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Related Collections Hematopoiesis and Stem Cells Immunobiology Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2001, Vol. 97, No. 1, pp. 183-191 IMMUNOBIOLOGY A soluble form of the murine common  chain is present at high concentrations in vivo and suppresses cytokine signaling Udo Meißner, Horst Blum, Markus Schnare, Martin Röllinghoff, and André Gessner From the Institute for Clinical Microbiology, Immunology and Hygiene, University of Erlangen-Nuremberg, Erlangen, Germany.     Abstract Top Abstract Introduction Materials and methods Results Discussion References The common gamma-chain (c) is a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signal transduction. Western blotting and a newly established enzyme-linked immunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble c (sc) in sera of murine inbred strains. It was demonstrated that purified immune cells, such as T, B, and natural killer cells, and macrophages released this protein after activation. Transfection experiments with cDNA encoding the full-length c showed that shedding of the transmembrane receptor led to the release of sc. The shedding enzymes, however, appeared to be distinct from those cleaving other cytokine receptors because inhibitors of metalloproteases (eg, TAPI) did not influence sc release. In vivo, superantigen-induced stimulation of T cells enhanced sc serum concentrations up to 10-fold within 6 hours. Because these findings demonstrated regulated expression of a yet unknown molecule in the immune response, further experiments were performed to assess the possible function(s) of sc. A physiological role of sc was indicated by its capacity to specifically inhibit cell growth induced by c-dependent cytokines. Mutational analysis revealed that the C-terminus and the WSKWS motif are essential for the cytokine inhibitory effect of the sc and for binding of the molecule to cytokine receptor-expressing cells. Thus, competitive displacement of the transmembrane c by excess sc is the most likely mechanism of cell growth inhibition. It was implied that naturally produced sc is a negative modulator of c-dependent cytokines. (Blood. 2001;97:183-191) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Materials and methods Results Discussion References The murine common gamma-chain (c) is a 64-kd type 1 transmembrane protein of the cytokine receptor family.1 Although the c alone is unable to bind to cytokines, it has been shown to be an essential component of the cell surface receptor complexes of IL-2, IL-4, IL-7, IL-9, and IL-15.1 Interaction of the respective cytokines with the c within the receptor complexes results in the activation of the Janus kinase JAK3,2 which subsequently interacts with Pyk2, a member of focal adhesion tyrosine kinases, leading to the phosphorylation of this protein.3 Recent studies have shown that the c is critical for lymphoid development because genetic defects of either this molecule4 or JAK35 in humans or targeted deletions of the c6,7 or JAK38-10 in mice severely impair the development of T and B cells, leading to severe combined immune deficiency syndromes (SCID).11 Soluble cytokine receptors and growth factor receptors are present as immunomodulatory molecules in body fluids of humans and mice (reviewed in references 12 and 14). Two major nonexclusive mechanisms are responsible for the generation of soluble cytokine receptors: proteolytic cleavage of transmembrane receptors catalyzed mostly by metalloproteases15-19 or de novo synthesis of alternatively spliced mRNAs encoding soluble receptor molecules lacking a transmembrane domain.20-28 Soluble cytokine receptors appear to be potent regulators of cytokine activities. Interference with the binding of cytokines to their membrane receptors and, thus, inhibition of cytokine signaling has been demonstrated to occur in vitro and in vivo.13,14,29 On the other hand, several soluble cytokine receptors have been shown to intensify the activity of their respective cytokines in vivoeg, by acting as carrier moleculesand to protect the cytokine against proteolytic cleavage30 or by forming agonistic complexes with their ligands able to interact with other signal-transducing molecules on cells.31,32 No reports concerning a murine soluble c (sc) have been published so far. In this study we investigated (1) whether sc is present in sera of mice, (2) which cells are able to release this molecule, and (3) what mechanism(s) are responsible for the production of sc. To assess the putative physiological function of the sc, we further analyzed (4) its effects on cytokine-induced cell proliferation and (5) generated recombinant, mutated forms of the sc to address the structure-function relationship of this molecule. Our findings imply that the newly identified sc of the mouse is released by several cell types of the immune system after activation and might be an important negative and highly selective regulator of cytokine responses.     Materials and methods Top Abstract Introduction Materials and methods Results Discussion References Mice and parasites Female mice of the inbred strains BALB/c, C57BL/6, AKR/J, FVB/N, and BALB/c-SCID were obtained from Charles River breeding laboratories (Sulzfeld, Germany). Gld mice were kindly provided by Dr H. Körner from our institute. ICR mice were obtained from RCC Ltd (Füllinsdorf, Switzerland), and c/ mice6 and sex- and age-matched control animals were a generous gift from Dr W. Müller (Institute of Genetics, University of Cologne, Germany). RAG2/ mice33 were a gift from Dr B. Arnold (DKFZ, Heidelberg, Germany). JAK3/ mice10 were a gift from Dr M. Lutz (Department of Dermatology, University of Erlangen-Nuremberg, Germany). Leishmania major promastigotes of the strain MHOM/IL/81/FEBNI were grown in vitro in blood agar cultures as previously described.34 Stationary-phase promastigotes were washed in phosphate-buffered saline, and 2.5 × 106 