Download The Ultrastructure of Sarcoma I Cells and

[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
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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
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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
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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.
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