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[CANCER RESEARCH 29, 301â€”317, February 1969]

Their Interaction in Vitro1

VelmaC. ChambersandRussellS. WeiserDepartment ofMicrobioiogy, University of Washington, Seattle, Washington 98105

SUMMARY INTRODUCTION

The ultrastructure of L cells and immune macrophages wasstudied at various intervals during their in vitro interaction,which leads to mutual cell destruction. When immune macrophages were added to a monolayer of L cells, they rapidlyadhered to the L cells and the adherence persisted. Electron

microscopic observations during the first 3 hours showednumerous contacts between immune macrophages and target Lcells in which the plasma membrane of each cell remainedintact, and no obvious degenerative changes were seen. How

ever, slender protuberances of L cell cytoplasm extended intosurface invaginations of the macrophage. Electron microscopic

evidence suggested that these protuberances may be pinchedoff and engulfed by the macrophage. Degenerative changeswere seldom observed prior to complete loss of cellular integrity. The occasional manifestations of degeneration included:disruption of polysomes and the random dispersion of ribosomes, patchy degeneration within the cytoplasm, and formation of blebs consisting of large bulbous protrusions of the cellsurface.

At 5 hours and later, many cells in an advanced stage ofdegeneration were seen. These cells often consisted of a degencrate nucleus surrounded by swollen vesicular cytoplasmiccomponents and little or no plasma membrane. The degeneration apparently progresses very rapidly once it has been mitiated.

These ultrastructural studies, when considered in the light ofother experimental data, suggest that the following sequenceof events occurs during the target L cell-immune macrophagereaction in vitro. The first stage consists of the rapid andstrong adherence of the immune macrophage to the target cellas the result of the reaction of the cytophiic antibody on the

surface of the macrophage and the antigens of the plasmamembrane of the L cell. During the second stage, the immunemacrophages are exposed to the antigens of the target cell bycontact and by phagocytosis of surface protuberances of thetarget cells. This event may provide a stimulus for the synthesis of a â€œmediatorâ€•for cell destruction by the macrophage, ormay act directly to bring about cell destruction.

1Supported in part by USPHS Research Grant CA-05698 from theNational Cancer Institute.

Received May 24, 1968; accepted October 7, 1968.

Granger and Weiser (7) showed that peritoneal macrophagestaken from C57BL/Ks mice that had been previously immunized with antigens of A/Jax mice caused specific â€œcontactdestructionâ€• of target cells in monolayer cultures. Cells derived from either A/Jax or C3H mice served effectively astarget cells since the two strains of mice have common H2histocompatibiity antigens. The first step in the interaction ofimmune macrophages and target cells involved the specificadherence of essentially all of the immune cells to the targetcell monolayer. A specific hemagglutinin on the surface of theimmune macrophages was found to be responsible for thisadherence (8). In the second stage of the reaction, both themacrophage and the target cell were destroyed. The nature ofthe destructive process is not known. Normal peritonealmacrophages treated with specific humoral isoantibody derived from immune donors were not effective in causing targetcell death. The exposure of immune macrophages to actinomycm D or to chloramphenicol inhibited cell destruction demonstrating the necessity of the biosynthetic activity of theimmune cells. Nonimmune macrophages and nonspecific immune macrophages showed a limited capacity to adhere totarget cells, and no cell destruction occurred (8).

In the present study, the ultrastructure of immune macrophages and target L cells was investigated during their interaction in vitro.

MATERIALS AND METHODS

Target Cells. Strain L mouse cells were used as target cellsfor the in vitro reaction with immune macrophages. Strain Lcells originated from the connective tissue of a C3H mouse in1940 (4). The cells have been in continuous cultivation in vitrosince that time. C3H mice and the A/Jax mice from whichSarcoma I originated have common major H2 histocompatibiity antigens (12).