parasites were injected in a volume of 50 µL intradermally into the right hind footpad. A lysate of L major (LmAg) was prepared by freezing and thawing the pellet 5 times. Borrelia burgdorferi spirochetes (strain N40) were a gift from Dr D. Postic (Institute Pasteur, Paris, France). Cytokines, antibodies, and reagents Recombinant murine IL-4 (LPS content less than 100 pg/µg protein) was purchased from ICChemikalien (Ismaning, Germany), and recombinant murine IL-2, IL-9, and IL-15 (LPS content less than 100 pg/µg protein) were obtained from R&D Systems (Wiesbaden, Germany). IL-3 was produced in supernatants (IL-3-BPV-SN) of transfected X63Ag8-653 cells.35 An antimurine CD3 monoclonal antibody (mAb; clone 145-2C11) was purchased from Pharmingen (Hamburg, Germany). Rat anti-c mAb TUGm2 was a generous gift from Dr Sugamura (Sendai, Japan). Purified, recombinant murine soluble IL-4 receptor was kindly provided by Dr F. Seiler Behringwerke AG (Marburg, Germany). A rat antimouse CD40 mAb (clone FGK) was a generous gift of Dr T. Winkler (Department of Immunology, University of Erlangen-Nuremberg). Streptavidin-fluorescein isothiocyanate (FITC) was purchased from Boehringer Mannheim (Mannheim, Germany). Concanavalin A (conA), PMA, and staphylococcal enterotoxin B were obtained from Sigma (Deisenhofen, Germany). Hygromycin, cyclosporin A, and rapamycin were purchased from Calbiochem (Bad Soden, Germany). The metalloprotease inhibitor TAPI was a kind gift of Dr R. Black (Immunex, Seattle, WA). Antipain, aprotinin, bestatin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), leupeptin, EGTA-Na2, EDTA-Na2, and phosphoramidon were obtained from Boehringer Mannheim. N-Ac-Leu-Leu-norleucine (ALLN) was obtained from Calbiochem, and [4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8,-tetrahydro-1naphtyl)alanine-N-methylamide (KB8301) was purchased from Pharmingen. RNA isolation, reverse transcription, and polymerase chain reaction After RNA extraction from murine lymphocytes with acidic guanidinium thiocyanate,36 cDNA was synthesized with reverse transcriptase (Pharmacia Biotech, Freiburg, Germany) as previously described.37 The cDNA was synthesized in a 40 µL reaction volume containing 50 mmol/L Tris HCl, pH 8.3, 2.5 mmol/L MgCl2, 10 mmol/L dNTP, 1 U Taq polymerase (Pharmacia Biotech), and 100 nmol/L primers during 35 cycles (1 minute denaturation at 94°C, 1 minute annealing at 58°C to 63°C, and 1 minute extension at 72°C). For amplification of c, primers used were c-sense primer 5'-CCCAGAGAAAGAAGAGCAAGCACC-3' and c-antisense primer 5'-AAGGATTGATGTTCAGGCTTCCGG-3'. The resulting polymerase chain reaction (PCR) product was subcloned into the vector pSPT18 (Boehringer Mannheim). For expression of the 255N-Stop variant of the sc, the coding region was amplified by using the c-sense primer and the 255N-Stop-antisense primer 5'-GAAGCTTTCAATTCTCCTCTACAGTATGACTCCC-3'. All samples were analyzed on 1.5% agarose gels containing 0.2 µg/mL ethidium bromide. Cloning, expression, and purification of murine sc The PCR fragment of the 255N-Stop variant was digested with PvuII and HindIII and was cloned into the PvuII/HindIII linearized eucaryotic expression plasmid pCEP4 (Invitrogen, Leek, Netherlands). The episomal pCEP-255N-Stop-sc expression plasmid was transfected into the human embryonic kidney cell line 293/EBNA (Invitrogen) by electroporation in 0.8 mL medium at 900 µF and 260 V using an Easyject electroporation unit (Eurogentec, Seraing, Belgium). Transfected cells, which were selected by the addition of 300 µg/mL hygromycin B and 250µg/mL neomycin, constitutively produced the recombinant pCEP4-255N-Stop-sc. For the expression of sc in SF9 insect cells, the plasmids pCEP4-255N-Stop-sc and pCEP4-GSKGS-255N-Stop-sc were linearized with KpnI, treated with T4 polymerase to generate blunt ends, and subsequently digested with BamHI. The resultant fragments were inserted into the plasmid pVL1392 (Pharmingen), which had been linearized with XbaI, treated with T4 polymerase, and digested with BamHI. The generation of recombinant baculovirus and the infection of SF9 cells were performed as recommended by the manufacturer (BaculoGold system; Pharmingen). For expression in Escherichia coli, the cDNA encoding murine c was amplified using the sense primer 5'-GAAGCTTTCAATTCTCCTCTACAGTATGACTCCC-3' and the antisense primer 5'-GAAGCTTTCAATTCTCCTCTACAGTATGACTCCC-3'. The resulting PCR fragment was purified by gel electrophoresis, digested with BamHI and HindIII, and cloned into the BamHI/HindIII linearized His-tag expression plasmid pQE30 (QIAGEN, Hilden, Germany). The resultant plasmid pQE30-255N-Stop-sc was transformed into the E coli strain C600 (Stratagene, Heidelberg, Germany). To analyze the expression of the 255N-Stop protein, transformed bacterial cells were induced with 2 mmol/L isopropyl--D-thiogalactoside for 3 hours, and total bacterial proteins were analyzed on 15% sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE). The bacterial cell pellets were mixed thoroughly in 2 vol homogenization buffer (50 mmol/L Tris HCl, pH 7.4-10 mmol/L MgCl2-0.2 mol/L KCl-5% glycerol) and passed through a French press (SLM Aminco, Rochester). Cell homogenates were centrifuged at 20 000g for 30 minutes at 4°C. Pellets were washed in 50 mmol/L Tris HCl, pH 7.4-10 mmol/L MgCl2 and treated with 6 mol/L guanidine HCl-0.1 mol/L NaH2PO4-0.01 mol/L Tris, pH 8.0, for 4 hours at room temperature. The extracted protein was purified by affinity chromatography on Ni-NTA-resin (QIAGEN) and eluted by acidification, as recommended by the manufacturer. Purified protein was dialyzed against PBS, pH 7.4. All cloned cDNAs were sequenced using the Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Warrington, United Kingdom) as recommended by the manufacturer. Site-directed mutagenesis Mutations of the 255N-Stop-sc variant were introduced by use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene) following the instructions of the