Molecular and Functional Characterization of a Soluble Form of Oncostatin M/Interleukin-31 Shared Receptor*>

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulin-like domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.

Activation of the signaling transduction pathways mediated by oncostatin M (OSM) requires the binding of the cytokine to either type I OSM receptor (leukemia inhibitory factor receptor/ gp130) or to type II OSM receptor (OSMR/gp130). In the present work we have developed an enzyme-linked immunosorbent assay detecting a soluble form of OSMR (sOSMR) secreted by glioblastoma, hepatoma, and melanoma tumor cell lines. sOSMR was also present in sera of healthy individuals, with increased levels in multiple myeloma. Molecular cloning of a corresponding cDNA was carried out, and it encoded for a 70-kDa protein consisting of a half cytokine binding domain containing the canonical WSXWS motif, an immunoglobulinlike domain, and the first half of a second cytokine binding domain with cysteines in fixed positions. Analysis of the soluble receptor distribution revealed a preferential expression in lung, liver, pancreas, and placenta. sOSMR was able to bind OSM and interleukin-31 when associated to soluble gp130 or soluble interleukin-31R, respectively, and to neutralize both cytokine properties. We have also shown that OSM could positively regulate the synthesis of its own soluble receptor in tumor cells.
The cytokines of the IL-6 family use two-or three-membrane subunit receptors to form high affinity receptor complexes able to mediate downstream signaling events (13)(14). These receptors belong to the type I cytokine receptors, characterized by the presence of at least one cytokine binding domain (CBD) with conserved cysteine positions and a WSXWS motif (15). All the receptor complexes belonging to the IL-6 cytokine family share the common gp130 signaling receptor subunit in the formation of their multimeric receptors (16). Depending on the ligand, gp130 can either homodimerize in the presence of IL-6 or IL-11 (17,18) or heterodimerize with related type I cytokine receptors such as LIFR, IL-27R, or OSMR when recruited by other members of the IL-6 family of cytokines (19 -21).
In humans, OSM signal transduction occurs via two distinct receptor complexes. The type I OSM receptor consists of the low affinity chain, LIFR, associated to gp130 (19). This type I receptor can indifferently bind LIF or OSM. Through this mechanism, OSM elicits biological activities overlapping with those induced by LIF, such as hepatocyte activation, bone renewal, or the in vitro maintenance of embryonic stem cell phenotype (22).
The type II OSM receptor, specifically recognizing OSM, associates gp130 and the OSMR subunit (23). OSMR is a 150-kDa protein composed in its external portion of a half CBD followed by an immunoglobulin-like domain, a second complete CBD, and then a region consisting of three FnIII domain repeats. The cytoplasmic domain of the receptor contains motifs required for the recruitment of Jak1, Jak2, and Tyk2 as well as of STAT1, STAT3, and STAT5 signaling pathways (24,25).
The interaction of OSM with its specific type II receptor mediates the unique functions of OSM that cannot be mimicked by LIF or other IL-6 family members. Signaling by the type II OSM receptor inhibits the proliferation of a number of tumor cells, including glioblastoma, melanoma, mammary, and prostatic cell lines (1, 2, 26 -28). In addition, OSM potently induces the proliferation of Kaposi sarcoma, fibroblastic, and smooth muscle cells (29 -31). It was recently reported that OSMR could also be recruited by IL-31, a novel cytokine with a skin tropism (32)(33)(34).
Soluble cytokine receptors are involved in the regulation of a number of physiological and pathological situations. They can behave either as agonists or antagonists of cytokine signaling depending on the particular family of cytokines. Soluble cytokine receptors can be generated by different mechanisms, * This study was supported in part by Grant 5176 from the Association pour la Recherche contre le Cancer and by the Post-Genome Program of the Ré gion Pays de la Loire. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. 1  including proteolytic cleavages of the receptor extracellular parts, alternative splicing of RNA transcripts, or cleavage of a glycosylphosphatidylinositol anchor (35). The soluble counterparts of ␣-membrane chains, such as soluble IL-6 or CNTF receptors, are able to potentiate the functional responses to their respective ligands (36,37). In contrast, ␤-chain-derived soluble receptors, such as soluble gp130, neutralize the response to IL-6, IL-11, or CNTF (38). In the present work we have molecularly and functionally characterized a soluble form of the OSM/IL-31 shared receptor.
