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[CANCER RESEARCH 32,413-419, February 1972] The Ultrastructure of Sarcoma I Cells and Immune Macrophages during Their Interaction in the Peritoneal Cavities of Immune C57BL/6 Mice Velma C. Chambers and Russell S. Weiser Department of Microbiology, University of Washington, Seattle, Washington 98195 SUMMARY C57BL/6 mice that had recently rejected a primary Sarcoma I (Sal) ascitic tumor were given a second i.p. dose of Sal cells. The interaction of the immune macrophages of the host and the Sal target cells was investigated by electron microscopy. The macrophages rapidly adhered to the target cells and spread over their surfaces. The interaction of the immune macrophages and Sal cells was manifested by interdigitation of their surfaces and the extension of Sal microvilli into deep invaginations of the immune macrophages. The spreading of the immune macrophages over the surfaces of the Sal cells resulted in the apparent phagocytosis of viable tumor cells. Such phagocytized Sal cells were contained within phagocytic vacuoles with the vacuolar membrane of the macrophage usually closely applied to the plasma membrane of the Sal cell. Amorphous material was observed in the vacuolar space, particularly in the mildly dilated areas. These findings suggest that the phagocytosis of Sal cells by immune C57BL/6 macrophages plays an important role in the rejection of the ascitic form of the tumor during the secondary immune response. INTRODUCTION The rejection of the ascitic form of Sal1 by the allogeneic C57BL/6 mouse is accompanied by the presence of large numbers of host macrophages in the peritoneal cavity (1). In earlier phase microscope studies, immune macrophages were observed to attach to the Sal cells and to spread over their surfaces (1). The adherence of immune macrophages to the target cells was later found to be due to the reaction of cytophilic antibody on the surface of the macrophage with antigens of the target cell (9, 10). Adherence per se did not cause destruction of the target cell. A step following adherence of the macrophages to target cells which demands metabolic activity of the macrophages appears to be necessary for target cell destruction (9, 10). It was later discovered that immune macrophages interacting with target cells in vitro release a SMC into the surrounding medium (11). The cell-free medium from such cultures contains sufficient SMC to destroy other cultures 1The abbreviations used are: Sal, Sarcoma I; SMC, specific macrophage cytotoxin. Received September 3, 1971; accepted November 8, 1971. FEBRUARY of target cells (11). Its possible contribution to target cell destruction is not known. However, the observation that killing in vitro is largely limited to target cells in close association with immune macrophages suggests that any SMC effect in vivo is probably restricted to target cells in close association with immune cells. The present study was concerned with the ultrastructure of immune macrophages and Sal target cells during their interaction following the injection of Sal cells into the peritoneal cavities of C57BL/6 mice that had recently rejected ascitic Sal tumor. MATERIALS AND METHODS The ascitic form of Sal was maintained in our laboratory by weekly serial transfer of tumor cells to the peritoneal cavities of mice of the A/Jax strain, the strain of tumor origin. Sal target cells were obtained from ascitic fluid taken from A/Jax mice on the 7th day after an i.p. injection of the tumor. The tumor cells were separated from the ascitic fluid by centrifugation. In one experiment, 36 X IO6 cells were suspended in 6 ml of cell-free immune ascitic fluid derived from C57BL/6 mice that had recently rejected Sal tumor. In another experiment, the sedimented cells were washed once in Medium 199, and 36 X IO6 cells were suspended in 6 ml of Medium 199. In each case the entire suspension of cells was injected i.p. into an immune C57BL/6 mouse that had received an initial inoculum of Sal cells 11 or 12 days earlier and had recently rejected the tumor. Cells were removed from the peritoneal cavities at 5, 10, and 20 min after the 2nd inoculation and were fixed in either s-collidine-buffered osmium tetroxide or in a combination of osmium tetroxide and glutaraldehyde buffered with sodium cacodylate. After fixation, the cells were dehydrated in ascending concentrations of ethyl alcohol and embedded in epoxy resin. Sections of the embedded cells were stained with lead citrate and uranyl acetate and were examined in an RCA 3G electron microscope. RESULTS When Sal cells were injected into the peritoneal cavities of C57BL/6 mice that had recently rejected a primary Sal ascitic tumor, the immune peritoneal macrophages rapidly adhered to the Sal cells (Fig. 1). Adherence was often accompanied by 1972 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. 