IL-6 and related cytokines such as OSM, (
)LIF, IL-11,
and ciliary neurotrophic factor play important roles in the immune
system, hematopoiesis, the nervous system, and acute phase
reactions(1) . These cytokines are structurally similar, having
a predicted four
-helical bundle structure, and show overlapping
spectra of individual activities (1, 2, 3, 4) . Their biological
activities are mediated through initial low affinity binding to cell
surface receptors, which are specific for their respective ligands yet
structurally
related(1, 2, 5, 6, 7) . In
addition, they share a receptor subunit, the transmembrane glycoprotein
gp130, which is responsible for initiating signal
transduction(3, 5, 8) . Once the receptor
complex is fully formed, the ligand binding affinity is much
increased(5) . For example, the K
of IL-6 for receptor alone is 1 nM but increases to 45
pM when gp130 is engaged(9) . The combination of
expression of specific cytokine receptors by particular cell types with
a sharing of the ubiquitously expressed gp130 (10) to enable
signal transduction provides some explanation for the observed
functional overlap or biological redundancy between members of this
cytokine family. The sharing of gp130 thus provides a useful target by
which to inhibit the function of this cytokine family(8) ,
members of which have been implicated in the pathology of several
diseases, such as AIDS, cancer, and autoimmune diseases(1) .
In this paper we describe the activity of a protein discovered in an
extract of the marine sponge Callyspongia, which inhibits the
formation of receptor complexes involving gp130, consequently
inhibiting the bioactivity of IL-6 and other members of this cytokine
family.
EXPERIMENTAL PROCEDURES
Cells and Reagents
U266 (human myeloma), HepG2
(human hepatoma), and SKW6.4 cells (human Epstein Barr
virus-transformed B cell) were all obtained from ATCC (Rockville, MD).
U266 and SKW6.4 cells were maintained in RPMI 1640 with 10% fetal
bovine serum and 1% each of penicillin, L-glutamine, and
streptomycin. HepG2 cells were maintained in Earle's minimal
essential medium supplemented with 10% fetal bovine serum, 1%
penicillin, streptomycin, L-glutamine, nonessential amino
acids, and sodium pyruvate (Life Technologies, Inc.). Human recombinant
cytokines, sIL-6R and sgp130, were purchased from R & D Systems.
Sponge Collection, Identification, and Preparation of
Extract
Sponges collected by scuba were identified by
microscopic evaluation of spicules and fiber architecture. The three
sponges used here were species of Callyspongia (Porifera,
Desmospongiae, Haplosclerida, Callyspongiidae). C. armigera (sample 3-XII-92-2-004) and C. vaginalis (sample
3-XII-92-4-002) (11) were collected at 100-foot and 20-foot
depths, respectively, off south Eleuthera, Bahamas. The third,
designated Callyspongia sp. (sample 16-VIII-90-1-003), was
collected at 50-foot depth off Ponta Santa Cruz, Madeira, and is most
closely related to C. simplex(12) . The major
difference is in the size/morphology of the oxeas; however, since this
can be influenced by environmental factors, the sponge may be
conspecific with C. simplex. Taxonomic vouchers for the three
sponges have been deposited in the Harbor Branch Oceanographic Museum
(catalog numbers 003:00923, 003:00924, and 003:00922, respectively).
Samples were stored at -20 °C until extraction. Except where
stated, all results pertain to experiments with Callyspongia
sp. extract. About 10 g wet weight of frozen sponge was
used to prepare extracts. With mortar and pestle set in a dry ice bath,
the sponge was powdered, 20 ml of ice-cold PBS-A was added, the
preparation was freeze-thawed 3 times, and centrifuged at 20,000
g for 30 min. The pellet was resuspended in 10 ml of
buffer and centrifuged again. Supernatants were pooled and frozen at
-20 °C. Total protein concentration of the mixture present in
the extract was estimated with the BCA assay (Pierce Chemical Co.),
using a BSA standard.
