Programmed cell death, or apoptosis, is a process that is wide-spread in nature and leads to non-necrotic (non-lytic) cell removal during tissue remodelling. It involves a number of active cell processes including characteristic DNA fragmentation, distinctive morphologic alterations and surface changes that can be recognized by phagocytic cells, especially macrophages, leading to uptake into the phagocytes. Apoptosis and subsequent cell removal is found in physiologic processes (e.g. remodelling in the uterus), in deletion of autoreactive lymphocytes in the immune system, as a process of cell removal during resolution of inflammation and in neoplasia. In this proposal we seek to determine the surface changes that occur on apoptotic cells and the mechanisms by which they are recognized by macrophages. We have identified at least two different recognition mechanisms to date: a macrophage integrin, the vitonectin receptor (VnR), and a specific receptor for phosphatidylserine (PS). We have found that a characteristic feature of the apoptotic changes appears to be the loss of normal membrane phospholipid asymmetry and the expression on the outer surface of the cell of the normally inner-leaflet confined PS. Elicited macrophages use the PS receptor but monocyte-derived cells use the VnR. However, murine bone marrow-derived macrophages (which engulf apoptotic cells via the VnR) can be stimulated with digestible particulate stimuli such as beta1-3 glucan to lose the VnR-mediated process and convert to phagocytosing via the PS receptor. This development of PS receptor activity coincides with, and is an excellent marker for, the development of what we have celled the 'inflammatory' macrophage phenotype, characterized by unique surface markers and production of increased lysosomal hydrolases and a variety of cytokines (e.g. PDGF and TGFbeta). Maturation to this phenotype is exclusive of development to cytocidal or IGF1-synthesizing phenotypes and appears to involve a multi-step activation process with autocrine/paracrine priming by lipid mediators and TGFbeta. We suggest therefore that macrophages can exhibit unique and mutually exclusive phenotypes, one of which expresses a PS receptor that is used for removal of apoptotic cells. Three main areas will be investigated: 1) The mechanisms of PS expression on apoptotic cells (neutrophils and lymphocytes) and their relationship to the development of apoptosis itself. The studies will also provide insight into the regulation of normal membrane phospholipid asymmetry. 2) The mechanisms leading to PS receptor induction in murine macrophages. this will, of necessity, include study of the development and control of the inflammatory macrophage phenotype. 3) We propose to clone the receptor as the likely simplest procedure for its detailed characterization and in order to obtain probes and antibody so that its distribution and function can be fully delineated. Techniques will include those of molecular biology, lipid biochemistry and cell biology and are familiar to us and our longstanding collaborators.
Showing the most recent 10 out of 23 publications