From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Inverse Expression of bcl-2 Protein and Fas Antigen in Lymphoblasts
in Peripheral Lymph Nodes and Activated Peripheral
Blood T and B Lymphocytes
By Tadashi Yoshino, Eisaku Kondo, Liu Cao, Kiyoshi Takahashi, Kazuhiko Hayashi,
Shintaro Nomura, and Tadaatsu Akagi
Toexamine the regulatorymechanismofapoptosisin
bcl-2 protein and less Far antigen. This inverse expression
lymphoid cells, expression both
of bcl-2 protein and
Fas antiof bcl-2protein and Far antigen was also shown
in activated
gen was investigated in reactivelymphnodes, in resting
from
peripheral
blood.
These
T and B lymphocytes
(PBLs), and inPBLs stimlymphocytes from peripheral blood
lymphoblasts fell into apoptosisdose-dependently in the
ulated with pokeweed mitogen, interleukin-4 (IL-4) + antipresence of anti-Fas monoclonal antibody, but resting lymIgM antibody, IL-2 + anti-CD3 antibody, phytohemagglutinin phocytes that expressed both bcl-2 protein and Far antigen
+ phorbol myristate acetate using immunohistochemistry
did not undergo apoptosis. These findings suggest
that beland flow cytometry. Germinal center cells expressed a large2 expression prevents the apoptosis of lymphoid cells inamount of Fas antigen, which is associatedwith the inducduced by tha Fas antigen-dependent mechanism and that
tion of apoptosis in lymphoid cell lines, in contrast to the
apoptosis of lymphocytes is exquisitely controlled, at least
lack of bcl-2 protein as an apoptosis inhibitor. On the other
in part, by regulationof the bcl-2 and Far genes.
hand, mantle zone lymphocytes expressed a high level of
0 1994 by The American Society of Hematology.
P
ROGRAMMED CELL DEATH or apoptosis plays an
important role in the physiologic turnover of normal
cells, in morphogenisis in embryonic development, and in
metamorphosis.’.’ In the immune system, immature thymic
T cells and B blasts in germinal centers undergo apoptosis
in response to antigenic and other stimuli, including glucocorticoids and irradiati~n.~.~
Anti-Fas monoclonal antibody MOA^)^ and anti- APO- 1
MoAb6 reportedly induce apoptotic cell death in sensitive
cell lines in vitro. Molecular cloning of these apoptosisinducing regulators has shown that Fas antigen and APO-l
antigen are identical and belong to the tumor necrosis factor
receptor (TNFR)/nerve growth factor receptor (NGFR) family.’ The bcl-2 protein, which was originally implicated in Bcell lymphomagenesis through its activation by the t( 14; 18)
chromosomal translocation, prevents apoptosis in hematopoietic cells (reviewed by Kor~meyer’.~).
We” and another group” have shown that germinal center
cells (GCCs) in the lymph node lack bcl-2 protein, despite
the expression of abundant bcl-2 transcripts. The regulatory
mechanism of expression of these two proteins in normal
lymphoid cells remains unclear.
Here, we studied Fas antigen expression in GCCs and in
peripheral blood lymphocytes (PBLs) stimulated with mitogen andother stimulators. In particular, bcl-2 protein expression was investigated, and we showed the inverse expression
of bcl-2 protein and Fas antigen in GCCs in reactive lymph
nodes and lymphoblasts. Both bcl-2 protein and Fas antigen
From the Department of Pathology, Okayama University School
of Medicine, Okayama; and the Department of Pathology, Osaka
University School of Medicine, Suita, Japan.
Submitted July 12, 1993; accepted November 23, 1993.
Address reprint requests to Tadashi Yoshino, MD, Department of
Pathology, Okayama University School of Medicine, Shikata-cho 25-1. Okayama, 700,Japan.
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.
0 1994 by The American Society of Hematology.
0006-4971/94/8307-0017$3.00/0
1856
may participate in clonal deletion and selection of lymphocytes through apoptosis in a reverse fashion.
