Formation of GM-CSF-autoantibody immune complexes in iPAP patients. (A) Detection of GM-CSF containing immune complexes in the BALF in iPAP by immunoprecipitation. Protein A Sepharose beads were incubated with BALF from a healthy control (lanes 1-2), iPAP patient (lanes 3-4), or, as a control, purified yeast-derived rhGM-CSF (lane 5). Bound and unbound fractions (indicated) were separated and subjected to SDS-PAGE under reducing conditions and Western blotting using a rabbit anti-GM-CSF antibody. Under these conditions, the rhGM-CSF standard consists of 3 molecular species migrating at 19.5, 16.8, and 15.5 kDa. (B) Detection of GM-CSF and IgG in BALF by Western blotting. BALF from a healthy control (lanes 2, 4), iPAP patient (lanes 3, 5), or rhGM-CSF (lane 1) was subjected to native PAGE and then Western blotting using a rabbit anti-GM-CSF antibody (lanes 1-3) or anti-IgG antibody (lanes 4-5). (C) Supershift of [¹²⁵I]-GM-CSF with iPAP-BALF. BALF from healthy controls (lanes 2-4) or iPAP patients (lanes 5-7) was incubated with [¹²⁵I]-GM-CSF and then subjected to native PAGE and autoradiography. As a control, [¹²⁵I]-GM-CSF alone was also included on the gel (lane 1).