413 Velma C. Chambers and Russell S. Weiser interdigitation of the protuberances of the cells (Fig. 2). Microvilli of the Sal cell sometimes extended into deep invaginations of the surface of the macrophage (Fig. 3) and occasionally appeared to be in the process of being pinched off. Many of the tumor cells in the samples removed from the peritoneal cavities at 10 and 20 min were surrounded (Fig. 4) or nearly surrounded (Fig. 5) in the plane of the section by 1 macrophage or more. Often a narrow rim of cytoplasm had spread over the surface of the tumor cell (Fig. 5). Serial sections sometimes clearly revealed that the rim of cytoplasm was of macrophage origin (Fig. 6). Some Sal cells that were undergoing phagocytosis by immune macrophages possessed the features of healthy cells (Fig. 5). These features included characteristic mitochondria, numerous aggregated ribosomes, and narrow cisternae of rough endoplasmic reticulum. Vesicles were often present in the peripheral cytoplasm of these cells in regions of contact with the immune macrophages (Fig. 5). Amorphous material resembling the interior of macrophage lysosomes was sometimes present in the narrow vacuolar space between the plasma membrane of the tumor cell and the membrane of the phagocytic vacuole (Figs. 4 and 7). These Sal cells were completely surrounded by a macrophage, at least in the plane of the section. The presence of amorphous material within the phagocytic vacuole was usually accompanied by degenerative changes in the phagocytized cell, such as vacuolation of the cytoplasm (Fig. 4), dilated endoplasmic reticulum (Fig. 7), and disaggregation of ribosomes (Figs. 4 and 7). Occasionally, protrusions of the macrophage cytoplasm invaded the phagosome and surrounded portions of degenerating Sal cytoplasm. This process led to the dispersal of Sal cytoplasm with secondary small phagosomes (Fig. 8). DISCUSSION In previous paper on the interaction of immune macrophages with target L-cells in vitro (7), we described the phagocytosis of microvilli of the target cells and suggested that this may provide a stimulus to macrophages for the synthesis of a mediator for target cell destruction or may act directly to kill target cells. A similar but more pronounced phagocytic activity of immune macrophages during the secondary response to L-cells was described in a recent publication (8). In the present study, the phagocytic activity of immune C57BL/6 macrophages during the secondary response to Sal cells was demonstrated. When the Sal cells were injected into the peritoneal cavities of immune C57BL/6 mice that had recently rejected Sal, the peritoneal cavities were heavily populated with immune macrophages that reacted rapidly with the target cells. The reaction involved the adherence of the immune macrophages to the Sal cells and interdigitation of their surfaces with the microvilli of the Sal cells extending into deep invaginations of the macrophage surface. The macrophage spread over the surface of the target cell, often surrounding much of the cell with a narrow rim of cytoplasm. In this manner an entire sarcoma cell became engulfed by a single macrophage. 