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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3494-3504
Interferon- Prevents Apoptosis in Epstein-Barr Virus-Infected
Natural Killer Cell Leukemia in an Autocrine Fashion
By
Shin-ichi Mizuno,
Koichi Akashi,
Koichi Ohshima,
Hiromi Iwasaki,
Toshihiro Miyamoto,
Naoyuki Uchida,
Tsunefumi Shibuya,
Mine Harada,
Masahiro Kikuchi, and
Yoshiyuki Niho
From the First Department of Internal Medicine, Faculty of Medicine,
Kyushu University, Fukuoka, Japan; the Department of Pathology,
Stanford Univesity School of Medicine, Stanford, CA; and the First
Department of Pathology, Fukuoka University, Fukuoka, Japan.
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ABSTRACT |
The significant function of cytokines includes maintenance of cell
survival as well as induction of cell differentiation and/or proliferation. We demonstrate here that interferon- (IFN-) plays a role for progression of Epstein-Barr virus (EBV)-infected natural killer cell leukemia (NK leukemia) through maintaining cell survival. NK leukemia cells obtained from 7 patients had clonal episomal forms of
EBV, indicating that the leukemic cells were of clonal origin. Although
normal NK cells constitutively expressed Bcl-2, the EBV-infected NK
leukemia cells lacked endogenous Bcl-2 expression and were
hypersensitive to apoptosis in vitro. The addition of IFN- to the
culture significantly inhibited their spontaneous apoptosis without
inducing cell proliferation or upregulation of Bcl-2. The NK leukemia
cells constitutively secreted IFN-, and the patients' sera
contained a high concentration of IFN-, levels that were high enough
to prevent NK leukemia cells from apoptosis. Bcl-XL was not
involved in the IFN--induced NK leukemia cell survival. These data
suggest that the acquisition of IFN--mediated autocrine survival
signals, other than Bcl-2 or BCL-XL, might be important for
the development of EBV-infected NK leukemia.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE LYMPHOPROLIFERATIVE diseases of
granular lymphocytes (LDGL) are classified into CD3+ LDGL
and CD3 LDGL.1-3 The CD3+
LDGL has been shown to clonally rearrange T-cell receptor (TCR) genes,
indicating their clonal T-cell origin (T-LDGL). On the other hand, the
CD3 LDGL belongs to the natural killer (NK) cell
lineage (NK-LDGL); the surface antigens expressed in NK-LDGL cells
include NK cell-associated antigens such as CD16, CD56, or CD57.
The NK-LDGL has been further classified into two categories according
to clinical manifestations that are seen in specified endemic areas;
the NK-LDGL is mainly observed in Asia and New Zealand and is an
aggressive malignant disorder characterized by hepatosplenomegaly,
pancytopenia, and disseminated intravascular coagulation due to
systemic invasion of NK cells.4 On the other hand, NK-LDGL
is prevalent in Europe or United States and mainly exhibits indolent
chronic proliferation of NK cells (chronic NK lymphocytosis).5,6 Recently, several studies have
demonstrated that cells from the former, but not from the latter, type
of NK-LDGL frequently possesses Epstein-Barr virus (EBV) genomic DNA in
a single episomal form.7,8 Accordingly, the agressive
EBV-infected (EBV+) NK-LDGL are of clonal origin and can be
diagnosed as NK leukemia.
EBV is capable of immortalizing B lymphocytes and epithelial cells of
the nasopharynx, at least through inhibiting apoptosis of target
cells.9 In these cases, EBV nuclear antigen-2 (EBNA-2) induces expression of EBV-associated latent membrane protein
(LMP-1),10 and the LMP-1 transactivates bcl-2
gene11 to promote survival of the infected
cells.12 Enforced expression of EBNA-1 in B cells results
in B-cell lymphoma in mice, indicating their oncogenic capacity.13 Therefore, it is reasonable to postulate that
these EBV-related proteins are involved in transformation of NK cells or in reinforcement of NK leukemia cell survival.
NK leukemia is frequently associated with a systemic activation of the
reticuloendothelial system called hemophagocytic lymphohistiocytosis (HLH).14-16 Macrophages and histiocytes in HLH are
activated by macrophage-activating lymphokines such as interferon-
(IFN-) and tumor necrosis factor- (TNF-)17,18 that
are released from CD2+ lymphocytes (ie, T cells and/or NK
cells).19 Clonal proliferation of EBV-infected T cells has
been found in HLH associated with EBV infection,20
suggesting the potential role of EBV to induce production of cytokines
such as IFN- and TNF- in the EBV-infected T cells.21
NK cells are also one of the major sources of IFN- and can produce a
high amount of IFN- in response to interleukin-2 (IL-2), IL-12,
and/or IL-15 in vitro.22,23 The total amount of IFN-
released from NK cells reached a plateau within 3 days, because the NK
cells undergo rapid apoptotic cell death after stimulation in
vitro.23 This spontaneous apoptosis is induced at least by
the TNF- that is released from the activated NK cells themselves and
might serve to limit the immune response.23,24 These data
collectively suggest that the production of cytokines, including
IFN- and/or TNF-, is deregulated or that the cells have an
altered response to these cytokines in the EBV+ NK leukemia.
