One of the important and so far poorly understood regulatory mechanisms controlling expression of some functionally important cell surface molecules (e.g. cytokine receptors adhesion molecules, membrane enzymes, etc.) is cleavage (shedding) by endogenous enzymes induced by external physiologic stimuli. Aberrant shedding of surface glycoproteins seems to be implicated in pathogenesis of some diseases, such as Alzheimer's disease. Intensity of shedding of some of these molecules may be useful as a diagnostic marker, for example, indicating progression of a pathologic process. In the present project, leukocyte surface molecules will be used as an experimental model to study shedding. The major questions to be addressed will be: 1) Which antigens are shed from leukocytes stimulated by various activators? 2) Which enzymes are involved in shedding? 3) Do these enzymes act as secreted soluble enzymes or are they membrane bound? 4) Is a universal enzyme responsible for the cleavage of several surface antigens from a single leukocyte subpopulation or does a group of specific enzymes, attacking only their specific target molecules, exist? 5) What is the physiologic role of soluble molecules produced by the cleavage of their membrane counterparts in vivo? To answer these questions, screening will be performed to detect functionally important molecules shed from stimulated leukocytes. the loss of molecules from the cell surface analyzed by flow cytometry and isolation of radioactive labeled soluble forms produced in vitro after cell stimulation will be used for identification of these molecules. Monoclonal antibodies prepared by immunizing mice with leukocytes together with antibodies already available recognizing various leukocyte enzymes will be tested for their capacity to block shedding. These antibodies immobilized to solid carrier will be employed for isolation of enzymes responsible for shedding. The isolated enzymes will be characterized biochemically. Their specificity will be examined using commercially available inhibitors as well as natural substrates. In addition, the properties of soluble forms of antigens shed in vitro will be compared to the soluble forms produced in vivo. Concentrations of soluble forms in human sera and other biologic fluids will be determined by sandwich ELISA and will be correlated with various pathologic conditions.
