Original Article
GM-CSF Autoantibodies and Neutrophil Dysfunction in Pulmonary Alveolar Proteinosis
N Engl J Med 2007; 356:567-579February 8, 2007
- 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.
Media in This Article
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 2
Effect of GM-CSF Autoantibodies on Neutrophil Dysfunction In Vitro and In Vivo.Article Activity
- Article
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
Neutrophil counts in blood and G-CSF levels in serum were normal in the 12 subjects with pulmonary alveolar proteinosis (Table 1Table 1
Clinical Characteristics of Subjects with Pulmonary Alveolar Proteinosis.). Neutrophils from subjects with pulmonary alveolar proteinosis and those from healthy control subjects had similar ultrastructure (Figure 1AFigure 1Ultrastructure and Functions of Neutrophils from Subjects with Pulmonary Alveolar Proteinosis (PAP), Healthy Control Subjects, GM-CSF−/− Mice, and Wild-Type Mice.), cell-surface differentiation markers (Table 2Table 2
Differentiation Markers and Functions of Neutrophils from Healthy Control Subjects, Subjects with Pulmonary Alveolar Proteinosis (PAP), Wild-Type Mice, and GM-CSF−/− Mice.), 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).
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 3Table 3
Neutrophil Functions in Control Subjects with Cystic Fibrosis or End-Stage Liver Disease, as Compared with Healthy Control Subjects.). 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
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 2AFigure 2
Effect of GM-CSF Autoantibodies on Neutrophil Dysfunction In Vitro and In Vivo.). 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.
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.
Source Information
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 bruce.trapnell@cchmc.org.
- References
References
1
Rosen SH ,Castleman B ,Liebow AA . Pulmonary alveolar proteinosis. N Engl J Med 1958;258:1123-1142
Full Text | Web of Science | Medline2
Seymour JF ,Presneill JJ . Pulmonary alveolar proteinosis: progress in the first 44 years. Am J Respir Crit Care Med 2002;166:215-235
CrossRef | Web of Science | Medline3
Trapnell BC ,Whitsett JA ,Nakata K . Pulmonary alveolar proteinosis. N Engl J Med 2003;349:2527-2539
Full Text | Web of Science | Medline4
Kitamura T ,Tanaka N ,Watanabe J , et al. Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor. J Exp Med 1999;190:875-880
CrossRef | Web of Science | Medline5
Uchida K ,Nakata K ,Trapnell BC , et al. High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 2004;103:1089-1098
CrossRef | Web of Science | Medline6
Shibata Y ,Berclaz PY ,Chroneos ZC ,Yoshida M ,Whitsett JA ,Trapnell BC . GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1. Immunity 2001;15:557-567
CrossRef | Web of Science | Medline7
Stanley E ,Lieschke GJ ,Grail D , et al. Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc Natl Acad Sci U S A 1994;91:5592-5596
CrossRef | Web of Science | Medline8
Dranoff G ,Crawford AD ,Sadelain M , et al. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 1994;264:713-716
CrossRef | Web of Science | Medline9
Ikegami M ,Ueda T ,Hull W , et al. Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation. Am J Physiol 1996;270:L650-L658
Web of Science | Medline10
Bonfield TL ,Raychaudhuri B ,Malur A , et al. PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor. Am J Physiol Lung Cell Mol Physiol 2003;285:L1132-L1136
Web of Science | Medline11
Seymour JF ,Presneill JJ . Pulmonary alveolar proteinosis: what is the role of GM-CSF in disease pathogenesis and treatment? Treat Respir Med 2004;3:229-234
CrossRef | Medline12
Seymour JF ,Lieschke GJ ,Grail D ,Quilici C ,Hodgson G ,Dunn AR . Mice lacking both granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF have impaired reproductive capacity, perturbed neonatal granulopoiesis, lung disease, amyloidosis, and reduced long-term survival. Blood 1997;90:3037-3049
Web of Science | Medline13
Gonzalez-Juarrero M ,Hattle JM ,Izzo A , et al. Disruption of granulocyte macrophage-colony stimulating factor production in the lungs severely affects the ability of mice to control Mycobacterium tuberculosis infection. J Leukoc Biol 2005;77:914-922
CrossRef | Web of Science | Medline14
Zhan Y ,Lieschke GJ ,Grail D ,Dunn AR ,Cheers C . Essential roles for granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF in the sustained hematopoietic response of Listeria monocytogenes-infected mice. Blood 1998;91:863-869
Web of Science | Medline15
Condliffe AM ,Kitchen E ,Chilvers ER . Neutrophil priming: pathophysiological consequences and underlying mechanisms. Clin Sci (Lond) 1998;94:461-471
Web of Science | Medline16
Fleischmann J ,Golde DW ,Weisbart RH ,Gasson JC . Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood 1986;68:708-711
Web of Science | Medline17
Graves V ,Gabig T ,McCarthy L ,Strour EF ,Leemhuis T ,English D . Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils. Blood 1992;80:776-787[Erratum, Blood 1993;81:1668.]
