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Epstein-Barr Virus Transformed B Lymphocytes Produce Interleukin-5
By Cassandra C. Paul, Jonathan R. Keller, James M. Armpriester, and Michael A. Baumann
Interleukin-5 (IL-5) has previously been isolated only as a
product of T lymphocytes. We have found that EpsteinBarr virus transformed B lymphocytes produce large
amounts of IL-5 activity in culture supernatants, inducing
proliferation of murine BCL, cells, and supporting the
selective growth of eosinophil colonies in semi-solid culture. Production of IL-5 messenger RNA by transformed
B-cell lines was verified by Northern analysis using a 3.2-
kilobase cloned DNA fragment containing the full-length
human IL-5 gene, and immunoreactive IL-5 was detected in
B-cell culture supernatants. These findings suggest a
possible expanded role for the B cell in the induction of
eosinophilia, and should serve as a focus for additional
investigation into possible roles for IL-5 in human B-cell
proliferation and differentiation.
0 1990 by The American Society of Hematology.
I
isolated after centrifugation of the buffy layer over Ficoll-Hypaque,
and the cells were resuspended at 4 x 105/mL in complete medium
consisting of RPMI-1640 containing 7.5% fetal calf serum, 2
mol/L
mmol/L 1-glutamine, 1 mmol/L sodium pyruvate, 5 x
2-mercaptoethanol, and 40 pg/mL gentamicin. Suspension cultures
were maintained in 75-cm’ tissue culture flasks and fed periodically
by replacement of one third of the medium. Transformed B-cell1
lines were seen to emerge at about 6 weeks, after which time they
rapidly became the only viable elements in the cultures. The identity
of the emerging cells was confirmed by flow cytometric analysis for
the B-cell specific antigens CD20 and CD19, and the EBV-induced
antigen CD23. Presence of EBV in the cell lines was confirmed by
immunofluorescent labeling with anti-EBNA (Chemicon International Inc, El Segundo, CA). All EBV-transformed cell lines studied
for growth factor activity had been maintained continuously in
culture for more than a year. The purity of the populations was
verified by flow cytometric analysis demonstrating absence of cell
types other than B cells. In some instances, immunoglobulin gene
rearrangement studies were performed, documenting the presence
after long-term culture of pure B-cell clones.
Detection of IL-5 activity in B-cell culture supernatants. Culture
supernatants were tested in two assays standardly used to detect
IL-5 activity: the induction of proliferation of the murine B-cell line
BCL,,’ and the support of selective growth of eosinophil colonies in
semi-solid cultures of human bone marrow.8 These assays are quite
specific for the detection of IL-5 activity, as no human growth factor
other than IL-5 has been shown to stimulate BCL, cells, and other
lymphokines capable of stimulating eosinophil colony growth, such
as granulocyte-macrophage colony-stimulating factor and IL-3, also
stimulate the growth of other hematopoietic lineages under the
culture conditions used.’
BCL, stimulation assay. BCL, cells, freshly obtained after in
vivo passage: were used for all assays. BCL, cells, 1 x lo5, were
cultured in 96-well flat-bottomed tissue culture plates in either 0.2
mL complete medium alone or with the addition of 25% vol/vol
medium conditioned by EBV-transformed B-cell lines. Quadruplicate cultures were incubated for 48 hours at 37OC before being
labeled with 0.5 pCi ’H-thymidine for an additional 6 hours. The
cultures were harvested onto glass fiber filters and the relative
amount of radiolabel incorporation was determined by liquid scintillation counting. IL-5 activity present in B-cell supernatants was
quantified by comparison to a reference curve constructed by
culturing BCL, cells as above in varying concentrations of recombinant human IL-5 (generously provided by Steven Clark, Genetics
Institute, Cambridge, MA).
Eosinophil colony formation assay. Bone marrow from four
normal volunteers was aspirated into heparinized syringes. The
mononuclear cell fraction was cultured using standard methodology’
in Iscove’s Modified Dulbecco’s medium with 0.8% methylcellulose
(final concentration), containing 20% heat-inactivated fetal calf
serum, 5 x IO-’ mol/L 2-mercaptoethanol, and 2 mmol/L
1-glutamine. Colonies were scored on day 14 and were identified by
characteristic morphology by inverted microscopy, with confirma-
NTERLEUKIN-5 (IL-5) is a hematopoietic growth factor that was initially studied in murine systems, where it
was found to induce proliferation and differentiation of
eosinophil precursors and promote proliferation and antibody
production by B cells.’ The gene coding for IL-5 was isolated
from a murine T-cell hybridoma, molecularly cloned, and
used to locate and clone the human IL-5 gene from a T-cell
leukemia
The murine and human IL-5 genes have
77% homology: and recombinantly produced human IL-5
has been found to induce the growth of human eosinophil
colonies,’,*but has lacked stimulatory effects in established
human B-cell functional assays.’