These data led us to analyze the expression of EBV-related proteins and
cytokines in the EBV+ NK leukemia. Unexpectedly, the
EBV+ NK leukemia cells had reduced levels of endogenous
Bcl-2 and spontaneously died in vitro. The EBV+ NK leukemia
cells constitutively produced high amounts of IFN-, and IFN-
prevented the spontaneous apoptosis of the EBV+ NK leukemia
cells without inducing cell proliferation. EBV-related proteins such as
LMP-1 and EBNA-2 do not appear to be involved in this process. We
propose that the acquisition of survival response to IFN- in an
autocrine fashion might be important for the progression of
EBV+ NK leukemia.
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MATERIALS AND METHODS |
Patients.
Seven patients with aggressive NK leukemia (cases no. 1 through 7) and
1 with chronic NK lymphocytosis (case no. 8) were enrolled in this
study (Table 1). NK leukemia cells bear a
morphology of large granular lymphocytes (Fig 1A) and
were positive for CD2 and CD56, but not CD16. Neither the TCR nor
the TCR gene was rearranged in any of the 8 cases (data not shown).
All 7 patients with NK leukemia presented with typical clinical
features such as hepatosplenomegaly and pancytopenia and died from
multiple organ failure within 6 months of diagnosis. There was
prominent phagocytosis of hematopoietic cells by marrow histiocytes and macrophages in 3 of the 7 patients with NK leukemia (cases no. 2, 6, and 7). In these 3 cases, the clinical variables met the criteria for
diagnosis as hemophagocytic lymphohistiocytosis.25 In case
no. 8, the diagnosis of chronic NK lymphocytosis was established by
persistent excess of blood NK cells according to the criteria proposed
by Tefferi et al.6
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| Fig 1.
Clonal origin of EBV-infected NK cell leukemia. (A) NK
leukemia cells in peripheral blood bear a morphology of large granular
lymphocytes (a) (May-Grünwald-Giemsa staining; original
magnification × 1,000); NK leukemia cells in peripheral blood (case
no. 1) (b) and in the liver (case no. 1) (c) expressed EBER1 RNA on in
situ hybridization, indicating that virtually all NK cells were
infected with EBV. (B) Southern blot analysis of DNA extracted
from NK-enriched PBMCs showed that each sample possessed a single
joined terminal sequences of the EBV genome (EcoRI digestion),
indicating their clonal origin from a single EBV-infected NK cell. The
numbers on the top of each lane correspond to case numbers listed in
Table 1. "C" is a positive control DNA from EBV-infected
lymphoepithelioma that contains a single episomal from of EBV.
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In all 8 patients, the serum EBV antibody titers showed that they had
been infected by EBV, positive for IgG-viral capsid antigen (VCA) and
EBV nuclear antigen, and negative for IgM-VCA. Antibodies to human
T-cell leukemia virus type I were not detected in any of the cases.
Preparation of NK leukemia cells.
Heparinized peripheral blood samples were taken from patients after
obtaining informed consent. Peripheral blood mononuclear cells (PBMCs)
were separated by centrifugation on a Ficoll-Hypaque density gradient
and incubated for 30 minutes in a plastic flask to remove monocytes,
and the nonadherent PBMCs were incubated with anti-CD3 and anti-CD19
mouse IgG monoclonal antibodies (MoAbs). The CD3+ T cells
and CD19+ B cells were removed by goat antimouse IgG-coated
magnetic beads (Dynabeads; Dynal, Oslo, Norway). More than 95% of NK
cell-enriched PBMCs were CD56+.
Phenotypic analysis and immunohistology.
Surface phenotypes was analyzed by flow cytometry using a FACScan
(Becton Dickinson, Mountain View, CA) or a highly modified FACS vantage
(Becton Dickinson).26 The MoAbs used were as follows: anti-CD2 (Leu5b), anti-CD3 (Leu4), anti-CD4 (leu3a), anti-CD5 (Leu1),
anti-CD7 (Leu9), anti-CD8 (Leu2a), anti-CD10 (CALLA), anti-CD19
(leu12), anti-CD20 (Leu16), anti-CD34 (HPCA), anti-CD16 (Leu11) and
anti-CD56 (leu19) were purchased from Becton Dickinson Imunocytometry
Systems; anti-CD13 (MY7), anti-CD14 (MY4), and anti-CD33 (MY9) were
from Coulter Immunology (Hialeah, FL); and anti-CD16 (OK-NK) was from
Ortho Pharmaceutical Corp (Raritan, NJ). For detection of
intracytoplasmic Bcl-2 and Bcl-XL proteins, cells were
fixed in 2% paraformaldehyde, permeabilized with 0.03% saponin, and
stained with either a fluorescein isothiocyanate (FITC)-labeled
anti-Bcl-2 (DAKO Japan Co Ltd, Kyoto, Japan) or a purified
anti-Bcl-XL MoAb27 (Transduction Laboratories,
Lexington, KY). An FITC-labeled goat antimouse IgG1 antibody (Caltag,
Burlingame, CA) was used to visualize Bcl-XL. Jurkat and
HL-60 human leukemia cell lines were used for positive and negative
controls for Bcl-XL staining, repectively. Cells are
stained for intracytoplasmic IFN- staining according to the method
reported by Buschle et al.28 Briefly, cells were incubated
with RPMI media with 10% fetal calf serum (FCS) and 2 µmol/L of
monensin for 3 hours and stained with an FITC-conjugated anti-IFN-
antibody (Pharmingen, San Diego, CA).