Web of Science | Medline18
Clark RA, Nauseef WM. Isolation and functional analysis of neutrophils. In: Bierer B, Coligan JE, Margulies DH, Shevach EM, Strober W, eds. Current protocols in immunology. New York: John Wylie, 2005:2-6.
19
Berclaz PY ,Zsengeller Z ,Shibata Y , et al. Endocytic internalization of adenovirus, nonspecific phagocytosis, and cytoskeletal organization are coordinately regulated in alveolar macrophages by GM-CSF and PU.1. J Immunol 2002;169:6332-6342
Web of Science | Medline20
Bainton DF ,Ullyot JL ,Farquhar MG . The development of neutrophilic polymorphonuclear leukocytes in human bone marrow. J Exp Med 1971;134:907-934
CrossRef | Web of Science | Medline21
Berclaz PY ,Shibata Y ,Whitsett JA ,Trapnell BC . GM-CSF, via PU.1, regulates alveolar macrophage Fcgamma R-mediated phagocytosis and the IL-18/IFN-gamma-mediated molecular connection between innate and adaptive immunity in the lung. Blood 2002;100:4193-4200
CrossRef | Web of Science | Medline22
Hassan NF ,Campbell DE ,Douglas SD . Phorbol myristate acetate induced oxidation of 2′,7′-dichlorofluorescin by neutrophils from patients with chronic granulomatous disease. J Leukoc Biol 1988;43:317-322
Web of Science | Medline23
DeForge LE ,Billeci KL ,Kramer SM . Effect of IFN-gamma on the killing of S. aureus in human whole blood: assessment of bacterial viability by CFU determination and by a new method using alamarBlue. J Immunol Methods 2000;245:79-89
CrossRef | Web of Science | Medline24
Boxio R ,Bossenmeyer-Pourie C ,Steinckwich N ,Dournon C ,Nusse O . Mouse bone marrow contains large numbers of functionally competent neutrophils. J Leukoc Biol 2004;75:604-611
CrossRef | Web of Science | Medline25
Rosenzweig SD ,Holland SM . Phagocyte immunodeficiencies and their infections. J Allergy Clin Immunol 2004;113:620-626
CrossRef | Web of Science | Medline26
Meager A ,Wadhwa M ,Bird C , et al. Spontaneously occurring neutralizing antibodies against granulocyte-macrophage colony-stimulating factor in patients with autoimmune disease. Immunology 1999;97:526-532
CrossRef | Web of Science | Medline27
Roberts AW . G-CSF: a key regulator of neutrophil production, but that's not all! Growth Factors 2005;23:33-41
CrossRef | Web of Science | Medline28
Treweeke AT ,Aziz KA ,Zuzel M . The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming. J Leukoc Biol 1994;55:612-616
Web of Science | Medline29
Hellmich B ,Csernok E ,Schatz H ,Gross WL ,Schnabel A . Autoantibodies against granulocyte colony-stimulating factor in Felty's syndrome and neutropenic systemic lupus erythematosus. Arthritis Rheum 2002;46:2384-2391
CrossRef | Web of Science | Medline30
Iwasaki H ,Somoza C ,Shigematsu H , et al. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation. Blood 2005;106:1590-1600
CrossRef | Web of Science | Medline31
Guthridge MA ,Powell JA ,Barry EF , et al. Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. EMBO J 2006;25:479-489
CrossRef | Web of Science | Medline32
Rosenbloom AJ ,Linden PK ,Dorrance A ,Penkosky N ,Cohen-Melamed MH ,Pinsky MR . Effect of granulocyte-monocyte colony-stimulating factor therapy on leukocyte function and clearance of serious infection in nonneutropenic patients. Chest 2005;127:2139-2150
CrossRef | Web of Science | Medline33
Krinner EM ,Raum T ,Petsch S , et al. A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF. Mol Immunol 2007;44:916-925
CrossRef | Web of Science | Medline34
Mann BS ,Chung KF . Blood neutrophil activation markers in severe asthma: lack of inhibition by prednisolone therapy. Respir Res 2006;7:59-59
CrossRef | Web of Science | Medline35
Koller DY ,Urbanek R ,Gotz M . Increased degranulation of eosinophil and neutrophil granulocytes in cystic fibrosis. Am J Respir Crit Care Med 1995;152:629-633
Web of Science | Medline36
