Labeling Rat Alveolar Macrophages with Fluorescent Microspheres

Abstract

I Alan HodgsonNational Radiological Protection Board, Chilton, Oxfordshire, United Kingdom Rats were exposed for various periods (5, 20, 60, and 720 min) to an aerosol of fluorescent polystyrene microspheres (FPM) with nominal diameter 7.07 p m . All the animals were killed 40 h later and the lungs were lavaged 70 times with physiological saline. Washings 7-2 and 3-70 were combined. The numbers of alveolar macrophages (AM) recovered by lavage were measured with a Coulter counter. Cytospin slides of cells in the lavage fluid were prepared and used to determine the fraction of recovered AM that contained FPM (labeling index, LO and the FPMIAM profile. The same parameters were also measured by flow cytometry. There was good agreement between determinations of LI and FPMIAM by manual scoring methods and by flow cytometry. Samples of lavage fluid were digested with sodium hypochlorite (bleach), and aliquots of the resulting digests were filtered. Examination of the filters with epifluorescence microscopy enabled the total numbers of FPM present in the various lung washes to be determined. The lavaged lungs were also digested with bleach, and the number of FPM remaining was estimated by manual scoring only. Flow cytometry could not be used for this purpose, due to the presence of cellular debris. After exposure to airborne FPM at the concentration used for 2 h, less than half the recovered AM contained FPM. For studies of AM kinetics it will be necessary to achieve higher LI values and numbers of FPM per cell than were obtained in the present study.

Are Macrophages Recovered from Mouse Lung by Bronchoalveolar Lavage Representative of the Alveolar Macrophage Pool?

“…Another possibility is that heavily loaded AM are more readily recovered by lavage. With the rat, there appear to be much smaller differences in the characteristics of macrophages recovered in the initial and later lung washes (Morgan et al, 1995).…”

“…When lavage was used to recover cells from the mouse lung following inhalation exposure to monodisperse fluorescent polystyrene particles (FP) it was found that AM recovered in washes 1 and 2 had a somewhat higher labeling index (LI, proportion of AM containing one or more FP) than AM recovered in subsequent washes and that this difference persisted until at least 8 days after exposure (Morgan et al, 1991). However, more recent studies of the recovery of macrophages from the rat lung following inhalation of FP showed that differences in the labeling index of AM recovered in the initial and subsequent lung washes were not significant (Morgan et al, 1995).…”

“…These include investigations of the uptake of particles by alveolar macrophages in vitro (Mueller & Guilmette, 1987a) and in vivo following administration either by inhalation (Mueller & Guilmette, 1987b;Morgan et al, 1991Morgan et al, , 1995 or by intratracheal instillation (Takahashi et al, 1992). They have also been used in studies of the clearance of particles from the lung (Velasquez & Morrow, 1984;Lehnert, 1992) and from the pleural cavity (Lehnert et al, 1988).…”