From the
Human
Present at high concentrations in human plasma (2-3
g/liter),
As soon as
it was identified as a foremost plasma-binding protein for several
growth factors and interleukins (reviewed in Refs. 7 and 8-12),
interesting hypotheses were proposed regarding the role of
Our study provides compelling evidence of an interaction
between purified human
We are gratefully indebted to Dr. Bernard Escudier
(Unité d'Immunothérapie, IGR, Villejuif, France)
for critical reading and helpful discussion. We also thank Lorna
Saint-Ange for kindly editing the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-macroglobulin (
M),
which irreversibly entraps proteinases through a drastic conformational
change, has also been reported to bind various cytokines. The meaning
of cytokine binding to native and/or transformed
M
molecules is, however, not understood. In an attempt to elucidate this
question, we have studied the interaction of radioiodinated recombinant
human interleukin-2 (
I-rhIL-2) with native and
chymotrypsin (
M-C)- or methylamine-transformed
(
M-MA)
M. Our results show that
native and
M-MA are able to bind
I-rhIL-2, with binding occurring only with the latter in
a covalent manner, whereas the labeled cytokine is proteolyzed when
incubated with
M-entrapped chymotrypsin. The
degradation of uncomplexed
I-rhIL-2 has also been
observed in the presence of trypsin, whereas
I-rhIL-2
bound to
M-MA is protected. Moreover, the
proliferative activity of this cytokine on responsive cells is still
maintained either with native
M- or
M-MA-complexed rhIL-2 in comparison with that observed
with the cytokine alone. Our results, which lead us to consider
M molecules as IL-2-binding proteins, emphasize the
possible role of these molecules as immune response regulators.
M
(
)
is a large
glycoprotein (720 kDa) widely distributed from arthropods
(1) to vertebrates
(2) . The main biological function now
attributed to
M within a unique trapping mechanism
(3) is its nonspecific endoproteinase inhibitory activity. A
proteolytic attack of each subunit of the tetrameric
M
molecule, leading to the breaking of internal thiol ester bonds,
induces a remarkable structural modification of this molecule.
Proteinases, whose activity is then confined to small substrates only
(4) , are irreversibly entrapped. A similar conformational
change in
M is also obtained after nucleophile
hydrolysis of these thiol ester bonds by primary amines, e.g. methylamine, in the absence of proteolytic cleavage. In
nondenaturing polyacrylamide gel electrophoresis (PAGE), native and
proteinase- or methylamine-transformed
M molecules,
which exhibit an obvious difference in mobility, are usually designated
as slow (S) and fast (F) forms, respectively
(5) . The two
existing F-forms cannot be distinguished by PAGE but are easily
differentiated by electron microscopy (EM)
(6) .
M in the regulation of cytokine functions:
M may act by scavenging them through its specific
receptor or by carrying and protecting them from proteinase-induced
degradation. Interleukin-2 (IL-2) is a 15 kDa glycoprotein, secreted by
antigen-activated T lymphocytes
(13) , which regulates the
growth and differentiation of cells belonging to the hematopoietic and
lymphoid lineages. As recombinant human IL-2 (rhIL-2) is now frequently
used as an immunotherapeutic agent in several trials against cancer
(14) , we felt that an important point worth investigating was
its possible interaction with
M. Recently, James and
co-workers
(15) , who demonstrated that
M-MA
could interfere with the detection of IL-2 using certain commercial
cytokine assays, suggested a possible interaction between this cytokine
and
M-MA. The present paper reports the first
demonstration by PAGE analysis of biochemical interactions between
I-rhIL-2 and different forms of
M.
Reagents
I-rhIL-2 (39-52
µCi/µg) was provided by Amersham (Little Chalfont, United
Kingdom). Unlabeled rhIL-2 (Proleukin
, Eurocetus,
Rueil-Malmaison, France) was a generous gift from Dr. Bernard Escudier
(Unité d'Immunothérapie, from the Institut
Gustave-Roussy). Porcine chymotrypsin and trypsin were purchased from
Boehringer (Mannheim, Germany), and methylamine was obtained from Merck
(Darmstadt, Germany). HEPES buffer was 20 m
M HEPES, pH 7.2, 50
m
M NaCl.
