From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Analysis of the Survival of Mature Human Eosinophils: Interleukin-5 Prevents
Apoptosis in Mature Human Eosinophils
By Yuji Yamaguchi, Toshio Suda, Shigeo Ohta, Kaoru Tominaga, Yasusada Miura, and Tadashi Kasahara
We and other groups have previously shown that interleukin-5 (IL-5) maintained the viability of mature eosinophils in
an in vitro liquid culture system. Mature eosinophils did not
proliferate but their survival was maintained in the presence
of IL-5. Using this culture system, we investigated the
mechanism of IL-Smediated survival. In the absence of
human IL-5 (hlL-5) mature eosinophils succumbed after 4
days, while in the presence of hlL-5 they survived up to 10
days. When DNA extracts of cultured eosinophils were
analyzed on an agar gel electrophoresis, marked DNA fragmentation was observed in the absence of hlL-5, while no
significant DNA fragmentation was observed in the culture
with hlL-5 for 48 hours. The DNA fragmentation appeared as
early as 6 to 12 hours after hlL-5 deprivation. Concomitantly,
IL-5 stimulated total RNA and protein synthesis, but did not
induce DNA synthesis in mature eosinophils. Because cycloheximide or actinomycin D impeded the protectionof apoptosir by hlL-5, some new RNA and protein synthesis appeared
to be required in this phenomena. These findings indicate
that IL-5 maintains survival of mature eosinophils with
induction of new RNA and protein synthesis, thus leading to
the inhibition of apoptosis.
o 1991 by The American Society of Hematology.
T
found that IL-5 maintained survival of eosinophils by
inhibiting apoptosis. The mechanism of eosinophil survival
by IL-5 is to be discussed.
HE SURVIVAL,, differentiation, and proliferation of
hematopoietic precursor cells and the functional maturation of blood cells are all under the influence of various
cytokines including colony-stimulatingfactors (CSFs). Many
hematopoietic cell lines have been established, and their
maintenance requires the exogenous addition of cytokines
such as interleukin-2 (IL-2), IL-3, IL-6, and CSFs. Most in
vitro cell lines do not survive on the removal of cytokines.
Cell death caused by the withdrawal of factors resembles
active cell death or apoptosis, which is characterized by the
condensation of nuclear chromatin, followed by DNA
fragmentati~n.”~
This programmed cell death is observed
first in thymocyte death exposed to glucocorticoids’.’ and
target cell destruction by cytotoxic T lymphocytes.’ In
addition, apoptosis has been observed in IL-Zdependent
T-cell clones on the removal of IL-2: in factor-dependent
continuous cell lines, Paterson Laboratories (FDCP-Mix)
cell clones on the removal of IL-3, granulocyte-CSF (GCSF), or granulocyte-macrophage CSF (GM-CSF),’ as well
as in erythroid progenitor cells on the removal of erythropoietin ( E ~ o ) These
.~
observations suggested that CSFs promote cell survival by suppressing this process.
We have previously shown that recombinant murine IL-5
(mIL-5) maintained the viability of mature murine eosinophils in an in vitro liquid culture system.’ Mature eosinophils did not proliferate but survived only in the presence of
IL-5. In the present study we have examined the mechanism
of action of IL-5 on the survival of mature eosinophils. We
From the Department of Medical Biology and Parasitology, Division
of Hematology, Department of Medicine, Department of Biochemistry,
Jichi Medical School, Tochigi-ken,Japan.
Submitted September 24, 1990; accepted July 8, 1991.
Supported by Grants-in-Aidfrom the Ministry of Education, Science
and Culture of Japan, from Sandoz Pharmaceuticals, Ltd, and from
the Ryoichi Naito Foundation for Medical Research.
Address reprint requests to Tadashi Kasahara, PhD, Dept. of
Medical Biology and Parasitology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken,329-04, 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 1991 by The American Society of Hematology.
0006-4971/91/7810-0003$3.00/0
2542
MATERIALS AND METHODS
Preparation and purification of eosinophils. Venous blood was
collected in a heparinized syringe from three patients with hypereosinophilic syndrome (HES) after obtaining their informed consent. Polymorphonuclear (PMN) leukocytes were separated by
Mono-Poly Resolving Medium (M-PRM; Japan Flow Laboratories
Ltd, Tokyo, Japan). PMN leukocytes separated by M-PRM were
layered onto discontinuous metrizamide gradients (Nycomed,
Oslo, Norway) as previously described by Vadas et a1.8 Enriched
eosinophil preparations used in the following experiments always
consisted of more than 90% eosinophils with the remainder being
neutrophils.
