(Received for publication, August 25, 1995; and in revised form, September 27, 1995)
From the
A stromal protein, designated restrictin-P, that specifically kills plasma-like cells was purified to homogeneity and shown to be identical with activin A. The specificity to plasma-like cells stemmed from the ability of restrictin-P/activin A to competitively antagonize the proliferation-inducing effects of interleukin (IL) 6 and IL-11. Restrictin-P further interfered with the IL-6-induced secretion of acute phase proteins by HepG2 human hepatoma cells and with the IL-6-mediated differentiation of M1 myeloblasts. A competition binding assay indicated that restrictin-P did not interfere with the binding of IL-6 to its receptor on plasma-like cells, suggesting that it may act by intervening in the signal transduction pathway of the growth factor. Indeed, concomitant addition of restrictin-P and IL-6 to cytokine-deprived B9 hybridoma cells was followed by sustained overexpression of junB gene until cell death occurred, while IL-6 alone caused a transient increase only. This altered response to IL-6 stimulation was accompanied by a moderate increase in STAT protein activation. Thus, in this study, we identified the plasmacytoma growth inhibitor, restrictin-P, as being activin A of stromal origin. It is shown that activin A is an antagonist of IL-6-induced functions and that it modifies the IL-6 signaling pattern.
Regulation of hemopoiesis is mediated by cytokines that act
through distinct mechanisms. Some, like colony-stimulating factors
(CSFs) ()promote accumulation of hemopoietic cells by
inducing proliferation coupled with differentiation(1) .
Others, like tumor necrosis factor, may cause cell cycle arrest and
thus limit cell accumulation(2) . The outcome of the
interaction between the growth factor and the cell often depends on the
nature of the target cell; as it is with transforming growth factor
(TGF)-
, the same cytokine may be stimulatory to one cell type and
inhibitory to the other (reviewed in (3) ). Whereas some
inhibitors operate by slowing down cell growth (4) or by
induction of terminal differentiation(5) , others cause cell
death (6) by inducing apoptosis (7, 8, 9, 10) . Restrictin-P has
formerly been described as an inhibitor of plasmacytoma cell
growth(11, 12) . The biological activity of this
factor was first noticed through the selective ability of primary
stromal cells to slow down the proliferation of plasmacytoma
cells(13, 14) . A similar function was exhibited by
trypsin-released proteins obtained by mild treatment of a bone
marrow-derived stromal cell line of mouse origin
(MBA-2.1)(12) . The released crude protein mixture inhibited
the growth of a series of plasmacytomas and hybridomas but did not have
significant effects on the growth of a variety of other leukemia cell
lines of lymphoid, erythroid, and myeloid origin(12) .
Similarly, no effect was observed on normal cell populations such as
bone marrow cells responding to colony-stimulating factors or spleen
cells induced by mitogens(12) . This unique specificity
prompted us to isolate the active component. However, factor(s)
mediating the growth inhibition were found to be produced by the stroma
cell line in minuscule amounts, and it was necessary to establish
conditions for large scale production of the factor. We found that the
producer cell line MBA-2.1 could be propagated on a three-dimensional
carrier of nonwoven fabric of polyester loaded in a bioreactor system
under complete protein-free conditions(15) . The study of such
bioreactors showed that the cells could be maintained under
protein-free conditions for up to 10 months while producing
restrictin-P activity along with TGF-
, macrophage (M)-CSF, and
IL-6. Restrictin-P obtained from the bioreactor system induced in its
target cells early G
/G
arrest, morphological
changes, and signs of cell damage characteristic of
apoptosis(11, 16) accompanied by intracellular ionic
changes(17) .
The present study was aimed at identifying restrictin-P by purifying it to homogeneity and at analyzing the mechanism by which restrictin-P exerts its specific inhibitory effect on plasma-like cells.
The inhibitory activity of restrictin-P, as detected in
conditioned media form MBA-2.1 cells, was specific to plasma-like tumor
cell lines (Table 1). A variety of other cell lines representing
different hemopoietic lineages and stages of maturation were only
slightly inhibited or were totally unaffected by this factor. To rule
out the possibility that restrictin-P activity could be ascribed to one
of the cytokines known to affect hemopoietic cells, we searched for
factors that might have restrictin-P-like activity. IL-1, IL-2, IL-3,
IL-4, IL-6, IL-7, IL-10, IL-11, M-CSF, G-CSF,
TGF-,platelet-derived growth factor, bovine fibroblast growth
factor, interferon
, and leukemia inhibitory factor were tested
over a range of concentrations. These cytokines were found to be devoid
of the ability to inhibit the growth of the MPC-11 plasmacytoma which
is highly sensitive to restrictin-P(12) . In addition,
neutralizing antibodies to TGF-
1 and -2, tumor necrosis factor,
IL-6, interferon
, and M-CSF did not reduce restrictin-P-like
activity in media conditioned by MBA-2.1 cells (results not shown).