To detect EBV-related proteins, cytocentrifuged preparations of NK
cell-enriched PBMCs and frozen or paraffin-embedded tissue sections of
liver biopsy specimens were stained with antibodies specific for EBNA2
and LMP-1 (DAKO) antibodies and with an avidin-biotin-alkaline phosphatase complex.14 Mouse IgG1 was used as a negative control.
Detection of EBV by Southern hybridization and in situ
hybridization.
High molecular weight DNA was extracted from NK cell-enriched PBMCs and
was digested with restriction enzyme EcoRI and BamHI (Takara Shuzou, Kyoto, Japan). The digested DNA was electrophoresed and
transferred to nylon membranes. The presence of EBV-specific DNA
sequences was determined by using BamHI-W (Bam W) fragments of
EBV DNA (ENZO, Hudson, NY), and the polymorphic fused termini of the
EBV genome were analyzed by using a probe containing the tandem
terminal repeated (TR) sequences of EBV genome (kindly provided by Dr
K. Hirai, Department of Virology and Immunology, Tokyo Medical and
Dental University, Tokyo, Japan).14,29
In situ hybridization of EBV RNA was performed on the liver specimen or
cytospin preparations using a 30-base oligonucleotide complementary to
a portion of EBV-encoded small nonpolyadenylated RNAs (EBER1)-specific
fragments, according to the method by Chang et al.30 The
sequence of the oligonucleotide was
5'-AGACACCGTCCTCACCACCCGGGACTTGTA-3'.
Cell culture.
Purified NK leukemia cells (106/mL) were cultured in
RPMI1640 (Flow Laboratories, Irvine, CA) containing 10%
heat-inactivated FCS at 37°C in 5% CO2. The following
cytokines were used: 10 U/mL IL-2 (Shionogi & Co, Osaka, Japan); 100 ng/mL IL-4 (Ono Pharmaceutical Co. Ltd., Osaka, Japan); 100 U/mL
IL-1, 50 ng/mL IL-6, 100 ng/mL stem cell factor (SCF),
and 10 to 1,000 U/mL IFN- (Kirin Brewery Co, Tokyo, Japan); and 10 to 100 ng/mL of IL-12 (Genzyme Co, Cambridge, MA). A neutralizing
anti-IFN- MoAb (Genzyme) was used at 200 ng/mL. At this
concentration, the antibody can neutralize 200 U/mL of IFN-.
Detection of viability, apoptosis, and proliferation.
Cell viability was assessed using an MTT assay31 as well as
a conventional trypan blue dye exclusion test. Briefly, 20 µL of 1 mg/mL MTT solution (Sigma Chemical Co, Ltd, Poole, UK) was added to
200-µL microcultures in a flat-bottomed 96-well microtiter plate, and
the plate was incubated for 4 hours. The formazan crystals that formed
were dissolved in 100 µL acid/alcohol (0.04 N HCl in isopropanol).
The optical density (OD) was measured with a dual-beam multiplate
reader (Titertek Multiscan, MCC; Flow Laboratories) using test and
reference wave lengths of 540 and 620 nm, respectively. Cell viability
was determined by calculating the change in the absorbance of the wells.
Apoptotic cells were identified by both light and electron microscopy.
Cytocentrifuged cells were stained with May-Grünwald-Giemsa stain, and cells were scored as apoptotic if they had condensation of
cytoplasm and chromatins and cytoplasmic fragmentation. In some cases,
the apoptotic cell death was confirmed on an electronmicroscopy. Fragmentation of DNA was also determined as reported
previously.32 DNA synthesis of NK leukemia cells was
evaluated by measuring [3H]thymidine([3H]TdR)
incorporation.33
Measurement of cytokines.