Stock cultures of L cells were trypsinized with 0.25% trypsinin calcium- and magnesium-free phosphate-buffered saline (pH7.4) for 10 minutes at 37Â°C.Trypsinized cells were seeded in60-mm plastic Petri plates, and 5 ml of complete tissue culturemedium were added. Complete tissue culture medium consistsof medium 199 containing 10% heat-inactivated newborn calfserum and antibiotics. The monolayers were incubated at 37Â°Cin a 5% CO2 atmosphere.

Immune Macrophages. The immune macrophages were ohtamed from the peritoneal cavities of C57BL/Ks mice that had

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Velma C. Chambers and Russell S. Weiser

been inoculated 10 to 14 days earlier with Sarcoma I cells. Bythe 10th day most of the Sarcoma I cells had disappeared andthe cellular component of the ascites consisted mainly ofmacrophages. The macrophages were washed 5 times in heparinized Hanks' balanced salt solution (HBSS) adjusted to pH7.4 with sodium bicarbonate. They were then suspended incomplete tissue culture medium to give between 1 and 6 million cells per ml. Aliquots of this suspension were seeded inp1astic Petri plates for later processing for electron microscopicobservation.

Inoculation of Immune Macrophages onto L-Cell Monolayers.Tissueculturemediumwasremovedfrom the L-cellmonolayers and 2 ml of fresh medium were added. One drop(approximately 0.05 ml) of the macrophage suspension wasdropped onto one or two marked areas of each L-cell monolayer. The cultures were carefully replaced in the CO2 incubator at 37Â°C. Medium was added or replaced as necessary tomaintain the cultures.

Electron Microscopy. Untreated monolayers and monolayers treated with immune macrophages were removed fromthe incubator after appropriate intervals. They were washedonce or twice with HBSS or with medium 199 without serum.Three ml of fixative were added. The fixative consisted ofequal parts of 4% osmium tetroxide and medium 199. Sodiumbicarbonate was added to adjust the pH to 7.5. The cells werefixed at room temperature for 40 minutes, dehydrated inascending concentrations of ethyl alcohol, and embedded in athin layer of Epon 812 embedding medium (10). After polymerization the embedded cells were observed under the lightmicroscope. Selected areas were cut out and mounted on previously polymerized Epon blocks. The cells were sectioned,stained with uranyl acetate and lead citrate, and observed in anRCA-EMU 3G electron microscope.

RESULTS

Ulfrastructure of L Cells. The L cells were cultivated inmonolayers in plastic Petri plates. The monolayers were fixedand embedded in situ, and thin sections were observed in theelectron microscope. The cells were often elongated (Fig. 1).The nucleus was large and round or oval. One or more prominent nucleoli were usually present. The mitochondria werelarge, oval or elongated, and contained prominent cristae and amatrix of low electron density (Fig. 2). Vesicular componentsincluded numerous smooth vesicles, several profiles of roughendoplasmic reticulum, and occasionally a prominent Golgicomplex. Ribosomes were abundant and usually occurred industers. Fine filaments and one or both centrioles were occasionally present in the section (Figs. 1, 2). The cell surfaceshowed a number of small projections. Virus-like particlesresembling those described by Dales and Howatson (3) occurved within and at the surface of many of the L cells (Figs.1â€”3). Some of these particles were in the process of buddingfrom the plasma membrane (Fig. 3). No cytopathic effect was

apparent in cells containing the virus-like particles.Ultrasfructure of Immune Macrophages Maintained in Mono

layer Cultures. Immune macrophages were obtained from theperitoneal cavities of C57BL/Ks mice that had been immunized against Sarcoma I cells. They were maintained in tissue

culture medium in plastic Petri plates for periods up to 48hours before use. Electron microscope observations were madeon sections of fixed and embedded cells at various intervalsduring this time. Regardless of the time in monolayer culture,the ultrastructural characteristics were similar to those observed in immune macrophages that were fixed and embeddedimmediately after removal from the peritoneal cavities ofC57BL/Ks mice. The cell outline was usually irregular (Fig. 4).Lipid bodies were commonly seen within the cytoplasm andwere sometimes numerous (Fig. 5). Lysosomes varied considerably in size and number and in electron density (Figs. 4, 5).Some sections contained a large Golgi complex (Fig. 4). Themitochondria were small, and the mitochondrial matrix wasdense (Figs. 4, 5) compared with the mitochondria of the Lcells (Figs. 1, 2). Clusters of free ribosomes, fine filaments,rough and smooth endoplasmic reticulum, and vacuoles variedfrom a paucity of these organelles in some macrophages to anabundance in others. The nucleus was often crescent-shaped,and the nuclear chromatin was sometimes clumped.

