Intraphagolysosomal pH in canine and rat alveolar macrophages: flow cytometric measurements.

Intracellular dissolution of inhaled inorganic particles is an important clearance mechanism of the lung and occurs in phagolysosomal vacuoles of phagocytes. Flow cytometric measurements of intraphagolysosomal pH in alveolar macrophages (AM) obtained from beagle dogs, Wistar rats, and from a baboon were made using fluorescein isothiocyanate-labeled amorphous silica particles (FSP). AM were obtained by bronchoalveolar lavage. FSP were phagocytized by AM in cell suspensions incubated in full media for 24 hr up to 6 days. Dual laser flow cytometry was performed and six-parameter list mode data were recorded from forward scatter, side scatter, and fluorescence intensities at 530 nm excited at 457 nm and 488 nm as well as logarithmic fluorescence intensity at wavelengths 630 nm excited at 488 nm. In this way it was possible to discriminate viable AM with phagocytized FSP from lysing AM with phagocytized FSP and from cells without FSP and from free FSP. Viable cells were distinguished from lysing cells by staining with propidium iodide immediately before the flow cytometric measurement. A calibration curve for the pH value was determined from FSP suspended in buffered media at pH values ranging from 3.5 to 7.5. First flow cytometrical results indicated that after an incubation time of 24 hr, the mean intraphagolysosomal pH of viable AM was 4.7 +/- 0.3 for dogs and 5.1 +/- 0.5 for rats. The intraphagolysosomal pH of the baboon AM was 4.5

Long-term clearance from small airways in subjects with ciliary dysfunction

The objective of this study was to investigate if long-term clearance from small airways is dependent on normal ciliary function. Six young adults with primary ciliary dyskinesia (PCD) inhaled (111 )Indium labelled Teflon particles of 4.2 μm geometric and 6.2 μm aerodynamic diameter with an extremely slow inhalation flow, 0.05 L/s. The inhalation method deposits particles mainly in the small conducting airways. Lung retention was measured immediately after inhalation and at four occasions up to 21 days after inhalation. Results were compared with data from ten healthy controls. For additional comparison three of the PCD subjects also inhaled the test particles with normal inhalation flow, 0.5 L/s, providing a more central deposition. The lung retention at 24 h in % of lung deposition (Ret(24)) was higher (p < 0.001) in the PCD subjects, 79 % (95% Confidence Interval, 67.6;90.6), compared to 49 % (42.3;55.5) in the healthy controls. There was a significant clearance after 24 h both in the PCD subjects and in the healthy controls with equivalent clearance. The mean Ret(24 )with slow inhalation flow was 73.9 ± 1.9 % compared to 68.9 ± 7.5 % with normal inhalation flow in the three PCD subjects exposed twice. During day 7–21 the three PCD subjects exposed twice cleared 9 % with normal flow, probably representing predominantly alveolar clearance, compared to 19 % with slow inhalation flow, probably representing mainly small airway clearance. This study shows that despite ciliary dysfunction, clearance continues in the small airways beyond 24 h. There are apparently additional clearance mechanisms present in the small airways

Mucociliary and long-term particle clearance in airways of patients with immotile cilia

Spherical monodisperse ferromagnetic iron oxide particles of 1.9 μm geometric and 4.2 μm aerodynamic diameter were inhaled by seven patients with primary ciliary dyskinesia (PCD) using the shallow bolus technique, and compared to 13 healthy non-smokers (NS) from a previous study. The bolus penetration front depth was limiting to the phase1 dead space volume. In PCD patients deposition was 58+/-8 % after 8 s breath holding time. Particle retention was measured by the magnetopneumographic method over a period of nine months. Particle clearance from the airways showed a fast and a slow phase. In PCD patients airway clearance was retarded and prolonged, 42+/-12 % followed the fast phase with a mean half time of 16.8+/-8.6 hours. The remaining fraction was cleared slowly with a half time of 121+/-25 days. In healthy NS 49+/-9 % of particles were cleared in the fast phase with a mean half time of 3.0+/-1.6 hours, characteristic of an intact mucociliary clearance. There was no difference in the slow clearance phase between PCD patients and healthy NS. Despite non-functioning cilia the effectiveness of airway clearance in PCD patients is comparable to healthy NS, with a prolonged kinetics of one week, which may primarily reflect the effectiveness of cough clearance. This prolonged airway clearance allows longer residence times of bacteria and viruses in the airways and may be one reason for increased frequency of infections in PCD patients