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Int. J. Cancer: 71, 1066–1076 (1997)
r 1997 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
AUTOCRINE INTERLEUKIN-1 RECEPTOR ANTAGONIST CAN SUPPORT
MALIGNANT GROWTH OF GLIOBLASTOMA BY BLOCKING
GROWTH-INHIBITING AUTOCRINE LOOP OF INTERLEUKIN-1
Elisabeth OELMANN1, Annette KRAEMER1, Hubert SERVE1, Birgit REUFI1, Dorothea OBERBERG1, Stephan PATT2,
Hermann HERBST3, Harald STEIN3, Eckhard THIEL1 and Wolfgang E. BERDEL1*
1Department of Hematology/Oncology, Benjamin Franklin Hospital, Freie Universität Berlin, 12200 Berlin, Germany
2Department of Neuropathology, Benjamin Franklin Hospital, Freie Universität Berlin, 12200 Berlin, Germany
3Department of Pathology, Benjamin Franklin Hospital, Freie Universität Berlin, 12200 Berlin, Germany
In situ hybridization (ISH) of human glioblastoma tissue
sections revealed expression of interleukin-1 (IL-1)a and/or b
and IL-1 receptor types I and II (IL-1R I and II) in the majority
of cases evaluable. To understand the function of IL-1-family
members in human glioblastomas, we have studied 6 glioblastoma cell lines. RT-PCR, ISH, ELISA and 125I-IL-1-binding
assays revealed expression of IL-1 and high-affinity receptors
for human (h)IL-1 in all but 1 cell line. Using a colony growth
assay in semi-solid media for testing serial plating efficacy (PE,
number of colonies per number of cells seeded in %), only the
IL-1R-negative cell line was not influenced by recombinant
human (rh)IL-1a or -b, whereas IL-1 down-regulated the
self-renewal of clonogenic cells of the other glioblastomas.
Tritiated thymidine uptake was down-regulated by rhIL-1 in
all cell lines studied. Cell viability remained unchanged by
rhIL-1. Wherever growth modulation by rhIL-1 was detected,
it could be reversed by either soluble IL-1R I or II or by rhIL-1
receptor antagonist (ra). IL-1ra not only was able to reverse
rhIL-1-induced growth modulation but alone could modulate
glioblastoma growth in comparison with control in cell lines
producing IL-1. Our results show the presence of public
autocrine loops for IL-1 leading to growth inhibition in some
glioblastomas. To understand these loops, we have studied
expression and function of IL-1ra in glioblastomas. ISH of
human glioblastoma tissue sections revealed expression of
hIL-1ra in all 8 cases evaluable. In 4 of 6 cell lines, IL-1ra was
found in the supernatant under constitutive conditions, the
IL-1R-negative line being among the 2 non-producers. The
other non-producing cell line, HTB 17, showed expression of
hIL-1R II. Most interestingly, a neutralizing antibody against
IL-1ra down-regulated growth of IL-1- and IL-1ra-producing
glioblastoma cells to approx. 30% of the controls. Thus, public
autocrine loops for IL-1 in human glioblastomas exist and
result in growth inhibition. An autocrine production of IL-1antagonizing molecules such as IL-1ra by these tumors can
counteract this IL-1 function and represent a basic escape
mechanism supporting malignant growth in some glioblastomas. Int. J. Cancer 71:1066–1076, 1997.
r 1997 Wiley-Liss, Inc.
IL-1a and IL-1b are pleiotropic cytokines with a multitude of
activities in a wide range of cell types and different tissues
(Dinarello, 1991, 1994, 1996; Platanias and Vogelzang, 1990;
Schmidt and Tocci, 1991). They have some amino-acid homology,
bind to the same type I and II cell surface receptors and share
biologic activities (Dinarello, 1991, 1994). Type I receptors are
important for signaling (Sims et al., 1993); type II receptors may be
released from cells and function as a decoy target for IL-1 (Colotta
et al., 1993; Symons et al., 1995). In addition to these shIL-1Rs,
naturally occurring antagonists for IL-1 have been described
(Arend, 1993; Dinarello, 1991; Dinarello and Thompson, 1991;
Larrick, 1989). The cDNA of the receptor antagonist IL-1ra has
been cloned (Eisenberg et al., 1990); the encoded protein shows
some amino-acid homology with IL-1a and IL-1b (Dinarello,
1991), binding to IL-1R without initiating IL-1 signal transduction
(Dripps et al., 1991), and can block a multitude of IL-1 effects
(Arend, 1993; Dinarello and Thompson, 1991). Thus, IL-1 activity
seems to be the result of a fine-tuned network of agonist and
antagonist molecules within the IL-1 family.
Diverse effects by IL-1 on the growth of cells from solid tumors
have been reported. The cytokine can exert growth-inhibitory
activity in some tumor cells (Bertoglio et al., 1987; Danforth and
Sgagias, 1993; Gaffney and Tsai, 1986; Kilian et al., 1991;
Lachman et al., 1986; Onozaki et al., 1985) but also stimulates
growth of several tumor cell lines (Hamburger et al., 1987; Ito et
al., 1993; Lahm et al., 1992), including autocrine growth stimulation (Zeki et al., 1993). Although these observations remain
contradictory, several clinical studies with IL-1 in cancer patients
are under way (Crown et al., 1991; Redman et al., 1994; Schuchter
et al., 1994; Smith et al., 1992; Triozzi et al., 1995). Detailed
studies have been published on the reduction of growth of myeloid
leukemic cells by IL-1ra (Estrov et al., 1991; Yin et al., 1992).
However, only few reports exist on the potential role of IL-1ra in
solid tumors. Expression of IL-1ra has been reported in endometrial cancer (Van Le et al., 1991) and bronchogenic carcinoma
(Smith et al., 1993). In both studies there was higher expression of
IL-1ra in tumor cells than in normal tissues, which was discussed as
being important in tumor evasion of host defense (Smith et al.,
1993). We have shown that modulation of colony formation by
rhIL-1 of some tumor cell lines was completely blocked by
rhIL-1ra (Oelmann et al., 1994).