414 In contrast to the phagocytosis of whole Sal cells, an entire L-cell was never seen within a single macrophage in the studies using L-cells as target cells (7, 8). The smaller size and lesser pliability of the Sal cell as compared with the L-cell may constitute factors influencing phagocytosis of whole target cells. Also, the kinds, distribution, and concentrations of antigens on the surfaces of target cells capable of reacting with cytophilic antibody of the macrophage may affect phagocytosis. The pinching off of bits of target cell membrane and cytoplasm occurs with both L-cells and Sal cells, albeit to a much greater extent with L-cells. This difference may likewise be due to the greater pliability of the L-cell and to the antigenic sites on its surface as compared with the Sal cell. In the present study, phagocytosis often involved Sal cells that had all the appearances of healthy cells (5). Phagocytosis was followed by the appearance of amorphous material within the phagocytic vacuole. The amorphous material strongly resembled the contents of lysosomes. Although lysosomes were usually numerous in the macrophages of the present study, direct evidence for their fusion with the phagocytic vacuole was rarely seen. The collection of vesicles at the periphery of the phagocytized tumor cell suggests that pinocytosis may continue for some time after ingestion. It is possible that the uptake of toxic material from the phagocytic vacuole may contribute to target cell death. As degeneration proceeded and dissolution of the plasma membrane of the target cell occurred, the phagosome was invaded by cytoplasmic processes of the macrophage. This invasion by macrophage processes led to compartmentalization of the degenerating cell material within small phagosomes for final digestion. Phagocytosis has been generally regarded to be a means of disposing of cellular debris rather than as a major mechanism for killing tumor cells. The extensive phagocytosis of apparently healthy tumor cells observed in the present studies suggests that in this system, using secondary challenge, phagocytosis is a major mechanism of tumor rejection. Its significance during the primary response is not known. The phagocytosis of viable Sal cells by immune macrophages was rarely observed in studies using a primary i.p. challenge and, therefore, was considered to be insufficient to account for tumor regression (6). The large amount of cell debris within phagosomes of macrophages removed from the peritoneal cavity during the latter part of tumor regression has previously been attributed to the phagocytosis of debris from tumor cells killed by complement action or by reaction with immune macrophages or lymphocytes. In the light of present knowledge, it is not possible to judge to what extent this debris represents the remains of cells phagocytized as dead or as living tumor cells. Since the rejection of a primary tumor occurs over a period of 2 to 4 days (from about the 6th to the 1Oth day after inoculation), the low percentage of mature and competent macrophages that can be observed to be engaged with tumor cells at any given time restricts the chances for observing phagocytosis of whole tumor cells, as was seen in the present experiments using secondary challenge. It is also possible that some other major mechanism operates early in tumor rejection and that phagocytosis becomes important only with time, as macrophages mature and cytophilic and CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. Interaction of Sarcoma I Cells and Immune Macrophage humoral opsonins become abundant. It is well established that humoral factors are of prime importance in the phagocytosis of whole Sal cells by macrophages (2—4). Phagocytosis may contribute to the killing of tumor cells in different ways. First, the macrophage may enclose the entire cell in a phagocytic vacuole where it is subjected to lysomomal enzymes and, possibly, SMC. Alternatively, the macrophage may attach to specific local surface areas of the target cell membrane and send out processes to surround and pinch off portions of the cell, untüeventually the target cell may be incapable of successfully repairing its membrane (7). REFERENCES 1. Baker, P., Weiser, R. S., lutila, J., Evans, C. A., and Blandau, R. J. Mechanisms of Tumor Homograf t Rejection: The Behavior of Sarcoma I Ascites Tumor in the A/Jax and C57BL/6K Mouse. Ann. N. Y. Acad. Sci., 101: 46-62, 1962. 2. Bennett, B. Phagocytosis of Mouse Tumor Cells In Vitro by Various Homologous and Heterologous Cells. J. Immunol., 95: 80-86, 1965. 3. Bennett, B. Specific Suppression of Tumor Growth by Isolated Peritoneal Macrophages from Immunized Mice. J. Immunol., 95: 656-664, 1965. 4. Bennett, B., Old, L. J., and Boyse, E. A. The Phagocytosis of Tumor Cells/n Vitro. Transplantation, 2: 183-202, 1964. 