ELECTRON MICROSCOPE STUDY OF ERYTHROPHAGOCYTOSIS phagocytosis vacuoles

The present study describes a submicroscopic surface fragmentation of erythrocytes which occurs in the ascitic fluid of rats bearing the Novikoff ascites hepatoma. The resulting fragments attach to the surface of macrophages and are phagocytized by pseudopod formation. Plasma membrane in the region of these phagocytosis vacuoles appears to condense into electron-opaque material, suggesting an alteration in its physicochemical state. Stages in intracellular digestion of intact erythrocytes or small fragments within the phagocytosis vacuoles are illustrated; no particles resembling ferritin are observed. The phagocytosis vacuoles possess high levels of acid phosphatase activity. They may be called phagosomes, a type of lysosome. There is no indication of a connection between phagosomes and other formed cytoplasmic organelles. Small vacuoles of the order of 80 m/z in diameter, which may represent pinocytosis vacuoles, are present in the cytoplasm and some appear to be in contact with the phagosome membrane, reminiscent of observations of Rose with HeLa cells. I N T R O D U C T I O N Although the phenomenon of erythrophagocytosis has been recognized and investigated for almost a century (1), the essential features of this process still remain obscure. Recent cytological studies have added little to the earlier descriptions, due largely to limitations in the resolving power of the light microscope. With the concept of the reticuloendothelial system formulated by Aschoff (2), physiologists have generally confined themselves to measuring the rates at which erythrocytes are cleared from the circulating blood, leaving unanswered direct questions concerning the fundamental nature of this important process. While it is generally agreed that only "aged," "worn," or otherwise "altered" erythrocytes are candidates for phagocytosis (3), opinion is divided on whether this represents the major pathway for their destruction. Recent studies (3) appear to support the earlier contention of Rous (4) that erythrocytes are eliminated primarily through fragmentation, but whether the resultant particles are subsequently phagocytized is not always clear. * Supported by grants from the United States Public Health Service (C-4131-C2) and the American Cancer Society (E-60 A). Other workers have not observed erythrocyte fragmentation (5). In any case, the sequential steps in the transport of erythrocytes or of their fragments across the surface membrane of the macrophage are not well known, nor is it clear what events occur in the intracellular digestion of this material. With the currently available procedures of electron microscopy, phagocytosis and other mechanisms operating at the cell surface can be revealed in much greater detail. This should make it possible to clarify the interrelatedness of such processes as phagocytosis, pinocytosis (6, 7), and cytopempsis (8). Cytochemical staining procedures can supplement electron microscopy in delineating the enzymatic machinery of the cytoplasmic structures involved in the ingestion and digestion of materials. The present work is concerned with erythrophagocytosis as it occurs in the hemorrhagic fluid within the peritoneal cavity of rats bearing the Novikoff ascites hepatoma (9). I t describes surface fragmentation of the erythrocyte. The resulting fragments are engulfed by formation of pseudopods of the macrophage with the result that a vacuole is formed limited by the plasma membrane of the macrophage. By staining procedures, acid phosphatase activity can be demonstrated within these