MATERIALS AND METHODS
Samples. Lymph nodes were surgically removed from patients
with reactive lymphoid hyperplasia. PBLs from healthy donors were
separated by Ficoll-Hypaque density gradient (Sigma, St Louis, MO)
and cultured in RPMI 1640 supplemented with10% fetal bovine
serum (GIBCO, Gaithersburg, MD). To get B-cell-rich and T-cellrich fractions, B-cell-rich fractions were obtained using neuraminidase-pretreated sheep red blood cell (RBC) depleting T cells (<2%)
and plate adherence depleting of monocytes (<2%) from PBLs, and
T cells were purified from peripheral blood mononuclear cells with
RBC-rosettes formation (>95%). Lymphoblasts were cultured in
RPMI 1640 supplemented with 10% bovine serum and 0.05% pokeweed mitogen (PWM; GIBCO) for 72 hours. The activated B cells
were obtained by the administration of 4 ng/mL interleukin-4 (IL4;Genzyme, Boston, MA) and 4 pg/mL anti-IgM antibody (DAKO,
Copenhagen, Denmark) for 3 days or of PWM to the B-cell fraction.
The activated T cells were obtained by the administration of IL-2
(Shionogi Phamacy, Tokyo, Japan) and 5 pg/mL anti-CD3 antibody
(DAKO), administration of 1 pg/mL phytohemagglutinin (PHA) and
20 ng/mL phorbol myristate acetate (PMA) for 3 days, or administrationof PWM to the T-cell fraction. Flow cytofluorometry using
propidium iodide staining confirmed the induction of proliferation
in the activated B- and T-cell preparations.
MoAb. The anti-Fas mouse MoAb, obtained from MBL (Nagoya, Japan), was established by Yonehara et al.’ Anti-bcl-2 protein
MoAb, designated Bcl-2 124a. was obtained from DAKO. AntiCD3 and CD19 MoAbs conjugated with fluorescein isothiocyanate
(FITC) were purchased from Becton Dickinson (Mountain View,
CA). MOPC 21 and TEK 183 for IgGl and IgM class match control
were obtained from Sigma.
Immunofuorescence. Indirect immunofluorescence was
performed on frozen tissue sections fixed with acetone and on cytosmears of PWM-treated or untreated PBLs. Samples were stained
with Bcl-2 124a MoAb at a dilution of 1:40 and with anti-Fas MoAb
at a concentration of 10 pg/mL for 60 minutes at room temperature.
As a second antibody, FITC-conjugated goat antimouse IgG ( y chain-specific; Zymed, South San Francisco, CA) and biotin-conjugated goat antimouse IgM (p-chain-specific) were used at a 1:20
dilution. After washing, phycoerythrin (PE)-streptavidin (Vector
Lab, Inc, Burlingame, CA) was further applied at a 1:lOO dilution
to biotin-labeled specimens. Some samples were double-stained with
anti-Fas MoAb (PE) and anti-CD3 or anti-CD19 MoAb (FITC).
Blood, Vol 83,No 7 (April l), 1994:pp 1856-1861
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1857
bcl-2 AND Fas IN LYMPHOBLASTS
Fig 1. (A)Doubleimmunofluorescence of reactive lymph node
hyperplasia using anti-Fasantigen MoAb (PE1and Bel-2 124a IFITC)
is shown. Most GCCs and a few scattered interfollicular blastic cells
reacted intensely with anti-Fas MoAb. The mantle zone and most
mature interfollicular cells were very faintly stained or negative.Inversely, anti-bcl-2 MoAb reacted mainly with the mantle zone and
interfollicular cells and not with GCCs. (B) Double immunofluorescence of PWM-treated PBL is shown. Blastic cells were intensely
positive for Fas antigen (PE), and most of them were negative for
bcl-2 protein (FITC). Nonblastic cells were positive for bcl-2 protein
and faintly positive for Fas antigen.
Aftera final wash,sectionswereobservedunderamicroscopic
fluorometer (MPM, Zeiss, Germany).
Flow cytomerry. One million PWM-stimulated and unstimulated
PBLs were incubated with 4 pg of anti-Fas MoAb for 30 minutes
at 4°C. were washed, and then were incubated with
20 pL of PEconjugated goat antimouse Ig (diluted1:80;Tago, Burlingame, CA)
for 20 minutes. These specimens were immediately fixed with 2%
paraformaldehyde in phosphate-buffered saline, applied to a FACScan, and analyzed with Consort30 (Becton Dickinson).