There are many studies on the role and functions of IL-1 in the
brain (Rothwell, 1991; Merrill, 1987; Smith, 1992). Of interest for
this investigation are the following findings. It has been shown
clearly that brain macrophages (microglia) produce IL-1 (Giulian
et al., 1986, 1988; Hetier et al., 1988; Malipiero et al., 1990).
Production is highest at the time of birth (Giulian et al., 1988).
Furthermore, there is expression of IL-1R located on neurons
throughout the brain (Dinarello, 1991; Rothwell, 1991), while
IL-1R on glial cells are expressed after brain injury (Rothwell,
1991). Thus, IL-1 appears to be important for brain development
and response to brain injury (Rothwell, 1991).
IL-1 is expressed by astrocytoma and glioma cell lines (Fontana
et al., 1982; Lee et al., 1989) and IL-1 mRNA was found in primary
brain tumors (Merlo et al., 1993). Although growth-promoting
effects of exogenous IL-1 have been reported in an astrocytoma cell
line (Bertoglio et al., 1987; Lachman et al., 1987), these early
observations must be interpreted with caution. The authors have
employed only short incubation times, and a detailed study
Abbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; h, human; HTCA, human tumorcloning assay; ICE, IL-1b-converting enzyme; IL-1, interleukin-1; IL-1ra,
IL-1 receptor antagonist; IL-1R, IL-1 receptor; ISH, in situ hybridization;
MAb, monoclonal antibody; PDH, pyruvate dehydrogenase; PE, plating
efficiency; rh, recombinant human; RT-PCR, reverse transcriptasepolymerase chain reaction; sh, soluble human.
*Correspondence to: Department of Hematology/Oncology, Benjamin
Franklin Hospital, Freie Universität Berlin, 30 Hindenburgdamm, 12200
Berlin, Germany. Fax: 49 30 8445 4479.
Received 9 December 1996; accepted 29 January 1997
INTERLEUKIN-1 NETWORK IN GLIOBLASTOMA
performed later on the same cell line reported only transient growth
stimulation but terminal differentiation of the cells after prolonged
incubation with exogenous IL-1 (Tanaka et al., 1994).
Up to now, there has been no comprehensive study on the
expression and functional role of agonist and antagonist members
of the IL-1 family in human glioblastoma. Using human glioblastoma tissue sections and cell lines, we show here the presence of
autocrine loops for IL-1 in glioblastomas which can lead to growth
inhibition and describe autocrine loops for IL-1-antagonizing
molecules such as IL-1ra in cell lines which, by counteracting IL-1
loops, represent an escape mechanism supporting malignant growth
of some glioblastomas.
MATERIAL AND METHODS
Tissue
Tissue specimens were obtained from glioblastoma patients of
our hospital during surgery according to our ethical board guidelines. Paraffin-embedded sections of glioblastoma multiforme were
studied by ISH.
Cells
HTB 14 and HTB 17 are human glioblastoma cell lines and were
purchased from the ATCC (Rockville, MD). All other human
glioblastoma cell lines were kindly provided by Dr. D. Stavrou
(Hamburg, Germany). Cell-culture techniques were performed
according to standard procedures, and cells were routinely checked
for Mycoplasma.
Cytokines
RhIL-1a was purchased from Genzyme (origin Escherichia coli,
105 units/µg; Cambridge, MA). RhIL-1b was from Genzyme
(origin E. coli, 5 3 105 units/µg). RhIL-1ra was from PeproTech
(origin E. coli; Rocky Hill, NJ). shIL-1R types I and II (origin
Chinese hamster ovary cells) were a kind gift of Dr. J.E. Sims
(Immunex, Seattle, WA).
Antibodies
Antibodies against IL-1a and -b were purchased from PeproTech (origin rabbit; catalog numbers 500-P21A and 500-P21B).
The mouse MAb against hIL-1R I (Genzyme, code 1592-01) is an
IgG1 antibody (expressed in C 127 cells). The rat MAb against
hIL-1R II (Genzyme, code 80-3503-01) is an IgG2b antibody.
Polyclonal antibodies neutralizing hIL-1ra were from either WakChemie (Bad Homburg, Germany, number AB-280-NA) or Genzyme (code 80-2975-01).
ISH
After linearization of plasmids (pGEM-3Z; Promega, Madison,
WI; or pCR II for IL-1ra; Invitrogen, San Diego, CA) containing
specific sequences of the genes for hIL-1a and -b (R&D Systems,
Minneapolis, MN), hIL-1R type I and type II (kindly provided by
Immunex) and hIL-1ra (produced by PCR from the common
sequence of hIL-1ra from fetal liver), 35S-labeled run-off anti-sense
and sense (control) transcripts were generated using Sp6 and T7
RNA polymerases. ISH for the detection of RNA transcripts was
performed as previously described (Herbst et al., 1992). In brief,
dewaxed and rehydrated paraffin sections were exposed to 0.2 N
HCl and 0.125 mg/ml pronase (Boehringer, Mannheim, Germany)
followed by acetylation with 0.1 M triethanolamine, pH 8.0/0.25%
(v/v), acetic anhydride and dehydration through graded ethanols.
Slides were hybridized to 2 to 4 3 105 cpm of labeled probes
overnight at 54°C. Washing and autoradiography were performed
as described by Milani et al. (1989).
All sections were processed in parallel using the same batches of
reagents and probes. Incubation of sections with Micrococcus
nuclease (Boehringer, Mannheim) prior to ISH resulted in extinction of the specific autoradiographic signal, establishing that RNA
sequences were targets of the hybridization procedure (Williamson,
1988).
1067
RT-PCR
Total RNA was isolated according to the instructions of the
supplier of RNAzol (Paesel and Lorei, Frankfurt, Germany). The
amount of RNA isolated was determined spectrophotometrically.