We used the above-conditioned media in an attempt to obtain some
clue as to the mechanism by which the inhibition of plasma-like cells
is mediated. The MBA-2.1 cell-conditioned medium inhibited the growth
of the MPC-11 plasmacytoma and, as detailed below, it also interfered
with the growth-promoting effect of IL-6 on B9 cells. To determine
whether these two functions were mediated by the same molecule, it was
necessary to purify restrictin-P to homogeneity. We therefore
constructed a bioreactor production system wherein restrictin-P
activity could be observed in media conditioned by the cells in
absolute protein-free conditions(15) . A batch of 600 liters of
conditioned medium was concentrated by diafiltration and subjected to a
further step of concentration by Amicon ultrafiltration followed by
fractionation using an automated FPLC column. The fractionation
included anion exchange chromatography, gel filtration, and, finally,
two steps of purification to homogeneity by reverse phase HPLC. Fig. 1summarizes the above purification steps. As can be seen,
the last stage yielded a single peak of protein that coincided with the
biological activity of restrictin-P, i.e. the capacity to
inhibit the growth of the plasmacytoma cell line, MPC-11. PAGE analysis
of this product, under reducing conditions, revealed a single protein
band at 15 kDa (Fig. 2A). This purified peptide was
N-terminally sequenced, and the first 36 residues were
indistinguishable from those of the precursor of the A subunit of
inhibin which gives rise to a 15-kDa polypeptide (37, 38, 39) (Fig. 2B). The
dimer of this subunit is known as activin A. Fig. 3A shows a
comparison between restrictin-P and recombinant activin A of bovine
origin. It is clear that these factors have an equal ability to
suppress the growth of MPC-11 cells. Considered together, these results
suggest that restrictin-P is identical with activin A.
Figure 1:
Purification
of restrictin-P from stromal cell conditioned medium. A,
Q-Sepharose anion exchange chromatography. Processed conditioned medium
of MBA-2.1 cell line (1.5 10
units) was loaded on a
Q-Sepharose column (100 ml, XK 50). The column was washed with 1200 ml
of the initial buffer (20 mM Tris-Cl, pH 7.8, 8 ml/min), and
restrictin-P was eluted with 400 ml of 0.05 M NaCl in the
initial buffer. B, Superdex 75 gel filtration. Fractions
containing restrictin-P from the Q-Sepharose column were concentrated,
dissolved in 20 mM Hepes (pH 7.8), and loaded onto a Superdex
75 column (20/60, Pharmacia). The column was washed with 2
phosphate-buffered saline (2 ml/min). C, C-8 reversed-phase
HPLC-I. Biologically active fractions from the Superdex 75 column were
loaded onto an Aquapore RP-300 column (7 µm, 4.6
100 mm
+ 4.6
30 mm precolumn). The adsorbed proteins were eluted
from the column with a multistep gradient of aqueous acetonitrile in
0.1% trifluoroacetic acid (0.5 ml/min). D, C-8 reversed-phase
HPLC-II. Partially purified restrictin-P was rechromatographed
(RP-HPLC) under essentially identical conditions as in C above, except for an additional isocratic step at the elution time
of the protein peak (at approximately 36% acetonitrile). Broken
lines indicate the absorption at 280 nm (A and B) or 214 nm (C and D); open circles and lines show the biological activity of restrictin-P as
measured by the MPC-11 assay, and lines indicate percentage of
acetonitrile (C and D).
Figure 2: A, SDS-PAGE analysis of highly purified restrictin-P. An aliquot from the active fraction (RP-HPLC-II, Fig. 1D) was loaded on 15% SDS-polyacrylamide gel under reducing conditions (lane 1). Protein bands were viewed by silver staining. Molecular mass markers, shown on the right-hand side, are: carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; and aprotinin, 6.5 kDa. B, N terminus amino acid sequence of purified restrictin-P compared to the known sequence of activin A monomer.
Figure 3:
A, inhibition of MPC-11 plasmacytoma cell
growth by purified restrictin-P and by recombinant activin A. Cells (4
10
/well) were seeded in 96-well Falcon microtiter
plates, in 100 µl of RPMI containing 10% FCS with serial dilutions
of restrictin-P (
), activin A (
), or control growth medium
(
). Cells were incubated for 4 days, and their viability was
estimated by the MTT assay. The bar lines represent the mean
of duplicate determinations ± S.D. B, lack of effect of
restrictin-P on MC/9 (
), 14M1.1 (
), and NFS (
)
cell lines dependent for growth on IL-3, M-CSF, and GM-CSF,
respectively. Cells were seeded at 5
10
,
10
, and 2.5
10
/well, respectively, with
the indicated amount of restrictin-P and specific growth factor, and
their viability was monitored as in A.