DNA labeling assay using pow cyfomefry. Toevaluatethe
or without mitoapoptosis inducedby anti-Fas MoAb on PBLs with
gen treatment, we performed DNA labeling with propidium iodide
(PI) and flow cytometric analysis. The method was essentially that
described by Nicolettiet al." Inbrief,the 200g centrifugedcell
pellet was gently resuspended in
1.2 mL hypotonic fluorochrome
solution of PI 50 pg/mL in 0.1 mL sodium citrate plus0.1 % Triton
X-100(Sigma). After 20 hours in the dark at 4"C, the samples were
applied to FACScan.
DNAfiagmenration assay. DNA fragmentation of PWM-stimulated or unstimulated samples was assessed following the method
describd previo~sly.'~
In brief, low molecular weight DNA was
extracted from the Supernatants of lysed cells with phenolkhloro-
..
.. -.
.. ,
.
.. . . .
... . .. .
-. ... . - -
Fig 2. Flow cytometry of Pm-treated PBLs is shown. Blasticend
nonblastic cells were examined by gating the forward scattering-aide
scattering channel (bottoml. Large blastic cells (b) were intensely
positive for anti-Fat MoAb as compared with nonblastic cells (a).
TEPC 183 was the class-matched control (fine broken lines), and unstained samples are symbolizedby coarse broken lines. Both the X(fluorescenceintensity) and Y-axes (cell number) in the upper figure
are linear. TheX- and Y-axes of the bottomfigure are forward and
side scattering,respectively.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1858
YOSHINO ET AL
form and precipitated with ethanoVammonium acetate. After digestion with RNase A, 5 pg of each DNA fragment was loaded on a
2% agarose gel containing 0.01% ethidium bromide and waselectrophoresed. Bands were visualized by means of a UV transilluminator
(Shoshin EM Cop, Okazaki, Japan).
RESULTS
Expression of Fas antigen and bcl-2 protein on germinal
center cells. As shown in Fig lA, Fas antigen was strongly
expressed on GCCs and on
a few scattered interfollicular
cells. Mantle zone mature lymphocytes and most interfollicular cells were faintly stained or mainly nonreactive with
anti-Fas MoAb.In contrast to Fas antigen, bel-2protein
was expressed on mantle zone lymphocytes andinmost
interfollicular cells. Most GCCs were negative for bel-2 pro-
tein. Double staining with anti-Fas MoAb and anti-CD3 or
anti-CD19 MoAb showed that most of Fas-positive cells in
germinal centers were B cells and that strongly Fas-positive
interfollicular blastic cells were composed of both T and B
cells (data not shown).
Expression of Fas antigen on resting and blastic PBLs.
As shown in Fig lB, Fas antigen was highly expressed on
PWM-stimulated lymphoblasts. Most small nonblastic PBLs
were faintly positive or negative for Fas antigen. Bcl-2 protein was expressed on nonblastic lymphocytes, and most
PWM-stimulated lymphoblasts were negative for bcl-2 protein. PWM-untreated PBLs showed the same staining pattern
as nonblastic cells in PWM-treated PBL cultures; they were
faintly positive or negative for Fas antigen and more positive
for bcl-2 protein. There was no difference in reactive pattern
Fig 3. DNA fluorosmnca histogramsof PI-atained PBL cultures after 24 hours of incubation
under various up.rim.ntal conditions isshown.
(a) PBLs with control mouse IgM antibody, apoptotk ratio (AR), 2%; (b) PBLs with 4 pg/mL of anti-Fa. MoAb, AR, 8%; (c) PWM-tnated PBLs
with controlMoAb, AR, 10%; Id) PWM-treated PBLs with 25 ng/mL of anti-Far MoAb, AR, 17%; (81 PWM-troated PBLs with 100 nglmL of antiFas MoAb, AR, 27%; (f) PWM-treated PBLs with 300 ng/mL of anti-Far MoAb, AR, 48%; (g) PWM-treated PBLs with 1 p g l m L of anti-Fas MoAb.
