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Expression of Interleukin-6 and Interleukin-6 Receptor in Hodgkin's Disease
By M. Jucker, H. Abts, W. Li, R. Schindler, H. Merz, A. Gunther, C. von Kalle, M. Schaadt, T. Diamantstein,
A.C. Feller, G.R.F. Krueger, V. Dieh1.T. Blankenstein,and H. Tesch
Interleukin-6 (IL-6) is a multipotent lymphokine that can
mediate differentiation of B cells into Ig-secreting cells,
stimulate the growth of plasmacytomas, hybridomas, and T
cells, and induce acute-phase proteins in liver cells. It has
been suggested that IL-6 is involved in the pathogenesis of
several diseases by autocrine or paracrine pathways. To
examine whether IL-6 is possibly involved in the pathophysiology of Hodgkin's disease (HD), w e analyzed the expression
of IL-6 and IL-6 receptor mRNA and protein in cell lines and
primary specimens from patients with HD. IL-&specific
transcripts were detected in three of six HD-derived cell lines
by Northern blot analysis. In the culture supernatants of four
HD-derived cell lines, IL-6 was detected by radioimmunoassay.kBiologic activity of IL-6 was confirmed by proliferation of
an IL-Mependent cell line. In situ hybridization experiments
showed IL-&specific transcripts in Hodgkin (H) and ReedSternberg (RS) cells in primary tissues of two patients. In
addition, mRNAs specific for the IL-6 receptor were detected
in five HD-derived cell lines. lmmunostaining experiments
showed expression of IL-6 receptor molecules on H and RS
cells in 8 of 16 cases with HD. Thus, our data suggest that IL-6
might be involved in the pathophysiology of HD.
o 1991 by The American Society of Hematology.
I
(AILD). The lymph nodes were partly formalin fixed and partly
frozen in liquid nitrogen. Paraffin sections, 5 p,m, were stained with
hematoxylin-eosin, Giemsa, and PAS as well as with Gomeri silver
impregnation. Frozen sections were used for immunophenotyping
and for in situ hybridization.
Northem blot. RNA was isolated using the guanidinium isothiocyanate method18 as described previo~sly.'~
Poly(A)+ RNA was
enriched by oligo (dT) chromatography. Poly(A)+ RNA, 5 kg, was
denatured for 30 minutes at 65°C and applied on 1% agarose gels
containing 1X MOPS (20 mmol/L 3-(N-morpholine)propanesulfonic acid) and 2.3 mol/L formaldehyde. Gels were stained with
ethidium bromide to verify that equal amounts of RNA were
loaded per lane. The RNA was transferred to nylon membranes by
blotting overnight in 20X SSC (1X SSC = 0.15 mol/L NaCI, 0.015
m o l b Na citrate pH 7.0). Filters were prehybridized, hybridized,
and washed as described previo~sly.'~
DNA probes were labeled by
random hexanucleotide primingz0with specific activities of 1 to 2 X
lo9 cpm/p,g DNA. The IL-6 probe was a 0.57-kb XbaI-EcoRI
fragment that was subcloned from BSF2.5.' The IL-6 receptor
probe was a 1.2-kb PstI-Xhol fragment that was subcloned from
~BsF2R.236.'~
The size of mRNAs was estimated from the
positions of 28s (5.0 kb) and 18s (2.0 kb) rRNA bands and by using
RNA markers (RNA ladder; Bethesda Research Laboratories,
Gaithersburg, MD).
In situ hybridizalion. Cryostate sections, 5 km, were placed on
2% 3-(Triethoxysilyl)-propylamin-activated slides and fixed in 4%
paraformaldehyde, 0.1% glutaraldehyde in phosphate-buffered
saline (pH 7.5) for 60 minutes at room temperature and stored in
70% ethanol. Rehydrated slides were acetylated in 0.1% triethanolamine (pH &a), 0.25% acetic anhydride for 10 minutes, fixed in
NTERLEUKIN-6 (IL-6), which is identical with B-cell
stimulatory factor-2,' 26-Kd protein: interferon 6-2
(IFN-p2),3 hybridoma growth f a ~ t o r ,and
~ hepatocytestimulating factor,' is a cytokine with several biologic
activities. The human IL-6 gene has recently been cloned. It
encodes a protein of 21 Kd consisting of 184 amino acids.'
