Influence of acridine tracer dyes on neutrophil function

A study was performed to elucidate the effect of two commonly used fluorescent dyes in in vivo microscopic studies, acridine orange (AO) and acridine red (AR), on the ability of phorbol myristate acetate (PMA)- or formyl peptide (fMLP)-stimulated human neutrophils to adhere to a bovine serum albumin matrix and to generate superoxide anions (SOX). Unlabeled stimulated human neutrophils showed 36 +/- 9% (PMA, 10(-7) M) and 11 +/- 7% (fMLP, 10(-7) M) adherence to the matrix. This adhesion was CD18 dependent as evidence by 98% and 92% reduction, respectively, when the anti-CD18 antibody IB4 was included. A dose-dependent inhibition of stimulated human neutrophil adhesion was evident after 30 min of in vitro dye labeling and the EC50 was approximately 70 micrograms/ml (AR) and 145 micrograms/ml (AO). SOX generation by PMA-stimulated neutrophils was unaffected up to 100 micrograms/ml AR and AO but was reduced by 40-60% at higher doses. Rabbit neutrophils labeled in vivo or in vitro with 100 micrograms/ml AR exhibited 41% and 61% lower SOX generation, respectively. The study indicate that neutrophil function, in terms of ability to adhere to a BSA matrix using CD11/CD18 integrins and to generate SOX upon stimulation, is reduced depending on the dose and choice of fluorescent dye. Caution should be exercised when using these compounds at high concentrations in studies of PMN function.