage colony-stimulating factor (GM-CSF) in blood and tissues, including pulmonary alveoli.4 These autoantibodies neutralize the biologic activity of GM-CSF.5 In mice, GM-CSF stimulates the terminal differentiation of alveolar macrophages, primarily through the action of the transcription factor PU.1.6 The homozygous deletion of GM-CSF genes causes pulmonary alveolar proteinosis in mice7,8 by impairing the clearance of pulmonary surfactant by alveolar macrophages that are dependent on GM-CSF.9 In patients with pulmonary alveolar proteinosis, PU.1 levels are reduced in alveolar macrophages, but levels increase in response to GM-CSF therapy.10 There is evidence that the regulation of alveolar macrophages by GM-CSF is similar in humans and mice.3,11

Infections, which are caused predominantly by opportunistic pathogens, account for 18% of reported deaths attributable to pulmonary alveolar proteinosis.2 Some of these infections occur at extrapulmonary sites, an indication that the predisposition to infection is systemic, rather than confined to the lungs.2 Similarly, in GM-CSF−/− mice, mortality from infection and susceptibility to bacterial, fungal, and mycobacterial pathogens are increased.11-13 These findings suggest that pulmonary alveolar proteinosis is characterized by defective immune function, especially because GM-CSF is important during infection.14

GM-CSF augments the antimicrobial functions of neutrophils by means of a mechanism known as priming.15,16 GM-CSF priming increases levels of the adhesion molecule CD11b, which promotes the adhesion of neutrophils to the vascular endothelium, a critical event in the recruitment of neutrophils into infected tissues.17 GM-CSF also primes phagocytosis, oxidative burst, and bactericidal activity in neutrophils. Since GM-CSF autoantibodies may impair these functions, we studied neutrophils from patients with pulmonary alveolar proteinosis and from GM-CSF−/− mice. We also evaluated the functions of normal human or mouse neutrophils after incubation with highly purified GM-CSF autoantibodies.

Methods

Participants

The institutional review board of the Cincinnati Children's Hospital Medical Center approved the study. All participants or their legal guardians gave written informed consent, and minors gave assent. Between July 24, 2004, and June 9, 2006, all 12 subjects with pulmonary alveolar proteinosis who were referred to our center for evaluation or treatment were enrolled in the study. We also studied 61 healthy control subjects, 6 control subjects with cystic fibrosis, and 6 control subjects with end-stage liver disease. (The case histories of the subjects with pulmonary alveolar proteinosis are given in the Supplementary Appendix, available with the full text of this article at www.nejm.org.) GM-CSF autoantibodies were quantified in serum with the use of an enzyme-linked immunosorbent assay (ELISA), as previously described.5 Granulocyte colony-stimulating factor (G-CSF) was quantified in serum by means of an ELISA (Quantikine Kit, R&D Systems).

Human Neutrophils

The isolation of human neutrophils in blood on Ficoll (MP Biomedicals) gradients was initiated within 1 hour after phlebotomy. After the lysis of red cells, neutrophils were resuspended in phosphate-buffered saline containing 10 mM d-glucose.18 Mean (±SE) viability, determined immediately after isolation by means of trypan-blue staining, was 97.5±1.2% in neutrophils from subjects with pulmonary alveolar proteinosis and 97.9±0.6% in neutrophils from healthy control subjects. In some experiments, neutrophils were isolated for evaluation with the use of Mono-Poly resolving medium (MP Biomedicals), washed in phosphate-buffered saline (without hypotonic red-cell lysis), and resuspended in Hank's balanced salt solution, 20% fetal bovine serum, and 25 mM HEPES buffer (pH 7.4); these neutrophils are referred to as washed neutrophils. Cell-surface markers of apoptosis were assessed by flow cytometry (Apoptosis Detection Kit I, Pharmingen).

Neutrophil differentiation was evaluated with the use of cell-surface differentiation markers and flow cytometry.6,19 We measured PU.1 messenger RNA levels in neutrophils using reverse transcription and quantitative polymerase-chain-reaction amplification6 and evaluated PU.1 protein using Western-blot analysis.6 Neutrophil ultrastructure was evaluated in 20 neutrophils from each of two subjects with pulmonary alveolar proteinosis (Patients 6 and 7) and from each of two healthy control subjects.20

The basal capacity of isolated neutrophils to phagocytose opsonized fluorescent microspheres was evaluated as previously described for macrophages,21 except that internalization of the microspheres was quantified with the use of confocal microscopy at a magnification of 63×.19 The phagocytic index was calculated as the percentage of neutrophils that had phagocytosed microspheres multiplied by the number of microspheres per cell. For each human (or mouse) studied, 400 neutrophils were evaluated.