5. Chambers, V. C., and Weiser, R. S. An Electron Microscope Study of Sarcoma I in a Homologous Host. I. The Cells of the Growing Tumor. Cancer Res., 24: 693-708, 1964. 6. Chambers, V. C., and Weiser, R. S. An Electron Microscope Study of Sarcoma I in a Homologous Host. II. Changes in the Fine Structure of the Tumor Cell during the Homograft Reaction. Cancer Res., 24: 1368-1390, 1964. 7. Chambers, V. C., and Weiser, R. S. The Ultrasctructure of Target Cells and Immune Macrophages during Their Interaction In Vitro. Cancer Res., 29: 301-317, 1969. 8. Chambers, V. C., and Weiser, R. S. The Ultrastructure of Target Cells and Immune Macrophages during Their Interaction In Vivo. Cancer Res., 31: 2059-2066, 1971. 9. Granger, G. A., and Weiser, R. S. Homograft Target Cells: Specific Destruction In Vitro by Contact Interaction with Immune Macrophages. Science, 145: 1427-1429, 1964. 10. Granger, G. A., and Weiser, R. S. Homograft Target Cells: Contact Destruction In Vitro by Immune Macrophages. Science, 151: 97-99,1966. 11. Mclvor, K. L., and Weiser, R. S. Mechanisms of Target Cell Destruction by Alloimmune Peritoneal Macrophages. II. Release of a Specific Cytotoxin from Interacting Cells. Immunology, 20: 315-322,1971. All figures are electron micrographs of thin sections of cells stained with uranyl acetate and lead citrate. The magnification of all electron micrographs is X 11,000. Bar in each figure represents 1 urn. Fig. 1. Macrophage (M) in contact with Sal cell (S) 5 min after Sal cells were injected into the peritoneal cavity of an immune C57BL/6 mouse. Fig. 2. Macrophage (M) in contact with Sal cell (S), showing interdigitation of protuberances of both cells. Fig. 3. Microvilli (arrows) of Sal cell (S) extending into invaginations of macrophages (M). Fig. 4. Sal cell (S) surrounded by an immune macrophage (M). The Sal cell shows vacuolation of the cytoplasm (V) and mild disaggregation of ribosomes (R). Amorphous material has accumulated in the vacuolar space between the plasma membrane of the Sal cell and the membrane of the phagocytic vacuole (arrows). Fig. 5. A healthy-appearing Sal cell (S) nearly surrounded by a narrow rim of macrophage cytoplasm (M). Collections of vesicles (arrows) resembling pinosomes are present at the periphery of the Sal cell. Fig. 6. A section close to the one shown in Fig. 5, showing continuity (X) between the narrow rim of cytoplasm around the Sal cell (5) and the macrophage (M). Fig. 7. Sal cell (S) surrounded by macrophage (M) shows degenerative changes, including dilated endoplasmic reticulum (ER), disaggregation of ribosomes, and clumping of nuclear chromatin. Amorphous material (arrows) has collected in the phagocytic vacuole. A 2nd phagocytic vacuole contains membrane-bound cytoplasm (5, ) from the same or another Sal cell. Dilated endoplasmic reticulum and disaggregation of ribosomes are also apparent in this body of cytoplasm. Fig. 8. The nucleus of a degenerating cell (S) is surrounded by macrophage cytoplasm (M). Much of the cytoplasm of the degenerating cell has been dispersed in small phagosomes (P). Part of the macrophage nucleus is seen at the lower left. FEBRTIARY 1972 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. 415 wîE ^4» ' Ã-?T;-.•C_ - M» /•< •¿ : :•.» :-V •¿â€¢N S. .JPft.. -( :*a. ^Ç '.^ '- ' Vr'• .- i v" ' \ ** tA- i K@; Si»»«»^' '/v>> «acSi'S-*; A-ïà ÇSt:''•¿ \f'- . J, •¿-. .^>'*.V : 416 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. Ultrastructure of Sarcoma I Cell and Macrophage ^ VJ .®o*iX b* #p '.-* 6"-»< /~xO FEBRUARY 1972 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. 417 Velma C. Chambers and Russell S. Weiser V M 4 A, x . 418 CANCER RESEARCH VOL. 32 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. -' . V-:: ìm J v '. ;i •¿ <•¿ •¿-^SJÓ?'%< »y r - •¿â€¢*« c t: FEBRUARY 1972 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research. 419 The Ultrastructure of Sarcoma I Cells and Immune Macrophages during Their Interaction in the Peritoneal Cavities of Immune C57BL/6 Mice Velma C. Chambers and Russell S. Weiser Cancer Res 1972;32:413-419. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/32/2/413 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1972 American Association for Cancer Research.