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thesis entitled

Murine Macrophage Activation After Cisplatin
or Carboplatin Treatment

presented by

JoAnn Paulette Palma

has been accepted towards fulfillment
of the requirements for

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rump-now

MURINE MACROPHAGE ACTIVATION
AFTER CISPLATIN OR CARBOPLATIN TREATMENT

By

JoAnn Paulette Palma

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
' for the degree of

MASTER OF SCIENCE
Department of Zoology

1992

ABSTRACT
MURINE MACROPHAGE ACTIVATION AFTER '
CISPLATIN OR CARBOPLATIN TREATMENT
By

JoAnn Paulette Palma

Peritoneal macrophages when treated with cisplatin [9 pg/mi] or carboplatin
[50 rig/ml] for 2 h are stimulated to form cytoplasmic extensions which seek out
tumor cells [8180] establishing contact. Over time these extensions shorten pulling the
tumor cells towards the macrophages. No contact is established with normal cells.
Confocal microscopy reveals an increase in the lysosomes and their transfer to the
tumor cells via cytoplasmic extensions musing tumor cell lysis. Extension formation
is inhibited by 2.5 mM EDTA. CaC12 [4 mM] in the medium by itself is able to
induce extension formation but no contact formation develops with tumor cells.
Studies using fluo-3 as the probe for calcium reveal that cisplatin and carboplatin
induce opposite intracellular calcium changes. Calcium seems to be involved in the
signalling of macrophage activation, however, cytosolic calcium does not seem to

influence this process.

Dedicated to the memories of my father, sister and brother.

iii

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to Dr. Surinder Aggarwal, my research
adviser, from whom I have learned a lot, both in science and non science related
matters. His guidance, support, and patience including the many hours spent in the
preparation of this thesis is greatly appreciated. I also wish to thank Drs. R. Neel
Band and Stanley Flegler for having served as committee members and Dr. Mukhta
Webber for her suggestions in the project.

I extend my gratitude to Drs. John Sell and Joanne Whallon as well as Stuart
Pankratz and Asmina Jiwa of Meridian Instruments for their technical assistance. Dr.
Ajit Sodhi, whose name I wish to mention, as his work has instilled in me an interest
in the subject of macrophage activation.

0 I would like to acknowledge all my friends who have certainly made graduate
school more interesting and lastly, to my family whose support both spiritually and

financially has enabled me to reach my goals.

iv

TABLE OF CONTENTS

Page

LIST OF FIGURES ............................................................................ vi
INTRODUCTION .............................................................................. 1
MATERIALS AND METHODS ............................................................. 3
Cell Cultures ............................................................................ 3

Cell Viability ............................................................................ 4
Macrophage-Tumor Cell Interaction ................................................ 4
Lysosomal Studies ..................................................................... 5
Calcium Studies ........................................................................ 5
RESULTS ................................................................................... ' ..... 8
Macrophage-Tumor Cell Interaction ............................................... 8
Lysosomal Studies ..................................................................... 9
Calcium Studies ........................................................................ 26
DISCUSSION ................................................................................... 43
LIST OF REFERENCES ...................................................................... 46

Figure l.
‘ Figure 2.
Figure 3.
Figure 4.

Figure 5.

Figure 6.
Figure 7.
Figure 8.

Figure 9.

Figure 10.
Figure 11.
Figure 12.
Figure 13.
Figure 14.

Figure 15.

LIST OF FIGURES

Page
Scanning electron micrographs [murine macrophages] ................. ll
Scanning electron micrographs [murine macrophages] ................. 13
Scanning electron micrographs [murine macrophages] ................. 15
Confocal light micrographs [lysosomes] .................................. 17
Phase contrast, confocal light micrograph [murine macrophage and
tumor cells] ..................................................................... 19
Confocal light micrographs [lysosomal transfer] ........................ 21
Confocal light micrographs [lysosomal transfer] ........................ 23
Confocal light micrographs [lysosomes] .................................. 25
Graph showing intracellular calcium levels .............................. 30
Electron micrographs [membrane bound calcium] ...................... 32
Light micrographs [extension formation] 34
Confocal light micrographs [lysosomes] .................................. 36
Confocal light micrographs [lysosomes] .................................. 38
Confocal light micrographs [lysosomes] .................................. 40
Confocal light micrographs [lysosomes] .................................. 42

vi

INTRODUCTION

Cisplatin a broad spectrum anticancer drug [39], is currently on the market for
the treatment of testicular and ovarian cancers [5,17,49]. It is now being tested for the
treatment of head, neck and lung cancers [18,23,36]. Although an effective anticancer
drug, it has severe toxic side effects of which gastrointestinal and nephrotoxicity are
the dose limiting factors [48].