AR, 47%; lh) PWM-treated PBLs with 4 pg/mL of a n t i - F r MoAb, AR, 57%; (i)IL-4 anti-lgM antibody-treatedPBLs with control antibody, AR,
9%; ljl IL-4 + anti-lgM antibody-treatedPBL. with 4 m / m L of anti-Fas MoAb, AR, 49%; (k)IL-2 + antl-CD3 antibody-treated PBL. with control
antibody, AR, 3%; (I) IL-2 + anti-CD3 antibody-treated PBLs with 4 pg/mL of anti-Fas MoAb, AR, 38%; lm) PHA + PMA-treated PBLs with
control antibody, AR, 23%; and In) PHA PMA-treated PBLs with 4 p g l m L of anti-Fas MoAb, AR, 56%.
+
+
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
bcl-2 AND Fas
IN
LYMPHOBLASTS
1859
Table 1. Anti-Fas MoAb-lnducd PBL Cultures Treated With Stimulants
~
B Cells
Whole PBLs
Untreated (%)*
IgM control (%)
Anti-Fas antibody (%)
(4 pg/mL)
T Cells
Untreated
IL-4 + Anti-lgM
PWM
Untreated
IL-2 + Anti-CD3
9
8
<5
<5
13
11
14
12
<5
15
<5
<5
17
20
<5
<5
48
9
42
40
9
32
40
7
PWM
PHA
+ PMA
Untreated
Apoptotic cells were evaluated with flow cytometry (see Materials and Methods). The number in each column is the average apoptotic
percentage of samples from four individuals.
between B and T lymphoblasts stimulated with PWM. The
numbers of B lymphoblasts induced by E - 4 + anti-IgM
antibody treatment and T lymphoblasts induced by IL-2 +
anti-CD3 antibody treatment were smaller than those induced by PWM treatment, but the inverse expression of Fas
antigen and bcl-2 protein was essentially the same (data not
shown).
Flow cytometry showed again that PWM-induced blasts
definitely expressed more Fas antigen than did nonblastic
cells (Fig 2). The cells between small-sized lymphocytes
(designated “a” in Fig 2) and large-sized blastic cells (“b”
in Fig 2) showed intermediate expression of Fas antigen.
PWM-untreated PBL samples expressed the same level of
Fas antigen as did nonblastic PBLs in PWM-treated samples.
Induction of apoptosis by anti-Fas MoAb on PWM-treated
PBLs. Anti-Fas MoAb (25 ng/mL to 4 pg/mL) was added
to the PBL cultures that had been treated with PWM for 3
days, at which time about 40% to 70% of lymphocytes became blastic. Anti-Fas MoAb was also added to the B-cell
cultures with IL-4 + anti-IgM treatment and to T-cell cultures with IL-2 + anti-CD3 antibody or PHA + PMA treatment. Three hours after culture with anti-Fas antibody, stimulator-induced blastic cells began to show pyknotic nuclei.
We checked the apoptosis with flow cytometric analysis. As
shown in Fig 3, anti-Fas MoAb induced apoptosis in PBL
cultures treated with PWM (Fig 3c through h), in IL-4 +
anti-IgM antibody-treated B cells (Fig 3i and j), in IL-2 +
anti-CD3 antibody-treated T cells (Fig 3k and l), and in PHA
+ PMA-treated T cells (Fig 3m and n). Only a small amount
of untreated PBLs underwent apoptosis (Fig 3a and b).
PWM-treated T cells also showed apoptosis (data not
shown). The data of apoptosis induced by anti-Fas MoAb
were also listed in Table 1. As shown in Fig 4, the degree
of apoptosis was dose-dependent at a range of 25 to 300 ng/
mL, and the ratio of apoptotic cells reached a plateau from
1 pg/mL to 4 pg/mL. There was an individual variation in
the degree of blastic response to stimulators. We examined
PBL cultures from five healthy donors, and the apoptotic
ratio was 37% to 72% (average, 48%) at the concentration
of 4 pglmL anti-Fas antibody. Most of the surviving cells
were nonblastic, and only a minor population of
lymphoblasts was distinct. Anti-Fas MoAb almost failed to
induce apoptosis in PBLs without PWM. More than 90% of
PWM-induced lymphoblasts were distinct at this time, unless
incubated with anti-Fas MoAb. As shown in Table 1, B
cells stimulated with IL-4 + anti-IgM antibody and T cells
stimulated with IL-2 + anti-CD3 antibody or with PHA +
PMA showed apoptosis at 42%, 32%, and 40%, respectively,
on average with 4 pg/mL anti-Fas antibody, respectively.
