Neutrophils (PMN) are activated via different stimulus-dependent, but still not full elucidated, mechanisms leading to the same end result, the release of their granules' cytotoxic contents. Parameters such as membrane potential changes, activation of phospholipases, kinases and ion-specific channels, changes in cytoplasmic [Ca2++], [K+], [Na+] and pH, and alterations in the cytoskeleton all can figure in the signal trans-duction events leading to this degranulation. During the past five years we have developed a new approach which permits kinetic measurements of changes in a number of these parameters simultaneously, in each cell, and correlation of these observations with receptor occupancy on the same cell. We have also developed new fluorescent probes for the measurement of oxidative burst and of phospholipase A activation, as well as a new technique for the isolation of the neutrophil's separate intact granules and of the plasma membrane (uncontaminated with any other fractions). The combination of these techniques now permits us to detect disproportionation in the responding population of cells, as well as to determine the temporal interdependence of each stimulus' elicited response and to analyze this response in terms of receptor identity (e.g., Fc receptor subclass) and occupancy on both the responding and non-responding subpopulations. We have found that, even when subsaturating doses of stimuli are used, the agonist binds uniformly and equally to all the cells. However, while each cell responds dose-dependently to subsaturating doses of chemotactic peptide (fMLP), only a dose-dependent portion of the cells responds to a subsaturating dose of insoluble immune complex (IIC), with each response being 100% of maximal. Thus, there is a disproportionation which is not attributable to any cooperativity of binding but, rather, indicates that the activation mechanism with IIC involves a bimolecular rate controlling step posterior to the initial receptor/ligand binding. We seek to identify this mechanism and the pathway by which each of these very different stimuli activates a neutrophil. Furthermore, we will examine the Fc receptor mediated activation by comparing multivalent with monovalent immune complexes as stimuli, by determining the specific role of FcRII and of FcRIII alone in the mechanism by which PMN are activated, using monoclonal antibody Fab fragments to block one class of receptors or the other. The requirement for Fc receptor crosslinking can be studied by using monovalent soluble immune complexes (SIC; Ag:Ab=2:1) and then using fluorescently labeled Fab2 fragments of the antibody to perform crosslinks. For all of these studies, we shall make use of oxidative product-indicating immune complexes (which also indicate binding) and Fab or Fab2 fragments (labeled with a variety of fluorescent groups), and/or the Bodipy phospholipase indicator; the correlation between binding and activating will be determined by using membrane potential, [Ca++]in or pHi indicators in the same cells. We shall also use our pure organellar preparations to investigate the role of separate right side out plasma membrane vesicles and of granules in the overall mechanism of PMN activation by soluble (fMLP and SIC) vs insoluble (IIC) stimuli.
Showing the most recent 10 out of 17 publications
