[CANCER RESEARCH 47, 47-50, January 1, 1987]
Induction of Release of Cytotoxin from Murine Bone Marrow Cells by
an Animal Lectin
Takafumi Okutomi, Yuki Nakajima, Fusao Sakakibara,
Hiroaki Kawauchi, and Masatoshi
Yamazaki1
Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01 [T. O., Y. N., M. Y.J, and Institute of Cancer Research, Tohoku College of
Pharmaceutical Sciences, Sendai, Miyagi 983 [F. S., H. K.J, Japan
ABSTRACT
determined by assay of killing of L929 cells, as described by Ruff and
Gifford (7). Briefly, L929 cells (8 x Kr'/well) were incubated with
various dilutions of sample and actinomycin D (0.2 ¿ig/well)for 18 h
in 96-well trays containing 200 u\ of medium (Eagle's minimal essential
An animal lectin purified from loach (Misgurnus anguillicaudatus)
eggs induced release of cytotoxin from fresh bone marrow cells from
mice, although the other lectins tested, wheat germ agglutinili, concana-
medium supplemented with 5% fetal calf serum). Then the cells were
stained with 0.1% crystal violet for IS min, washed with water, and
treated with 100 u\ of 0.5% sodium dodecyl sulfate. The absorbance at
590 nm of the solubilized material was measured. The survival ratio
was calculated as the ratio of the absorption of the test culture to that
of the control without sample. The dilution of the sample giving the
half-survival ratio (50% effective dose) was obtained from a doseresponse curve. The cytotoxic activity (units) in each test plate was
calculated as the ratio of the 50% effective dose of the culture super
natant to that of rabbit tumor necrosis serum, as described previously
(8). The cytotoxic activity of this rabbit tumor necrosis serum was
equivalent to recombinant human tumor necrosis factor, 6 x 10' units/
valin A, and phytohemagglutinin
did not. The cytotoxin released from
bone marrow cells was a heat-labile protein with a molecular weight of
70,000. The main cells responsible for release of M, 70,000 cytotoxin
seemed to be of macrophage lineage, since they adhered to plastic and
were sensitive to certain antibodies for markers of macrophages. How
ever, they did not express asialo GMI antigen which is expressed by
activated macrophages. Removal of cells that phagocytized iron did not
diminish but rather enhanced the release of cytotoxin. Therefore, active
bone marrow cells appeared to be immature, not mature macrophages.
These data suggest that immature bone marrow cells that are not specif
ically activated have a cytolytic potency against tumor cells and that
internal animal lectins may induce release of the cytotoxin from these
cells.
ml (kindly provided by Asahi Chem. Ind., Tokyo, Japan).
Agents. Loach (Misgurnus anguillicaudatus) egg lectin was purified
by chromatography on two types of ion-exchange resin and affinity
chromatography on L-rhamnose coupled with epoxy-activated Sepharose 6B. This lectin appeared homogeneous on polyacrylamide gel
electrophoresis and was identified as a glycoprotein of A/, 50,000 with
an isoelectric point of pH 6.6 (9). No contaminating I I'S' was detected
INTRODUCTION
Studies on cytotoxic effector cells against tumor cells have
been focused on peripheral cells such as cytotoxic T-lymphocytes (1), natural killer cells (2), lymphokine-activated killer
cells (3), macrophages (4), and polymorphonuclear leukocytes
(5). These cells are all known to show cytocidal activity against
tumor cells in vitro. However, it is unknown at which stage of
maturation these cells become cytotoxic or whether fresh bone
marrow cells, which are precursors of peripheral cells, show
cytotoxicity against tumor cells. Therefore, we have focused
attention on the cytotoxicity of bone marrow cells, namely
central cells, and found that fresh bone marrow cells from
normal mice can lyse syngeneic tumor cells in the presence of
appropriate ligands (6). In this study, in an attempt to determine
how fresh bone marrow cells cause cytolysis, we examined
whether a soluble cytotoxin participated in tumor killing. This
paper shows that bone marrow cells released a cytotoxin in the
presence of an animal lectin and that the cells responsible for
its release may be immature macrophages.
in this lectin preparation (10). Wheat germ agglutinin, concanavalin A,
and phytohemagglutinin were purchased from E. Y. Laboratories, Inc.
(San Mateo, ÇA).BCG was from Mitsui Seiyaku Co. (Tokyo, Japan).
LPS and trypsin (1:250) were purchased from Difco Laboratories
(Detroit, MI).
