Regulation of GM-CSF signaling by GM-CSF autoantibodies in healthy subjects and patients with PAP. (A) The serum GM-CSF–neutralizing capacity of GM-CSF autoantibodies was measured using the TF-1 cell-proliferation assay.²⁰  Equal volumes (30 μL) of serum from healthy subjects (△) or a PAP patient (■) (as a positive control), IVIG reconstituted at physiologic concentration (◇), or culture media (●) (as a negative control) were evaluated. The neutralizing capacity of purified GM-CSF autoantibodies from HC serum or IVIG (dashed line) was intermediate between that of autoantibodies isolated from serum the patient with PAP, which contains high concentrations of GM-CSF autoantibody, and control media, which contains none. (B) Neutrophils isolated from healthy subjects were incubated with various concentrations of GM-CSF affinity-purified autoantibodies isolated from IVIG or with control antibody (1 μg/mL) and stimulated with 10 ng/mL GM-CSF for 15 minutes and total and phosphorylated STAT5 (pSTAT5) was measured by immunoblotting. (C) The signaling activity of free and autoantibody-bound GM-CSF was measured by quantifying the level of STAT5 phosphorylation in isolated neutrophils by immunoblotting (shown as the ratio of phosphorylated STAT5 to total STAT5) as described in “Methods.” The signaling activity of GM-CSF complexed to autoantibody was markedly lower than free GM-CSF (0.142 ± 0.1 vs 2.192 ± 0.2 pg/mL, respectively; n = 3 each; *P < .001). (D) The typical pattern of GM-CSF–stimulated increase in CD11b levels on neutrophils in whole blood (CD11b stimulation index) is shown for a healthy subject (HC) and a patient with PAP (PAP). The amount of exogenous GM-CSF (dashed lines) required to stimulate an increase in neutrophil CD11b levels to the threshold value (dotted line) was lower in HCs than in patients with PAP. The median (IQR) GM-CSF concentration required to reach this stimulation threshold (inset) was significantly higher in patients with PAP than in healthy subjects (120 [80-347] ng/mL, n = 5; and 3.96 [1.07-4.86] ng/mL; n = 12; respectively; P < .002, Mann-Whitney). (E) Concentration-dependent reduction in the CD11b stimulation index by GM-CSF autoantibody purified from IVIG () or patients with PAP (■) and incubated with fresh whole blood at various concentrations. Each bar represents the results of 3 separate determinations. *Significant decrease (P < .001) from baseline determined in the absence of GM-CSF autoantibody. (F) Specificity of purified GM-CSF autoantibody. Neutrophils were incubated in the presence of GM-CSF (10 ng/mL) or IL-8 (100 ng/mL) and in the absence or presence of 1 μg GM-CSF autoantibody or control IgG as indicated. Data represent the level of CD11b in stimulated cells—the level in unstimulated cells. GM-CSF autoantibodies markedly inhibited the GM-CSF–stimulated (■), but resulted in levels of inhibition by IL-8 () that were significantly lower and similar to control (IgG, □). *A significant difference (P < .001) compared with inhibition of the GM-CSF–stimulated increase by GM-CSF autoantibody (■). (G) The CD11b stimulation index was measured in fresh blood from healthy control subjects (○), patients with PAP with active disease (◇), or patients with PAP in clinical remission of the lung disease (♦). The range of serum GM-CSF autoantibody levels separating healthy subjects and patients with PAP with active disease evaluated with this assay is indicated (3.2-24 μg/mL, ▨). Each symbol represents the results of triplicate determinations for one subject. The median (IQR) free serum GM-CSF level in healthy subjects was 0.00 (0.00-0.390) pg/mL and did not correlate with the CD11b stimulation index (P > .05), whereas the median (IQR) GM-CSF autoantibody level (0.90 [0.58-1.19] μg/mL) correlated with CD11b stimulation index (R² = 0.46, P = .03) (Spearman rank order correlation).