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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2744-2753
 2-Microglobulin Identified as an Apoptosis-Inducing
Factor and Its Characterization
By
Masaki Mori,
Yasuhito Terui,
Masayuki Ikeda,
Hiroshi Tomizuka,
Masaya Uwai,
Tadashi Kasahara,
Nobuyuki Kubota,
Takehito Itoh,
Yuji Mishima,
Miyuki Douzono-Tanaka,
Muneo Yamada,
Seiichi Shimamura,
Jiro Kikuchi,
Yusuke Furukawa,
Yukihito Ishizaka,
Kazuma Ikeda,
Hiroyuki Mano,
Keiya Ozawa, and
Kiyohiko Hatake
From the Department of Hematology and the Divisions of Molecular
Hematopoiesis and of Genetic Therapeutics, Center for Molecular
Medicine, Jichi Medical School, Tochigi, Japan; the Biochemical
Research Laboratory, Morinaga Milk Industry Co Ltd, Kanagawa, Japan;
the Department of Biochemistry, Kyouritsu Pharmaceutical College,
Tokyo, Japan; the Immunochemistry System Department, Eiken Chemical Co
Ltd, Tochigi, Japan; the Department of Intractable Diseases,
International Medical Center of Japan, Tokyo, Japan; and the Department
of Transfusion, University of Okayama, Okayama, Japan.
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ABSTRACT |
Major histocompatibility complex (MHC) molecules play an important
role in antigen presentation for induction of tumor as well as cellular
and humoral immunities. Recent studies using anti-MHC antibodies
demonstrated that antibodies specific for HLA class I molecules induced
cellular activation and a type of apoptosis that may be distinct from
Fas-dependent or TNFR (tumor necrosis factor- receptor)-dependent
processes. We purified a previously untested apoptosis-inducing factor
from HL-60 human leukemic cell-conditioned media to homogeneity and
sequenced it. It was identified as  2-microglobulin
(2m), which has been previously known as thymotaxin and
is a part of the HLA class I antigen complex. 2m acts on
both T-leukemic cells and myeloid leukemic cells to induce apoptosis,
which then activates caspase 1 and 3. Cross-linking studies showed that
biotinilated 2m recognized an epitope distinct from
those recognized by the anti-HLA class I antibody, as reported previously. We demonstrated that 2m plays a previously
unrecognized and important role in regulating the elimination of tumor
cells, which occurs as a result of the action of 2m as
an apoptosis-inducing factor.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MAJOR HISTOCOMPATIBILITY antigen (MHC)
molecules, including HLA class I, are heterodimers made of a 45-kD  chain with 3 extracellular domains noncovalently associated with the
invariant 12-kD 2-microglobulin (2m).
They play an important role in regulating tumor immunity as well as
cellular and humoral immunities.1,2 They function in the
presentation of proteasome-generated antigens to CD8+ T
cells.3
In addition to their role in antigen presentation, HLA class I
molecules expressed on T cells may also be involved in the signal
transduction.4-9 Intracellular signal transduction after HLA class I ligation includes protein tyrosine phosphorylation and an
increase in intracellular free calcium.9-12
In addition, several studies have shown that HLA class I ligation on
the surface of T cells may result in growth arrest, anergy, and,
eventually, apoptosis induction.13-15 The expression of HLA class I antigens has been observed in some tumor cells. Chemoprevention agents, such as all-trans retinoic acid and retinamide, induce upregulation of HLA class I antigens and other factors (reactive oxygen
radicals).16,17 Upregulation of HLA class I antigens explains how the enhancement of tumor immunity may occur. On the other
hand, some kinds of viral infections, including adenovirus, cytomegalovirus, and human immunodeficiency virus (HIV), induce downregulation of HLA class I antigen expression and may be a factor in
the immunodeficient state after infection with these viruses.18-20
In the immune system, apoptosis induced by surface molecules, such as
Fas (CD95)21 and tumor necrosis factor receptor (TNFR), is
the main mechanism for the homeostasis of T and B
cells22,23 and for the lysis of target cells by cytotoxic T
lymphocytes (CTL) and natural killer (NK) cells.21-23 In
human T cells, apoptosis can be triggered not only by several membrane
receptor families, including Fas, TNFR, and CD30, but also by CD2,
CD45, CTLA4, and HLA class I molecules, especially in activated T
cells.21-31 Recent reports indicate that cross-linking
monoclonal antibodies (MoAbs), which recognize the 2m or
the 3 domain of the heavy chain, can induce T-cell apoptosis, and
this pathway may be distinct from Fas- or TNFR-induced
apoptosis.32 Two antibodies against an epitope of the 1
domain of the HLA class I heavy chain may also induce apoptosis of
activated T cells. These reactions do not involve Fas/Fas-ligand
interaction.33,34 However, identification of specific
ligands for the MHC class I complex has not yet been achieved. An
interaction between human 2m35,36 and MHC I
heavy chains has been studied by mutational analysis of the heavy
chains.37
2m, a chemotactic protein previously known as
thymotaxin, plays an important role in T-cell precursor colonization in
the thymus.38,39 We purified an apoptosis-inducing factor
from a conditioned media of HL-60 cells stimulated by phorbol
12,13-dibutyrate (PDBu).40,41 One of the purified proteins
has been identified as human 2m. A serum
concentration of 2m induced apoptosis in both myeloid
and lymphoid leukemia cell lines. We demonstrate a previously unknown
function of 2m as an apoptosis-inducing factor in
lymphoid cells.
