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Mechanisms by which macrophages discriminate between "mature-self" and "senescent-self" were investigated using the human red blood cell (RBC) system as a model. Conditions simulating those encountered in situ were adhered to as closely as possible by using short term culture techniques and incubating with autologous macrophages and immunoglobulins (Ig). It Very little is known about the mechanisms by which macrophages of the reticuloendothelial system recognize and phagocytize effete or senescent cells. Macrophages distinguish between "self" and "non-self," that is, they phagocytize foreign cells while leaving their own alone. Since they also phagocytize senescent self cells, they must be capable of distinguishing between "mature self" and "senescent self." This study was designed to investigate and elucidate the mechanism by which macrophages recognize senescent cells. The human red blood cell (RBC) system was used as a model since macrophages of the RES routinely phagocytize RBC at the end of their 120-day lifespan.The few published studies directed toward macrophage recognition of senescent cells limited themselves to RBC aged in vitro. Numerous studies on the phagocyte's surface have attempted to define the receptor(s) which allow them Abbreviations: RBC, red blood cell(s); Y-, 0-, and "O"-RBC, young, old, and "aged" in vitro RBC, respectively; PNH, pooled normal human; SEM, scanning electron microscopy; SV40, simian virus 40; KLH, keyhole limpet hemocyanin; VCN, Vibrio cholerae neuraminidase.to bind immunoglobulin (Ig) G, IgM, and complement (1-10). These studies may not be applicable to the present problem because they: (1) employed phagocytes from one species and RBC from another (1-6); (2) coated RBC deliberately with antisera, generally unfractionated and from another species (1, 2, 4-6) although allogeneic antisera have occasionally been employed (3, 7); (3) employed multivalent cation coupling reagents, such as chromic chloride, which can aggregate and couple proteins (in this case Ig) to RBC by nonimmunological methods (7); (4) treated RBC with tannic acid, periodate, polylysine, colloidal silica, glutaraldehyde, formalin, etc. (8,(10)(11)(12), which are insults not encountered in situ; and/or (5) evaluated phagocytosis by quantitating attachment of macrophages to RBC (5, 7, 9, 10) based on the assumption that phagocytosis then follows attachment which, unfortunately, is not always the case (7).The few studies which attempted to investigate the mechanism by which macrophages recognize damaged or senescent RBC had defects in their experimental design in that they employed approaches 4 and 5 cited above (9, 10), and cells were "aged" by storing them in serum or Hanks' solution for 48 hr at 370 (9).In short, these previously reported experiments do not seem to simulate conditions in situ. Therefore, it would be prudent to be cautious in extrapolating from these studies on artificially aged RBC the proposed mechanisms whereby macrophages recognize and remove senescent or damaged cells in situ....

To remove senescent red blood cells (RBCs) from the circulation, macrophages must distinguish them from mature RBCs. That is achieved by a specific recognition system. An antigen that develops on the surface of a senescing RBC is recognized and bound by the Fab region of an IgG autoantibody in the serum. Subsequently the Fc region of the autoantibody is recognized and bound by a macrophage, which proceeds to phagocytose the RBC. The antigenic molecule can be extracted from senescent but not young RBCs with Triton X-100 (ref. 4), although 10--30% as much antigen can be extracted from middle-aged as from senescent RBCs. I have now used IgG autoantibodies eluted from senescent RBCs to isolate and purify the IgG-binding antigen on senescent RBCs, and to detect the antigen on other somatic cells. The antigen is a congruent to 62,000-Mr protein which is present on stored platelets, lymphocytes and neutrophils, and on cultured human adult liver and embryonic kidney cells, as well as senescent RBCs.