[CANCER RESEARCH 53, 362-367. January 15, 19931
A Mechanism for Neutrophil-mediated Lysis of Human Neuroblastoma Cells I
Edward Barker and Ralph A. Reisfeld 2
Department of lmmunology, The Scripps Research Institute, 10666 North Torrey Pines Road, IMMI3, La Jolla, California 92037
ABSTRACT
sequently cannot produce superoxide anions (16) are still able to
mediate the lysis of anti-GD2 antibody-coated neuroblastoma cells
(5, 15).
We report here on a nonoxidative mechanism by which antibodycoated human neuroblastoma cells are lysed by neutrophils. We indicate the importance of neutrophil degranulation in the lysis of human
neuroblastoma cells and demonstrate that GM~CSF increases antibody-dependent lysis of these cells by enhancing degranulation. We
also identified cathepsin-G and defensins as molecules within neutrophil granules that are responsible for mediating the lysis of human
neuroblastoma cells.
Neutrophils mediate the lysis of human neuroblastoma cells coated with
human/mouse chimeric anti-GD2 ganglioside antibody ch14.18. This study
examined the mechanism(s) by which this occurs. Neutrophil degranulation was found to be a required step for lysis, since release of granular
enzymes from neutrophils correlated with the lysis of antibody-coated
neuroh!astoma cells. In addition, agents which block degranulation specifically inhibited this process. Antibody-dependent lysis of neuroblastoma
cells was enhanced by exposing neutrophils to granulocyte-macrophage
colony stimulatory factor. An increased release of lytic granular molecules
was found to be responsible for this lymphokine-mediated phenomenon.
Among the molecules released from neutrophil granules that were shown
to be involved in neuroblastoma cell lysis were defensins, Mr 30011-4000
neutrophil granular proteins which are known to bind and permeabilize
tumor cells. In addition, cathepsin-G, a neutrophil granular protease, was
demonstrated for the first time to mediate the lysis of human neuroblastoma cells. The enzymatic activity of cathepsin-G was found to be required
for the lysis of these tumor cells, since phenylmethyisulfonyl fluoride
blocks the lytic ability of this protein.
MATERIALS
INTRODUCTION
It is well established that neutrophils are involved in the destruction
of microbial pathogens. Several reports indicate these cells to be
capable of mediating the lysis of leukemic as well as solid tumor cells
(1-7). The mechanisms utilized by neutrophils to mediate tumor cell
lysis include the generation of reactive oxygen intermediates (2, 8)
and/or the release of lytic molecules prepackaged in their granules
(9, 10). The lysis of tumor cells by neutrophils also was shown to be
enhanced by various cytokines (6, 7, 11-14).
Recently we (7) as well as others (5, 6) reported that mouse monoclonal antibodies directed against disialoganglioside Gt)2 surface
antigens can mediate the lysis of human neuroblastoma cells by neutrophils. We also observed that h u m a n / m o u s e chimeric anti-GD2 antibody ch14.18 mediates the lysis of human neuroblastoma cells by
neutrophils more effectively than its mouse counterpart, 14.G2a,
which recognizes the same antigen (7). In addition, we (7) and others
(5, 6) have demonstrated that G M - C S F 3 can enhance antibody-mediated lysis of human neuroblastoma cells by neutrophils.
Although the mechanism(s) by which neutrophils lyse neuroblastoma cells and G M - C S F enhances this process is still unknown, it has
been reported that reactive oxygen intermediates are not involved in
the lysis of neuroblastoma cells. This conclusion is based on the
observation that scavengers of oxygen metabolites do not affect the
ability of neutrophils to lyse antibody-coated neuroblastoma cells
(5, 15). In addition, neutrophils from individuals with chronic granulomatous disease which are deficient in N A D P H oxidase and con-
Received 8/3/92; accepted 11/5/92.
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.
l Supported by NIH Grant IR35CA42508. This is Scripps Research Institute manuscript number 7173-IMM.
2 To whom requests for reprints should be addressed, at Departmentof Immunology,
IMMI3, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA
92037.
3 The abbreviations used are: GM-CSF, granulocyte-macrophagecolony-stimulating
factor; ADCC, antibody-dependentcellular cytotoxicity; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; DMX, 1,3-dimethylxanthine;ISO, l-(3',4'-dihydroxyphenyl)2-isopropyl aminoethanol; HBSS, Hanks' balanced salt solution; PMSF, phenylmethylsulfonyl fluoride.