In vivo, cisplatin is known to cause hypocalcemia and hypomagnesemia
[20,40,41]. Various calcium channel blockers like nifedipine cause an increase in
induced cytotoxicity in cisplatin resistant cell lines [31]. Cisplatin has been shown to
inhibit cell division in tumor cells by depolymerization of actin like filaments [43]
known to be under the influence of calcium [35]. It seems evident that calcium plays
apartin the mechanism of action of cisplatin, however, this is
not yet very well understood.

Cisplatin has recently been shown to activate murine peritoneal macrophages
in vitro [42]. These activated macrophages selectively and efficiently seek out tumor
cells through the formation of cytoplasmic extensions and lysosomal transfer to these
target cells causing cell death. The role of the immune system in the regression of
tumors has been widely accepted [7,12,19,29] and various activating agents as
mm mm, calcium ionophore A23187 and lymphocyte mediators have
been used to study macrophage activation [l,9,ll,33,37,45]. In addition to inhibition
of DNA synthesis through interstrand or intrastrand crosslinking [24,38,44,51],

1

2
inhibition of transport enzymes [4] or inhibition of cytokinesis through
depolymerization of microfilaments of the tumor cells [2,3,26], activation of
macrophages by cisplatin may be one of the major mechanisms of action for tumor
regression.

Carboplatin, a second generation analogue of cisplatin has been demonstrated
to be less toxic but just as effective an anticancer agent utilizing much higher dosages
compared to cisplatin [14]. The prospect of a drug which is capable of enhancing the
immune system with less severe toxic side effects is very appealing thus this
investigation of carboplatin as a macrophage activator. In order to understand the
mechanism of action of cisplatin and carboplatin, the role of calcium in macrophage

activation was evaluated.

MATERIALS AND METHODS

11 1

Swiss Webster mice [Charles River Laboratories, Mass] were sacrificed by
cervical dislocation and peritoneal macrophages were isolated by injection of 5 ml chilled
minimal essential medium [MEM] without serum containing penicillin G sodium [1000
U/ ml], streptomycin sulfate [10000 mcg] , and amphotericin B into the peritoneal cavity.
After gently massaging the abdominal wall, cells were aspirated and seeded into modified
35 mm petri dishes made by boring a hole [10 mm] in the middle and gluing a clean
coverglass with silicon glue [Dow] underneath. After 2 h of incubation at 37°C, cultures
were washed vigorously to remove nonadherent cells leaving only macrophages. These
cultures were washed three times and incubated in fresh culture medium with 10% heat
inactivated fetal calf serum 3-5 days in a 5% CO2 incubator resulting in a well spread
macrophage monolayer at a density of 2—4 x 10‘ cells/m1.

Ascites sarcoma-180 [CCRFS-180II: American Type Culture Collection,
Rockville, MD. 20852] were maintained by serial transplantation in Swiss Webster mice.
Cells were obtained by sacrificing mice whenever needed. Such cells were washed with
Hank’s balanced salt solution [HBSS]. After centrifugation [1000 g, 5 min] cells were
resuspended in MEM at a concentration of 3 x 105 cells/ml of the
medium. These cells served as ‘ target cells for macrophages.
Normal hepatocytes were obtained by mincing a small piece of the liver through a

fine wire mesh, 105x105 pm in size ['I‘etko, Incorporated, Rolling Meadow, IL] and

4
mouse 3T3 fibroblasts [American Type Culture, Rockville, MD 20852] were

maintained in culture for a short time as target cells for the macrophages. In all

experiments an effector: target cell ratio of 1:10 was maintained.

Viability of various cells after co-incubation with macrophages [both treated

and untreated] was monitored by the trypan blue exclusion test [8].