Lymphokines. We used purified recombinant factors as follows:
recombinant human IL-2 (rhIL-2; specific activity, 1 x lo7 U/mg
protein; provided by Shionogi Pharmaceutical Co, Osaka, Japan),
rhIL-3 (specific activity, 21,900 U/mL; provided by the Genetic
Institute, Cambridge, MA),9 rhIL-5 (purity is more than 80% by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDSPAGE]; specific activity, 1to 2 x 10’ U/mg protein assayed by IgM
secretion on murine B-cell lymphoma (BCL,) cells; provided by
Suntory Central Research Institute, Osaka, Japan),” rhG-CSF
(specific activity, 2.5 x lo7 U/mg protein; provided by Chugai
Pharmaceutical Co, Tokyo, Japan),” and rhGM-CSF (specific
activity, 1 x lo9U/mg protein; provided by Sumitomo Pharmaceutical Co, Osaka, Japan).I2 The optimal concentration of each factor
was determined by titration using human normal bone marrow
(BM) cells.
Cell culture. Mature human eosinophils were cultured in 24well tissue culture plates (Coster, Cambridge, MA). Each well
contained 1 mL RPMI 1640 supplemented with 20% fetal calf
serum (FCS), lo6 eosinophils, and various CSFs. At various times,
viable cells were counted by the eosin dye exclusion test.
Analysis of DNA fragmentation. DNA fragmentation in mature
human eosinophils and cytotoxic T-cell line (CTLL-2) cells was
determined according to the method described previously by Nieto
and Lopez-Rivas! Briefly, 1 to 5 x lo6cells were incubated at 37°C
for 4 hours in 500 kL of lysing buffer (200 mmol/L Tris, pH 8.5,lOO
mmol/L EDTA, 50 kg/mL proteinase K, 1% SDS). The DNA was
phenol-extracted and then dialyzed for 12 hours against 10mmol/L
Tris (pH 7.5), 1 mmol/L EDTA. After dialysis the DNA solution
was incubated for 5 hours at 37°C with 50 kg/mL of RNase A, and
further incubated with 150 kg/mL of proteinase K for 5 hours at
37°C. The DNA was extracted with phenol and chloroform and
precipitated with ethanol. Each sample was electrophoresed on
Blood, Vol78, No 10 (November 15L 1991:pp 2542-2547
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2543
IL-5 BLOCKS EOSINOPHIL APOPTOSIS
RESULTS
Days h c u b e
Fig 1. In vitro survival of mature human eosinophils in the presence of 40 ng/mL hG-CSF (A), 2 ng/mL hlL-3 (=), 1 ng/mL hGM-CSF
(A),and 50 ng/mL hlL-5 ( 0 ) Control
.
means the absence of CSFs. At
various times, the viable cell number was counted by eosin exclusion.
Each value is the mean of data from duplicate cultures.
agarose gels (1%) with TBE buffer (89 mmol/L Tris, 89 mmol/L
borate, 2 mmol/L EDTA) containing 0.5 pg/mL ethidium bromide.
DNA, RNA, and protein synthesis hv matitre human eosinophib
Assay culture was performed as described elsewhere." In brief,
enriched mature human eosinophils (1 x IO"/mL) in RPMI 1640
and 10% FCS were preincubated for 1, 2, and 3 days with various
lymphokines and/or 2 pg/mL actinomycin D (Sigma Chemical Co,
St Louis. MO) or 10 pg/mLcycloheximide (Sigma). Tubes containing 2.5 x IO-' cells and factors were preincubated and the
incorporation of 740 kBq/mL 'H-thymidine (specific activity, 185
GBq/mmol; Amersham Japan, Tokyo, Japan). 2.8 x 10' kBq/mL
'H-uridine (specific activity. 1.37 GBq/mmol; Amersham Japan),
and 10' kBq/mL 'H-leucine (specific activity, 2.74 TBq/mmol;
Amersham Japan) into trichloroacetic acid (IO%)-insoluble materials for the last 5 hours was measured by a Packard liquid
scintillation counter (Packard Instrument Co, Meriden, CT).