manufacturer. For creating a 244P-Stop-sc, the sense primer 5'-GTAAATGGAGCCAGCCTTAGCACTGGGGGAGTCATACTG-3' and the antisense primer 5'-CAGTATGACTCCCCCAGTGCTAAGGCTGGCTCCATTTAC-3' were used. The 235S-Stop-sc variant was generated with the sense primer 5'-CCCAATCTGTGGAAGTTAGCAACAGTGGAGTAAATGG-3' and the antisense primer 5'-CCATTTACTCCACTGTTGCTAACTTCCACAGATTGGG-3'. For exchange of the WSKWS motif we used the sense primer 5'-GTTCTCAACAGGGGAGTAAAGGGAGCCAGCCTG-3' and the antisense primer 5'-CAGGCTG GCTCCCTTTACTCCCCTGTTGAGAAC-3'. Transient transfection of COS-7.1 cells The cDNA encoding the complete transmembrane murine c was amplified, and the PCR fragment was subcloned into the pSPT18 vector as described above. After digesting the vector with HindIII and SmaI and after treatment of the 5'-end using Klenow enzyme (Pharmacia Biotech), the c-encoding fragment was ligated into the HindIII/NotI-linearized eucaryotic expression plasmid pCDM7.38 After confirmation of the nucleotide sequence, 15 µg purified receptor plasmid DNA was electroporated into 1 × 107 COS-7.1 cells kindly provided by Dr S. Rose-John (Mainz, Germany) in 0.8 mL medium at 900 µF and 260 V in an Easyject electroporation unit (Eurogentec). Transiently transfected cells were used for analysis of c expression 48 to 72 hours after transfection. Control cells were transfected with vector only. Cell culture and proliferation assays EL-4 cells39 were used for FACS analysis of membrane-bound c and cultured as described below. L1/1,40 MC-9,41 and CTLL-242 cells were used for cell proliferation studies. The cells were grown in complete medium (Clicks/RPMI medium; Life Technologies, Eppenstein, Germany), supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 2 mmol/L L-glutamine, 10 mmol/L HEPES, 100 µg/mL penicillin, 60 ng/mL streptomycin, 13 mmol/L NaHCO3, and 5 × 105 mol/L 2-ME, and stimulated in the presence or absence of various concentrations of IL-2, IL-3, IL-4, IL-9, IL-15, and the purified and concentrated supernatants containing the sc or an equally treated control supernatant, respectively, in 96-well flat-bottom microtiter plates (Nunc, Wiesbaden, Germany). The cells were pulsed after 48 hours of culture with [3H] thymidine (18.5 kBq/well; Amersham, Braunschweig, Germany) for 16 hours and processed for beta-counting. B-cell and natural killer-cell enrichment by magnetic cell sorting and analysis by flow cytometry B cells were purified from naive murine spleen cells by magnetic separation with a MACS column (Miltenyi Biotech, Bergisch Gladbach, Germany) according to the manufacturer's instructions. The purity of B cells negatively selected with anti-CD4, anti-CD8, and anti-CD11b mAb-coupled microbeads (Miltenyi Biotech) was 95% to 97% as analyzed by flow cytometry. Purified splenic B cells (1 × 106) were cultured in the presence or absence of stimuli as indicated. Natural killer (NK) cells were purified from naive C57BL/6 spleen cells by magnetic separation using biotinylated DX-5 mAb (Pharmingen) and streptavidin-conjugated Dynabeads (Deutsche Dynal, Hamburg, Germany) according to the manufacturer's instructions. More than 90% of the isolated cells were NK cells, and no T cells were detectable by flow cytometry. Purified splenic NK cells (2.5 × 105) were cultured in the presence or absence of stimuli as indicated. The concentrations of sc were measured by enzyme-linked immunosorbent assay (ELISA) as described below. Preparation and stimulation of macrophages Macrophage monolayers were prepared and tested for purity as described elsewhere.43 Briefly, thioglycolate-elicited peritoneal exudate cells were seeded at a concentration of 1 × 106/mL, and nonadherent cells were removed after 4 hours of incubation by 3 washings. Macrophages were then stimulated with total lysate of B burgdorferi (bacteria-cell ratio, 10:1), conA, or PMA in the respective medium from 6 to 72 hours. Measurements of the sc in the cell culture supernatants were made using ELISA. In vitro stimulation of spleen cells Spleen cells from BALB/c mice were prepared as single-cell suspensions and cultured in vitro for 48 to 72 hours at densities indicated in complete medium in the presence or absence of various concentrations of conA and PMA, respectively. After 12 to 96 hours of culture, the release of the sc into the supernatant was determined by ELISA. SDS-PAGE and Western blot analysis Column elutions containing sc from sera or concentrated culture supernatants containing the recombinant sc were separated under reducing conditions using 15% SDS-PAGE. Immunodetection of sc on nitrocellulose blots was performed with the rabbit antiserum K20 (Santa Cruz Biotechnology, Heidelberg, Germany) followed by a goat antirabbit immunoglobulin conjugated to horseradish peroxidase (Dianova, Hamburg, Germany) and the enhanced chemiluminescence Western blotting system (Amersham). sc ELISA The concentrations of murine sc in sera or supernatants of cells were measured by a 2-site ELISA. Monoclonal antibody 4G3 (Pharmingen) served as capture antibody. Bound sc was detected by rabbit-antimouse c antiserum K20 followed by biotinylated donkey-antirabbit IgG (Dianova), using StreptAB (DAKO, Hamburg, Germany) and PNPP (Sigma) for visualization in an ELISA reader (Dynatech, Denkendorf, Germany). This ELISA detects murine sc in the range of 0.1 to 20 ng/mL. We used recombinant sc expressed in 293/EBNA cells and purified by affinity chromatography at known concentrations for the standardization of this ELISA. sIL-4R ELISA Monoclonal antibodies were raised against BHK-derived recombinant murine sIL-4R by standard techniques and were used for a sandwich ELISA for quantitative determination of murine sIL-4R. This ELISA has a working range of 50 to 2000 pg/mL, as described previously.23 Recombinant murine sIL-4R at known concentrations was used as a standard. Flow cytometry analysis The surface expression of murine c was determined by staining with rat-antimouse c mAb TUGm2 and FITC-labeled