We propose to investigate, by cytochemistry and immunocytochemistry the localization within cell-populations and subcellular compartments of the oxidative systems involved in phagocytosis and killing of microorganisms and target-tumor cells by leucocytes. Cytochemical techniques will be developed for localization of the superoxide radical which is generated during phagocytosis in polymorphonuclear leucocytes (PMN). Aldehyde oxidase which has been demonstrated in guinea pig PMN will be studied by a correlated biochemical and cytochemical approach to investigate the potential importance of this enzyme in these cells. Development of appropriate cytochemical techniques will be required. Comparison of the cytochemical localization of H2O2 in PMN from different species and under different conditions of activation (i.e., particulate vs non-particulate stimuli) will be pursued. Morphological and cytochemical techniques will be used to examine the proposition that it is H2O2 or another activated oxygen species that is the basis of tumoricidal activity, particularly at the site of cell-cell interaction. In addition to localization of the products of these oxidative enzymes, we will localize the enzymes per se by immunocytochemistry. Comparison of results obtained with enzyme cytochemistry and immunocytochemistry should provide much more definitive information about the localization of these oxidative systems. Cytochemical techniques will also be employed to study the distribution of Ca ions ions in leucocytes under various states of activation (i.e., phagocytosis, chemotaxis, etc.). The subcellular localization of the calcium regulatory protein calmodulin will be investigated by immunocytochemistry. Comparisons will be made between the localization of Ca ions and calmodulin in order to better understand the role of Ca ions metabolism in leucocytes. Cytochemistry and immunocytochemistry have proved valuable approaches for determining the location of cellular components. Correlation of such cytological data with biochemical data should lead to further better understanding of the structure-function relationships in leucocytes.
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