Reverse transcription was carried out using 1 µg of total RNA, 0.8
µM oligo-p(dT)15 primer, 0.125 mM of each dNTP, 13.5 units of
RNAse inhibitor (Promega) and 200 units of MMLV reverse
transcriptase in 13 final concentration of the reverse transcriptase
buffer (Life Technologies, Eggenstein, Germany) and supplemented with DTT to yield a final concentration of 10 mM. The final
reaction volume was 20 µl. The mixture was incubated at 37°C for
60 min and subsequently for 5 min at 95°C to inactivate the reverse
transcriptase. PCR was performed in a total volume of 50 µl
containing 4 µl of the RT reaction mixture, 10 mM Tris-HCL (pH
8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 0.125 mM
dNTPs, 1 µM of each up- and down-stream primer and 1.4 units of
Taq polymerase (Angewandte Gentechnologie Systeme, Heidelberg, Germany). Amplification was carried out in a Perkin Elmer
(Norwalk, CT) 9600 Thermocycler using a modified ‘‘hot-start’’
technique. The following RNA transcripts were detected via
amplification of the corresponding cDNAs: (i) the b-subunit of
PDH, using the primer pair composed of the (1) primer 58-GGT
ATG GAT GAG GAC CTG GA-38 and the (2) primer 58-CTT
CCA CAG CCC TCG ACT AA-38, yielding an amplicon of 105 bp;
(ii) hIL-1a, using specific primer pairs (Clontech, Palo Alto, CA)
which amplify a 491-bp fragment; (iii) hIL-1b, using specific
primer pairs (Clontech) which amplify an 802-bp fragment; (iv)
hIL-1R type I, using specific primer pairs (Clontech) which
amplify a 300-bp fragment; (v) hIL-1R type II, using specific
primer pairs composed of the (1) primer 58-GAA GAG ACC ATT
CCT GTG ATC-38 and the (2) primer 58-GAA AGT CTT GAT
GAT GAG GCC-38, which amplify an expected fragment of 481
bp; (vi) intracellular form of hIL-1ra, using a primer pair (Haskill et
al., 1991) composed of the (1) primer GM 397 and the (2) primer
GM 368, yielding a 512-bp PCR product, common sequence of
hIL-1ra: (1) primer 58-TTA ACA TCA CTG ACC TGA GCG AGA
ACA G-38 and (2) primer 58-CCT GGA AGT AGA ATT TGG
TGA CCA TGA C-38, producing a 201-bp amplicon; (vii) ICE,
using specific primer pairs (Thornberry et al., 1992) composed of
modified primer pairs: p10 (1) primer 58-GCT ATT AAG AAA
GCC CAC ATA GA-38 and (2) primer 58-TTC AGT GGT GGG
CAT CTG CG-38, amplifying a 222-bp fragment and p20 (1)
primer 58-GAC AAC CCA GCT ATG CCC AC-38 and (2) primer
58-CGG CTT GAC TTG TCC ATT ATT G-38, producing a 128-bp
amplicon.
The cycle program for each primer pair was preceded by an
initial denaturation at 95°C for 4 min, a specific annealing
temperature according to the Tm of each primer, and followed by a
final extension at 72°C for 10 min. The cycle program comprised
35 cycles of 95°C for 1 min, annealing for 1.5 min and 72°C for 1.5
min. Amplification of transcripts of the PDH b-subunit was
efficient using this cycle program. Amplification of the PDH
b-subunit was used to judge DNA contamination in the RNA
samples of the different cell lines examined and, moreover, to
justify the comparability of amplification results of the specific
target regions in the different cell lines.
Amplicon identities were verified by Southern hybridization
with the cDNA probes as mentioned above (details not shown).
ELISA
Cells were washed in RPMI 1640 medium without serum and
incubated for 48 hr at pH 7.2, 37°C, 5% CO2 and high humidity in
RPMI 1640 medium containing 0.2% BSA without serum. Cell
concentrations at the beginning of the 48-hr incubation period were
1 to 2 3 106 cells/ml. Subsequently, cell supernatants were
harvested and assayed for hIL-1a, hIL-1b, hIL-1b precursor or
hIL-1ra, respectively. We have used ELISA kits obtained from
Endogen (hIL-1a, code EH-IL1A; hIL-1b, code EH-IL1B; Boston,
MA), R&D Systems (hIL-1ra, code DRA 00) and Cistron Biotech.
(hIL-1b precursor, code 03-1000; Pine Brook, NJ).
1068
OELMANN ET AL.
FIGURE 1 – Expression of hIL-1a (a), hIL-1b (b), IL-1 receptor type
I (c), hIL-1 receptor type II (d) and hIL-1ra (e) in tissue sections
(f, example for sense control) of human glioblastomas (results, see g) as
shown by ISH. Black grains represent expression of specific messages.
INTERLEUKIN-1 NETWORK IN GLIOBLASTOMA
FIGURE 2 – RT-PCR analysis of hIL-1a, hIL-1b and ICE transcripts
(a) and of hIL-1R types I and II transcripts (b) in human glioblastoma
cell lines. M, molecular markers. For bp sizes see ‘‘Material and
Methods’’.
Binding assay
Dissociation constants (Kd ) of hIL-1-binding sites were determined by Scatchard analysis of (3-[125I]-iodotyrosyl)-labeled hIL-1a
(Amersham, Aylesbury, UK; specific activity, 2,000 Ci/mmol or 74
TBq/mmol) binding (Oelmann et al., 1995) with the following
modifications. Cells were plated in 6-well plates at 1.0 to 10.0 3
105 cells/well 1–5 days before the binding assay to allow adherence. Cells were counted on the day of the assay. After washing the
cells once with serum-free DMEM containing 2.5 mg/ml BSA,
125I-labeled hIL-1a (10 pM–1 nM) was added in 0.5 ml DMEM/
BSA to duplicate wells and incubated for 60 min at 37°C.
Non-specific binding was determined at each concentration step by
adding 100 3 molar excess hIL-1a to duplicate wells. Cells were
then washed 3 times with ice-cold DMEM/BSA and removed from
the wells by lysis with 2 3 0.5 ml 1 N NaOH. Radioactivity was
counted in a Berthold gamma counter. Experiments were repeated
3–4 times.
HTCA
For evaluation of anchorage-independent clonal colony growth
of the cell lines, a newly developed HTCA using mixtures of
methylcellulose and agar (Oelmann et al., 1994; Topp et al., 1993)
was used with the following modification: cells were detached by
1069
trypsinization and washed with their own growth medium, resuspended with 1 ml RPMI 1640 medium (GIBCO, Glasgow, UK)
plus 10% FCS and counted by Trypan blue staining to yield a final
concentration of 3 3 104 cells/ml. Viability of the cells .80% was
required before cells were taken for an experiment. Methylcellulose solution was produced by boiling 0.5 I distilled water with 21 g
methylcellulose (code M 0512; Sigma, Deisenhofen, Germany).
