Mouse monoclonal antibodies (MAb) raised against purified human neutrophils identified two granulocyte specific antigens the binding of which had opposing effects on antibody dependent neutrophil-mediated killing of tumor targets. One MAb recognized a surface structure with an approximate Mr of 110,000 and stimulated killing, whilst the other recognized a structure with Mr of 95,000 and inhibited this function. The analysis of structure and mechanism of action of these two granulocyte-specific functional antigens is the main aim of this project. Further evidence for the role of these antigens in neutrophil function will be sought by examining the effect of the MAbs on a series of other functions (e.g. degranulation, random and directed movement, phagocytosis and capacity to kill organisms such as Candida albicans). Functional studies on cells with different distributions of these antigens will also be carried out to gain an understanding of the basis of functional heterogeneity of granulocytes. Purification of the antigens will be attempted by affinity chromatography and HPLC. Fragments of the purified material will be subjected to sensitive micro-sequencing techniques. At the same time transfection experiments with DNA from human neutrophils will attempt to isolate the chromosome fragment coding for these antigens. The human DNA will then be cloned and sequenced using established techniques. The relevance of these antigens to granulocyte maturation will be studied in colony-forming assays to identify the stage in differentiation where these antigens are first expressed. The clinical relevance of these antigens will then be sought by correlating the amount of antigen on neutrophils with unstimulated function and the capacity to become stimulated by strong enhancers of function such as colony stimulating factor. The long-term aim of this project is the understanding of steps and structures involved in the regulation of function initially of neutrophils, but subsequently all granulocytes with special reference to basophils and mast cells. It is hoped that this will lead to a rational approach to the assessment of susceptibility not only to infections but also to allergic diseases.