The concentrations of cytokines were measured in serum samples obtained
from all patients and in cultured supernatants of NK leukemia cells
harvested after plating at a concentration of 106 cells/mL
and incubating for 24 hours at 37°C with 5% CO2. The concentrations of IFN-, IL-2, and TNF- were measured by using radioimmunoassay kits that could detect human IFN-, IL-2, and TNF- (Ire-Medgenix, Fleurus, Belgium), respectively. IL-12 p70 was
measured by using PREDICTA IL-12p70 enzyme-linked immunosorbent assay
(ELISA) kit (Genzyme). These assay systems could detect concentrations
as low as 0.1 U/mL IFN-, 0.1 U/mL IL-2, 5 pg/mL TNF-, and 2 pg/mL
IL-12.
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RESULTS |
EBV+ NK leukemia cells are of clonal origin.
The Bam W sequence of EBV was detected in all 7 cases of NK leukemia
(cases no. 1 through 7) by Southern blot analysis, but not in case no.
8, the patient with chronic NK lymphocytosis
(Table 2). The liver specimens showed a
diffuse aggressive infiltration of large granular lymphocytes in all 3 cases studied (cases no. 1, 2, and 4). In situ hybridization analysis
to detect EBER1 RNA showed that virtually all NK leukemia cells
expressed EBER1 in all 5 cases studied (Table 2 and Fig 1A). Southern
blot analysis of the joined terminal repeats of the EBV genome
contained in the NK cell-enriched PBMCs showed a single band by
EcoRI digestion in all 7 cases (Fig 1B), indicating that NK
leukemia cells contained a single episomal form of EBV. These data
indicate that EBV+ NK leukemia cells were originated from a
single EBV-infected NK cell in all 7 cases.
To determine the possible role of EBV-related proteins, we analyzed the
expression of these proteins by immunohistochemical stainings. The
EBV+ NK leukemia cells did not express LMP-1 and EBNA-2 in
all 5 cases studied (Table 2).
EBV+ NK leukemia cells lack endogenous Bcl-2
expression.
Because the infection of EBV has been known to induce Bcl-2 in B
cells,12 we evaluated the cytoplasmic Bcl-2 expression in
the EBV+ NK leukemia cells. Normal NK cells constitutively
expressed Bcl-2 at levels that are detectable upon immunohistochemical
and flow cytometric analyses
(Fig 2), as reported
previously.34 NK cells from 1 patient with chronic NK
lymphocytosis (case no. 8) also expressed Bcl-2. However, unexpectedly,
cytoplasmic Bcl-2 was undetectable in all EBV+ NK leukemia
cases; circulating NK leukemia cells did not express detectable levels
of Bcl-2 on both flow cytometric and immunohistochemical assays in all
7 cases (Fig 2 and Table 2). Bcl-2 could also not be detected in the NK
leukemia cells that infiltrated into the liver in all 3 cases studied
(Table 2). Bcl-XL was undetectable by a flow cytometry in
normal controls as well as in all 4 cases with NK leukemia studied
(cases no. 1, 2, 4, and 5; data not shown). These data indicate that
the expression of Bcl-2 is impaired in the EBV+ NK leukemia
cells.
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| Fig 2.
Impaired expression of endogenous Bcl-2 in the
EBV-infected NK leukemia cells. Analysis of Bcl-2 expression in either
normal NK cells, NK leukemia cells, or NK cells from chronic NK
lymphocytosis on immunohistochemical (upper panels) and flow cytometric
(lower panels) analyses. On both assays, endogenous Bcl-2 was
undetectable in NK leukemia cells, although NK cells from normal
controls and a case of chronic NK lymphocytosis constitutively
expressed Bcl-2. On the immunohistochemical method, Bcl-2 is visualized
as diffuse red staining in cytoplasm in normal NK cells and chronic NK
lymphocytosis (upper left and right panels). Results are summarized in
Table 2.
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EBV+ NK leukemia cells are hypersensitive to
apoptosis in vitro.
The absence of Bcl-2 in the NK leukemia cells strongly suggests that
these cells might be sensitive to apoptosis. To evaluate this, we
cultured the cells in vitro and over 3 days determined the percentages
of viable cells as well as apoptotic cells. As shown in
Fig 3A, NK leukemia cells rapidly lost
their viability as determined by a trypan blue dye exclusion test;
approximately 40% of cells died as early as 24 hours after incubation
and approximately 60% of cells died after 48 hours. The spontaneous
apoptotic death of NK leukemia cells was most prominent in the first 24 hours, but almost reached its plateau after 48 hours. The decrease in viable NK leukemia cells during the culture was correlated with the
gradual increases in the apoptotic cells (Fig 3B) that were characterized by condensed and fragmented nuclei and a loss of cell
volume (Fig 4A). The mean percentage of
viable NK leukemia cells evaluated 48 hours after the initiation of
culture by a trypan blue dye exclusion test (42%) was almost equal to
that of viability determined on an independent MTT assay (44%;
Fig 5). The DNA also became more fragmented
during the culture of NK leukemia cells (Fig 4B). In contrast, greater
than 95% of NK cells from 6 normal donors were viable and did not
undergo apoptosis even 72 hours after the initiation of culture (Fig
3). NK cells from 1 patient with chronic NK lymphocytosis (case no. 8)
also did not undergo significant apoptosis during the culture (data not
shown).