The Ultrastructure of L Cells and Immune Macrophages during their Interaction in Vitro. Observations of L Cell-Macrophage Contacts Prior to Degeneration. When immune macrophages were added to the L-cell monolayer, they rapidlyadhered to the L cells. Electron microscopic observationsshowed many contacts between L cells and macrophages in theearly specimens (Fig. 6). Contacts between the two cell typeswere also seen at 24 hours (Fig. 7) and at 48 hours (Fig. 8).The plasma membranes of both the macrophages and the Lcells were intact when viewed in cross section. Often the membranes were sectioned obliquely and the condition of the

membranes was not discernible (Fig. 9). Numerous pinocytoticvesicles were observed at the surface of some of the L cells inthe region of the contact with an immune macrophage (Fig.10). During the first hour of the reaction, macrophages occasionally exhibited a zone of cytoplasm along the area of contact with an L cell which was free of cytoplasmic organellesother than fine fibrillar material (Fig. 11).

Slender protuberances of L cell cytoplasm sometimes cxtended into invaginations of the macrophage cytoplasm (Fig.12). This relationship was observed as early as 20 minutes afterinoculation of the immune macrophages onto the L cell monolayer (Fig. 12) and also in later specimens (Figs. 13â€”15). InFig. 13 the invaginations occur at regular intervals along thearea of contact between the L cell and macrophage. Someelectron microscopic images suggest that the surface projections of L cells may be pinched off and become phagocytizedby the macrophage (Fig. 14). A budding virus particle occasionally occupied at the tip of a projection that appeared to beundergoing phagocytosis (Fig. 15). Similar virus particles thatoccurred within vesicles in some of the macrophages hadapparently been phagocytized (Fig. 14).

There was little evidence that extensions of macrophagecytoplasm were phagocytized by the L cells.

Degenerative Changes. Degenerating cells were seldom ohserved in specimens prepared before 3 hours following theaddition of immune macrophages to the target cells. At 3 and4 hours a few degenerating cells were observed, and the number increased progressively in the later specimens. At 24 and48 hours many of the degenerating cells had become detached

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Target Cells and Immune Macrophages

from the plastic Petri plate leaving a plaque or partially denuded area in the monolayer at the site of inoculation. Somecells still remained attached to the Petri plate in the plaquearea. Most of the attached cells were in an advanced stage ofdegeneration and could not be identified. However, a few Lcells and macrophages that showed no obvious degenerativechanges were also present.

Mild degenerative changes were rarely observed. In a few Lcells that were in contact with macrophages, patches of moderately electron-dense material occurred in the cytoplasm (Fig.16). The cytoplasmic patches were composed of fine fibrillarmaterial (Fig. 17). Occasionally a cluster of small dense bodiesor vesicles approximately 210 A in diameter was observed inthe vicinity of the fibrillar material (Fig. 17). The occasionalassociation of these structures with lysosomes suggest thatthey may either coalesce with lysosomes or that they areformed from lysosomes.

Degenerating L cells often formed blebs consisting of largebulbous protuberances of cytoplasm (Fig. 18). The blebsusually contained numerous dispersed ribosomes, a few profiles of endoplasmic reticulum, and occasionally mitochondria.The plasma membrane of the blebs and the cell proper wasoften intact. Some of the blebs apparently became detachedfrom the cell and were phagocytized by macrophages (Fig.19).