DNA-jragrnentation assay. PWM-stimulated lymphocytes showed DNA fragmentation when incubated with antiFas MoAb, whereas nontreated lymphocytes did not show
fragmentation. Control samples treated with class-matched
control sera also showed no DNA fragmentation (Fig 5).
DISCUSSION
T cells are differentiated and programmed within the thymus; most cortical immature thymocytes die by apoptosis.13
B cells proliferate mainly in the germinal centers of peripheral lymphoid organs. They respond to antigens and undergo
somatic mutation of Ig variable region genes. Autoreactive
B cells or those that failed to achieve appropriate avidity to
the antigen undergo apoptosis accompanied by scavenging
macrophage^.'^,'^ Most GCCs lack bcl-2 protein, which has
been thought to be an antidote to apoptosis.lo3”Most PWMstimulated lymphoblasts from PBLs lack bcl-2 protein as
well, despite high levels of bcl-2 transcripts.16 Therefore,
with respect to bcl-2 regulation for apoptosis, PWM-induced
lymphoblasts seem to be analogous to GCCs.
Recently, MBller et all7 described that APO-Was antigen
is expressed on GCCs (IgD- and CD10+) and is not detected
on mantle zone surface (s)IgD+ lymphocytes. Miyawaki et
all8 have described that Fas antigen is expressed exclusively
on CD45RO+ memory or previously activated T cells and
on sIgD- B cells in PBLs that increase in number according
to age. Our results using lymph nodes partly support this
notion; Fas antigen was expressed on sIgD- GCCs and not
on sIgD+ mantle zone lymphocytes. Interfollicular mature
lymphocytes were faintly positive or negative, and interfollicular scattered T blasts were intensely positive for Fas
antigen. Nonstimulated PBLs were weakly positive for Fas
antigen, but, after activation with PWM, IL-4 + anti-IgM
antibody, IL-2 + anti-CD3 antibody, or PHA PMA, not
only B cells but also T cells became intensely positive and
underwent apoptosis in the presence of anti-Fas MoAb. This
result is consistent with a previous report” which showed
that Fas-resistant PBLs became sensitive after exposure to
IL-2. Therefore, activation of lymphocytes seemedto be
associated with upregulation of Fas antigen and the simultaneous downregulation of bcl-2 protein, and these findings
seemed to be associated with most blastic cells undergoing
apoptosis for clonal deletion and selection. Most blastic cells
downregulated bcl-2 protein in the presence of PWM, but
+
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
YOSHINO ET AL
1860
5
-a
P
c
o.
0
n
0
0.0
1 .o
2.0
3.0
antl-ha antlbody and IgM control (uglml)
some lymphoblasts were highly positive for bcl-2 protein
and escaped apoptosis.I6
Fas antigen may functionally regulate programmed cell
death, because anti-Fas MoAb induced apoptosis in
lymphoblasts in a dose-dependent manner. The most distinctive cells among PWM-stimulated PBLs incubated with antiFas MoAb were small lymphocytes. These results contradict
a previous report" which showed that the lymphocytes stimulated in vitro were not significantly changed by anti-Fas
MoAb. We used adult PBLs for in vitro stimulation, which
may explain the discrepancy with the previous study" that
used neonatal PBLs. Interestingly, anti-Fas MoAb could not
induce apoptosis in nontreated PBLs, although at least some
of them were positive for Fas antigen. This may be related
f'
4.0
Fig 4. The apoptotic effect of anti-Far MoAb on
PWM-treated-untreated P B b
is
shown. PWMtreated or untreated
PBLs were incubated with various concentrations of anti-Fas MoAb 10 t o 4 pg/mL)
at 37°C for 24 hours. Apoptosis was evaluated by
DNA-labeling assay using flow cytometry. A centrifuged cell pellet was resuspended in 1.2 mL hypotonic fluorochromesolution.'* After 20 hours at 4°C.
the sample was applied t o a FACScan. Apoptosis of
PBL cultures with or without stimulant was examined with samples from five healthy donors: (-1,
PWM-treated PBLs; (---l, untreated PBLs; (01, PBL
cultures with anti-Far MoAb; (01. PBL cultures with
TEPC 183. Y-axis bar is thestandard deviation of the
examined five samples.
