The Ultrastructure of Sarcoma I Cells and Immune...

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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. 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 FEBRUARY 1972 413 Research. on August 10, 2019. © 1972 American Association for Cancer cancerres.aacrjournals.org Downloaded from

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[CANCER RESEARCH 32,413-419, February 1972]

The Ultrastructure of Sarcoma I Cells and Immune Macrophagesduring Their Interaction in the Peritoneal Cavities ofImmune 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 SarcomaI (Sal) ascitic tumor were given a second i.p. dose of Sal cells.The interaction of the immune macrophages of the host andthe Sal target cells was investigated by electron microscopy.The macrophages rapidly adhered to the target cells and spreadover their surfaces. The interaction of the immunemacrophages and Sal cells was manifested by interdigitation oftheir surfaces and the extension of Sal microvilli into deepinvaginations of the immune macrophages. The spreading ofthe immune macrophages over the surfaces of the Sal cellsresulted in the apparent phagocytosis of viable tumor cells.Such phagocytized Sal cells were contained within phagocyticvacuoles with the vacuolar membrane of the macrophageusually 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 suggestthat the phagocytosis of Sal cells by immune C57BL/6macrophages plays an important role in the rejection of theascitic form of the tumor during the secondary immuneresponse.

INTRODUCTION

The rejection of the ascitic form of Sal1 by the allogeneic

C57BL/6 mouse is accompanied by the presence of largenumbers of host macrophages in the peritoneal cavity (1). Inearlier phase microscope studies, immune macrophages wereobserved to attach to the Sal cells and to spread over theirsurfaces (1). The adherence of immune macrophages to thetarget cells was later found to be due to the reaction ofcytophilic antibody on the surface of the macrophage withantigens of the target cell (9, 10). Adherence per se did notcause destruction of the target cell. A step following adherenceof the macrophages to target cells which demands metabolicactivity of the macrophages appears to be necessary for targetcell destruction (9, 10). It was later discovered that immunemacrophages interacting with target cells in vitro release a SMCinto the surrounding medium (11). The cell-free medium fromsuch 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.

of target cells (11). Its possible contribution to target celldestruction is not known. However, the observation thatkilling in vitro is largely limited to target cells in closeassociation with immune macrophages suggests that any SMCeffect in vivo is probably restricted to target cells in closeassociation with immune cells. The present study wasconcerned with the ultrastructure of immune macrophages andSal target cells during their interaction following the injectionof Sal cells into the peritoneal cavities of C57BL/6 mice thathad recently rejected ascitic Sal tumor.

MATERIALS AND METHODS

The ascitic form of Sal was maintained in our laboratory byweekly serial transfer of tumor cells to the peritoneal cavitiesof mice of the A/Jax strain, the strain of tumor origin. Saltarget cells were obtained from ascitic fluid taken from A/Jaxmice on the 7th day after an i.p. injection of the tumor. Thetumor cells were separated from the ascitic fluid bycentrifugation. In one experiment, 36 X IO6 cells weresuspended in 6 ml of cell-free immune ascitic fluid derivedfrom C57BL/6 mice that had recently rejected Sal tumor. Inanother experiment, the sedimented cells were washed once inMedium 199, and 36 X IO6 cells were suspended in 6 ml of

Medium 199. In each case the entire suspension of cells wasinjected i.p. into an immune C57BL/6 mouse that had receivedan initial inoculum of Sal cells 11 or 12 days earlier and hadrecently rejected the tumor.

Cells were removed from the peritoneal cavities at 5, 10,and 20 min after the 2nd inoculation and were fixed in eithers-collidine-buffered osmium tetroxide or in a combination ofosmium tetroxide and glutaraldehyde buffered with sodiumcacodylate. After fixation, the cells were dehydrated inascending concentrations of ethyl alcohol and embedded inepoxy resin. Sections of the embedded cells were stained withlead citrate and uranyl acetate and were examined in an RCA3G electron microscope.

RESULTS

When Sal cells were injected into the peritoneal cavities ofC57BL/6 mice that had recently rejected a primary Sal ascitictumor, the immune peritoneal macrophages rapidly adhered tothe Sal cells (Fig. 1). Adherence was often accompanied by

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Velma C. Chambers and Russell S. Weiser

interdigitation of the protuberances of the cells (Fig. 2).Microvilli of the Sal cell sometimes extended into deepinvaginations of the surface of the macrophage (Fig. 3) andoccasionally appeared to be in the process of being pinchedoff.