In vitro sriniwl of human matiire eosinophils in the
presence of hematopoietic cytokines. It is well known that
normal mature eosinophils separated from peripheral blood
do not survive more than 4 days in vitro without the
addition of cytokines. When the culture medium was
supplemented with hIL-5 (50 ng/mL) or hGM-CSF (1
ng/mL), eosinophils survived up to 10 days. Similarly, hIL-3
(2 ng/mL) supported 50% survival up to 6 days (Fig 1).
hG-CSF (40 ng/mL) did not maintain viability. By removing
hIL-5 from the culture medium, the number of cosinoFhils
began to decrease rapidly and few viable cells were detected 4 days after the withdrawal.
IL-5 inhibits the DNA fragmentation and cell death of
matitre eosinophils. It is of a great interest to know
whether the death of mature eosinophils accompanies
DNA fragmentation, and if so, whether hIL-5 inhibits this
process. We performed DNA fragmentation analysis of
mature eosinophils on agarose gel electrophoresis. As
shown in Fig 2, marked DNA fragmentation occurred in
eosinophils incubated for 48 hours without IL-5, whereas
DNA fragmentation was prevented in the presence of 50
ng/mL hIL-5. As a control, DNA from an IL-2-dependent
cell line, CTLL-2, was similarly analyzed (Fig 2; CTLL-2).
After the withdrawal of hIL-2, DNA from CTLL-2 cells
exhibited the fragmentation rapidly, ie, as early as 4 hours
after the withdrawal of hIL-2. Figure 3 shows the kinetic
changes of DNA fragmentation in mature eosinophils in
the absence of hIL-5. The DNA fragmentation in mature
eosinophils appeared from 6 to 12 hours after hIL-5
deprivation, which preceded by 24 to 48 hours the morphologically observed death of eosinophils as shown in Fig 1.
Protection against DNA fiapentation in matitre eosinophils by IL-5 reqitires de novo RNA and protein .ynthesi.s.
eo
Fig 2. Apoptosis in mature human eosinophils
produced by hlL-5 deprivation (eo) and by hlL-2
deprivation of an IL-Z-dependent cell line (CTLL-2).
Mature eosinophils were incubated in the presence
or absence of hlL-5 for 48 hours. CTLL-2 cells were
incubated in the absence of hlL-2 for various periods
(0,4,6,8, and 10 hours).
947 bp
564 bp
CTLL-2
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
YAMAGUCHI ET AL
2544
L
L
a
tn
fi
I
622
404
242
110
- 2100 bp
-5000
-2000
1400
947
564
i4 --
bpbpbpbp
I
bp
bP
bp
bp
bp
Fig 3. Analysis in agarose gels
of DNA extracted from mature
human eosinophils incubated in
the absence of hlL-5 after various
periods (0, 3, 6, 12, 24, and 48
hours).
-
We then examined whether some metabolic inhibitors
affect the IL-5-induced survival of eosinophils to discover
the role of IL-5. Figure 4 shows that viability of mature
eosinophils was not supported by hIL-5 in the presence of 2
pg/mL actinomycin D (Act D), an inhibitor of transcription, or 10 pg/mL cycloheximide (CHX), an inhibitor of
protein synthesis. This observation indicated that the hIL-5induced survival of mature eosinophils seemed to require
de novo RNA and protein syntheses. Simultaneously, we
determined whether IL-5 induces new RNA and protein
syntheses in mature eosinophils. DNA synthesis was not
observed in mature eosinophils incubated with hIL-5 or
with other lymphokines, at least during 3-day incubation
period (Fig 5A). However, the enhanced RNA synthesis
occurred in eosinophils incubated with hIL-5 or hGM-CSF
(Fig 5B). Total RNA synthesis in mature eosinophils
reached a maximal level 1 day after incubation with either
hIL-5 or hGM-CSF, and declined thereafter. Likewisc, a
nearly threefold increase in protein synthesis was observed
in mature eosinophils incubated with hIL-5, hGM-CSF, or
hIL-3 (Fig 5C), reaching a maximal level at 2 days. The
hIL-5-induced RNA or protein synthesis in mature eosinophils described above was completely inhibited in the
prcsence of 10 pg/mL CHX or 2 pg/mL Act D.
Eflect of metabolic inhibitors on the IL-Sinduced protection of DNA fragmentation. Under the conditions described above, we tested how the metabolic inhibitors
affected the DNA fragmentation of eosinophils. Figure 6
shows that DNA fragmentation was prevented by the
incubation with hIL-5, whereas marked DNA fragmentation occurred in eosinophils incubated with IL-5 in the
presence of CHX or in the presence of Act D. Neitl-.erCHX
nor Act D alone prevented the spontaneously occurring
DNA fragmentation.