Phagocytosis by neutrophils in whole blood (hereafter called the phagocytic capacity) was measured by flow cytometry21 to eliminate the potential influence of reduced adhesion. Triplicate samples of heparinized human blood (200 μl) were incubated (at 37°C for 60 minutes) with IgG-opsonized fluorescent microspheres (7.5×106, prepared as previously described21) in capped, siliconized microcentrifuge tubes with gentle orbital rotation (Thermomixer, Eppendorf). After red-cell lysis, flow cytometry was performed to evaluate neutrophils. Phagocytic capacity was calculated as the percentage of neutrophils containing internalized microspheres multiplied by the mean fluorescence intensity of phagocytic neutrophils (both determined with the use of flow cytometry) and multiplied by the neutrophil count in the blood.

Cellular adhesion was evaluated by seeding isolated neutrophils into low-adhesion plastic dishes (Corning, catalog no. 3471). After 2 hours, the plates were washed twice with phosphate-buffered saline, and adherent cells were counted with the use of a microscope.6 The production of hydrogen peroxide was measured in neutrophils in whole blood as previously described.22 All data for the basal phagocytic index, phagocytic capacity, oxidative burst, and cellular adhesion were normalized by dividing by the mean value for the healthy control group and multiplying by 100.

The amount of Staphylococcus aureus killed (American Type Culture Collection no. 49476) in 1 hour by neutrophils in whole blood or by isolated neutrophils was determined as previously described,18,23 except that interferon-γ and lysostaphin, respectively, were omitted.

We studied neutrophil functions after GM-CSF priming by incubating heparinized whole blood for 30 minutes in the absence or presence of 10 ng of human GM-CSF per milliliter (Leukine, Berlex) or 10 ng of mouse GM-CSF per milliliter (R&D Systems). The increase in CD11b levels on neutrophils after GM-CSF priming (CD11b stimulation index) was calculated as the mean fluorescence intensity of CD11b on neutrophils primed by GM-CSF minus that of CD11b on nonprimed neutrophils, divided by the mean fluorescence intensity of nonprimed neutrophils and multiplied by 100. Phagocytic capacity after GM-CSF priming was calculated as the phagocytic capacity of neutrophils primed by GM-CSF minus that of nonprimed neutrophils.

Mouse Neutrophils

Experiments in mice were conducted according to protocols approved by the local institutional animal care and use committee. GM-CSF−/− mice were backcrossed for more than 10 generations into C57BL/6J mice, which served as the wild-type control mice. Mouse neutrophils were isolated and evaluated as described for human neutrophils, except as follows. Neutrophils were isolated from bone marrow on Percoll (Amersham) gradients24; more than 90% of the isolated cells were neutrophils, and more than 95% were viable. Phagocytic capacity was determined with the use of 100 μl of blood, 30-minute periods of incubation, and immunostaining of neutrophils with Ly6G.

Effects of GM-CSF Antibody

We made two GM-CSF autoantibody preparations using GM-CSF affinity chromatography as previously described5: one from a single subject with pulmonary alveolar proteinosis (Patient 6) and one from serum pooled from 11 subjects with the disease (purified GM-CSF autoantibodies and purified pooled GM-CSF autoantibodies, respectively). Affinity-purified autoantibodies were concentrated with the use of ultrafiltration (Microcon 30 K, Amicon), resuspended in phosphate-buffered saline, and assessed for purity by means of electrophoresis on polyacrylamide gels. We measured the ability of GM-CSF autoantibodies to neutralize GM-CSF, using TF1 cells as previously described.5 For in vitro studies, purified GM-CSF autoantibody or human immune globulin (Gammagard, Baxter) was incubated with heparinized blood or washed neutrophils (0.5 μg per milliliter, except as noted) before CD11b levels or phagocytic capacity was assessed. Since human GM-CSF and mouse GM-CSF are not immunologically cross-reactive, for in vivo studies, monoclonal anti–mouse GM-CSF antibody (22E9, Endogen) or isotype-control antibody (rat IgG2a, Pharmingen; 200 μg per mouse) was injected intraperitoneally into C57BL/6J mice (five in each group). The phagocytic capacity of neutrophils was assessed 3 days after injection.