Five hundred milliliters of cold Iscove’s modified Dulbecco’s
medium (double concentrated; GIBCO, code 041-90132) was
added to the methylcellulose after cooling down to 37.0°C. The
mixture was kept in 3.6 ml aliquots at 220°C. Agar was dissolved
by boiling 3 g Difco agar (Agar Noble; Difco, Detroit, MI) in 100
ml distilled water for 30 min, and consecutively 10 ml of the
boiling agar were added to 20 ml RPMI 1640 (37.0°C). The
incubation mixture was made up of 3.6 ml methylcellulose
solution, 2.7 ml FCS (Hyclone, Logan, UT; code A-1111-D), 0.06
ml mercaptoethanol (8.4 3 1023 M mercaptoethanol in distilled
water; GIBCO; code 043-01350 D), 0.3 ml cell suspension, 0.8 ml
Iscove’s medium (Gibco; code 041-01980M) and 1.6 ml agar/
RPMI 1640 mixture. This incubation mixture was vortexed thoroughly and kept in the dark at 37.0°C for 20 min. RhIL-1 or control
vehicle was added to Lux dishes (suspension culture dishes,
35 3 10 mm, code 174926; Miles, Naperville, IL) as a solution of
0.1 ml PBS (Gibco; code 14190-094) plus 0.1% BSA and rhIL-1.
An aliquot of 1 ml of the incubation mixture was then added to the
dishes. This final incubation mixture contained the final cytokine
concentrations, as indicated in the results; thus, tumor cells were
exposed to the cytokine for the complete assay period. For all
experiments in which rhIL-1ra, shIL-1R or antibodies were used
together with rhIL-1, these agents were incubated for 1 hr at room
temperature or 37.0°C with the cells or the cytokines as indicated
before being added to the assay. The number of cells finally seeded
per dish was 1 3 103. Colony formation was evaluated with an
inverted microscope before (to exclude cell clumping) and after an
incubation period of 10 days at pH 7.2, 37°C, in an atmosphere of
5% CO2 and high humidity.
PE was defined as the number of colonies per number of cells
seeded, expressed as a percentage. PE1 represents PE observed at
the completion of 1 HTCA. Serial PE was assayed by removing
single colonies from either control or experimental cultures with
micropipettes, obtaining single-cell suspensions by mechanical
means, washing these cells and directly replating them at identical
numbers per dish into HTCA. PE2 was calculated on the basis of
the numbers of colonies counted at the end of a second HTCA.
HTCA was used since this assay has been shown to reliably
detect growth modulation of tumor cells by cytokines and to be
predictive for in vivo tumorigenicity and modulation of in vivo
tumor growth by cytokines (Freedman and Shin, 1974; Gross et al.,
1988; Topp et al., 1993).
Tritiated thymidine uptake
Before the assay was started, cells were starved by reducing the
serum concentration to 1.0% for 24 hr in order to obtain a
synchronization effect on the cell cycle. Thymidine uptake kinetics
under different serum concentrations had been obtained before and
the serum concentrations used for starving allowed for 3–50% of
tumor cell thymidine uptake over 24 hr compared with uptake in
10% FCS. For the tritiated thymidine uptake assay, 100-µl aliquots
of the cytokines or antibodies in test medium (usual growth media
of the tested cell lines) were seeded into 6 wells per test group of
96-well flat-bottomed microtiter plates (Greiner, Nürtingen, Germany) at indicated concentrations. The wells already contained 100
µl of the cell suspension (0.5 to 2 3 104 cells/well, depending on
the growth kinetics of the cell line used). Controls contained 100 µl
of pure test medium instead of the cytokine or antibody. Wherever
antagonist members of the IL-1 family, such as rhIL-1ra or shIL-1R
or neutralizing antibodies, were used in addition to rhIL-1,
pre-incubation conditions with either the cells (e.g., rhIL-1ra,
antibody against IL-1R) or the recombinant cytokine (e.g., shIL-1R
types I or II, antibodies against IL-1 or IL-1ra) were as stated for
OELMANN ET AL.
1070
FIGURE 3 – Specific binding of 125I-labeled hIL-1a to human glioblastoma cell lines. (a) X-axis, concentrations of
Scatchard analysis of the binding data is shown as inset. (b) Kd values, sites per cell and correlation co-efficients.
HTCA. Plates were incubated at 37°C, pH 7.2, in an atmosphere of
5% CO2 and high humidity for up to 72 hr. Cultures were pulsed for
the last 6 hr with 1.0 µCi of [3H] thymidine (specific activity 5.0
Ci/mmol; Amersham) per well. Samples were processed and
counted in an LKB Betaplate system (LKB Pharmacia, Freiburg,
Germany). Values are given as means 6 SD.
Cell numbers and viability
All cell lines were incubated in 6-well plates (5 3 104 cells/well)
in 3 ml medium plus 10% FCS with the cytokine concentrations as
indicated for 24–168 hr in the above conditions (tritiated thymidine
uptake assay). Subsequently, cells were detached by trypsinization
and stained with Trypan blue to evaluate cell number and viability.
Statistics
Results were statistically evaluated by the Kruskal-Wallis test or
the Mann-Whitney test as indicated; p values , 0.05 were
interpreted as indicating significant differences.
RESULTS
Expression of IL-1 and IL-1R in human glioblastoma tissue
sections in situ
In a first set of experiments utilizing ISH, we assayed 10 human
glioblastoma sections for the presence of hIL-1 and hIL-1R
expression. The majority of human glioblastoma tissue sections
evaluable expressed mRNA for hIL-1a, hIL-1b, and hIL-1R types
I and II (Fig. 1). The main reason for being not evaluable was high
125I-labeled
hIL-1a.
FIGURE 4 – (a) Influence of rhIL-1a on colony formation (PE1 ) of
human glioblastoma cell lines in HTCA. Values are expressed as % of
controls (tumor cells only) and represent means 6 SD of 6-fold assays.