AND METHODS
Reagents. DIDS, DMX, and ISO were purchased from Sigma Chemical
Co. (St. Louis, MO) and prepared for use in the lytic assay as described by
Dallegri et al. (4). PMSF was also obtained from Sigma Chemical Co. and
prepared as described elsewhere (17). Elastase, cathepsin-G, lysozyme, and
myeloperoxidase, purified from human neutrophils, were purchased from ICN
Biochemical Co. (Irvine, CA). The purity of cathepsin-G was >98% by SDSPAGE. The purified defensin molecule HNP-1 was kindly provided by Dr.
Robert I. Lehrer (University of California, Los Angeles, CA). Recombinant
human GM-CSF was a gift from Behring Werke (Marburg, Germany).
Cell Lines and Monocional Antibody. The human neuroblastoma cell line
NMB-7 was a gift from Dr. S. K. Liao (McMaster University, Hamilton,
Ontario, Canada) and has been maintained in our laboratory for more than
10 years. The human neuroblastoma cell lines LAN-1 and LAN-5 were kindly
provided by Dr. R. Seeger (University of Southern California, Los Angeles,
CA). These cell lines were grown as described previously (7). Human/mouse
chimeric anti-GD2 antibody ch 14.18 was constructed by Dr. Stephen D. Gillies
(Abbott Biotech, Boston, MA) as described elsewhere (18).
Isolation of Neutrophils. The source of human neutrophils was venous
blood obtained from healthy donors. The blood was collected into 7-ml vacutainer tubes (Becton Dickinson, Mountain View, CA) containing the potassium
salt of EDTA as an anticoagulant (19). Neutrophils were isolated according to
the method of Wright (20). The number of neutrophils recovered varied from
1 X 106 to 2 x 106 cells/ml of whole blood. Percentage viability always
exceeded 98% as determined by exclusion of trypan blue dye (0.4%; Sigma
Chemical Co.).
Isolation of Neutrophil Granular Proteins. Neutrophil granular proteins
were isolated according to the methods described by Okrent et aL (21) and
Ganz et al. (22), which were applied with some modifications. Human neutrophils (0.5-1 X 101~ were routinely obtained from a single healthy donor by
leukopheresis. Contaminating erythrocytes were removed by dextran sedimentation and hypotonic lysis (20). Neutrophils were suspended at a final concentration of 108 cells/ml in Ca2+-free HBSS (Irvine Scientific Co., Santa Ana,
CA) containing 2.5 rnM MgC12 (Mallinckrodt, Inc., Paris, KY), pH 7.4. The cell
suspension was placed in a nitrogen bomb and pressurized to 750 psi for
20 min at 4~ The suspension was released dropwise into Ca 2§ and Mg2+-free
HBSS (Irvine Scientific Co.) containing 5 rnM EDTA (Sigma Chemical Co) at
pH 7.4, while stirring. This was followed by centrifugation (Technospin R;
Dupont Co., Burbank, CA) at 250 x g for 10 min to remove nuclear and
cellular debris. More than 90% of neutrophils were lysed by this procedure as
determined by exclusion of trypan blue dye (0.4%; Sigma Chemical Co.). The
supernatant was centrifuged (L3-50 ultracentrifuge; Beckman Instruments,
Fullerton, CA) at 27,000 x g for 30 min. The pellet was resuspended in either
0.4 Msodium acetate (JT Baker, Inc., Phillipsburg, NJ), pH 4.0, or 35 rnM acetic
acid (Mallinckrodt, Inc.) at a final concentration of 2 • 108 cell equivalents/ml.