Statistical Analysis

We evaluated the numerical data for a normal distribution using the Kolmogorov–Smirnov test and for equal variance using the Levene median test; parametric data are presented as means (±SE) and nonparametric data are presented as medians and interquartile ranges. Statistical comparisons of parametric data were made with Student's t-test for two-group comparisons and with one-way analysis of variance with post hoc analysis according to the Holm–Sidak method for multiple-group comparisons. Nonparametric data were compared with the use of the Mann–Whitney rank-sum test. P values of less than 0.05 were considered to indicate statistical significance. All experiments were repeated at least twice, with similar results.

Results

Neutrophil Counts and Differentiation Markers

Table 1. Table 1. Clinical Characteristics of Subjects with Pulmonary Alveolar Proteinosis. Figure 1. Figure 1. Ultrastructure and Functions of Neutrophils from Subjects with Pulmonary Alveolar Proteinosis (PAP), Healthy Control Subjects, GM-CSF−/− Mice, and Wild-Type Mice.

Electron photomicrographs of neutrophils show their ultrastructure (Panel A). The scale bar represents 2 μm. Confocal photomicrographs (Panel B) show neutrophil phagocytosis of microspheres (shown in yellow); F-actin is shown in red and nuclei in blue. Panel C shows flow-cytometry histograms of phagocytosis by neutrophils in whole blood, as well as the percentage of phagocytic neutrophils (calculated over the area indicated by the horizontal black bar). Each successive peak represents the number of cells with an increased number of microspheres per cell (e.g., the first peak after 102 is for 1 microsphere per cell; and the second peak is for 2 microspheres per cell). Panel D shows the similarity of impairment in various corresponding neutrophil functions in GM-CSF−/− mice and in subjects with PAP. The dashed line indicates equal impairment; the data are from Table 2. Panel E shows the CD11b stimulation index for neutrophils in a blood specimen from a healthy control subject and one from a subject with PAP after GM-CSF priming. The CD11b stimulation index was calculated as the mean fluorescence intensity of CD11b on neutrophils primed by GM-CSF minus that of CD11b on nonprimed neutrophils, divided by the mean fluorescence intensity of nonprimed neutrophils and multiplied by 100. Panel F shows the phagocytic capacity of neutrophils from a subject with PAP and a healthy control subject before and after GM-CSF priming. Phagocytic capacity was calculated as the percentage of neutrophils containing internalized microspheres multiplied by the mean fluorescence intensity of phagocytic neutrophils (both determined with the use of flow cytometry) and multiplied by the neutrophil count in the blood. In Panels D and F, I bars and T bars indicate standard errors.

Table 2. Table 2. Differentiation Markers and Functions of Neutrophils from Healthy Control Subjects, Subjects with Pulmonary Alveolar Proteinosis (PAP), Wild-Type Mice, and GM-CSF−/− Mice.

Neutrophil counts in blood and G-CSF levels in serum were normal in the 12 subjects with pulmonary alveolar proteinosis (Table 1). Neutrophils from subjects with pulmonary alveolar proteinosis and those from healthy control subjects had similar ultrastructure (Figure 1A), cell-surface differentiation markers (Table 2), and levels of PU.1 expression (data not shown). Differentiation markers on neutrophils from GM-CSF−/− and wild-type mice were also similar (Table 2).

Neutrophil Function

Patients

The basal phagocytic functions of isolated neutrophils, evaluated with the use of confocal microscopy, were reduced in subjects with pulmonary alveolar proteinosis as compared with healthy control subjects (Figure 1B and Table 2). In whole blood, the percentage of phagocytic neutrophils and the number of phagocytosed microspheres per neutrophil were also reduced in subjects with pulmonary alveolar proteinosis (Figure 1C and Table 2). Basal cellular adhesion, oxidative burst, and bactericidal activity were reduced in subjects with pulmonary alveolar proteinosis as compared with healthy control subjects (Table 2).