PE values for controls were 87-HG-31, 10.8%; HTB 17, 2.2%; HTB
14, 11.2%; 86-HG-39, 10.7%; 87-HG-28, 1.6%; 88-HG-14, 11.6%.
Since 103 cells were seeded per dish, the numbers of colonies per dish
can be calculated as PE 3 10. *, p . 0.05 (indicating nonsignificant
differences) when compared with the tumor cell (only) controls
(Mann-Whitney test). All other values were significantly ( p , 0.05)
different from controls. (b) Influence of rhIL-1 on the secondary plating
efficiency (PE2 ) of human glioblastoma cell lines in HTCA. PE1 values
after the first HTCA were calculated from means of 6-fold assays
showing significant ( p , 0.05, Mann-Whitney test) stimulation (87-HG31) or inhibition (88-HG-14) of colony formation by rhIL-1a/b in
comparison with control, given as 100%. Single colonies from either
the controls or the rhIL-1-incubated cultures of the first HTCA were
removed by micropipettes, gently agitated to yield single-cell suspensions, washed and immediately replated without rhIL-1 into a second
HTCA at equal cell numbers per dish for calculation of PE2 from means
of 6-fold assays. PE2 values from control colonies of the first HTCA did
not decrease significantly when compared to PE1, indicating no toxicity
of the replating procedure itself. All PE2 values obtained from
rhIL-1-incubated cultures of the first HTCA were significantly
( p , 0.05, Mann-Whitney test) lower than those from control colonies
of the first HTCA. (c) Blocking of the rhIL-1 effect by either rhIL-1ra
or shIL-1R I using the 88-HG-14 cell line in HTCA. Values represent
means 6 SD of 6-fold assays. Mann-Whitney tests were performed
comparing control values with rhIL-1 condition (*) or comparing
rhIL-1 condition with rhIL-1 plus inhibitor condition (**). Asterisks
represent p values , 0.05.
FIGURE 4
OELMANN ET AL.
1072
TABLE I – CONCENTRATION OF IL-1 NETWORK MEMBERS IN THE SUPERNATANTS OF HUMAN GLIOBLASTOMA CELL
LINES AFTER 48 HR OF SERUM-FREE INCUBATION
Cytokine
Cell lines
86-HG-39
88-HG-14
87-HG-28
87-HG-31
HTB 14
HTB 17
hIL-1a1
11.3 6 3.65 64.8 6 55.7 129.2 6 80.7 5.7 6 1.0 60.3 6 42.2 19.3 6 3.3
hIL-1b precursor2
60.66
40.0
50.0
20.0
480.0
10.0
hIL-1b3
8.6 6 7.15 42.7 6 21.9 54.9 6 35.8 8.6 6 8.6 73.7 6 69.9 4.1 6 1.0
hIL-1ra4
n.d.6
2,800.0
14,500.0
12.8
9,500.0
n.d.
1Detection limit of ELISA at 13 pg/ml hIL-1a.–2Detection limit of ELISA at 50 pg/ml hIL-1b
precursor.–3Detection limit of ELISA at 4 pg/ml hIL-1b.–4Detection limit of ELISA at 6.5 pg/ml
hIL-1ra.–5Means 6 standard error of 2 or 3 experiments with 4-fold or 5-fold assays each, pg/ml.–6Means
of one 4-fold assay, pg/ml. n.d., not detectable.
non-specific background staining in the sense controls, which led to
exclusion of the case. IL-1 message–positive sections and IL-1R
message–positive sections were from identical patients. Signals
could be located clearly over the tumor cells, however, some
stromal cells also showed expression. For both IL-1 a/b and IL-1R
I/II, mRNA expression was not equally distributed in all tumor
cells of 1 section but occurred in patches of positive cells and/or in
single cells distributed throughout the tumor (Fig. 1).
Expression of IL-1 and IL-1R and functionality of specific
IL-1-binding sites (receptors) in 6 human glioblastoma cell lines
Next, we used RT-PCR, ISH and ELISA techniques to examine 6
human glioblastoma cell lines for expression of IL-1 a/b and IL-1R
I/II.
RT-PCR revealed message for IL-1R I in all cell lines, with
86-HG-39 showing only a faint band (Fig. 2). Type II receptor
message expression was minimal in 2 lines, including 86-HG-39,
and present in all other cell lines (Fig. 2). Both IL-1a and IL-1b
messages were detected in all but 2 cell lines, and all cell lines
expressed message for ICE (Fig. 2). Bands detected by RT-PCR
were checked using Southern blotting to identify sequence homology with the sequences of interest (details not shown). Amplification of the PDH b-subunit was used to exclude DNA contamination
in the RNA samples and to justify the comparability of amplification results of the specific target regions in the different cell lines
(details not shown). In addition, data derived from ISH agreed
almost exactly with RT-PCR and showed that mRNA expression
for both IL-1 a/b and IL-1R I/II was not equally distributed in all
tumor cells of 1 line but occurred in patches of positive cells and/or
in single cells distributed throughout the cell line (details not
shown).
ELISA revealed the presence of either IL-1a or IL-1b or both in
the supernatants of all cell lines, with 87-HG-31 being at the
detection limit (Table I). There was some variability of IL-1 a/b
production when ELISAs were performed at different times and
numbers of cell passages, with 87-HG-31 ranging from below to
clearly above detection limits for IL-1a/b (e.g., IL-1b range not
detectable to 45.6 pg/ml; detection limit, 4 pg/ml).
To test our cell lines for the presence of functional hIL-1R, we
performed 125I-hIL-1-binding assays. These experiments revealed
the presence and function of high-affinity receptors for hIL-1, with
Kd values of 95–178 pM in all but 1 cell line (Fig. 3). Numbers of
IL-1R ranged approx. 400–5,500 per cell. Only 86-HG-39 showed
no specific binding of hIL-1. These experiments revealed a good
overall agreement between IL-1 and IL-1R expression on the
mRNA and the protein levels.
Studies on the functional role of IL-1 in glioblastoma cell lines
Having established the existence of expression of both IL-1 and
IL-1Rs in tissue sections and cell lines of human glioblastomas, we
were interested to understand the functional role of the IL-1
network in glioblastomas. Thus, we performed experiments using
different assays for cell growth under the influence of rhIL-1 family
members.