Purification of Human Plasma
Human native M
M was prepared
from fresh plasma (Centre National de la Transfusion Sanguine, Les
Ulis, France) by zinc affinity chromatography
(16) .
Chymotrypsin (
M-C)- and methylamine-transformed
(
M-MA)
M molecules were obtained, as
described previously
(17) . The purity and homogeneity of
M solutions were systematically controlled by EM
observation
(6) .
Characterization of
Samples were run on 4% native PAGE in 0.1
M Tris
borate buffer, pH 8.0, for 2-3 h at 250 V. For SDS-PAGE analysis,
samples were treated with 3% SDS in the absence or not of 60
m
M DTT and incubated for 1 h at 37 °C prior to
electrophoretic migration. Gels were stained with Coomassie Brilliant
Blue in methanol:acetic acid (40:10), dried, and autoradiographed.
M-
I
rhIL-2 Complexes by Native
PAGE
Studies on the Binding of
Two and four pmol of
S- and F-forms, respectively, were incubated for 2 h at 37 °C with
0.04 pmol of I-rhIL-2 to
Native and Transformed
M
I-rhIL-2 in a total volume of 10 µl of
HEPES buffer and then subjected to gel electrophoresis.
Effect of Trypsin Treatment on the Binding of
I-rhIL-2 to
M-MA
M-MA (400 n
M)
was first incubated for 2 h at 37 °C with
I-rhIL-2 (4
n
M). The mixture was subsequently treated or not with various
amounts of trypsin for 1 h at 37 °C in HEPES buffer (15 µl
final volume) and run onto nondenaturing PAGE, as described previously.
Control of proteinase digestion was performed in the same conditions
with
I-rhIL-2 alone.
Biological Activity of the
IL-2 activity of the complexes was determined with a
standard T-cell proliferation assay by measuring the uptake of
tritiated thymidine in the IL-2-dependent cell line CTLL-2
(18) , kindly provided by Dr. Didier Fradelizi (INSERM U283,
Hôpital Cochin, Paris, France). Cells were seeded at 4 M
rhIL-2
Complexes
10
cells/well in 100 µl of RPMI 1640 culture medium
(Life Technologies, Inc., Paisley, Scotland) supplemented with 7% fetal
calf serum, 2 m
M
L-glutamine, 1%
penicillin/streptomycin, and 5
10
M
-mercaptoethanol in 96-well flat-bottomed microtiter trays.
Incubations of rhIL-2 were first performed at 37 °C for 3 h in 100
µl either with culture medium or with native and chymotrypsin- or
methylamine-transformed
M. Mixtures were then added to
each well in triplicate. After 24 h of culture at 37 °C in 5%
CO
, the incubation was prolonged for 18 h in the presence
of 100 µCi (10 µl) of [6-
H]thymidine (5
Ci/mmol) (Amersham). Cells were collected on filters with a cell
harvester and the radioactivity was measured in a Beckmann liquid
scintillation counter.
M and
I-rhIL-2.
Indeed, we have observed that
I-rhIL-2 binds to native
M (Fig. 1, lanes 1 and 2) and
to
M-MA ( lanes 5 and 6), whereas
I-rhIL-2 is degraded in the presence of purified (with no
free chymotrypsin)
M-C, as demonstrated by the absence
of radioactivity either in the wells (nonmigrating
I-rhIL-2) or associated with the
M-C
band ( lanes 3 and 4). The remaining proteinase
activity of chymotrypsin was due to the entrapment of the proteinase
within the
M molecule via a mechanism which does not
involve its active site
(3) . As a consequence,
M-complexed proteinases would still be able to degrade
or activate some substrates by proteolysis as already demonstrated for
the conversion of proinsulin into insulin
(19) . Our findings
are therefore at variance with those of Heumann and Vischer
(20) who observed undegraded
I-rhIL-2 in the
presence of
M-chymotrypsin for a broad spectrum of
M-proteinases in which
M-entrapped
trypsin was only efficient when proteolyzing
I-rhIL-2.