Antibodies. Antimacrophage monoclonal antibodies (B12) were pre
pared as follows. An 8-week-old Wistar rat was immunized with 7.5 x
IO6 glycogen-elicited peritoneal cells from C3H/He mice. On day 21,
the rat received further injections of cells, SxlO6 cells i.v. and 5x10*
cells i.p. Three days after the second immunization, a spleen cell
suspension of the immunized rat was fused with P3U1 mouse myeloma
cells in a ratio of 4:1. The supernatants of growing hybrids were tested
10-14 days after fusion by complement-dependent cytotoxic assay.
Hybrids that secreted antibody with selective reactivity for macrophages
were cloned by limiting dilution. Antibodies were obtained from culture
supernatants of selected hybridomas. This antibody reacts with resident
and variously elicited peritoneal macrophages but not with other types
of cells.3
Anti-asialo GMI antibody was purchased from Wako Pure Chemical
Industries (Osaka, Japan). Anti-I-Ak and anti-Mac-1 monoclonal anti
MATERIALS AND METHODS
bodies were from Cedarlane Laboratories (Hornby, Ontario, Canada)
and Sera-lab (Sussex, England), respectively.
Column Chromatography. Gel filtration was carried out in a highperformance liquid chromatography system (LC-6A; Shimadzu) fitted
with a 0.75- x 60-cm column of G3000 SW (Toyo Soda Manufacturing
Co.). Material was eluted with phosphate-buffered saline at a flow rate
of 1 ml/min.
Bone Marrow Cells. Inbred male C3H/He mice were obtained from
Shizuoka Experimental Animal Farm (Shizuoka, Japan) and used at 8
weeks of age. Bone marrow cells were collected from femoral shafts by
flushing the marrow cavity with Hanks' balanced salt solution. The
dispersed cells were filtered through nylon mesh, washed with two
changes of cold phosphate-buffered saline and suspended in RPMI
medium with 5'V heat-inactivated fetal calf serum. Cell viability was
counted with 0.3% trypan blue and nucleated cells were counted with
1% Turk stain. About 70% of the bone marrow cells from normal mice
were nucleated and their viability was about 90%.
Cytotoxic Assay. The cytotoxic activity of culture supernatants was
RESULTS
Induction of Cytotoxin Release from Bone Marrow Cells by
Loach Egg Lectin. To start with, the ability of various lectins
2The abbreviations used are: LPS, lipopolysaccharide; BCG, Bacillus Calmette-Guérin.
3T. Okutomi, M. Satoh, H. Oshima. and M. Yamazaki. Antigens associated
with various cytotoxic activities of murine peripheral macrophages, submitted for
publication.
Received 3/11/86; revised 6/12/86, 8/27/86; accepted 9/10/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
47
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CYTOTOXIN
FROM BONE MARROW
consist of many types of cells, such as erythrocytes, polymorphonuclear leukocytes, monocytes, lymphocytes, and their im
mature cells. To determine which types of cells release cyto
toxin, we separated bone marrow cells into plastic-adherent
and nonadherent cells. Fig. 5 shows that both adherent and
nonadherent bone marrow cells released cytotoxin in the pres
ence of loach egg lectin but that the release by adherent cells
was several times greater than that by nonadherent cells in
terms of activity per cell (Fig. 5, D). Therefore, the activity of
the nonadherent fraction may be ascribed to contaminating
adherent cells in it. Adherent cells (about 30% of the bone
marrow cells) consist of morphologically heterologous popula
tions, but with few typical monocytes/macrophages
(less than
10% of the adherent cells).
In an attempt to determine the antigens expressed on the
surface of adherent cells, the ability of five antibodies to detect
surface antigens were tested in the presence of complement. As
shown in Fig. 6, the release of cytotoxin from adherent cells
to induce cytotoxin release from bone marrow cells of mice was
examined. LPS was used as a control. Bone marrow cells from
normal mice were incubated in vitro with various lectins or
LPS, and then the cell-free culture media were tested for
cytotoxic activity against L929 cells. Fig. 1 shows that LPS and
loach egg lectin induced cytotoxin release from bone marrow
cells into the medium, whereas wheat germ agglutinin, concanavalin A, and phytohemagglutinin did not. Culture supernatants
from bone marrow cells alone did not show any cytotoxicity.
Loach egg lectin alone at below 100 Mg/ml also had no cytotox
icity against L929 cells.