to the fact that more than 50% of PBLs were positive for
bcl-2 protein." Mapara et al" have described that APO-I/
Fas-antigen-mediated apoptosis in chronic B-lymphocytic
leukemia cells is correlated with downregulation of bcl-2
mRNA. The signalling mechanism in apoptosis by this exquisite and inverse regulation of Fas antigen and bcl-2 protein will be clarified by further examination.
cDNA cloning for Fas/APO-I antigen showed that this
apoptosis-associated molecule belongs to the TNFIUNGFR
family.' Itoh et a17 have shown, using a transfection assay,
that anti-Fas MoAb directly induces apoptosis by binding to
its receptor. Fas maynot be the only apoptosis inducer,
because TNF also induces tumor regression with apoptosis.
BcI-2 protein may not bethe only apoptosis inhibitor either,
S1 2 3 4 5 6
Fig 5. DNA fragmentation in
PWM -stimulated unstimulated
cells in the presence or absence
of anti-Fas MoAb. Anti-Fas
MoAb was added 3 days after
PBLs were culturedwith or without 0.5% PWM. Twenty-four
hours later, DNA was extracted
from the supernatants of lysed
calls with
phanollchloroform
andprecipitated with ethanol/
ammonium acetate. After digestion withRNase A, 5 p g of DNA
fragments was loaded on a 2%
agarose gel containing 0.01%
ethidium bromide and resolved
by electrophoresis. Lane l,untreated PBLs; lane 2, P B b w i t h
TEPC183; lane 3, PBLs with antiFas MoAb; lane 4, PWM-treated
PBLs without antibody; lane 5,
PWM-treatedP8Ls with lEPC183;
and lane 6, PWM-treated PBLs
with anti-Fas MoAb.
-
26451605676350-
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
bcl-2 AND Fas IN LYMPHOBLASTS
because Hashimoto et a12* described the adenovirus oncogene Elb confers cellular resistance to cytolysis by anti-Fas
MoAb. Our results, however, suggested that Fas-antigedbcl2-protein regulation plays an important role in the apoptotic
events in GCCs and in PWM-stimulated PBLs.
there
Natural ligands to Fas antigen remain unclarified, but
might be some natural ligands mimickingthe anti-Fas or
Apo-l antibodies. Antigen-primed lymphocytes might express other apoptosis-inducing factors. It has been reported
that human memoryT cells show enhancedlevels of various
accessory molecules?3s24P%"-stimulated PBLsbegan to
show blastic changes after 3 days. At that time, blastic cells
did not show apoptosis but did show apoptosis after 7 days
in the presence of PWM.I6 This finding suggests that some
ligands for the Fas antigen or for other unknown apoptosisassociated receptors are presentin culture medium. The
finding that GCCs lack bcl-2 protein and had upregulated
expression of Fas antigen suggests that the inverse expression of bcl-2 and Fas antigen also plays an important role
in vivo.
In conclusion, the blastic changes of lymphoid cells induced by antigen stimulation or mitogens seem to be associated with upregulation of Fas antigen and with the inverse
downregulation of bcl-2 protein, which may be related to
the clonal deletion of unfavorable clones through apoptosis.
REFERENCES
1 . Wylie AH, Kerr JFR, Cunie AR: Cell death: The significance
of apoptosis. Int Rev Cytol 68:251, 1980
2. Walker NI, Harmon BV, Gob6 GC, Kerr JFR: Patterns of cell
death. Methods Achiev Exp Pathol 13:18, 1988
3. Cohen JJ, Duke RC: Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J
Immunol 132:38, 1984
4. Smith CA, Davis T, Anderson D, Solam L, Beckmann MP,
Jerzy R, Dower SK,Cosman D, Goodwin R G A receptor for tumor
necrosis factor defines an unusual family of cellular and viral proteins. Science 248:1019, 1990
5 . Yonehara S , Ishii A, Yonehara M: A cell-killing monoclonal
antibody (anti-Fas) to a cell surface antigen co-downregulated with
the receptor of tumor necrosis factor. J Exp Med 169:1747, 1989
6. Trauth BC, Klas C, Peters A M J , Matzku S , Moller P, Falk W,
Debatin K-M, Krammer PH: Monoclonal antibody-mediated tumor
regression by induction of apoptosis. Science 245301, 1989
7. Itoh N, Yonehara S,Ishii A, Yonehara M, Mizuhima S, Sameshima M, Hase A, Seto Y, Nagata S: The polypeptide encoded by
the cDNA for human surface antigen Fas can mediate apoptosis.