Bioassays
Functional activity of IL-6 was assessed
through the increase in haptoglobin secretion by HepG2 cells and
induction of IgM secretion by SKW6.4 cells. The HepG2 assay was adapted
from methods already described(13) : HepG2 cells grown to
confluency in 96-well plates were treated for 4 h with 10 ng/ml IL-6 in
100 µl of their normal growth medium, with or without extract, in
quadruplicate. Controls were included where no IL-6 was added. After a
further 48 h of culture in normal medium, the medium was removed for
assay of haptoglobin. The assay using SKW6.4 cells was set up in an
analogous fashion, using cells in log phase growth and 10,000 cells/200
µl of medium; again, cells were treated with cytokine and inhibitor
for 4 h and after a further 72-h incubation, the medium was removed for
IgM assay. Cell proliferation was measured (MTT) using a CellTiter96
assay kit (Promega).
Immunoassays
Haptoglobin was measured by
radioimmunoassay as described previously(13) . The working
range was 0.1-10 ng/ml haptoglobin. IgM was measured using a
``sandwich'' enzyme-linked immunosorbent assay method. Goat
anti-human IgM (Boehringer Mannheim Biochemicals) was bound to 96-well
plates as first antibody, samples or standard IgM was incubated in the
wells for 2 h; the same antibody conjugated to alkaline phosphatase was
used to detect bound IgM, and the working range was 50-500 ng/ml.
Binding of
I-IL-6 to Cells
HepG2
cells were plated into 35-mm Petri dishes and grown to almost 100%
confluency (2
10
cells). All procedures were
carried out with 1 ml of normal cell growth medium. After 2 washes,
binding of 10 pM
I-IL-6 (approximately 100
µCi/µg; DuPont NEN) was carried out at 4 °C for 4 h. Cells
were washed 3 times, then solubilized at 37 °C in 1 ml of 1 M NaOH with 0.1% SDS before counting. For U266 cells, a suspension
cell line, binding (10 pM
I-IL-6) to 10
cells in a 250-µl volume was compared at 4 °C for 4 h or
at 20 °C for 2 h. Unbound IL-6 was removed by rapid filtration
through Whatman GF/C glass fiber filters presoaked in 2% nonfat dried
milk solution. The filters were washed twice in cold 0.9% NaCl and
assessed for radioactivity. Results were found to be very similar for
both conditions, and the 2-h binding method was selected for all
further U266 experiments. In typical experiments, K
values for HepG2 and U266 cells were determined as 119 pM and 70 pM respectively, similar to values already
published by others for the high affinity site for IL-6 binding on U266
cells(9) . In contrast to this study, however, Scatchard
analysis demonstrated no significant numbers of low affinity sites on
our clone of U266 cells (data not shown).
Binding Conditions for Forming Complexes of
I-IL-6, sIL-6R, and sgp130
A constant input (7
pM, 20,000 cpm) of
I-IL-6 was mixed with varying
amounts of sIL-6R from 1 nM to 2 pM in 200 µl of
PBS-A containing 1% BSA. Binding of IL-6 to sIL-6R (2 h/20 °C) was
assessed by immunoprecipitation using an excess of non-neutralizing
mouse anti-IL-6R (Biosource) bound to rabbit anti-mouse IgG coupled to
PMPs (Ciba Corning Diagnostics). PMPs were washed 3 times with 0.1 M Tris-HCl, pH 7, + 0.1% Tween 20 before counting.
Background binding, assessed by omitting sIL-6R, was around 700 cpm. A
concentration of 125 pM sIL-6R gave total binding around 2,000
cpm; this was then used to measure the increase in IL-6 binding when
sgp130 was included. Through a similar titration method, 540 pM sgp130 was found to increase total binding to about 3,000 cpm.
These conditions were used to examine the mechanism of action of
inhibitors of IL-6 function. In some experiments, when sgp130 was
present, receptor-ligand complexes were pulled down using polyclonal
goat anti-gp130 (R & D Systems) on rabbit anti-goat IgG PMPs
(Perseptive Diagnostics); this method gave a similar pattern of results
to experiments with anti-IL-6R PMPs.
RESULTS AND DISCUSSION
Inhibition of Binding
The Callyspongia
sp. extract (estimated total protein 0.5 mg/ml) inhibited
I-IL-6 binding to HepG2 cells in a dose-dependent manner
as illustrated in Fig. 1, giving 50% inhibition at 1:6,200. This
extract, and those of the two other Callyspongia organisms
tested, showed similar inhibition of IL-6 binding in U266 binding
(IC
of 1:1,600 for C. armigera, and 1:3,200 for C. vaginalis, compared with 1:5,900 for Callyspongia
sp.). The extract had no effect on the binding of
I-IL-1 to its receptor on EL4.6.1 cells or of
I-IL-4 to the IL-4R of Raji cells (data not shown),
indicating ligand/receptor specificity.