Macrophagmmgr cell 111' when studies

To study macrophage tumor cell interaction, macrophage monolayers were
treated with either cisplatin [9 ug/ ml] or carboplatin [50 pg/ ml] for 2 h at 37°C. The
medium was then replaced by normal medium and target cells were added. These
cultures of macrophage and target cells were co-incubated for 10, 20, 30 min, 1, 2,
24, and 48 h. Sarcoma 180 cells and normal cells [hepatocytes or fibroblasts] were
also treated with cisplatin [9 [Lg/I111] or carboplatin [50 ug/ml] for 2 h at 37°C and
then presented to the untreated macr0phages for varying time intervals as stated
above. Coverslips seeded with macrophages and target cells were fixed with 1.5 %
glutaraldehyde in 0.05M phosphate buffer [pH 7.2] at room temperature for 30 min.
Samples were dehydrated in a graded series of ethanol, critical point dried, sputter
coated with gold [thickness ~200A; vacuum ~l x 10‘ Torr] using the EmscOpe
sputter coater and viewed under the JEOL ISM 35CF Scanning electron microscope
operated at 15 kv. Morphological criteria used for defining lymphocyte-mediated

death of a target cell have already been established [21,22].

Lysosomal Smdies

The quantitation of lysosomes before and after various treatments was achieved
by exposing macrophage cultures to fresh medium containing acridine orange [5
rig/ml] for 30 min at 37°C in the dark [36]. After careful washing, these cultures
were exposed to unlabelled tumor cells. As controls, tumor cells were incubated l h
in medium from which labelled macrophages were cultured for l h. These
macrophage-tumor cell cultures were examined under the ACAS 570 Interactive Laser
Cytometer with a confocal outfit after 5, 10, 30, 60 min intervals. Results were
analyzed using the ACAS 570 Workstation.

In order to study the transfer of lysosomes and to demonstrate cytoplasmic
continuity between the macrophage and tumor cell, confocal microscopy was used to
obtain optical sections [0.3 pm intervals] at 0, 30, and 60 min intervals using the
Zeiss Universal Confocal Laser Scanning Microscope [LSM 10]. Micrographs were
prepared at 0, 30, and 60 min after co-incubat ion of macrophages and tumor

cells using Kodak TMax film.

i ie
Macrophage monolayers were washed three times with HBSS for 5 min at
room temperature. Fluo-3AM [27] and pluronic acid F—127 [32,34] [Molecular
Probes, Inc. Eugene, OR 97402] were added at a final concentration of 4 uM each
for 45min at 37 °C. This was achieved by mixing 1:1 fluo-3AM [0.002 g/ml DMSO]
and pluronic acid F-l27 [0.0758 g/ml dH20], then adding 8 ul of this stock solution

to 1 ml of H888. After loading, cells were washed thoroughly and examined under

6
the ACAS 470 Interactive Laser Cytometer [Meridian Instruments, Okemos, MI] in

arbitrary units and percent differences were computed. Such cells already loaded with
the fluo—3AM and pluronic acid were exposed to cisplatin [9 ug/ ml] or carboplatin [50
ug/ml]. This calcium indicator is nonfluorescent until hydrolyzed by nonspecific
esterases within the cell [15]. An increase in fluorescence [40 fold] was observed as
calcium binds. The optimal excitation wavelength used was 506 nm and the maximum
emission wavelength after cytosolic calcium binding was 526 nm [47]. Fluorescent
measurements were made before and after drug treatments preferably on the same
cells for up to 2 h at 5 min intervals. A 10% neutral density filter was used to prevent
excessive photobleaching which may occur during repeated and lengthy experiments.
A discontinous strategy was applied to lengthy experiments deterring the effects of
any pho tob 1 each i n g . A 100x oil immersion lens was used for all experiments.
All data was analyzed using the ACAS 570 Workstation. Unlabelled macrophages

' were analyzed to check for autofluorescence.

Membrane bound calcium was investigated by fixing macrophages (both
control and treated macrophages) in 1% glutaraldehyde and 1% potassium
pyroantimonate for 4 h at room temperature. Controls included fixing macrophages in
1% glutaraldehyde with EGTA [2.5 mM] 1 h before being treated with 1% potassium
pyroantimonate. All cells were osmicated in 1% osmium tetroxide for 1 h before
being processed for electron microscopy. Thin sections [700 A] stained with uranyl
acetate, lead citrate were viewed under the Philips CM-lO transmission electron
microscope operated at 80 kV. Calcium pyroantimonate granules associated with the

membrane were counted and subjected to statistical analysis using the Student’s t—test

[two tailed] [13].