Proteins from the neutrophil granules were extracted over a 12-h period at 4~
with gentle stirring. The residue was removed by centrifugation at 27,000 x g
for 30 min, and the supernatants were collected and stored at -70~ Extraction
of granular proteins from the pellet was repeated at least twice. The extracts
obtained by acetic acid treatment of neutrophil granules were pooled and
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NEUTROPH1L-MEDIATED I.YSIS OF NEUROBLASTOMA CELLS
concentrated by lyophilization (Virtis, Gardiner, NJ). Extracts obtained by
acetate treatment of neutrophil granules were concentrated by ultrafiltration
(YC-05 filter; Amicon Corporation, Beverly, MA). The two different extraction
methods were chosen since defensins are most effectively extracted by acetic
acid, a procedure which was reported to destroy the activity of the enzymes in
the various fractions (23). Each extract was concentrated to a final volume of
5.0 ml and layered on top of a Sephadex G-100 (Pharmacia, Piscataway, NJ)
gel filtration column (200 cm • 1.5 cm; BioRad, Richmond, CA). Elution of
proteins was achieved with 0.4 M sodium acetate, pH 4.0, containing 0.1 M
CaCI2 (Sigma Chemical Co.) or 17.5 mM acetic acid, respectively. Fractions
(10 ml) were collected and measured at 280-nm wavelength with a UV-160
spectrophotometer (Cole Scientific, Chicago, IL). Essentially the same A~so
profiles were observed as those described by others (21, 22). The respective
Kd values at which each fraction eluted were calculated as:
Kd-
v~-v0
V,- Vo
where Ve is the elution volume, V0 is the void volume (208.5 ml), and Vt is the
total column volume (567 ml). The Kd value for the fractions combined from
the column eluted with sodium acetate buffer were 0.22--0.28 (fraction A);
0.40-0.41 (fraction B); 0.62-0.63 (fraction C). A Ko value of 0.80-0.82 was
calculated for the fraction obtained from the column eluted with acetic acid that
contained defensins. Fractions corresponding to these respective Ka values that
represented the major peaks in the A28o profiles of the gel filtration columns
were pooled and concentrated by either ultrafiltration or lyophilization and
then dialyzed against phosphate-buffered saline (Irvine Scientific Co.), pH 7.4,
in spectapore 3 tubing with a molecular weight cut-off of 2000 (Spectrum,
Houston, TX).
Protein Determination. Protein concentration was measured with the BCA
protein assay system (Pierce Chemical Co., Rockford, IL) using chicken egg
lysozyme (ICN Biochemical Co.) as the standard. Each fraction as well as
purified neutrophil proteins were subjected to electrophoresis on 12.5% polyacrylamide gels in 6.25 M urea at a final pH of 4.0.
Lytie Assays. Assays of ADCC were performed as described previously
(7). In cases where inhibitors were used, 0.05 ml of neutrophils (ranging from
0.5 X 10 6 tO 2 • 10 6 cells) were incubated with 0.05 ml of each inhibitor in
U-bottomed 96-well plates (Coming Glass Works, Coming, NY) for 1 h at
37~ prior to adding antibody and target cells. Fetal bovine serum (Gibco,
Grand Island, NY) was omitted from lytic assays which evaluated the ability
of either proteins extracted from neutrophil granules or purified neutrophil
proteins to lyse neuroblastoma cells. Although the percentage of specific lysis
achieved by neutrophils and their granular proteins varied somewhat from
experiment to experiment, the overall pattern of results remained the same.
Degranulation Assay. Degranulation was measured by the release of
elastase in an assay described by Boros et al. (24), which was applied with
slight modifications. Briefly, neutrophils (2 x 10 6 cells) were suspended in
HBSS without phenol red (Irvine Scientific Co.) containing 0.1 mg/ml bovine
serum albumin (Sigma Chemical Co.) and added to 24-well tissue culture
plates (Linbro; Flow Laboratories, McLean, VA). Next, 1 • 105 NMB-7
neuroblastoma cells were added to each well. Either 0.025 ml of HBSS-bovine
serum albumin or ch14.18 were placed in the appropriate wells. The final
concentration of the antibody in each well ranged from 0.01 to 1 pg/ml. In
cases where GM-CSF was used, 0.025 ml of the cytokine was added to the
appropriate wells to a final concentration of 100 ng/ml. Each well was adjusted
to 1 ml total volume with HBSS-bovine serum albumin. Control wells consisted of those containing neutrophils without antibody, tumor cells, or GMCSE Maximal release of elastase was achieved by adding 0.1% Nonidet P-40
(Sigma Chemical Co.) in 0.125 ml phosphate-buffered saline (Irvine Scientific
Co.) to neutrophils instead of antibody and tumor cells. Each treatment group
was tested in triplicate. All plates were incubated at 37~ for 3 h.
Enzyme release was quantified by taking 0.1-ml aliquots of supematant
from the 24-well plates and placing them into V-bottomed 96-well plates
(Flow Laboratories). These plates were then centrifuged for 5 min at 250 x g
(TJ-6 Centrifuge; Beckman Instruments) to remove any cells that were present
in the supernatants. Aliquots of 0.025 ml were removed from each well and
placed into fiat-bottomed 96-well microtiter plates (Flow Laboratories).