The phagocytic capacity of washed neutrophils incubated in Hank's balanced salt solution without added GM-CSF declined during a 3-hour period (100.0±0.6 at 0 minutes, 92.3±0.3 at 90 minutes, and 71.8±5.0 at 180 minutes; 3 determinations per time point) (90 minutes vs. 0 minutes, P=0.04; 180 minutes vs. 0 minutes, P<0.001). The decline was not due to apoptosis, which occurred in less than 2% of neutrophils at each time point. In Patient 6, who had pulmonary alveolar proteinosis and received GM-CSF therapy for 13 weeks, the basal phagocytic capacity of neutrophils (normalized to the capacity in 20 healthy control subjects) improved from 44.1±2.2% before therapy to 104.5±1.9% after therapy. Although we studied only three children with pulmonary alveolar proteinosis (Patients 2, 3, and 5), their pattern of neutrophil impairment was similar to that of the adult subjects.

GM-CSF priming in vitro increased CD11b levels on neutrophils in the blood of healthy control subjects to maximum levels at low levels of GM-CSF (Figure 1E and Table 2). In contrast, GM-CSF priming of CD11b levels on neutrophils from subjects with pulmonary alveolar proteinosis was severely impaired, with levels increasing by only modest amounts at high GM-CSF levels. The phagocytic capacity of neutrophils after GM-CSF priming was also severely impaired in the subjects with pulmonary alveolar proteinosis (Figure 1F and Table 2).

Table 3. Table 3. Neutrophil Functions in Control Subjects with Cystic Fibrosis or End-Stage Liver Disease, as Compared with Healthy Control Subjects.

We also examined basal and GM-CSF–primed neutrophil functions in control subjects with cystic fibrosis or end-stage liver disease, neither of which is associated with GM-CSF autoantibodies. The basal phagocytic capacity and GM-CSF priming of CD11b levels of neutrophils was normal in both disorders (Table 3). Thus, neutrophil dysfunction is not a characteristic feature of these chronic diseases.

GM-CSF−/− Mice

Basal neutrophil functions were reduced in GM-CSF−/− mice in a pattern similar to that among subjects with pulmonary alveolar proteinosis. Phagocytosis was reduced the most, followed by adhesion, oxidative burst, and bactericidal activity (r2=0.94, P=0.03) (Figure 1D and Table 2).

In contrast to basal functions, GM-CSF–primed neutrophil functions — including CD11b levels and phagocytic capacity — were not impaired in GM-CSF−/− mice (Table 2). This finding contrasts with that in subjects with pulmonary alveolar proteinosis in the absence of GM-CSF priming, but the mechanism underlying the disruption of GM-CSF functions differs: in GM-CSF−/− mice, it is the absence of GM-CSF production; in humans, it is the high levels of neutralizing GM-CSF autoantibodies.5

Effect of GM-CSF Autoantibodies

Figure 2. Figure 2. Effect of GM-CSF Autoantibodies on Neutrophil Dysfunction In Vitro and In Vivo.

Panel A shows the results of polyacrylamide-gel electrophoresis of GM-CSF autoantibodies isolated from subjects with pulmonary alveolar proteinosis. Panel B shows the CD11b stimulation index normalized by dividing by the mean value for the pooled human immune globulin control. Panel C shows the mean phagocytic capacity of neutrophils from healthy control subjects after GM-CSF priming and incubation with GM-CSF autoantibodies. The dashed line indicates the capacity of nonprimed neutrophils; phagocytic capacity after GM-CSF priming was calculated as that of primed neutrophils minus that of nonprimed neutrophils. Panel D shows the phagocytic capacity of washed neutrophils from healthy control subjects incubated in Hank's balanced salt solution for 90 minutes as a percentage of the normal phagocytic capacity at 0 minutes (dashed line). All comparisons indicated by brackets in Panel D were significant (P<0.001). Panel E shows the basal phagocytic capacity of neutrophils in whole blood. In Panels B, C, D, and E, T bars and I bars indicate standard errors.