To evaluate the effect(s) of calcium channel blockers and calmodulin
antagonist on macrophage extension formation macrophage monolayers were treated
with cisplatin [9 [Lg/ml], carboplatin [so rig/m1], verapamil [105 M], nifedipine [10"
M], chlorpromazine [10‘ M], cisplatin plus verapamil, nifedipine or chlorpromazine,
or carboplatin plus verapamil, nifedipine or chlorpromazine for 2 h. Cells were
washed 3x and transferred to normal medium. Control cultures received the vehicle
only. S180 cells were added after the various treatments. Cells were co-incubated for
4 days while moni toring the macrophages for cytoplasmic extensions
and target cell cytolysis at regular intervals of 10 min up to 6 h and then 8 h intervals
for up to 4 days. Cisplatin or carboplatin treated macrophages [2 h] were left in
medium containing verapamil, for up to 4 days while monitoring extension formation.
In order to study the effect of low or high calcium in the incubation medium, cisplatin
or carboplatin treated macrophages [2 h] were incubated in medium containing either
disodium ethylene tetraacetic acid [EDTA] at a concentration of 2.5 mM or CaCl2 at a
concentration of 4 mM and monitored for extension formation through 4 days. In
addition, CaCl2 and verapamil treated macrophages [with or without cisplatin or
carboplatin treatment] were co-incubated with 8180 cells for 0, 30 min, I, 2, 6, 12,
24 and 48 h intervals and processed for scanning electron microscopy. These
macrophages were also stained with acridine orange for lysosomes and examined

under the ACAS 570 as previously described.

RESULTS

Macrophage-target Ell interaction

Normal mun'ne peritoneal macrophages [Fig. 1A] when treated with cisplatin
[9 ng/ ml] or carboplatin [50 pg/ ml] for 2 h at 37°C in culture developed cytoplasmic
extensions within 10 min [Fig. 1B]. Untreated macrophages when cultured with tumor
cells, did not show any evidence of extensions or contact formation for up to 24 h
[Fig. 1C]. Carboplatin treated macrophages with long extensions developed contacts
and cytoplasmic continuity with the tumor cells in the form of bridges within 30 min
of co-incubation [Fig. 1D]. Often times a macrophage developed contact with many
tumor cells. Within 1-2 h of co-incubation, cytoplasmic extensions were observed to
shorten pulling the tumor cells towards the macrophages [Fig. 2A]. After 6-48 h of
co-incubation, lysis of tumor cells occurred with eventual phagocytosis [Fig. 28].
Cisplatin or carboplatin treated macrophages seemed to discriminate between normal
cells [hepatocytes and fibroblasts] and tumor cells, for no extensions or contacts were
established with the hepatocytes or fibroblasts [Fig. 3A and B]. However, the treated
macrophages developed close contact with the tumor cells [Fig. 3C] and excluding
normal cells [hepatocytes] [Fig. 3D]. Untreated macrophages when co-cultured with
tumor cells that had been exposed to either cisplatin [9 pg/ ml] or carboplatin [50
ug/ml] for 2 h did develop extensions. Within 1-2 h these macrophage
extensions established contact with the tumor cells similar to the macrophages that had

been stimulated by cisplatin or carboplatin directly. When exposed to hepatocytes or

9

fibroblasts and tumor cells simultaneously, cisplatin or carboplatin treated

macrophages established contact only with the tumor cells.

W

Based on fluorescence measurements after acridine orange labelling, there was
observed an increase of about 59% in the number of lysosomes in the macrophages
after 2 h of cisplatin treatment while there was an 83% increase after 2 h of
carboplatin treatment [Fig. 4].

Cytoplasmic bridges between the macrophages and the tumor cells were
demonstrated with the confocal microscope in the phase contrast mode [Fig. 5]. Such
cytoplasmic bridges were not evident when viewed in the fluorescence mode after
acridine orange labeng because of low cytoplasmic fluorescence. A gradual transfer
of lysosomes from the macrophages into the tumor cells was observed over a 60 min
co-incubation period [Fig. 6A-C and 7A-H]. At times large accumulations of
lysosomes could be observed at the distal ends of the macrophage extensions [Fig.
6C] during the process of transfer to the tumor cells. Lysosomes in 8180 cells appear
to be very low [Fig. 8 A,C]. There was no leakage of acridine orange from the
labelled macrophages. Incubation of tumor cells in culture medium in which acridine
orange labelled normal macrophages were cultured for l h [Fig. 8 B and D] also did
not show any uptake of acridine orange by the tumor cells demonstrating that any
fluorescence of the tumor cells could only have come through a direct transfer from

the macrophages via cytoplasmic connections.