IL-6 is involved in the final maturation of B cells into
antibody-producing cells: in the growth of plasmacytoma,'
and in the proliferation of human B cells infected with
Epstein-Barr virus (EBV).8 It promotes proliferation and
IL-2 production in activated T cells,910and induces differentiation of cytotoxic T cells." Expression of IL-6 was detected in several types of lymphoid and nonlymphoid
tissues, eg, B cells, T cells, monocytes, fibroblasts, and
epithelial cell^.'*^^^ Cloning of the human IL-6 receptor gene
showed that it belongs to the C2 set of the Ig superfamily
like other receptors for cyt~kines.'~The mature IL-6
receptor protein has a molecular weight of 80 Kd and
consists of an extracellular, a transmembrane, and a cytoplasmic domain. IL-6 receptors are expressed on a wide
variety of cells such as resting and activated T cells,
activated B cells, B-lymphoblastoid cell lines, myeloma cell
lines, and myeloid leukemia cell lines. However, IL-6
receptors are not detectable on resting B cells or Burkitt's
lymphoma cell lines."
It has been suggested that IL-6 is involved in the
pathogenesis of several diseases." In Hodgkin's disease
(HD) the expression of IL-6 has been investigated in one
case by immunostaining in which a weak expression of IL-6
was observed.I6 However, the investigators did not state
whether IL-6 staining occurred in Hodgkin (H) and ReedSternberg (RS) cells or in the large number of reactive cells.
To analyze whether IL-6 is involved in the pathophysiology of HD, we investigated the expression of IL-6 and IL-6
receptor in HD-derived cell lines and in primary specimens.
Our results demonstrate expression of both IL-6 and IL-6
receptor by H and RS cells.
MATERIALS AND METHODS
Cell lines andprimary tissues. HD-derived cell lines L428, L540,
L591, CO, KMH2, and HDLM2 were described previo~sly.'~
All
cell lines were cultured in RPMI 1640 medium supplemented with
10% heat-inactivated fetal calf serum (FCS) at 37°C in a humidified, 5% CO, atmosphere. Primary biopsies of lymph nodes were
obtained from patients with HD and peripheral T-cell lymphoma
Blood, Vol77, No 11 (June 1). 1991: pp 2413-2418
From I. Medizinische Klinik and Immunpathologisches Labor,
Institut fur Pathologe, Universitat Koln; Institut fur Immunologie,
Klinikum Steglitz, Freie Universitat Berlin; Abteilung fur Nephrologie,
Med. Hochschule Hannover; and Abteilungfur Pathologie, Universitat
Wunburg, Germany.
Submitted September IO, 1990; accepted January 24,1991.
Supported by the Deutsche Krebshilfe, Mildred Scheel Stiftung e. F,
and the Deutsche Forschungsgemeinschaft. M.J. is a recipient of a
fellowship from the Boehringer Ingelheim Fonds, Germany.
Address reprint requests to H. Tesch, MD, Medizinische Klinik I,
Universitat K o l , J.-Stelzmannstr. 9, 0-5000Koln 41, Germany.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
O I991 by The American Sociery of Hematology.
0006-49711911771I-00I4$3.00/0
2413
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JOCKER
2414
4% paraformaldehyde for 10 minutes, dehydrated in graded
ethanol, and finally air dried. Hybridization was performed in a
solution containing 4X SSC, 10% dextran sulfate, 20 mmol/L
vanadyl-ribonucleoside-complex,200 pg/mL yeast tRNA, 200
pg/mL salmon-sperm DNA, 500 pg/mL bovine serum albumin,
50% deionized formamide, 10 mmol/L Tris-HCI (pH 8.0), 1
mmol/L EDTA. Five nanograms of a ['SS]-UTP-labeled antisense
or sense riboprobes (specific activity: 1 to 2 x 10' cpm/pg) was
added to 20 KLof hybridization solution. Incubation time was 4 to
16 hours in a humidified chamber at 50°C. Washing was performed
in 4X SSC at room temperature for 10 minutes, 2X SSC at 52°C for
30 minutes, 2X SSC, 20 pg/mL RNAse A at 37°C for 30 minutes,
0.2X SSC, 10 mmol/L dithiothreitol (DlT) at 52°C for 30 minutes.