Affinity-purified GM-CSF autoantibodies produced a single band similar in size to purified human IgG on polyacrylamide gels under nonreducing conditions and two bands corresponding to the expected sizes of immunoglobulin heavy and light chains under reducing conditions (Figure 2A). The amounts of autoantibodies required to inhibit the activity of GM-CSF by 50% were similar with purified GM-CSF autoantibodies from a single patient and with purified pooled GM-CSF autoantibodies (10.3 and 10.6 mol of IgG per mole of GM-CSF, respectively); these values were also similar to previously reported values.5 In vitro incubation of the GM-CSF autoantibodies with whole blood from healthy control subjects or from wild-type mice blocked GM-CSF priming of CD11b levels on human neutrophils but not mouse neutrophils (Figure 2B). Phagocytic capacity primed by GM-CSF was also reduced in a dose-dependent fashion by the presence of GM-CSF autoantibodies (Figure 2C). A low GM-CSF level (10 pg per milliliter) maintained normal neutrophil phagocytic capacity at 90 minutes as compared with 0 minutes (P=0.11) (Figure 2D). The addition of GM-CSF autoantibodies reduced the phagocytic capacity of washed neutrophils and blocked the ability of GM-CSF to maintain phagocytic functions in neutrophils during short-term, ex vivo incubation (Figure 2D). Purified GM-CSF autoantibodies from a single patient and purified pooled GM-CSF autoantibodies had similar inhibitory effects on CD11b levels primed with GM-CSF (mean of three replicates, 9.61±4.90 and 0.00±4.83, respectively; P=0.24) and on GM-CSF–primed phagocytic capacity (mean of three replicates, −0.84±6.17 and 4.21±0.53, respectively; P=0.46).

Injection of a monoclonal mouse GM-CSF antibody into wild-type mice reproduced the impaired neutrophil phagocytic capacity observed in GM-CSF−/− mice (Figure 2E). The mean serum GM-CSF antibody level at the time of evaluation was 69.3±8.1 μg per milliliter.

Discussion

Our study showed impairment of basal phagocytosis, adhesion, oxidative burst, and bactericidal activity of neutrophils from subjects with pulmonary alveolar proteinosis and neutrophils from GM-CSF−/− mice. Neutrophils from the subjects also had impaired responses to GM-CSF priming. The dysfunction was reproduced in normal human neutrophils by incubating them with highly purified GM-CSF antibodies from subjects with pulmonary alveolar proteinosis and also by injecting mouse GM-CSF autoantibodies into normal mice. The observed constellation of neutrophil abnormalities can explain the increased risk of infection, especially infection with opportunistic organisms, that is associated with pulmonary alveolar proteinosis2,3 and with other phagocyte immunodeficiencies,25 as well as the increased mortality from infection in GM-CSF−/− mice.12

Neutrophil dysfunction in patients with pulmonary alveolar proteinosis was associated with high levels of GM-CSF autoantibodies and could not be attributed to a nonspecific effect of chronic illness. Neutralizing GM-CSF autoantibodies have been reported in patients with myasthenia gravis,26 but the antibody levels are relatively low, and pulmonary alveolar proteinosis has not been reported in this setting.

G-CSF can also prime the antimicrobial function of neutrophils27 by means of a mechanism distinct from that in GM-CSF priming.28 In our study, however, serum G-CSF levels and blood neutrophil counts, which are regulated by G-CSF,27 were similar in subjects with pulmonary alveolar proteinosis and in healthy subjects. In some patients with Felty's syndrome, G-CSF autoantibodies are thought to cause neutropenia by neutralizing G-CSF activity29; however, pulmonary alveolar proteinosis has not been reported in these patients. Therefore, G-CSF and GM-CSF have distinct effects on neutrophil functions. Levels of macrophage colony-stimulating factor (M-CSF), which has regulatory effects on myeloid cells that overlap with the regulatory effects of GM-CSF, are increased in patients with pulmonary alveolar proteinosis and in GM-CSF−/− mice.3,6,10 However, since M-CSF receptors are not present on neutrophils, elevated M-CSF levels in patients with pulmonary alveolar proteinosis are unlikely to influence neutrophil functions.