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MATERIALS AND METHODS |
Cell lines and reagents.
The human cell lines used in this study were nonlymphocytic cell lines
(K562 [chronic myelocytic], HL-60 and NB-4 [acute promyelocytic], THP-1 [acute monocytic], U937 [myelomonocytic], and KG-1 [acute erythrocytic]), B-lymphocytic cell lines (SKW, BALL, and Daudi), and
T-lymphocytic cell lines (Jurkat, TALL, and CCRF-CEM). All of these
cell lines were cultured in GIT media (Wako Co, Tokyo, Japan), which
does not contain proteins such as 2m or cytokines, as
demonstrated in our previous studies.40,41 Human
2m was purified from human urine to homogeneity (its
sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]
analysis showed a single band) and was used in the early experiments
(data not shown). Endotoxin was not detectable by Limulus assay
(data not shown). Recombinant human 2m consisting of 99 amino acids (Oriental Yeast Co, LTD, Shiga, Japan) was used for the
same purposes as urine-derived 2m. Reducing SDS-PAGE
analysis of the recombinant product showed homogeneity with a single
band. The concentrations of 2m in supernatants of PDBu
(Sigma, St Louis, MO) -treated HL-60 cells and in several maintained
cell lines were measured by enzyme-linked immunosorbent assay (ELISA).
In addition, anti-Fas MoAb (IgM, clone CH-11; MBL, Nagoya, Japan),
recombinant human TNF- (Calbiochem, La Jolla, CA), and endothelial
interleukin-8 (IL-8; R&D System, Minneapolis, MN) were used as
apoptosis-inducing factors in control studies.
Purification and sequencing of 2m as an
apoptosis-inducing factor (AIF).
The HL60 cells were resuspended at 2 × 105 cells/mL
in HamF12/Dulbecco's modified Eagle's medium (DMEM; GIBCO, Rockville,
MD), which contains 10% GIT medium and 50 nmol/L PDBu and were
incubated at 37°C for 3 days as in our previous
studies.40,41 After 3 days, the supernatant was collected
and precipitated in ammonium sulfate. The preparation was then purified
further in a process using cation exchange fast protein liquid
chromatography (FPLC) and gel filtration high performance liquid
chromatography (HPLC). Additionally, the pool of active fraction eluted
from the gel filtration HPLC column was added to reverse-phase HPLC.
When the concentration of acetonitrile reached approximately 40%, the
highest peak of AIF activity was eluted and added to the reverse-phase HPLC. The N-terminal sequence of the protein was determined by Edman
degradation on an automated Applied Biosystems 473A sequencer (Applied
Biosystems, Foster City, CA), as in our previous
study.40,41
MTT and TUNEL assays for the detection of apoptosis.
Each of the human cell lines was seeded at 1 × 105
cells/mL in HamF12/DMEM with 10% GIT medium, and 2m was
added to the media at 10 µg/mL. After 2 days, the capacity to reduce
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
was determined. After adding 10 µL of MTT solution (5 mg/mL MTT in
phosphate-buffered saline [PBS]), the preparation was incubated at
37°C for 3 hours. Cells with MTT formazan were dissolved in a
solution of 2-propanol and 0.04 mol/L of HCl, and color absorbance was
measured at 595 nm by a microplate reader (Bio-Rad, Hercules, CA).