10

Fig. 1 Scanning electron micrographs showing murine peritoneal macrophages
before and after carboplatin [50 pg/ ml] treatment and co—culturing with S 180 cells:

A. Normal macrophages showing no cytoplasmic extensions; B. Macrophages after
carboplatin treatment [2 h]. Note the long cytoplasmic extensions [arrow]; C.
Untreated macrophage [arrow] and S 180 cell after 24 h of co-incubation. Note the
absence of contact between the two cells; and D. Carboplatin-treated macrophage
co-incubated with S 180 cells for 30 min. Note the presence of long cytoplasmic
extensions [arrow] in contact with 8180 cells. Final Mag. x 700 [A,B,D]; x 1300 [C]

 

12

Fig. 2 Seanning electron micrographs of macrophages treated with carboplatin
[50 ug/ml] [m] for 2 h and co—incubated with 8180 cell[s]: A. Note the shortening
of the cytoplasmic extensions after 1 h of co-incubation; and B. Lysis of 8180
cells [arrow] after 6 h of co-incubation. Final Mag. x 1300 [A]; x 700 [B]

l3

 

14

Fig. 3 Scanning electron micrographs of macrophages treated with carboplatin-
[50 ug/ ml] macrophages for 2 h and co-incubated for 24 h with A. normal
hepatocytes [arrow]; B. fibroblasts [arrowhead]. Note the absence of contact
formation; C. Note the association of tumor cells [arrowheads] and the treated
macrophages [m] in comparison to; D. macrophages [m] incubated with normal
hepatocytes [arrows] showing no contact formation. Final Mag. x 720 [A]; x 1300
[B]; x 480 [C and D].

 

16

Fig. 4 Fluorescent images taken from the ACAS 570 of macrophages labelled
with acridine orange [5 ng/ ml] showing cytoplasmic fluorescence and lysosomal
fluorescence in the untreated [A and C] and carboplatin-treated [2h] macrophages [B
and D]. Note the increase in lysosomal fluoresence after carboplatin treatment. Bar =
5 pm.

17

 

18

Fig. 5 Confoeal section of a carboplatin-treated [50 ug/ ml] macrophage [m]
for 2 h and after 2 h of co-incubation with 8180 cells [5]. Note the cytoplasmic
continuity [arrow] between the macrophage and tumor cells [5]. Final Mag.

x 5800

l9

 

20

Fig. 6 Fluorescent images taken from the LSM 10 of macrophages [arrows]
treated with carboplatin [50 ug/ ml] for 2 h and labelled with acridine orange [5
jig/ml] and co-incubated with unlabelled 8180 cells [S] for 0, 30, and 60 min. A.
Note the S 180 cells are not visible at 0 min of co-incubation due to the lack of
fluorescence; B. There is an increase in the number of lysosomes both in the
macrophages and the tumor cells, however, no lysosomes are visible in the
macrophage extensions; C. Lysosomes are clear in the cytoplasmic extensions
caught in the process of transfer [arrowheads] to the tumor cells. Final Mag. x 400
Bar = 5pm.

21

 

22

Fig. 7 Fluorescent images taken from the ACAS 570 of macrophages [m]
treated with carboplatin [50 pg/ ml] for 2 h and labelled with acridine orange [5
jig/ml] after co-incubation with 8180 cells [3] for 5, 30, 60, and 120 min. A-D
represent images as seen with the green detector [cytoplasmic fluorescence] and
corresponding images [E-H] as seen with the red detector [lysosomal fluorescence].
Note the absence of red fluorescence in the 8180 cell at 5 min of cooincubation [E].
There is a gradual increase in the number of lysosomes in the 8180 cells after 30 [F],
60 [G] and 120 [H] min of co-incubation. Arrows point to the lysosomes probably in
the process of transfer from the macrophages to the tumor cells. Bar = 5 um.

