Figure 7

Molecular diagnosis and mechanism. Peripheral neutrophils were isolated by means of density-gradient centrifugation with Ficoll (GE Healthcare, Munich, Germany and assessed in the presence and absence of GM-CSF for the expression of the GM-CSF Ra and Rb chain, and CD11b. The purity of neutrophils was greater than 95% as assessed by differential cell counts of Pappenheim cytospin preparations. Cell viability was greater than 95% using trypan-blue exclusion method. 106 neutrophils were treated with 50 ng/ml GM-CSF or the same volume of the vehicle buffer (aqua a.i.) for 30 min at 37°C. In the absence of GM-CSF stimulation, GM-CSF-Ra chain expression in the parents was only 50% of that of the controls (Figure. 3 A), whereas GM-CSF-Rb chain and CD11b expression were normal. After stimulation with GM-CSF, GM-CSF-Ra in the patient remained low, whereas the parents' level increased to almost control level. GM-CSF-Rb chain and CD11b stimulation were normal (Figure. 3 A). CD11b expression on the neutrophils was used to assess signal transduction after GM-CSF stimulation. In the patient, similar as blocked by auto-antibodies from a subject with auto-immune PAP, no dose-dependent stimulation of CD11b was observed, demonstrating interruption of signalling (Figure. 3 B).