Many of the cells that were in an advanced stage of degeneralion had lost their plasma membranes (Fig. 20). The identityof these cells was uncertain. Fragments of membranes fromdegenerating cells were occasionally seen adhering to the

face of macrophages (Figs. 21, 22). In some instances it wasnot clear whether the membrane fragments were within deepinfoldings of the cell surface or whether they were enclosedwithin a long flat vacuole (Fig. 22).

During the immune cell-target cell interaction, the immune

macrophages often exhibited large masses of engulfed cellularmaterial (Fig. 16). As the reaction progressed, lysosomes became less numerous in many of the macrophages, and thenumber of lipid bodies increased (Fig. 19). Advanced degenerative changes included vacuolation of the cytoplasm and disintegration of the plasma membrane (Fig. 19). At this stagethe identification of the cell was questionable.

DISCUSSION

The first stage in the immune macrophage-target cell reaction was shown by Granger and Weiser (8) to consist of arapid and strong adherence of the immune macrophages to thetarget cell monolayer. Cytophiic antibody coating the surface

of the immune macrophage was shown to be responsible forthe adherence. The present electron microscope study demonstrates some morphologic details of the close association of theimmune cell and target cell. Apparently adherence often persisted for several hours during which the plasma membrane ofboth cells remained intact, and no obvious degenerativechanges in ultrastructure occurred. The interdigitation of thetwo cell surfaces and the number of projections and infoldingssuggest that the cell membranes were intensely active in the

area of contact. The large number of vesicles in the region ofcontact indicates that pinocytosis probably continues for sometime after contact. The significance of the paucity of organ

des in the cytoplasm of macrophages in the area of contact isnot known. Macrophages sometimes surrounded and apparently engulfed the surface projections of the target cells. Itremains to be determined whether these events are induced bythe cytophiic antibody on the surface of the macrophages. Ithas been shown that humoral antibody directed against surfaceantigens of cells has a stimulating effect on the surface activityof these cells causing infoldings and outfoldings of the surfaceof individual cells and interdigitation of the surface of adjoining cells (5, 6). It is also possible that the virus-like particles onthe surface of the L cells may have stimulated the formationof slender proturberances which were then phagocytized bythe macrophages. This seems unlikely, however, because manyof the surface projections were free of virus-like particles.

The early close contact between the immune macrophageand the L cell with the plasma membrane of both cells remaining intact, the rare observance of mild degenerativechanges, and the advanced degeneration in cells of the 16- to48-hour specimens suggest that a substantial period of contactis essential before severe injury is initiated or at least becomesevident, and that, once initiated, degeneration proceeds rapidly to death of the cell. This course of events differs fromthat of immune cytolysis following exposure of cells to specific humoral antibody and complement. Degenerative changeswere usually far advanced within an hour after specific humoral antibody and complement were added to a monolayerof L cells (unpublished observations). The ultrastructure ofthese cells was similar to that described by Goldberg andGreen (6) and others (14) and showed swollen cells with theirlimiting plasma membrane, swollen mitochondria, dilatedendoplasmic reticulum, and loss of ribosomes and other cytoplasmic macromolecules.

Granger and Weiser (7) described the mutual destruction ofboth the immune macrophage and the target cell in the secondstage of the reaction. They found that immune macrophagespretreated with either actinomycin D or chloramphenicol retamed their capacity to adhere to the target cell but were nolonger effective in causing cell destruction (8). This observation indicated that biosynthetically active immune macrophages were necessary for the second stage of the reaction toproceed. The need for biosynthetically active macrophages andthe delay in appearance of degenerative changes after contactof immune macrophages with target cells is in agreement withthe hypothesis that the contact stimulates the macrophages tosynthesize and/or release a â€œmediatorâ€•which somehow causesdestruction of the target cell. In addition to surface contactbetween the immune cell and target cell, the phagocytosis ofsurface projections of the L cell may be an essential event forproviding the stimulus for synthesis or release of the â€œmediator.â€• Recent observations by Mclvor and Weiser (unpublishedresults) provide further evidence that pinocytosis by themacrophage plays an important role early in the second stageof the reaction following adherence of the immune cell to thetarget cell. When pinocytosis was blocked with sodium fluoride within one hour after adherence, the macrophage was nolonger capable of killing the target cell. The blocking of pinocytosis may prevent the engulfment of L cell material whichmay be needed to trigger the synthesis of the â€œmediatorâ€•bythe immune macrophage.