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| Fig 3.
Sequential analysis of spontaneous apoptosis of
EBV-infected NK leukemia cells in vitro. NK leukemia cells (NKL;  )
from cases no. 1 through 7 and normal NK cells from 6 normal controls
( ) were cultured in vitro, and percentages of viable cells (A) and
percentages of apoptotic cells (B) were sequentially determined on a
trypan blue dye exclusion test and on morphology under an microscope,
respectively. The NK leukemia cells underwent apoptotic cell death
immediately after initiation of the culture. Note that apoptotic cell
death reached a plateau approximately 48 hours after initiation of the
culture. The addition of IFN- (100 U/mL) to the culture
significantly inhibited the progression of spontaneous apoptosis of NK
leukemia cells on both tests (NKL+IFN;  ). Data are shown as the
mean ± SD (error bars).
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| Fig 4.
Evidences of apoptotic cell death in
EBV-infected NK leukemia cells. (A) Apoptotic NK leukemia
cells 48 hours after the in vitro culture. Morphology of the apoptotic
cells with May-Grünwald-Giemsa staining (original magnification × 1,000) (a) and with an electron microscopy (original magnification × 15,000) (b). (B) Fragmentation of DNA from NK leukemia cells (case
no. 1) cultured in vitro. DNA ladder gradually became evident during
the cultures in the absence of cytokines. The addition of either
IFN- or IL-2 significantly inhibited the amounts of DNA
fragmentation.
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| Fig 5.
Effects of IFN-, IL-2, and IL-12 on viability of
EBV-infected NK leukemia cells. Cells (106/mL) were
cultured in the presence or absence of either IFN- (10 and 100 U/mL), IL-2 (10 U/mL), or IL-12 (10 ng/mL), and viability of cells was
determined on MTT assay 48 hours after the initiation of cultures. Data
are shown as percentages of viability determined by OD after the
culture/OD before the culture. Numbers in open circles correspond to
case numbers in Table 1. (A) The addition of IFN- to the culture
significantly increased viablity of NK leukemia cells in a
dose-dependent manner. The antiapoptotic effects of 1,000 U/mL of
IFN- did not significantly differ from those of 100 U/mL of IFN-
(data not shown). Percentages of viability reached more than 100% in
some cases cultured with IL-2, which was due to the cell proliferation
during the culture with IL-2, whereas IFN- did not stimulate
proliferation of NK leukemia cells (see text). *A significant
difference between the groups (P < .05) determined on
Wilcoxon signed-rank tests. (B) The addition of IL-12 inhibited
spontaneous apoptosis of NK leukemia cells in only 1 of 4 cases
studied. *A significant difference (P < .05) determined on
Student's t-tests.
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EBV+ NK leukemia cells avoid apoptotic cell
death by constitutive secretion of IFN-.
Various cytokines such as IFN-, TNF-, and IL-2 are involved in
the activation and subsequent spontaneous apoptotic cell death in
normal NK cells.23 We evaluated the effect of IFN- and
IL-2 on the spontaneous apoptotic cell death of NK leukemia cells in
vitro. As shown in Fig 3, the addition of 100 U/mL of IFN- in the
culture resulted in the significant increase in percentages of viable
cells and the significant decrease in the percentages of apoptotic
cells, evaluated by a tripan-blue exclusion test and a morphology-based
apoptotic cell determination, respectively. The spontaneous DNA
fragmentation was inhibited by the addition of either IL-2 (10 U/mL) or
IFN- (100 U/mL; Fig 4B). Furthermore, the evaluation of viability of
the NK leukemia cells on an MTT assay also showed that the addition of
either IFN- (10 to 1,000 U/mL) or IL-2 (10 U/mL) in these cultures
significantly promoted their viability in both 24-hour (data not shown)
and 48-hour cultures (Fig 5A). This survival-promoting effect of
IFN- on NK leukemia cells was dose-dependent (Fig 5A) and reached
its plateau at 100 U/mL. IFN- did not induce cell proliferation in
vitro, because the absolute numbers of cells remained unchanged, and
3H-thymidine was not incorporated during the cultures in
all 7 cases cut (Table 3). In contrast,
IL-2 induced proliferation of NK leukemia cells as well as cell
survival, because numbers of cells significantly increased after the
48-hour culture, and the 3H-thymidine was significantly
incorporated during the culture (Table 3).