DISCUSSION
I
2
3
4
5
days
Fig 4. In vitro survival of mature human eosinophils in the presence of 50 ng/mL hlL-5 alone, 50 ng/mL hlL-5 plus 2 pg/mL Act D or
10 pg/mL CHX. Control means the absence of CSFs. At various times,
the viable cell number was counted by eosin exclusion. Each value is
the mean of data from duplicate cultures.
The viability of both human and murine mature eosinophils is maintained in the presence of IL-5, as has been
previously rep~rted.'.'~Usually, deprivation of growth factors results in cell death of factor-dependent cell lines, such
as CTLL-2 and FDCP-mix, with morphologic changes
called apopto~is.~.'
Apoptosis is characterized by condensation of nuclear chromatin and DNA fragmentation into
nucleosome-length fragments.I5 This type of cell dcath is
also found in the glucocorticoid-induced death of immature
thymocytes, cytotoxic T-cell-mediated cytolysis, and/or
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2545
11-5 BLOCKS EOSINOPHIL APOPTOSIS
A
I: hlL-8
?: hlL-SrCHX
3: hlL-5+AOl.D
4: hGU-CSC
5: hlL-5
% hG-CBC
7: OO"l.01
, 2 3 4 , 1 1 7
dl
1 2 3 4 1 8 7
a2
1 2 3 4 1 e 7
d3
-
C
'2
n
'2
E
n
Fig 5. DNA, RNA, and protein syntheses in mature
human eosinophils stimulated with various factors.
(A), (E), and (C) show the incorporation of 'Hthymidine, 3H-uridine,and 3H-leucine,respectively. 1,
50 ng/mL hlL-5; 2, 50 ng/mL hlL-5 plus 10 pg/mL
CHX; 3, 50 ng/mL hlL-5 plus 2 pg/mL Act D; 4, 1
ng/mL hGM-CSF; 5, 2 ng/mL hlL-3; 6, 40 ng/mL
hG-CSF; 7, medium alone (control).
!
,
I
ai
irradiation of lymphocytes."' In the present liquid culture
system, human mature eosinophils did not proliferate and
were only maintained in the presence of hIL-5 or hGMCSF. Therefore, this culture system has been thought to
c
c
c
c
21 Kbp
5 Kbp
2 Kbp
1.4 Kbp
947 bp
564 bp
Fig 6. Analysis in agarose gels of DNA extracted from mature
human eosinophils incubated in the presence of 50 nglmL hlL-5, 10
pg/mL CHX, 2 pg/mL Act D, hlL-5 plus CHX, and hlL-5 plus Act D for
24 hours.
a?
a3
' 3 . 1 8 7
a3
provide one of the most suitable systems for the elucidation
of survival and cell death in mature blood cells.
Eosinophilopoietic factors such as IL-5, GM-CSF, and
IL-3 are capable of supporting eosinophil s~rvival?'~.'~
The
order of potency of the factors on eosinophil survival was
hIL-5, hGM-CSF, and hIL-3. This order of potency may
correlate with the number of receptors for each factor on
mature human eosinophils.The outstanding feature of IL-5
is that it prevents the DNA fragmentation of mature
eosinophils that inevitably proceeds in the absence of IL-5.
Thus, the prolonged survival of mature eosinophils by '1IL-5
may be due to the inhibition of DNA cleavage. This survival
was not maintained in the presence of Act D or CHX.
Presumably, some proteins inhibiting endonuclease activity
might be produced in mature eosinophils in response to
stimulation by hIL-5. It has been reported that the half-life
of human blood eosinophils is 13 to 18 hour^.''.^^ However,
the survival time of eosinophils in tissues is not known. The
blood eosinophil half-life is prolonged during eosinophilia,
which may be due to the prevention of apoptosis in mature
eosinophils by increased IL-5 level.
We determined whether DNA, RNA, and protein synthesis occurred in mature eosinophils incubated with hIL-5,
hGM-CSF, or hIL-3. In these cells, significant RNA and
protein synthesis were observed but no DNA synthesis was
seen. The absence of DNA synthesis indicates that mature
human eosinophils do not proliferate. On the other hand,
RNA and protein syntheses did not take place in mature
eosinophils incubated with hIL-5 plus actinomycin D or
cycloheximide, which resulted in cell death. In addition, no
RNA and protein synthesis was observed in eosinophils
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2546
YAMAGUCHI ET AL
incubated with hG-CSF, which did not maintain survival.
