©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for the Binding of a Biologically Active Interleukin-2 to Human -Macroglobulin (*)

Luc G. Legrès (1)(§), Franois Pochon (2), Martine Barray (1), Franoise Gay (3), Salem Chouaib , Etienne Delain (1)

From the (1) Laboratoire de Microscopie Cellulaire et Moléculaire, URA-147, Centre National de la Recherche Scientifique, Institut Gustave-Roussy, Rue Camille-Desmoulins, F-94805 Villejuif Cedex, France, (2) U350, Institut National de la Santé et de la Recherche Médicale, Institut Curie Biologie, Université de Paris-Sud Orsay, F-91400 Orsay, France. (3) Groupe Cytokines et Immunité Antitumorale, CJF 94-11, Institut National de la Santé et de la Recherche Médicale, Institut Gustave-Roussy, Rue Camille-Desmoulins, F-94805 Villejuif Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human -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.


INTRODUCTION

Present at high concentrations in human plasma (2-3 g/liter), 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) .

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 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.


EXPERIMENTAL PROCEDURES

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 M

Human native 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 M-IrhIL-2 Complexes by Native PAGE

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.

Studies on the Binding of I-rhIL-2 to Native and Transformed M

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 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 MrhIL-2 Complexes

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 10cells/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.


RESULTS AND DISCUSSION

Our study provides compelling evidence of an interaction between purified human 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 10µ 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) .


FOOTNOTES

*
This work was supported in part by Grant CRE 920905 from the Institut National de la Santé et de la Recherche Médicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of two fellowships, from the Institut Gustave-Roussy (CRC number 92-11) and from La Fondation Jean Dausset (Association des Amis Sciences, 16 Rue Mazarine, Paris, France), successively. To whom correspondence should be addressed.

The abbreviations used are: 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.


ACKNOWLEDGEMENTS

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.


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