To study the mechanism by which restrictin-P/stromal activin A inhibits plasma-like cell growth, we examined whether its effect was mediated by reversible cytostasis or through a mechanism that involves cell destruction. Restrictin-P in its unpurified form appeared to induce ionic changes that are associated with apoptosis(16) . This mode of cell death is known to occur in hemopoietic cells deprived of their specific growth factor. The B9 hybridoma is dependent for growth on IL-6 and was therefore used to examine the possibility that restrictin-P causes growth cessation by interfering with the action of the growth factor. Extensive proliferation of B9 cells was induced by 0.1 IU/well of IL-6, and only moderate growth stimulation is observed upon addition of increasing concentrations of the purified cytokine. Addition of restrictin-P to B9 cell cultures stimulated by 0.01-0.1 IU/well of IL-6 caused almost complete growth inhibition. This inhibitory effect was gradually reduced with increasing the concentration of IL-6 and was almost abolished at 200 IU/ml of IL-6 (Fig. 4A). In contrast to the ability of restrictin-P to antagonize the growth-stimulating effect of IL-6 it had no effect on the growth of 14M1.1, NFS, and MC/9 cells which are dependent for growth on M-CSF, GM-CSF, and IL-3, respectively (Fig. 3B).
Figure 4:
Restrictin-P antagonizes IL-6- and
IL-11-induced proliferation of B9 cells. Growth factor-deprived B9
cells were washed (3 times) with growth medium (RPMI + 5% FCS) and
were seeded in 96-microtiter well plates (5 10
cells/200 µl/well) in growth medium supplemented with the
indicated dilutions of recombinant human IL-6 (A, open
symbols) or with the same IL-6 dilutions and 1.5 units/well of
purified restrictin-P (A, closed symbols). A similar
experiment was set with serial dilutions of recombinant human IL-11 (B, open symbols) or with IL-11 and 0.018 unit/well
of restrictin-P (B, closed symbols). Cells were
incubated for 48 h and then pulsed with
[
H]thymidine (1 µCi/well) for 12 h. Values
represent the mean of triplicate determinations ±
S.D.
IL-11 is an additional stimulator of plasma-like cells(40) . As shown in Fig. 4B, restrictin-P inhibited the growth of IL-11-stimulated B9 cells, and this inhibition was competed out by increasing the titer of IL-11. Thus, restrictin-P counteracted the growth-stimulating effect of both IL-6 and IL-11, and these cytokines at high titers overcame the effect of restrictin-P.
Plasmacytomas are but one target cell type that
responds to IL-6 signaling. The HepG2 hepatoma release acute phase
proteins under the influence of IL-6(41) . As shown in Fig. 5, the secretion of both -acid glycoprotein and
haptoglobin induced in HepG2 cells by IL-6 was markedly reduced by
addition of restrictin-P (Fig. 5). M1 (clone 11) myeloblastic
cells differentiate into adherent monocytes under the influence of
IL-6(28, 29) . Following IL-6 induction, M1 cells
exhibit high mitochondrial activity and growth inhibition. As shown in Table 2, restrictin-P abolished the IL-6-induced effect.
Figure 5:
Restrictin-P interferes with the
IL-6-induced secretion of the acute phase proteins -acid
glycoprotein (AGP) and haptoglobin (HP). HepG2
hepatoma cells were grown to confluence and stimulated with 100
units/ml IL-6 or an equal amount of IL-6 with 312 units/ml restrictin-P (RP). Conditioned media were collected at 24 h, subjected to
PAGE (25 µl/well, 10% and 7% gels for AGP and HP, respectively) and
were tested by Western blotting for the acute phase proteins using the
corresponding antibodies.