These findings strongly suggest that the prolonged survival
of mature eosinophils by hIL-5 is associated with some
newly synthesized proteins induced by hIL-5 stimulation.
Although no particular endonuclease involved in DNA
fragmentation has been defined, a set of nuclear proteins
that increases during the apoptosis of T-cell line induced by
dexamethasone has been identified as histone proteins.”
On the other hand, Vaux et almand Nunez et a1” reported
that transfection of proto-oncogene bcl-2 prolonged the
survival of IL3-dependent cell lines deprived of a facThus, it is possible that such bcl-2 gene expression or
altered histone proteins may also be directly or indirectly
involved in IL-5-induced suppression of apoptosis in eosinophils.
All cellular activities in the human body are driven by
energy obtained through the breakdown of ATP. The major
energy-supplying enzyme is ATP synthase in inner mitochondrial membranes. Therefore, we attempted to look at
whether hIL-5-modulated gene transcription encoding some
mitochondrial ATP synthases, ie, cytochrome c oxidase
subunit 11, which is encoded by mitochondrial DNA,” and
ATP synthase p subunit and the cytochrome c oxidase
subunit VI,, which are encoded by nuclear
Northern blot analysis showed, however, that the expression of
these mitochondrial protein genes was increased variably
among HES patients and that no specific correlation
between hIL-5 stimulation and particular enzyme gene
expression has been obtained so far (data not shown). On
the other hand, it has been pointed out that mitochondrial
gene transcripts increased in immortalized cell lines with
transfection of polyoma large T protein, adenovirus E,A,
and myc oncogenes.25Mitochondrial gene expression study
remains one of the interesting issues to be explored in the
eosinophil survival.
Thus, we have shown that IL-5 maintains survival of
mature eosinophils presumably by inhibiting apoptosis and
that it was associated with newly synthesized proteins. No
definitive information regarding the nature of the proteins
or enzymes involved in apoptosis is yet available, but it must
be characterized soon.
ACKNOWLEDGMENT
We thank Prof Kohei Nakano for his suggestions and Kyoko
Kokuho for her help with the manuscript.
REFERENCES
1. Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is
associated with endogenous endonuclease activation. Nature 284:
555,1980
2. Cohen JJ, Duke R C Glucocorticoid activation of a calciumdependent endonuclease in thymocyte nuclei leads to cell death. J
Immunol132:38,1984
3. Ucker DS: Cytotoxic T lymphocytes and glucocorticoids
activate an endogenous suicide process in target cells. Nature
32752,1987
4. Nieto MA, Lopez-Rivas A: IL-2 protects T lymphocytes from
glucocorticoid-induced DNA fragmentation and cell death. J
Immunol143:4166,1989
5. Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor DR:
Haemopoietic colony stimulating factors promote cell survival by
suppressing apoptosis. Nature 343:76,1990
6 . Koury MJ, Bondurant MC: Erythropoietin retards DNA
breakdown and prevents programmed death in erythroid progenitor cells. Science 248:378, 1990
7. Yamaguchi Y, Hayashi Y, Sugama Y, Miura Y, Kasahara T,
Kitamura S, Torisu M, Mita S, Tominaga A, Takatsu K, Suda T
Highly purified murine interleukin 5 (IL-5) stimulates eosinophil
function and prolongs in vitro survival: IL-5 as an eosinophil
chemotactic factor. J Exp Med 167:1737,1988
8. Vadas MA, David JR, Butterworth A, Pisani NT, Siongok
T A A new method for the purification of human eosinophils and
neutrophils, and a comparison of the ability of their cells to damage
Schistosomula of Schbtosoma mansoni. J Immunol 122:1228, 1979
9. Yang Y-C, Ciarletta AB, Temple PA, Chung MP, Kovacic S,
Witek-Giannoti JS, Leary AC, Kriz R, Donaheu RE, Wong GG:
Human IL-3 (multi-CSF): Identification by expression cloning of a