Figure 6 shows the production of these
cytokines from the NK leukemia cells. Significant production of IFN-
was seen in the 24-hour culture supernatants of the NK leukemia cells
in all cases (2.0 to 18 U/mL; Fig 6A), whereas neither TNF- nor IL-2
was detectable (data not shown). Intracytoplasmic stainings of
cytokines showed that a majority of NK leukemia cells possessed
intracytoplasmic IFN- (Fig 6B), but not IL-2 in all 4 cases studied
(cases no. 1, 2, 4, and 5). However, IL-2 further promoted the
production of IFN- from NK leukemia cells in vitro in all 3 cases
studied (cases no. 1, 2, and 5; Table 4), suggesting that IL-2
maintained cell survival at least by inducing high amounts of IFN-
from NK leukemia cells themselves.
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| Fig 6.
Constitutive expression of IFN- in the EBV-infected NK
leukemia cells. (A) Concentrations of IFN- in 24-hour culture
supernatants of NK leukemia cells. The culture supernatants of NK
leukemia cells (open circles 1 through 7) contained significant levels
of IFN-, whereas those of normal NK cells did not (). (B)
Intracytoplasmic staining of IFN- in the NK leukemia cells on flow
cytometric analysis. The cells were incubated with monensin (2 µmol/L) for 3 hours before the analysis. The majority of NK leukemia
cells expressed intracytoplasmic IFN- protein. (C) Concentrations of
IFN- in patients' sera. Significant levels of serum IFN- were
seen in all NK leukemia patients (), whereas in normal controls
(), levels of IFN- were below detectable levels (<0.1 U/mL).
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In contrast, NK cell-enriched PBMCs from normal controls do not secrete
detectable levels of either IFN- or IL-2. These antiapoptotic effects on NK leukemia cells were not observed in other cytokines, including IL-1, IL-4, IL-6, and SCF (data not shown).
Because 24 hours after initiation of culture the NK cell leukemia cells
secreted high enough levels of IFN- to prevent apoptosis, the
apoptosis of the cells in vitro might be at least partially prevented
by the IFN- released from NK leukemia cells themselves. To confirm
this, we added neutralizing anti-IFN- antibodies to the culture. As
expected, in all 3 cases studied (cases no. 1, 2, and 5), the
anti-IFN- antibodies significantly promoted the spontaneous
apoptosis of NK leukemia cells, resulting in significant decreases in
OD value on MTT assays evaluated 24 hours after initiation of the
culture (Fig 7).
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| Fig 7.
Neutralization of IFN- in culture media accelerates
spontaneous apoptosis of EBV-infected NK leukemia cells in vitro. Data
are shown as mean OD values on MTT assay 24 hours after the culture
that correlate with viability of the cells. In all 3 cases studied, the
addition of neutralizing anti-IFN- antibodies to the culture media
resulted in an accelerated loss of viability of NK leukemia cells in
vitro. *A significant difference between the groups (P < .05)
determined on Student's t-tests.
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IL-12 is not involved in the IFN--mediated autocrine
survival loop of NK leukemia cells.
We were interested in whether IL-12 is involved in the
IFN--mediated autocrine survival loop of NK leukemia cells, because IL-12 is known to be a potent inducer of IFN- in normal NK cells. Figure 5B and Table 4 show the effect of
IL-12 on the NK leukemia cell survival and on the production of IFN-
or IL-2 from NK leukemia cells, respectively. In only 1 of 4 cases
studied (case no. 1), the addition of IL-12 to the culture
significantly promoted the survival of NK leukemia cells (Fig 5B).
IL-12 also stimulated IFN- production from NK leukemia cells only in
case no. 1, but not in the other 2 cases studied (Table 4).
Furthermore, NK leukemia cells did not produce detectable levels of
IL-12 in the absence or presence of either IL-2 or IFN- in all 3 cases studied including the case no. 1 (Table 4). Accordingly, although
responsiveness to IL-12 (to produce IFN-) might be preserved in some
cases of NK leukemia, IL-12 does not play a role in the
IFN--mediated autocrine survival loop of NK leukemia cells.
The sera from patients with EBV+ NK leukemia
contains high concentrations of IFN-, but not IL-2 or
IL-12.
Because IFN- maintains survival of NK leukemia cells in an autocrine
fashion in vitro, we measured concentrations of IFN- in NK leukemia
patients' sera to test whether serum IFN- prevent circulating NK
leukemic cells from apoptosis in vivo. As shown in Fig 6C, all 7 NK
leukemia sera showed an increased levels of IFN- (2.8 to 620 U/mL;
mean, 156.2 U/mL). On the other hand, IFN- was undetectable in sera
from 5 healthy volunteers and 1 patient from chronic NK lymphocytosis
(case no. 8). Neither IL-2, IL-12, nor TNF- was detectable in all
sera from EBV+ NK leukemia cases as well as those from
normal controls.
Antiapoptotic effect of IFN- on
EBV+ NK leukemia cells does not involve
upregulation of either Bcl-2 or Bcl-XL.