Additionally, residual cells were incubated with a digoxigenin-UTP
terminal deoxynucleotide transferase mixture. Subsequently, they were
stained with a peroxidase-conjugated antibody for digoxigenin by using
an in situ Apoptosis Detection Kit (Apop Tag Plus; Oncor, Gaithersburg,
MD); they were then counter-stained with 1% methyl green in sodium
acetate (pH 4.0) and finally mounted. Specimens were examined and
photographed with a microscope as in our previous
study.40,41 Statistical analysis was performed using a
Student's t-test.
MoAbs.
The mouse anti-2m MoAbs (IgG, clone NK1, NK2, and NK3)
were derived from hybridoma cells and purified in a PBS together with ammonium sulfate. K562 cells and CCRF-CEM cells, at a concentration of
1 × 105 cells/mL were exposed in 1 mL of HamF12/DMEM
to a 10% GIT medium for 48 hours at 37°C after preincubation of
100 µg of anti-2m MoAb with 10 µg of
2m for 15 minutes at room temperature (RT). Inhibition
of the 2m-induced apoptosis in these cells were detected using the MTT assays and TUNEL method as described above. The anti-Fas
MoAb (IgG2b, clone SM1/23; Chemicon Int Inc, Temecula, CA) and
anti-TNFR(p55) MoAb (IgG; AUSTRAL Biologicals, San Ramon, CA) at a
concentration of 1 µg/mL were added to show whether
2m-induced apoptosis was inhibited. In addition, the
anti-IL-8 MoAb (IgG1, clone #6217.11; R&D Systems), anti-IL-8
receptor A (CDW128), MoAb (IgG2b, clone 5A12; Pharmingen, San Diego,
CA), and anti-IL-8 receptor B (CXCR2) MoAb (IgG1, clone 6C6;
Pharmingen) were added at a concentration of 5µg/mL to show whether
2m-induced apoptosis was inhibited. Ten micrograms per
milliliter each of TU149 MoAb (IgG2a, anti-HLA-B, C, and some A;
Caltag, Burlingame, CA), YTH862 MoAb (IgG2b, anti-HLA-A, B, and C;
Serotec, Oxford, UK), and W6/32 MoAb (IgG2a, anti-HLA-A, B, and C;
Serotec) were added at 10 µg/mL to study the correlation with
apoptosis induced by HLA-class I. The mouse anti-RecA MoAb (IgG2b,
clone ARM414; MBL) was used as a negative control. These anti-HLA class
I MoAbs and anti-RecA MoAb were used after dialysis against PBS.
Caspase inhibitors.
The IL-1-converting enzyme (ICE)-like protease inhibitors
Ac-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) and
Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK) as well as the
CPP32/Apopain-like protease inhibitor Ac-Asp-Glu-Val-Asp-aldehyde
(Ac-DEVD-CHO) were purchased from Calbiochem-Novabiochem Co (San Diego,
CA). These peptides were dissolved separately in dimethylsulfoxide
(DMSO; Wako Co, Tokyo, Japan) and 10 or 100 µmol/L of each peptide
was added to the cell suspensions with final concentrations of 0.1% or
0.3% DMSO as performed in our previous study.42
Fluorescence-activated cell sorter (FACS) analysis.
Fluorescein isothiocyanate (FITC)-conjugated mouse IgG1 (DAKO,
Glostrup, Denmark) was used as a negative control. FITC-conjugated anti-HLA class I MoAb (clone W6/32; Serotec) was added to the solution
to detect the presence of HLA class I on the cell surface membrane.
2m was biotinilated with an ECL protein biotinylation module (Amersham, Little Chalfont, Buckinghamshire, UK). Avidin R-PE
(Caltag) was added. Cells from various cell lines were centrifuged at
2,000 rpm for 1 minute and washed once in PBS. The cells were exposed
to the biotinilated-2m (2µg) for 30 minutes, put on
ice, washed twice in PBS, and exposed to avidin R-PE- and
FITC-conjugated mouse anti-HLA class I MoAb simultaneously for 30 minutes on ice. The cells were analyzed using the FACScan (Nippon
Becton Dickinson Co, Tokyo, Japan) as performed in our previous
study.43 Each data point was derived from an analysis of 1 × 104 cells.
Protein cross-linking.