Slides were then rinsed twice in 70% ethanol, 200 mmol/L
ammoniumacetate (pH 5.4) for 10 minutes, dehydrated, and air
dried. Slides were subsequently dipped in Ilford K2 Emulsion
(Ilford, Neu Isenburg, Germany), stored for 3 days at 4"C, developed in a Kodak D19 developer (Kodak, Stuttgart, Germany),
and Giemsa stained.
IL-6 analysis. A radioimmunoassay (RIA) specific for human
IL-6 was performed as described previously?' The biologic activity
of IL-6 was measured by proliferation of the mouse plasmacytoma
cell line TEPC 1033.' The growth of TEPC 1033 cells is dependent
on exogenous IL-6. Cells do not respond to IL-1, IL-2, IL-3, IL-4,
IL-5, and IFN-yF2 5 x lo' cells per 0.2 mL were cultured in
microtiter wells for 48 hours in RPMI 1640 medium, plus 10%
FCS, 50 pmol/L 2-ME, and serial dilutions of standard human IL-6
or conditioned media were to be tested. Cells were pulse-labeled
with 0.5 pCi/well ['HI-thymidine during the last 4 hours of culture
and DNA synthesis was determined by ['HI-thymidine incorporation. Standard recombinant human IL-6 was purchased from
Boehringer Mannheim (Mannheim, Germany). Half maximal proliferation of TEPC 1033cells is defined as 1U of IL-6 in our results
which is equal to 100 U of IL-6 detected in the 7TDl assay."
Immunophenotypic analysis. Immunophenotyping was performed on frozen sections according to the alkaline phosphataseantialkaline phosphatase meth~d.~'
MT18 is a monoclonal antibody (MoAb) against the IL-6 receptor?' For control staining, two
MoAbs with an IgG, and IgG,b isotype were used.
RESULTS
Expression of IL-6mRNA in HD-derived cell lines. The
expression of IL-6 was analyzed in HD-derived cell lines
L428, L540, L591, CO, KMH2, and HDLM2 by Northern
blot analysis. These cell lines are characterized as lymphoid
cells by rearrangementsof Ig (L428, L591, KMH2) or T-cell
receptor genes (L540, CO, HDLM2). All cell lines express
antigens (CD30, CD15, HLA-DR) that are regularly detected on H and RS cells in primary lesions. IL-&specific
mRNAs of 1.3 kb were detected in HD-derived cell lines
L428, L591, and HDLM2, but not in L540, CO, and KMH2
cells (Fig 1). The same blot was rehybridized with a p-actin
probe to show that equal amounts of RNA were loaded in
each lane.
Expression of IL-6in the supernatants of HD-derived cell
lines. Significant amounts of IL-6 were detected in the
culture supernatants of LA28, L591, HDLM2, and KMH2
cells by RIA (Table 1). The concentrations of IL-6 varied
from 2,250 pg/mL (L591) to 580 pg/mL (KMH2). IL-6
activity in the culture supernatants of HD-derived cell lines
was measured by proliferation of the IL-wependent cell
line TEPC 1033. Figure 2 shows [3H]-thymidineincorpora-
ET AL
6l
4
r
c
.* -1.3kb
.
p
Ik6R
actin,
7
?
Fig 1. Northern blot analysis of 11-6 and 11-6 receptor (IL-6R) in
HD-derived cell lines. Poly(A)+ RNA of the given cell lines, 5 pg,
size-separated on 1% agarosdformaldehyde gels, transferred to
nylon membranes, and hybridized with [UP]-labeled probes of IL-6,
IL-6R. and pactin. The size of the specific transcripts and the
positions of 28s and 18s ribosomal RNAs are indicated. Exposure
times for hybridization with IL-6 was 21 days, and 4 days for IL-6R
(L540, KMH2, HDLM2, and L591) and 21 days (CO and L428).
tion of TEPC 1033cells in the presence of serial dilutions of
standard recombinant human IL-6 and culture supematants of HD-derived cell lines. IL-6 activity was detected in
the culture supernatants of cell lines L591 and HDLM2
(Fig 2).
