This proposal will use fluorescent chemotactic factors and techniques of flow cytometry and quantitative video intensification microscopy (QUAVIM) to study human neutrophil (PMN) and monocyte function and dysfunction. Fluorescein derivatives of C5a, casein and f-Met-Leu-Phe-Lys (fMLP-Lys) have been prepared and similar probes labeled with red fluorochromes will be developed. The first goal is to examine the cycling and modulation of distinct chemotactic receptors for casein, fMLP-Lys and C5a on PMN and monocytes using fluorescent derivatives of these factors. This will include determining the temporal pattern and cellular mechanism involved in receptor expression, how these receptors are modulated during phagocytosis or cell activation by soluble stimuli and if these receptors are regulated independently. The presence of each of these receptors on primary and secondary granules will be examined and a new method of granule isolation using flow cytometry will be developed. QUAVIM on single cells will be used to complement flow cytometry to directly visualize and quantitate receptor distribution, mobilization and re-expression. Multiparameter flow cytometry will be used to define subsets of PMN and monocytes using fluorescent chemotactic factors and functional assays (phagocytosis, H202 production, membrane depolarization, NADPH content and degranulation). The second goal is to define the sequence of functional maturation and chemotactic factor expression using HL-60 and U937 cell lines as models of PMN and monocyte differentiation. Studies will determine if these lines express any or all 3 chemotactic receptors, when in cell differentiation receptors appears and if subsets of cells identified by function and receptor expression are found in human bone marrow. The third objective is to apply single-cell assays of cell function and receptor expression to the study of patients with acute myeloid leukemia and patients undergoing chronic hemodialysis where extensive complement activation occurs. These studies will provide a unique approach to evaluation of the stage of leukemic cell differentiation and its relationship to treatment and survival, as well as information pertaining to the sequence of normal cell differentiation and receptor expression. Studies with hemodialysis patients will determine how in vivo C5a activation may modulate both receptor expression and cell function and provide a better understanding of leukocyte dysfunction in these patients.
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