The results of our binding study are shown briefly. Biotinilated
2m was incubated with 5 × 106 K562
cells or CCRF-CEM cells for 30 minutes at 4°C. After resuspension in 450 µL PBS, cells were washed twice. After removal of PBS, 1 µL
of 120 µg/mL biotinilated 2m was added. As a cold
competitor, 100× 2m was added. Ten microliters of
1 mmol/L d-biotin (Sigma) was added and incubated at 4°C for 1 hour
with occasional shaking. As for the manufacturer's protocol, 1 × 106 cells of K562 cells were suspended in a total volume of
100 µL PBS and solubilized with 2.0% (vol/vol) Nonidet P
40.44,45 After removing insoluble material by
centrifugation, 0.5 mL of the detergent extract was mixed with 5 µL
disuccinimidyl suberate (DSS; Pierce, Rockford, IL) solution (20 mmol/L
in DMSO; final concentration, 1 mmol/L) and allowed to react for 30 minutes at 14°C. The reaction was stopped by adding 2 µL of 1 mol/L Tris-HCl, pH 7.5.
SDS-PAGE and immunoblotting.
After further centrifugation to remove precipitates from this sample,
the supernatant was loaded onto an SDS-PAGE and immunoblotted. Protein
samples were submitted to SDS-PAGE analysis on a 4% to 20% gradient
gel under standard conditions using a mini-Protean II system (Bio-Rad),
followed by silver staining. For immunoblotting, the proteins were
transferred to polyvinylidene difluoride (Bio-Rad) membrane filters and
were blocked in 2% (wt/vol) bovine serum albumin in washing buffer
Tween 20 in PBS.
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RESULTS |
2m was detected as a type of AIF.
HL-60 cells at a concentration of 2 × 105 cells/mL
were cultured with 50 nmol/L of PDBu at 37°C for 3 days and
differentiated into the monocyte/macrophage lineage. We investigated
whether apoptosis was induced in K562 cells when the supernatant from PDBu-treated HL-60 cells was added to a culture media of K562 cells.
PDBu itself did not induce apoptosis in K562 cells (data not shown),
and we did not detect any AIF activity in conditioned media from
unstimulated HL-60 cells. To detect whether proteins were the AIFs, the
protein in the supernatant was precipitated from a solution of 95%
ammonium sulfate. The preparation was then purified further in a
process using cation exchange (FPLC; Fig 1A) and gel filtration (HPLC; Fig 1B). Additionally, the pool of active
fractions eluted from the gel filtration column was applied to
reverse-phase HPLC. The purified AIF activity was eluted when the
concentration of acetonitrile reached approximately 40% in
reverse-phase HPLC (Fig 1C). We obtained specific activity of 1 × 105 U/mg of protein (1 U AIF inhibited 50% of maximum
reduction, as indicated by MTT reducing activity) and with a final
purification of 270-fold, calculated from the first supernatant
concentration.40 The overall yield was 0.05%.
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| Fig 1.
The apoptosis-inducing factor was purified from the
supernatant of PDBu-treated HL60 cells and recognized as human
2m. The supernatant of PDBu-treated HL60 cells collected
as in Materials and Methods was purified in a process using (A) the
cation exchange FPLC (SP Sepharose HP; Pharmacia, NJ) and
(B) the gel filtration HPLC (TSKgel G3000SW; Tosoh, Tokyo,
Japan). The pool of active fraction eluted from gel
filtration HPLC was applied to (C) the reverse-phase HPLC. The column
(µBondasphere; Waters, MA) was eluted through a
90-minute linear time gradient from 28% to 40% acetonitrile. The flow
rate was maintained at 2 mL/min. (D) The N-terminal sequence of the
fraction with AIF activity. It is closely homologous with the
N-terminal sequence of human 2m. X shows an undetermined
amino acid sequence. (E) The concentration of 2m in the
supernatant of PDBu-treated HL60 cells gradually increased over time
for 72 hours.
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We sequenced the protein that showed a single peak with AIF activity.
From an analysis of the N-terminal sequence of the AIF, we found that
it was strikingly (97%) homologous to the N-terminal sequence of human
2m (Fig 1D). In addition, using ELISA, the concentrations of 2m in the supernatants from the
PDBu-treated HL-60 were measured and found to increase in relation to
time (Fig 1E). We did not detect 2m in unstimulated
HL-60 cell-conditioned media (data not shown). The expression of
2m mRNA in PDBu-treated HL-60 cells correlated with the
concentration of 2m in the supernatant (data not shown).