It was concluded, therefore, that IL-6 and restrictin-P are competing on some target machinery used to generate a signaling pathway in at least 3 completely different target cell types. A candidate target molecule for restrictin-P action was the IL-6 receptor complex. We studied the possibility that restrictin-P is a receptor antagonist by testing its ability to compete with radiolabeled IL-6 for binding to its receptor on the surface of B9 cells. Fig. 6shows that ``cold'' IL-6 competed out the binding of radiolabeled IL-6 to its receptor as expected. On the other hand, restrictin-P, at a concentration that would completely abolish the growth-stimulating effect of IL-6, failed to reduce the binding of radiolabeled IL-6 to its receptor. Thus, restrictin-P does not seem to interfere with ligand binding and may therefore interfere with postreceptor event(s) within the IL-6 signaling pathway. B9 cells stimulated by IL-6 following a period of cytokine deprivation showed a transient increase in expression of the early response gene junB (Fig. 7). Restrictin-P added to such cells caused increased and sustained expression of the junB gene until 24 h post-treatment at the time cell death already occurs. A similar augmented expression of junB mRNA was observed in the MPC-11 cell line. The expression of another early response gene TIS11 was increased in B9 cells incubated with restrictin-P but was unaffected in MPC-11 cells. The effect of restrictin-P on junB expression was also observed in HepG2 hepatoma cells (not shown). In these cells, restrictin-P did not interfere with the JAK/STAT pathway (42) (Fig. 8). The data further suggest that restrictin-P moderately increased STAT activation (Fig. 8). This is surprising in view of the fact that restrictin-P abrogated the IL-6-induced secretion of acute phase proteins by HepG2 hepatoma (Fig. 5). It is implied therefore that a separate, possibly unknown pathway exists which allows cross-talk between the restrictin-P and IL-6 signaling cascades.
Figure 6:
Restrictin-P does not interfere with the
binding of IL-6 to B9 cells. B9 cells were weaned from IL-6 in the
growth medium 24 h before binding assay. A, specific high
affinity binding of a constant amount of r-Mu-I-IL-6
competed with varying amounts of crude concentrated mouse IL-6 (
)
or partially purified restrictin-P (
). Mean total r-Mu-IL-6 bound
was 1120 ± 48 cpm. B, specific high affinity binding of
a constant amount of r-Hu-
I-IL-6 mutein (
) or
purified restrictin-P (
). The mean total r-Hu-IL-6 binding was
2350 ± 68 cpm which was competed to 123 ± 30 with a
200-fold excess of crude concentrated murine IL-6. Results of the
competition binding studies are plotted as a function of fold excess
where 1 unit of restrictin-P inhibits 1 unit of IL-6 by 50% on B9
cells. Error bars indicate standard deviation of
replicates.
Figure 7: Restrictin-P causes augmented expression of junB mRNA in plasma-like cells. The IL-6-dependent B9 cells were incubated overnight without IL-6 and were then induced by 2 units/ml of this cytokine for 20 min before addition of restrictin-P (RP) (200 units/ml) or 10 mM Tris-HCl (0 min) (A). MPC-11 plasmacytoma cells were seeded with or without IL-6 and with restrictin-P as above (B). Following the time point indicated, RNA was extracted and examined by Northern blotting for junB and TIS11 transcripts.
Figure 8:
Effect of restrictin-P on STAT activation.
HepG2 cells were incubated with human recombinant IL-6 (10 units/ml)
and restrictin-P as indicated. Either IL-6 and restrictin-P were added
simultaneously or restrictin-P was added 30 min or 16 h prior to IL-6.
15 min after addition of IL-6 to the medium, the cells were harvested
and nuclear extracts were prepared. 10 µg of protein was then
analyzed in a gel retardation assay using a P-labeled
oligonucleotide probe that contained a high affinity mutant of the
c-fos promoter sis-induced element (SIE). The
positions of DNA-protein complexes containing either Stat3 and
Stat1
homodimers or Stat3/Stat1
heterodimers are
indicated.
We investigated the nature of the activity, designated as
restrictin-P(11) , found in media conditioned by stromal
cells(12) , which causes growth arrest and subsequent cell
death of mouse plasmacytomas and hybridomas. This activity was mediated
by a protein that was purified to homogeneity from medium conditioned
by the stromal cell line MBA-2.1 and was found to have an N-terminal
amino acid sequence indistinguishable from that of activin A (37, 38, 39) which is also known as
follicle-stimulating hormone releasing protein or erythroid
differentiation
factor(39, 43, 44, 45, 46) .
Activin A was found to be expressed by stromal cells(47) . The
molecular mass of monomeric restrictin-P, as deduced from PAGE, was 15
kDa, a size similar to that of monomeric activin A (A-inhibin).
Like activin A, restrictin-P is a dimer of 25 kDa under nonreducing
conditions and loses its biological activity upon reduction (not
shown). Furthermore, recombinant activin A was inhibitory to the MPC-11
plasmacytoma to the same extent as was restrictin-P. It is therefore
concluded that these two molecules are identical.