Next, we examined the dose response of loach egg lectin and
the kinetics of cytotoxin release. Loach egg lectin induced
concentration-dependent
cytotoxin release from bone marrow
cells (Fig. 2). Fig. 3 shows that the cytotoxin activity induced
by loach egg lectin reached a peak after 3 h and then decreased.
Thus the release of cytotoxin was transient and the cytotoxin
was unstable.
Some Properties of Cytotoxin from Bone Marrow Cells In
duced by Loach Egg Lectin. The temperature stability of a crude
preparation of the cytotoxin released from bone marrow cells
was examined. As shown in Table 1, the cytotoxin was heat
labile, losing about 90% of its activity in 15 min at 70°C.The
a6
cytotoxin also lost its activity on treatment with trypsin.
Next, we passed the culture supernatant through a highperformance liquid chromatography column to estimate the
molecular weight of the cytotoxin. The cytotoxin induced by
loach egg lectin was mainly eluted in the fraction corresponding
to a molecular weight of about 70,000 (Fig. 4).
Cells Responsible for Cytotoxin Release. Bone marrow cells
o
CELLS
0.3
Cytotoxic activity (units/ml)
ai
0.2
03
IO
Incubation
LPS (lua/mD
Loach egg lectin
(lOug/mU
OOug/ml)Con
WGA
(lOuQ/mOPHA
A
(IOug/ml)None1lJ]]3
15
time (hri
20
25
Fig. 3. Time course of cytotoxin release from bone marrow cells. Cytotoxin
release from bone marrow cells (8 x 10*cells/ml) stimulated by loach egg lectin
(10 Mg/ml) was assayed. Culture supernatants were harvested after the indicated
times of incubation.
Fig. 1. Induction of release of cytotoxin from bone marrow cells. Bone marrow
cells (X x 10" cells/ml) were incubated with LPS (1 Mg/ml) or various lectins (10
Mg/ml) and the culture supernatants were harvested after 4 h. WGA, wheat germ
agglutinin; Con A, concanavalin A; I'll l. phytohemagglutinin.
Table I Stability of cytotoxin from bone marrow cells
Supernatants were obtained from cultures of bone marrow cells (8x10* cells/
ml) with loach egg lectin (20 Mg/ml) and were heated as indicated or treated with
0.125% trypsin for 30 min at 37'C.
Residual activity (%)
Treatment
None37'C,
min56'C,
30
min70'C,
30
15minTrypsin1001005680
3.0
§2JO
S
2 1.0
IO
5
IO
Looch egg lecm
20
tig/mi)
30
IS
20
Fraction
25
No.
Fig. 4. Elution profile with high-performance liquid chromatography of cy
totoxin. Bone marrow cells were stimulated with loach egg lectin (20 Mg/ml) for
2 h. The culture supematants were concentrated by ultrafiltration and subjected
to high-performance liquid chromatography on a G3000 SVVcolumn (0.7 x 65
cm). J, molecular weights of standards. K, thousands.
Fig. 2. Dose-response curve of loach egg lectin. Bone marrow cells (8x10*
cells/ml) were incubated with the indicated concentrations of loach egg lectin and
the culture supernatants were harvested after 2 h.
48
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CYTOTOXIN
Cytotoxic activity
I.O
2O
FROM BONE MARROW
CELLS
Therefore, mature phagocytes, which phagocytize iron powder,
are not the main cells in bone marrow responsible for cytotoxin
release and may in fact cause inhibitory control of the active
cells.
(units/ml)
3.0
4O
Total
DISCUSSION
Adherent
Nonadherent
02
Cyfotoxic
0.4
activity
06
0.8
(units/celDXIO
Fig. 5. Cytotoxin release from adherent and nonadherent fractions of bone
marrow cells. Bone marrow cells (1.6 x 1o" udì)were incubated in flat-bottomed
96-well plates for I h at 37°Cto allow the cells to adhere to the plates. Then the
adherent and nonadherent cells were stimulated with loach egg lectin (20 Mg/ml).
The culture supernatants were harvested after 2 h and assayed for cytotoxicity.
20
(%! Inhibition
40
60
80
100
Anti-M0s (BI2)
Anti-Moc-l
Anti-la
Anti-asialoGMi
Fig. 6. Effects of various antibodies on cytotoxin release from adherent bone
marrow cells. Adherent bone marrow cells were incubated with various antibodies
(10-fold final dilution) for 30 min at 37'C and for 20 min at 37'C after addition
of low-tox-M rabbit complement. Then the medium was removed and the adher
ent cells were washed twice. These cells were stimulated with loach egg lectin (20
(ig/ml) and the culture supernatants were harvested after 2 h. GMI, ( ¡M1.