goat-antirat antibody (Dianova), analyzed by flow cytometry (FACScan; Becton Dickinson, Heidelberg, Germany). A rat IgG2b antibody of unknown specificity (R35-38; Pharmingen) was used as isotype control. Affinity chromatography Mice were bled from the retro-orbital veinplexus. After allowing the whole blood to coagulate at 4°C for 4 hours, it was centrifuged for 15 minutes at 3000g. Serum was collected and stored at 20°C. Five milliliters serum was diluted in 95 mL PBS. The sc from serum or from supernatants of transfected 293/EBNA cells or infected SF9 insect cells was bound to a Hi-Trap affinity column (Pharmacia Biotech) coupled with anti-c mAb 4G3 (Pharmingen) and eluted with 100 mmol/L glycine, pH 2.7. Concentration of serum-free cell supernatants Serum-free supernatants of transfected 293/EBNA cells were collected and concentrated 10- to 20-fold by ultrafiltration using YM10 membranes (Amicon, Witten, Germany). The concentration of sc was measured using ELISA. Sodium iodide I 125-sc-binding assays Twenty-five micrograms recombinant murine sc was iodinated using Iodogen-coated glass tubes (Pierce Chemical, Rockford, IL) according to the manufacturer's instructions. Iodinated sc was repurified by the use of an anti- c affinity column. Equilibrium binding of 125I-sc to murine IL-4R expressed on human TF-1 cells44 was measured after the incubation of 2 × 106 cells in the presence of 125I-sc and 100 ng/mL IL-4, in a final volume of 200 µL in microfuge tubes at 4°C for 120 minutes. To separate nonbound 125I-sc from cell-bound 125I-sc, the reaction mixture was centrifuged through an oil gradient.44 Specificity of binding was determined by the addition of a 200-fold excess of unlabeled sc.     Results Top Abstract Introduction Materials and methods Results Discussion References Sc can be detected in sera of inbred mouse strains To identify soluble c in the body fluids of mice, BALB/c serum was subjected to affinity chromatography using an anti-c mAb, and eluted proteins were analyzed by SDS-PAGE followed by Western blot analysis. A signal representing a protein with a molecular weight of approximately 56 kd was detected with an anti-c antiserum K20 (Figure 1, lane 1) and with the anti-c mAb 4G3 (data not shown). Bands of comparable length were detected with these antibodies when culture supernatants of conA-stimulated murine spleen cells or supernatants of EL-4 cell stimulated with phorbol esters were tested (data not shown). The molecular weight of the natural sc is approximately twice as high as the calculated mass of the extracellular domain of the c (27.6 kd), which might result from the presence of 6 potential N-glycosylation sites within the extracellular part of the c.45 Therefore, we analyzed the migration behavior of 2 recombinant variants of the sc in parallel. The coding region of the extracellular domain of the c, with a stop codon introduced after Asn255 (2-amino acid N-terminal of the transmembrane region), was inserted into a eucaryotic and a procaryotic expression vector. Recombinant 255N-Stop-sc expressed in eucaryotic cells had a molecular weight similar to that of natural sc (Figure 1, lane 2), whereas the protein expressed in E coli lacking N-glycosylation migrated as a single-band 28 kd (Figure 1, lane 3). View larger version (76K): [in this window] [in a new window]   Figure 1. Visualization of natural and recombinant soluble forms of the membrane-bound c by Western blot. Lane 1: naturally occurring sc concentrated and purified from serum of BALB/c mice by affinity chromatography using the 4G3 mAb. Lane 2: Rsc (255N-Stop-sc) expressed in 293/EBNA cells. Lane 3: Rsc expressed in E coli. Molecular mass markers are indicated on the left. Reduced concentrations of sc in sera of immunocompromised mice as compared with immunocompetent mice Sera taken from at least 5 mice per strain were tested in an sc-ELISA using a recombinant protein of known concentration as a standard (Figure 2). BALB/c, C57BL/6, FVB/NJ, AKR/J, ICR, and gld mice had similar levels of circulating sc (200-250 ng/mL) that were independent of age and sex (data not shown). Immune-deficient animals with severe T- and B-cell defects, such as BALB/c-SCID, JAK3/, or RAG2/ mice, had 2- to 5-fold reduced concentrations of sc in their sera. The specificity of the newly developed sc-ELISA was convincingly demonstrated by the complete absence of detectable protein in the sera of c/ mice (Figure 2). View larger version (12K): [in this window] [in a new window]   Figure 2. Serum levels of the sc in mice. Mice were bled from retro-orbital veins, and sera were analyzed by sc ELISA. Bars represent the mean of at least 5 mice per strain. Sc is released after activation by different subsets of immune cells To define the potential cellular source(s) of the sc, different cell types were stimulated in vitro, and the kinetics of sc release were determined by ELISA. The Th2 cell clone L1/1 released sc with nearly identical kinetics after stimulation with the respective specific antigen (LmAg, mitogens conA, PMA) (Figure 3A), whereas only a slight increase of sc was observed in control cultures. Purified primary splenic B cells released sc with a somewhat later maximum at 72 hours after stimulation with lipopolysaccharide (LPS) (Figure 3B) and after stimulation using an anti-CD40 mAb (clone FGK) (data not shown). Because sera of mice devoid of T and B cells (Figure 2) had reduced but still clearly detectable levels of sc, NK cells were analyzed for their capacity to produce sc. NK cells purified from spleens of mice showed an enhanced production of sc only in the presence of PMA but not with conA, arguing against the presence of significant numbers of contaminating T cells (Figure 3C). Thioglycolate-elicited peritoneal exudate macrophages showed an enhanced release of sc in the presence of LPS and B burgdorferi spirochetes (Figure 3D). Interestingly, the presence of the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15, using the c in their receptor complexes for signal transduction, did not alter the release of sc with any of the cell types tested (data not shown). View larger version (20K): [in this window] [in a new window]   Figure 3. Kinetics of release of the sc into culture supernatants after stimulation of immune cells analyzed by ELISA. (A) Th2 cells of the L major- specific clone L1/1 (4 × 106 cells/mL) were stimulated with PMA (500 ng/mL), conA (5 µg/mL), or soluble L major antigen (LmAg) (3 × 106/well). (B) B cells purified by MACS from spleens of BALB/c mice (1 × 106 cells/mL, 95%-97% purity) were stimulated with PMA (500 ng/mL) or LPS (10 µg/mL). (C) NK cells from C57BL/6 mice were purified using Dynabeads yielding purity greater than 90%. 