We found that GM-CSF priming of neutrophil functions was blocked in patients with pulmonary alveolar proteinosis but not in GM-CSF−/− mice, although the pattern of basal neutrophil dysfunction was similar between the two. In humans and mice, abrogation of GM-CSF signaling (by means of gene knockout in mice and by means of antibodies in patients with pulmonary alveolar proteinosis) reduces, but does not abolish, multiple neutrophil functions. Moreover, GM-CSF appears to be unnecessary for neutrophil differentiation: neutrophil morphology, cell-surface differentiation markers, and levels of expression of PU.1 (a transcription factor that is critical for neutrophil differentiation30) were similar in subjects with pulmonary alveolar proteinosis and healthy control subjects. This evidence contrasts with the fact that GM-CSF stimulates the terminal differentiation of alveolar macrophages, primarily by increasing levels of PU.1 expression.6

The GM-CSF receptor mediates the dose-dependent effects of GM-CSF in a mutually exclusive, reciprocal fashion through two β-chain residues: Ser585 at low GM-CSF levels (in general, <300 pg per milliliter) and Tyr577 at high levels (>300 pg per milliliter).31 Thus, the GM-CSF receptor acts as a binary switch that promotes cell survival at low GM-CSF levels but also stimulates proliferation and antimicrobial functions (including an increased CD11b level on the cell surface) at high levels. This arrangement of the GM-CSF receptor places the level of GM-CSF bioactivity at the center of the mechanism that regulates neutrophil functions.

Our findings, together with those in previously published reports,13,32 provide support for the feasibility of GM-CSF therapy to augment innate immune defenses in patients with serious infections. Conversely, therapy with a humanized monoclonal GM-CSF antibody33 could be of use in reducing neutrophil priming (and functional capacity) in patients with chronic inflammatory disorders characterized by increased numbers of activated neutrophils, such as severe neutrophilic asthma,34 cystic fibrosis,35 and the adult respiratory distress syndrome.36 It is relevant that GM-CSF antibodies reduce neutrophilic pulmonary inflammation in a dose-dependent fashion in endotoxin-exposed mice.37 Finally, assessment of blood neutrophil functions may provide convenient, functional surrogate-outcome measures for use in clinical trials evaluating new therapies for pulmonary alveolar proteinosis.

Funding and Disclosures

Supported in part by grants from the National Heart, Lung, and Blood Institute (HL071823 and HL69459, to Dr. Trapnell, and T32HD043005, to Dr. Puchalski) and the National Center for Research Resources and the National Institutes of Health Office of Rare Diseases (RR019498, to Dr. Trapnell).

No potential conflict of interest relevant to this article was reported.

We thank Drs. Elizabeth Allen (Ohio State University, Columbus), Hugh Black (Carolinas Medical Center, Charlotte, NC), J.-P. Clancy (University of Alabama, Birmingham), David Dortin (Medical Associates of Cincinnati, Cincinnati) Jeffrey Hammersley (Medical University of Ohio, Toledo), David Kamelhar (New York University Medical Center, New York) Vijay Patel (Newnan Hospital, Newnan, GA), and Robert Smith (Dayton Chest Medicine, Dayton, OH) for referring their patients to us; Drs. Robert Wood (Cincinnati Children's Hospital Medical Center, Cincinnati), John Howington, and Michael Reed (University of Cincinnati Medical Center, Cincinnati) for contributions to the care of these patients; Diane Black for technical support; Susan Radtke and Carrie Stevens (Translational Research Trials Office, Cincinnati Children's Research Foundation, Cincinnati) for help with the clinical protocols; Carrie Jennings (University of Cincinnati Medical Center, Cincinnati) for identifying patients with chronic liver disease; Dr. Glenn Dranoff (Harvard Medical School, Boston) for providing the GM-CSF−/− mice; and Drs. David Williams and Jeffrey Whitsett (Divisions of Experimental Hematology and Pulmonary Biology, respectively, Cincinnati Children's Hospital Medical Center, Cincinnati) for critical reading of the manuscript.

Author Affiliations

From the Divisions of Pulmonary Biology (K.U., T.Y., S.A., M.K.S., B.C.C., S.E.W., D.M.H., B.C.T.), Pulmonary Medicine (P.-Y.B., J.T.P., B.C.T.), Experimental Hematology (M.-D.F.), and Gastroenterology (L.A.D.), Cincinnati Children's Hospital Medical Center; and the Division of Pulmonary, Critical Care, and Sleep Medicine, University of Cincinnati College of Medicine (D.C.B., J.T.P., B.C.T.) — both in Cincinnati.

Address reprint requests to Dr. Trapnell at the Division of Pulmonary Biology, Rm. 4029, TCHRF, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039, or at .