Our results, however, indicate that
M could indeed be
an rhIL-2-binding protein and extend the previous observations which
described its relationship with various cytokines
(7) .
Figure 1:
Studies on the binding of
I-rhIL-2 to native and transformed
M.
Samples were processed as described under ``Experimental
Procedures.'' Lane C corresponds to
I-rhIL-2 alone. Lanes 1, 3, 5 and 2, 4, 6 correspond to 2 and 4 pmol, respectively, of native ( lanes 1
and 2) and chymotrypsin ( lanes 3 and 4)- and
methylamine-transformed ( lanes 5 and 6)
M.
The
following results are noteworthy for they avoid ambiguous
interpretations. Using outdated M-MA (stored for
months at 4 °C), we observed another slower migrating band which
could be confused with a native S-form
M in
nondenaturing PAGE (Fig. 2 A). Using EM to control this
old
M-MA preparation we detected associated molecules
of this F-form of
M (Fig. 2 B), which
could account for this slower migration. This additional band
containing dimeric forms of
M-MA was unable to bind
I-rhIL-2, whereas single molecules did. In the same
setting, using outdated
M-C, proteolytic degradation
of
I-rhIL-2 was not observed. Seemingly, the
entrapped-chymotrypsin molecules had lost their activity with time.
I-rhIL-2, which was only detectable in the wells, could
not, however, be bound by
M-C (data not shown). The
above observations underscore the need to both clearly characterize the
conformational state of the
M molecules with which
cytokines react and to verify the purity and homogeneity of the
different
M preparations. EM, associated with PAGE
analysis, should therefore be considered as a valid tool for such
quality control.
Figure 2:
Slow bands are not always the S-form
M. Outdated
M-MA and
I-rhIL-2 were incubated in duplicate. A, PAGE
analysis ( lanes 1 and 2) shows two bands, a faster
band corresponding to
M-MA ( F-form) which
binds
I-rhIL-2 and a slower one which does not ( lanes
3 and 4). B, the EM picture demonstrates that
this old
M-MA preparation contains polymers of
M-MA ( arrows). Scale = 50
nm.
Characterization of the binding of
I-rhIL-2 with SDS-PAGE showed that radioactivity was
still present in the
M-MA band of the sample which had
not been treated with DTT (Fig. 3, lane 3), but was
absent from the reduced sample (Fig. 3, lane 4). We
conclude that the cytokine is covalently bound only to
M-MA through a disulfide bond. It has been established
(21, 22) that, among the three cysteine (Cys) residues
present in the mature form of human IL-2, two are involved in an
intramolecular disulfide bond (at positions 58 and 105) and are
essential for biological activity
(23) , whereas the Cys residue
at position 125, which contains a free sulfhydryl group, is not
important
(23, 24) . Native
M is
transformed by methylamine through a slow process which generates four
SH groups. The Cys-125 residue probably reacts with one of these groups
to form a covalent bond, without compromising the biological activity
of IL-2.
Figure 3:
Covalent
binding. For SDS-PAGE analysis, samples were treated with 3% SDS in the
absence ( lanes 1 and 3) or not ( lanes 2 and
4) of 60 m
M DTT. Lanes 3 and 4 are
autoradiograms. In the presence of SDS, M-MA molecules
migrate as half-molecules (360 kDa), and under reducing conditions, we
observe the complete conversion to the subunits (180
kDa).
To examine whether M-MA is able to protect
rhIL-2 from proteolysis, both were first allowed to form complexes and
then incubated with various amounts of trypsin. Fig. 4shows that
I-rhIL-2 resisted trypsin activity when complexed to
M-MA ( lanes 4-6), whereas free
I-rhIL-2 was completely degraded in the wells ( lanes
4-6) and when incubated alone with the proteinase ( lane
2). We postulate that some trypsin-sensitive sites on the IL-2
molecule become inaccessible when it is bound to
M-MA.
Similar results, already demonstrated for the pro-inflammatory mediator
IL-6
(25) and nerve growth factor
(12) , suggest that
cytokines could be protected by
M from the onslaught
of proteinases.