Serum Samples-Sera from healthy humans were obtained from informed volunteers, and the sera of multiple myeloma-suffering patients were obtained from people who gave their informed consent in agreement with French legislation.
Cloning of sOSMR Spliced Form-Total RNAs from WRL68 hepatoma cell line were extracted by the TRIzol method according to the manufacturer's instructions (Invitrogen). cDNA was amplified with the Advantage polymerase (Clontech, Palo Alto, CA) using 10 nM OSMR sense primer CGGC-CTGCCTACCTGAAAAC and an oligo(dT) primer (Invitrogen). PCR products were cloned in the pGEMT vector (Promega), and the sequencing was performed using an automatic DNA sequencer (Beckman Coulter). To express the recombinant sOSMR, the 3Ј-end of the cDNA was replaced by a V5-His tag and subcloned in the pcDNA3.1D/TOPO-V5-His mammalian expression vector (Invitrogen).
Protein Expression and Purification-The human embryonic kidney 293 cell line was stably transfected with sOSMR-V5-His pcDNA3.1D/TOPO plasmid using the Exgen transfection reagent (Euromedex, Souffelweyersheim, France). Cell superna-  18 anti-OSMR mAbs were tested either as capture antibody (10 g/ml) or as tracer antibody (1 g/ml) to detect soluble OSMR-Fc. In black, signal detected for a 10-ng/ml concentration of OSMR-Fc. In gray, signal detected for a 100-ng/ml concentration of OSMR-Fc. In white, absence of signal. B, ELISA standard curve obtained using the AN-A2 mAb as a capture antibody and the AN-V2 mAb as a tracer antibody. C, absence of cross-reactivity with related or distant cytokine receptors. Drosophila Toll-Fc, CLF-Fc, soluble LIFR, sgp130, soluble CNTFR, sIL-6R, sIL-4R, and OSM were added at 50 ng/ml. tants were submitted to an anion exchange column (Amersham Biosciences) before an affinity purification step using Ni 2ϩ -Sepharose column chromatography (Amersham Biosciences). Purified fractions were desalted by size exclusion before being submitted to SDS-PAGE silver staining and Western blotting analyses. The OSMR-Fc was a fusion protein made of the extracellular part of the membrane receptor coupled to the Fc portion of human IgG1 (34). In Figs. 8B and 9E a fusion protein consisting of the first 428 residues of sOSMR fused to a 40-residue linker and to the Ig domain of gp130 (amino acids 767-817) was used. The Fc and fusion proteins were expressed in COS and human embryonic kidney 293 cell lines, respectively. The culture supernatants were collected and loaded either on a protein A-Sepharose or on a Ni 2ϩ -Sepharose column and the recombinant proteins eluted. Purity and protein determination analyses were carried out by SDS-PAGE and by silver staining.
Reverse Transcription PCR Analyses-cDNAs were synthesized from 2 g of total RNA by random hexamer primers using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Reverse transcription products were subsequently amplified by 35 cycles of PCR using the primers for sOSMR (sense TCTGGGGAAAAGAAACTTTGTAC, antisense TAAG-CAGGGTTCTTACTTGC), by 25 cycles of PCR using the primers for OSMR (sense TCTGGGGAAAAGAAACTTTGTAC, antisense GTACTCGCGCCATGTACTCT), and by 20 cycles of PCR using the primers for GAPDH (sense ACCACAGT-CCATGCCATCAC, antisense TCCACCACCCTGTTGCT-GTA). Amplified products were analyzed by 2% agarose gel electrophoresis.
Real-time Quantitative PCR-Quantitative real-time PCR was carried out using the LightCycler-FastStart DNA Master-PLUS SYBR Green I kit (Roche Diagnostics). The cDNA tissue samples were obtained from Clontech. The reaction components were 1ϫ FastStart DNA MasterPLUS SYBR Green I and 5 nM of the forward and reverse primers for sOSMR (forward CCTTTT-TAACCTGACTCATCG, reverse AGCAGGGTTCTTACTTG-CAT), for OSMR (forward AGATTGAACTCCATGGTGAA, reverse GCTTCAAGTGTGGTGAAGTT), and for GAPDH (forward GAAGGTGAAGGTCGGAGTC, reverse GAAGATGGT-GATGGGATTTC). Data analysis was performed as indicated by Roche using the "Fit Point Method" in the Light Cycler software 3.3 (Roche Diagnostics). Quantification was made using GAPDH as a housekeeping gene.