2.5 × 105 cells/mL were stimulated with PMA or conA, as above. (D) Peritoneal exudate cells (1.5 × 106 cells/mL) of BALB/c mice were stimulated with PMA, conA, LPS as above, or B burgdorferi (10 spirochetes per macrophage). In vivo activation of T cells leads to the release of sc BALB/c mice were treated with staphylococcal enterotoxin B to induce polyclonal T-cell activation in vivo. As shown in Figure 4A, there was a rapid increase of sc in the sera of mice 1 hour after injection that reached a maximum of approximately 3 µg/mL after 6 to 24 hours. These 10-fold elevated sc concentrations returned to basal levels within 6 days. To address the question whether T cells activated during a primary immune response are capable of releasing sc in an antigen-specific manner, BALB/c mice were infected subcutaneously with L major promastigotes. Cells of the lymph nodes draining the site of infection released sc, cumulating in their supernatants during 96 hours of in vitro culture. There was a steep increase from day 3 to day 9 after infection (Figure 4B), a period in which T-cell activation has been shown to take place in this experimental infection. Furthermore, the addition of parasite lysates (LmAg) to cell suspensions already containing parasites enhanced sc production. Together with our finding that L major-specific CD4 T cells respond to their antigen with sc production (Figure 3A), these data strongly argue for sc release by T cells after priming in vivo. View larger version (21K): [in this window] [in a new window]   Figure 4. Release of sc after activation of immune cells in vivo. (A) Increase of sc in sera of BALB/c mice after intraperitoneal injection of 10 µg staphylococcal enterotoxin B per mouse. Sera of 3 mice per time-point were analyzed for their sc content by ELISA. Bars indicate the means of 3 mice. (B) Sc concentrations in supernatants of lymph node cell cultures obtained from BALB/c mice during the early course of an infection with L major. Mice were infected with 2 × 106 L major promastigotes subcutaneously into the right hind footpad. At the time-points indicated, the popliteal lymph nodes were obtained, and their cells (2 × 106 cells/mL) were cultured for 48 and 96 hours in the presence or absence of LmAg. Concentrations of sc were determined by ELISA. Proteolytic shedding of membrane-anchored c molecules is responsible for the release of the sc We next analyzed the mechanisms of sc release. When EL-4 thymoma cells, constitutively expressing high numbers of c on their surfaces (Figure 5A), were stimulated with PMA, up to 40 ng/mL sc were released into the culture medium during the first 6 hours (Figure 5B). A corresponding decrease in the number of cell surface c molecules was demonstrated by the 2-fold reduction of the mean fluorescence in flow cytometry analysis (Figure 5C). A complete re-expression of the transmembrane c was detected within 24 hours of washing the cells to remove the PMA (Figure 5C). As expected, because of the relatively rapid reappearance of c, there was no influence on cytokine responsiveness of T cells after PMA-induced shedding in proliferation tests, which lasted 48 to 72 hours (data not shown). View larger version (35K): [in this window] [in a new window]   Figure 5. Sc is generated by proteolytic shedding of membrane-bound c. (A) On the surfaces of EL-4 cells, c expression was detected by flow cytometry with an isotype-matched control mAb (white graph) or TUGm2 mAb (gray graph). (B) PMA induces the release of sc by EL-4 cells. 2 × 106 cells/mL were cultured in the presence or absence of PMA (250 ng/mL), and at the time-points indicated the sc concentrations in the culture supernatants were measured by ELISA. (C) Decreased expression of membrane-bound c of EL-4 cells in the presence of PMA and re-expression after PMA withdrawal. In parallel to the sc measurements in the supernatants (see B), cell surface expression of c was analyzed by flow cytometry. Forty-eight hours after stimulation, the cells were washed and the re-occurrence of the c was measured. Ratios of the mean fluorescence (MF) of stimulated cells divided by the MF of nonstimulated cells are depicted. (D) Expression of full-length transmembrane c by transfected COS-7.1 cells. Graphs appear as described for panel A. (E) Enhanced release of sc by COS-7.1/c, but not control transfected COS-7.1, cells in the presence of PMA (300 ng/mL). (F) PMA dose-dependent decrease of the MF after staining the surface-bound c on COS-7.1/c cells. Because these findings were compatible with shedding as the main mechanism of sc production, we tested this possibility by transfection experiments. COS-7.1 cells were transiently transfected with an expression vector containing the non-spliceable cDNA for the transmembrane c. The transfectants expressed cell surface c as detected after staining with specific mAb (Figure 5D). Stimulation of the transfected, but not the control COS-7.1, cells resulted in sc production (Figure 5E) accompanied by a reduction of cell surface c expression, as shown by flow cytometry in a PMA dose-dependent manner (Figure 5F). Proteolytic shedding appears to be independent of metalloproteases responsible for the cleavage of other