Many of the tumor cells in the samples removed from theperitoneal cavities at 10 and 20 min were surrounded (Fig. 4)or nearly surrounded (Fig. 5) in the plane of the section by 1macrophage or more. Often a narrow rim of cytoplasm hadspread over the surface of the tumor cell (Fig. 5). Serialsections sometimes clearly revealed that the rim of cytoplasmwas of macrophage origin (Fig. 6).

Some Sal cells that were undergoing phagocytosis byimmune macrophages possessed the features of healthy cells(Fig. 5). These features included characteristic mitochondria,numerous aggregated ribosomes, and narrow cisternae of roughendoplasmic reticulum. Vesicles were often present in theperipheral cytoplasm of these cells in regions of contact withthe immune macrophages (Fig. 5). Amorphous materialresembling the interior of macrophage lysosomes wassometimes present in the narrow vacuolar space between theplasma membrane of the tumor cell and the membrane of thephagocytic vacuole (Figs. 4 and 7). These Sal cells werecompletely surrounded by a macrophage, at least in the planeof the section. The presence of amorphous material within thephagocytic vacuole was usually accompanied by degenerativechanges in the phagocytized cell, such as vacuolation of thecytoplasm (Fig. 4), dilated endoplasmic reticulum (Fig. 7), anddisaggregation of ribosomes (Figs. 4 and 7). Occasionally,protrusions of the macrophage cytoplasm invaded thephagosome and surrounded portions of degenerating Salcytoplasm. This process led to the dispersal of Sal cytoplasmwith secondary small phagosomes (Fig. 8).

DISCUSSION

In previous paper on the interaction of immunemacrophages with target L-cells in vitro (7), we described thephagocytosis of microvilli of the target cells and suggested thatthis may provide a stimulus to macrophages for the synthesisof a mediator for target cell destruction or may act directly tokill target cells. A similar but more pronounced phagocyticactivity of immune macrophages during the secondaryresponse to L-cells was described in a recent publication (8). Inthe present study, the phagocytic activity of immune C57BL/6macrophages during the secondary response to Sal cells wasdemonstrated. When the Sal cells were injected into theperitoneal cavities of immune C57BL/6 mice that had recentlyrejected Sal, the peritoneal cavities were heavily populatedwith immune macrophages that reacted rapidly with the targetcells. The reaction involved the adherence of the immunemacrophages to the Sal cells and interdigitation of theirsurfaces with the microvilli of the Sal cells extending into deepinvaginations of the macrophage surface. The macrophagespread over the surface of the target cell, often surroundingmuch of the cell with a narrow rim of cytoplasm. In thismanner an entire sarcoma cell became engulfed by a singlemacrophage.

In contrast to the phagocytosis of whole Sal cells, an entireL-cell was never seen within a single macrophage in the studiesusing L-cells as target cells (7, 8). The smaller size and lesserpliability of the Sal cell as compared with the L-cell mayconstitute factors influencing phagocytosis of whole targetcells. Also, the kinds, distribution, and concentrations ofantigens on the surfaces of target cells capable of reacting withcytophilic antibody of the macrophage may affectphagocytosis. The pinching off of bits of target cell membraneand cytoplasm occurs with both L-cells and Sal cells, albeit toa much greater extent with L-cells. This difference maylikewise be due to the greater pliability of the L-cell and to theantigenic sites on its surface as compared with the Sal cell.

In the present study, phagocytosis often involved Sal cellsthat had all the appearances of healthy cells (5). Phagocytosiswas followed by the appearance of amorphous material withinthe phagocytic vacuole. The amorphous material stronglyresembled the contents of lysosomes. Although lysosomeswere usually numerous in the macrophages of the presentstudy, direct evidence for their fusion with the phagocyticvacuole was rarely seen. The collection of vesicles at theperiphery of the phagocytized tumor cell suggests thatpinocytosis may continue for some time after ingestion. It ispossible that the uptake of toxic material from the phagocyticvacuole may contribute to target cell death. As degenerationproceeded and dissolution of the plasma membrane of thetarget cell occurred, the phagosome was invaded bycytoplasmic processes of the macrophage. This invasion bymacrophage processes led to compartmentalization of thedegenerating cell material within small phagosomes for finaldigestion.