Using a colony growth assay in semi-solid media (HTCA), we
compared the activity of rhIL-1a with rhIL-1b and found both to
TABLE II – EFFECT OF rhIL-1a ON TRITIATED THYMIDINE UPTAKE OF HUMAN
GLIOBLASTOMA CELL LINES IN VITRO
rhIL-1a
(ng/ml)
Control
0.1
1.0
10.0
Cell lines
88-HG-14
100.0 (3,071.3 6
101.0
63.02
27.02
87-HG-31
605.5)1
100.0 (5,467.1 6 943.3)
97.0
49.02
49.02
Cells were grown at 1% FCS for 24 hr for synchronizing, detached
with EDTA and incubated with vehicle (control) or rhIL-1a for 48 hr.
Tritiated thymidine was added for the last 6 hr before freezing/thawing
and processing/counting (for details, see ‘‘Material and Methods’’).
1Values are shown as means of 6-fold assays 6 SD (SD , 20%)
expressed as percentage of controls (controls in cpm in parentheses).–2p , 0.01, when compared with control (Mann-Whitney test).
be equally effective (details not shown; for examples, see Fig. 4b).
For most of the following experiments, therefore, we used rhIL-1a.
The IL-1R-negative cell line 86-HG-39 was the only cell line not
influenced by rhIL-1, whereas the first plating efficiency (PE1 ) was
significantly and dose-dependently down-regulated by rhIL-1 in -2
and up-regulated in the remaining 3 cell lines (Fig. 4a).
Formation of a colony generally can reflect self-renewal of
clonogenic cells or mitosis with consecutive growth arrest and/or
differentiation. The same end point of an HTCA can indicate more
than one distinct biological event with consequent fundamental
differences for tumor biology. Thus, this read-out system must be
interpreted with caution, particularly when an experimental condition yields higher numbers of colonies over controls. To address
this question, we removed single colonies from rhIL-1-incubated
and control cultures with micropipettes and directly and serially
replated them as single-cell suspensions. Testing serial PE of 1 cell
line (87-HG-31) up-regulated by rhIL-1 in PE1 and 1 downregulated (88-HG-14) cell line by this method, we observed
uniform and drastic reduction of colony formation by rhIL-1 in
both lines already in PE2 (Fig. 4b). This reduction reached almost 1
log step. We therefore conclude that IL-1 generally down-regulates
the self-renewal of clonogenic glioblastoma cells.
To show IL-1 specificity of these observations, we used rhIL-1ra
and soluble human IL-1R types I and II for blocking experiments.
All 3 agents were able to dose-dependently reverse modulation of
colony formation by rhIL-1 (Fig. 4c).
Tritiated thymidine uptake was down-regulated by rhIL-1 in the
cell lines studied (Table II), and this effect was uniform for
87-HG-31 and 88-HG-14. We did not perform tritiated thymidine
uptake assays with HTB 17 and HTB 14 since terminal differentiation by rhIL-1 has been described for these cell lines (Tanaka et al.,
1994). To clearly link this inhibition of proliferation by rhIL-1 to
ligand–receptor interaction, we used rhIL-1ra also in this assay
system. This receptor antagonist was able to completely reverse the
rhIL-1 effects when up to 100-fold molar excess concentrations
over rhIL-1 were used (Table III). IL-1 antagonism by IL-1ra in
other biological systems also requires more than a 10-fold molar
excess of the receptor antagonist (our HTCA results above and
INTERLEUKIN-1 NETWORK IN GLIOBLASTOMA
Arend et al., 1990; Dinarello, 1991; Granowitz et al., 1991a, 1992).
As in the HTCA system, rhIL-1ra was able to override rhIL-1
activity and stimulated proliferation as measured by tritiated
thymidine uptake in a cell line producing high amounts of IL-1
(88-HG-14; Table III).
We counted cells to measure the activity of rhIL-1 during
incubation periods of up to 168 hr. In rapidly growing cell cultures,
rhIL-1 induced a significantly ( p , 0.05, Mann-Whitney test)
lower increase of cell numbers in comparison with controls (e.g.,
87-HG-31: mean [6-fold assay] number of cells 3 104 per
well 6 SD; control/rhIL-1 cultures after 24 hr, 7.8 6 1.8/4.9 6 1.8;
after 72 hr, 44.8 6 5.0/37.3 6 2.6; after 168 hr, 68.9 6 6.4/
51.0 1 9.4). Where counting the cells after Trypan blue dye
staining in parallel cultures after incubation times of up to 168 hr,
we observed that cell viability remained unchanged by rhIL-1
(details not shown). FACS analysis of a few cell lines also has not
revealed cell death after short-term incubation with rhIL-1 (details
not shown).
Together, these experiments indicate that IL-1 acts merely
cytostatically and antiproliferatively but does not induce cell death.
Expression and function of IL-1ra in glioblastomas
Since we have shown expression and production of IL-1 and
IL-1R in glioblastomas and growth modulation by rhIL-1 in the
same tumors, our results suggest the presence of public autocrine
loops for IL-1 leading to growth inhibition in some glioblastomas.
The existence of such growth-inhibiting loops in a rapidly growing
malignant tumor is hard to understand. Thus, we have looked for
possible escape mechanisms. In several experiments, rhIL-1ra not
only was able to reverse rhIL-1-induced growth modulation but
alone could modulate glioblastoma growth in comparison with
controls in cell lines producing IL-1 (see above), making this
member of the IL-1 family a possible candidate for promotion of
malignant growth.
Using ISH, we found expression of hIL-1ra message in all 8
evaluable (for reasons of being non-evaluable, see above) glioblastoma tissue sections (Fig. 1) and in the majority of our glioblastoma
cell lines also expressing hIL-1 and hIL-1R (details not shown).
Expression was not equally distributed in all tumor cells but
occurred in patches of positive cells and/or in single cells distributed throughout the tumor (Fig. 1). Details of the distribution
of positive cells within a tumor are under further investigation. ISH of tissue sections revealed signals for hIL-1ra after
shorter exposition time (21 days) and of higher intensity in the
positive cells than for IL-1 or the receptors. However, some
members of the IL-1 network, such as the type II receptor, revealed a much higher percentage of positive cells (details not
shown).
