Presence of GM-CSF autoantibodies in healthy subjects. (A) Total IgG was isolated from the serum of healthy control subjects (HC) or patients with PAP (PAP) or from pharmaceutical grade IVIG by protein G chromatography and subjected to ultrafiltration under acidic conditions to remove bound GM-CSF. GM-CSF autoantibodies were then isolated by GM-CSF affinity chromatography and evaluated by far-Western analysis probed with ¹²⁵I-GM-CSF. Shown are Coomassie Blue–stained electrophoresis gels (bottom panels) and corresponding far-Western blots (top panels). Each numbered lane represents the corresponding bound (left panels) and unbound (right panels) chromatography fractions from 1 subject or sample. (B) Serum GM-CSF–binding proteins from 6 healthy human subjects were fractionated individually on gels as in panel A, and proteins in the 180-kDa band from each were extracted, subjected to liquid chromatography and tandem mass spectroscopy, and the results evaluated by comparison to Mascot database as described under “Methods.” Only matches with a probability-based Mowse score greater than 64 (indicating a P value < .05) were considered in the analysis. The percentages of immunoglobulin (Ig) and non-Ig peptides among the top 50 peptide fragment matches identified for each sample are shown. (C) GM-CSF autoantibodies were isolated by GM-CSF affinity chromatography from serum of healthy subjects (HC; n = 10), patients with PAP (PAP, n = 4), or from pharmaceutical IVIG (n = 1 clinical grade vial), and the percentage of IgG subtypes was measured by ELISA. (D) Serum GM-CSF autoantibody concentrations in healthy subjects (HC), patients with PAP (PAP), or IVIG (reconstituted at 9.94 mg/mL in PBS). Serum GM-CSF autoantibody levels in healthy subjects (median (interquartile range [IQR]) = 1.04 [0.63-1.7] μg/mL, n = 72) were lower than in patients with PAP (median [IQR] = 59.8 (27.4–116.5) μg/mL; n = 21; P < .001). Median values (HC, PAP) are indicated by a horizontal bar. (E) Serum GM-CSF autoantibody levels in males (n = 15) and females (n = 57). Data are shown as whisker plots indicating the interquartile range (upper and lower borders of box), the 90th and 10th percentile (error bars), the 95th and 5th percentile (upper and lower open symbols), the median (solid horizontal line in box), and mean (dashed line in box) values of GM-CSF autoantibody levels. (F) Serum GM-CSF autoantibody levels in healthy women (△) and men ( □ ) of various ages (n = 72). Regression analysis did not reveal a significant correlation GM-CSF autoantibody levels with age (R² = 0.08).