Figure 1:
Inhibition of
I-IL-6
binding to HepG2 cells by dilutions of the sponge extract. Input was
30,000 cpm, maximum binding (no extract) was 2,300 cpm; results are
expressed as percent of total binding. Each point was carried out in
triplicate, and the error bars represent the
S.D.
Activity in Functional Assays for IL-6
HepG2 cells
respond to cytokines such as IL-6 and IL-1 by increased production of
certain acute-phase proteins, e.g. haptoglobin. IL-1 has been
reported to up-regulate haptoglobin production by HepG2 cells less
effectively than IL-6(14) , which we also observed; however,
maximum up-regulation of haptoglobin by IL-6 or IL-1 was found to occur
at about 10 ng/ml for both cytokines (data not shown). To discover
whether inhibition of binding by the extract was due to the presence of
an agonist or an antagonist for IL-6, HepG2 cells were incubated for 4
h with extract dilutions plus 10 ng/ml IL-1or IL-6, or neither. After a
further 48 h, haptoglobin was measured (Fig. 2A). The
extract inhibited IL-6-stimulated up-regulation of haptoglobin in a
dose-dependent fashion, with an IC
of 1:430, but did not
affect the basal production of haptoglobin when present without IL-6,
indicating that the extract was neither cytotoxic to HepG2 cells nor a
general protein synthesis inhibitor. As expected, IL-1 increased the
level of haptoglobin secretion to a lesser extent than IL-6; however,
this increase was not responsive to the presence of extract. In other
experiments (Fig. 2B), the extract inhibited the
IL-6-induced production of IgM by SKW6.4 cells, with an IC
of 1:1,050. These cells were also tested using the MTT assay, and
the extract did not change the rate of cell proliferation or affect
their viability.
Figure 2:
Inhibition of cellular responses to IL-6
by sponge extract. A, HepG2 cells were incubated in the
presence of extract dilutions and 10 ng/ml IL-6 (
), IL-1
(
), or no cytokine (
). Haptoglobin produced in the absence
of sponge extract was 38.9 ± 1.2 ng/ml (+IL-6), 22.5
± 1.3 ng/ml (+IL-1), and 17.8 ± 1.5 ng/ml (no
cytokine). Each point is the mean of 4 replicates; error bars represent S.D. B, SKW6.4 cells were incubated in the
presence of extract dilutions and 10 ng/ml IL-6 (
) or no cytokine
(
); each point is the mean of 4 replicates, and the S.D. was
always less than 12%. Mean IgM produced in the absence of extract was
824 ng/ml (+IL-6) and 62 ng/ml (no
IL-6).
The results in functional assays indicate that the
inhibition of IL-6 binding to cells was due to an antagonist present in
the extract. Using
I-IL-6 binding to U266 cells to follow
activity, it was shown that the inhibition was completely lost after
heating the extract for 5 min at 100 °C and after treatment at 37
°C with 1% trypsin for 10 min. Activity was >80% retained by
ultrafiltration membranes (Amicon) with M
retention of 10,000 and by cellulose dialysis membranes of
12,000-14,000 M
cut-off. Activity could be
concentrated by precipitation with 50% saturated ammonium sulfate
solution, with a 40% recovery of activity in the precipitate. In
addition, the heated extract was checked in the HepG2 functional assay
and found to be completely inactive. These data indicated that the
activity was due to a protein present in the sponge extract.
HepG2
cells were preincubated for 1 h at 4 °C with extract at 1:1000,
which was then removed, and the cells were washed. Subsequent binding
of
I-IL-6 to these cells was inhibited by an amount (63.7
± 0.9%) similar to that achieved by a 4-h co-incubation with
extract (Fig. 1). Furthermore, the inhibitory activity contained
in the extract taken off the cells was found to be progressively
reduced in subsequent 1-h incubations at 4 °C with fresh HepG2
cells, from 63.7 ± 0.9% to 59.1 ± 0.9% after the first
absorption, to 46.4 ± 2.8% after a second. This experiment
suggested that the inhibitor bound to a component of the complex on the
cell surface rather than to IL-6 itself.