Therefore, 2m was recognized as a candidate AIF.
2m induces apoptosis in K562 cells in a
dose-dependent manner, with this apoptosis increasing in relation to
time.
We initially examined whether 2m would induce apoptosis
in K562 cells. The proliferation of K562 cells was suppressed in a
dose-dependent manner in the presence of 2m at
concentrations of more than 10 µg/mL, as detected by the MTT assay
(Fig 2A). Simultaneously, we detected the
induction of apoptosis in K562 cells with the TUNEL assay (Fig 2C and
D), and we found that 2m induced apoptosis in these
cells in a dose-dependent manner (Fig 2B). In the presence of
2m for 48 hours, the fraction of apoptotic K562 cells
increased from 21.0% ± 2.2% (at 10 µg/mL) to 42.7% ± 4.5%
(at 20 µg/mL) and to 43.3% ± 1.7% (at 100 µg/mL). Time course
studies showed both inhibition of proliferation and induction of
apoptosis most markedly between 48 and 96 hours (Fig 2E and F).
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| Fig 2.
Inhibition of cell growth and induction of apoptosis in
K562 cells by human 2m. (A) The proliferation of K562
cells (1 × 105 cells/mL) was suppressed in the presence
of more than 10 µg/mL of 2m for 48 hours, and (B)
apoptosis was induced. (C and D) The percentage of apoptotic cells was
determined microscopically by counting 200 cells on in situ-stained
slides. (C) Control K562 cells cultured without 2m or
(D) with 10 µg/mL of 2m at 37°C for 48 hours.
Apoptotic cells were detected using in situ staining with Apop Tag PLUS
(Oncor), which gives a dark contrast to the insoluble precipitate,
indicative of genomic fragmentation, as described. Arrows indicate
apoptotic cells (original magnification × 200). (E) The proliferation
of K562 cells was suppressed, and after more than 48 hours of
incubation with 10 µg/mL of 2m apoptosis was induced
(F). Results are expressed as the mean ± SE of 3 independent
experiments. Standard deviations are shown by horizontal bars.
P values of less than .05 are shown as (*) and less than .01 as
(**).
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2m induces apoptosis in human leukemic
and lymphoma cell lines.
Based on the detection of 2m-induced apoptosis in K562
cells, various human leukemic and lymphoma cell lines were cultured at
1 × 105/mL in the presence of 10 µg/mL of
2m for 48 hours at 37°C. Proliferation and
percentage of apoptotic cells were studied. We found that 2m induced apoptosis in more than 10% of U937, BALL,
and CCRF-CEM cells (Fig 3A and B) as well
as K562 cells (Fig 3C). Moreover, apoptosis both in K562 cells and in
CCRF-CEM cells was induced by recombinant human 2m (Fig
3D and E). FasL/Fas interaction is known to strongly and rapidly
initiate apoptosis in human leukemic cell lines such as Jurkat cells.
2m does so more slowly, eg, over a few days instead of a
few hours. K562 cells and CCRF-CEM cells were especially useful in this
study of the mechanism of 2m-induced apoptosis, because
apoptosis is not regularly induced in their natural in vitro growth
patterns.
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| Fig 3.
2m induces apoptosis in other human
leukemic and lymphoma cell lines. (A) Control CCRF-CEM cells were
cultured without or (B) with 10 µg/mL 2m at 37°C
for 48 hours. Apoptotic cells were detected using in situ staining with
Apop Tag PLUS (original magnification × 200). (C) Induction of
apoptosis by 2m in various cell lines. Nonlymphocytic
cell lines (HL-60, NB4, THP-1, U937, and KG-1), B-lymphocytic cell
lines (SKW, BALL, and Daudi), and T-lymphocytic cell lines (Jurkat,
TALL, and CCRF-CEM) cells (1 × 105 cells/mL) were
cultured with 10 µg/mL of 2m at 37°C for each or
48 hours, and TUNEL assays were performed. In the controls of U937
cells and BALL cells, apoptosis was induced in more than 5% of the
cells. Therefore, they did not prove useful ([ ] control; [ ]
additive of 2m). (D and E) Apoptosis in K562 cells (D)
and in CCRF-CEM cells (E) was induced by recombinant human
2m in the same way as with urine-derived human
2m.
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Anti-2m MoAb inhibits the
2m-induced apoptosis.