Testing our cell lines with RT-PCR, we found expression of
message of the intracellular or secreted forms of IL-1ra in all but 2
1073
cell lines, the IL-1R-negative line (86-HG-39) being among the 2
non-transcribers (details not shown). Using ELISA, IL-1ra was
found also in the supernatant under constitutive conditions in 4 of 6
lines, the IL-1R-negative line again being among the 2 nonproducers (Table I). There was good correlation between PCR and
ELISA results. HTB 17 was the only cell line not producing IL-1ra
but secreting IL-1 and possessing IL-1R, including expression of
type II receptors.
To study the functional role of autocrine IL-1ra secretion, we
performed HTCA and tritiated thymidine uptake experiments with
neutralizing antibodies against hIL-1ra. This neutralizing antibody
against IL-1ra significantly down-regulated growth of IL-1- and
IL-1ra-producing glioblastoma cells (88-HG-14) in both assays
(see Table IV for results of tritiated thymidine uptake). In contrast,
this antibody showed no non-specific toxicity in cell lines not
producing IL-1ra (not shown). We have interpreted these results as
reconstituting efficacy of growth-inhibiting IL-1 loops by blocking
autocrine IL-1ra activity. These results convincingly show autocrine loops for IL-1ra which counteract the negative growth
regulation by autocrine IL-1 loops in some glioblastomas. Further
evidence for this conclusion comes from experiments showing
inefficiency of neutralizing antibodies against hIL-1a or hIL-1b in
growth modulation of glioblastoma cells producing autocrine
IL-1ra (Table IV, almost identical HTCA results not shown in
detail). Thus, the autocrine production of IL-1-antagonizing molecules such as IL-1ra represents a basic escape mechanism
supporting malignant growth in some glioblastomas.
DISCUSSION
Our results show that the majority of human glioblastomas
express IL-1a, IL-1b or both, as well as IL-1R types I or II or both,
in situ (Fig. 1). This also holds true for human glioblastoma cell
lines (Fig. 2), which can produce and secrete either IL-1a or IL-1b
or both (Table I) and possess functional high-affinity receptors for
IL-1 (Fig. 3). Addition of exogenous rhIL-1 down-regulates growth
of IL-1R-positive cell lines in a variety of assays (Fig. 4, Tables II,
III), and this can be blocked by antagonist members of the IL-1
family, such as rhIL-1ra (Table III, Fig. 4). This receptor antagonist
alone can modulate glioblastoma growth in comparison with
controls in the cell lines producing IL-1 (Table III).
Our results clearly demonstrate the presence of public autocrine
loops for IL-1 in human glioblastomas leading to growth inhibition.
Interestingly, autocrine production of antagonist members of the
IL-1 network, such as IL-1ra, can block this IL-1 activity (Tables I,
IV). Thus, by blocking growth inhibition of autocrine IL-1, this
represents a basic escape mechanism allowing and supporting
malignant growth of some glioblastomas (Tables III, IV). Caution,
however, is necessary when using the term ‘‘autocrine’’ since the
distribution of the single IL-1 family members and the receptors is
not homogeneous and paracrine activity may be operative as well.
TABLE III – BLOCKING OF THE rhIL-1a (10 ng/ml) EFFECT ON TRITIATED
THYMIDINE UPTAKE INTO HUMAN GLIOBLASTOMA CELL LINES BY rhIL-1ra
Condition
Control
rhIL-1a
rhIL-1a 1 rhIL-1ra
Cell lines
88-HG-14
87-HG-31
4,016.9 6 293.6
(100.0)1
15.02
137.02
26,478.3 6 1,452.0
(100.0)
78.02
103.0
Cells were grown at 1% (88-HG-14) or 10% (87-HG-31) FCS for 24
hr, detached with EDTA and incubated with vehicle (control), rhIL-1a
(10 ng/ml) or rhIL-1a plus 100-fold molar excess (over IL-1a)
concentration of rhIL-1ra for 24 hr. Tritiated thymidine was added for
the last 6 hr before freezing/thawing and processing/counting (for
details, see ‘‘Material and Methods’’).
1Values are shown as means of 6-fold assays 6 SD for the controls in
cpm (percentage in parentheses) and as percentage of controls for all
other conditions (SD , 20%).–2p , 0.01, when compared with control
(Mann-Whitney test).
TABLE IV – INFLUENCE OF NEUTRALIZING ANTIBODIES AGAINST hIL-1ra AND
hIL-1 ON TRITIATED THYMIDINE UPTAKE OF A HUMAN GLIOBLASTOMA CELL
LINE (88-HG-14) PRODUCING IL-1ra AND IL-1
Antibody
Cell line 88-HG-14
Control
Anti-IL-1ra
Anti-IL-1a
Anti-IL-1b
37,169 6 3,698 (100.0)1
30.32
82.0
86.0
Cells were grown at 1% FCS for 24 hr for synchronizing, detached
with EDTA and incubated with vehicle (control) or with antibody at
concentrations of up to 1 mg/ml each for a further 24 hr. Tritiated
thymidine was added for the last 6 hr before freezing/thawing and
processing/counting (for details, see ‘‘Material and Methods’’).
1Values are shown as means of 6-fold assays 6 SD for the controls in
cpm (percentage in parentheses) and as percentage of controls for the
antibody conditions (SD , 10%).–2p , 0.01, when compared with
control (Mann-Whitney test).
1074
OELMANN ET AL.
There have been several reports on growth stimulation and
growth inhibition of glioblastomas caused by addition of IL-1
(Bertoglio et al., 1987; Lachman et al., 1987; Tanaka et al., 1994),
the reports on growth inhibition and terminal differentiation clearly
prevailing. Other reports have noted production of IL-1 by
astrocytoma and glioma cell lines (Fontana et al., 1982; Lee et al.,
1989) and expression of IL-1 mRNA in primary brain tumors
(Merlo et al., 1993). IL-1ra mRNA expression has been observed in
some human glioblastoma tissue sections (Tada et al., 1994). We
present here a comprehensive study on the expression and functional role of agonist and antagonist members of the IL-1 family in
human glioblastoma.
