Measurement of basal, substrate induced and total P-glycoprotein activity in bronchoalveolar lavage T-cell subsets.

ABSTRACT

BACKGROUND: P-glycoprotein (P-gp) is a member of the ABC transporter superfamily. P-gp activity can be detected by measuring efflux of fluorescent substrates such as rhodamine 123 (R123). Our objectives were to evaluate P-gp activity in T cells freshly isolated from bronchoalveolar lavage (BAL) and to develop a strategy to distinguish between basal, in vitro substrate-induced, and total P-gp activities. METHODS: Cells were obtained from blood (n = 44) and BAL (n = 34), stained for expression of CD3, CD4, CD8, and CD14, and incubated with R123 (0.13 microM) +/- cyclosporine (5 microM), a specific P-gp inhibitor. P-gp activity was detected as median fluorescence intensity (MFI) and percentage of cells falling below a pre-established cutoff. RESULTS: BAL T cells displayed significant basal P-gp activity, which was most apparent when measured as percentage below the cutoff. Induced activity (difference between P-gp activity measured after load and efflux) was determined equally well when using the MFI or the percentage below cutoff parameter. Total activity was represented by the efflux parameters (MFI or percentage below cutoff) or by the activity-time area under the curve (AUC) method. The two efflux parameters correlated well but were insensitive to the time-dependent nature of dye efflux. In the AUC method, two samples with identical R123 brightness or percentage below cutoff after dye efflux can have very different total activities, depending on their basal activity. The AUC method was also most sensitive in distinguishing between P-gp activity in peripheral blood and resident lung T cells. Application of this methodology to the comparison of P-gp activity in BAL and peripheral CD8+ T cells best revealed the elevated total P-gp activity in BAL T cells. CONCLUSIONS: We have systematically evaluated several methodologies for analysis of P-gp activity and arrived at a novel and robust strategy amenable to standardization and evaluation of the effects of P-gp modulators in vivo and in vitro.