High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis

Abstract

Deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mice results in pulmonary alveolar proteinosis (PAP) from impaired surfactant catabolism by alveolar macrophages (AMs). Recently, we have shown that neutralizing anti-GM-CSF autoantibodies develop specifically in patients with idiopathic pulmonary alveolar proteinosis (iPAP). Analogous to murine PAP models, it is plausible that the autoantibodies reduce GM-CSF activity, resulting in AM dysfunction and surfactant accumulation. To examine this hypothesis, we estimated the neutralizing activity of the autoantibodies in the lungs of patients and characterized their biologic properties. GM-CSF bioactivity was completely abrogated in the bronchoalveolar lavage fluid (BALF) of patients with iPAP but not in healthy subjects. Autoantibodies were present in the alveoli in high concentrations and colocalized with GM-CSF. They recognized human GM-CSF with high avidity (K AV ‫؍‬ 20.0 ؎ 7.5 pM) and high specificity, reacting with its superstructure and neutralizing GM-CSF activity to a level 4000 to 58 000 times the levels of GM-CSF normally present in the lung. Although target epitopes varied among patients, GM-CSF amino acids 78 to 94 were consistently recognized. Thus, autoantibodies bind GM-CSF with high specificity and high affinity, exist abundantly in the lung, and effectively block GM-CSF binding to its receptor, inhibiting AM differentiation and function. Our data strengthen the evidence associating anti-GM-CSF autoantibodies with the pathogenesis of this disease.

High titer autoantibodies to GM-CSF in patients with AML, CML and MDS are associated with active disease

“…To demonstrate GM-CSFspecific neutralizing activity of antibodies, rescue of TF-1 cell proliferation with IL-3 (10 ng ml À1 ) was performed. A competition assay with epitope-specific MAb 3092 15,26 was used to test epitope specificity of antibodies. 15 Briefly, the GM-CSF-binding activity of MAb 3092 in the presence of GM-CSF-binding Igs from patients was measured by direct ELISA.…”

“…A competition assay with epitope-specific MAb 3092 15,26 was used to test epitope specificity of antibodies. 15 Briefly, the GM-CSF-binding activity of MAb 3092 in the presence of GM-CSF-binding Igs from patients was measured by direct ELISA. Additionally, the influence of MAb 3092 on binding of patient-derived IgG, IgA and IgM to GM-CSF was tested.…”

“…Inhibition of TF-1 proliferation was estimated according to the method of Uchida. 15 Neutralizing antibody concentrations at 50% growth inhibition (IC 50 ) were obtained from dosedependent growth inhibition curves. To demonstrate GM-CSFspecific neutralizing activity of antibodies, rescue of TF-1 cell proliferation with IL-3 (10 ng ml À1 ) was performed.…”

“…11,12 Immunity to GM-CSF is a potential problem, because autoantibodies to GM-CSF are implicated in the pathogenesis of idiopathic pulmonary alveolar proteinosis (PAP) where they inhibit GM-CSF-mediated endocytosis of surfactant in alveoli. 14,15 The clinical relevance of induced antibodies is not established; however, recent findings indicate that they may weaken the biological activity of endogenous and pharmacological GM-CSF. 16, 17 Disis et al 18 showed that subcutaneous co-administration of tumor-derived peptides and GM-CSF was effective in generating tumor-specific cytotoxic lymphocytes in mice, and GM-CSF has been extensively used as an adjuvant in clinical trials of anti-tumor vaccines.…”

Plasmapheresis for treatment of pulmonary alveolar proteinosis: Fig. 1—

“…However, it was unable to reduce levels to ,130 mg?mL -1 , despite 10 sequential plasmapheresis treatments over a 2-month period. This resulting concentration is higher than the mean¡SEM serum GM-CSF autoantibody level in 158 autoimmune PAP patients with active lung disease (113¡7 mg?mL -1 ) [1] measured using the same ELISA method [5]. It is also considerably higher than the critical threshold predicted to increase the risk of developing the lung manifestations of autoimmune PAP [6].…”