Elastase activity of neutrophils was assayed by cleavage of N-t-BOC-ala-pronva thiobenzyl ester (Sigma Chemical Co.) in the presence of Ellman's reagent
[5,5'-dithio-bis-(2-nitrobenzoic acid)] (Pierce Chemical Co.). Each aliquot of
supematant was combined with a reaction mixture (0.075 ml) consisting of
4.5 mM peptide and 0.33 mM Ellman's Reagent in phosphate-buffered saline
(lrvine Scientific Co.), pH 7.4, and 10% dimethyl sulfoxide (Sigma Chemical
Co.). The plates were incubated at room temperature for 3-5 h and then read
at 405 nm in an EL-310 enzyme-linked immunosorbent assay microplate
reader (Biotek Instruments, Burlington, VT).
Percentage of degranulation was calculated as:
Absorbance of experimental release
- mean absorbance of spontaneous release
X 100
Mean absorbance of maximal release
- mean absorbance of spontaneous release
Although the percentage of specific degranulation achieved by neutrophils
from various individuals varied from experiment to experiment, the overall
pattern of results remained the same.
RESULTS
Ability of Antibody-coated Neuroblastoma Cells to Mediate
Neutrophil Degranulation. We have previously reported that human
neutrophils lyse monoclonal antibody ch 14.18-coated neuroblastoma
cells in a 4-h 5~Cr release assay (7). We now determined the extent of
neutrophil degranulation in this lytic process and demonstrated its
importance (Fig. 1). Here a clear correlation is seen between the
magnitude of degranulation (Fig. la) and the extent of tumor cell lysis
(Fig. lb). In the absence of antibody, the NMB-7 neuroblastoma cells
were unable to induce degranulation.
Effect of lnhibitors of Neutrophil Degranulation on Neutrophilmediated Lysis of Antibody-coated Neuroblastoma Cells. Since
a correlation was observed between degranulation and ADCC induced by antibody-coated human neuroblastoma cells, we determined
whether agents which inhibit degranulation would also effect ADCC.
For this purpose neutrophils were treated with 1 rnM of DIDS, ISO, or
DMX 1 h prior to the lytic assay. At this concentration DMX, ISO, and
DIDS inhibited the release of elastase by 90%, 85%, and 50%, respectively (data not shown). These same inhibitors markedly decreased neutrophil-mediated lysis of chl4.18-coated NMB-7 cells
(Fig. 2). Thus degranulation is clearly of importance for the ability of
neutrophils to lyse antibody-coated neuroblastoma cells.
Effect of GM-CSF on Degranulation of Neutrophils following
Their Exposure to Antibody-coated Neuroblastoma Cells. We
demonstrated previously that recombinant human GM-CSF enhanced
human neutrophil-mediated lysis of neuroblastoma cells coated with
monoclonal antibody ch14.18 (7). Since a correlation was observed
between the magnitude of degranulation of neutrophils and their ability to effect tumor cell lysis, it was determined whether GM-CSF
would actually enhance the level of neutrophil degranulation. The data
illustrated in Fig. 3 indicate that the same concentration of GM-CSF
(100 ng/ml) which enhanced neutrophil-mediated lysis of neuroblastoma cells also increased the magnitude of neutrophil degranulation.
The degree of lytic enhancement correlated with the concentration of
antibody used. GM-CSF alone had no effect on neutrophil degranulation, although this cytokine increases neutrophil-mediated lysis of
antibody-coated neuroblastoma cells by enhancing the ability of these
effector cells to degranulate.
Lytic Activity of Neutrophil Granular Proteins against Neuroblastoma Cells. Since degranulation is an important step in the process by which neutrophils lyse antibody-coated neuroblastoma cells,
an attempt was made to assess which granular protein(s) may be
responsible for this lysis. Proteins from neutrophil granules were
extracted and purified by techniques described in "Materials and
Methods." This resulted in the isolation of 4 major protein fractions that were essentially the same as those reported previously by
others (21, 22). The various fractions obtained from gel filtration
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Research.
NEUTROPHIL-MEDIATED
LYSIS OF N E U R O B L A S T O M A
oxidase and lysozyme, did not lyse the NMB-7 neuroblastoma cells
significantly. Purified myeloperoxidase and lysozyme from neutrophils also failed to lyse the neuroblastoma cells (data not shown). The
lytic activity of acetic acid-extracted neutrophil granular proteins resided essentially in the fraction containing the low-molecular-weight
(Mr 3000--4000) defensins. This fraction was able to mediate the lysis
of NMB-7, LAN-5, and LAN-1 human neuroblastoma cells (Fig. 6).