Some cytokines have been shown to directly upregulate endogenous Bcl-2
to maintain survival of target cells; for example, we have shown that a
principal role of IL-7 for T-cell development is to maintain cell
survival through upregulating (at least) Bcl-2.26 To
clarify a mechanism of survival-promoting effect of IFN- on the
EBV+ NK leukemia cells, we evaluated intracytoplasmic Bcl-2
and Bcl-XL levels after 24 hours of incubation with either
IFN- (100 U/mL) or IL-2 (10 U/mL) by flow cytometry. Although both
cytokines could block the spontaneous apoptosis of NK leukemia cells,
both Bcl-2 and Bcl-XL remained undetectable in the
IFN-- or IL-2-treated NK leukemia cells in all 4 cases studied
(cases no. 1, 2, 4, and 5; data not shown). Accordingly, the
survival-promoting effects of IFN- and IL-2 on EBV+ NK
leukemia cells might be independent of either Bcl-2 or
Bcl-XL.
| |
DISCUSSION |
NK cells are innate immune effector cells. The production of IFN-
from NK cells plays a pivotal role for the initiation of an immune
response to various infections through activating monocytes to
eliminate pathogens. However, this immune response of NK cells is
self-limiting; NK cells undergo apoptotic cell death approximately 3 days after activation, probably through producing apoptosis-inducing cytokines (ie, TNF-) in an autocrine fashion.23,24 In
the present report, we demonstrated that this self-limited process of
activation of NK cells is disrupted in the EBV+ NK leukemia cells.
The EBV+ NK leukemia cells were hypersensitive to apoptosis
when they were transferred into liquid culture. The majority of NK
leukemia cells constitutively secreted IFN-. These features of NK
leukemia cells closely resemble those of normal NK cells activated by
IL-2 and IL-12.23 However, in NK leukemia, the addition of
IFN- at the initiation of liquid cultures significantly inhibited
the spontaneous apoptosis. The EBV+ NK leukemia cells
gradually ceased to undergo apoptosis in the liquid culture without
adding IFN-, because the relative numbers of apoptotic cells reached
the plateau after 48 hours. This probably results from the accumulation
of IFN- released from the NK leukemia cells, because concentrations
of IFN- in the 24-hour culture supernatant had already reached
levels that protected NK leukemia cells from apoptosis, and
neutralization of the secreted IFN- significantly promoted the
apoptosis of NK leukemia cells in vitro. The NK leukemia cells probably
receive this survival signal from IFN- in vivo, because the
concentration of IFN- in patients' sera raise to the effective
levels. Neither IL-2, IL-12, nor TNF- was detectable in either
culture supernatants or patients' sera. Thus, the NK leukemia cells
might become responsive to IFN- to maintain cell survival through
the transformation process, resulting in the acquisition of an
autocrine loop for survival. IL-12 seems not to be involved in the
autocrine loop. It is possible that lack of TNF--mediated
apoptosis-inducing signals23,24 may augment the
survival-promoting effect of IFN-.
Another significant intracellular change in NK leukemia cells is an
impaired expression of the survival protein, Bcl-2.11 This
might account for the hypersensitivity to apoptosis of the NK leukemia
cells. Because normal NK cells do not downregulate Bcl-2 after they are
activated by IL-2 and IL-12,23 the reduction of Bcl-2
expression in the NK leukemia cells might result from EBV-related
transforming events. The aquisition of the autocrine loop via IFN-
might be required to compensate the loss of Bcl-2 in NK leukemia cells.
Bcl-XL was not detected in normal NK cells or NK leukemia
cells, and neither IFN- nor IL-2 could induce Bcl-XL in
both populations. Therefore, the survival-promoting effect of IFN-
on the NK leukemia cells is independent of Bcl-2 or Bcl-XL.
The impairment of Bcl-2 expression and the autocrine survival-promoting
loop via IFN- are exclusively seen in EBV+ NK leukemia,
but not in EBV chronic NK lymphocytosis or normal NK
cells. Furthermore, the aggressive clinical course in EBV+
NK leukemia markedly differs from the relatively indolent clinical features in EBV chronic NK
lymphocytosis.6 Accordingly, it is reasonable to assume
that EBV may cause alterations of intracellular events specific for
EBV+ NK leukemia, such as constitutive expression of
IFN-, loss of Bcl-2 expression, and IFN--mediated survival
response, and that the acquisition of these alterations might lead to
aggressive phenotypes in EBV+ NK leukemia. It is curious
that hypersensitivity to apoptosis with loss of Bcl-2 was seen in
EBV+ NK leukemia cells; EBV-infection in Hodgkin's
lymphoma cells and B cells has been shown to induce bcl-212
and/or bcl-2 homologue, BHRF-1,35,36 to protect the cells
from apoptosis. Because EBNA-2 and LMP-1 were undetectable in
EBV+ NK leukemia cells, it appears that these EBV-related
proteins do not have anything to do with the prolonged survival of
EBV+ NK leukemia cells in response to IFN-. This was not
unusual, because both EBNA-2 and LMP-1 are largely undetectable in
other EBV-related neoplasms such as Burkitt's lymphoma, nasopharyngeal carcinoma,37 and T-cell neoplasms.38
Other similarities between EBV+ NK leukemia and Burkitt's
lymphoma are that both are hypersensitive to apoptotic cell
death39 and have an impaired expression of
Bcl-2.40 Because EBNA-1 is reportedly expressed in
Burkitt's lymphoma,41 further studies of other EBV-related
proteins or genes, including EBNA-1, concerning the regulation of Bcl-2
and/or IFN- expression and the modulation of downstream events of
IFN- receptors are warranted in Burkitt's lymphoma as well as
EBV+ NK leukemia.
