Studies of chemotactic factor-induced polarity in human neutrophils : Lipid mobility, receptor distribution and the time-sequence of polarization

Differences in membrane composition between the anterior and posterior poles of human blood neutrophils on exposure to chemoattractant were studied using a laser microscope, and the effects of attractant concentration and time on the ability of neutrophils to polarize were determined. The findings were as follows. (1) The fluorescein-labelled chemotactic hexapeptide fLeu-Norleu-Phe-Norleu-Tyr-Lys was asymmetrically distributed on polarized cells with the highest concentration at the anterior pole of the cell. (2) Differences in membrane lipid behaviour were studied by fluorescence recovery after photobleaching (FRAP) using the probe 5-N-(octadecanoyi)-aminofluorescein. Recovery curves suggested that the proportion of mobile lipid probe at the anterior pole of the cell was higher than at the posterior. However, no difference was found in the rate of recovery between the two poles. (3) Studies of the time-course of polarization were undertaken using neutrophils in suspension exposed to different isotropic concentrations of fMet-Leu-Phe (fMLP). At low concentrations (≤10-9M), the cells polarized immediately on exposure to the attractant. At a high concentration (10-7M) they assumed multipolar morphologies and polarized very poorly, suggesting that ligand binding was too rapid for the cells to form a dominant pole. At the optimal concentration, 10-8M fMLP, the cells assumed irregular, ruffled, morphologies after 30 s, but showed an increasing proportion of polarized forms over the next 30 min. These results suggest that, while cells exposed to low concentrations of attractant may respond by polarizing towards the site of a first signal from ligand, at optimal attractant concentrations a dominant pole is formed by integration of responses to ligand-binding at multiple sites. The polarization response is likely to be a stochastic process determined by chance events such as asymmetry or fluctuations in ligandreceptor binding.