IL-1ra amounts found to be produced by some of the cell lines
are rather high in comparison with IL-1 (Table I). In this respect, it
is of interest that IL-1 antagonism by IL-1ra in other biological
systems also requires more than a 10-fold molar excess of the
antagonist (Arend et al., 1990; Dinarello, 1991; Granowitz et al.,
1991a, 1992). Additionally, peak plasma concentrations of IL-1ra
in endotoxinemia were found to be approximately 100-fold higher
than IL-1 (Fischer et al., 1992; Granowitz et al., 1991b). However,
cell lines may acquire altered properties in vitro and may not be
totally representative for the tumor biology in situ. ISH of tissue
sections revealed IL-1ra signals after shorter exposition time and of
higher intensity in the positive cells than for IL-1 or the receptors,
but some members of the IL-1 network, such as the type II receptor,
revealed a much higher percentage of positive cells. Higher
expression of IL-1ra message in the cell lines over the tissue
sections prompted the hypothesis of the cell lines having developed
from IL-1ra-positive cells of a tumor. Additionally, 1 cell line
expressing the IL-1 loop (HTB 17) did not produce detectable
amounts of IL-1ra, but did express mRNA for hIL-1R II. Further
studies are needed to show whether other escape mechanisms, such
as the production of soluble IL-1Rs, may be operative in this line.
Soluble extracellular domains of IL-1R II have been described to
be produced and secreted and to act as antagonist members of the
IL-1 network (Colotta et al., 1993; Symons et al., 1995). It is not
possible from our study to comparatively judge the importance of
the single antagonist members of the IL-1 network in situ.
Further studies must test the hypothesis of antagonist members
of the IL-1 network, such as IL-1ra, as possessing proto-oncogene/
oncogene function for these tumors but may also shed some light
on the concept of tumorigenesis as representing aberrant wound
healing (Marshall et al., 1992). Normal brain expresses IL-1 and
IL-1R, and IL-1 has been discussed as being important for brain
development and response to brain injury (Giulian and Lachman,
1985; Rothwell, 1991) including wound healing. Glioblastoma
development in scar tissue has been reported (Gruss et al., 1993),
though there is no sound statistical basis for a correlation. IL-1 has
a role in wound healing (Giulian and Lachman, 1985), and genetic
alterations leading to over-expression and production of IL-1ra
could represent an important step toward malignant aberration.
These observations and hypotheses do not exclude the existence
of other mechanisms allowing or facilitating malignant growth of
glioblastoma. Interestingly, we have observed 1 cell line which has
lost IL-1 control and expresses neither the receptor nor the ligands
(86-HG-39). Differentiating autocrine loops have been described
for TGF-b in glial progenitor cells (McKinnon et al., 1993). With
the exception of some morphological changes, we have no clear
evidence for induction of differentiation or senescence by autocrine
IL-1 in our experiments, and further studies in this area are
necessary. This (McKinnon et al., 1993) also draws attention to the
fact that multiple growth factors can influence glioblastoma
growth, some of them acting synergistically (Merzak et al., 1995).
In this respect, it is of interest that our glioblastoma cell lines
constitutively express messages for cytokines known to act antiproliferatively such as TGF-b and TNF (data not shown). The
meaning of this observation in a rapidly growing cell line must be
further studied.
Expression of IL-1ra has been reported in endometrial cancer
(Van Le et al., 1991) and bronchogenic carcinoma (Smith et al.,
1993). In both studies there was higher expression of IL-1ra in
tumor cells than in normal tissues (Smith et al., 1993; Van Le et al.,
1991). Thus, our studies in glioblastoma may serve as a model, and
we have begun to investigate the role of IL-1 antagonists such as
IL-1ra in other tumor histologies. However, the activity of IL-1 in
malignant disease is diverse and at times contradictory. Thoughtful
and detailed studies clearly indicate a role for this cytokine in
stimulating proliferation in tumors of epithelial origin (Hamburger
et al., 1987; Ito et al., 1993; Lahm et al., 1992; Woodworth et al.,
1995; Zeki et al., 1993), and each tumor type may be regulated
individually. Indirect effects of this cytokine, such as augmentation
of metastasis (Bani et al., 1991) through altered integrin expression
(Garofalo et al., 1995) and their blockade by IL-1ra (VidalVanaclocha et al., 1994), must be taken into consideration when the
role of the IL-1 network in progression of malignant disease in vivo
is assessed.
There is one more aspect of our results. Human tumor-cloning
assays are widely used to estimate the cytostatic and cytotoxic
potential of new drugs (Von Hoff, 1990). HTCA has been used in
our study since it has been shown to reliably detect growth
modulation of tumor cells by cytokines and to be predictive for in
vivo tumorigenicity and modulation of in vivo tumor growth by
cytokines (Freedman and Shin, 1974; Gross et al., 1988; Topp et
al., 1993). However, as with other biological read-out systems,
caution is necessary when HTCA results are interpreted, and this
holds particularly true when any kind of stimulation is observed. As
for hemopoiesis, careful studies are necessary to distinguish
between stimulation of self-renewal and stimulation of mitotic
divisions before growth arrest or during differentiation. Such
studies unequivocally have revealed negative growth regulation by
IL-1 in our as well as in other investigations (Tanaka et al., 1994)
with glioblastomas. Thus, equating up- and down-regulation of
colony formation by cytokines such as IL-1 in varying percentages
with ‘‘growth’’ without further in-depth investigation of the
biological meaning of this read-out may be misleading (Hanauske
et al., 1992; Koch et al., 1995).
In conclusion, we have shown that autocrine IL-1ra is overexpressed by some human glioblastoma cell lines and can support
malignant growth of these tumors by blocking growth-inhibiting
autocrine loops of IL-1. Our finding must be further studied in this
and other tumor entities to learn more about the role of the IL-1
network in malignant disease, particularly since therapeutic interventions, e.g., with ribozymes against IL-1 antagonists such as
IL-1ra, can be envisaged.
ACKNOWLEDGEMENTS
The authors thank Dr. J.E. Sims (Immunex, Seattle, WA) for
kindly supporting this work.
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