Cytotoxic
activity
(units/ml)
0246
a) Untreated cells
b) Rr treated
eels
c) Fe-phagocytosed
eels
d) R»-unphogocytos«d cell*
Fig. 7. Effect of iron powder treatment on cytotoxin release from bone marrow
cells. Bone marrow cells (8 x 10*cells/ml) were incubated with (h it) or without
(a) iron powder (8 x lOVml) for 1 h at 37*C and then iron-phagocytized cells (c)
were obtained by magnet. Residual cells did not phagocytize iron powder (d).
Then the cells were adjusted to 8 x IO6 cells/ml and incubated with loach egg
lectin (20 jig/ml). The culture supernatants were harvested after 2 h and assayed
for cytotoxicity.
was inhibited about 80% by an tim:., opinine monoclonal anti
body (B 12) and about 50% by anti-Mac-1 antibody. Anti-la
antibody caused different extents of inhibition in four experi
ments, and so its effect was not clear. Anti-asialo GMI antibody
did not inhibit cytotoxin release. These results suggest that the
adherent cells responsible for cytotoxin release may be of mac
rophage lineage.
Next, we examined whether mature phagocytes could release
the cytotoxin. As shown in Fig. 7, removal of cells that phagocytized iron did not diminish but rather enhanced the release.
We have shown that fresh bone marrow cells can release a
cytotoxin on stimulation with loach egg lectin as well as with
LPS. Loach egg lectin was similar to LPS in its ability to induce
cytotoxin, but we have shown that it does not contain LPS (10).
Therefore, the induction of cytotoxin release by loach egg lectin
is not due to contaminating LPS. This cytotoxin is rather
unstable, since cytotoxic activity was observed within 5 h after
stimulation and disappeared within 15 h (Fig. 3).
The main cells responsible for cytotoxin release seemed to
be of macrophage lineage, since they adhered to plastic (Fig.
5), and their ability to release cytotoxin was lost when they
were treated with antimacrophage or anti-Mac-1 antibodies
(Fig. 6). However, these cells did not express asialo GMi antigen
(Fig. 6), which is expressed by activated macrophages such as
BCG-elicited macrophages (11). Moreover, these cells did not
show phagocytic activity (Fig. 7). These results suggest that the
bone marrow cells that release the cytotoxin are immature and
not mature macrophages. However, further studies are required
to determine whether these cells are monoblasts, premonocytes,
or monocytes.
Much attention has been paid to the role of peripheral cells
in host defense. Most of the cells responsible for the host
defense mechanisms are thought to originate in the bone mar
row and to become effector cells after differentiation, matura
tion, and activation. But more immature cells were reported to
be involved in defense against infection (12) and tumors (13)
and in allograft rejection (14, IS). In this study we also found
that bone marrow cells that were not already specially activated
had cytotoxic activity like that of peripheral cells. The amount
of cytotoxin released per adherent cell was equivalent to that
released by BCG-elicited peritoneal macrophages (data not
shown). We have reported that loach egg lectin could induce
cytotoxin release from peritoneal macrophages only when they
were activated (10). In contrast, immature bone marrow cells
without activation have cytolytic potency against tumor cells.
Since there is a large pool of bone marrow cells, their partici
pation must be taken into account when considering the host
defense system against tumors.
We reported that macrophages ( 16), polymorphonuclear leu
kocytes (17, 18), and bone marrow cells (6) from mice lyse
tumor cells in the presence of a plant lectin (lectin-dependent
cellular cytotoxicity). Some selected animal lectins could also
induce lectin-dependent cellular cytotoxicity (19-22). There
fore, internal lectins may be involved in tumor recognition and
destruction. Active lectins bind effector cells to tumor cells (23,
24) and at the same time stimulate effector cells to release
cytotoxin (10). Thus induction of cytotoxin may be one role of
internal lectins. We speculate that cytotoxic factors are pro
duced in situ by internal lectins and are effective in the host
defense mechanism.
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Induction of Release of Cytotoxin from Murine Bone Marrow
Cells by an Animal Lectin
Takafumi Okutomi, Yuki Nakajima, Fusao Sakakibara, et al.
Cancer Res 1987;47:47-50.
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