novel hematopoietic growth factor related to murine IL-3. Cell
47:3,1986
10. Azuma C, Tanabe T, Konishi M, Kinashi T, Noma T,
Matsuda F, Yaoita Y, Takatsu K, Hammarastrom L, Smith CIE,
Severinson E, Honjo T Cloning of cDNA for human T-cell
replacing factor (interleukin-5) and comparison with the murine
homologne. Nucleic Acids Res 14:9179,1986
11. Nagata S, Tsuchiya M, Asano S, Kaziro Y, Yamazaki T,
Tamamoto 0,Hirata Y, Kubota N, Oheda M, Nomura H, Ono M:
Molecular cloning and expression of cDNA for human granulocyte
colony-stimulating factor. Nature 319:415,1986
12. Wong GG, Witek JS, Temple PA, Wilkins KM, Leary AC,
Luxenberg DP, Jones SS, Brown EL, Kay RM, Orr EC, Shoemaker
C, Golde DW, Kaufman RJ, Hewick RM, Wang EA, Clark SC:
Human GM-CSF Molecular cloning of the complementary DNA
and purification of the natural and recombinant protein. Science
228:810,1985
13. Kasahara T, Nakano K, Osawa T: Correlation between
mature and immature thymocytes. Immature thymocytes potentiating factor produced by mature thymocytes. Int Arch Allergy 48:452,
1975
14. Owen WF, Rothenberg ME, Petersen J, Weller PF, Silverstein D, Sheffer AL, Stevens RL, Soberman RJ, Austin KF:
Interleukin 5 and phenotypically altered eosinophils in the blood of
patients with the idiopathic hypereosinophilic syndrome. J Exp
Med 170:343,1989
15. Wyllie AH, Kerr JFR, Currie AR: Cell death: The significance of apoptosis. Int Rev Cytol68:251,1980
16. Begley CG, Lopez AF, Nicola NA, Warren DJ, Vadas MA,
Sanderson CJ, Metcalf D: Purified colony-stimulating factors
enhance the survival of human neutrophils and eosinophils in vitro:
A rapid and sensitive microassay for colony-stimulating factors.
Blood 68:162,1986
17. Panvaresch MR, Walle AJ, Arndt D: The peripheral kinetics of human radiolabelled eosinophils. Virchows Arch [B] 2157,
1976
18. Steinbach KH, Shick P, Trepel F, Raffler H, Dohrmann J,
Heilgeist G, Hetzel W, Li K, Past W, Lamge JA, Them1 H, Fliedner
TM, Begemann H: Estimation of kinetic parameters of neutrophilic, eosinophilic, and basophilic granulocytes in human blood.
Blut 39:27, 1979
19. Baxter GD, Smith PJ, Lavin MF: Molecular changes associated with induction of cell death in a human T-cell leukemia line:
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
IL-5 BLOCKS EOSINOPHIL APOPTOSIS
Putative nucleases identified as histones. Biochem Biophys Res
Commun 162:30,1989
20. V a w DL, Cory S, Adams JM: Bcl-2 gene promotes haematopoietic cell survival and cooperates with c-myc to immortalize
pre-B cells. Nature 335:440,1988
21. Nunez G, London L, Hockenbery D, Alexander M, Mckearn
JP, Korsmeyer SJ: Deregulated Bcl-2 gene expression selectively
prolongs survival of growth factor-deprived hemopoietic cell lines.
J Immunol144:3602,1990
22. Anderson S, Bankier AT, Barrel BG, DeBruijin MHL,
Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger
F, Schreier PH, Smith AJH, Staden R, Young EG: Sequence and
2547
organization of the human mitochondrial genome. Nature 290457,
1981
23. Ohta S, Kagawa Y Human F,-ATPase: Molecular cloning of
cDNA for the beta subunit. J Biochem 99:135,1986
24. Otsuka M, Mizuno Y, Yoshida M, Kagawa Y, Ohta S:
Nucleotide sequence of cDNA encoding human cytochrome c
oxidase subunit VIc. Nucleic Acids Res 16:10916,1988
25. Glaichenhaus N, Leopold P, Cuzin F Increased levels of
mitochondrial gene expression in rat fibroblast cells immortalized
or transformed by viral and cellular oncogenes. EMBO J 5:1261,
1986
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1991 78: 2542-2547
Analysis of the survival of mature human eosinophils: interleukin-5
prevents apoptosis in mature human eosinophils
Y Yamaguchi, T Suda, S Ohta, K Tominaga, Y Miura and T Kasahara
Updated information and services can be found at:
http://www.bloodjournal.org/content/78/10/2542.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.