Subpopulations of human polymorphonuclear leukocytes (PMN) have been reported based on variations in rosetting to IgG coated erythrocytes, chemotaxis, binding of FMLP and membrane potential charge upon stimulation. No size differences have been noted among these subpopulations. Counterflow centrifugal elutriation (CCE) separates cells based on differences in size and density. Phase I of the proposal will evaluate volume dependent subpopulations of blood PMN isolated by CCE. Analysis of the early activation events of receptor ligand binding, membrane potential change, and alteration in membrane bound and cytosolic calcium upon stimulation will be performed. Superoxide has already been shown to be greater on a per cell basis in larger subgroups. Evaluation will be done to assess PMN subpopulation lag time and rate of the oxidative burst after stimulation. To determine if specific granule contents have a role in PMN subpopulations, Vit B12 binding protein will be analyzed. This analysis will assess the hypothesis that subpopulations of PMN exist in varying states of """"""""readiness for activation"""""""". Phase II will assess the contribution of storage pool PMN to PMN subpopulations. PMN from bone marrow samples will be compared to peripheral blood PMN. PMN will also be isolated before and after a subcutaneous injection of epinephrine to assess the marginating pool of PMN. To evaluate the role of the spleen in storage of PMN and in volume dependent PMN subpopulations patients undergoing elective splenectomy will have PMN isolated before and after this procedure. These PMN will be compared to peripheral blood PMN by the methods utilized in Phase I. Analysis of shifts in subpopulations of the storage pool PMN will be performed utilizing CCE. Phase III will evaluate volume dependent PMN subpopulations of patients with altered host defenses. Wiskott-Aldrich syndrome, chronic granulomatous disease, burn patients, a patient with PMN lactoferrin deficiency, and sickle cell disease will be assessed. We believe that this information will aid in the understanding of PMN activation.