Immunoprecipitation and Western Blotting Experiments-Cytokines and soluble receptors were preincubated at a final concentration of 5 nM each for 2 h at 37°C before being added to the cells for 10 min. Cells were lysed in SDS sample buffer, sonicated, submitted to SDS-PAGE, and transferred onto an Immobilon membrane. The membranes were subsequently incubated overnight with the primary antibody before being incubated with the appropriate peroxidase-labeled secondary antibody for 1 h. The reaction was visualized by ECL detection according to the manufacturer's instructions (Amersham Biosciences). Membranes were stripped in 0.1 M glycine, pH 2.8, for 2 h before reblotting.
For the co-precipitation experiments, the cytokines were incubated at a concentration of 5 nM in the presence of the same concentration of soluble receptors. After an overnight contact, the proteins were precipitated using Ni 2ϩ beads and analyzed by Western blotting as described above.
Endoglycosidase Treatment-The sOSMR receptor was diluted in 1% Brij 96 lysis buffer and treated with 25 units/ml of N-glycosidase-F (Roche) for 12 h at 37°C before Western blotting analysis.   the culture and the incorporated radioactivity determined by scintillation counting (Packard Topcount luminometer, Meriden, CT).
Flow Cytometry Analysis-Cells were incubated for 30 min at 4°C with the AN-N2 anti-OSMR antibody or an isotype control antibody (IgG1) (10 g/ml) before an incubation step with a phycoerythrin-conjugated anti-mouse antibody. Fluorescence was subsequently analyzed on a FACScalibur flow cytometer from BD Biosciences.

RESULTS
Detection of a Soluble Form of OSMR by ELISA-Eighteen monoclonal antibodies recognizing the soluble OSMR part of an Fc fusion protein were generated and characterized. Each of them was tested in pairs to develop a sandwich ELISA (Fig. 1A). We selected the AN-A2 anti-OSMR mAb as a coating antibody and the AN-V2 anti-OSMR mAb as a tracer antibody. The ELISA allowed the detection of 2.5 ng/ml recombinant soluble OSMR (Fig. 1B) and did not react with any other related soluble receptors (LIFR, gp130, CNTFR, CLF, IL-6R) or irrelevant receptors (soluble Drosophila Toll, soluble human IL-4R) (Fig.  1C). Moreover, addition of OSM to the samples did not interfere with the detection of soluble OSMR in the developed ELISA.
We next looked for a native form of sOSMR by screening culture supernatant from different cell lines. All the tested glioblastoma, hepatoma, and melanoma cell lines secreted high amounts of a soluble form of OSMR with concentrations ranging from 5 to 50 ng/10 6 cells (Fig. 2). In contrast, we could not observe the secretion of sOSMR in neuroblastoma-, choriocarcinoma-, and lymphocyte-derived cell lines. In addition, high levels of sgp130 were detected in the cell lines secreting sOSMR.
We also looked for sOSMR in sera from healthy individuals and from patients suffering from multiple myeloma, a pathology known to induce increased levels of circulating soluble cytokine receptors (41). An average value of 77 ng/ml circulating sOSMR was measured in normal situations that reached a concentration of 288 ng/ml in multiple myeloma patients (Fig. 3). These results revealed the existence of a circulating form of OSMR that can be positively modulated in pathologic situations.

Molecular Cloning and Expression of a Soluble OSMR Splice Variant-
The cloning of a soluble form of OSMR was carried out by reverse transcription PCR starting from the WRL68 hepatoma cell line. A 1185-bp cDNA was isolated and encoded a truncated soluble form of OSMR of 394 amino acids diverging from the membrane form of OSMR by 16 amino acids encoded at the end of exon 8 (Fig. 4, A-C). The soluble receptor sequence included an N-terminal potential hydrophobic signal peptide (exons 1 and 2), a half CBD containing a WSXWS motif (exons 3 and 4) followed by an Ig-like domain (exon 5), and then a half CBD presenting four conserved cysteine residues in fixed position (exons 6 to 7).