Rivkind AI ,Siegel JH ,Littleton M , et al. Neutrophil oxidative burst activation and the pattern of respiratory physiologic abnormalities in the fulminant post-traumatic adult respiratory distress syndrome. Circ Shock 1991;33:48-62
Medline37
Bozinovski S ,Jones J ,Beavitt SJ ,Cook AD ,Hamilton JA ,Anderson GP . Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4. Am J Physiol Lung Cell Mol Physiol 2004;286:L877-L885
CrossRef | Web of Science | Medline
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Carolina Althoff Souza, Edson Marchiori, Letícia Pereira Gonçalves, Gustavo de Souza P. Meirelles, Gláucia Zanetti, Dante L. Escuissato, Julia Capobianco, Arthur Soares Souza. (2012) Comparative study of clinical, pathological and HRCT findings of primary alveolar proteinosis and silicoproteinosis. European Journal of Radiology 81:2, 371-378
CrossRef2
J. Carlier, H. Martin, B. Mariame, B. Rauwel, C. Mengelle, H. Weclawiak, A. Coaquette, C. Vauchy, P. Rohrlich, N. Kamar, L. Rostaing, G. Herbein, C. Davrinche. (2011) Paracrine inhibition of GM-CSF signaling by human cytomegalovirus in monocytes differentiating to dendritic cells. Blood 118:26, 6783-6792
CrossRef3
Megan Stuebner Devine, Christine Kim Garcia. (2011) Genetic Interstitial Lung Disease. Clinics in Chest Medicine
CrossRef4
Charles M. Samson, Ingrid Jurickova, Erin Molden, William Schreiner, Joshua Colliver, Erin Bonkowski, Xiaonan Han, Bruce C. Trapnell, Lee A. Denson. (2011) Granulocyte-macrophage colony stimulating factor blockade promotes ccr9+ lymphocyte expansion in Nod2 deficient mice. Inflammatory Bowel Diseases 17:12, 2443-2455
CrossRef5
Doris S C Shim, Heidi C Schilter, Michelle L Knott, Ranni A Almeida, Robert P Short, Charles R Mackay, Lindsay A Dent, William A Sewell. (2011) Protection against Nippostrongylus brasiliensis infection in mice is independent of GM-CSF. Immunology and Cell Biology
CrossRef6
Alba Llop-Guevara, Josip Marcinko, Ramzi Fattouh, Manel Jordana. 2011. Interleukin 3, Interleukin 5, and Granulocyte-Macrophage Colony-Stimulating Factor. , 187-196.
CrossRef7
Jason C Lenzo, Amanda L Turner, Andrew D Cook, Ross Vlahos, Gary P Anderson, Eric C Reynolds, John A Hamilton. (2011) Control of macrophage lineage populations by CSF-1 receptor and GM-CSF in homeostasis and inflammation. Immunology and Cell Biology
CrossRef8
Glenn Dranoff. (2011) Granulocyte-Macrophage Colony Stimulating Factor and Inflammatory Bowel Disease: Establishing a Connection. Gastroenterology 141:1, 28-31
CrossRef9
Cade M Nylund, Sharon DʼMello, Mi-Ok Kim, Erin Bonkowski, Jan Däbritz, Dirk Foell, Jon Meddings, Bruce C Trapnell, Lee A Denson. (2011) Granulocyte Macrophage-Colony-stimulating Factor Autoantibodies and Increased Intestinal Permeability in Crohn Disease. Journal of Pediatric Gastroenterology and Nutrition 52:5, 542-548
CrossRef10
Marc Ansari, Anne-Laure Rougemont, Françoise Le Deist, Hulya Ozsahin, Michel Duval, Martin A. Champagne, Jean-Christophe Fournet. (2011) Secondary pulmonary alveolar proteinosis after unrelated cord blood hematopoietic cell transplantation. Pediatric Transplantationno-no
CrossRef11
Miller, Amy Leigh, Schissel, Scott, Levy, Bruce D., Loscalzo, Joseph, . (2011) A Crazy Cause of Dyspnea. New England Journal of Medicine 364:1, 72-77
Full Text12
Sarah K Browne, Steven M Holland. (2010) Immunodeficiency secondary to anticytokine autoantibodies. Current Opinion in Allergy and Clinical Immunology 10:6, 534-541
CrossRef13
Sarah K Browne, Steven M Holland. (2010) Anticytokine autoantibodies in infectious diseases: pathogenesis and mechanisms. The Lancet Infectious Diseases 10:12, 875-885
CrossRef14
Laia Egea, Yoshihiro Hirata, Martin F Kagnoff. (2010) GM-CSF: a role in immune and inflammatory reactions in the intestine. Expert Review of Gastroenterology & Hepatology 4:6, 723-731
CrossRef15