The mouse antihuman 2m MoAbs (clone NK1, NK2, and NK3)
were derived from hybridoma cells and purified in PBS together with ammonium sulfate (Kubota et al, manuscript in
preparation). K562 cells were grown in HamF12/DMEM to a
10% GIT medium for 48 hours at 37°C after preincubation of 100 µg of anti-2m MoAb with 10 µg of 2m
for 15 minutes at RT. The anti-2m MoAb (clone NK2) completely blocked both the suppression of cell proliferation (data not
shown) and the induction of apoptosis in K562 cells (Fig 4A) and coordinately blocked both the
suppression of cell proliferation (data not shown) and the induction of
apoptosis in CCRF-CEM cells (Fig 4B). The anti-2m MoAb
(NK2) inhibited any interaction with either biotinilated
2m or the cell surface of CCRF-CEM cells (Fig 4C). This
result suggests that 2m induces apoptosis through an
interaction with a receptor on the cell's surface.
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| Fig 4.
Inhibition of induction of apoptosis in K562 cells and in
CCRF-CEM cells by anti-2m MoAb. (A)
2m-induced apoptosis in K562 cells was suppressed by the
addition of 100 µg anti-2m MoAb derived from hybridoma
clone NK2. Anti-2m MoAbs (clone NK1 and NK3) were the
negative controls at the same concentration. (B)
2m-induced apoptosis in CCRF-CEM cells was suppressed by
preincubation with anti-2m MoAb (clone NK2). Apoptotic
cells were assayed by TUNEL. Data are expressed as the mean ± SE of 3 independent experiments. (C) The effects of anti-2m
MoAbs for the interaction of biotinilated 2m and the
cell surface on CCRF-CEM cells were analyzed using FACS (dotted line,
control; thin line, no additive of anti-2m MoAb; thick
line, additive of anti-2m MoAb).
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2m-induced apoptosis is mediated by
neither the FasL/Fas nor the TNF-/TNFR systems.
To investigate whether 2m-induced apoptosis is mediated
by the FasL/Fas or TNF-/TNFR systems, we examined the effect of an
anti-Fas neutralizing MoAb and an anti-TNFR neutralizing MoAb each at a
concentration of 1 µg/mL. Blocking Fas/FasL interaction by an
antagonistic anti-Fas MoAb did not interfere with
2m-induced apoptosis in either K562 or CCRF-CEM cells
(Fig 5A and B). Similarly, blocking
TNF-/TNFR interaction also did not interfere in either cell line
(Fig 5A and B). We propose that 2m-induced apoptosis is
dependent on neither the FasL/Fas system nor the TNF-/TNFR system.
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| Fig 5.
In K562 cells, apoptosis is induced, which is distinct
from the interaction of FasL/Fas, TNF-/TNFR, and IL-8/IL-8R. (A and
B) After 2 hours of preincubation with 1 µg of anti-Fas MoAb (IgG2b,
clone SM1/23) or 1 µg of anti-TNFR(p55) MoAb (IgG), K562 cells (A)
and CCRF-CEM cells (B) at a concentration of 1 × 105
cells/mL were exposed to 10 µg/mL 2m or at 37°C
for 48 hours. 2m-induced apoptosis in both cell lines
were prevented by neither anti-Fas MoAb nor anti-TNFR MoAb. (C and D)
The preincubations with each 5 µg/mL of anti-IL-8 MoAb and
anti-IL-8 receptor antibodies (receptor A [RA] and receptor B
[RB]) did not inhibit the induction of apoptosis in K562 cells (C)
and in CCRF-CEM cells (D) as described above. In addition, 10 ng/mL of
anti-Fas MoAb, 10 ng/mL of recombinant human TNF-, and 20 ng/mL of
endothelial IL-8 (data not shown) were used as apoptosis-inducing
factors in control studies. Apoptotic cells were assayed by TUNEL. Data
are expressed as the mean ± SE of 3 independent
experiments.
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2m-induced apoptosis is independent of
IL-8-induced apoptosis.
We previously reported that endothelial IL-8 is purified as an AIF from
PDBu-conditioned media of HL-60 cells and induces apoptosis in human
leukemic cell lines.40,41 To confirm that 2m
induces apoptosis in human leukemic cell lines distinct from the
pathway of IL-8-induced apoptosis, we examined the effect of an
anti-IL-8 neutralizing MoAb and 2 anti-IL-8 receptors neutralizing MoAbs, each at a concentration of 5 µg/mL. These 3 antibodies could
not inhibit 2m-induced apoptosis in either K562 (Fig 5C) or CCRF-CEM cells (Fig 5D). In addition, IL-8 was not detected in the
purified human urine-derived 2m by ELISA (data not shown).