23

 

24

Fig. 8 Fluorescent images of 8180 cells taken from the ACAS 570.
Representing cytoplasmic fluorescence [A,B] and right panel micrographs representing
lysosomal fluorescence [C,D] after acridine orange [5 [Lg/1111] staining. Note the low
number of lysosomes [C]; B,D. 8180 cell 1 h after incubation in the medium in
which acridine orange labelled macrophages were incubated for 1 h. Note the absence
of any fluorescence indicating no leakage of the dye from the macrophages and that
any fluorescence in the S180 cells could only be due to the transferred lysosomes
from the macmphages. Bar = 5 pm.

WW I "”1?ij .‘t

    

   

 

2 6
i m die
Using fluo-3AM as the probe there was observed a sudden increase in the

intracellular calcium levels within 5 min of exposure to carboplatin by the
macrophages [Fig. 9]. This increase gradually returned to near baseline levels after 60
min and remained so for up to 120 min when observations ceased. However,
cisplatin-treated macrophages demonstrated a decline in intracellular calcium levels
reaching about 20% of the normal levels after 60 min with a steady increasing trend
thereafter. These intracellular calcium levels were only 25 % of the normal levels by
120 min when observations ceased. Untreated macrophages did not show any
signifieant fluctuations in intracellular calcium levels. Loss of fluorescence due to
photobleaching was less than 10 % and there was no autofluorescence observed.

Using potassium pyroantimonate technique to measure membrane bound
calcium under the electron microscope there was observed a decrease in macrophage
membrane bound calcium after cisplatin [9 pg/ ml] or earboplatin [50 pg] ml] treatment
of 2 h compared to untreated macrophages [Fig 10]. Calcium pyroantimonate granule
counts on the membrane of normal macrophages was 14.8 i 2.8 per cell vs. 3.7 i
1.64 and 4.8 i 1.93 for cisplatin and carboplatin treated cells respectively. This was
found to be statistically significant compared to controls [p < 0.05]. No membrane
bound calcium antimonate granules were observed in macrophages incubated in
EGTA [2.5mM]. The typical morphology of normal macrophages is depicted in
Figure 10A. The mitochondria appear dense, normal Golgi and rough surfaced
endoplasmic reticulum. However, after cisplatin treatment mitochondria appeared

swollen, endoplasmic reticulum and Golgi membranes bloated and prominent

27

vesiculation from the outer nuclear envelope [Fig. 108]. Similarly, carboplatin
induced swelling of the endoplasmic reticulum and the vesiculation from the outer
nuclear membrane, however, the mitochondria did not show marked swelling [Fig.
10C].

Normal macrophages when cultured in normal culture medium did not show
any significant extension formations for up to 4 days [Fig. 11A]. Macrophages when
treated with cisplatin [9 rig/ml] or carboplatin [50 jig/ml] for 2 h and placed in
normal medium developed prominent extensions [Fig. 11B]. Macrophages treated
with verapamil for 2 h and placed in normal medium, demonstrated no extensions for
up to 2 days. However, such cells demonstrated extensive extension formation soon
after through 4 days [Fig. 11C]. Macrophages treated with cisplatin or carboplatin
plus verapamil [2 h] also did not demonstrate extension formation for up to 2 days,
but did develop extensions after 2 days [Fig. 11D]. Results after verapamil treatment
could also be duplicated with other calcium channel blockers like nifedipine or
calmodulin antagonist chlorpromazine at similar concentrations. Cisplatin or
carboplatin treated [2 h] macrophages when left in MEM + EDTA [a calcium
chelator at a concentration of 2.5 mM] extension formation was completely inhibited
for up to 4 days when the observations ceased. When macrophages were exposed to
medium containing CaCl2 [4 mM] these did develop extensions but only after 2 days
of exposure. When CaC12 and verapamil treated macrophages were stained with
acridine orange for lysosomes, no apparent increase in the lysosomes were seen,
though extension formation was observed [Fig. 12]. However, when these

macrophages were treated with either cisplatin or carboplatin, an increase in the

28

lysosomes and their transfer to 8180 cells was observed [Fig. 13]. Interestingly,
EDTA treated macrophages, even when treated with cisplatin or carboplatin did not

show any lysosomal increase and transfer to 8180 cells [Fig. 14 and 15].