Phagocytosis has been generally regarded to be a means ofdisposing of cellular debris rather than as a major mechanismfor killing tumor cells. The extensive phagocytosis ofapparently healthy tumor cells observed in the present studiessuggests that in this system, using secondary challenge,phagocytosis is a major mechanism of tumor rejection. Itssignificance during the primary response is not known. Thephagocytosis of viable Sal cells by immune macrophages wasrarely observed in studies using a primary i.p. challenge and,therefore, was considered to be insufficient to account fortumor regression (6). The large amount of cell debris withinphagosomes of macrophages removed from the peritonealcavity during the latter part of tumor regression has previouslybeen attributed to the phagocytosis of debris from tumor cellskilled by complement action or by reaction with immunemacrophages or lymphocytes. In the light of presentknowledge, it is not possible to judge to what extent thisdebris represents the remains of cells phagocytized as dead oras living tumor cells. Since the rejection of a primary tumoroccurs over a period of 2 to 4 days (from about the 6th to the1Oth day after inoculation), the low percentage of mature andcompetent macrophages that can be observed to be engagedwith tumor cells at any given time restricts the chances forobserving phagocytosis of whole tumor cells, as was seen in thepresent experiments using secondary challenge. It is alsopossible that some other major mechanism operates early intumor rejection and that phagocytosis becomes importantonly with time, as macrophages mature and cytophilic and

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Interaction of Sarcoma I Cells and Immune Macrophage

humoral opsonins become abundant. It is well established thathumoral factors are of prime importance in the phagocytosisof whole Sal cells by macrophages (2—4).

Phagocytosis may contribute to the killing of tumor cells indifferent ways. First, the macrophage may enclose the entirecell in a phagocytic vacuole where it is subjected to lysomomalenzymes and, possibly, SMC. Alternatively, the macrophagemay attach to specific local surface areas of the target cellmembrane and send out processes to surround and pinch offportions of the cell, untüeventually the target cell may beincapable 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 ofSarcoma 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 byVarious Homologous and Heterologous Cells. J. Immunol., 95:80-86, 1965.

3. Bennett, B. Specific Suppression of Tumor Growth by IsolatedPeritoneal Macrophages from Immunized Mice. J. Immunol., 95:656-664, 1965.

4. Bennett, B., Old, L. J., and Boyse, E. A. The Phagocytosis ofTumor Cells/n Vitro. Transplantation, 2: 183-202, 1964.

5. Chambers, V. C., and Weiser, R. S. An Electron Microscope Studyof Sarcoma I in a Homologous Host. I. The Cells of the GrowingTumor. Cancer Res., 24: 693-708, 1964.

6. Chambers, V. C., and Weiser, R. S. An Electron Microscope Studyof Sarcoma I in a Homologous Host. II. Changes in the FineStructure 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 TargetCells 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 TargetCells 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: SpecificDestruction In Vitro by Contact Interaction with ImmuneMacrophages. Science, 145: 1427-1429, 1964.

10. Granger, G. A., and Weiser, R. S. Homograft Target Cells: ContactDestruction In Vitro by Immune Macrophages. Science, 151:97-99,1966.

11. Mclvor, K. L., and Weiser, R. S. Mechanisms of Target CellDestruction by Alloimmune Peritoneal Macrophages. II. Release ofa 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 electronmicrographs 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 thephagocytic 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 themacrophage (M).

Fig. 7. Sal cell (S) surrounded by macrophage (M) shows degenerative changes, including dilated endoplasmic reticulum (ER), disaggregation ofribosomes, and clumping of nuclear chromatin. Amorphous material (arrows) has collected in the phagocytic vacuole. A 2nd phagocytic vacuolecontains membrane-bound cytoplasm (5, ) from the same or another Sal cell. Dilated endoplasmic reticulum and disaggregation of ribosomes arealso 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 hasbeen dispersed in small phagosomes (P). Part of the macrophage nucleus is seen at the lower left.

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1972;32:413-419. Cancer Res   Velma C. Chambers and Russell S. Weiser  C57BL/6 Miceduring Their Interaction in the Peritoneal Cavities of Immune The Ultrastructure of Sarcoma I Cells and Immune Macrophages

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