Bronchoscopy with BAL and lung function
Bronchoscopy with BAL was performed as previously
described34. Patient number 4 became obstructive after
the first bronchoscopy and patient number 5 was excluded due to a
possible adverse event and the procedure was therefore not repeated in
these two patients. Cells in BALF were separated from recovered fluid by
centrifugation, fixed on cytospin slides and stained with Giemsa for
calculation of leucocyte differential count. Mast cells were counted in
10 visual fields in x16 magnification. The percentage of CD4+ and CD8+
T-cells, respectively were measured by triple-laser, eight-colour flow
cytometry using a FACS Fortessa X-20 (Becton-Dickinson). The following
antibodies were used: CD3-FITC clone UCHT1 (BD, Pharmingen, CA, USA);
CD4-APC-H7 clone SK3, CD8-AmCyan clone SK1, CD69-PE-Cy7, clone FN50 (BD
Biosciences, CA, USA); FoxP3-PE clone PCH 101 (eBioscience, San Diego,
CA, USA). Spirometry was performed using a SensorMedics system
(SensorMedics, Yorba Linda, CA, USA).