29

Fig. 9 Graph showing the effects of cisplatin [9 pig/m1] and carboplatin [50
ug/ ml] treatment on intracellular levels of calcium in macrophages as measured with
the dye Fluo-3AM [4 uM] using the ACAS 470 Interactive Laser Cytometer. Results
are mean of twelve cells per treatment expressed as fluorescence intensities in
arbitrary units. There is an abrupt 40% increase after carboplatin treatment [+]
within 5 min reaching normal levels after 60 min. In cisplatin treated cells [I] there
is a steady decline through 60 min after treatment with an upward trend thereafter.
Normal cells did not show any significant changes [0 ].

30

FLUORESCENCE EMISSION 0F FLUO-SAM
iN MURINE PERITONEAL MACROPHAGES

 

 

 

 

 

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0 I I I I I I I

 

0 5 10 15 30 45 60 90 105 120

MINUTES AFTER TREATMENT
I CDDP 1' CBDCA 0 N0 DRUG

31

Fig. 10 Electron micrographs showing the distribution pattern of calcium
pyroantimonate granules on the plasma membrane of: A. untreated macrophage; B.
cisplatin treated [9 [lg/[Ill] macrophage after 2 h; and C. earboplatin treated [50
jig/mi] macrophage atier 2 h. Note the decreased number of granules [arrows] in
treated macrophages as compared to the controls. The bloating of mitochondria [>],
swelling of the nuclear membrane [v] and endoplasmic reticulum [*] is prominent in
the cisplatin treated macrophage. Some of the mitochondria although swollen, the
momhology of the nuclear membrane and the endoplasmic reticulum after carboplatin
seems closer to normal. Final Mag. x 10500. Bar= 2 pm.

32

 

33

Fig. 11 Light micrographs showing macrophages at 4 days in normal medium
after: A. no treatment; B. cisplatin [9 jig/ml] for 2 h; C. verapamil [105 M] for 2
h; and D. cisplatin [9 jig/ml] plus verapamil [105 M] for 2 h. Note the extension
formation after cisplatin, verapamil or cisplatin plus verapamil treatments. Final Mag.
x 300. Bar=30 um.

34

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e e. e. \ p \‘ .
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. .. . . 3A haw...“
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35

Fig. 12 Fluorescentimages taken from the ACAS 570 of macrophages cultured
in 4mM CaClz for 4 days and stained with acridine orange [5 ug/ ml]. Left panel
pictures [A and B] represent cytoplasmic fluorescence and right panel pictures
represent lysosomal fluorescence [C and D]. A and C. Untreated macrophage; B
and D. Carboplatin treated [50 jig/m1] macrophage for 2 h. Note the increase in
lysosomal fluorescence in the carboplatin treated macrophage. Bar = 5 um.

 

37

Fig. 13 Fluorescent images taken from the ACAS 570 of macrophages [M]
cultured in CaCl, for 4 days, treated with carboplatin [50 jig/ml] for 2 h, labelled
with acridine orange [5 ug/ml] and co-incubated with 8180 cells [S] for 15, 30 and 60
min. Left panel micrographs [A—C] represent cytoplasmic fluorescence and right panel
micrographs [D-F] represent lysosomal fluorescence. Note the absence of red
fluorescence in the 8180 cell at 15 min of co-incubation [A and D.] and increasing
red fluorescence in the 8180 cell after 30 [B and E] and 60 [C and F] min ofco-
incubation. Bar = 5 pm.

38

 

39

Fig. 14 Fluorescent images taken from the ACAS 570 of macrophages cultured
in EDTA for 4 days and stained with acridine orange [5 ng/ ml]. Left panel
micrographs [A and B] represent cytoplasmic fluorescence and right panel
micrographs [C and D] represent lysosomal fluorescence. A and C. Untreated
macrophage; B and D. Carboplatin treated macrophages [50 rig/ml] for 2 h. Note
that there is no apparent increase in the lysosomes after carboplatin treatment. Bar =
5 um.

 

41

Fig. 15 Fluorescent images of macrophages cultured in EDTA for 4 days,
treated with carboplatin [50 ug/ ml] for 2 h, stained with acridine orange [5 rig/ml]
and co-incubated with 8180 cells [not visible] for 30 [A and C] and 60 min [C and
D]. Left panel pictures [A and B] represent cytoplasmic fluorescence and right panel
pictures [C and D] represent lysosomal fluorescence. Note that S 180 cells are not
visible due to lack of fluorescence and that there is very low lysosomal number in the
macrophages even after carboplatin treatment. Bar. = 5 pm.