C. Basset-Léobon, L. Lacoste-Collin, J. Aziza, J. C. Bes, S. Jozan, M. Courtade-Saïdi. (2010) Cut-off values and significance of Oil Red O-positive cells in bronchoalveolar lavage fluid. Cytopathology 21:4, 245-250
CrossRef16
Gary P. ANDERSON. (2010) Prospects for an asthma vaccine. Respirology 15:6, 902-908
CrossRef17
Masato Watanabe, Kanji Uchida, Kazuhide Nakagaki, Bruce C. Trapnell, Koh Nakata. (2010) High avidity cytokine autoantibodies in health and disease: Pathogenesis and mechanisms. Cytokine & Growth Factor Reviews 21:4, 263-273
CrossRef18
Brenna Carey, Bruce C. Trapnell. (2010) The molecular basis of pulmonary alveolar proteinosis. Clinical Immunology 135:2, 223-235
CrossRef19
Jeffrey A. Whitsett, Susan E. Wert, Timothy E. Weaver. (2010) Alveolar Surfactant Homeostasis and the Pathogenesis of Pulmonary Disease. Annual Review of Medicine 61:1, 105-119
CrossRef20
K. Uchida, B. Carey, T. Suzuki, K. Nakata, B. Trapnell. (2010) Response: Granulocyte/macrophage colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy persons. Blood 115:2, 431-433
CrossRef21
Sakagami, Takuro, Uchida, Kanji, Suzuki, Takuji, Carey, Brenna C., Wood, Robert E., Wert, Susan E., Whitsett, Jeffrey A., Trapnell, Bruce C., , Luisetti, Maurizio, . (2009) Human GM-CSF Autoantibodies and Reproduction of Pulmonary Alveolar Proteinosis. New England Journal of Medicine 361:27, 2679-2681
Full Text22
Sarah B. Redmond, Phongsakorn Chuammitri, Claire B. Andreasen, Dušan Palić, Susan J. Lamont. (2009) Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis. Veterinary Immunology and Immunopathology 132:2-4, 129-134
CrossRef23
Ann L. Cornish, Ian K. Campbell, Brent S. McKenzie, Simon Chatfield, Ian P. Wicks. (2009) G-CSF and GM-CSF as therapeutic targets in rheumatoid arthritis. Nature Reviews Rheumatology 5:10, 554-559
CrossRef24
Bruce C Trapnell, Brenna C Carey, Kanji Uchida, Takuji Suzuki. (2009) Pulmonary alveolar proteinosis, a primary immunodeficiency of impaired GM-CSF stimulation of macrophages. Current Opinion in Immunology 21:5, 514-521
CrossRef25
Zhihao XU, Jiyong JING, Huiying WANG, Feng XU, Jiaoli WANG. (2009) Pulmonary alveolar proteinosis in China: A systematic review of 241 cases. Respirology 14:5, 761-766
CrossRef26
U. Keilholz, A. Letsch, A. Busse, A. M. Asemissen, S. Bauer, I. W. Blau, W.-K. Hofmann, L. Uharek, E. Thiel, C. Scheibenbogen. (2009) A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS. Blood 113:26, 6541-6548
CrossRef27
Xiaonan Han, Kanji Uchida, Ingrid Jurickova, Diana Koch, Tara Willson, Charles Samson, Erin Bonkowski, Anna Trauernicht, Mi-Ok Kim, Gitit Tomer, Marla Dubinsky, Scott Plevy, Subra Kugathsan, Bruce C. Trapnell, Lee A. Denson. (2009) Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies in Murine Ileitis and Progressive Ileal Crohn's Disease. Gastroenterology 136:4, 1261-1271.e3
CrossRef28
K. Uchida, K. Nakata, T. Suzuki, M. Luisetti, M. Watanabe, D. E. Koch, C. A. Stevens, D. C. Beck, L. A. Denson, B. C. Carey, N. Keicho, J. P. Krischer, Y. Yamada, B. C. Trapnell. (2009) Granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects. Blood 113:11, 2547-2556
CrossRef29
José Antonio Rodríguez Portal, Eulogio Rodríguez Becerra, Antonio Sánchez Garrido. (2009) Proteinosis alveolar. Respuesta al tratamiento con factor estimulante de colonias de granulocitos y macrófagos por vía inhalada. Archivos de Bronconeumología 45:3, 150-152
CrossRef30
Carina de Lemos Rieper, Pia Galle, Morten Bagge Hansen. (2009) Characterization and potential clinical applications of autoantibodies against cytokines. Cytokine & Growth Factor Reviews 20:1, 61-75
CrossRef31
Boris W. Kramer, Suhas Kallapur, John Newnham, Alan H. Jobe. (2009) Prenatal inflammation and lung development. Seminars in Fetal and Neonatal Medicine 14:1, 2-7
CrossRef32
Christopher A. French. 2009. Respiratory Tract. , 65-103.