Infection of human B cells by the Epstein-Barr virus
(EBV) results in a permanent state of activation, in which
the B cell is induced to express a number of activation
antigens and to secrete substances with apparent growth
factor a ~ t i v i t y It
. ~ is thought that an autocrine mechanism
fuels the perpetual proliferation of EBV-transformed B cells
in vitro.5 We have found that EBV transformation of human
B cells consistently results in the induction of production and
secretion of large amounts of IL-5. This phenomenon may
have physiologic relevance, since it suggests a possible
expanded role for the B cell in the promotion of eosinophilia
during type 1 immune responses. In addition, these findings
may serve as a focus for further investigation into possible
activities of IL-5 on human B cells.
MATERIALS AND METHODS
Establishment of EBV-transformed B-cell lines. Peripheral blood
was obtained after informed consent on several occasions from each
of two subjects with known previous exposure to EBV. We had
determined previously that spontaneous EBV-transformed B-cell
lines would emerge from suspension cultures of peripheral blood
mononuclear cells of these subjects.‘ The mononuclear fraction was
From the Department of .Medicine, Wright State University
School of Medicine and VA Medical Center. Dayton. OH: and
Program Resources, Inc. Frederick Cancer Research Facility, MD.
Submitted November 30,1989: accepted January 3.1990.
Supported by a Merit Review Grant from the Veterans Administration, and by the American Heart Association. Ohio ABliate.
Address reprint requests to Michael A . Baumann, MD, Department of Medicine 1 1 1 W , 4100 W Third St. Dayton, OH 45428.
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 I8 U.S.C.section 1734 solely to
indicate this fact.
o I990 by The American Society of Hematology.
0006-4971/90/7507-0027$3.00/0
1400
Blood, Vol 75,No 7 (April l), 1990:pp 1400-1403
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1401
8 CELLS PRODUCE IL-5
tion by Wright-Giemsa staining of colonies picked off the semi-solid
culture and allowed to dry onto a glass microscope slide. Background
colony numbers were obtained from cultures not supplied with any
exogenous growth factor. Positive controls were incubated with 10 U
of recombinant human (rh) IL-5. Experimental cultures contained
50 pL of sterile-filtered medium conditioned by EBV-transformed
B-cell lines. All cultures were performed in quadruplicate, except
unsupplemented cultures used for comparison.
Northern analysis for IL-5 messenger R N A (mRNA) expression
by B-cell lines. Northern analysis was performed using standard
methodology." Cells were lysed in 4 mol/L guanidinium solution
and the lysate was ultracentrifuged through 5.5 mol/L cesium
chloride and 0.1 mol/L EDTA for 18 hours. The RNA pellet was
resuspended in 10 mmol/L Tris.Cl (pH 7.4), 5 mmol/L EDTA, and
1% sodium dodecyl sulfate, ethanol-precipitated, and dissolved in
diethylpyrocarbonate-treated water. Total cellular RNA (15 pg/
lane) was electrophoresed through a 1.3% denaturing agarose gel.
Visualization of the 28s and 18s ribosomal RNA bands by ethidium
bromide staining was used to ascertain that similar amounts of RNA
were loaded in each lane before blotting to a nylon transfer
membrane (Nytran, Schleicher & Schuell, Keene, NH). A 3.2kilobase (kb) cloned DNA fragment containing the full-length
human IL-5 gene" (generously provided by Steven Clark) was
)*P-labeled by the random oligonucleotide primer method,'* and
hybridized with the blot. The blot was then washed to greater than
90% stringency and autoradiographed at -7OOC using an intensifying screen.
Western analysis for IL-5. Separation by polyacrylamide gel
electrophoresis was performed using the PhastSystem (Pharmacia,
Inc, Piscataway, NJ), with an 8% to 25% gradient, followed by
diffusion blotting at 7OoC of separated proteins to nitrocellulose. The
filter was blocked in phosphate-buffered saline containing 20% fetal
calf serum. The primary antibody was the rat monoclonal anti-IL-5,
TRFK-5') (gift of Donna Rennick, DNAX, Palo Alto, CA). As this
antibody is known to react preferentially with dimeric IL-5 (personal
communication, Dr Michael Grace, Schering Corp, November
1989), electophoresis conditions were nonreducing. The secondary
antibody was a goat anti-rat immunoglobulin (Ig) G alkaline
phosphatase conjugate (BRL, Inc, Gaithersburg, MD), and the
substrate was 5-bromo-4-chloro-3-indolylphosphate
p-toluidine salt
and nitroblue tetrazolium chloride (BRL).
B
3 -
n
x
-
a
A
C
Fig 2. Removal of BCL, stimulation by preincubation of three
EBV-B-cell supernatants with anti-IL-5. (A) BCL, cells alone; (B)
BCL, cells
B-cell supernatant; ( C ) BCL, cells
antibodypreincubated B-cell supernatant.
+
+
liferation of murine BCL, cells in short-term culture (Fig 1).
In a repeat experiment, the activity of B-cell supernatants on
BCL, cells was completely removed by preincubation of the
supernatants for 2 hours at 22OC with the monoclonal IL-5
neutralizing antibody, TRFK-513 (Fig 2).