cytokine receptors Pharmacologic inhibitors for several proteases and for immunosuppressive agents and kinase inhibitors were used to define the molecular mechanisms and enzyme(s) responsible for the shedding of membrane-bound c. Spleen cells obtained from BALB/c mice were stimulated with conA, PMA, or anti-CD3 mAb in the presence or absence of inhibitors in nontoxic concentrations, as determined by trypan blue exclusion. As shown in Table 1, none of the compounds tested significantly reduced the concentrations of sc in the respective supernatants. Other inhibitors, such as aprotinin (1 µg/mL), leupeptin (2 µg/mL), pepstatin (1.3 µg/mL), and PMSF (5 µmol/L), also were unable to inhibit the shedding process of c. Of special interest, neither staurosporin, a kinase inhibitor, nor TAPI, a hydroxamic acid-based inhibitor of zinc-metalloproteases,15 influenced the sc release, whereas they clearly suppressed the release of sIL-4R as measured in the same supernatants.                                View this table: [in this window] [in a new window]   Table 1. Influence of protease and kinase inhibitors on the release of sc Inhibition of c-dependent cell growth by a recombinant form of sc To test sc for its biologic functions, a recombinant form of this molecule comprising the extracellular part lacking only the 2 last C-terminal aa (255N-Stop-sc) was analyzed in cell proliferation tests. When sc was added 30 minutes before the c-dependent cytokines, such as IL-2 (Figure 6A), IL-4 (Figure 6B), IL-9 (Figure 6C), and IL-15 (data not shown), a dose-dependent inhibition of cytokine-induced proliferation of T (Figure 6A) and MC-9 mast cells (Figure 6B-C) was observed, irrespective of whether the sc was expressed in 293 EBNA or SF9 insect cells. Proliferation of MC-9 cells after stimulation with IL-3, a c-independent cytokine, remained unaffected, excluding unspecific toxicity of the 255N-Stop-sc preparations. The inhibitory effects of sc were absent when the recombinant protein was added later than 60 minutes after the cytokines (data not shown). View larger version (33K): [in this window] [in a new window]   Figure 6. Recombinant sc inhibits cytokine-induced proliferation in a dose-dependent and specific manner. Cells were preincubated with serial dilutions of concentrated supernatants of 293/EBNA cells, expressing 255N-Stop-sc (black bars) or the equally treated supernatants of vector-transfected cells (control, open bars) 30 minutes before the addition of cytokines. (A) L1/1 T cells were stimulated using IL-2 (2 ng/mL). (B) MC-9 mast cells were stimulated using IL-4 (1 ng/mL), (C) IL-9(1 ng/mL), or (D) IL-3 (1.25% IL-3-BPV-SN). After 48 hours, [3H]thymidine was added, and 16 hours later, cells were harvested and radioactivity was measured in a -counter. The data presented are representative of at least 5 comparable assays per cytokine tested. Bars marked with w/o represent cytokine proliferation in the absence of inhibitor (100% control value). Cytokine-inhibitory effects of sc require the C-terminus and the WSKWS motif of the protein To analyze the structural requirements for cytokine inhibition, we cloned and expressed mutants of the biologically active 255N-Stop-sc, as shown schematically in Figure 7A. Because we considered the interaction of sc with other membrane-bound receptors and not the direct binding of cytokines as the most likely mechanism, the C-terminus of the protein was subjected to site-directed mutagenesis. Three mutated forms of the sc expressed in 293 EBNA cells were secreted, displayed the expected molecular sizes in Western blots, and were bound by the conformation-dependent mAbs 4G3 and 3E12 (not shown). These facts argue against the possible misfolding of these mutated receptor proteins. As shown in Figure 7B, deletion of the last 11 aa (244P-Stop-sc) completely abrogated the growth-inhibitory effect of sc. Of special interest, the GSKGS mutant of the 255N-Stop-sc also did not suppress cytokine-induced cell growth, illustrating the functional importance of the WSKWS motif conserved among the cytokine receptor family.45,46 View larger version (30K): [in this window] [in a new window]   Figure 7. Requirement of the C-terminus and the WSKWS motif of the sc for cytokine-inhibitory activity and binding to cells. (A) Two C-terminal deletions (244P, 235S), and a double-point mutant (GSKGS instead of WSKWS) were generated by site-directed mutagenesis and expressed in 293 EBNA cells. tm, transmembrane domain. (B) CTLL-2 cells were preincubated with different sc variants at concentrations indicated or equally treated control supernatant 30 minutes before adding IL-2 (2 ng/mL). Cell proliferation was determined as described in the legend to Figure 6. (C) Affinity-purified 125I-sc (255N) and IL-4 were added to human TF-1 cells expressing murine IL-4R in the presence or absence of 200-fold excess amounts of unlabeled 255N-Stop-sc, 244P-Stop-sc, or GSKGS-255N-Stop-sc, respectively. The percentages of cell-bound 125I-sc were determined as described in "Materials and methods." Data given are the mean (± SEM) of triplicate determinations. To evaluate the mechanism of cytokine inhibition by the sc cells, binding studies were performed. It has been shown47 by surface plasmon resonance that the extracellular domain of the c binds to a preformed binary IL-4R/IL-4 complex, but not to immobilized IL-4R or IL-4 alone. Thus, affinity-purified sc was labeled with 125I and added together with saturating concentrations of IL-4 to transfected TF-1 cells,44 expressing approximately 20 000 molecules of the mouse IL-4R per cell. As depicted in Figure 7C, 125I-sc bound to the cells in a specific manner because binding competition was seen with excess of the wild-type sc (255 N-Stop-sc). In contrast, an excess of biologically inactive mutants of the sc (244P-Stop-sc, GSKGS mutant) did not interfere with 125I-sc binding to the cells. Thus, our data, together with the findings of Letzelter et al,47 support the hypothesis that interaction of the sc in trimolecular complexes with IL-4 and the IL-4R results in competitive displacement of the transmembrane c, which, in turn, results in an inhibition of cytokine response.     