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The disruption of the normal configuration of polysomesinto numerous randomly dispersed ribosomes was observed insome of the degenerating L cells of the present study (Fig. 16)and has also been observed in regressing Sarcoma I cells in theperitoneal cavities of immune C57BL/Ks mice (2). The patchyareas of degeneration seen in some of the L cells of the presentstudy may represent further disintegration of polysomes andribosomes. These observations together with recent studies ofMclvor and Weiser (unpublished results) support the view thatthe polysome may be attacked early in the second stage of thereaction, thus blocking protein synthesis in the L cell as wasproposed earlier (2). Such a block in protein synthesis mayrepresent the initial lesion in target cell destruction or it maybe secondary to an earlier â€œlesionâ€•such as a block in production of messenger RNA by the target cell or the effect of aâ€œmediatorâ€•on the plasma membrane of the target cell.

Although the present study provides little evidence that theinitial lesion occurs in the plasma membrane of the target cell,neither does it rule out this possibility. Changes in membranepermeability and the presence of lesions or â€œholes,â€•such asthose demonstrated in negatively stained membranes of erythrocytes subjected to the action of humoral antibody and complement (1, 11), probably would not have been observed inthe electron micrographs of thin sections. Although the invitro immune macrophage-target cell system was supposedlycomplement-free, it is possible that some components of cornplement or some other active substance may have been supplied by the immune macrophages. The macrophage has beenreported to be a source of certain components of complement(13).

An alternative hypothesis is that the phagocytosis of L cellmembranes is the key to target cell destruction. It is possiblethat the reaction of cytophilic antibody with antigens of the Lcell membrane promotes pinocytosis and causes slender protuberances to develop at the surface of the target cell. Phagocytosis of these structures may weaken the target cell membrane and result in â€œballooningâ€•and the formation of thelarge surface blebs with accompanying cell degeneration. Thisattack against target cells by rnacrophages engaged in â€œmembrane phagocytosisâ€• may be similar to the contact activities ofmacrophages in causing sphering and fragmentation of erythrocytes coated with Rh antibody recently reported by LoBuglioet al. (9). The ingested complex of target cell membrane andantibody could be toxic to macrophages and account for theirdestruction.

304 CANCER RESEARCH VOL.29

ACKNOWLEDGMENTS

The authors gratefully acknowledge the technical assistance of Mrs.Jean Greenaway and Miss Kristine Stokoe.

REFERENCES

1. Borsos, T., Dourmashkin, R. R., and Humphrey, J. H. Lesions inErythrocyte Membranes Caused by Immune Haemolysis. Nature,202: 251â€”252, 1964.

2. Chambers, V. C., and Weiser, R. S. An Electron Microscope Studyof Sarcoma I in an Homologous Host. II. Changes in the Fine Structure of the Tumor Cell during the Homograft Reaction. CancerRes., 24: 1368â€”1390,1964.

3. Dales, S., and Howatson, A. F. Virus-like Particles in Associationwith L Strain Cells. Cancer Res., 21: 193â€”197, 1961.

4. Earle, W. R. Production of Malignancy In Vitro. IV. The MouseFibroblast Cultures and Changes Seen in Living Cells. J. Nail Cancer Inst., 4: 165â€”212, 1943.

5. Easton, J. M., Goldberg, B., and Green, H. Immune Cytolysis:Electron Microscopic Localization of Cellular Antigens with Ferritin-Antibody Conjugates. J. Exptl. Med., 115: 275â€”287, 1962.