Eosinophil colony formation assay. Eosinophil colonies
were obtained in bone marrow cultures supplemented with
EBV-transformed B-cell conditioned medium in numbers
comparable with those obtained in cultures receiving 10 U
rhIL-5. Only rare colony forming unit-granulocyte macrophage (CFU-GM) or CFU-M colonies were seen in cultures
receiving no supplementation, and these colonies were present
in similar numbers in supplemented cultures (Table 1).
Northern analysis for ZL-5 mRNA. The DNA probe
containing the full-length human IL-5 gene specifically
hybridized to a 3.2-kb fragment of RNA in all four EBVtransformed B-cell lines tested (Fig 3).
Western analysis for IL-5. Immunoreactive bands were
found at a molecular weight of approximately 45 Kd, as has
been reported for native human IL-5,I4 in culture supernatants of 3 of 4 EBV-transformed B-cell lines studied (Fig 4).
RESULTS
BCL, proliferation assay. Culture supernatants of five
EBV-transformed B-cell lines markedly augmented the pro-
DISCUSSION
The finding that B cells can be induced to produce IL-5 by
EBV infection is cause for re-evaluation of the interactions
Table 1. Bone Marrow Colony Formation in Response to rhlL-5
and EBV-Transformed B-Cell Culture Supernatant
Bone Marrow
1
2
3
4
BCLl
---- + B-Cell Supernatants---
Fig 1. Effect of EBV-transformed B-cell supernatants on proliferation of BCL, cells. Results graphed as mean 2 SD of four
replicates. CPM, counts per minute.
CFU-EO
CFU-GM or
CFU-EO
CFU-GM or
CFU-EO
CFU-GM or
CFU-EO
CFU-GM or
Unsupplemented
0
M
M
M
M
1
0
2
0
2
0
2
+ 10 U rhlL-5
+B-Cell CM
5.4,8.4
0.2,o. 1
8,7,6,9
1.2.1 ,o
5.8.6.8
1.0.0.0
5.4.5.3
0,l ,o,o
8,4.9.8
1,o, 1,o
8,10,10,12
1.0.0.2
9.11.15.10
0,2.0.0
6.8.8.10
1,o,o,o
Cultures supplemented with rhlL-5 or E8V-transformed B-cell conditioned medium were performed in quadruplicate. Single unsupplemented
cultures were done for comparison.
Abbreviation: CM, conditioned medium.
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PAUL ET AL
1402
a
20s
b
c
d
e
D
3-2kb m
10s m
~
b.
'
'
rk
I.
+2 1
4 14
Fig 4. W e "
0 ~ l y . hfor
- i
Culture W W M of
h"n
IL-6. (Lane 1)
m
p
h
m
l
"
I
-
cdl'. (Lane 2,
human IL-"
and 4,
Culture supermtant of two EBV-transformed B-cell lines. In
addfihl
of cell lines
dawable IL-6.
Fig 3. Northern amtysis for IL-6 mRNA showing apecitic bands a 3.2 kb for four distlnct
EBV-transformed &cell lines (lanes b through e).
(Lane a) WS1 human fibroblast cdls (neggaive
control). (Right) Ethidiumbromidestainedgel showing comparable amounts of total cellular RNA
loaded into each lane.
that occur in the induction of eosinophilia and in B-cell
activation, growth, and differentiation. Transformation of B
cells by EBV results in the induction of expression of several
cell-surface antigens. including the CD23 structure that
functions as a low-affinity IgE receptor." It is now known
that a variety of activation stimuli, including Stuphylmmcus
uuteus Cowan strain I, TPA (12-0-tetra-decanoylphorbol
13-acetate), and IL-4 will induce normal B lymphocytes to
express the CD23 activation antigen.I6
Thus, B-cell expression of an IgE receptor seems to be a general feature of B-cell
activation, and may be coupled, a t least in EBV-transformed
cells, with the production and secretion of large amounts of
IL-5. If IL-5 production is found to be inducible in B cells by
activation stimuli other than EBV, it will be necessary to
consider the possibility that B cells may be capable of
amplifying the IL-5 signal in the inductionof eosinophilia in
type 1 immune responses.
In murine systems, IL-5 has been shown to induce B cells
to produce Ig and cause augmented proliferation of B cells
costimulated with a mitogen such as dextran sulfate.' Murine B cells have been found to display an IL-5 receptor that
is upregulated in response to lipopoiysaccharidestimulati~n.'~
Although no B a l l growth activity of IL-5 has yet been
convincingly detected in human systems,' the finding that
human B cells may be induced to produce and secrete IL-5
provides a focus for future investigations of the interaction of
IL-5 with human B lymphocytes, including possible autocrine mechanisms operative during B-cell proliferation and
differentiation.
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G Molecular and cellu-
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1990 75: 1400-1403
Epstein-Barr virus transformed B lymphocytes produce interleukin-5
CC Paul, JR Keller, JM Armpriester and MA Baumann
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