Northern blot analysis of IL-6receptor in HD-derived cell
lines. The expression of IL-6 receptor mRNA in HDderived cell lines was analyzed by Northern blot experi-
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2415
INTERLEUKIN-6 IN HODGKIN’S DISEASE
Table 1. Expression of IL-6 and IL-6 Receptor in Hodgkin Cell Lines
Northern Blot
IL-6
Receptor
Cell
Line
IL-6
+
L428
L540
-
co
Bioassay
IL-6
[UlmL)
840
<1.0
<1.0
<1.0
72
1.3
<1.0
+
+
+
< 100
+
2,250
980
580
< 100
+
+
+
L591
HDLM2
KMH2
RIA
IL-6
(pg/mL)
-
-
ments. Very different amounts of IL-6 receptor-specific
mRNAs were detected in L428, L540, L591, CO, and
HDLM2 cells, but not in KMH2 cells (Fig 1).
Detection of IL-6 and IL-6 receptor mRNA by in situ
hybridization. Cryostate sections of primary biopsies from
lymph nodes from two untreated patients with HD and one
patient with a T-cell lymphoma (AILD) were investigated
by in situ hybridization for expression of IL-6 and IL-6
receptor mRNAs. IL-6-specific mRNA was detected in
both cases of HD in a moderate number of lymphocytes
(20% to 40%) and in some H and RS cells (Fig 3). The
expression of IL-6 mRNA was not detectable in the case of
AILD. These results were confirmed by Northern blot
analysis of total RNA from the same tissues. In both cases
of HD, but not in the case of AILD IL-6, specific transcripts
were detected (data not shown).
The same specimens were analyzed for expression of IL-6
receptor mRNA by in situ hybridization. In a high to
moderate number of lymphocytes and in some H and RS
100
80
60
40
20
1
10
100
1000
10000
DILUTIONS
Fig 2. IL-6 activity in HD-derived cell lines. Proliferation of TEPC
1033 cells was analyzed in the presence of serial dilutibns of standard
recombinant human IL-6 (D-W) and conditioned media of HD-derived
cell lines L591 (+-+) and HDLMZ (*-I). The results represent the
mean value of triplicate well analysis.
cells mRNAs for the IL-6 receptor were expressed, whereas
in the case of AILD no hybridization could be detected
(data not shown).
Expression of IL-6 receptors in primaly tissues of HD.
Cryostate sections of lymph nodes from 16 patients with
H D were investigated for expression of IL-6 receptor by
immunophenotypic analysis using MT18 MoAbs. In 8 of 16
cases with HD, a positive staining of some H and RS cells
and of varying groups of surrounding lymphocytes were
detected with the MT18 antibodies but not with control
antibodies (Fig 4).
DISCUSSION
The characteristic histology of H D in which low numbers
of H and RS cells are surrounded by a large number of
reactive cells suggests a functional relationship between
these cells, possibly mediated by cytokines. Indeed, expression of several lymphokines has been demonstrated in H D
in
or in vi^^.^^,^^,^^ To study the role of IL-6 in the
pathophysiology of H D we investigated the expression of
IL-6 and IL-6 receptor in HD-derived cell lines and in
primary specimens.
Expression and biologic activity of IL-6 was analyzed in
six HD-derived cell lines by Northern blot analysis, RIA,
and proliferation of an IL-6-dependent cell line. The
results are summarized in Table 1. The level of expression
of IL-6, as determined by Northern blot assay, correlates
well with the IL-6 activity detected by RIA and bioassay.
However, IL-6 was detected in one cell line (KMH2) by
RIA in contrast to the negative result obtained from
Northern blot assay, suggesting a higher sensitivity of the
RIA. In addition, most HD-derived cell lines express
mRNA for the IL-6 receptor.
Expression of IL-6 and IL-6 receptor mRNA was also
detected in lymph nodes of patients with HD by in situ
hybridization. In two specimens we detected mRNA for
IL-6 and its receptor in some H and RS cells. Cells
surrounding the H and RS cells as well as additional
lymphoid cells in the tissue section were positively stained
for IL-6 and IL-6 receptor mRNA, whereas a T-cell
lymphoma was negative for both. By immunostaining we
detected IL-6 receptors on H and RS cells in 50% of the
cases.