Restrictin-P in
its purified form killed the factor-dependent hybridoma cell line B9 by
competing with externally added IL-6 or IL-11. On the basis of the
inability of a 270-fold excess of partially purified restrictin-P to
compete with r-Mu-I-IL-6 binding (Fig. 6A) or a 340-fold excess of highly purified
restrictin-P to compete with r-Hu-
I-IL-6 (mutein) (Fig. 6B) binding, it is concluded that restrictin-P
does not exert its effect by competing with IL-6 for high affinity IL-6
ligand binding sites.
The antagonistic effect of restrictin-P is specific to IL-6 and IL-11 since restrictin-P did not affect the growth of other cytokine-dependent cell lines such as 14M1.4 macrophages that depend on M-CSF for growth, MC/9 mastocytoma which are IL-3-dependent or NFS-60, GM-CSF-dependent cells (Fig. 3B).
The strict specificity of killing by restrictin-P of plasmacytomas and hybridomas suggested that the factor detects some molecular machinery characteristic to this cell type. The growth dependence on IL-6 is common to many plasmacytomas and hybridomas. We show here that restrictin-P inhibits the growth of B9 cells by competing with the growth factors obligatory for the survival of the hybridoma. However, some cells, like the MPC-11 clone, are cytokine-independent, but are nonetheless growth-inhibited by restrictin-P. The question raised is whether the mechanism of action of restrictin-P in the case of IL-6-dependent B9 cells is different from that in MPC-11 cells. An alternative possibility is that the restrictin-P/activin A receptor (48) transduces a signal that interferes with IL-6 signaling downstream in the pathway. It has been shown that human myelomas possess intracellular IL-6 and IL-6 receptor, and their growth is triggered by an internal autocrine loop(49) . Restrictin-P may interfere with such a hypothetical internal loop in the MPC-11 cell. One piece of evidence seems to support this notion, i.e. the increased junB expression induced in both B9 and in MPC-11 cells by restrictin-P. We further utilized the HepG2 cells in an attempt to identify a possible interference of restrictin-P in the JAK/STAT pathways involved in IL-6 signaling(42) . Restrictin-P did not have such an effect, and we further noted an increase in STAT activation.
IL-6 is a pleiotropic cytokine that affects cells in different tissues and organs(50, 51, 52, 53, 54) . It is therefore expected that the activity of IL-6 would be tightly regulated. This may occur on the level of expression of the IL-6 gene as a result of activity of other cytokines or a variety of mediators(55, 56, 57) . A more refined control involves interference with the biological activity of IL-6. This may occur due to inactivation of the protein. In U937 cells, membranal peptidyltransferases inactivated the IL-6 fragments by dimerizing them into 16-kDa complexes(58) . A different regulatory mechanism involves a variety of agents that diminish the ability of cells to respond to IL-6 (59, 60, 61, 62, 63) . Natural receptor antagonists to IL-6 have not been isolated, to the best of our knowledge. However, it has been reported that oncostatin M, at high doses, is an IL-6 antagonist in a hepatoma cell model(64) . These cells do not harbor oncostatin M receptors and the factor binds through the gp130 signal transduction transmembrane receptor which is part of the IL-6 receptor complex. A recent study of alveolar macrophages from smokers showed that these cells release upon lipopolysaccharide stimulation a mediator that antagonized the ability of IL-6 to support the growth of B9 cells(65) . This activity was not biochemically isolated. Our results indicate that the pleiotropic cytokine activin A is identical with restrictin-P and that this molecule is an antagonist of IL-6 and IL-11. Since in our experiments restrictin-P acted in subnanogram amounts, it is likely that the function we describe has physiological significance.
Unrelated cell systems were studied, i.e. proliferating hybridomas, differentiating myeloblasts, and activated hepatoma cells. In these different cell systems, the inducer molecule IL-6 caused different biological outcomes, but, nonetheless, restrictin-P similarly antagonized the IL-6 functions implying that restrictin-P may be a universal antagonist of IL-6.
Cytokines that affect hemopoiesis are pleiotropic both from the point of view of target cells and in the biological consequence of their actions. Lineage-specific inducers are rare, and the explanation for the formation of tissue sites in which only one cell type accumulates requires an alternative explanation. It is conceivable that the interplay between inducer molecules and corresponding inhibitors would determine which cell type predominates in a particular tissue site. The theory of restrictins maintains that the growth of each hemopoietic cell type is negatively regulated by a lineage-specific inhibitor (66, 67, 68, 69) . This may be a mechanism to prevent specifically the survival of cells in nondesirable sites. The identification of restrictin-P as a stromal activin A and the demonstration that it is an antagonist of IL-6 and IL-11, which are growth factors for plasma-like cells, provides support to this notion. The antagonist is specific for the growth factor and through this property it would not be restrictive to other cells that depend on alternative growth factors.