 

DISCUSSION

Macrophages have been shown to mediate cell cytotoxic mechanisms in the
destruction of tumor cells [1,7,9,11,28,29,33,37,45]. Macrophages are most often
first activated with a biological response modifier or activator. Cisplatin is able to
activate peritoneal macrophages [42] in vitro and cause lysis of the tumor cells.
Carboplatin, a second generation analogue of cisplatin, also has this capability to
activate macrophages in vitro. This was demonstrated by contact formations
established with tumor cells in much less time than when untreated macrophages are
co-incubated with tumor cells. Extensions develop after cisplatin or carboplatin
treatment even in the absence of tumor cells which suggests that their presence is not
a prerequisite in the development of such extensions. However, the attraction of
activated macrophages seems to be specific for the tumor cells only, as no contact
formation was seen when normal cells [hepatocytes or fibroblasts] were co—incubated
with untreated or treated macrophages. Discrimination between non-neoplastic and
neoplastic cells is typical of activated macrophages [28,33,42,43].

In the study of macrophage activation, lysosomes have been suggested to take
part in the process of tumor cell death [6,16]. In cisplatin activated macrophages, an
increase in the number and their transfer to the tumor cells via the extensions have
been documented. Tumor cell lysis has been proven to be due to these transferred
lysosomes [42]. CarbOplatin induces an increase of lysosomes in the macrophages that

are transferred to the tumor cells through the extensions after contact has been

43

44

established. Activation of macrophages seems to be a multi-step process. There is the
development of cytoplasmic extensions, an increase in the lysosomes, recognition of
tumor cells leading to contact formation, a transfer of the lysosomes from the
macrophage to the tumor cells through these extensions and finally tumor cell lysis
leading to tumor cell death.

Calcium has been demonstrated to have an important role in macrophage
activation [42.50]. The use of calcium channel blockers or calmodulin antagonist
inhibited extension formation in cisplatin or carboplatin treated macrophages for 2
days, however, extensions developed soon after. CaC12 [4 mM] by itself is able to
induce extension formation in the macrophages while 2.5 mM EDTA inhibits such
extension formations even when used with cisplatin or carboplatin. These results seem
to indicate the role of calcium in macmphage activation, speci f i cal ly in
extension formation. There does not seem to be any relationship between intracellular
‘ calcium and extension formation as both drugs induce extension formation in
macrophages and diagonally opposite changes in the intracellular calcium levels.
MacrOphages incubated in CaCl, or EDTA seem to suggest that extension formation
may be regulated by calcium specifically. When CaClz incubated macrophages are co-
cultured with tumor cells, no contact formation occurs nor any lysosomal increase or
their transfer to the tumor cells can be demonstrated. EDTA inhibits all extension
formations. Further, CaCl2 incubated macrophages when treated with either cisplatin
or carboplatin, not only establish contact formation with the tumor cells, but also
cause an increase in the lysosomes and their transfer to the tumor cells. EDTA

inhibits all extension formation, lysososmal increase and transfer in the cisplatin or

45

carboplatin treated macrophages. Calcium therefore seems to be regulating the process
of extension formation only. It may be possible that calcium is acting as a priming
signal for macrophage activation [30,46] by cisplatin or carboplatin. Although both
cisplatin and carboplatin induce depletion of membrane bound calcium in
macrophages to a different degree yet both induce activation of the macrophages and
its involvement in the process is not clear.

Cisplatin and earboplatin induce murine macrophage activation resulting in the
expedition of tumor cell recognition through the development of cytoplasmic
extensions establishing contact only with tumor cells. An increase in lysosomal
content in the activated macrophages and the eventual transfer of these lysosomes via
the extensions to tumor cells seem to be responsible for tumor cell death. Although
calcium is involved in the formation of extensions, our studies seem to demonstrate
that intracellular calcium may not be playing any major role in the macrophage
activation process. It is possible that calcium acts as a priming signal and that actin
may be affected. Our study suggests the enhancement of the immune system through
the activation of macrophages as one of the probable mechanism(s) of action of the

antitumor agents cisplatin and carboplatin.

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