Soluble OSMR Tissue Distribution-Tissue distribution was analyzed by real-time quantitative RT-PCR using poly(A) ϩ RNA from different human tissues. Two pairs of primers were used, one specifically detecting the soluble form of receptor and the second one only amplifying the membrane form of OSMR. The specificity of amplified products was controlled on a large panel of cell lines and tissues, and in each case a single band corresponding to the expected molecular weight was observed on gels (data not shown). The soluble form of OSMR was expressed in pancreas, lung, liver, and placenta and weakly in skeletal muscle (Fig. 5). With the exception of heart, preferentially expressing the membrane receptor, the distribution of both forms of OSM receptors was similar in the tested tissues.
Soluble OSMR Expression and Binding of OSM to sgp130-sOSMR Complex-The sOSMR was expressed as a tagged protein (V5-histidine tags) and purified by affinity from culture supernatants of stably transfected cells (Fig. 6A). SDS-PAGE gels and Western blotting analyses of the purified fraction evidenced a 75-kDa polypeptide, corresponding, after subtracting the tag molecular mass, to a mature protein of 70 kDa. Deglycosylation experiments were carried out using the N-glycosidase-F. A shift of 25 kDa was observed in agreement with the predicted occupation of 10 N-glycosylation sites (Fig. 6B).
We next studied interaction between the OSM, sOSMR, and sgp130 (Fig. 7). The soluble receptors were added to OSM or LIF, and the tagged sOSMR was precipitated using Ni 2ϩ beads. The samples were assayed by Western blotting using an anti-OSM antibody to detect its association to sOSMR. The experiments show that OSM specifically recognized sOSMR, with a slight increase of the binding in the presence of sgp130.  DECEMBER 1, 2006 • VOLUME 281 • NUMBER 48

JOURNAL OF BIOLOGICAL CHEMISTRY 36677
Soluble OSMR Neutralizes STAT3 Signaling-We tested the possibility for sOSMR to behave as an antagonist for its ligands. Experiments were carried out using the GO-G-UVM and T98G glioblastoma and A375 melanoma cell lines previously reported to be responsive to OSM (Fig. 8A) (1, 28). Treatment of the cells with OSM plus sgp130 showed a very slight or no decrease in STAT3 phosphorylation compared with the signal observed in the presence of OSM alone, in agreement with the published studies (42). Importantly, the combined addition of both soluble receptors induced a marked decrease of STAT3 phosphorylation in the studied cell lines. This was not observed in the presence of LIF or IL-6, used as controls (Fig. 8B). Recent studies have demonstrated the OSMR involvement, together with Gp130-like receptor (GPL), also known as IL-31R, in the formation of a functional IL-31 receptor complex (32)(33)(34). We tested the possibility for sOSMR to also neutralize an IL-31 response in the T98G glioblastoma cell line (Fig. 8C). Similarly to that observed for OSM, a decrease in STAT3 recruitment by IL-31 was observed when simultaneously adding sOSMR and soluble GPL/IL-31R to the cells. Altogether, these results indicate that a combination of truncated OSMR and gp130, or GPL/ IL-31R, could trap and neutralize OSM and IL-31 responses, respectively.
Soluble OSMR Combined with Soluble gp130 Neutralizes Both Type I and Type II OSM Receptors-In the next experiment, murine IL-3-dependent BA/F3 cells engineered to specifically express type I (gp130/LIFR) or type II (GP130/OSMR) OSM receptors on their surface were used (39). Both cell lines proliferated in a similar manner with the addition of OSM to the cultures (Fig. 9, A and B). A 50% inhibition of the signal was measured when sOSMR and sgp130 were added together to the type I or type II OSM receptor-expressing BAF/3 cell cultures (Fig. 9, C and D). In contrast, no induced inhibition could be detected when type I receptor-expressing cells were grown in the presence of LIF (Fig. 9E). These experiments show that the FIGURE 6. Purification of sOSMR. A, SDS-PAGE analysis of sOSMR purified from a Ni 2ϩ -agarose column. Gel was silver stained, and a Western blotting analysis was performed on parallel lanes using the monoclonal anti-V5 tag antibody. B, Western blot was performed on sOSMR treated for 12 h at 37°C with 1 unit of N-glycosidase-F (N-glyco-F). WB, Western blot. FIGURE 7. OSM-sOSMR interaction requires the presence of sgp130. 5 nM soluble receptors were incubated at 4°C overnight in the presence of 5 nM OSM or LIF as control cytokine before the pull down of V5-His-tagged sOSMR by using Ni 2ϩ beads. Associations to OSM or LIF were detected by using biotinylated polyclonal anti-OSM or anti-LIF antibodies. An anti-V5 antibody coupled to peroxidase was used to control the loading of sOSMR. WB, Western blot. FIGURE 8. sOSMR associated to sgp130 or sGPL/IL-31R neutralizes OSM and IL-31, respectively. A, OSM (5 ng/ml) was preincubated with sIL-4R, sgp130, sOSMR, or sgp130 and sOSMR (200 ng/ml each) for 2 h at 37°C. The GO-G-UVM glioblastoma and the A375 melanoma cell lines were stimulated with the studied proteins for 10 min, and the STAT3 tyrosine phosphorylation levels were determined. B, a fusion protein made of sOSMR linked to a gp130 soluble form neutralized specifically the OSM response. Cytokines (10 ng/ml) were preincubated with 100 ng/ml OSMR-L-gp130 for 2 h at 37°C. Preincubated samples, or cytokines alone, were added for 10 min to the GO-G-UVM glioblastoma cell line, and the STAT3 tyrosine phosphorylation levels were determined. C, IL-31 (10 ng/ml) was preincubated with soluble Fc-tagged GPL (250 ng/ml), sOSMR (200 ng/ml), or a combination of both for 2 h before being added to T98G, an IL-31-responsive glioblastoma cell line. After a 10-min contact, the STAT3 tyrosine phosphorylation levels were determined.