2m induces apoptosis through the caspase
cascade.
Recent studies have proposed that various types of apoptosis, such as
Fas-mediated apoptosis, depend on the activation of caspases. To
investigate whether a caspase cascade participates in
2m-induced apoptosis, we studied the inhibition of
apoptosis through pretreatment with caspase inhibitors. Induction of
apoptosis in K562 cells was blocked by Ac-DEVD-CHO, Ac-YVAD-CHO, and
z-VAD-fmk 3 hours before adding the 2m
(Fig 6A). The reduction of
2m-induced apoptosis in K562 cells was efficient in the
presence of each of the 3 caspase inhibitors at concentrations of 10 µmol/L (data not shown). Ac-YVAD-CHO significantly inhibited
apoptosis at 100 µmol/L, achieving more than 90% inhibition.
Simultaneously, preincubation of CCRF-CEM cells with each of the 3 kinds of caspase inhibitors inhibited 2m-induced
apoptosis (Fig 6B). The reduction of apoptosis in CCRF-CEM cells from
our control was insufficient with concentrations of 10 µmol/L of each
inhibitor (data not shown), but effective with 100 µmol/L of each.
Z-VAD-fmk almost completely inhibited apoptosis at 100 µmol/L. We
concluded that 2m-induced apoptosis depends on the
activation of both ICE-like protease and CPP32-like protease.
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| Fig 6.
The effect of Ac-DEVD-CHO (CPP32/Apopain-like protease
inhibitor), Ac-YVAD-CHO (ICE-like protease inhibitor), and Z-VAD-FMK
(pancaspase inhibitor) on induction of apoptosis by 2m.
(A) K562 cells were preincubated for 3 hours in the presence of 3 caspase inhibitors separately, each at 100 µmol/L. After that, these
cells were exposed at 1 × 105 cells/mL to 10 µg/mL of
2m at 37°C for 48 hours. (B) CCRF-CEM cells were
similarly preincubated with the 3 caspase inhibitors separately and
exposed to 2m. Control cells were cultured with the
addition of 0.3% DMSO. Apoptotic cells were assayed by TUNEL. Data are
expressed as the mean ± SE of 3 independent experiments.
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Chemical cross-linking of 2m.
Addition of the amine-reactive, cross-linker, DSS, to Triton-X extracts
of K562 cell membranes resulted in the formation of high molecular
broad bands (its average complex showed a molecular weight [MW] of
150 kD) that were specifically inhibited by the addition of 100×
unlabeled human purified 2m
(Fig 7). Using anti-2m MoAb,
Western blot showed an approximately 150-kD 2m-binding protein complex (data not shown).
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| Fig 7.
DSS cross-linking of 2m on the K562
leukemia cell line. Broad high molecular mass complexes of 150 kD
(2m and its binding protein complex) were generated by
cross-linking. Several nonspecific bands were also stained; however, a
band of appropriately 150 kD was specifically suppressed by the
addition of 100× unlabeled 2m in lane 3. b2m, biotinilated 2m.
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| |
DISCUSSION |
MHC class I antigens, or HLA antigens, play an important role in tumor
immunity. Loss of HLA molecules in B-cell lymphomas is associated with
an aggressive clinical effect, such as lung cancer.16,17,46
In melanoma, whose tumor cells often lack HLA class I antigen
expression, distinct mutations in the 2m gene have been
identified.47 Antibodies against MHC class I antigen
complexes induced apoptosis in activated T cells and T-cell leukemia
cell lines. However, it is still not clear which epitope is responsible
for apoptosis and which ligand binds to the responsible molecule on HLA
class I antigens. We observed that human 2m itself induced apoptosis in various kinds of leukemic cells and
2m-enhanced antibody induced MHC complex-apoptosis in
the T-cell leukemia cell line CCRF-CEM. We used K562 cells as target
cells because they showed low background in MTT and TUNEL assay.