Supplementary Material

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    Figures/Media

    1. Table 1. Clinical Characteristics of Subjects with Pulmonary Alveolar Proteinosis.
      Table 1. Clinical Characteristics of Subjects with Pulmonary Alveolar Proteinosis.
    2. Figure 1. Ultrastructure and Functions of Neutrophils from Subjects with Pulmonary Alveolar Proteinosis (PAP), Healthy Control Subjects, GM-CSF−/− Mice, and Wild-Type Mice.
      Figure 1. Ultrastructure and Functions of Neutrophils from Subjects with Pulmonary Alveolar Proteinosis (PAP), Healthy Control Subjects, GM-CSF−/− Mice, and Wild-Type Mice.

      Electron photomicrographs of neutrophils show their ultrastructure (Panel A). The scale bar represents 2 μm. Confocal photomicrographs (Panel B) show neutrophil phagocytosis of microspheres (shown in yellow); F-actin is shown in red and nuclei in blue. Panel C shows flow-cytometry histograms of phagocytosis by neutrophils in whole blood, as well as the percentage of phagocytic neutrophils (calculated over the area indicated by the horizontal black bar). Each successive peak represents the number of cells with an increased number of microspheres per cell (e.g., the first peak after 102 is for 1 microsphere per cell; and the second peak is for 2 microspheres per cell). Panel D shows the similarity of impairment in various corresponding neutrophil functions in GM-CSF−/− mice and in subjects with PAP. The dashed line indicates equal impairment; the data are from Table 2. Panel E shows the CD11b stimulation index for neutrophils in a blood specimen from a healthy control subject and one from a subject with PAP after GM-CSF priming. The CD11b stimulation index was calculated as the mean fluorescence intensity of CD11b on neutrophils primed by GM-CSF minus that of CD11b on nonprimed neutrophils, divided by the mean fluorescence intensity of nonprimed neutrophils and multiplied by 100. Panel F shows the phagocytic capacity of neutrophils from a subject with PAP and a healthy control subject before and after GM-CSF priming. Phagocytic capacity was calculated as the percentage of neutrophils containing internalized microspheres multiplied by the mean fluorescence intensity of phagocytic neutrophils (both determined with the use of flow cytometry) and multiplied by the neutrophil count in the blood. In Panels D and F, I bars and T bars indicate standard errors.

    3. Table 2. Differentiation Markers and Functions of Neutrophils from Healthy Control Subjects, Subjects with Pulmonary Alveolar Proteinosis (PAP), Wild-Type Mice, and GM-CSF−/− Mice.
      Table 2. Differentiation Markers and Functions of Neutrophils from Healthy Control Subjects, Subjects with Pulmonary Alveolar Proteinosis (PAP), Wild-Type Mice, and GM-CSF−/− Mice.
    4. Table 3. Neutrophil Functions in Control Subjects with Cystic Fibrosis or End-Stage Liver Disease, as Compared with Healthy Control Subjects.
      Table 3. Neutrophil Functions in Control Subjects with Cystic Fibrosis or End-Stage Liver Disease, as Compared with Healthy Control Subjects.
    5. Figure 2. Effect of GM-CSF Autoantibodies on Neutrophil Dysfunction In Vitro and In Vivo.
      Figure 2. Effect of GM-CSF Autoantibodies on Neutrophil Dysfunction In Vitro and In Vivo.

      Panel A shows the results of polyacrylamide-gel electrophoresis of GM-CSF autoantibodies isolated from subjects with pulmonary alveolar proteinosis. Panel B shows the CD11b stimulation index normalized by dividing by the mean value for the pooled human immune globulin control. Panel C shows the mean phagocytic capacity of neutrophils from healthy control subjects after GM-CSF priming and incubation with GM-CSF autoantibodies. The dashed line indicates the capacity of nonprimed neutrophils; phagocytic capacity after GM-CSF priming was calculated as that of primed neutrophils minus that of nonprimed neutrophils. Panel D shows the phagocytic capacity of washed neutrophils from healthy control subjects incubated in Hank's balanced salt solution for 90 minutes as a percentage of the normal phagocytic capacity at 0 minutes (dashed line). All comparisons indicated by brackets in Panel D were significant (P<0.001). Panel E shows the basal phagocytic capacity of neutrophils in whole blood. In Panels B, C, D, and E, T bars and I bars indicate standard errors.

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