Taken together, the data from these experiments indicate that neutrophil granular protein fractions which contained either cathepsin-G/
elastase or defensins were able to lyse human neuroblastoma cells.
Effect of PMSF on the Ability of Proteins in Fraction B to Lyse
Neuroblastoma Cells. PMSF inhibits the enzymatic activities of cathepsin-G and elastase (17, 25). Therefore we determined whether the
lytic activity of the protein fraction that contained mainly these enzymes (fraction B) could be inhibited with PMSE Since PMSF completely prevented the material in fraction B from lysing neuroblastoma
cells (Fig. 7), we concluded that the enzymatic activity of either
cathepsin-G and/or elastase is required for lysis.
Ability of Purified Cathepsin-G and/or Elastase to Lyse Neuroblastoma Cells. Since both cathepsin-G and elastase are major
proteins of fraction B, it was determined whether either one or both
are responsible for mediating the lysis of neuroblastoma cells. For
this experiment enzymes were used which were purified from human neutrophils. Purified cathepsin-G was found to lyse NMB-7
neuroblastoma cells effectively; however, purified elastase had very
little if any lytic effect (Fig. 8). These data also indicate that elastase
does not enhance the ability of cathepsin-G to lyse the NMB-7 neuroblastoma cells. The ability of cathepsin-G to lyse neuroblastoma
+l
m
C
.=
ol
G)
"O
.2
.m
O.
u)
o~
0.01
0.1
Concentration of ch 14.18 (~g/rnl)
d
tti
+l
5O
40
.~_
>.,
-o
,=_
30
o
o.
20
lO
0
0
0.01
0.1
CELLS
1
a
Concentration of ch 14.18 (~g/ml)
Fig. 1. Correlationbetweenneutrophildegranulation(a) and lysis of chl4.18-coated
neuroblastoma cells mediatedby these effector cells (b). Effector:target ratio for both
assays was 100:1.
60
a."
(/)
+l
r
>,
--
.2
50
o
40
o~
60
40
.=
20
30
0.1
20
1
Concentration of ch 14.18 (]~g/ml)
10
Untreated
DIDS
ISO
18
DMX
Fig. 2. Inhibitionof neutrophil-mediatedlysis of ch 14.18-coated neuroblastomacells
by agents that block neutrophildegranulation.The concentrationof monoclonalantibody
ch 14.18 was 0.1/ag/ml.Numbers in parentheses, percentageinhibitionof specificlysis of
treated versus untreated groups.
d
u~
+1
~o'J
9
chromatography of neutrophil granule extracts contained the following major proteins as demonstrated by acid-urea polyacrylamide gel
electrophoresis (Fig. 4): myeloperoxidase (fraction A); cathepsin-G
and elastase (fraction B); lysozyme (fraction C); and defensins
(fraction D).
The proteins present in each of these major fractions as well as the
unfractionated material were tested for their ability to lyse neuroblastoma cells in a 4-h 5 ~Cr release assay. The lytic profile of the acetateextracted neutrophil granular protein fractions (Fig. 5) indicates that
cathepsin-G and elastase (fraction B) were able to lyse the NMB-7
neuroblastoma cells. Fractions A and C, containing mostly myeloper-
"0
u
~
.g
is
9
6
o.
~
3
0
.........T..
0
0.1
1
Concentration of ch ! 4.18 (~g/ml)
Fig. 3. GM-CSF enhancementof neutrophil-mediatedlysis of chl4.18-coatedneuroblastoma cells (a) correlates with its ability to enhance neutrophil degranulation
(b). Effector:targetratio for both assays was 100:1, and concentrationof GM-CSF was
100 ng/ml.[], untreatedneutrophils;1~, GM-CSF treated neutrophils.
364
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Research.
NEUTROPHIL-MEDIATED LYSIS OF NEUROBLASTOMA CELLS
1
3
4
5
DISCUSSION
MPO
The results of this study indicate that degranulation is an important
step in the process by which neutrophils lyse antibody-coated human
neuroblastoma cells. This was shown both by a correlation of tumor
cell lysis with neutrophil degranulation and by the inhibition of this
lysis by agents known to block degranulation. Both cathepsin-G and
defensins were implicated as components of neutrophil granules that
are involved in this lytic process. In addition, we demonstrated that
the mechanism by which GM-CSF enhances neutrophil-mediated lysis of antibody-coated neuroblastoma cells involves the ability of this
cytokine to increase the release of lytic molecules from neutrophil
granules.