The expression of I G 6 and IL-6 receptors in HD-derived
cell lines and in primary tissues of HD indicates a putative
role of this cytokine in the pathophysiology of HD, as it has
been suggested for several other diseases, such as plasmacytoma,7.34,35 Lennert’s T-cell
Castleman’s disease,37 rheumatoid arthritis,38 and cardiac myxoma? in
which IL-6 possibly acts as an autocrine or paracrine growth
factor.
How could IL-6and IL-6receptor be involved in HD? In
a conceivable first model, H and RS cells stimulate their
own proliferation by an autocrine loop via IL-6 and IL-6
receptor. In three HD-derived cell lines and in two primary
tissues of HD we detected expression of both IL-6 and IL-6
receptor. However, we do not know yet whether the same H
and RS cells in the primary tissues expressed both the
cytokine and its receptor.
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2416
JUCKERET AI
Fig 3. In situ hybridization
with PSI-UTP-labeled antisense
(A) or sense (6) RNA probes of
IL-6 on a frozen section of a
lymph node from a patient with
HD. Autoradiographic grains
were detected on H cell (arrow)
and on 20% t o 40% of surrounding lymphocytes.
Asecond model takes into account that the cells surrounding H and RS cells are mostly CD4 positive T lymphocytes,
which carry activation markers.@ Activated T cells were
shown to produce IL-6 after antigenic stimulation." We
detected expression of IL-6 mRNA in some cells surrounding H and RS cells and IL-6 receptor molecules on H and
RS cells in 50% of cases investigated. Thus, it may be
possible that activated T cells surrounding the H and RS
Fig 4. Immunohistologic investigation on a frozen section
from a malignant lymphoma, HD
type, mixed cellularity variant,
for expression of IL-6 receptor.
(A) Incubation with MoAb MT18,
which is directed against the IL-6
receptor, shows diffuse reaction
of the RS cell (arrow) and membrane reaction of the H cell (double arrow) and of surrounding
small lymphocytes. (E) Control
staining on a tissue section of
the same lymph node.
cells produce IL-6, which stimulates the growth of H and
RS cells in a paracrine pathway.
In a third model, two lymphokines, IL-6 and IL-2, act
synergistically. It is known that activated T cells can be
induced by IL-6 to secrete IL-2" and that H and RS cells
express IL-2 receptor molecules in most cases.4''" In the
HD-derived cell line L540 it has been shown that the high
affinity form of the IL-2 receptor is expressed and IL-2 is
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2417
INTERLEUKIN-6 IN HODGKIN'S DISEASE
internalized after binding to the cells." Thus, it seems
possible that IL-6 secreted by H and RS cells triggers
surrounding T cells to produce IL-2, which in turn stimulates H and RS cells via IL-2 receptor.
However, it is unlikely that a possible autocrine or
paracrine stimulation of H and RS cells by IL-6 is sufficient
for the transformation of H and RS cells. Transgenic mice
carrying the human IL-6 gene conjugated with the Ig
enhancer developed polyclonal plasmacytosis but not plasmacytomas," indicating that in addition to deregulated IL-6
production, additional genetic changes like the activation of
oncogenes may be required for full transformation of these
cells.
The heterogeneity of HD with respect to cell surface
antigen expression,4'" presence of EBV and human herpes
virus-6 i n f e ~ t i o n , ~ ~Ig. ~and
'
T-cell receptor rearrangement~:*-~' proto-oncogene expres~ion,'~
and presence of
bc12 trans location^^^ might indicate that HD encompasses
different lymphomas rather than represents one disease
entity. The incoherent expression of IL-6 and its receptor in
H D may hint to the same direction.
ACKNOWLEDGMENT
We are grateful to J. van Snick for the TEPC 1033 cells; to D.B.
Jones, H.G. Drexler, and H. Kamesaki for providing HD-derived
cell lines; to T. Kishimoto for MoAbs against IL-6 receptor; and to
T. Hirano for the IL-6 and IL-6 receptor probes.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1991 77: 2413-2418
Expression of interleukin-6 and interleukin-6 receptor in Hodgkin's
disease
M Jucker, H Abts, W Li, R Schindler, H Merz, A Gunther, C von Kalle, M Schaadt, T Diamantstein
and AC Feller
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