CrossRef33
K. Uchida, K. Nakata, T. Suzuki, M. Luisetti, M. Watanabe, D. E. Koch, C. A. Stevens, D. C. Beck, L. A. Denson, B. C. Carey, N. Keicho, J. P. Krischer, Y. Yamada, B. C. Trapnell. (2009) Granulocyte/macrophage-colony-stimulating factor autoantibodies and myeloid cell immune functions in healthy subjects. Blood 113:11, 2547
CrossRef34
F.-C. Lin, Y.-C. Chen, S.-C. Chang. (2008) Clinical Importance of Bronchoalveolar Lavage Fluid and Blood Cytokines, Surfactant Protein D, and Kerbs von Lungren 6 Antigen in Idiopathic Pulmonary Alveolar Proteinosis. Mayo Clinic Proceedings 83:12, 1344-1349
CrossRef35
T. Suzuki, T. Sakagami, B. K. Rubin, L. M. Nogee, R. E. Wood, S. L. Zimmerman, T. Smolarek, M. K. Dishop, S. E. Wert, J. A. Whitsett, G. Grabowski, B. C. Carey, C. Stevens, J. C.M. van der Loo, B. C. Trapnell. (2008) Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA. Journal of Experimental Medicine 205:12, 2703-2710
CrossRef36
L. D. Notarangelo, I. Pessach. (2008) Out of breath: GM-CSFR mutations disrupt surfactant homeostasis. Journal of Experimental Medicine 205:12, 2693-2697
CrossRef37
M. Martinez-Moczygemba, M. L. Doan, O. Elidemir, L. L. Fan, S. W. Cheung, J. T. Lei, J. P. Moore, G. Tavana, L. R. Lewis, Y. Zhu, D. M. Muzny, R. A. Gibbs, D. P. Huston. (2008) Pulmonary alveolar proteinosis caused by deletion of the GM-CSFR gene in the X chromosome pseudoautosomal region 1. Journal of Experimental Medicine 205:12, 2711-2716
CrossRef38
Donn Spight, Bruce Trapnell, Bin Zhao, Pierre Berclaz, Thomas P. Shanley. (2008) GRANULOCYTE-MACROPHAGE-COLONY-STIMULATING FACTOR-DEPENDENT PERITONEAL MACROPHAGE RESPONSES DETERMINE SURVIVAL IN EXPERIMENTALLY INDUCED PERITONITIS AND SEPSIS IN MICE. Shock 30:4, 434-442
CrossRef39
Hermann Neugebauer, Pia Hartmann, Sebastian Krenn, Thomas Glück, Jürgen Schölmerich, Rainer Straub, Reiner Wiest. (2008) Bacterial translocation increases phagocytic activity of polymorphonuclear leucocytes in portal hypertension: priming independent of liver cirrhosis. Liver International 28:8, 1149-1157
CrossRef40
A Sergeeva, Y Ono, R Rios, J J Molldrem. (2008) High titer autoantibodies to GM-CSF in patients with AML, CML and MDS are associated with active disease. Leukemia 22:4, 783-790
CrossRef41
Veronika Kleff, Ursula R Sorg, Carsten Bury, Takuji Suzuki, Ina Rattmann, Moran Jerabek-Willemsen, Christopher Poremba, Michael Flasshove, Bertram Opalka, Bruce Trapnell, Uta Dirksen, Thomas Moritz. (2008) Gene Therapy of βc-Deficient Pulmonary Alveolar Proteinosis (βc-PAP): Studies in a Murine in vivo Model. Molecular Therapy
CrossRef42
(2007) GM-CSF Autoantibodies in Pulmonary Alveolar Proteinosis. New England Journal of Medicine 356:19, 2001-2002
Full Text43
Doerschuk, Claire M., . (2007) Pulmonary Alveolar Proteinosis — Is Host Defense Awry?. New England Journal of Medicine 356:6, 547-549
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