A novel finding of this study is that lysis of antibody-coated neuroblastoma cells by neutrophils can be mediated by cathepsin-G and
defensins which are prepackaged in the cytoplasmic granules of these
effector cells (26). We also established that the contents of neutrophil
granules are important for the lysis of antibody-coated neuroblastoma
cells. A direct correlation was found between the ability of neutrophils
to lyse neuroblastoma cells and the amount of granular components
released by these effector cells upon interaction with antibody-coated
tumor cells. In addition, agents that block neutrophil degranulation
were shown to inhibit the ability of neutrophils to lyse antibodycoated neuroblastoma cells. At the concentrations used in this study,
ELASTASE
CATHEPSIN-G
HNP-1
LYSOZYME
Fig. 4. Acid-urea polyacrylamide gel electrophoresis of acetate and acetic acid extracts
of neutrophil granular proteins. Lane 1, 10 ~ag of unfractionated protein from acetate
extracts of neutrophil granules; Lanes 2-5, contain 10 lag of fraction A-D, respectively.
MPO, myeloperoxidase; HNP-I, defensin.
100
80
e~
60
r
+1
r
+t
6O
M
._o
r
_>,
o
0
40
40
o
o
O.
2O
M
2O
0
NMB-7
None
A
B
C
LAN-1
LAN-5
Fig. 6. Lysis of neuroblastoma cells by acetic acid-extracted neutrophil granular proteins which contained mostly defensins. The final concentrations of the proteins used in
the lytic assay were 50 lag/ml ([7) and 25 lag/ml (l~l).
UnfracUonated
matedal
Fractions
Fig. 5. Lysis of NMB-7 neuroblastoma cells by acetate-extracted neutrophil granular
proteins (A-C). The final concentration of each protein fraction used in the lytic assay was
50 lag/ml.
25
cells was not limited to NMB-7 cells, since this enzyme also lysed
LAN-5 and to a smaller extent LAN-I neuroblastoma cells. The
percentage of specific lysis mediated by 1 laM cathepsin-G was 34.72
+ 1.07 (SD) for NMB-7; 35.39 + 1.19 for LAN-5; and 13.07 + 5.59
for LAN-1.
Effect of Purified Defensin HNP-1 on the Lysis of Neuroblastoma Cells. Since defensins are present in the lytic fraction of the
acetic acid-extracted material, we determined whether the purified
defensin molecule HNP-1 is able to mediate the lysis of neuroblastoma cells. As can be seen from the data shown in Fig. 9, HNP- 1 lysed
neuroblastoma cells at concentrations ranging from 1.7 to 17 tUM
(5--50 lag/ml). These concentrations are similar to those reported by
others to be necessary for lysis of a variety of leukemic and lymphoma
cells (9, 10). Our data suggest that in addition to cathepsin-G, defensins are also capable of lysing human neuroblastoma cells.
20
CO
+I
w
u~
_>,
.2
o
15
10
0
Q.
u)
N
s
O ~
None
1 mM PMSF
Fig. 7. PMSF inhibits the lysis of neuroblastoma cells by the lytic fraction (fraction B)
of the acetate-extracted neutrophil granular proteins. The final concentration of fraction B
was 25 lag/ml, and that of PMSF was 1 laM.
365
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NEUTROPHIL-MEDIATED
LYS1S
OF
~1
d.
30"
u)
-H
a
cO
q-
O.
o)
10'
I
9
0.0
.
0.5
,
.
'
1.0
1.5
i
2'.0
2.5
Enzyme concentration (~M)
Fig. 8. Effect of cathepsin-G and elastase, purified from neutrophils, on the lysis of
NMB-7 neuroblastoma cells, ll, cathepsin G-treated target cells; O, elastase-treated target
cells; &, treatment with both enzymes.
50
40
30
2O
10
0
0
i
I
5
10
i
15
20
Concentration of HNP-1 (j~M)
Fig. 9. Lysis of NMB-7 neuroblastoma cell by purified defensin protein HNP- 1.
these inhibitors did not affect the viability of the effector cells (data
not shown). Nor have these inhibitory agents been found to interfere
with the ability of neutrophils to conjugate with antibody-coated tumor cells (4). These same inhibitors of degranulation also do not affect
the ability of neutrophils to form oxygen metabolites (27). Taken
together these findings indicate that neutrophil granular proteins are
required for these effector cells to lyse neuroblastoma cells.