Cell 66:233, 1991
8. Korsmeyer SJ: Bcl-2: A repressor of lymphocyte death. Immuno1 Today 13:285, 1992
1861
9. Korsmeyer SJ: Bcl-2 initiates a new category of oncogenes:
Regulators of cell death. Blood 80:879, 1992
10. Kondo E, Nakamura S , Onoue H, Matsuo Y, Yoshino T, Aoki
H, Hayashi K, Takahashi K, Minowada J, Nomura S , Akagi T:
Detection of bcl-2 protein and bcl-2 messenger RNA in normal and
neoplastic lymphoid tissues by immunohistochemistry and in situ
hybridization. Blood 80:2044, 1992
11. Chleq-Deshamps CM, LeBrun DP, Huie P, Besnier DP,
Wamke RA, Sibley RK, Cleary ML: Topographical dissociation of
bcl-2 messenger RNA and protein expresion in human lymphoid
tissues. Blood 81:293, 1993
12. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi
C: Arapid and simple method for measuring thymocyte apoptosis by
propidium iodide staining and flow cytometry. J Immunol Methods
139:271, 1991
13. Smith CA, Williams GT, Kingston R, Jenkinson El, Owen
JJT: Antibodies to CD3E-cell receptor complex induce death by
apoptosis in immature T cells in thymic cultures. Nature 337:181,
1989
14. Goldstein P, Ojcius DM, Young JD-E: Cell death mechanisms
and the immune system. Immunol Rev 121:29, 1991
15. Cohen JJ, Duke RC: Apoptosis and programmed cell death
in immunity. Annu Rev Immunol 10267, 1992
16. Kondo E, Yoshino T, Nomura S , Nakamura S, Takahashi K,
Teramoto N. Hayashi K, Akagi T: bcl-2 regulation in normal resting
lymphocytes and lymphoblasts. Jpn J Cancer Res 1994 (in press)
17. Moller P, Henne C, Leithauser F, Eichelmann A, Schmidt A,
Briiderlein S, Dhein J, Krammer PH: Coregulation of the APO-1
antigen with intercellular adhesion molecule-l (CD54) in tonsillar
B cells and coordinate expression in follicular center B cells and in
follicle center and mediastinal B-cell lymphomas. Blood 81:2067,
1993
18. Miyawaki T, Uehara T, Nibu R, Tsuji T, Yachi A, Yonehara
S, Taniguchi N: Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations inhuman peripheral blood. J
Immunol 149:3753, 1992
19. Owen-Schaub LB. Yonehara S, Crump WL 111, Grim EA:
DNA fragmentation and cell death and selectively triggered in activated humanlymphocytes by Fas antigen engagement. Cell Immunol
140:197, 1992
20. Reed JC, Miyashita T, Cuddy M, Cho D: Regulation of p26Bcl-2 protein levels in human peripheral blood lymphocytes. Lab
Invest 67:443, 1992
21. Mapara MY,BargouR,Zugck
C.DGhnerH,
Ustaoglu F,
Jonker RR, Krammer PH, Darken B: APO-I mediated apoptosis or
proliferation in human chronic B lymphocytic leukemia: Correlation
with bcl-2 oncogene expression. Eur J Immunol 23:702, 1993
22. Hashimoto S , Ishii A, Yonehara S: The Elb oncogene of
adenovirus confers cellular resistance to cytotoxicity of tumor necrosis factor and monoclonal anti-Fas antibody. Int Immunol 3:343,
1992
23. Beverley PCL: Is T-cell memory maintained by crossreactive
stimulation? Immunol Today 11:203, 1990
24. Akbar AN, Salmon M, Janossy G: The synergy between naive
and memory Tcells during activation. Immunol Today 12:184, 1991
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1994 83: 1856-1861
Inverse expression of bcl-2 protein and Fas antigen in lymphoblasts
in peripheral lymph nodes and activated peripheral blood T and B
lymphocytes
T Yoshino, E Kondo, L Cao, K Takahashi, K Hayashi, S Nomura and T Akagi
Updated information and services can be found at:
http://www.bloodjournal.org/content/83/7/1856.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.