interaction between OSM and sOSMR/sgp130 was sufficient to lower the functional signals mediated by either type I or type II OSM receptors.
OSM Up-regulates Its Own Soluble Receptor-We studied the possibility of tumor cells modulating their sOSMR expression in response to OSM. Experiments were carried out using the A375 melanoma cell line and three glioblastoma cell lines. The expression levels of soluble and membrane forms of OSMR were first determined by reverse transcription PCR (Fig. 10A). A clear induction of the RNA coding for the soluble form of receptor was observed when treating A375, GO-G-UVM, and U87MG cells with OSM. Importantly, no variation in the expression level of the membrane form of the receptor could be evidenced in any studied cell line, suggesting that OSM preferentially up-regulated its soluble form of receptor ( Fig. 10B and data not shown). Similar experiments were then performed at the protein level with the GO-G-UVM and A375 cell lines (Fig. 10C). The obtained results showed an increased sOSMR secretion when the cells were grown in the presence of at least 12.5 ng/ml OSM.
Further experiments were carried out to test the possibility for additional gp130 signaling cytokines (IL-6, IL-11, LIF, CT-1), pro-inflammatory cytokines (interferon ␥, IL-1␣), or anti-inflammatory mediators (dexamethasone, transforming growth factor ␤, IL-10) to modulate sOSMR secretion (Fig.  10D). A positive modulation of sOSMR secretion was only observed in response to its cognate ligand. Together these results demonstrate that OSM positively regulates the secretion of sOSMR, opening the possibility for tumor cells to display a reduced sensitivity to the static activity of the cytokine during an immune response.

DISCUSSION
In the present work we identified an alternatively spliced form of OSM receptor leading to the generation of a soluble form of receptor. Soluble type I cytokine receptors can be generated by different mechanisms, including alternative splicing of mRNA transcripts and proteolytic shedding of receptor ectodomains. Cleavage of glycosylphosphatidylinositol-anchored receptor by phosphatidylinositol-specific phospholipase C was also reported in the case of CNTFR ␣-chain (37).
Soluble IL-6 receptor is generated two different ways, shedding of the external IL-6R portion or alternative splicing, resulting in the secretion of a soluble form of receptor that lacks the transmembrane domain (43)(44)(45). Two large size signaling receptors belonging to the IL-6 family, gp130 and LIFR, have also been described as soluble products (38,46). In these latter cases the soluble receptors have been identified as splice variants of membrane forms. In the present work we have highlighted the existence of a soluble OSMR generated by alternative splicing in intron 8 leading to a stop codon after residue 394. In parallel experiments, we also explored the possibility for soluble OSMR to be simultaneously generated by shedding processes. Preliminary results show an induction of sOSMR, after a phorbol ester cell contact, that is counteracted by a metalloprotease inhibitor treatment. 4 This suggests that, in addition to the exon splicing mechanism, sOSMR can also be generated by proteolytic cleavage, similarly to that previously reported for the soluble IL-6R (43)(44)(45).