6. Goldberg, B., and Green, H. The Cytotoxic Action of ImmuneGamma Globulin and Complement on Krebs Ascites Tumor Cells.I. Ultrastructural Studies. J. Exptl. Med., 109: 505â€”510,1959.

7. Granger, G. A., and Weiser, R. S. Homograft Target Cells: SpecificDestruction In Vitro by Contact Interaction with Immune Macrophages. Science, 145: 1427â€”1429, 1964.

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

9. LoBuglio, A. F., Cotran, R. S., and Jandl, J. H. Red Cells Coatedwith Immunoglobulin G: Binding and Sphering by MononuclearCellsinMan. Science,158: 1582â€”1585,1967.

10. Luft, J. H. Improvement in Epoxy Resin Embedding Methods. J.Biophys. Biochem. Cytol., 9: 409â€”414, 1961.

11. Rosse, W. F., Dourmashkin, R., and Humphrey, J. H. Immune Lysisof Normal Human and Paroxysmal Nocturnal Hemoglobinuria(PNH) Red Blood Cells. III. The Membrane Defects Caused byComplement Lysis. J. Exptl. Med., 123: 969â€”984, 1966.

12. Snell, G. D., Hoecker, G., Amos, D. B., and Stimpfling, J. H. ARevised Nomenclature for the Histocompatibiity -2 Locus of theMouse. Transplantation, 2: 777â€”784,1964.

13. Stecher, V. J., and Thorbecke, G. J. Sites of Synthesis of SerumProteins. HI. Production of Big, Bie and Transferrin by Primateand Rodent Cell Lines. J. Immunol., 99: 660â€”668, 1967.

14. Walford, R. L., Latta, H., and Troup, G. M. The Reaction BetweenHuman Lymphocytes and Allogeneic Antisera: A Serologic andElectron Microscopic Study. Ann. N. Y. Acad. Sci., 129: 490â€”499,1966.




Figs. 1â€”22. All figures are electron micrographs of thin sections of cells stained with uranyl acetate and lead citrate. The bar in all figures except

Figs. 3 and 17 represents 1 micron; in Figs. 3 and 17 it represents 0.5 micron.

Fig. 1. L cells from a monolayer culture. x 7300.Fig. 2. A portion of an L cell from a monolayer culture showing the edge of the nucleus (N), numerous Golgi vesicles (C), mitochondria (Mi),

numerous clusters of ribosomes (R), and rough endoplasmic reticulum (ER). Some profiles ofER appear as narrow channels and others appear asdilated spheres. Virus particles (V) are seen within two ER cisternae. X 19,000.

Fig. 3. Extracellular virus and several virus particles budding from the plasma membrane of an L cell (7). x 56,000.Fig. 4. A macrophage from a C57BL/Ks mouse 12 days after an intraperitoneal inoculation with Sarcoma I cells. The section shows a

crescent-shaped nucleus (N), abundant rough endoplasmic reticulum (ER), a large Golgi zone (G), and several electron dense lysosomes (Ly). x12,000.

Fig. 5. A macrophage from a C57BL/Ks mouse 14 days after an intraperitoneal inoculation with Sarcoma I cells. Several large lipid bodies (Li),lysosomes (Ly), a few filaments (F), a few profiles of rough endoplasmic reticulum (ER), numerous clusters of ribosomes (R) and a number ofsmall, dense mitochondria (Mi) are seen within the cytoplasm. x 14,000.

Fig. 6. Two macrophages (M) in contact with an L cell (L) 20 minutes after immune macrophages were added to an L cell monolayer. Theplasma membranes of both cells are intact and no degenerative changes are apparent. X 7,900.

Fig. 7. An area of contact between a macrophage (M) and an L cell (L) 24 hours after the immune macrophages were added to the L cells. Theplasma membrane of both cells is intact. The mitochondria (Mi) of the macrophage are small and show greater electron density than themitochondria of the L cell. Several lipid bodies (Li) are present in the macrophage. x 18,000.