Figure 4:
Effect
of trypsin treatment on cytokine binding. Complex form between
M-MA and
I-rhIL-2, observed on
autoradiograms before trypsin treatment ( lane 3), is
maintained after treatment with 1.25, 2.50, or 5.00 pmol of trypsin
( lanes 4-6). The profiles of
I-rhIL-2
alone or treated with 2.50 pmol of trypsin are shown as controls in
lanes 1 and 2,
respectively.
Using increasing amounts of unlabeled rhIL-2, we
inhibited I-rhIL-2 binding to
M-MA in a
dose-dependent competitive manner (Fig. 5). Surprisingly, the
data presented in Fig. 6demonstrate a poor
M-MA
binding ratio, for only half of these molecules were able to bind one
rhIL-2 molecule. We wondered whether the rhIL-2 complexed to
M molecules was still biologically active or not.
Results, which are depicted in Fig. 7, show first that whatever the
M form used and without an additional amount of
cytokine, CTLL-2 proliferation was not induced (Fig. 7 A, Exp.
1). When incubated with rhIL-2 ( Exp. 2), as described
under ``Experimental Procedures,'' native
M
or
M-MA stimulated cell proliferation to the same
extent as rhIL-2 alone, whereas a loss of rhIL-2 biological activity
was observed in the presence of
M-C. This last result
further corroborated our preliminary biochemical data which evidenced
cytokine degradation by
M-entrapped chymotrypsin. In
order to exclude any biological effect of nonmigrating (uncomplexed)
rhIL-2, described in our PAGE experiments, native
M or
M-MA was incubated overnight at room temperature
together with a large rhIL-2 molar excess and rhIL-2-complexed
M molecules were then isolated by gel filtration. We
demonstrate that the proliferative activity of either the native
M- or the
M-MA-complexed rhIL-2 is
maintained on CTLL-2 ( B), in a similar manner to that obtained
with the cytokine alone, and we conclude that
M is
potentially able to bind a biologically active rhIL-2.
Figure 5:
Binding competition. Incubation of
M-MA (1.33 µ
M) and
I-rhIL-2
(13.3 n
M) was carried out as described under
``Experimental Procedures'' at a constant
I-rhIL-2:
M-MA (1:100) molar ratio in the
presence of 100-, 200-, 300-, 400-, 1000-, and 1600-fold molar excess
of unlabeled rhIL-2 ( lanes 2-7). Control experiments
with
I-rhIL-2 alone and without unlabeled rhIL-2 are
shown in lane C and lane 1,
respectively.
Figure 6:
Distribution of I-rhIL-2
during
M-MA binding. A mixture of cold rhIL-2 (6.66
µ
M) and
I-rhIL-2 (4.69
10
µ
M) as a tracer was incubated with
M-MA (1.66 µ
M) at 37 °C for 3 h in
HEPES buffer (total count: 58,560 cpm) prior to loading on an AcA-54
gel filtration column (1
15 cm) (Sofracor, Paris, France).
I-rhIL-2 ( open square) co-eluted with a single
peak of
M ( closed square) which was
controlled by EM. The concentration of
M-MA in sample
7 (600 µl) was determined to be 0.38 mg/ml by absorbance at 280 nm
with an associated radioactivity of 2221
cpm.
Figure 7:
CTLL-2
proliferative assay. The biological activity of
M-complexed rhIL-2 was determined by incubation of the
cells with each of the three unpurified
M/rhIL-2
mixtures ( A) and with purified rhIL-2-complexed
M ( B), as described in the legend to Fig. 6.
In histograms A and B,
M
concentration is 240 mg/l. A: Exp. 1, without rhIL-2;
Exp. 2, with rhIL-2 (10 ng/ml).
, medium;
,
native
M;
,
M-MA;
,
M-C. B: a, without rhIL-2;
b, with rhIL-2 (1 ng/ml); c, with purified
rhIL-2-complexed
M.
, medium;
, native
M;
,
M-MA.
Autoradiographic analysis revealed that uncomplexed
I-rhIL-2 did not migrate into the 4% nondenaturing gel.