The binding of soluble IL-6R-IL-6 complex to membrane gp130 confers an IL-6 signaling capability named "trans-signaling" (47). This phenomenon is also reported for IL-11 or CNTF (37,48). In contrast, sgp130 or soluble LIFR abrogate the signaling mediated by the membrane form of receptors in response to the IL-6 family members (38,46). The present results show that sOSMR also behave as a neutralizing receptor for OSM. 4 C. Diveu, unpublished observations. FIGURE 9. sOSMR associated to sgp130 neutralizes OSM-induced proliferation of OSMR type I-or type II-expressing BA/F3 cell lines. Proliferative response of BA/F3 cells transfected with type II (gp130/OSMR) (A) or with type I (gp130/LIFR) (B) receptors to OSM. Cells were cultured in triplicate with 3-fold dilutions of indicated cytokines. C and D, type II and type I receptor-transfected BA/F3 cells were cultured in the presence of 0.6 ng/ml OSM, 1.5 g/ml sgp130, and 1.5 g/ml sOSMR as indicated. E, 100 ng/ml OSMR-L-gp130 fused protein and 0.1 ng/ml OSM (in black) or 0.1 ng/ml LIF (in gray) were added to type I and type II receptorexpressing BA/F3 cells. After 48 h of culture, a [ 3 H] thymidine pulse was carried out and the incorporated radioactivity determined.
The receptor-transducing chains of the IL-6 family, gp130, LIFR, and OSMR, have a modular organization containing in their ectodomain at least one Ig-like domain, a CBD, and FnIII domains (49 -51). Structural analyses of this family of receptors revealed that high affinity binding of the cytokine to its receptor complex involves on one side the CBD of a first receptor subunit and on the other side the Ig-like domain of the second receptor component (50 -54). We previously reported that OSM binds to the CBD of gp130 and to the Ig-like domain of OSMR (52,55). In the soluble form of OSMR, the CBD located downstream from the Ig-like domain was truncated and stopped after half of the module, suggesting that this truncated portion of receptor is not able to bind a cytokine. The present work supports the idea that the Iglike domain of the soluble OSMR, which remains intact, contributes to its neutralizing potential.
Recent studies from Zymogenetics researchers and from our group reported that OSMR also associate to a gp130-like receptor, GPL or IL-31R, to generate a functional receptor for IL-31 (32)(33)(34). In the present study we have shown that sOSMR together with GPL/IL-31R could also neutralize IL-31. Because the GPL/IL-31R receptor is devoid of Ig-like module and binds IL-31 through its CBD, we can hypothesize that sOSMR on its side might also recognize IL-31 through its Iglike domain.
sOSMR circulating levels have been detected in healthy individuals with an average value of roughly 80 ng/ml, similar to that reported for sgp130 (38). Interestingly, a clear increase was observed in sera from multiple myeloma-suffering patients, a pathology with an important inflammatory component. Analysis of circulating sOSMR in additional inflammatory or leukemia situations would bring further information about these diseases.
The results demonstrate that sOSMR requires association with sgp130 to induce a potent neutralizing response. Interestingly, in tested tumor cell lines a co-expression of both forms of soluble receptors was observed, which was further increased in the presence of OSM. This opens the possibility for tumor cells to protect themselves against the static activities of OSM secreted by T lymphocytes and monocytes during the immune response. A similar process has already been described for Fas ligand. In this case, lung and colon tumor cells inhibited Fas ligand-induced apoptosis by overproducing a decoy receptor (56). A specific neutralization of sOSMR in tumor experimental models might improve the anti-tumoral response to OSM (28).
On the other hand, OSM has been demonstrated to induce severe inflammatory responses, notably by recruiting the hepa- tocytes. Recently, cytokines of the IL-6 family were successfully neutralized either by using antibodies or by fused proteins encompassing truncated soluble receptors or by their association to immunoglobulin Fc fragments, opening the possibility of new treatments in inflammatory pathologies (58 -61). The development of an sOSMR-sgp130 association might also be useful to contribute to reducing the inflammatory response in acute or chronic diseases.
In conclusion, the present work identified a soluble form of OSMR that displays neutralizing properties toward OSM and IL-31 when associated to sgp130 or GPL/IL-31R, respectively. Because OSMR or IL-31R gene inactivation only led to a mild decrease in platelet number or to an absence of phenotype in mice (32,57), the development of neutralizing strategies based on the use of sOSMR in chronic pathologies should bring useful reagents with potentially weak side effects.