Fig. 8. An area of contact between a macrophage (M) and an L cell (L) in a 48-hour specimen. The plasma membrane of both cells is intact. X14,000.

Fig. 9. An oblique section through adjacent plasma membranes of a macrophage (M) and L cell (L) in close contact. The membranes (7) areindistinct because of the obliquity of the section. Vesicles are absent in the area of contact. x 14,000.

Fig. 10. Numerous vesicles (7) in the L cell (L) cytoplasm in the region ofcontact with a macrophage (M). x 14,000.Fig. 1 1. A contact between a macrophage (M) and an L cell (L) showing a paucity of cytoplasmic organelles in the macrophage in the region of

contact (7). x 14,000.Fig. 1 2. A contact between a macrophage (M) and L cell (L) in a 20-minute specimen showing a surface projection of the L cell extending into

an invagination of the macrophage (7). The cytoplasm ofthe macrophage in the area of contact is sparsely populated with cytoplasmic organelles. x16,000.

Fig. 13. A contact between a macrophage (M) and an L cell (L) in a 24-hour specimen. There appear to be four surface projections of the L cellextending into invaginations in the macrophage at regular intervals along the area of contact (2'). The lower three projections may be undergoingphagocytosis. x 18,000.

Fig. 14. Surface projections of an L cell (L) extend into invaginations of the macrophage (M) and appear to be undergoing phagocytosis (7'). Avirus particle (II within a vesicle has apparently been phagocytized. x 14,000.

Fig. 15. A long slender process of an L cell (L) with a budding virus particle (V) at its tip extends into an invagination of a macrophage (M). Thecytoplasm of the macrophage appears to be pinching off the end of the L cell process. x 14,000.

Fig. 16. A macrophage (M) in close association with a degenerating L cell (L). Patches of moderately electron dense material appear in the L cellcytoplasm (7'). The endoplasmic reticulum (ER) is dilated and the ribosomes (R) are dispersed. Large membrane-bound bodies containing celldebris are present in the macrophage. The bodies, (A) and (B), resemble detached blebs from degenerating L cells (see Figs. 18, 19). Aphotographic enlargement of the outlined area is presented in Fig. 17. x 14,000.

Fig. 17. A photographic enlargement of the area indicated in Fig. 16 showing the fibrillar nature of the electron dense material and theassociated dense bodies or vesicles. Two small bodies similar in size and in electron density to those in the cluster are closely associated with alysosome (7'). x 55,000.

Fig. 18. A degenerating L cell which has formed large bulbous blebs of cytoplasm at its surface. Some of the blebs have either separated fromthe cell proper or they are connected in another plane. The blebs contain dispersed ribosomes (R), endoplasmic reticulum (ER) and occasionallymitochondria (Mi). Several of the mitochondria are swollen. x 18,000.

Fig. 19. An inclusion (A) within a macrophage (M) has the structural characteristics of a bleb from an L cell. Another â€œblebâ€•(B) is partially

surrounded by the same macrophage (M). A portion of a second possible macrophage (M2) shows advanced degenerative changes including many

lipid bodies (Li), several cytoplasmic vacuoles (x), and beginning disintegration ofthe plasma membrane (7). x 14,000.Fig. 20. A portion of a degenerating cell lying in close proximity to a nondegenerating L cell (L). The degenerating cell is completely devoid of

plasma membrane in the surface area shown. x 28,000.Fig. 21. A macrophage with a closely adhering membrane (7') of a degenerating cell. Other membrane fragments are seen at the left. X 14,000.Fig. 22. Membranes adhering to the surface of a macrophage (/â€˜).The membranes often occur as two parallel membranes. Some membrane

fragments appear to be enclosed within cytoplasmic vacuoles (X). x 14,000.

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1969;29:301-317. Cancer Res Velma C. Chambers and Russell S. Weiser

in Vitroduring Their Interaction The Ultrastructure of Target Cells and Immune Macrophages

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the ultrastructure of target cells and immune macrophages ... · cells (figs. 1, 2). clusters of...

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