SCF has been shown to maintain survival of NK cells through
upregulating Bcl-2.42 However, SCF is not involved in the
maintenance of NK leukemia cells, because the NK leukemia cells did not
express the SCF receptor, c-Kit, and the spontaneous apoptosis was not inhibited in the presence of SCF in vitro. IL-2 could induce both proliferation and survival of NK leukemia cells. The survival-promoting effect of IL-2 on NK leukemia cells is independent of Bcl-2 or Bcl-XL and might be mediated by IFN- released from
proliferating NK leukemia cells, as shown in vitro (Table 4). Although
IL-2 was not detected in either culture supernatants or patients'
sera, it is possible that localized production of IL-2 from cells other than NK leukemia cells, ie, normal T cells, plays an important role for
the stimulation of proliferation of the NK leukemia cells at the sites
of invasion such as liver and spleen. Other NK cell-stimulating cytokines, including IL-12 and IL-15, may be involved in the
proliferation of NK leukemia cells, because activated monocytes can
produce IL-1243 and IL-15.22 However, our study
showed that NK leukemia cells retain responsiveness to IL-12 in only a
minority of cases.
To clarify the oncogenesis of EBV to NK cell lineage, the initial
target cells of EBV infection in EBV+ NK leukemia is needed
to be determined. Although CD21, a receptor for the EBV envelope
protein, was reportedly expressed in a EBV+ NK cell line
established from lymphoblastic lymphoma44 and a case of
EBV+ nasal lymphoma with an activated NK cell
phenotype,45 de novo EBV+ NK leukemia cells
have not been demonstrated to express CD21.7 In agreement
with previous reports, we could not detect CD21 expression in all 3 cases analyzed (data not shown). It is of interest to examine
susceptibility to EBV infection in NK cell progenitors, including
lymphoid-restricted common lymphoid progenitors46,47 as
well as a minority of mature NK cells that reportedly express CD21.45
The autocrine loop for maintenance of cell survival in EBV+
NK leukemia demonstrated here may not directly connect the oncogenesis of the disease, but might be related to its aggressive phenotype. The
maintenance of cell survival is one of the important functions of
cytokines,26,48,49 and the conversion into aggressive
phenotypes or the accumulation of malignant cells could result from the
acquisition of prolonged cell survival in some hematological
malignancies.50-52 In the case of EBV+ NK
leukemia, although malignant cells lack endogenous Bcl-2 expression and
are susceptible apoptotic cell death, the cells maintain survival by a
positive autocrine loop via IFN-. The high levels of IFN- released from NK leukemia cells might also contribute to activation of
macrophage and histiocytes, triggering the occurrence of fatal HLH.19,45 Therefore, this study demonstrates that clinical maneuvers directed at inhibition of IFN- in vivo may be one of the
potential therapeutic strategies for patients with this disease. It is
important to evaluate the role of responsible survival proteins other
than Bcl-2 and Bcl-XL, such as Bcl-X53 on
the IFN--mediated survival loop in NK leukemia cells. The
participation of EBV-related genes for the oncogenesis of
EBV+ NK leukemia remains unclear. Understanding the effect
of EBV-related genes other than LMP-1 and EBNA-2 to NK cells on the
regulation of Bcl-2 and IFN- expression and/or on the modulation of
the IFN- signaling pathway will help to clarify developmental
mechanisms of EBV+ NK leukemia.
| |
ACKNOWLEDGMENT |
The authors are indebted to Drs Akazawa Kouhei and Naoko Kinukawa
(Department of Medical Informatics, Faculty of Medicine, Kyushu
University) for statistical analysis of the data. We thank Drs T. Okamura, Y. Takamatsu, T. Eto, Y. Ohno, and H. Gondo for providing
samples of NK leukemia cells and Dr A. Schlageter for critically
reviewing this manuscript.
| |
FOOTNOTES |
Submitted June 5, 1998; accepted January 8, 1999.
Supported by a Grant-in-aid from the Ministry of Education, Science and
Culture and in part by the Jose Carreras International Leukemia
Foundation to K.A.
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 Koichi Akashi, MD, Departments of
Pathology, B-261 Beckman Center, Stanford Univeristy School of
Medicine, Stanford, CA 94305; e-mail: Akashi{at}Darwin.Stanford.edu.
| |
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ABSTRACT
INTRODUCTION