Dissection of the Receptor Complex
In order to
find out which part of the receptor complex was being inhibited by the
extract, the soluble forms of the IL-6R and gp130 were used to set up a
I-IL-6 binding assay. We used anti-IL-6R on PMPs to
immunoprecipitate complexes of IL-6
sIL-6R or of
IL-6
sIL-6R
sgp130 formed in the presence or absence of
extract (Fig. 3A). No change in binding of IL-6 to
sIL-6R could be detected in the presence of extract; however, it
abolished the enhancement of IL-6 binding conferred by sgp130 in a
dose-dependent manner. Precipitation of complexes containing sgp130
with anti-gp130 PMPs also demonstrated dose-dependent inhibition of
binding by extract (Fig. 3B).
Figure 3:
Immunoprecipitation of
IL-6
sIL-6R
gp130 complexes. A,
I-IL-6
was incubated with sIL-6R with (
) or without (
) sgp130, in
the presence of extract dilutions. Immune complexes were pulled out of
solution with anti-IL-6R on PMPs. Nonspecific binding was determined by
running parallel determinations excluding sIL-6R, for each dilution of
extract, and this was subtracted from the results. B,
I-IL-6 was incubated with sIL-6R and sgp130 (
), in
the presence of extract dilutions. Immune complexes were pulled out of
solution with anti-gp130 on PMPs. Nonspecific binding was determined by
running parallel determinations using PMPs with no anti-gp130 antibody,
for each dilution of extract, and this was subtracted from the results.
For A and B, points were run in triplicate, and the error bars represent S.D.
Activity in Functional Assays with Other IL-6 Cytokine
Family Members
Since OSM and LIF have both been demonstrated to
increase acute-phase protein production by hepatoma cells and also
utilize gp130 to transduce signals(8) , we examined whether the
sponge extract would also inhibit these cytokines in the HepG2 assay.
We found that a 4 h ``pulse'' of OSM and LIF up-regulated
haptoglobin production by HepG2 cells in extent and dose range closely
analogous to IL-6, so the effect of the extract on the stimulation of
HepG2 cells by IL-6, OSM, or LIF, all at 5 ng/ml, was compared (Fig. 4). All three cytokines were inhibited by the extract, but
the maximum degree of inhibition of OSM and LIF achieved in these
experiments (around 35%) was lower than for IL-6 (nearly 90%). IL-6
requires a disulfide-linked homodimerization of two gp130 molecules to
elicit signal transduction (15) , whereas the current evidence
regarding OSM and LIF function indicates that only one gp130 molecule
is included in the receptor complex(1) . This suggests that,
even if the site of interaction of the various receptor-ligand
complexes with gp130 were identical and is blocked by the extract
protein, it would be more difficult for an IL-6
IL-6R complex to
compete with a gp130 inhibitor and acquire sufficient interactions with
gp130 to complete the signaling circuit than it would be for OSM or
LIF. Thus, the extract protein would be expected to be more inhibitory
for IL-6 than for OSM or LIF, and this is borne out by our experimental
findings.
Figure 4:
HepG2 cell stimulation by other cytokines.
HepG2 cells were incubated with 5 ng/ml IL-6 (
), OSM (
), or
LIF (
) in the presence of extract dilutions, and haptoglobin
production was expressed as percent of haptoglobin produced in response
to each cytokine when no extract was present (37.1 ± 4.4 ng/ml
for IL-6, 38.1 ± 2.1 for OSM, and 41.7 ± 5.1 ng/ml for
LIF). Each point represents the mean of at least 4 determinations; S.D.
was <12% of the mean.
The overlapping structural relationships of cytokine
families and of their corresponding receptors have in the last few
years excited much interest and speculation concerning their
evolution(2, 7) . Sponges are of an ancient lineage of
multicellular vertebrates which have existed for about 500 million
years. It is quite possible that the Callyspongia protein
whose activity we describe, which connects with the IL-6 family of
cytokines through interaction with gp130, will turn out to be
structurally related to receptor or cytokine. Thus, identification of
this sponge protein will provide not only a possible lead for a
functional inhibitor for these cytokine and receptor families, but may
in addition yield valuable information concerning their evolutionary
pathways.