Quantitation of characteristics of polymorphonuclear leukocytes (PMNL) from patients with acute bacterial infections (ABI) has been limited in that standard assays measure mean activity of a bulk cell population. Such studies have suggested variable alterations in toxic PMNL; PMNL from most patients demonstrated augmented (""""""""activated"""""""") responses, but some patient's PMNL had decreased responses to stimulation in vitro. The variability of the previous data is probably due to 1) toxic PMNL comprise a quantitatively variable subpopulation of circulating PMNL in patients with ABI, 2) even among the toxic PMNL subpopulation, these cells may differ in their degree of """"""""toxicity"""""""", extending from activated to deactivated. We will employ a new, quantitative, biochemically defined flow cytometric assay to monitor oxidative product formation by normal PMNL, PMNL from patients with ABI and from patients with other causes of neutrophil leukocytosis, to define the presence of PMNL subpopulatins of varying oxidative metabolic capability. We will also examine the cellular mechanisms which hypothetically couple membrane stimulation to functional responses, e.g., transmembrane potential depolarization and increased cytosolic free Ca++ concentration. Using dual beam fluorescence flow cytometry, we will perform correlative analysis of any two of these variables at the same time within individual cells, to ascertain relationships between these putatively casually related events in stimulated normal and toxic PMNL. This should define the functional/biochemical alterations of toxic PMNL with a precision previously not attainable. With this definition of the alterations of toxic PMNL, especially those with augmented (activated) responsiveness in vitro, we will examine whether defined humoral agents caused similar activation of normal PMNL during co-incubation in vitro. Agents to be examined will include highly purified chemotactic factors (C5a, N-formyl-methionyl peptides), substances released by stimulated neutrophils (in particular hydroxylated derivatives of arachidonic acid and platelet activating factor), and substances released during acute inflammation by mononuclear cells (in particular interleukin 1 and colony stimulating factor). The ability of these substances to alter each step in the stimulus-response coupling sequence will be quantitatively analyzed.