Discussion Top Abstract Introduction Materials and methods Results Discussion References In this study we have shown that a soluble form of the murine c is present in blood at high concentrations. This molecule is released after activation by all immune cells analyzed and is able to inhibit specifically cell proliferation induced by c-dependent cytokines. The size of the newly identified, naturally occurring sc, as determined after purification from sera by Western blotting, was indistinguishable from that of a recombinant form of the nearly complete extracellular domain of the c expressed in eucaryotic cells. Thus, as previously shown for other soluble cytokine receptors, the sc represents a molecule with a C-terminus located in proximity to the transmembrane domain of the cell surface anchored receptor.13,14 To investigate the regulation of sc expression, we developed an ELISA-based assay. The specificity of this assay was convincingly demonstrated by the fact that sc was undetectable in sera and in cell culture supernatants of c/ mice. The constitutive concentration of sc in the sera of immunocompetent mice (more than 200 ng/ mL) was 10- to more than 100-fold higher than those reported previously for the soluble forms of the IL-4R (1-2 ng/mL),44 IL-6R (less than 10 ng/mL),48 or the tumor necrosis factor receptors (TNF-Rs; p55 60 pg/mL, p75 5 ng/mL),49 respectively. Dummer et al50 reported that preparations of soluble IL-2R and IL-2R molecules, enriched from human sera by affinity chromatography, contained proteins showing reactivity with antibodies specific for the c. Thus, it can be assumed that a soluble form of the human c in sera is at least partially complexed with other cytokine receptor molecules. The c is expressed on hematopoietic cells such as granulocytes, B, T, and NK cells,51 and dendritic and Langerhans cells,52 which, therefore represent potential sources for the sc. Indeed, all immune cell types tested in vitro were capable of releasing high amounts of sc after appropriate stimulation. TCR-mediated activation of primary or cloned T cells and stimulation of B cells or macrophages by bacteria or their products was followed by an enhanced release of sc. A very potent stimulus, previously shown to enhance the proteolytic shedding of several other cytokine receptors,15,18,23 is the phorbol ester PMA, demonstrating the critical involvement of PKC-dependent pathways in the process of sc release. Importantly and in contrast to other soluble cytokine receptors, the presence of c-dependent cytokines such as IL-2, IL-4, IL-7, IL-9, and IL-15 did not influence sc production over wide range of concentrations and in all cell types tested. All c-dependent cytokines have been shown to mediate their cell activation by the induction of JAK3 tyrosine phosphorylation.53 Thus our finding that the sc levels in sera of JAK3/ mice were similar compared to those of other mice with severe B- and T-cell defects, such as SCID and RAG2/ mice (Figure 2) support the notion that signaling by the c is not essential for the release of its soluble receptor fragment. The observation that despite the absence of B and T cells sc was detectable in the sera of immunodeficient mice at concentrations up to 100 ng/mL is most likely a consequence of production by NK cells and macrophages/monocytes capable of releasing this molecule (Figure 3). Nevertheless, cells of the specific immune system appear to be the main producers of sc in vivo. The importance of activated T cells as a source for sc was shown by the rapid 10-fold rise in sc levels after polyclonal activation in vivo using the superantigen staphylococcal enterotoxin B. Of special interest, the kinetics of increase was significantly slower than that of cytokines, such as IL-2 and TNF, which show their concentration maximus within the first 4 hours after staphylococcal enterotoxin B injection returning to baseline levels after 8 hours.54 These findings suggest that the processes involved in the up-regulation of sc appear to be distinct from those of cytokine up-regulation and that sc might possess a longer t1/2 in vivo than typical cytokines. However, the elucidation of the exact pharmacokinetics of the sc awaits further studies. Using the well-established infection model of mice with the protozoon parasite L major, we studied the kinetics of sc up-regulation during an anti-infectious immune response. Although only a slight increase of 30% to 50% of sc in sera of infected mice was detected (data not shown), we observed a dramatic increase of sc production when cells of lymph nodes draining the site of infection were analyzed in vitro. The kinetics of expression and the enhancement of sc production after the addition of soluble Leishmania antigens to the cultures strongly argue for parasite-specific T cells being the main producers of the sc. In agreement with this idea, cloned L major-specific Th2 cells were stimulated to release sc after recognition of their specific antigen (Figure 3A). Two main nonmutually exclusive mechanisms for the production of solubilized cell surface molecules have been described. The receptors for TNF, IL-1, IL-2, macrophage-colony-stimulating factor (M-CSF), platelet-derived growth factor (PDGF), and nerve growth factor (NGF) appear to be subject to proteolytic cleavage, resulting in the shedding of the extracellular part of the respective molecules.13,14 On the other hand, alternative splicing of the receptor mRNAs resulting in proteins lacking a transmembrane domain have been described for several cell surface molecules, including the receptors for GM-CSF, G-CSF, IL-4, IL-5, IL-7, IL-9, EGF, LIF, erythropoietin, and thrombopoietin.13,14 However, as analyzed in detail for the sIL-4R of