Correlation of GM-CSF autoantibody level and basal neutrophil function in vivo, and effects of GM-CSF autoantibody level on neutrophil function in vitro. (A) The phagocytic capacity of unstimulated neutrophils in whole blood was measured in healthy subjects (○), patients with PAP with active disease (◇), and patients with PAP in clinical remission of the lung disease (♦) by quantifying the uptake of IgG-opsonized latex microspheres as described under “Methods.” The range of serum GM-CSF autoantibody levels separating healthy subjects and patients with PAP with active disease evaluated with this assay is indicated (3.2-39 μg /mL, ▨). Each symbol represents the results for triplicate determinations for one subject. The mean (IQR) free serum GM-CSF in healthy subjects was 0.00 (0.00-0.267) pg/mL serum and did not correlate with the neutrophil phagocytic capacity (P > .05), whereas GM-CSF autoantibody levels (1.07 [0.74-1.66] μg/mL) correlated with neutrophil phagocytic capacity (R² = −0.70, P = .001) (Spearman rank order correlation). (B) Neutrophil chemotaxis was measured as described in “Methods.” In brief, neutrophils were placed in the upper chamber of a transwell culture plate and IL-8 (10 ng/mL) was placed in the lower chamber, both the upper and lower chambers (chemokinesis [CK] control) or was omitted (No IL-8) and GM-CSF autoantibody (0.5, or 1.0 μg/mL) or isotype control antibody (0.5 or 1.0 μg/mL) was placed in the upper chamber. Each bar represents the mean (± SE) for results from 3 determinations. Compared with the respective isotype antibody controls, increasing concentrations of GM-CSF autoantibody reduced neutrophil chemotaxis in rheostatic fashion (*P < .05; **P < .005).