This phenomenon is partly explained by a similarity between the pH of
the electrophoretic buffer and the isoelectric point of IL-2 which, as
a consequence, fails to enter the gel. Unexpected radioactive bands
were also observed on the autoradiograms as intermediate or faster than
M-MA migrating bands. We felt that these bands merely
reflected a particular type of behavior of
I-rhIL-2 in
our experimental conditions. We therefore performed similar binding
experiments either at pH 6.5 or at pH 9.0 (Fig. 8). We show first
that the labeled cytokine still remained in the wells at both pH
values, but with a reduced radioactive signal at pH 9.0 which is
interpreted as a loss of labeled cytokine from the gel. At pH 6.5
(Fig. 8 A), autoradiography demonstrated lesser
radioactive intensity for the native ( lane 2) than for the
methylamine-transformed ( lane 3)
M bands. We
previously observed that native
M does not bind the
cytokine in a covalent manner. We suggest that
I-rhIL-2
could be released from
M molecules in an acidic
environment, as already described for other cytokines
(26, 27) . In addition, at this pH value, other faster
bands appeared on autoradiography (not detectable in Coomassie
staining) in the presence of native
M. These bands
could be related to the dissociation of
M molecules
into subunits, as already demonstrated by Pochon et al. (28) . In our experiment at pH 8.0 (Fig. 1, lanes
1 and 2), we have also observed an extra radioactive
faster band, not detectable in Coomassie staining, which is also
capable of binding the cytokine. At the present time we have no other
explanation than the possibility of native
M molecules
dissociation. At pH 9.0 (Fig. 8 B), migrating bands, slower
than the native corresponding band, appeared on autoradiography
( lanes 2 and 3) and were comparable with the control
where a similar band was detected ( lane 1). However, cytokine
binding to
M-MA was seemingly unmodified either at pH
6.5 or at pH 9.0. It should be noted that this commercial
I-rhIL-2 is in a nonglycosylated form and thus could be
aggregated and form unspecific radioactive bands, as described above.
These observations, which are relevant to rhIL-2 binding to
M, suggest that environmental variations could
physiologically influence the binding or the release capacity of
M, thus providing further evidence that
M plays a regulatory role in the immune system.
Figure 8:
Unspecific radioactive bands.
M forms (400 n
M) were incubated with
I-rhIL-2 (4 n
M). Native PAGE were processed as
already described either at pH 6.5 ( A) or at pH 9.0
( B). Lanes 1,
I-rhIL-2; lanes
2, native
M +
I-rhIL-2;
lanes 3,
M-MA +
I-rhIL-2.
The
conformational change of the M molecule into the
F-form is accompanied by the exposure of receptor-binding sites
(29) . The internalization of
M is mediated
through high affinity receptors present on the surface of a variety of
cells, including macrophages
(30) . Only
M
F-forms can bind to these receptors and be cleared from the circulation
(31) or delivered to macrophages, resulting in enhanced
M-bound antigen presentation and antibody production,
as related by Pizzo and co-workers
(32, 33) . Cancer
treatments using rhIL-2, which now tends to be administered in lower
doses, either injected alone or combined with immunocompetent cells for
adoptive immunotherapy
(34) , is still hampered by considerable
cytokine degradation and cytokine-induced toxicity. Interactions
between IL-2 and
M have already been reported in a
biological system. Although it has been shown by Hubbard et al. (35) that T-cell proliferation is inhibited when
M-bound trypsin is added to human mixed lymphocyte
cultures, suggesting a decrease in IL-2 biological activity, two other
papers
(36, 37) have reported that the loss of IL-2
activity is due to
M-bound trypsin residual activity
and not the result of a trypsin-induced conformational change in
M. Our study, which at the present time throws further
light on the role of human plasma
M in the regulatory
functions of cytokines, could be of significant clinical interest in
cancer immunotherapy, particularly as rhIL-2 is currently employed in
the treatment of metastatic renal cell carcinoma and melanoma
(34, 38) .
M,
-macroglobulin;
M-MA,
M-methylamine;
M-C,
M-chymotrypsin; rhIL-2, recombinant human
interleukin-2; CTLL-2, cytotoxic T-lymphocytes,
interleukin-2-dependent; DTT, dithiothreitol; EM, electron microscopy;
PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.