the mouse,23 for one soluble receptor both mechanisms can occur in the same cell but appear to be activated by distinct pathways. Several considerations and observations make shedding the most likely mechanism responsible for sc production. No alternatively spliced message has been described so far, and we found no up-regulation of the c-encoding mRNA by RNAase protection assays after stimulation of T cells, B cells, or mixed spleen cell populations in vitro (Blum and Meiner, unpublished data). Furthermore, there was a reproducible inverse correlation between activation-induced decrease of cell surface c and increasing amounts of sc in the cell culture supernatants. To test directly whether sc is generated by shedding, COS-7.1 cells were transfected with complete cDNA for the transmembrane c. Because these cells produced sc with a molecular weight indistinguishable from that of the recombinantly expressed extracellular domain of the c (255N-Stop-sc) (data not shown), alternative splicing could be excluded as being involved in sc generation at least in the transfectants. Numerous sheddases responsible for the secretion of other cytokine receptors are activated after protein kinase C activation by phorbol esters.55 However, it is still unclear whether the sheddase(s) are directly activated by phosphorylation events56 or whether the phosphorylation of the receptor molecule itself renders it accessible for proteolytic enzymes, as has been suggested for the TNFR.57 We used a number of protease inhibitors to define the sheddase(s) responsible for the cleavage of c. Although TAPI and KB8301, well-characterized inhibitors of zinc-metalloproteases, markedly suppressed sIL-4R production, no inhibition of sc production was observed in the same cultures. Because of a PEST-like sequence in the intracellular domain of the c and a recent publication showing that the protease calpain mediates the intracellular cleavage of c at the PEST-like site,58 we used the calpain inhibitors ALLN and antipain to analyze whether this protease is also involved in the release of the sc. None of the agents tested led to a significant reduction of sc release. Thus, based on these experiments with inhibitors, the shedding protease(s) responsible for c cleavage appears to be distinct from other enzymes identified as essential for the proteolysis of other receptors and cytokines (eg, TNF alpha-converting enzyme).16 Our future experiments aiming to define the C-terminus of the naturally occurring sc and using transfected mutants of the c will help to elucidate the shedding mechanism. An important function of the newly identified sc was found when we analyzed the influence of this molecule on cytokine-induced cell proliferation. Specific inhibition of c-dependent cytokines was observed with sc concentrations similar to those occurring in vivo. In line with a potent immune-regulatory role of the sc, this growth suppression was accompanied by the inhibition of cytokine-induced JAK-STAT activation (Gessner et al, manuscript in preparation). Because no direct interaction of the c with cytokines takes place in the absence of additional cytokine receptor molecules,59 cytokine neutralization in solution by this molecule appears to be unlikely. Soluble gp 130 is the only other cytokine receptor described so far that shows cytokine-suppressive capacity despite its inability to bind to the respective ligands directly. Narazaki et al60 reported that the sgp130 in human sera inhibited IL-6 effects, and they proposed the competition of sgp130 with membrane-bound gp130 in IL-6R complexes as the most likely mechanism of cytokine suppression. To address this possibility, we cloned and expressed variants of the inhibitory sc (255N-Stop-sc) carrying mutations not in the N-terminal, presumably ligand-interacting, but in the C-terminal part. These recombinant proteins were unable to reduce cytokine-induced proliferation, supporting our hypothesis that competitive replacement of the membrane-bound c in excess of the sc leads to a loss of cytokine responsiveness of the cell. The WSXWS motif, previously shown to be critical for the function of the human membrane-bound c,61 also appears to be essential for the interaction of soluble and membrane-bound cytokine receptors. As shown recently by Letzelter et al47 using biosensor techniques, the extracellular domain of the c interacts with binary complexes consisting of IL-4 and IL-4R chain, whereas no binding of the c to the IL-4R chain was detected in the absence of IL-4. Along these lines, we detected specific binding of purified and iodinated sc to cells expressing large numbers of the mouse IL-4 R when IL-4 was present. Thus, sc-mediated cytokine inhibition is most likely due to an association of sc with cytokine-cytokine receptor complexes already preformed on the cell surface. Additional experiments are necessary to precisely define the mechanisms and kinetics of the inhibitory interaction of the sc with cytokine-responsive cells. In conclusion, the shedding of c is a regulated process with 2 important functional consequences, the transient depletion of a cell surface molecule required for the signaling of at least 5 cytokines and the generation of a soluble inhibitor capable of modulating an ongoing immune response. Because the initiation of the c shedding process appears to be independent of cytokines, this mechanism represents paracrine and retrocrine modes of controlling cytokine functions in vivo.     Acknowledgments We thank Dr H. Körner and Dr P. Curley for critical reading of the manuscript.     Footnotes Submitted September 9, 1999; accepted July 28, 2000. Supported by the Deutsche Forschungsgemeinschaft (SFB263, A6) and the Graduiertenkolleg, Immunologische Mechanismen bei Infektion, Entzündung und Autoimmunität (U.M.). U.M. and H.B. have contributed equally to this work. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. 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