The difference in magnitude of inhibition of lysis achieved by
DMX, ISO, and DIDS may be due in part to the respective differences
in their mechanisms of action. Thus whereas DMX and ISO increase
intracellular levels of adenosine 3',5'-monophosphate and inhibit microtubule formation (4), DIDS acts as an anion channel blocker (27).
Thus it is possible that the differences observed in the ability to block
lysis of neuroblastoma cells may be due in part to the mechanism by
which the various inhibitors block neutrophil degranulation.
Since our study clearly demonstrates a correlation between degranulation and tumor cell lysis, it is conceivable that the role of the
antibody molecule in neutrophil-mediated lysis of neuroblastoma cells
may be to initiate a cascade of events in the effector cells, which
eventually leads to the release of lytic molecules from neutrophil
granules. In this regard, Kushner and Cheung (6) reported that 3G8, a
monoclonal antibody directed against Fc3~III receptor, was able to
NEUROBLASTOMA
CELLS
block the antibody-dependent lysis of neuroblastoma cells mediated
by neutrophils.
Several studies indicated that GM-CSF can enhance neutrophilmediated lysis of antibody-coated neuroblastoma cells (5-7). Here we
report for the first time that exposure of neutrophils to GM-CSF
increases the cytolysis of antibody-coated tumor cells by enhancing
the ability of these effector cells to degranulate. These findings are in
general agreement with the observation made by Kowanko et al. (28)
that enhanced killing of antibody-coated fungi, Torulopsis glabrata,
by neutrophils exposed to GM-CSF was due in part to the ability of
the cytokine to enhance degranulation.
Defensins are well-characterized Mr 3500-4000 proteins which
constitute 5-8% of the neutrophil's total protein content (29) and
30-50% of the primary granule's proteins (30). Four of these molecules, which have been identified in human neutrophils (HNP-1, 2, 3,
and 4) were found to have similar lytic ability (31) when tested against
a variety of leukemic and lymphoma cells (9, 10). Here we demonstrate for the first time that a solid tumor like human neuroblastoma is
also sensitive to one of the defensins, HNP-1, at concentrations ranging from 1.7 to 17 ~ (5-50 lag/ml). Although the exact mechanism by
which defensin HNP-1 lyses neuroblastoma cells is not known, it may
be similar to the one described by others for leukemia and lymphoma
cells (10, 32, 33), where defensins form dimers which bind to the
target cell membrane and create small ion channels (32). The effiux of
such ions then disrupts the electric potential of the target cell's membrane, leading to cell disruption (33).
Okrent et al. (21) reported that a fractionate from an acetate extract
of neutrophils, containing mainly cathepsin-G and elastase, lysed
human umbilical cord endothelial cells. However, these investigators
did not identify the molecule(s) involved in this process. We could
demonstrate that cathepsin-G but not elastase contained in a similar
fraction obtained from an acetate extract was responsible for the lysis
of neuroblastoma cells, since only purified cathepsin-G was able to
achieve this lysis.
One mechanism by which the enzymatic activity of cathepsin-G
may mediate the lysis of neuroblastoma cells is by activating phospholipase A2. In this regard phospholipase A2 was reported to convert
membrane phosphatidylcholine into detergent-like lysolecithin and as
such lyse tumor cells (34), including neuroblastoma cells (35). Since
neuroblastoma cells express phospholipase A2 (36, 37), it is conceivable that its activity may increase upon exposure of the zymogen form
of the enzyme to cathepsin-G, which is able to produce more active
molecules.
Although the detailed mechanisms by which either defensins or
cathepsin-G can lyse neuroblastoma cells are not completely known,
our findings indicate that these two molecules are involved in the lytic
process, although they have completely different biological functions.
Thus, while defensins form pores in the membranes of their target
cells (32), the lytic effect of cathepsin-G appears to be linked to its
enzymatic activity.
ACKNOWLEDGMENTS
We would like to thank Dr. Tomas Ganz for his advice on the isolation of
neutrophil granular proteins.
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A Mechanism for Neutrophil-mediated Lysis of Human
Neuroblastoma Cells
Edward Barker and Ralph A. Reisfeld
Cancer Res 1993;53:362-367.
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