Although K562 cells have no MHC class I antigen complex (data not
shown),48,49 2m induced apoptosis even in
these cells. We strongly suggest that human 2m uses an
apoptosis pathway in both T cells and myeloid leukemia cells distinct
from the MHC class I antigen complex-induced apoptosis and Fas- or
TNFR-mediated apoptosis pathways. Our report is the first evidence
demonstrating the existence of human 2m-induced apoptosis. Neutralizing antibodies against 2m resulted
in 2m-induced apoptosis being completely blocked. So
far, we have not determined the epitopes that might be involved in cell
death signaling. Our cross-linking study suggested the presence of a MW
150-kD complex including presumably a binding protein and
2m. Altered expressions of those genes that control
apoptosis may lead to autoimmunity, malignant cell growth,
neurodegenerative diseases, prolonged survival of cells during latent
viral infections, and enhanced cell death during acquired
immunodeficiency syndrome (AIDS). For example, Fas-ligand and Fas
antigen, which control apoptosis and may lead to autoimmunity, are
known to be mutations of lpr and gld, respectively, in
mice.21,50-52 Christianson et al53
reported that the phenotype in 2m null mice was
predisposed to develop chronic lupus erythematosus and to
have defective antibody responses. Thus, in addition to the role of
lpr and gld in autoimmunity, gene knock-out technology showed the importance of the 2m gene in this disease.
Obviously, a defective mechanism in apoptosis leads to autoimmunity,
and 2m may be involved. As for the relationship between
FasL and Fas antigen, the binding protein for 2m will be
important. 2m-deficient and TAP-1-deficient mice show
self-tolerance of NK cells due to low expression levels of MHC class I
protein.54
TCR antigen-induced cell death occurs from the late G1
phase of the cell cycle studies55 and is dependent on pRb
(retinoblastoma).55 In the future, we will investigate
whether pRb or cdk-2 (cyclin-dependent kinase) activation is also
involved in our system.
Compared with the action of the Fas- or TNFR-dependent pathway, the
dose and time frame required for 2m-induced apoptosis is
large. 2m actions require a specific physiological
concentration measured in micrograms (which corresponds to serum
levels) to induce apoptosis.
In our system, 2m's binding protein in the apoptosis
pathway or its signaling pathway remains to be clarified. Ceramide, which is known as a second messenger, is produced by caspase-dependent apoptosis after activation by Fas or HLA class I antigen.56 In Fas- and HLA class I-mediated peripheral T-cell apoptosis, caspase-dependent ceramide production, which acts downstream of the
mitochondria, is important.32 Like the HLA class I-mediated pathway, our system also involves at least 2 different caspases. Table 1 shows the characterization of
2m-induced apoptosis and various types of HLA class
I-induced apoptosis. The presence of antibody for HLA class I antigen
induced more apoptosis in CCRF-CEM cells by 2m shown in
TUNEL assay (data not shown). A recent report suggests that
Fas-dependent apoptosis is dependent on ceramide.57
Using specific MoAbs against HLA class I, 2 domain, which binds to
the TCR, the results of several studies suggest that 2m induces Fas-independent cell death.58 This pathway does not involve ICE-like proteases or CPP32-like proteases.
The cell surface regulator Toso has been discovered to be a regulator
of Fas-induced apoptosis in T cells and blocks the Fas pathway.59 In 2m-induced apoptosis, the
molecule that blocks the 2m pathway has not yet been
identified. A recent study using primary embryonal fibroblasts from
transgenic mice expressing murine H-2 and a swine class I transgene,
transformed with the highly oncogenic Ad12, showed abnormal turnover of
2m and suggests the existence of a novel
2m-binding molecule that sequesters 2m.60 One or more specific regulators or
inhibitors for 2m-induced apoptosis remain to be
discovered. However, the signaling pathway is addressed in our system
to the extent that 2m-induced apoptosis is a distinct
pathway from the Fas-dependent or MHC class I-dependent and the
TNFR-dependent pathways of apoptosis.
| |
ACKNOWLEDGMENT |
The authors thank S. Kurokawa, K. Sato, and H. Ishikawa for technical
assistance and we appreciate Dr Y. Miura.
| |
FOOTNOTES |
Submitted September 3, 1998; accepted May 30, 1999.
Supported by a grant-in-aid from the Ministry of Education, Science and
Culture of Japan; Research on Advanced Medical Technology, from the
Ministry of Health and Welfare; the Japanese Foundation for
Multidisciplinary Treatment of Cancer; and Jichi Medical School Young
Investigator Award.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kiyohiko Hatake, MD, PhD, Associate
Professor, Department of Hematology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan;
e-mail:kiyohiko{at}jichi.ac.jp.
| |
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